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• Published: 04 April 2017

Quantification of bioluminescence from the surface to the deep sea demonstrates its predominance as an ecological trait

• Séverine Martini 1 &
• Steven H. D. Haddock 1  

Scientific Reports volume  7 , Article number:  45750 ( 2017 ) Cite this article

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• Biooceanography
• Marine biology

The capability of animals to emit light, called bioluminescence, is considered to be a major factor in ecological interactions. Because it occurs across diverse taxa, measurements of bioluminescence can be powerful to detect and quantify organisms in the ocean. In this study, 17 years of video observations were recorded by remotely operated vehicles during surveys off the California Coast, from the surface down to 3,900 m depth. More than 350,000 observations are classified for their bioluminescence capability based on literature descriptions. The organisms represented 553 phylogenetic concepts (species, genera or families, at the most precise taxonomic level defined from the images), distributed within 13 broader taxonomic categories. The importance of bioluminescent marine taxa is highlighted in the water column, as we showed that 76% of the observed individuals have bioluminescence capability. More than 97% of Cnidarians were bioluminescent, and 9 of the 13 taxonomic categories were found to be bioluminescent dominant. The percentage of bioluminescent animals is remarkably uniform over depth. Moreover, the proportion of bioluminescent and non-bioluminescent animals within taxonomic groups changes with depth for Ctenophora, Scyphozoa, Chaetognatha, and Crustacea. Given these results, bioluminescence has to be considered an important ecological trait from the surface to the deep-sea.

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Introduction.

In marine environments, the presence of light plays a major role in the spatial distribution of marine communities. However, in the photic zone during nighttime and in the deeper parts of the water column, where sunlight has been absorbed, animals live in perpetual dim light or darkness 1 . Light emitted by organisms is called bioluminescence; this emission of cold light is due to a biologically generated chemiluminescent reaction. It is an active ability to communicate, in contrast to the passive traits of fluorescence or phosphorescence in which photons are absorbed by a tissue or structure and then re-emitted at a different wavelength. Moreover, bioluminescence is known to play many roles in intra- and inter-specific interactions 2 . Due to the wide diversity of organisms using this process, bioluminescence has also been utilized to detect biological activities in the deep ocean 3 , 4 , the presence of pelagic animals 5 , 6 , 7 , 8 , 9 , 10 , 11 , and to evaluate biomass for oceanographic studies 12 . A robust description of the abundance and distribution of organisms able to emit light and their ecological niches in the water column is needed to accurately perform such surveys. To our knowledge, the most complete catalog of known bioluminescent organisms was compiled by Herring 13 and updated more recently 2 , 14 . For coastal environments less than 2.5% of the species are estimated to be bioluminescent 15 , while for pelagic environments, this percentage is considerably higher. Indeed, the earliest studies estimate that bioluminescence occurs in approximately 70% of fish species 16 , and by number of individuals, 90% of fishes observed below 500 m depth in the eastern North Atlantic were said to be bioluminescent 16 , 17 . For decapod shrimp (Crustacea) 80% of individuals from the surface to 500 m depth and 41% between 500 and 1,000 m depth were said to be bioluminescent 17 , 18 . Later studies described bioluminescence capability for a range of jellyfish (Cnidaria) 2 , 19 and established a value of about 90% for species of planktonic siphonophores 20 and ctenophores 21 . Because of this surprisingly high percentage, these estimates for pelagic species have been intensively used since, in scientific publications 22 , 23 , 24 , 25 and for outreach on marine biology. However, these studies were limited by: focusing on certain restricted taxonomic categories (siphonophores, ctenophores, jellyfish); being based on general phylogenetic description of bioluminescence ability within taxa; or using non-quantitative approximations. Well documented data sets on diverse taxa are still needed to evaluate the importance of this capability across marine diversity. A primary interest of understanding the distribution of bioluminescent and non-bioluminescent taxa is to examine ecologic niches with co-occurring bioluminescent taxa, and establish the niches shared with taxa where biological interaction or avoidance could be due to light emission. Another interest of collating this information across a large number of taxa is to identify gaps in our understanding of bioluminescence capability for marine ecosystems. Because depth is the main spatial variable driving the distribution of organisms, the development of deep-sea technologies such as cameras, remotely operated vehicles (ROVs), and non-destructive sampling methods has increased the number of dives and in situ observations in the pelagic ocean.

This study is based on in situ video observations performed by the MBARI ROVs over the last 17 years in the eastern Pacific. Each observation in the videos has been identified taxonomically and its bioluminescence capability has been associated from the literature. Using this information, we quantify the distribution of bioluminescent and non-bioluminescent organisms from the surface to the deep-ocean across 13 taxonomic categories (8 phyla), and 553 phylogenetic concepts (species, genera, families) of organisms observed. Finally, this study shows that bioluminescence is important in the water column, as an ecological trait, spanning the range of depths and the diversity of organisms.

Taxonomic observations

A total of 350,536 water-column observations were annotated from videos taken during 240 ROV dives between 1999 and 2016, with about 3/4 of the data between 2006 and 2012. This large amount of data in number, depth, and the large time coverage gives reliable patterns of the vertical distribution of bioluminescence features, which may be considered largely representative of the deep-ocean. The data set, having been gathered during periodic cruises and only during the day, is less suitable for analysis of seasonal or long-term trends, or subtleties like vertical migrations. This data set focuses only on planktonic or pelagic species; any predominantly benthic organisms, such as echinoderms, anthozoans, and ascidians were pre-filtered from the data set, although they can include bioluminescent entities. For analyzing trends, organisms were grouped into broader taxonomic categories, based on functional groupings and broader bioluminescent patterns (see Materials and Methods) section. For example Cnidaria were split into hydromedusae, siphonophores, and scyphozoans because these three groups are readily identifiable and have different patterns with depth. Chordates were sorted into three categories because fishes are functionally very different from urochordates, and within the urochordates, appendicularians are mainly luminous while Thaliacea (salps and doliolids) are mainly non-luminous. Initially organisms were placed into 14 of these taxonomic categories, but the group Nemertea (about 0.1% of the data set), representing 3 concepts, was excluded from further analyses because of its low numbers of observations. The rest of the 553 concepts belonging to 13 broader taxonomic categories ranged from 0.2% (Pteropoda) to 17.9% (Hydromedusae) of the observations ( Fig. 1 ), and these were further analyzed for trends.

In this Figure, the monophyletic taxa are Cnidaria (Hydromedusae, Siphonophora, and Scyphozoa) with 32.7% of the data, Mollusca (Pteropoda and Cephalopoda) with 1.5% of the data and, Urochordata (Thaliacea and Appendicularia) with 23.9% of the data. Scyphozoa silhouette from http://phylopic.org , by Mali’o Kodis, photograph by Ching ( http://www.flickr.com/photos/36302473@N03/ ) (license https://creativecommons.org/licenses/by/3.0/ ). All other are in the public domain, accessible at http://phylopic.org .

Crustacea are pelagic planktonic species mainly represented in our dataset by mysids, decapods, and euphausiids. Infrequently annotated copepods were excluded due to inability to identify them from videos and inconsistencies in how well organisms smaller than a few mm have been annotated. Ctenophores and cnidarians are the most abundant gelatinous organisms. Within cnidarians, Siphonophora, Hydromedusae, and Scyphozoa have been divided into different taxa in this study based on known differentiated behaviors and distributions. Appendicularia are pelagic tunicates producing a feeding structure called a “house”, known to contain bioluminescent inclusions and to be a significant fraction of the organic material sinking to the oceans depths. Fishes mainly represent the marine ray-finned fishes, but for this analysis we have included sharks, while the Pteropoda is a place-holder for pelagic gastropods and also includes heteropods (non-luminous) and pelagic nudibranchs (often luminescent).

Distribution of bioluminescence over depth

The total number of counts per hour is represented ( Fig. 2a ), for probably bioluminescent and probably non-bioluminescent organisms, after normalization of the data set. The total sum of counts per hour (including bioluminescent, non-bioluminescent from Fig. 2 and undefined, not shown) increased from the surface to the maximum of 411 counts per hour at 350 m depth (bioluminescent and non-bioluminescent, respectively 265 and 131 counts per hour, Fig. 2 , and undefined with 15 counts per hour). Then, the number of counts per hour decreased with depth to the lowest value of 14 counts per hour of operation at 3,650 m depth.

( a ) Number of observations (counts per hour) through the water column for probably non-bioluminescent (non-bioluminescent and unlikely) and probably bioluminescent (bioluminescent and likely) organisms. ( b ) Proportion of bioluminescence capability distributed over depth. In the lower box, the overall percentage of bioluminescent organisms is represented as 76%. The variability of this percentage of bioluminescent capability, depending on the how undefined animals might be assigned, is added on the yellow bar (from 69 to 78%, see text for more details).

Bioluminescent and likely bioluminescent organisms were dominant in the entire water column (in blue, Fig. 2b ), ranging between 48 and 77% of the organisms observed. The non-bioluminescent and unlikely bioluminescent organisms represented a small portion of the observations (in dark grey, Fig. 2b ), ranging between 2 and 35%. These numbers do not total 100% due to animals undefined for bioluminescence, accounting for between 2 and 43% of the observations. Raw numbers of observations in each depth bin were normalized using the amount of time spent at each depth. As might be expected, the upper part of the water column has a lower percentage of undefined organisms than the less known deeper waters. However, when omitting the undefined organisms, in the global data set, probably bioluminescent organisms accounted for 76% of all observations (down, Fig. 2b ), and the probably non-bioluminescent reached 24%. The variability of these percentages over depth is low, and the variability due to undefined organisms is also relatively constant. Indeed, the percentage varies only a small amount, from a low of 69%, if all undefined animals are assumed to be non-luminous, to 78% if they are all assigned as bioluminescent.

Distribution of bioluminescence within taxa

The proportion of bioluminescent observations calculated for each of the 13 main taxonomic categories showed clear taxon-specific trends ( Fig. 3 ). In this part of the analysis, the undefined observations were not taken into account when computing the percentage of probably bioluminescent observations within each taxon. As with the water-column calculations, these numbers are based on in situ observations of organism abundance (total counts), and not on the number of species within each group, meaning that the abundance of some numerically dominant organisms could drive the observed trends. For example, within the Polychaeta, Poeobius meseres , which is bioluminescent, was observed in high abundance at all the sampling stations and dives, especially in the deeper waters, and thus was largely responsible for the pattern seen in the Polychaeta. This worm was found with a maximum abundance at about 1,800 m depth. Other bioluminescent-dominant taxa included Appendicularia (94.2% of probably bioluminescent), Polychaeta (92.9%), Ctenophora (91.8%), and all the subgroups of cnidarians i.e .: Siphonophora (99.7%), Hydromedusae (100.0%) and Scyphozoa (97.6%) were bioluminescent dominant.

The percentages only represent the probably bioluminescent organisms relative to the sum of probably bioluminescent and probably non-bioluminescent ones. The undefined organisms were not taken into account in these percentages. The color of the typography represents the dominance of the capability. Grey bounding boxes show larger taxonomic groups: Cnidaria (Hydromedusae, Siphonophora and Scyphozoa), Mollusca (Pteropoda and Cephalopoda) and Urochordata (Thaliacea and Appendicularia). Scyphozoa silhouette from http://phylopic.org , by Mali’o Kodis, photograph by Ching ( http://www.flickr.com/photos/36302473@N03/ ) (license https://creativecommons.org/licenses/by/3.0/ ). All other are in the public domain, accessible at http://phylopic.org .

In contrast, Rhizaria (34.7% probably bioluminescent), Chaetognatha (11.4%), Pteropoda (6.1%) and Thaliacea (2.4%) were mainly probably non-bioluminescent with a low diversity of identified luminous species. For Chaetognatha, the recently studied Caecosagitta macrocephala and Eukrohnia fowleri were the only two bioluminescent species observed 26 . Only two species of bioluminescent Pteropoda (Haddock, pers. obs.) and four species of bioluminescent Thaliacea were also observed ( Doliolula equus, Paradoliopsis harbisoni, Pseudusa bostigrinus and Pyrosoma atlanticum ).

Each of the three sub-groups of cnidarians are clearly bioluminescent dominant (no Cubozoa, which are probably all non-bioluminescent, were observed). The two groups of urochordates, Thaliacea and Appendicularia, show completely contrasting dominance for bioluminescence capability (2.4 and 94.2% of probably bioluminescent, respectively). It also has to be noted, for Crustacea and fishes, a substantial fraction of the observations remain undefined, representing together about 15% of the total observations for those groups. We explain this further in the discussion.

Distribution of bioluminescence over depth and taxa

The proportion of observations within each taxon shows variability over depth ( Fig. 4 ), taking into account only the probably bioluminescent and probably non-bioluminescent entities.

On the left, taxonomic make-up of the probably non-bioluminescent (including unlikely) observations. On the right, taxonomic components of the probably bioluminescent observations (including likely). The total number of observations differs between the two panels and across depths (see Fig. 2a ), but the proportion between 0 and 1 of each group is represented over depth (0 to 3,900 m) using bins of 100 m. Silhouettes are in the public domain, accessible at http://phylopic.org .

The photic zone, above 100 m depth, shows a unique distribution of taxa for both probably non-bioluminescent and for probably bioluminescent organisms, compared to deeper depths, which have a more uniform taxonomic make-up. For the probably non-bioluminescent taxa, this shallow layer was mainly composed of Thaliacea, Chaetognatha, Ctenophora, and Pteropoda while for the probably bioluminescent taxa Siphonophora, Ctenophora, and Hydromedusae were dominant. Below 100 m, the probably non-bioluminescent taxa were dominated by Chaetognatha, Thaliacea and Crustacea. Chaetognatha were dominant almost continuously, from 0 to 3,900 m depth, while Thaliacea were mainly present shallower than 2,100 m, and Crustacea below 2,100 m. For the probably bioluminescent taxa, below 100 m, a succession of dominant taxa appeared from the surface to the meso- and bathypelagic zones. Firstly, Siphonophora was the most represented bioluminescent group from the surface to about 500 m. Then, the Hydromedusae dominated the distribution from 500 m to 1,500 m followed by Polychaetes, dominant down to 2,250 m depth. In the deepest layer, below 2,250 m depth, the Appendicularia were the most represented bioluminescent taxon, although they were also well represented throughout the entire water column.

Within each taxonomic category, the proportion of observations belonging to each of the 5 classes of bioluminescence capability was tabulated within depth bins across the full range of depth ( Fig. 5 ). Siphonophora, and Polychaeta had a homogeneous distribution with no clear pattern that varied with depth, mainly due to the fact that more than 99% of them are probably bioluminescent with a low portion of undefined ( Fig. 3 ). In contrast, Ctenophora, Scyphozoa, Pteropoda, Chaetognatha, Crustacea, and Thaliacea show large changes in the distribution of bioluminescence capability through the depths. A clear pattern is observed for Crustacea: bioluminescent Crustacea were mainly observed above 500 m depth (krill, mesopelagic shrimp), while the non-bioluminescent ones (isopods, decapods) were observed below 2,500 m with a gap in between. For Ctenophora, Scyphozoa, and Pteropoda the non-bioluminescent observations predominate in the upper part of the water column (above 500, 200 and 1,500 m respectively). Cephalopoda, Polychaeta, and fishes show the same proportion for probably bioluminescent and non-bioluminescent over depth, with differences in the numbers of counts only ( Fig. 6 ). The probably non-bioluminescent Ctenophora and Scyphozoa (and to some extent Chaetognatha on Fig. 6 ) are strongly represented in the epipelagic zone and above 500 m but almost absent below. For Ctenophora, this is attributable to the two non-luminous genera ( Hormiphora and Pleurobrachia ) being constrained to shallow depths, and for Chaetognatha, the only two luminous species mainly occur below 700 m. For Scyphozoa, Chrysaora fuscescens is the main non-bioluminescent species exclusively observed in the upper part of the water column (above 200 m). Figure 6 , showing the number of observations over depth, gives a quantification of the distribution patterns. Although the proportion of non-bioluminescent Scyphozoa is high in the upper part of the water column, the absolute numbers of this taxon remain low ( Figs 1 and 6 ). Similarly, the Crustacea show strong proportional patterns, but the absolute counts of deep observations (below 1,000 m) are relatively low.

The proportion between 0 and 1 of each group is represented using bins of 100 m from 0 to 3,900 m.

The number of counts of animals is normalized per hour for each group and is represented over depth (0 to 3,900 m) using bins of 100 m.

Finally, it is notable that most of the undefined animals, particularly in Hydromedusae, Polychaeta, and fishes occurred in the deeper parts of the water column ( Fig. 5 ), mirroring the pattern seen in the combined observations (Yellow bars in Fig. 2b ). Moreover, because bioluminescent organisms are strongly represented in Hydromedusae and fishes ( Fig. 3 ), it will be of interest to examine deeper representatives of these groups collected in good condition, to discover previously undocumented bioluminescence capabilities.

ROV detection and associated biases

The use of ROVs fitted with high-definition cameras is a powerful way to conduct observations and surveys over long time scales in the deep-sea 27 , 28 . This method does require a great deal of effort, including time at sea aboard a support ship, video annotation, and organism identification, but it provides an unparalleled view of the abundance and diversity of macroscopic deep-sea organisms. Indeed, ROVs provide a large coverage in space, time, and depth with the ability to investigate the deep sea over a dive lasting several hours 29 . Moreover, the accuracy of the data recorded by video camera is also highly valuable for exploration, in several ways. It allows classification to more precise taxonomic levels than acoustical and other methods, and there is the potential to update annotations over time to keep pace with evolving classifications and species descriptions. The archived images also allow the verification of the data, with recognition of artifactual annotations of organisms (dead or sinking animals, misidentification or identification revised by experts) during the data processing.

One shortcoming is that for a few active species, in particular some fishes, crustaceans and cephalopods, the lights (and sounds) of the vehicle may lead to avoidance behavior, and rarely to attraction 30 , 31 , 32 . Such behaviors are known to be a reaction to bright artificial light, motor noise, electrical fields and vehicle-induced water motion 33 , 34 . In particular, potential reaction to light has to be taken in consideration for bioluminescence studies. The degree to which this can bias quantification is variable and dependent on species. In 2016, Ayma et al . 35 , found that fishes would freeze and become motionless in the presence of an ROV, rather than being attracted or fleeing. Some bias is present in our survey, with potentially not all organisms detected by the video cameras. However, the large amount of data collected and the consistency of instruments (3 ROVs and 4 cameras) used over 17 years, and the lack of avoidance response for the majority of organisms analyzed, reinforce the reliability of the survey conducted in this study with the most suitable instrumentation for exploration of the large deep-sea fauna.

When using ROV for camera-based surveys another limitation is the minimum size of organisms that can be recognized. With high-definition video, and depending on the species, this size can be as small as a few millimeters, but typically animals should be larger than a centimeter. This minimum size evolved through the time-span of this study, due to upgrades of the camera sensor and recording at HD resolution. Due to this limit of the ROV for the smallest organisms, this study focused on organisms bigger than one cm. Based on this limitation, copepods (Crustacea) have been removed from the dataset. Indeed, most copepods can only be identified upon close microscopic examination. While this group includes both bioluminescent and non-bioluminescent species, their inclusion would have increased the “undefined’’ group without providing more information on depth-related bioluminescence capability. Ostracods are another group of abundant crustaceans that contain bioluminescent species, but which are too small to be identified using this methodology.

Bioluminescence description for taxa in the literature

While some estimates of the proportion of bioluminescent organisms in the open ocean have been previously published 16 , 17 , 18 , 27 , to our knowledge, there has been no study based on a thoroughly quantified data set for the full range of midwater taxa. Moreover, the most complete list of bioluminescent taxa in the literature was published 30 years ago 13 with few addition since 2 , 14 . Because there is a fairly low-level of activity in deep-sea research, and even less work on potentially bioluminescent organisms in good condition, the rate of discoveries of bioluminescent taxa occurs at a very slow decadal rate. Such a slow rate and lack of studies on bioluminescence as an ecological capability is given the high estimates of bioluminescence capabilities (up to 90%) for fauna living in the deep ocean 27 . Our study quantifies that 76% of the organisms observed have the ability to emit bioluminescence. This value is remarkably consistent throughout a 3,900-meter depth range. Although our work is limited to organisms above one cm in size, and may miss some especially reclusive fish, crustacea and cephalopods due to the escape behavior, this value is the most accurate and consistent current estimate across a very broad depth range. Our results also highlight that for some taxa such as Ctenophora and cnidarians (including Siphonophora, Hydromedusae, and Scyphozoa) this percentage was higher than 90%. In contrast, Chaetognatha, Pteropoda, and Thaliacea showed the opposite pattern, with less than 15% of organisms observed being bioluminescent, and most of those newly documented. In fact, until recently, chaetognaths, doliolids and pteropods were considered to be the three main planktonic groups that had no bioluminescent representatives 13 . Based on the prevalence of bioluminescence capability within Hydromedusae, and because about 9% of observations are organisms with undefined capability, it will be interesting to continue exploration of the full extent of luminescence in this group. One of the interesting patterns was that the uppermost layer contained the predominance of non-bioluminescent species of scyphozoan jellyfish. This shallow group includes the most commonly encountered medusae such as Aurelia (moon jellies) and Chrysaora (sea nettles), which are not bioluminescent. Although they are abundant shallow, they are not a significant portion when considering the water-column as a whole. Deeper, the most abundant scyphozoans are coronate medusae which have been shown to have dramatic bioluminescent displays 36 . It will be very interesting in the future to examine the bioluminescent capabilities of the recently discovered deep Ulmaridae (Scyphozoa), relatives of the moon jellies, such as Tiburonia 37 , Deepstaria , and Stellamedusa 38 .

Representativeness of the water column

The sampled area is located offshore of central California, and stations extent out into the California current, one of the major coastal currents affiliated with upwelling zones 39 and blooms 40 . The California Current is part of the North Pacific Gyre, occupying the northern basin of the Pacific. One of the distinctive features of the sampled zone is the distance to the continental shelf break. In this stretch of coast, the shelf is relatively narrow, so that the abyssal seafloor is within a few hundred km from shore. These results, therefore, which are not limited to one particular canyon, should be somewhat representative of other deep-sea waters, comparable to other well-studied areas such as the Porcupine Abyssal Plain station in the North Atlantic (49°N 16.5°W; 4,800 m) 41 .

Our results, interestingly, showed a low variability in the percentages of probably bioluminescent and probably non-bioluminescent organisms over depth. This study is restricted to daytime observations, the dives being almost exclusively conducted between 06:00 and 19:00 local time. Organisms occurring above the oxygen minimum zone (above 700 meters) may undergo day/night vertical migration through the water column. Interestingly, the organisms living in the twilight zone actively use their bioluminescence during day and night. They do not undergo daily changes in their bioluminescent capabilities like surface-dwelling dinoflagellates and crustaceans. Our results, therefore, should be considered to apply to the daytime depths of these shallower deep-sea taxa, while still representative of the deeper and non-migrating taxa. Examining the effects of organism migrations and chronobiological rhythms will be interesting material for future studies.

Several studies measuring bioluminescence intensity using bathyphotometers 42 , or high sensitivity video cameras 43 , 44 , 45 found a decrease in the bioluminescence intensity recorded with depth. One important implication of our results is that if the proportion of bioluminescent organisms remains stable over depth, as we found, then such decreases are principally related to the decrease of biomass ( Fig. 2a ). In future works, the relationship between such decrease of abundance and the decrease of bioluminescence measured in situ over depth will be interesting to investigate. These future investigations, based on our results, could assess the effectiveness of bulk bioluminescence measurements as a reliable proxy for water-column biomass.

Conclusions

Bioluminescence is frequently viewed as an exotic phenomenon, but its widespread occurrence and the high diversity of organisms with this capability support that it serves many important ecological roles 2 . Our study found that 76% of oceanic marine organisms observed in deep waters offshore of California have the capability of bioluminescence. This percentage is surprisingly stable throughout the water column, from the surface to the deep sea, although the dominant taxonomic groups contributing to this proportion change over depth. In situ measurements of bioluminescence profiles, which decline with depth, are potentially a powerful proxy to detect the changes in biomass with depth and in different water masses. The full extent of bioluminescence capability is yet to be established, especially in the deep sea where continued discoveries await. However, given that the deep ocean being the largest habitat on earth by volume, bioluminescence can certainly be said to be a major ecological trait on earth.

Materials and Methods

Sampling using remotely operated vehicles (rovs).

The data were recorded off California, from nearshore waters to 300 km offshore, (latitude from 34.23° to 37.00°N and longitude from 125.02° to 121.73°W) during 240 cruises exploring down to 3,900 m depth ( Fig. 7 ). The study covered the water column within diverse areas from the Monterey canyon to the abyssal plain, and from relatively shallow coastal waters to deep pelagic habitats. During the 17 years sampled, from March 1999 to June 2016, several ROVs were used (Tiburon, Doc Ricketts, Ventana) and 4 cameras were mounted (Panasonic 3-chip, Sony 3-chip, Ikegami HDL40 and Sony HDTV). The focus distance was defined as 1.5 m from the camera. The typical volume observed by the camera varied between 1.2 and 3 m 3 during this period, although our normalization method is not dependent on this value. The ROV video transects were annotated by staff experts using the Monterey Bay Aquarium Research Institute’s open source Video Annotation Reference System (VARS) 46 for database entry. Each observation of an animal was logged as a concept, defined at its most specific phylogenetic level observable from video, along with concurrent physical parameters (depth, location). After filtering and quality checks, the final analyzed data set included 350,536 entities within 553 taxonomic concepts (species or higher).

Map of the eastern Pacific Ocean and California coast showing the sampling stations (orange dots) from March 1999 to June 2016 in the greater Monterey Bay region. The map is based on NOAA bathymetry ( https://maps.ngdc.noaa.gov/viewers/bathymetry/ ), and sampling stations have been represented using R software 47 .

Data treatment

The observations’ depths were discretized into 100-m bins from 0 to 3,900 m. Because the ROV spends less time at the deepest depths, in order to obtain comparable values over depth independent of the time spent for observation, the data were normalized by the total time spent (in hour) per 100-m depth bin. This study focuses on the water column, so benthic taxa were removed from the observations. Data treatment has been performed using Python scripts for retrieval and normalization, and R version 3.3.1 47 for stats and plotting.

Bioluminescence capability attribution

A database of concepts (taxonomic entities) was annotated for the capability of bioluminescence. The capability was classified into one of the following five categories: bioluminescent, likely, undefined, unlikely, and non-bioluminescent, Table 1 . Those descriptions are mainly based on previous literature 2 , 13 , 14 and supplemented with additional unpublished discoveries and observations since (Haddock, pers. obs.). They have been collated for each taxon and are accessible online through the “Deep Sea Guide’’ from MBARI ( http://dsg.mbari.org/dsg/home ).

For classifying an organism’s capability, as an example, Aequorea was defined as bioluminescent 48 . On the opposite end, for the non-bioluminescent category, Pleurobrachia was described as non-bioluminescent based on the literature 21 . An example from the undefined category is the Hydromedusa Ptychogastria that has never been described for this capability. The categories with the most likely and unlikely observations are Ctenophora (comb jellies), Chaetognatha (arrow worms), and Appendicularia (larvaceans). In the case of Ctenophora, all members examined are luminous except for certain benthic species (not included in this study) and the genera Pleurobrachia and Hormiphora , which are restricted to fairly shallow waters. The deep-sea ctenophores that could not be identified to a precise taxonomic level are mostly species that have not been given names yet. These are all luminous, so when there are undefined ctenophores from deeper depths it is likely that they are luminous species. For chaetognaths, the inverse is true: nearly all are non-luminous except for two orange-colored deep-living species. If a chaetognath therefore was not specifically identified, then it is unlikely to be one of these two distinct luminous species, and therefore it is catalogued as unlikely . Non-specific appendicularians observed are mainly small animals, which are most abundantly in the luminous genus Oikopleura , and they are visible due to the presence of their mucus house, which acts as a particle accumulator. For these there is a likely probability that the observed and non-described appendicularians are bioluminescent.

In this work and several of the subsequent plots, the bioluminescent and likely-bioluminescent were grouped into “probably bioluminescent’’ and the non-bioluminescent and unlikely-bioluminescent were grouped into “probably non-bioluminescent’’. Data sets and the script (Rmarkdown under R-Studio) of the representations are available as Supplementary Information (S1 to S3 datasets).

Additional Information

How to cite this article: Martini, S. and Haddock, S. H. D. Quantification of bioluminescence from the surface to the deep sea demonstrates its predominance as an ecological trait. Sci. Rep. 7 , 45750; doi: 10.1038/srep45750 (2017).

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Acknowledgements

The authors thank Kyra Schlining, Susan Von Thun, Brian Schlining, Nancy Jacobsen Stout, and Linda Kuhnz for annotations and the implementation of the VARS database, and the ROV and ship crews for their expert surveys. Monique Messié, Darrin Schultz, Manabu Bessho, and Anela Choy provided helpful discussions. This work is supported by the David and Lucile Packard Foundation and S. Martini is supported in part by a grant from the Bettencourt-Schueller Foundation.

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S.H. conducted the dives and collated the dataset, S.M. analyzed the data, S.M and S.H. wrote the manuscript.

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Martini, S., Haddock, S. Quantification of bioluminescence from the surface to the deep sea demonstrates its predominance as an ecological trait. Sci Rep 7 , 45750 (2017). https://doi.org/10.1038/srep45750

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Original research article, bioluminescence of the largest luminous vertebrate, the kitefin shark, dalatias licha : first insights and comparative aspects.

• 1 Marine Biology Laboratory, Earth and Life Institute, Université catholique de Louvain – UCLouvain, Louvain-la-Neuve, Belgium
• 2 National Institute of Water and Atmospheric Research (NIWA), Wellington, New Zealand

Bioluminescence has often been seen as a spectacular yet uncommon event at sea but considering the vastness of the deep sea and the occurrence of luminous organisms in this zone, it is now more and more obvious that producing light at depth must play an important role structuring the biggest ecosystem on our planet. Three species of deepwater sharks ( Dalatias licha , Etmopterus lucifer , and Etmopterus granulosus ) were collected from the Chatham Rise, off New Zealand, and for the first time, we documented their luminescence. Comparison of glowing shark pictures, combined with histological description of light organs and hormonal control analysis, highlight the evolutive conservation of the bioluminescence process within Dalatiidae and Etmopteridae. A special emphasis is placed on the luminescence of D. licha , the largest known luminous vertebrate. This first experimental study of three luminous shark species from New Zealand provides an insight into the diversity of shark bioluminescence and highlights the need for more research to help understand these unusual deep-sea inhabitants: the glowing sharks.

Introduction

Bioluminescence, defined as the production of visible light by living organisms, is a widespread phenomenon mainly encountered among various marine taxa ( Widder, 1999 ; Haddock et al., 2010 ). This living light, also called cold light, occurs through a biochemical reaction; the oxidation of a substrate, a luciferin, by an enzyme, the luciferase, or through a stabilized complex called photoprotein ( Shimomura, 2006 ). Among Squaliformes, bioluminescence is documented for two deep-sea families: Dalatiidae and Etmopteridae ( Claes and Mallefet, 2009b ; Straube et al., 2015 ). A third family, Somniosidae was recently suggested to also contain a luminous species, Zameus squamulosus (Günther, 1877), based on density and upper view of putative light organs (i.e., photophores) ( Straube et al., 2015 ), new results brought clear evidence Z. squamulosus being a luminous species ( Duchatelet et al., 2021 ). The first mentions of shark light emission date back to the nineteenth century ( Bennett, 1840 ; Johann, 1899 ), but it is only recently that bioluminescence studies, focusing on physiological control, and photophore morphology and function, have been developed. These studies investigated bioluminescence in three etmopterids, Etmopterus spinax (Linnaeus, 1758), Etmopterus molleri (Whitley, 1939), Etmopterus splendidus (Yano, 1988), and one dalatiid, Squaliolus aliae (Teng, 1959) (e.g., Claes and Mallefet, 2009b , c , 2015 ; Claes et al., 2010a , 2011b , 2012 ; Renwart et al., 2014 , 2015 ; Duchatelet et al., 2019b , 2020b ). Luminous sharks appear to produce blue-green light (between 455 and 486 nm; Claes et al., 2014a ) for multiple purposes, such as counterillumination ( Claes et al., 2010a ), aposematism ( Claes et al., 2013 ; Duchatelet et al., 2019b ), and conspecific recognition ( Claes et al., 2014a , 2015 ). Luminescence is achieved via thousands of photophores located within the epidermis. Each photophore is composed of a cup-shaped layer of pigmented cells encapsulating one to more than twelve photogenic cells (i.e., photocytes) and topped by one or more lens cells. In E. spinax , a guanine crystal reflector structure is located between the cup-shaped pigmented layer and the photocyte ( Renwart et al., 2014 , 2015 ). Photophores also display an iris-like structure (ILS), composed mainly of chromatophores, between the photocytes and the lens cells ( Renwart et al., 2014 ; Duchatelet et al., 2020b ). Recently, studies of the luminous system of E. spinax failed to identify the reactive compounds underlying the emission of light (i.e., luciferin/luciferase or photoprotein) ( Renwart and Mallefet, 2013 ). Moreover, it has been demonstrated that shark luminescence is not due to symbiotic luminous bacteria ( Duchatelet et al., 2019a ). Therefore, the nature of the shark luminous system remains enigmatic.

In Metazoans, sharks are the only known bioluminescent organisms to hormonally control light emission. For the studied species, researchers have demonstrated the involvement of several hormones in the control of light emission: melatonin (MT) triggers light production, while alpha-melanocyte-stimulating (α-MSH) and adrenocorticotropic hormones (ACTH) inhibit it ( Claes and Mallefet, 2009c ; Duchatelet et al., 2020b ). Prolactin triggers brighter and faster light emission than MT in Etmopteridae ( Claes and Mallefet, 2009c ; Claes et al., 2011b ), while this hormone inhibits light production in S. aliae ( Claes et al., 2012 ). More recently, in silico mRNA sequences and expression sites of MT and α-MSH/ACTH receptors were highlighted within the photophores, but neither mRNA sequences nor protein presence was found for the prolactin receptor ( Duchatelet et al., 2020a ). Other molecules, such as nitric oxide or γ-aminobutyric acid, also exhibited modulatory effects on light emission in some Etmopteridae ( Claes et al., 2010b , 2011a ). Finally, an extraocular opsin (Es-Opn3) has been demonstrated to be involved in a secondary control targeting the ILS and modulating the aperture of this pigmented structure acting as a light organ shutter ( Duchatelet et al., 2020c ). To establish the conservation of photophore morphology and the control of hormonal light emission in the evolution of luminous Squaliformes, increasing the knowledge on bioluminescent sharks is crucial.

While the majority of Squaliformes never reach more than 60 cm in adulthood, the kitefin shark (also named seal shark or black shark), Dalatias licha (Bonnaterre, 1788), can grow to 180 cm ( Compagno, 1984 ; Roberts et al., 2015 ). This giant holobenthic dalatiid has a worldwide distribution at depths ranging from 50 to 1800 m but it is usually found in depths below 300 m ( Compagno, 1984 ; Roberts et al., 2015 ). Recently, through baited-remote video and muscle enzymatic activity analysis, D. licha was suggested to be one of the slowest moving elasmobranch species ( Pinte et al., 2020 ). Reif (1985) assumed that this shark is luminous as it presents pavement-like placoid scales at the ventral side of the body like the related cookie cutter shark, Isistius brasiliensis (Quoy and Gaimard, 1824) ( Reif, 1985 ; Widder, 1998 ; Delroisse et al., 2021 ). Nevertheless, no clear evidence has been put forward to confirm its luminescence status.

The diet of the kitefin shark is mainly composed of small demersal sharks such as lanternsharks (Etmopteridae), gulpersharks (Centrophoridae), and catsharks (Scyliorhinidae), followed by demersal fishes, crustaceans, and cephalopods ( Macpherson, 1980 ; Matallanas, 1982 ; Dunn et al., 2010 ; Navarro et al., 2014 ). Chunks of large fast swimming epipelagic fishes have been also reported in the stomach contents of kitefin sharks ( Matallanas, 1982 ), similar to what is observed for I. brasiliensis ( Jones, 1971 ; Muñoz-Chápuli et al., 1988 ; Papastamatiou et al., 2010 ).

Along the coast of New Zealand, D. licha inhabit waters where at least six lanternshark species have been reported: E. lucifer (Jordan and Snyder, 1902), E. granulosus (Günther, 1880), Etmopterus molleri , Etmopterus pusillus (Lowe, 1839), Etmopterus unicolor (Engelhardt, 1912), and Etmopterus viator (Straube, 2011) ( Roberts et al., 2015 ). Photophores have been observed for these species ( Ohshima, 1911 ; Last and Stevens, 1994 ; Tracey and Shearer, 2002 ; Straube et al., 2011 ), but bioluminescence has only been confirmed for Etmopterus molleri ( Claes and Mallefet, 2015 ). The blackbelly lanternshark ( E. lucifer ) and the southern lanternshark ( E. granulosus ) are the most common shark by-catch species in New Zealand deep-sea trawl fisheries ( Blackwell, 2010 ). Studying light emission of the kitefin shark, the blackbelly lanternshark, and the southern lanternshark, might increase our understanding of their bioluminescence functions, and possible prey-predation relationships between these species.

Here, organization, morphology, density, and physiological control of kitefin shark photophores were investigated. To determine if this species displays the same photophore structure and hormonal control, a comparative analysis was performed on the two most abundant New Zealand lanternshark species, E. lucifer and E. granulosus . Results are compared to previously studied dalatiids and etmopterids. Homogeneity of light emission control among luminous elasmobranch and photophore structures among each shark families are observed, strengthening a conservative evolution of light emission capabilities among sharks. These observations and results raise questions on the luminescence role for the largest luminous vertebrate. The use of counterillumination for this giant luminous shark is here suggested to be co-opted for a camouflage-type approach as a predatory tool.

Materials and Methods

Specimen sampling.

Shark specimens were captured during the Chatham Rise Trawl survey by the R.V. Tangaroa in January 2020 off the coast of eastern New Zealand. The survey used the same eight-seam hoki bottom trawl and survey methodology that was used on previous surveys ( Hurst et al., 1992 ; Stevens et al., 2018 ). The net has 100 m sweeps, 50 m bridles, 12 m backstrops, 58.8 m groundrope, 45 m headline, and 60 mm codend mesh. The trawl doors were Super Vee type with an area of 6.1 m 2 .

The following depth range information are available: D. licha – mean maximal depth 678 ± 26 m (min-max 443–997 m); E. lucifer – mean maximal depth 542 ± 8 m [min-max 235–1078 m]; E. granulosus – mean maximal depth 903 ± 13 m (min-max 498–1269 m).

A total of 37 D. licha [40.9–138.0 cm total length (TL)], 304 E. lucifer (16.2–53.2 cm TL), and 281 E. granulosus (19.3–75.6 cm TL) were captured on the survey, of which 13 D. licha , 7 E. lucifer , and 4 E. granulosus were used for bioluminescence studies. Each specimen was maintained in a tank with fresh cold sea water in a dark cold room until manipulation. Each shark was sexed, measured, weighed ( Supplementary Table 1 ) and photographed in dim daylight and in dark conditions using Sony α7SII camera before having a full incision of the spinal cord at the level of the first vertebrae, according to the European regulation for animal research handling. Ventral skin of a specimen of S. aliae and I. brasiliensis , collected, respectively, as in Delroisse et al. (2021) and Duchatelet et al. (2020b) , were used for dalatiid comparative photophore histology.

Photophore Histology and Density

Skin patches of 3 cm 2 were dissected from different locations along the body of D. licha specimens (i.e., rostral, mandibular, pecto-ventral, pectoral, ventral, dorsal, dorsal fin, pelvic, flank, infra-caudal, precaudal, and caudal zones; Figure 1A ) to assess photophore presence, size and densities. Skin patches were fixed in 4% formalin at least overnight before being transferred to phosphate buffer saline (PBS). Skin patches were observed and photographed under a transmitted light microscope (Leitz Diaplan, Germany) coupled with a ToupCam camera (UCMOS Series C-mount USB2.0 CMOS camera, ToupTek, Zhejiang, China). Photophore densities (per mm 2 ) and mean diameter ( n = 30 or 50 zones per species) were also measured on the two etmopterid species using the same protocol ( Supplementary Figure 1 ).

Figure 1. Dalatias licha photophore visualization and density measurements. (A) Photophore densities for each studied zone along the shark body. (B) Representation of the dorso-ventral photophore density gradient. Black-dotted photophores (red arrowhead) observed between the placoid scales (delimited areas) at the (C) rostral and (D) ventral areas. Rostral area presents specific leaf-shaped placoid scales, while ventral area harbors typical pavement type placoid scales. (E) Close-up of the black circular-shaped photophores within the integument surrounding the ventral placoid scales. d, placoid scale; e, epidermis; m, melanophore; p, photophore. (F) Photophore density variation across the studied zones. Different lettering indicates statistical differences. All density values are expressed as mean ± SEM.

In parallel, skin patches of D. licha , E. lucifer , E. granulosus , S. aliae , and I. brasiliensis were used to perform histological sections across the photogenic organ. Skin tissues were bathed for 7 days in decalcifying solution (OsteoRAL, Fast decalcifier for Large Anatomical Specimens, RAL Diagnostics, France) with constant agitation and renewal of the solution every 2 days, rinsed in PBS, and placed in PBS with increasing concentrations of sucrose (10% for 1 h, 20% for 1 h, and 30% overnight). Tissues were then embedded in optimal cutting temperature compound (O.C.T. compound, Tissue-Tek, Netherlands) and rapidly frozen at −80°C. Sections of 10 μm were obtained with a cryostat microtome (CM3050S, Leica, Solms, Germany). Sections were placed on coated Superfrost slides (Thermo Scientific) and left overnight to dry. All sections were observed under a transmitted light microscope (Leitz Diaplan) equipped with a ToupCam camera (ToupTek).

Pharmacological Studies

In addition to the skin patches used for histology, round skin patches were dissected from the ventral luminous area of each shark using a metal cap driller (6 mm diameter) as described in Duchatelet et al. (2020b) . Freshly dissected patches were rinsed and kept in shark saline [292 mmol L –1 NaCl, 3.2 mmol L –1 KCl, 5 mmol L –1 CaCl 2 , 0.6 mmol L –1 MgSO 4 , 1.6 mmol L –1 Na 2 SO 4 , 300 mmol L –1 urea, 150 mmol L –1 trimethylamine N-oxide, 10 mmol L –1 glucose, 6 mmol L –1 NaHCO 3 ; total osmolarity: 1.080 mOsmol; pH 7.7 ( Bernal et al., 2005 )] at 4°C in dark conditions before being used for pharmacological tests.

Hormones known to trigger or inhibit light emission in luminous elasmobranchs were applied ( Claes and Mallefet, 2009c ; Duchatelet et al., 2020b ). Here, evaluations of the effect of MT, α-MSH and ACTH were conducted for the first time on the dalatiid species, D. licha , and the etmopterid species, E. lucifer , and E. granulosus .

Experiments were first conducted on 10 D. licha specimens. To obtain a dose response curve for MT application, three different concentrations of MT (i.e., 10 –6 , 10 –7 , 10 –8 mol L –1 ) were used. Skin patches were immersed in 200 μL of MT solution (either 10 –6 , 10 –7 , 10 –8 mol L –1 ). To analyze the effect of α-MSH and ACTH on the light emission of D. licha , another set of skin patches were subjected to an immersion in 100 μL of MT 10 –6 mol L –1 followed after 5 min by an application of 100 μL of either α-MSH 10 –6 mol L –1 or ACTH 10 –5 mol L –1 . Luminescence of ventral skin patches subjected to the various treatments was measured using a FB12 tube-luminometer (Titertek-Berthold, Pforzheim, Germany) calibrated as in Duchatelet et al., 2020b . Lights emissions were recorded through FB12- Sirius, multiple kinetics software (Titertek-Berthold) for at least 30 min with a measurement every 58 s. For comparative purposes, similar treatments were performed on seven specimens of E. lucifer (same experiments) and four E. granulosus specimens (MT dose response and α-MSH treatments). In parallel, for D. licha and E. lucifer , photophore aperture and closure were observed after drug application by taking a time-lapse series of pictures (every 10 min) with a Sony α7SII camera mounted on a binocular microscope.

Luminescence measurements were characterized as follows ( Duchatelet et al., 2020b ): the maximum intensity of light emission [Lmax, in megaquanta per second (Mq s –1 )], the total amount of light emitted during experimentation [Ltot, in Gigaquanta per hour (Gq h –1 )] and the time to reach maximum light intensity [TLmax, in seconds (s)]. Inhibitory actions of α-MSH and ACTH were measured as the total amount of light emitted after the second drug application [Ltot app , in Gq h –1 ]. All light parameters were standardized according to the surface area of each skin patch (in cm 2 ). A second treatment (α-MSH or ACTH) was added to the first one when the light intensity plateau was reached with the MT application, each timing being species-specific. Results of the luminescence decrease were expressed as a percentage of the maximal luminescence value (i.e., plateau MT) measured before the second application.

To evaluate the putative evolutive conservation of the hormonal control of light emission in dalatiids and etmopterids, pharmacological data on shark luminescence were extracted from literature.

Statistical Analyses

All analyses were performed with the software R studio (version 1.1.383, 2009, R Studio Inc., United States). Variance normality and homoscedasticity assumptions were tested by Shapiro-Wilk and Levene’s test, respectively, before running ANOVA which reveals significant differences between skin photophore densities or pharmacological treatments. When these parametric assumptions were not met, a non-parametric Kruskal-Wallis ANOVA was used. Post hoc Tukey’s tests or Wilcoxon tests allowed pair-wised comparison of means, attributing different letters to significantly different values ( P -value < 0.05).

Luminous Pattern and Photophore Morphology

A blue glow was observed on the ventral surface of D. licha , E. lucifer , and E. granulosus specimens kept in a fully dark environment ( Figures 2A,D,E ). D. licha also emit a faint blue glow from the lateral and dorsal areas and at the two dorsal fins ( Figure 3 – Mallefet personal observation). Both etmopterids present a more complex pattern of light emission with flank marks, and lateral, dorsal, and rostral patterns ( Figure 4 ; E. granulosus – Mallefet personal observation). Skin patches observed in toto present black round-shaped photophores distributed between placoid scales for all the observed sharks ( Figures 1C–E and Supplementary Figure 1 ). The mean photophore diameters are 83.9 ± 9.5, 122.4 ± 10.8, and 132.3 ± 14.5 μm, for D. licha , E. lucifer , and E. granulosus , respectively. No statistical differences in photophore diameter were observed between the zones presenting large amount of photophores (ventral, pecto-ventral and infra-caudal) ( D. licha ANOVA: F (2,183) = 1,1928, P -value = 0,3057; E. lucifer ANOVA: F (2,183) = 0,1014, P -value = 0,9036; E. granulosus ANOVA: F (2,183) = 0.1376, P -value = 0.8716).

Figure 2. Dalatiidae and Etmopteridae ventral luminous pattern and photophore histology. Picture of the lateral side in daylight, ventral luminescent pattern and section across ventral integument photophore of (A) Dalatias licha , (B) Isistius brasiliensis , (C) Squaliolus aliae , (D) Etmopterus lucifer , (E) Etmopterus granulosus , and (F) Etmopterus spinax . Ventral luminescence in dalatiid shows a homogenous pattern, while etmopterids show a heterogenous pattern with different zones. Photophores histology highlights a single photocyte within small photophores in dalatiids, while etmopterids harbor bigger and more complex photophores. c, connective tissue; e, epidermis; i, iris-like structure cells; l, lens cell; p, photocyte; s, pigmented sheath. In toto shark picture scale bar: 10 cm; photophore section scale bar: 100 μm.

Figure 3. Lateral and dorsal luminescent pattern of Dalatias licha . (A) Lateral daylight view and luminescent pattern highlighting the dorso-ventral luminous pattern. (B) Dorsal daylight view and luminescent pattern. Luminescence of the second dorsal fin is observable on this specimen (red arrowhead). Scale bar: 10 cm.

Figure 4. Lateral and dorsal luminescent pattern of Etmopterus lucifer . (A) Lateral daylight view and luminescent pattern. The species-specific flank mark is indicated by a red arrowhead (B) dorsal daylight view and luminescent pattern with specific luminous lines. Scale bar: 10 cm.

Analyses of photophore density along the D. licha body show an increasing dorso-ventral repartition of photophores reaching up to 20.14 ± 4.01 photophores per mm 2 at the ventral side of the shark ( Figures 1A,B,F ). The lowest densities were observed for the caudal and dorsal areas with a mean density of 2.85 ± 1.11 and 4.85 ± 1.70 photophores per mm 2 , respectively ( Figure 1A ). Statistical differences [ANOVA: F (11,514) = 128.64, P -value < 2.2 × 10 –16 ] in photophore densities are illustrated Figure 1F and Supplementary Table 2A . The scales of the rostral area were leaf-like in shape while the scales of the remaining body parts were pavement-like in shape ( Figures 1C,D ).

For both studied etmopterids, a high density of photophores was observed at the pectoral zone with 34.00 ± 6.20, and 15.63 ± 2.50 photophores per mm 2 for E. lucifer and E. granulosus , respectively. E. granulosus also have a high density of photophores at the infra-caudal and caudal zones. Conversely, for both species, only a few photophores were spread within the dorsal epidermis. Both species have a well-defined flank mark with photophores. All the remaining photophore densities and their respective statistical differences [ E. lucifer ANOVA: F (9,490) = 263.39, P -value < 2.2 10 –16 ; E. granulosus ANOVA: F (10,298) = 175.16, P -value < 2.2 10 –16 ] are reported in Supplementary Figure 1 and Supplementary Tables 2B,C . Both etmopterids present needle-shaped placoid scales in all the studied zones ( Supplementary Figure 1 ).

Histological sections across photogenic skin highlight the structure of D. licha photophores. Each light organ is embedded in the stratified squamous epidermis and is composed of a cup-shaped pigmented sheath containing a unique photocyte, topped by a lens cell with a few diffuse pigmented cells between the photocyte and lens cell ( Figure 2A ). This structural organization is similar to that found in S. aliae and I. brasiliensis photophores ( Figures 2B,C ).

Photophore morphologies of E. lucifer and E. granulosus are consistent with those already described for other etmopterids (i.e., E. spinax , Etmopterus molleri , and Etmopterus splendidus ) ( Figures 2D–F ). They are composed of a cup-shaped pigmented sheath embedded with luminous cells and topped a with lens. They are similar to dalatiid photophores, but they harbor a higher number of photocytes, a larger iris-like structure area, and more lens cells (up to 3) ( Figures 2D–F ).

Light Emission Control

The effect of MT on D. licha , E. lucifer , and E. granulosus was tested through a dose-dependent response. For the studied species, MT 10 –6 mol L –1 triggered a long-lasting light emission, significantly different from the MT 10 –8 mol L –1 application ( P -value < 0.05; Figures 5A–C and Supplementary Tables 3A , 4 ), while MT 10 –7 mol L –1 triggered an intermediate light emission and Ltot value ( Figures 5A–C and Supplementary Tables 3A , 4 ). All treatments were significantly different from the shark saline control, except for the MT 10 –7 and 10 –8 mol L –1 treatments of E. granulosus ( P -value < 0.05; Supplementary Tables 3A , 4 ). Although the total amount of light emitted under MT 10 –6 mol L –1 treatment was significantly different [Kruskal-Wallis χ 2 (2) = 10.14, P -value = 0.0063], E. lucifer produced a mean total amount of light during the experiment 2.5 and 5 times higher than D. licha and E. granulosus , respectively. Similar patterns of bioluminescence were observed for the three species ( Figures 2 , 5 ).

Figure 5. Effect of MT on the studied species luminescence. Time course of the mean light emissions (Mq s –1 cm –2 ) and total amount of light produced (Gq h –1 cm –2 ) from ventral skin patches under hormonal treatments (MT 10 –8 to 10 –6 mol L –1 ) for (A) Dalatias licha ( n = 10), (B) Etmopterus lucifer ( n = 7), and (C) Etmopterus granulosus ( n = 4). Different lettering indicates statistical differences [Kruskal-Wallis ANOVA: D. licha χ 2 (3) = 23.95, P -value = 2.56 × 10 –5 ; E. lucifer χ 2 (3) = 23.823, P -value = 2.72 × 10 –5 ; E. granulosus χ 2 (3) = 8.5368, P -value = 0.0361]. Error bars correspond to SEM.

The effect of α-MSH was evaluated for the three species after reaching the Lmax triggered through MT 10 –6 mol L –1 application. Application of α-MSH 10 –6 mol L –1 induced a rapid decrease of light emission for the studied luminous sharks ( Figures 6A–C and Supplementary Table 3B ). After MT-induced bioluminescence, Ltot app values of α-MSH were statistically significant compared with the MT 10 –6 mol L –1 control ( Figures 6A–C and Supplementary Tables 3B , 5 ).

Figure 6. Effect of ACTH and α-MSH on luminescence induced by MT in the studied species. Time course of the light produced (expressed as percentage of maximal melatonin control value), and total amount of light produced (Gq h –1 cm –2 ) after melatonin pretreatment from ventral skin patches under melanocortin treatments (ACTH 10 –5 mol L –1 /α-MSH 10 –6 mol L –1 ) for (A) Dalatias licha ( n = 10), (B) Etmopterus lucifer ( n = 7), and (C) Etmopterus granulosus ( n = 4 – no ACTH treatment). Hormonal treatments are expressed in mol L –1 . Different lettering indicates statistical differences [ANOVA: D. licha F (2,33) = 2.585, P -value = 0.0437; E. lucifer F (2,18) = 14.482, P -value = 0.0002; Kruskal-Wallis ANOVA: E. granulosus χ 2 (1) = 3.857, P -value = 0.0495]. Error bars correspond to SEM.

The effect of ACTH was evaluated on D. licha and E. lucifer bioluminescence. Similar to the results obtained for α-MSH, ACTH 10 –5 mol L –1 applications rapidly induced a decrease in light emission ( Figures 6A,B and Supplementary Table 3B ). Each Ltot app value of ACTH 10 –5 mol L –1 was not significantly different from those of α-MSH 10 –6 mol L –1 , respectively, but were statistically different from the MT 10 –6 mol L –1 control ( Figures 6A,B and Supplementary Tables 3B , 5 ). Mean values of Lmax, TLmax, Ltot, Ltot app are presented in Supplementary Table 3 .

The time-course of light emission in D. licha under MT 10 –6 mol L –1 stimulation revealed a concomitant opening of the photophore ILS within 15 min of luminescence, in which the ILS stayed open for the next 30 min while the light level remained high ( Figure 7A ). In the case of E. lucifer MT-induced luminescence, a rapid opening of the photophore ILS was observed within 8 min followed by a slow decrease during which a closure of the ILS was visible ( Figure 7B ). Aperture and closure of photophores showed pigment movements concomitant to light emission.

Figure 7. Time-course of MT-induced luminescence, and time-lapse of photophore pigment movements. Luminescence in relative light unit (RLU) recorded during a 40 min MT 10 –6 mol L –1 application on ventral skin patches and time-lapse pictures (times: 0, 10, 20, 30, and 40 min, respectively) of photophore pigment movements of (A) Dalatias licha , (B) Etmopterus lucifer .

The three studied shark species inhabit the mesopelagic zone ( Roberts et al., 2015 ), therefore they face an environment with no place to hide, hence the need for glowing camouflage or counterillumination, first proposed by Clarke, 1963 . The mesopelagic zone, often called the twilight zone, ranges from 200 to 1000 m depth (maximal depth of solar light penetration) and is the realm of bioluminescence ( Martini and Haddock, 2017 ; Martini et al., 2019 ). At 200 m the residual solar light is considered too weak to initiate photosynthesis but organisms living there are well adapted to see in low light conditions ( Nicol, 1978 ). Mesopelagic cephalopods, sharks and bony fishes have large eyes with specialized structures such as a large iris, a tapetum, huge rod density, high content of opsins (rhodopsin and chrysopsin), and an elevated integration rate at the optical nerve which allows them to perceive very low light levels down to 800 m depth ( Douglas et al., 1998 ; Warrant, 2004 ; Warrant and Locket, 2004 ; Claes et al., 2014a , b ).

Luminescent Pattern

The light emission pattern observed in D. licha is similar to that observed in previously studied dalatiids i.e., S. aliae and I. brasiliensis ( Claes et al., 2012 ; Delroisse et al., 2021 ). The dorso-ventral gradient and the relative homogeneity in ventral photophore densities suggest the luminescence is used for counterillumination. The luminous pelvic zone of D. licha reveals a sexual dimorphism but, contrary to E. spinax and Etmopterus molleri ( Claes and Mallefet, 2010b ; Duchatelet et al., 2020c ), it is not brighter than the rest of the ventral body, suggesting it is less important for sexual signaling. The kitefin shark D. licha , like other dalatiids, does not have flank marking or specific dorsal patterns. The lack of these luminescent patterns, previously suggested to be used as conspecific signaling for group aggregation, swimming, or hunting in etmopterids ( Claes et al., 2015 ; Duchatelet et al., 2019b ), rules out this function in D. licha . The aposematic function described for etmopterids ( Claes et al., 2013 ; Duchatelet et al., 2019b ) is also ruled out for D. licha luminescence due to the absence of dorsal fin defensive spines. Nevertheless, D. licha is the first shark with fully luminous dorsal fins ( Figures 1A , 3 ), which raises questions about its luminescence function.

The light emission patterns of E. lucifer and E. granulosus , are similar to that of previously studied etmopterids. The dorsal photophores, flank markings, and brighter pectoral fin and claspers are likely to be used for intraspecific communications while the ventrally emitted light is likely to be used for counterillumination. These functions have been documented for E. spinax ( Claes and Mallefet, 2009a ; Claes and Mallefet, 2010a ), Etmopterus molleri ( Claes and Mallefet, 2015 ), and Etmopterus splendidus ( Claes et al., 2011b ). However, a bioluminescence aposematic function through specific spine-associated photophores ( Claes et al., 2013 ; Duchatelet et al., 2019b ) was not documented for E. lucifer and E. granulosus.

Reif (1985) postulated that a trade-off exists between the space occupied by placoid scales and luminous organs, and that four different types of placoid scales have evolved to allow this trade-off: pavement, cross-, bristle/needle-, and hook-shaped placoid scales. A new type of squamation with overlapping leaf-shaped placoid scales is present in the luminous rostral area of D. licha. This new bioluminescent-associated squamation was observed in the somniosid, Zameus squamulosus , which is assumed to be luminous ( Straube et al., 2015 ). This new type of bioluminescence-associated placoid scale needs to be highly translucent or possess specific physical characteristics to allow efficient light transmission. The use of Reif placoid scale types to assess the bioluminescent status of a shark species is not a decisive character as shown by a recent study of Ferrón et al. (2018) ; highlighting the presence of bioluminescent-like squamation in a galeomorph shark, Apristurus ampliceps , a species not known to be luminous.

Photophore Morphology Conservation

Histology revealed an evolutive conservation of photophore morphology across each family. Kitefin shark photophores are larger (mean diameter 83.9 μm) than those observed in S. aliae and I. brasiliensis [i.e., 50 and 56 μm, respectively ( Claes et al., 2012 ; Delroisse et al., 2021 )] while the internal structure of typical dalatiid photophores is conserved. Here, D. licha photophores are depicted as morphologically similar to those of S. aliae, S. laticaudus and I. brasiliensis ( Seigel, 1978 ; Delroisse et al., 2021 ).

E. lucifer and E. granulosus showed typical etmopterid photophore histology ( Claes and Mallefet, 2009b , 2015 ; Claes et al., 2011b ; Renwart et al., 2014 ; Duchatelet et al., 2020b ). These observations provide further insights on the evolutive conservation of light organ morphology across luminous squaliform radiation ( Straube et al., 2015 ).

Luminescence Control Evolutive Conservation

The effect of hormones on light emission in D. licha , E. lucifer and E. granulosus are consistent with increasing literature on light emission control in sharks ( Claes and Mallefet, 2009c ; Claes et al., 2012 ; Duchatelet et al., 2020b , d ): MT, and α-MSH/ACTH, have been demonstrated as the main triggering and inhibiting agents of shark luminescence, respectively. Similar to observations of E. spinax and Etmopterus molleri photophores ( Claes and Mallefet, 2010a ; Duchatelet et al., 2020b ), aperture and closure of D. licha and E. lucifer photophores involved pigment motion within the ILS cells. Simultaneities of curves kinetics and pigment motions highlight the evolutive conservation of hormonally controlled pigment motion regulating luminescence. These data strongly suggest that luminous etmopterids and dalatiids share a common luminescence control mechanism, involving at least MT, and α-MSH/ACTH hormones. This control is assumed to have been successfully and evolutionary co-opted from shark melanophore pigment motion control by a common ancestor of these two squaliform families. For both families, luminescence appears to be dually controlled at the level of ( i ) the photocyte, site of luminescent reaction, and ( ii ) the ILS cells, acting as a diaphragm capable of occluding light produced by the photocytes, via melanophore-associated pigment movements ( Duchatelet et al., 2020b , d ). This was recently demonstrated within ILS cells of the lanternshark, E. spinax ( Duchatelet et al., 2020d ) i.e., transduction pathways that activate cellular motors such as dynein and kinesin, leading pigment movements within ILS melanophores. The bioluminescence control mechanisms in the two studied etmopterids, as well as in D. licha , might share common features. Moreover, the involvement of extraocular photoreception events in the light emission control of photophores ( Duchatelet et al., 2020d ), remains to be deciphered for these sharks. Further research are necessary to fully demonstrate the evolutive conservation of luminescence control within etmopterids and dalatiids.

Luminescence of Dalatias licha

The question remains concerning bioluminescence in the largest luminous vertebrate; why does D. licha emit light ventrally to counterilluminate when it has few or no predators? Pinte et al. (2020) , analyzed the swimming speed of several New Zealand deep-sea sharks, and found that D. licha possesses one of the slowest cruise swimming speeds ever measured in sharks. Conversely, this species is assumed to possess a high burst capability ( Pinte et al., 2020 ). Stomach content analyses have revealed that this shark species hunts and eats etmopterids, which have a higher cruise swimming speed. Therefore, there are two hypotheses which might explain the ventral luminescence of this holobenthic species: luminescence might be used ( i ) to illuminate the ocean floor while searching and hunting for prey; or ( ii ) to stealthily approach toward prey, using counterillumination camouflage, before striking fast when close enough ( Zintzen et al., 2011 ), allowing them to predate etmopterids. In both cases, the principle of counterillumination would have been distorted to serve as a predation tool instead of an avoidance mechanism, a hypothesis already proposed for the cookie cutter shark, I. brasiliensis ( Widder, 1998 ). However, to validate such hypotheses for these dalatiid species, in vivo observations and behavioral studies are essential.

Through a histological and pharmacological approach, the bioluminescence of three different shark species was investigated. Our results support evolutive conservation of light organ morphology and luminescence control. For the first time, luminescence was recorded and analyzed for the largest luminous vertebrate, D. licha and two lanternsharks, E. lucifer and E. granulosus . Dalatiid photophores are similar between species and are structurally composed of a single photocyte embedded in a cup-shaped pigmented cell and surmounted by lens cells. The same observation was made for etmopterids, which showed a conservation of photophore structure between species. Etmopterid photophores are slightly more complex than those of dalatiids, with several photocytes and a well-developed ILS between the lens cells and the photocytes. Through this study, the action of MT and α-MSH/ACTH in the bioluminescence control in these two families was shown to be identical and seem to have been co-opted during evolution from the regulation of skin pigment movements. With these data, we can assume that the common luminous ancestor of etmopterids and dalatiids likely had hormonal control of its luminescence and had luminous organs similar to those of the dalatiids (i.e., the simplest structure) for counterillumination.

Data Availability Statement

The original contributions presented in the study are included in the article/ Supplementary Material , further inquiries can be directed to the corresponding author/s.

Ethics Statement

Ethical review and approval was not required for this study. The shark specimens were captured as bycatch of a fisheries assessment survey for the New Zealand Ministry for Primary Industries.

Author Contributions

JM and DS collected the samples. JM collected the bioluminescence pictures and performed pharmacological studies and fixations on the survey. LD performed the classical histology, pattern, and pharmacological analyses. LD and JM were major contributors to the initial manuscript that was improved by DS revisions. All authors approved the final manuscript.

This work was supported by an F.R.S.– FNRS Grant (T.0169.20) awarded to the Université Catholique de Louvain Marine Biology Laboratory and the Université de Mons Biology of Marine Organisms and Biomimetics Laboratory. JM received a travel grant (35401759) from F.R.S.– FNRS Belgium.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors acknowledge R. O’Driscoll, Program Leader – Fisheries Monitoring NIWA, the scientific staff, and the skillfull crew of R.V. Tangaroa on voyage TAN2001 (Chatham Rise fish survey, NIWA). The authors thank Dr. Nicolas Pinte and Constance Coubris for the help during statistical analyses. JM is Research Associate F.R.S.– FNRS. This study is the contribution BRC #276 of the Biodiversity Research Center (UCLouvain) from the Earth and Life Institute Biodiversity (ELIB) and the “Centre Interuniversitaire de Biologie Marine” (CIBIM).

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars.2021.633582/full#supplementary-material

Supplementary Figure 1 | External features and densities of photophores in Etmopterus lucifer and Etmopterus granulosus . Black-dotted photophores observed at the ventral side (A) , flank mark (B) , and pectoral (C) specific area of E. lucifer . Dotted line corresponds to the flank mark boundaries. (D) Measured photophore densities for the studied zones of E. lucifer ( n = 50 for each zones). Black-dotted photophore observed at the ventral (E) , infra-caudal (F) and rostral (G) areas of E. granulosus . (H) Measured photophore densities for the studied zones of E. granulosus ( n = 30 for each zones). Different lettering indicates statistical differences. Values are expressed as mean ± SEM. Scale bars: 750 μm.

Supplementary Table 1 | Experimental specimens. Morphometrics measurements of Dalatias licha , Etmopterus lucifer , E. granulosus , Squaliolus aliae and I. brasiliensis studied specimens. ♀, female; ♂, male.

Supplementary Table 2 | Photophore density, statistical analyses. Results of Tukey’s test for the photophore density of (A) D. licha , (B) E. lucifer , and (C) E. granulosus different skin zones. Gray-shaded cases represent not significant differences.

Supplementary Table 3 | Hormone-induced luminescence parameters (mean maximal light intensity: Lmax; time to reach the Lmax: TLmax; total amount of emitted light: Ltot; total amount of emitted light after second drug application: Ltot app ). (A) luminescence recorded parameters for the melatonin (MT) dose response treatments for Dalatias licha ( n = 12), Etmopterus lucifer ( n = 7) and E. granulosus ( n = 4). ∗ indicate significant differences ( P -value < 0.05) from the shark saline control experiment. (B) Ltot app for each treatment and each shark species. ∗ indicate differences ( P -value < 0.05) from the melatonin 10 –6 mol L –1 control experiment. All data are means ± SEM.

Supplementary Table 4 | MT dose response, statistical analyses. Kruskal-Wallis ANOVA and pairwise Wilcoxon test results for the MT dose response of the three studied sharks. Gray-shaded cases represent not significant differences.

Supplementary Table 5 | α-MSH and ACTH effects, statistical analyses. ANOVA and Tukey’s test results for the decrease of light triggered by α-MSH and ACTH treatments (except for E. granulosus non-parametric test). Gray-shaded cases represent not significant differences.

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Keywords : Dalatiidae, Etmopteridae, light emission control, photophore, shark

Citation: Mallefet J, Stevens DW and Duchatelet L (2021) Bioluminescence of the Largest Luminous Vertebrate, the Kitefin Shark, Dalatias licha : First Insights and Comparative Aspects. Front. Mar. Sci. 8:633582. doi: 10.3389/fmars.2021.633582

Received: 25 November 2020; Accepted: 05 February 2021; Published: 26 February 2021.

Reviewed by:

Copyright © 2021 Mallefet, Stevens and Duchatelet. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Jérôme Mallefet, [email protected]

† These authors have contributed equally to this work

This article is part of the Research Topic

Bioluminescence from Land to the Oceans: Its Role in Evolution, Communication, and Ecology

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Peer-reviewed

Research Article

Repeated and Widespread Evolution of Bioluminescence in Marine Fishes

* E-mail: [email protected]

Affiliation St. Cloud State University, St. Cloud, MN 56301, United States of America

Affiliation American Museum of Natural History, New York, NY 10024, United States of America

Affiliation University of Kansas, Lawrence, KS 66045, United States of America

• Matthew P. Davis, 
• John S. Sparks, 
• W. Leo Smith

• Published: June 8, 2016
• https://doi.org/10.1371/journal.pone.0155154
• Reader Comments

Bioluminescence is primarily a marine phenomenon with 80% of metazoan bioluminescent genera occurring in the world’s oceans. Here we show that bioluminescence has evolved repeatedly and is phylogenetically widespread across ray-finned fishes. We recover 27 independent evolutionary events of bioluminescence, all among marine fish lineages. This finding indicates that bioluminescence has evolved many more times than previously hypothesized across fishes and the tree of life. Our exploration of the macroevolutionary patterns of bioluminescent lineages indicates that the present day diversity of some inshore and deep-sea bioluminescent fish lineages that use bioluminescence for communication, feeding, and reproduction exhibit exceptional species richness given clade age. We show that exceptional species richness occurs particularly in deep-sea fishes with intrinsic bioluminescent systems and both shallow water and deep-sea lineages with luminescent systems used for communication.

Citation: Davis MP, Sparks JS, Smith WL (2016) Repeated and Widespread Evolution of Bioluminescence in Marine Fishes. PLoS ONE 11(6): e0155154. https://doi.org/10.1371/journal.pone.0155154

Editor: Erik V. Thuesen, The Evergreen State College, UNITED STATES

Received: July 10, 2015; Accepted: April 25, 2016; Published: June 8, 2016

Copyright: © 2016 Davis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Data accessibility. Alignment available in S1 Text . The previously published nuclear genes come from a diversity of studies. Sequences of Sagamichthys abei , Xenodermichthys copei , and Yarrella blackfordi were taken from DeVaney [ 34 ]. Sequences of Archoplites interuptus , Elassoma zonatum , and Percopsis omiscomaycus were taken, in part, from Near et al. [ 35 ]. Sequences of Albula vulpes , Aldrovandia affinis , Alepocephalus agassizii , Ameiurus natalis , Amia calva , Ammodytes hexapterus , Anguilla rostrata , Antennarius striatus , Aphredoderus sayanus , Aplochiton taeniatus , Archoplites interuptus , Argentina silus , Argyropelecus gigas , Assurger anzac , Astyanax mexicanus , Ateleopus japonicus , Barbourisia rufa , Bathylaco nigricans , Bathylagus euryops , Bathypterois atricolor , Bothus lunatus , Callionymus bairdi , Caranx crysos , Chaetodon striatus , Chanos chanos , Chaunax suttkusi , Chelmon rostratus , Chitala chitala , Chologaster cornuta , Conger oceanicus , Coregonus clupeaformis , Coryphaena hippurus , Cromeria nilotica , Cyclothone microdon , Cyttopsis rosea , Denticeps clupeoides , Echeneis naucrates , Elassoma zonatum , Elops saurus , Esox lucius , Eurypharynx pelecanoides , Fistularia petimba , Galaxias maculatus , Galaxiella nigrostriata , Gephyroberyx darwini , Gnathonemus petersii , Gonorynchus greyi , Gymnorhamphichthys petiti , Halieutichthys aculeatus , Halosauropsis macrochir , Helostoma temminckii , Heteroconger hassi , Heteromycteris japonicus , Himantolophus sagamius , Hiodon tergisus , Histiophryne cryptacanthus , Hypomesus pretiosus , Hypoptychus dybowski , Ijimaia loppei , Lates niloticus , Lepidogalaxias salamandroides , Lepidogobius lepidus , Lepomis macrochirus , Luvarus imperialis , Macropinna microstoma , Macroramphosus scolopax , Megalops atlanticus , Melamphaes polylepis , Mola mola , Monocentris japonica , Nansenia ardesiaca , Neochanna burrowsius , Neonesthes capensis , Neoscopelus microchir , Ogcocephalus nasutus , Opsanus pardus , Opsariichthys uncirostris , Osmerus mordax , Pachypanchax playfairii , Paratrachichthys sajademalensis , Percopsis omiscomaycus , Polypterus ornatipinnis , Porichthys notatus , Psettodes erumei , Rachycentron canadum , Ranzania laevis , Retropinna semoni , Rondeletia loricata , Saccopharynx ampullaceus , Salvelinus alpinus , Samariscus latus , Scopelengys tristis , Searsia koefoedi , Selenotoca multifasciata , Sphyraena barracuda , Stokellia anisodon , Stylephorus chordatus , Symphurus atricaudus , Syngnathus fuscus , Tetraodon miurus , Thymallus brevirostris , Trachipterus arcticus , Triacanthus biaculeatus , Umbra limi , Xiphias gladius , Zanclus cornuta , and Zeus faber were taken in whole or in part from Near et al. [ 20 ]. Sequences of Aeoliscus strigatus , Anarhichas lupus , Aplodinotus grunniens , Apogon lateralis , Arrhamphus sclerolepis , Aulostomus maculatus , Betta splendens , Brotula multibarbata , Cataetyx lepidogenys , Centropomus undecimalis , Cephalopholis argus , Chromis cyanea , Cottus carolinae , Cubiceps baxteri , Diodon holocanthus , Dissostichus eleginoides , Eleotris pisonis , Etheostoma atripinne , Forcipiger flavissimus , Gambusia affinis , Gasterosteus aculeatus , Gazza minuta , Halichoeres bivittatus , Heteroconger hassi , Hoplostethus atlanticus , Labrisomus multiporosus , Leiognathus equulus , Lophius americanus , Maccullochella peelii , Meiacanthus grammistes , Menticirrhus littoralis , Monopterus albus , Morone chrysops , Parapercis clathrata , Paratilapia polleni , Polymixia japonica , Pseudopleuronectes americanus , Ptychochromis grandidieri , Rheocles wrightae , Rhinesomus triqueter , Ruvettus pretiosus , Sarda sarda , Scatophagus argus , Sebastes fasciatus , Sebastolobus alascanus , Seriola dumerili , Serranus tigrinus , Stegastes leucostictus , Stereolepis gigas , Toxotes jaculatrix , Trachinotus carolinus , Triacanthodes anomalus , and Xenentodon cancila were taken from Wainwright et al. [ 36 ]. Sequences of Abalistes stellatus , Acanthaphritis unoorum , Acanthurus nigricans , Acropoma japonica , Ameiurus natalis , Anomalops katoptron , Anoplogaster cornuta , Antennarius striatus , Antigonia capros , Argentina silus , Argyropelecus gigas , Assurger anzac , Ateleopus japonicus , Aulotrachichthys prosthemius , Banjos banjos , Barathronus maculatus , Barbourisia rufa , Bathymaster signatus , Beryx decadactylus , Brosmophycis marginata , Callionymus bairdi , Cantherhines pullus , Capros aper , Carapus bermudensis , Caranx crysos , Cataetyx lepidogenys , Ceratias holboelli , Cetostoma regani , Chaetodon striatus , Chanos chanos , Chaunax suttkusi , Chelmon rostratus , Chiasmodon sp., Chologaster cornuta , Coryphaena hippurus , Cromeria nilotica , Cryptopsaras couesi , Cyttomimus affinis , Cyttopsis rosea , Diretmichthys parini , Diretmus argenteus , Echeneis naucrates , Elassoma zonatum , Electrona antarctica , Fistularia petimba , Gephyroberyx darwini , Gigantactis vanhoeffeni , Glaucosoma hebraicum , Gonorynchus greyi , Helostoma temminckii , Heteromycteris japonicus , Himantolophus sagamius , Histiophryne cryptacanthus , Histiopterus typus , Howella zina , Hygophum proximum , Hypomesus pretiosus , Icichthys lockingtoni , Kali normani , Kurtus gulliveri , Lachnolaimus maximus , Lampris guttatus , Lamprogrammus niger , Lates niloticus , Liparis mucosus , Lota lota , Luvarus imperialis , Macroramphosus scolopax , Macrourus sp, Malakichthys elegans , Melamphaes polylepis , Mene maculata , Mola mola , Monocentris japonica , Muraenolepis microps , Naso lituratus , Neonesthes capensis , Neoscopelus microchir , Nezumia bairdii , Onuxodon parvibrachium , Ophioblennius atlanticus , Opsanus pardus , Opsariichthys uncirostris , Osmerus mordax , Ostracoberyx dorygenys , Pachypanchax playfairii , Paragalaxias mesotes , Paraliparis meganchus , Paratrachichthys sajademalensis , Pempheris schomburgkii , Pempheris schwenkii , Pentaceros japonicus , Peprilus triacanthus , Polymixia lowei , Porichthys notatus , Porichthys plectrodon , Protomyctophum choriodon , Psenes maculatus , Pseudopentaceros pectoralis , Rachycentron canadum , Ranzania laevis , Rathbunella hypoplecta , Regalecus russelii , Rondeletia loricata , Salvelinus alpinus , Scopelengys tristis , Siganus spinus , Sphyraena barracuda , Stylephorus chordatus , Symphurus atricaudus , Tetraodon miurus , Triacanthus biaculeatus , Xiphias gladius , Zanclus cornuta , Zenopsis conchifera , and Zeus faber were taken, in whole or in part (often just ENC1 from species taken from Near et al. [ 20 ], from Near et al. [ 37 ]. Sequences of Aphredoderus sayanus and Chologaster cornuta were taken in part from Niemiller et al. [ 38 ]. Newly accessioned sequences are on GenBank (KX227793-KX228066) and listed in S1 Table . GenBank accession information is available for mitochondrial gene fragment cytochrome oxidase I in S2 Table , as data for this fragment was taken from various sources.

Funding: Financial support was provided by National Science Foundation grants (DEB) to M.P.D. (1543654, 1258141), J.S.S. (0444842, 1257555), and W.L.S. (1060869, 1258141), with support from the Dalio Family Foundation, KU, and SCSU.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Bioluminescence, the production and emission of light from a living organism, is a fascinating phenomenon that is documented in over 700 genera of metazoans across the tree of life, with the vast majority living in the ocean [ 1 – 3 ]. Among vertebrates, bioluminescence has evolved in cartilaginous (Chondrichthyes) [ 1 – 4 ] and ray-finned fishes (Actinopterygii) [ 1 – 3 ], and it is not observed in any lobe-finned fishes or tetrapods (Sarcopterygii). Previous survey studies [ 1 – 2 ] have identified bioluminescence in 11 orders of marine fishes; however, the phylogeny and classification of fishes has changed considerably since these previous studies, and the authors of these earlier studies did not investigate this phenomenon in a phylogenetic framework, identify independent evolutionary events of bioluminescence, or explore macroevolutionary patterns of bioluminescent lineages. Broad studies of bioluminescence have typically counted fishes as a single evolutionary event among the 40 independent higher-level evolutionary events of bioluminescence documented across the tree of life [ 2 – 3 ]; therefore, a focused study of the bioluminescent ray-finned fishes is critical to determine the number and identity of bioluminescent fish clades.

Bioluminescence is produced in living organisms following a chemical reaction between a substrate (luciferin) and an enzyme (luciferase) that results in a visible photon [ 2 – 3 ]. Among fishes, bioluminescence is generated intrinsically (e.g., stomiiform dragonfish barbels and photophores) [ 1 – 3 , 5 ] or through bacterially mediated symbiosis (e.g., leiognathid [ponyfish] esophageal pouches, anomalopid [flashlightfish] subocular organs) [ 6 – 7 ]. The functions of bioluminescence are diverse and engrossing, exemplified by remarkable morphological specializations that range from anatomically complex species-specific luminescent structures to variation in the biochemistry of the bioluminescent systems themselves [ 1 – 13 ]. In ray-finned fishes, bioluminescent structures are variously used for camouflage, defense, predation, and communication [ 1 – 4 , 7 – 11 ].

Here we present the first investigation of the evolution and distribution of bioluminescence across ray-finned fishes in a phylogenetic context. Recent work indicates that bioluminescence evolved once or twice within chondrichthyans (e.g., Etmopteridae and Dalatiidae) [ 4 , 14 ]; however, the phenomenon is considerably more widespread, anatomically variable and complex, and biochemically diverse in ray-finned fishes [ 1 – 13 ]. Our objectives in this study were to determine the number of independent evolutionary origins of bioluminescence in ray-finned fishes, infer the ages of the phenomenon across this assemblage, and investigate patterns of diversification in bioluminescent lineages. Previous studies have suggested that bioluminescence may play a role in diversification within marine environments, particularly in deep-sea lineages, and specifically among taxa that are hypothesized to use bioluminescence for communication [ 8 ]. We further examine whether any bioluminescent lineages of ray-finned fishes exhibit exceptional species richness given their clade age for taxa living both in the deep sea, where there are few obvious physical barriers to reproduction, and shallow water habitats, to provide a roadmap for future macroevolutionary work.

Materials and Methods

To investigate the evolution of bioluminescence across ray-finned fishes, we inferred a phylogeny from ten nuclear (enc1, Glyt, myh6, plagl2, Ptr, rag1, SH3PX3, sreb2, tbr1, zic1) and one mitochondrial (COI) gene fragments. Taxonomic sampling includes 301 taxa (297 genera, S1 Table ). The data matrix is 80% complete and includes 274 newly collected gene fragments ( S1 Table , GenBank KX227793-KX228066, with sequences aligned with MAFFT [ 15 ]). The previously published nuclear genes were obtained from a diversity of studies, as described in the data accessibility section. GenBank accession information is available for mitochondrial gene fragment cytochrome oxidase I in S2 Table , as data for this gene fragment were taken from various sources.

Evolutionary relationships were inferred using maximum likelihood in GARLI v2.01 [ 16 ] with 33 partitions (one for each codon position in each gene). Bootstrap values supporting clades are indicated in S1 Fig following the recommendation of Wiley et al. [ 17 ]. Codon positions were assigned models of nucleotide substitution from Akaike information criterion tests. Models of molecular evolution were identified by the program jModelTest v.2.1 [ 18 ] with the best fitting model under the Akaike information criterion (AIC): cytochrome oxidase I (GTR+Γ, GTR+I+Γ, GTR+Γ), ectodermal-neural cortex 1-like gene (GTR+Γ, GTR+I+Γ, GTR+I+Γ), glycosyltransferase (GTR+I+Γ, HKY+I+Γ, GTR+I+Γ), myosin heavy chain 6 alpha (GTR+I+Γ, GTR+I+Γ, GTR+Γ), pleiomorphic adenoma gene-like 2 gene (GTR+I+Γ, GTR+Γ, GTR+I+Γ), ptr hypothetical protein (GTR+I+Γ, GTR+I+Γ, GTR+I+Γ), recombination activating gene 1 (GTR+I+Γ, GTR+I+Γ, GTR+I+Γ), SH3 and PX3 domain-containing 3-like protein gene (GTR+I+Γ, GTR+I+Γ, GTR+I+Γ), brain super conserved receptor gene (GTR+I+Γ, GTR+I+Γ, GTR+I+Γ), T-box brain 1 gene (GTR+Γ, GTR+I+Γ, GTR+I+Γ), and zic family member protein (GTR+I+Γ, GTR+Γ, GTR+I+Γ).

Five independent likelihood analyses were conducted, and the tree with the maximum likelihood score was stored and used as a fixed-topology prior to generate a distribution of temporal (ultrametric) trees for character evolution analyses in BEAST v.1.8 [ 19 ]. The relative divergence times of representative fishes were estimated by incorporating 21 previously published fossil calibrations [ 20 – 21 ] with lognormal priors ( S1 Text , S1 Fig ) and builds heavily on previous phylogenetic work [ 20 ]. Parameters and tree topologies from BEAST analyses converged on a stationary distribution. A 50% maximum clade credibility (mean heights) tree was generated from the posterior tree distribution and was subsampled down from 45,000 to 5,000 trees (Figs 1 and 2 ).

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Evolutionary relationships and divergence times of ray-finned fishes inferred from 11 gene fragments. Letters at nodes correspond to clades indicated in Fig 4 . Branch colors indicate the presence of bioluminescence and whether the mechanism of bioluminescence is intrinsic, bacterially mediated, or unknown. Examples of bioluminescent ray-finned fishes include the A: midshipman ( Porichthys : intrinsic), and B: flashlight fish ( Anomalops : bacterially mediated).

https://doi.org/10.1371/journal.pone.0155154.g001

Numbers at nodes correspond to ancestral-character-state-reconstruction distributions of the evolution of bioluminescence indicated in Fig 3 . Blue branches and taxa labels indicate the presence of bioluminescence, all of which occur in marine habitats. Green taxa labels indicate additional marine taxa. Pink labels indicate lineages with marine and freshwater taxa, and white labels indicate lineages that are predominantly found in freshwater habitats.

https://doi.org/10.1371/journal.pone.0155154.g002

Bayesian ancestral-character-state reconstructions for the evolution of bioluminescence (0:Absent; 1:Present), coded from known and previously published occurrences in ray-finned fishes [ 1 – 3 , 5 – 7 ], were performed in BayesTraits v2.0 MultiState [ 22 ] using Markov chain Monte Carlo (MCMC) approaches to infer ancestral states at nodes in the phylogeny across a distribution of topologies (500 trees subsampled from 5,000 post burn-in trees) where the branches have varying lengths relative to time ( Fig 3 ). Each transformation from absence to presence in the Bayesian ancestral-states reconstruction was counted as an independent evolution of bioluminescence among ray-finned fishes. The GEIGER module in R [ 23 ] was used to calculate a 95% confidence interval of the expected number of species within a clade given a net diversification rate (r), a relative extinction rate, and crown clade age [ 24 ]. Rates for net diversification and relative extinction were estimated with MEDUSA [ 25 ] ( Fig 4 ), with species richness [i.e., the number of currently valid described species for each clade ( S3 Table )] generated from the Catalog of Fishes [ 26 ]. Following its use in recent studies [ 7 – 8 ], we identify and highlight lineages as having exceptional species richness if their known species diversity, given hypothesized clade age, lie outside the upper confidence interval bounds of expected species richness.

Bayesian ancestral-character-states reconstruction of bioluminescence across a distribution of 500 trees that resulted from the Bayesian inference of divergence times. Each rectangle includes 500 individual reconstructions across this distribution of 500 trees. Blue indicates the presence of bioluminescence and black indicates absence.

https://doi.org/10.1371/journal.pone.0155154.g003

Temporal hypothesis of the relationships of ray-finned fishes with net diversification rates and relative rates of extinction estimated by MEDUSA. Species richness curves indicate the 95 percent confidence interval for the expected number of species given clade age given a net diversification rate and relative rate of extinction. Letters indicate bioluminescent lineages of fishes in Fig 1 .

https://doi.org/10.1371/journal.pone.0155154.g004

Bioluminescence is inferred to have evolved independently at least 27 times among 14 major fish clades (Figs 1 and 2 , S1 Fig ). Intrinsic bioluminescence, in which a fish produces and emits light without the aid of bacterial symbiosis, evolved eight times (Figs 1 and 2 ). Of the approximately 1,510 species of known bioluminescent fishes, more than half (~785 species) exhibit intrinsic bioluminescence (Figs 1 and 2 , S1 Fig ). Bacterially mediated bioluminescence through symbiosis has evolved at least 17 times (Figs 1 and 2 , S1 Fig ), representing approximately 48% of all bioluminescent fishes (~725 species, Fig 4 ). All occurrences of bioluminescence across ray-finned fishes evolved from the Early Cretaceous (150 Ma) through the Cenozoic ( Fig 1 ), with the oldest occurrence in Stomiiformes ( Fig 1 ). Six orders (Alepocephaliformes, Myctophiformes, Stomiiformes, Batrachoidiformes, Beryciformes, and Acanthuriformes), representing 57% of all bioluminescent fishes (~814 species), include lineages that exhibit exceptional species richness given clade age ( Fig 4 ).

Bioluminescence is widespread across ray-finned fishes that occupy marine environments, and 27 independent evolutionary events of bioluminescence are identified (Figs 1 and 2 ). These 27 groups are distributed across 14 major lineages of ray-finned fishes (Figs 1 and 2 , S1 Fig ) that occupy deep-sea (e.g., lanternfishes, anglerfishes), inshore (e.g., ponyfishes, croakers), and coral reef (e.g., cardinalfishes, pineconefishes) habitats. Our findings demonstrate that the number of independent evolutionary events of bioluminescence across the tree of life is significantly higher than previous summaries suggest (40) [ 2 – 3 ] and highlight the need to explore the evolution of this phenomenon phylogenetically in bioluminescent lineages across Metazoa. By combining our findings with the inference that squaliform sharks have evolved bioluminescence once or twice [ 1 , 4 , 14 , 27 ], we can infer that bioluminescence has evolved at least 29 times in vertebrates alone. This significant increase in the number of independent origins of bioluminescence in vertebrates is found exclusively among fishes living in marine environments. At present, the only known terrestrial animals capable of bioluminescence are arthropods (e.g., fireflies, millipedes) [ 1 ]; whereas in marine environments, bioluminescence has evolved across the tree of life from bacteria to vertebrates (e.g., Ctenophora, Mollusca, Crustacea, Tunicata, Vertebrata) [ 1 – 3 ].

Of the 27 evolutionary events of bioluminescence in ray-finned fishes, bacterially mediated symbiosis has evolved 17 times (Figs 1 and 2 ), particularly among acanthomorph (spiny-rayed) lineages. All bioluminescent bacteria that are symbiotic with fishes are vibrionaceans [ 28 ], and there is little to no host specificity between species of bioluminescent bacteria and fishes, which acquire bacteria from their local environment [ 6 – 7 ]. Fishes that live in symbiosis with bioluminescent bacteria exhibit a vast array of anatomical structures to focus, broadcast, or even restrict the light these bacteria produce [ 7 , 10 ]. Multiple fish orders with bioluminescent bacteria contain lineages that exhibit exceptional species richness given clade age ( Fig 4 ), including Beryciformes (flashlightfishes) and Acanthuriformes (ponyfishes), with Ceratioidei (deep-sea anglerfishes) exhibiting exceptional species richness in the younger range of its estimated age of divergence. Ponyfishes (Leiognathidae) have evolved a complex array of sexually dimorphic muscular shutters and species-specific translucent windows to control the light emitted by the symbiotic bacteria living in a specialized pouch derived from esophageal tissue [ 7 , 10 ], and deep-sea anglerfishes have evolved complex, species-specific bioluminescent dorsal-fin escas (lures) that are presumably used for communication and prey attraction [ 29 ]. It is likely that the number of independent symbiotic relationships between fishes and bioluminescent bacteria could be higher than those estimated herein, given more fine scale species-level sampling of some lineages. For example, a densely sampled phylogeny of the diverse order Gobiiformes [ 30 ] suggests that bacterial bioluminescence may have independently evolved more than once among the cardinalfishes (Apogonidae), although bioluminescence was not explicitly optimized in the gobiiform study.

Across ray-finned fishes, intrinsic bioluminescence evolved at least eight times (Figs 1 and 2 , S1 Fig ) in some of the most species-rich lineages of deep-sea fishes (Figs 3 and 4 ), including dragonfishes (Stomiiformes, 426 species) and lanternfishes (Myctophiformes, 256 species). One genus of anglerfishes, the netdevils ( Linophyrne ), has even evolved an intrinsic bioluminescent chin barbel to complement their bacterially illuminated escal lure [ 29 ]. Despite evolving less frequently than bacterially mediated bioluminescence, intrinsic bioluminescence notably accounts for more than half of all known bioluminescent fish species and nearly 90 percent of bioluminescent species that exhibit exceptional species richness given their clade age ( Fig 4 ). A recent study hypothesized that bioluminescence functions as a species-specific communication/identification system among species-rich lineages (lanternfishes, dragonfishes) and that this system has played a significant role in their diversification in the deep sea, a region devoid of obvious physical barriers to reproduction [ 8 ]. The current study corroborates those findings and also indicates that other lineages with intrinsic bioluminescence and the potential for bioluminescent communication (as opposed to camouflage) have increased rates of diversification, including both inshore and deep-sea bioluminescent lineages that have more recently evolved (Batrachoidiformes and Alepocephaliformes, respectively, Fig 4 ). It is still unclear how most fishes with intrinsic bioluminescence obtain the necessary substrates to produce light. For at least one lineage of fishes ( Porichthys , midshipmen), luciferin is obtained from their diet [ 2 – 3 ].

We show that bioluminescence has repeatedly evolved in ray-finned fishes at varying times in Earth’s history (Figs 1 and 2 ), spanning the Mesozoic (150 to 65 Ma) and Cenozoic (65 Ma to present day). This suggests bioluminescence was present in Cretaceous seas and may have played an early role in the diversification of some deep-sea lineages that are exceptionally species rich given their clade age (lanternfishes and dragonfishes). Notably, none of the bioluminescent ray-finned fish lineages that possess exceptional present day species richness are thought to use bioluminescence exclusively for camouflage, with many of these lineages possessing species-specific anatomical structures that are thought to aid in communication, predation, and reproduction [ 7 – 8 ]. This pattern is also observed in squaliform sharks, where the two deep-sea bioluminescent lineages, Etmopteridae and Dalatiidae, are hypothesized to have also evolved during the Cretaceous and exhibit elevated rates of diversification [ 27 ]. As observed in the species-rich lanternfishes and dragonfishes [ 8 ], these sharks have species-specific bioluminescent structures and patterns [ 31 ]. Recent studies have shown that luminescent systems other than bioluminescence, such as biofluorescence, have repeatedly evolved and are phylogenetically widespread throughout the evolution of marine fishes [ 32 ]. Biofluorescence, like bioluminescence, may have a signaling function in marine fishes [ 32 – 33 ]. Our findings, and these additional studies investigating the evolution and function of bioluminescence and biofluorescence in marine systems, highlight how much remains to be discovered regarding the potential impacts of bioluminescence, and luminescent signaling in general, on the evolutionary history and ecology of marine fishes.

Supporting Information

S1 fig. maximum likelihood topology of the evolutionary relationships of ray-finned fishes..

Numbers at nodes indicate fossil calibrations. Black dots indicate bootstrap support value less than 60. All other nodes have bootstrap support values greater than 60.

https://doi.org/10.1371/journal.pone.0155154.s001

S1 Table. GenBank Accession Numbers for Newly Collected Sequences.

https://doi.org/10.1371/journal.pone.0155154.s002

S2 Table. GenBank Accession Numbers for COI Sequences.

https://doi.org/10.1371/journal.pone.0155154.s003

S3 Table. Classification of Vertebrates.

Classification of vertebrates ( www.classification.fish ) with known species diversity [ 26 ].

https://doi.org/10.1371/journal.pone.0155154.s004

S1 Text. Fossil Calibrations for Divergence Time Analyses.

https://doi.org/10.1371/journal.pone.0155154.s005

Acknowledgments

We thank the following people and institutions for providing specimens and tissue loans: A. Bentley (KU), H.J. Walker (SIO), K. Hartel and A. Williston (MCZ), C. McMahan, S. Mochel, and K. Swagel (FMNH), C. Baldwin (USNM), A. Graham (CSIRO), and M. Miya (CBM). Financial support was provided by National Science Foundation grants (DEB) to M.P.D. (1543654, 1258141), J.S.S. (0444842, 1257555), and W.L.S. (1060869, 1258141), with support from the Dalio Family Foundation, KU, and SCSU.

Author Contributions

Conceived and designed the experiments: MPD JSS WLS. Performed the experiments: MPD JSS WLS. Analyzed the data: MPD JSS WLS. Contributed reagents/materials/analysis tools: MPD JSS WLS. Wrote the paper: MPD JSS WLS.

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• 34. DeVaney SC (2008) The interrelationships of fishes of the order Stomiiformes. PhD Thesis, University of Kansas.

Terrestrial and marine bioluminescent organisms from the Indian subcontinent: a review

• Published: 05 November 2020
• Volume 192 , article number  747 , ( 2020 )

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• Ramesh Chatragadda   ORCID: orcid.org/0000-0002-8838-0583 1  

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The inception of bioluminescence by Harvey (1952) has led to a Nobel Prize to Osamu Shimomura (Chemistry, 2008) in biological research. Consequently, in recent years, bioluminescence-based assays to monitor toxic pollutants as a real-time marker, to study various diseases and their propagation in plants and animals, are developed in many countries. The emission ability of bioluminescence is improved by gene modification, and also, search for novel bioluminescent systems is underway. Over 100 species of organisms belonging to different taxa are known to be luminous in India. However, the diversity and distribution of luminous organisms and their applications are studied scarcely in the Indian scenario. In this context, the present review provides an overview of the current understanding of various bioluminescent organisms, functions, and applications. A detailed checklist of known bioluminescent organisms from India’s marine, terrestrial, and freshwater ecosystems is detailed. This review infers that Indian scientists are needed to extend their research on various aspects of luminescent organisms such as biodiversity, genomics, and chemical mechanisms for conservation, ecological, and biomedical applications.

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Acknowledgments

Author thanks the Director, NIO, for his support and encouragement. This is CSIR-NIO’s contribution reference number 6610. Special thanks to my friend, Dr. Tonlong Wangpan for informing me about the luminous spider. I thank Paresh Bagi for providing Mycena image.

This work is funded by the National Institute of Oceanography (CSIR-NIO), Goa.

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Chatragadda, R. Terrestrial and marine bioluminescent organisms from the Indian subcontinent: a review. Environ Monit Assess 192 , 747 (2020). https://doi.org/10.1007/s10661-020-08685-5

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Applications of bioluminescence in biotechnology and beyond †

First published on 18th March 2021

Bioluminescence is the fascinating natural phenomenon by which living creatures produce light. Bioluminescence occurs when the oxidation of a small-molecule luciferin is catalysed by an enzyme luciferase to form an excited-state species that emits light. There are over 30 known bioluminescent systems but the luciferin–luciferase pairs of only 11 systems have been characterised to-date, whilst other novel systems are currently under investigation. The different luciferin–luciferase pairs have different light emission wavelengths and hence are suitable for various applications. The last decade or so has seen great advances in protein engineering, synthetic chemistry, and physics which have allowed luciferins and luciferases to reach previously uncharted applications. The bioluminescence reaction is now routinely used for gene assays, the detection of protein–protein interactions, high-throughput screening (HTS) in drug discovery, hygiene control, analysis of pollution in ecosystems and in vivo imaging in small mammals. Moving away from sensing and imaging, the more recent highlights of the applications of bioluminescence in biomedicine include the bioluminescence-induced photo-uncaging of small-molecules, bioluminescence based photodynamic therapy (PDT) and the use of bioluminescence to control neurons. There has also been an increase in blue-sky research such as the engineering of various light emitting plants. This has led to lots of exciting multidisciplinary science across various disciplines. This review focuses on the past, present, and future applications of bioluminescence. We aim to make this review accessible to all chemists to understand how these applications were developed and what they rely upon, in simple understandable terms for a graduate chemist.

1. Introduction

Naturally occurring light originates from two main kinds of systems – bioluminescent systems which comprise of distinct luciferase enzymes and luciferin moieties, and photoproteins in which the light-emitting chromophore is part of the protein itself and light emission is triggered by changes in the protein's environment. The discovery of the mechanisms underpinning photoproteins such as the green fluorescent protein (GFP), aequorin, kaede and pholasin and their subsequent applications have been previously reviewed and will not be discussed in this review. 5–8

Whilst there are more than 40 known bioluminescent systems, the structures of the luciferin and luciferase have only been elucidated for 11 of them. The quest for the mechanistic characterisation of luciferin and luciferase pairs is an active area of research as is the search for new pairs. 9–11 The bioluminescent reaction generally requires a luciferase enzyme, its luciferin substrate and an oxidant which is often molecular oxygen. Some systems require energy in the form of ATP or NADH as well. One of the earliest luciferin structures to be elucidated were those of D -luciferin found in fireflies, reported in the mid 1900s. 12 About twenty years later the luciferin coelenterazine and its luciferase were discovered from the deep-sea shrimp Oplophorus gracilirostris . 13

Since then, due to the low toxicity, high quantum yield and high sensitivity of both of these reactions, these molecules and their luciferase enzyme partners have found wide use as in vitro reporters of analytes and metabolites – for example the firefly luciferase (FLuc) and D -luciferin system are widely used in biochemical assays to determine ATP levels. The last decade or so has seen luciferins and luciferases to reach previously uncharted applications fuelled by great advances in protein engineering, synthetic chemistry, physics and light capture technology. We will first cover some of the basic qualities and characteristics of the most widely used natural luciferin/luciferase pairs, with a brief mention of their uses as well as their shortcomings that make them less than ideal candidates for certain applications. This then leads up to the developments in synthetic luciferins and engineered luciferases and their improved properties and uses. The applications of bioluminescence are then extensively discussed including ATP sensing, hygiene control in the fish and milk industries, mapping pollution in ecosystems using bioluminescence based assays, culture and heritage in the form of art work preservation, the sensing of pH, metal ions, membrane potential, drug molecules, other metabolites, gene assays, the detection of protein–protein interactions, high-throughput screening in drug discovery and in vivo imaging of tumours as well as infections. Apart from sensing applications, the discussion will then move to how new applications are now trying to make use of the light from bioluminescence for various purposes such as to effect healing in the form of bioluminescence based photodynamic therapy (PDT) 14 or using the light for bioluminescence-induced photo-uncaging of small molecules, 15,16 and the use of bioluminescence to control neurons. 17 Exciting recent blue-sky research is also discussed such as the engineering of a light emitting plants of various types. 18 We aim to make this review accessible to all chemists to understand how these applications were developed and what they rely upon, in simple understandable terms for a graduate chemist. Hence, a stepwise journey across the various applications of bioluminescence has been taken, and how protein-engineering, synthetic chemistry and chemical biology tools have fed into the development of novel, state-of-the-art applications.

Whilst bioluminescence has found utility in various fields including medicine, biology, physics and engineering and led to exciting multidisciplinary science across all of them, we also discuss the current limitations in the state of the art, as well as prospects and future directions for the research community for the future development of applications of the luciferin–luciferase reaction from a chemical biology tool to something more widely used in industry, daily life or in a clinic.

2. Natural luciferin, their luciferases and mechanisms

In this section the mechanisms of bioluminescence with respect to D -luciferin, coelenterazine and bacterial luciferin have been looked at in greater detail as these luciferins and their respective luciferases have found the most utility in various applications. The remaining eight known luciferin–luciferase pairs have been included and discussed briefly towards the end of the section, as their understanding and inclusion would help foster future applications.

2.1 D -Luciferin

The activation of D -luciferin by ATP prior to oxidation is a unique feature of D -luciferin bioluminescence that is not shared by other luciferins such as coelenterazine. This feature bestows a variety of unique benefits upon the D -luciferin/firefly luciferase system that are not enjoyed by other bioluminescent systems. For example, as D -luciferin requires activation in the form of adenylation, it is less susceptible to auto-oxidation and more stable in solution, leading to less background chemiluminescence. 40 ATP is widely known as the ‘energy currency’ of a cell and found in varying amounts in virtually all eukaryotic and prokaryotic cells. 41 As the D -luciferin/Fluc assay produces ATP dependent light output, this assay has been widely adapted to measure ATP concentration in the double digit nanomolar region in various systems for both quality control and hygiene, often to determine the extent of bacterial contamination. 42,43 It is also used to monitor both ATP-forming reactions 44–47 and ATP-degrading reactions. 48–50 With the advent of more sensitive luminometers and improved assay techniques, ATP levels as low as attomole levels – corresponding to a single bacterial cell – can be routinely detected now using commercially available ATP bioluminescence reagents and kits. 42 More details on these assays are discussed in Section 4.1.

It is known that D -luciferin has greater aqueous solubility 51 and lower toxicity than coelenterazine. 52 Moreover D -luciferin has the most impressive quantum yield amongst all known luciferin/luciferase systems (41.0 ± 7.4% compared to 15–30% for most luciferase/luciferin pairs) 39,53 and the longest emission wavelength amongst them ( λ max = 558 nm 39 ), it is more ideal for imaging applications where blood is involved. Haemoglobin and tissues absorb light most strongly of wavelength <500 nm. 54 Mammalian cells and other biological entities of interest such as bacteria, fungi, protozoa and viruses can be genetically encoded with the luciferase gene of interest 55,56 and introduced in to a small mammal for proliferation, tracking and imaging. From naturally occurring luciferins and luciferases, D -luciferin and the firefly luciferase from the North American firefly, Photinus pyralis are the most common pair used for in vivo imaging (Table 1) . 57

D -Luciferin is commercially available in its carboxylic acid form (CAS Number: 2591-17-5) as well as its sodium salt (CAS Number: 103404-75-7). It can also be readily prepared in up to >50 mg scale batches using multi-step organic synthesis – preparations up to 50 mg scale have been reported although it can be envisaged that larger batches of >200 mg could be readily prepared too. 58,59 The plasmids and vectors used to express firefly luciferase are also readily available.

Although the light from D -luciferin bioluminescence is red-shifted compared to that from other naturally occurring luciferins, it is still strongly absorbed by blood and tissue. Near infra-red light (650–900 nm) has better penetration through blood and tissue. 60 Whilst there is a portion of light in the broad emission spectrum of D -luciferin within this desirable range of wavelengths, more red-shifted light would allow more sensitive imaging. 54

There are other factors as well that make D -luciferin a less than ideal candidate for bioluminescence imaging. For example, D -luciferin has modest cell permeability 61 and hence has to be dosed in large amounts for in vivo experiments. 62 Studies in which the 14 C-labelled radioactive D -luciferin substrate has been used have also demonstrated the inhomogeneous bio-distribution of the substrate in rodents. 63 Moreover, there is poor uptake of the probe in some organs of interest such as the brain. 64 Whilst D -luciferin is capable of emitting light of different wavelengths on interaction with various mutants of Fluc; 65 the range of wavelengths emitted does not render it suitable for in vivo multi-parametric imaging particularly in deep tissue imaging because the most red-shifted λ max obtained by D -luciferin and Fluc mutants is 620 nm. 66,67 The light at this wavelength suffers from absorption, attenuation and scatter in tissues making spectral unmixing from different luciferases more challenging. 68 Some of these set-backs have been overcome with the design, synthesis and testing of novel D -luciferin analogues, which have led to brighter, red-shifted emission in some cases. 69 Details on these can be found in Section 3.1.

There has also been a strong case for engineering the firefly luciferase enzyme to obtain better properties more suited for various applications. For example, in order to detect a variable ATP concentration, it is essential that the luciferase concentration remains constant during the duration of the measurement and luciferase is not inactivated by other factors such as pH, temperature, the concentration of metal ions, detergents and other soluble proteins such as bovine serum albumin, in the assay mixture. The optimum pH for wild-type luciferase activity is pH 7.8. 70,71 A workable pH of 6–8 can be used for analytical applications using the wildtype enzyme. 46 However, the colour of emission from some wild-type firefly luciferases such as P. pyralis and H. parvula changes from yellow-green to red when the pH is lowered below optimum. 72–74 The reason for this has been proposed to be the disruption of a key hydrogen-bonding network involving key water molecules and amino acid residues in the enzyme's active site around the oxyluciferin emitter. It is believed that disruption at lower pH enhances the delocalisation of the phenolate negative charge that increases red shifted emission. 75 This rendered these wild-type luciferases not as useful for comparative bio-analytical applications where often photons of a specific wavelength of light are being quantified. Moreover, most wild-type luciferases are thermolabile at even room temperature, whilst work on mammalian cells often requires temperatures of around 37 °C. 76 Indeed, wild-type luciferase has a half-life of only 3–4 h in mammalian cells due to both thermal inactivation and proteolysis. 77 Consequently, there has been significant work done in protein engineering to create mutant firefly luciferases of greater pH stability, thermostability and proteolytic stability. Indeed, a small change in the sequence of the enzyme can lead to changes significant changes in the emission and properties of the enzyme. For example, the mutant S286N of the Japanese Firefly Luciferase has red-shifted emission at λ max = 605 nm compared to the wild-type at λ max = 560 nm ( Fig. 2A and B ). This is because Ser 286 is involved in a key hydrogen-bonding network that is disrupted when it is mutated to an asparagine residue. Moreover, this also causes a change in the conformation of Ile 288 close to the emitter making the hydrophobic pocket more flexible ( Fig. 2C and D ). For further details on engineered Fluc enzymes please see Section 3.1.

2.2 Coelenterazine

Coelenterazine is commercially available (CAS Number: 55779-48-1) and can be synthesised in up to >200 mg scale batches using multi-step synthesis. 85 However, coelenterazine is a larger molecule than D -luciferin, has poor water solubility, greater toxicity and is also susceptible to auto-oxidation leading to chemiluminescence in solution as it does not need activation in the form of adenylation. 40 Moreover, coelenterazine has also been reported to be transported into cells through other mechanisms. For example, the multidrug resistance P-glycoprotein (MDR1 Pgp) was reported to mediate the transport of coelenterazine into cell lines. This led to greater amounts of coelenterazine being transported into cancer cells expressing greater quantities of MDR1 Pgp. This could lead to an inaccurate representation of tumours in small mammal in vivo imaging i.e. tumours that do not express MDR1 PGp would not be detected. 86 Coelenterazine gives out blue light which is strongly absorbed by blood and tissue, making it a poor candidate for in vivo imaging when used alone without the red-shifting effects of BRET. 54 To overcome these short-comings, several synthetic coelenterazine analogues have been prepared, of which some are commercially available. Details of these analogues are presented in Section 3.2.

In terms of size, both Rluc (∼34 kDa), Gluc and Mluc (both ∼20 kDa) are smaller than Fluc (∼62 kDa). This makes them more suitable for applications involving small vectors and/or proteins. In the sea pansy, Renilla reformis the luciferase Rluc is closely associated with a green fluorescent protein (GFP) and the blue light emitted by the luciferase is coupled through resonance energy transfer to the fluorophore of the GFP allowing it to form an excited-state species which emits a photon of green light ( λ max 510 nm). 87 This principle of resonance energy transfer led to the development of bioluminescence resonance energy transfer (BRET) and several associated applications where the BRET light emission has be used as a measure for the spatial proximity of two proteins. 88

Most coelenterazine utilising luciferases possess several disulphide bonds in the protein structure which often help in protein-folding and confer these enzymes with greater thermal stability than firefly luciferases. However, these disulfide bonds also make the enzymes sensitive to any reducing agents in buffer solutions as it is vital for the cysteine residues to be in the correct oxidation state for native protein folding. Moreover, the optimum conditions for activity of these luciferases often mimic their natural marine environment. So, most of these luciferases are halophilic reflecting the saltiness of seawater and some forms – for example the Mluc2 isoform of Metridia longa luciferase – is psychrophilic i.e. has a very low optimum temperature reflecting the low temperatures at the bottom of the ocean ( Table 2 ). 89 Copepod luciferases Gluc and Mluc are the only known luciferases that are naturally secreted from eukaryotic cells. 90 These unique properties make them potentially useful for a unique set of applications such as high throughput studies as cell-lysis is not required.

2.3 Bacterial luciferin

The mechanism of bacterial bioluminescence is well understood (Fig. 4) . 104 Flavin mononucleotide (FMN) reductase reduces oxidised FMN 12 to form reduced FMN 13 which reacts with molecular oxygen to form an FMN-hydroperoxide species 14 , possibly through a single electron transfer (SET) process as suggested for adenylated D -luciferin and oxygen. 35,105,106 In the absence of long-chain aldehyde 15 , intermediate 14 is fairly stable and was characterised by UV-vis absorption and NMR spectroscopy. 107,108 It has been proposed that the long-chain aldehyde 15 adds to 14 to form the FMN-4 a -peroxyhemiacetal species 16 which collapses to form the carboxylic acid 17 and FMN-4 a -hydroxide 18 in an excited state. 106 The exact mechanism of formation of the excited-state species is still under debate. A few different mechanisms have been proposed for the breakdown of FMN-4 a -peroxyhemiacetal species 16 , including the proposed formation of a dioxirane intermediate 109 or a flavin-mediated intra-molecular electron transfer mechanism initiating the collapse of 16 . There is greater computational 110 and experimental data 111 to support the intra-molecular flavin-mediated electron transfer from the 5-N of the isoalloxazine ring to the distal oxygen atom of FMN-4 a -peroxyhemiacetal species 16 . Light emission occurs when the flavin mononucleotide species FMN-4 a -hydroxide 18 relaxes to its ground state 19 and emits a photon of bluish-green light ( λ max 490 nm) (Table 3) . The long-chain aldehyde is oxidised to the corresponding carboxylic acid as part of the cycle.

In essence, the entire light emission machinery including luciferase production, luciferin biosynthesis and recycling machinery is genetically encoded for together in a single operon, so transgenic auto-luminescence is possible. Exogenous administration of luciferin is not required in any imaging applications, unlike what is observed in firefly bioluminescence imaging and coelenterazine bioluminescence imaging. This is of great value in synthetic biology, especially as the administration of expensive and unstable luciferins in some hosts such as plants is difficult. 112,113 However, considerable work and optimisation of the genetic make-up was required to make bacterial operons useful in eukaryotic cells and machinery. Although in early studies only luxA and luxB were expressed in plant cells such as tobacco and carrots to form functional luciferase in them, autoluminescent tobacco plants were later designed using the operons form Photobacterium leighognathi lux operon. 114,115 Although previously, the bacterial lux operon had been used to produce a weakly autoluminescent human cell line ( HEK293 cells), more recently codon-optimised lux sequences have been developed that produce bright, autonomous bioluminescence in mammalian cells. 116,117 Further details of the codon-optimisation and enzyme engineering involved are presented in Section 3.3.

Bioluminescent bacteria are often used as biosensors in ecotoxicological studies as these can often be tuned to detect concentrations of a variety of different organic (alcohol, carboxylic acids, aromatic compounds) and inorganic substances (heavy metal ions). 118–120 Moreover, bioluminescent bacteria are also being used to create unique glowing artwork and proposals have been suggested to use them in indoor aquariums as part of aesthetic architecture in skyscrapers. 121

As both bacterial luciferase and luciferin are encoded for in the lux operon, this has led to limited mutations in the native luciferase structure and no mutations to the luciferin, although shorter-chain aldehydes are also tolerated and as expected result in no change in wavelength of the light emitted. 105

2.4 Other known luciferins

The discovery and characterisation of yet-unknown luciferins and luciferases is an area of active research and recent highlights include reports on the luciferin–luciferase systems of the New Zealand glow-worm Arachnocampa luminosa and the diptera Orfelia fultoni from the United States. 151,152 Both systems emit blue light and although structures of the luciferins have been proposed based on NMR spectroscopy, mass spectrometry and isotopic labelling studies although, the proposed structures need to be validated through chemical synthesis.

3. Designer luciferins and their luciferases

3.1 novel d -luciferin analogues and luciferase mutants.

The chemical synthetic routes towards luciferin analogues were succinctly compiled by Podsiadly et al. in 2019. 69 Herein, we briefly highlight synthetic luciferin analogues, particularly those that have found use in applications, or have useful and interesting properties such as brightness that could potentially make them useful in applications. As the primary use of red-shifted D -luciferin analogues is in in vivo applications, we particularly highlight analogues that emit in the near infra-red region (>650 nm), and of those analogues, specifically those that have been successfully tested in vivo ( Table 5 ). It is important to note that although the Fluc/ D -luciferin combination emits at λ max = 558 nm in vitro , there is a spectral red-shift observed in in vivo imaging, with λ max ∼ 610 nm due to attenuation through tissue. 153

Of all the synthetic luciferin analogues reported to-date, BtLH 2 28 was reported to have the highest bioluminescence quantum yield (70%) relative to that of D -luciferin 1 with wildtype Fluc. Although BtLH 2 28 , had a bioluminescence λ max of 523 nm which was around 20 nm blue-shifted, it was also reported to have a longer-lasting and sustained bioluminescence signal compared to that of D -luciferin. 154 This could possibly make it a better candidate for applications that require blue-shifted and longer-lasting light emission. One of the earliest reported synthetic luciferins was the aminoluciferin 29 by White et al. This synthetic luciferin was red-shifted ( λ max = 594 nm) compared to D -luciferin ( λ max = 558 nm) but only about 10% as bright in vitro . 61,155 Since then, several other synthetic aminoluciferin analogues were reported, and although all of them are dimmer than D -luciferin 1 , with the wild-type Fluc, some have useful properties for in vivo imaging. For example, aminoluciferin analogues 30 and 31 have emission around λ max ∼ 600 nm. Both analogues 30 and 31 was reported to have better penetration than D -luciferin through the blood–brain barrier in mice. 156,157 Moreover, analogue 31 CycLuc1 was reported to have brighter bioluminescence output from cells at lower substrate concentrations than D -luciferin 1 , indicating that it had better cell-permeability than D -luciferin 1 . 62 Both analogues 30 and 31 are commercially available. The Prescher group reported a brominated luciferin analogue, that was red-shifted ( λ max = 625 nm) and had an appreciable relative bioluminescence quantum yield of 46% compared to D -luciferin 1 at 100 μM substrate concentration and 1 μg of Fluc. 158 At these concentrations they reported aminoluciferin 29 to be 61% as bright as D -luciferin 1 .

A handful of synthetic luciferin analogues truly emit in the nrIR region (>650 nm). The analogues 32–34 were reported by Maki et al. and have been successfully taken on to in vivo detection of tumours in mice. 159–161 In particular, the hydrochloride salt of Akalumine 32 was used with the engineered firefly luciferase Akaluc in single-cell bioluminescence imaging (BLI) of deep-tissue in the lungs of live, freely-moving mice and to image small numbers of neurones in the brains of live marmosets. 64 The analogues Akalumine 32 and Sempei 34 are now commercially available from Merck. The analogues 34 and 36 were also reported by Maki et al. as significantly dimmer substrates than Akalumine 32 but whose emission was ∼700 nm. 162 Anderson et al. reported the analogue iLH 2 38 as a significantly red-shifted luciferin that showed enhanced tumour burden in deep-seated tumours such as liver metastasis in mice due to less scattering and greater penetration of the near-infrared (nrIR) light. 66 The luciferin iLH 2 38 was designed to emit different colours of light with different Fluc mutants through retention of the phenol group which is deemed necessary to modulate the colour emission of D -luciferin analogues. This ability of iLH 2 38 to emit different colours of light with different Fluc mutants made it unique, and it was proposed that iLH 2 38 was a suitable analogue for multiparametric imaging and tomography. Recently, the suitability of iLH 2 38 also demonstrated in a report by Anderson and co-workers in which racemic iLH 2 38 together with stabilised colour mutants of firefly luciferase (Fluc_green ∼ 680 nm and Fluc_red ∼ 720 nm) were shown to be a suitable system for nrIR dual in vivo bioluminescence imaging in mouse models where they simultaneously monitored both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model. 68 The Anderson lab also reported the analogue PBIiLH 2 37 as a racemic compound, which was prepared as a conformationally restrained infra-luciferin analogue. 163 Although PBIiLH 2 37 was only tested in in vitro assays and found to be less bright than racemic iLH 2 38 , it did demonstrate an increased bimodal emission with increasing pH. A primary bioluminescence peak at 608 nm was observed with Fluc x11 and a secondary peak at 714 nm of increasing intensity. This emission pattern could potentially be used to monitor pH, although the work does not build on this possibility. 164 In 2018, Mezzanote et al. reported the most red-shifted luciferin analogue 40 and sister compound 39 with mutant CBR2opt luciferases without the use of resonance transfer. 165 Both analogues 39 and 40 were tested in HEK-293 cell lines expressing CBR2opt, and as the analogue 39 gave higher light output than the analogue 40 , the analogue 39 was tested in in vivo mouse studies. The most useful output from this study appeared to be the development of the mutant luciferase CBR2opt as the D -luciferin 1 and CBR2opt combination was demonstrated to be the brightest and most useful in all the experiments reported in the work, whilst both analogues 39 and 40 were dimmer than D -luciferin with CBR2opt.

In the area of luciferase engineering, several firefly and beetle luciferase mutants have been reported with improved properties such as increased stability, increased substrate affinity, and increased brightness over the years and these were comprehensively reviewed in 2016 by Yampolsky et al. 10 Some highlights since then include engineered luciferases for improved light output of specific synthetic substrates such as Akaluc for Akalumine 32 and CBR2opt for the analogue 39 . 165,166 Other highlights include work by Miller et al. on mutants that have a significantly higher K m for D -luciferin and ATP than for cyclic amino-luciferins. The rate of reaction when the enzyme is saturated with substrate is the maximum rate of reaction, V max . For practical purposes, K m is the concentration of substrate which permits the enzyme to achieve half V max . An enzyme with a high K m for a particular substrate has a low affinity for that substrate and requires a greater concentration of the substrate to achieve V max . Hence the mutants developed by Miller et al. are more selective for cyclic aminoluciferins than for D -luciferin and this allowed substrate-selective BLI in mouse-brain. 167 The Prescher group reported an elegant piece of work in which they prepared a library of 159 mutant luciferases by mutations of 23 key residues near the active site of the enzyme. 168 These were then screened against 12 synthetic luciferins to identify orthogonal luciferin–luciferase pairs. Three of the ‘ hit ’ pairs from this analysis were taken up for in vivo mouse studies of mammary carcinoma. 169 Imaging conditions are sensitive to a variety of factors, such as concentration of the imaging agent, the type of cell line used and the type of mouse model and tumour or infection studied. Although a number of synthetic luciferin analogues and mutant luciferases have been reported, there are very few studies reported that compare these against each other to match the best luciferin analogue and its complementary luciferase for a particular application in one study.

3.2 Coelenterazine analogues and luciferase mutants

A host of luciferases including Renilla luciferase (Rluc) from the sea pansy, Gaussia luciferase (Gluc) from the marine copepod and Oplophorus gracilirostris luciferase (Oluc), from the deep-sea shrimp use coelenterazine 7 as their substrate. 24,25,79–84

A number of key developments using protein engineering were carried out on these coelenterazine utilising luciferases, which resulted in useful imaging and visualisation tools. For example, Nagai and co-workers developed the Nano-lantern, which was the brightest luminescent protein reported at the time in 2012. This was a chimera of Rluc8 (a brighter and more stable Rluc mutant) 93 and a fluorescent protein called Venus which has high BRET efficiency. 170 They then further developed this work by using different fluorescent proteins as BRET acceptors of Rluc8 and other Rluc mutants to develop a suite of Nano-lanterns that emit light of different colours, including the most red-shifted Nano-lantern ReNL ( λ max 585 nm). 171–173 The Nano-lantern series was shown to have broad applicability in both in vitro and in vivo imaging, as well as in the detection of Ca 2+ ions.

In another ground-breaking development, the catalytically active portion of Oluc was identified and mutated using a combination of both rational mutagenesis and random mutagenesis for enhanced thermal stability and light output by Promega. 174 This small 19 kDa mutant enzyme was called Nanoluc and it was optimised to perform best with the synthetic substrate furimazine (Fz) 42 Table 6 . 175 Both Nanoluc and furimazine are now commercially available. However, the Nanoluc-furimazine combination emits blue light ( λ max ∼ 456 nm) which makes it unsuitable for in vivo applications, although the fact that this system has ATP-independent emission has led to possible advantages in some applications over the firefly bioluminescence system. 176

Following the development of Nluc, there were reports of chimeric proteins that use Nluc as the BRET donor together with a fluorescent protein as the BRET acceptor. For example, the LumiFluor series was developed by creating chimeras of Nluc with bright, fluorescent proteins such as eGFP to get emission of around ∼460–508 nm or with an orange light emitting GFP variant LSSmOrange for emission ∼572 nm. 177 The LumiFluor series was shown to be useful in the in vivo imaging of tumours as well. In a complimentary approach to the development of Nano-lanterns, small-molecule fluorophores could also be appended to Nluc through the development of Nluc-Halotag fusion proteins. 178,179 BRET then occurs from the furimazine oxyluciferin to the fluorophore.

A number of coelenterazine analogues including 43 and 44 were reported by Shimomura et al. and these were tested against the wild-type luciferases that naturally utilise coelenterazine. 180 The analogue e-CTZ 43 was reported to be ∼1.4 times brighter than CTZ 7 with Rluc and had a 7.5 times higher initial peak intensity than CTZ 7 . This could potentially be due to the fact that this analogue is a conformationally restrained analogue of CTZ 7 . The analogue v-CTZ 45 was reported to be 0.73 times brighter than CTZ 7 with Rluc but had a 6.4 times higher initial peak intensity than CTZ 7 . It was also unsurprisingly more red-shifted than CTZ 7 , possibly due to extended conjugation of the π-electon system. This led to an emission of λ max ∼ 512 nm. Both these analogues have been used with modified and optimised Rluc luciferase systems in the in vivo imaging of small mammals and are commercially available. 181,182

Recently, some furimazine analogues such as 45 and 47 have been reported which have red-shifted emission with Nanoluc in the absence of any resonance transfer fluorophore. 183 However, these analogues were about 10 2 –10 4 times dimmer in in vitro assays than the Fz-Nanoluc combination. Consequently, Nanoluc was mutated further in attempts towards red-shifted emission. These attempts had limited success, with light emission being red-shifted only up to 509 nm which is still blueish-green light. 184 In an attempt to create bright and red-shifted reporters, Nanoluc was fused with CyOFP1, a bright, engineered, orange-red fluorescent protein that is excitable by cyan light (497–523 nm), to develop a BRET-based genetically encoded reporter called Antares. The Fz-Antares combination was reported to be the brightest in vitro and in vivo when compared with D -luciferin-Fluc, Fz-Nanoluc and Fz-Orange Nanolantern combinations. 185

Building on from this work, Ai et al. created another fusion protein Antares2 in which random mutations were introduced in NanoLuc across the gene using error-prone PCR. 184 From this, they identified a Nanoluc mutant (Nanoluc-D19S/D85N/C164H) with a 5.7-fold enhancement of DTZ bioluminescence and named it teLuc as it gave teal coloured emission ( λ max ∼ 502 nm). The teLuc fusion with CyOFP1 was named Antares2. This BRET based reporter utilised DTZ 46 rather than Fz 42 and emitted 3.8 times more photons above 600 nm than Antares. Although this analogue is reported to be brighter than D -luciferin 1 and Akalumine 33 in vitro , it suffers from poor solubility in aqueous media and poor stability as like all coelenterazine analogues it is prone to auto-oxidation. This makes in vivo studies particularly challenging. In order to address these challenges, Ai et al. reported a number of pyridyl analogues of DTZ 46 including 8-pyDTZ 48 that had ∼13 times better aqueous solubility and bioavailability than DTZ 46 . 186 In their work they reported the development of a teLuc mutant called LumiLuc which was through a series of error-prone PCR experiments on teLuc resulting in a total of 12 mutations, The emission from 8-pyDTZ/LumiLuc was ∼5 times brighter than 8-pyDTZ/teLuc and had emission around λ max ∼ 525 nm. LumiLuc was then fused to a fluorescent protein mSCarlet-I to form LumiScarlet which was useful in BRET based BLI and had emission around λ max ∼ 600 nm. The emission from 8-pyDTZ/LumiScarlet in in vivo imaging was reported to be comparable to that by Akalumine/Akaluc.

Two new substrates hydrofurimazine 49 (HFz) and fluorofurimazine 50 (FFz) were also reported recently to address the challenges of solubility and bioavailability in coelenterazine analogues. The analogue HFz 49 exhibited similar brightness to AkaLuc with its substrate Akalumine 33 , whilst a second substrate, FFz 50 with even higher brightness in vivo . The FFz-Antares combination was used to track tumour size in vivo whilst Akalumine-AkaLuc combination was used to visualise CAR-T cells within the same mice. 187

3.3 Bacterial bioluminescence

A breakthrough in this area was reported by Gregor, Hell and co-workers, who engineered the ilux operon. 195 Prior to their work, bacterial bioluminescence resulted in a weakly autoluminescent mammalian cell line. 116 Hell and co-workers chose the luxCDABE operon from P. luminescens due to its thermostability and systematically carried out studies to identify the cause of poor luminescence in mammalian cells. This led to codon optimisation and enzyme engineering, including supplementing the FMN reductase in P. luminescens with that from V. campbellii followed by error-prone PCR to select brighter mutants. The final result ilux contains a total of at least 15 mutations and around 8 times brighter than the original construct allowing single-cell imaging of bacterial cells for extended periods of time in vitro . 195 This work was then further developed by codon optimisation of ilux for mammalian cells, which led to brighter emission by around 3 orders of magnitude compared to previous approaches. The light output was also reported to be comparable to that of a D -luciferin/Fluc system in HeLa cells.

4. Applications

4.1 atp sensing.

Once the reagents are mixed at 25 °C, there is a 0.25 ms time lag until light emission is observed after which light output peaks at around 300 ms. This is followed by a rapid decay in light output and then finally slow, sustained light emission. 197 This phenomenon is known as ‘burst kinetics’ and the decay in light output is thought to be due to inactivation of the enzyme or if a significant proportion of the substrates D -luciferin and ATP are consumed per minute in the reaction, when their concentration is low compared to that of the luciferase. 42 Inactivation of the luciferase can occur if the luciferase is bound to surfaces or there is a significant concentration of an inhibitor such as oxyluciferin – the product of the reaction, or contaminants in the D -luciferin preparation such as L -luciferin and dehydroluciferin. Inactivation of the enzyme can be counteracted by using a highly pure D -luciferin sample to avoid contaminants, and by the addition of stabilising substances such as bovine serum albumin (BSA), neutral detergents and osmolytes for the protein, so that stable light output is obtained. 198 In most ATP sensing assays, a fixed amount of D -luciferin is added to the assay mixture, which is in excess of ATP levels. At high luciferase concentrations, the peak light output is proportional to the amount of luciferase, as ATP is depleted in a first-order reaction. When luciferase concentration is low, ATP is slowly depleted and so light emission is stable and proportional to the ATP concentration, when the ATP concentration is significantly below the K m of the enzyme i.e. ATP < 0.1 mM L −1 , as the rate of reaction and hence light output are proportional to the ATP concentration below K m . 48 This is useful to monitor ATP forming and ATP degrading reactions including kinetic and end-point assays of enzymes and metabolites. 42,48,199

Through the careful manipulation of all of these factors, a series of ATP-bioluminescence reagents have been made commercially available as simple-to-use kits which include the luciferin and luciferase preparations. These reagents are of two main types based on the intensity and duration of light-emission. Constant light emitting reagents have moderate sensitivity towards ATP (working range: 10 −6 to 10 −11 M ATP). The constant light signal is useful for kinetic studies of enzymes and metabolic studies, or if coupled enzymatic assays are applied. Such assays have been used to determine the amount of ATP in various diseased and healthy cell lines using both lysed human HeLa cells, mouse MEF cells and in worms such as the round worm, as well as intact cells or isolated mitochondria. 200–203 This type of reagent can also be used to determine the activity of enzymes such as the activity of H + -ATP synthase from live isolated mitochondria. 204

The second type of ATP-bioluminescence reagents are high sensitivity light emitting reagents. These have a higher concentration of luciferase and exploit the ‘burst kinetics’ phenomenon where the peak height is proportional to the amount of ATP in the sample and dependent on the concentration of luciferase. These reagent combinations have higher sensitivity towards ATP (working range: 10 −5 to 10 −12 M ATP), although reagents that report even lower detection limits are available in the market. These reagents are often sold packaged with cell lysis reagents, and are suitable for use in luminometers where automatic injection of the reagents is possible such as in tube luminometers and microplate luminometers (Table S1, ESI † ). 48

It is important to note that different types of cells have varying levels of ATP. For example, bacteria have lower levels of ATP compared to fungi or mammalian cells. 48 It is important to pre-treat the sample effectively, and to use aseptic conditions to ensure that ATP levels from the correct desired source are detected. For example, a clinical urine sample may contain 3 different pools of ATP – extracellular ATP, ATP in mammalian cells, and ATP in bacterial cells and the purpose of an ATP bioluminescence measurement might be to estimate bacterial levels. The level of ATP from all 3 sources can be detected by using an appropriate kit containing ATP-degrading enzyme, neutral detergent, strong extractant, ATP reagent, and ATP standard ( Fig. 5 and Table S1, ESI † ). 48

Recently, there have been developments in luciferase engineering that allow ATP bioluminescence technology to reach unchartered territories. For example, Branchini et al. reported a red-emitting chimeric firefly luciferase that has a low K m for ATP and D -luciferin and would reach half of the maximum rate of the bioluminescence reaction at lower levels of ATP and D -luciferin than the wild-type enzyme making it suitable for in vivo imaging in low ATP cellular environments. 205 Moreover Pinton et al reported protocols for the in cellulo and in vivo the use of chimeric luciferases that ensure the specific cellular localisation of the luciferase in a cell i.e. in the mitochondrial matrix and the outer surface of the plasma membrane to determine the ATP concentration in those areas. 206 Viviani and co-workers also reported a blue-shifted luciferase that has the lowest reported K m for ATP, highest catalytic efficiency, and thermal stability among beetle luciferases that was suitable for ratiometric ATP, metal and pH biosensing assays. 207 Metal sensing bioluminescence assays are covered in greater detail in Section 4.3, while pH sensing assays are covered in Section 4.5. There have also been advances in the development of more sensitive and portable luminometers and sensing devices. 208,209 More recently, Roda and co-workers reported a low-cost wax-printed nitrocellulose paper biosensor that immobilised luciferase/luciferin reagents, and whose light signal could be detected and analysed by a smart phone to detect E. coli levels in urine samples. 210 This low-cost, readily available technology would be ideal for use in developing countries where access to a luminometer and other specialist kit would be limited.

4.2 Hygiene control

Several studies have been reported on the use of swab taking and ATP bioluminescence as a quick and objective way of monitoring the cleanliness of hospital surfaces, including those of large objects such as tables and benches and small pieces of equipment such as tweezers and other kit. Swabs that are impregnated with buffer are often commercially available as part of ATP-bioluminescence kits. These are used to sample surfaces, and then processed with the ATP-bioluminescence reagent in a portable luminometer. The swabs and portable luminometer are often sold from the same supplier and complement each other. However, this technique is still poorly standardised at an international level and the difference in kit and reagents used in different studies is one reason why significant differences in ATP levels are reported. 215,216 Despite these limitations over the comparability of results, ATP bioluminescence remains a quick and cost-effective measure for surface cleanliness and hygiene control in hospitals and routinely informs the cleaning practices of housekeeping and healthcare staff. 217 More recently these assays have been used to monitor the cleanliness of not just surfaces but surgical instruments and dentures as well. 218,219

ATP bioluminescence measurements are routinely used to monitor quality control and hygiene in the food industry. 212 In particular, fish processing plants have used it for decades to determine contamination levels. 220 Recently a study was reported to determine the contamination levels in various fish processing environments i.e. different lines of production including different fish types such as trout and cod, different types of meat such as protein-rich loin meat or fat-rich belly meat and different levels of processing such as slaughtered or cooked products, and the results were compared against conventional culture and plating techniques. 221 It was established that it is essential to set up critical limits after a period of validation and calibration that are specific to each processing plant, type of ATP-bioluminescence kit used, specific areas, types of fish and fish meat and different hygiene zones, to obtain more robust, consistent and meaningful results. The dairy industry also benefits greatly from ATP bioluminescence assays as these are used to determine the quality of milk by selectively measuring the ATP from somatic cells and milk spoilage by determining ATP levels from bacteria and other microorganisms both before and after UHT treatment to estimate shelf life. 222

The lux operon which is responsible for bacterial bioluminescence has also found great utility in the food industry. 214 The lux genes responsible for bioluminescence can be genetically encoded onto bacteria that are not naturally bioluminescent, and the localisation, population size and environment of these bacteria can be monitored in real time. As all known bioluminescent bacteria are Gram-negative, there were initial challenges in obtaining a good level of light output and gene expression in Gram positive bacteria. However, this was overcome by introducing translational signals optimized for Gram positive bacteria in front of luxA , luxC and luxE genes. 223,224 To date bioluminescent E. coli and Listeria have been used to monitor the survival of these bacteria post-processing in yoghurt and cheese. 225 A number of lux -tagged bacteria such as Campylobacter jejuni and Salmonella enteritidis have also been used to determine egg-shell penetration and colonisation. 225,226 Lux -tagged bacteria have also been used to monitor the development of bacterial infection in plant seedlings so interventions can be made at the appropriate time. 227 The lux -based gene expression system has also been fused to genes of bacterial toxin production such as the promoter of the cereulide toxin gene ces in B. cereus to determine the ability of various foods to support toxin formation. 228 Lux-tagged bacteria have been administered to mice for in vivo imaging of the resultant developing bacterial infection – for example lux -tagged L. monocytogenes were shown to grow and localise in the gallbladder of mice and cause re-infection in the intestines when bile was released. 229 However, as bacterial bioluminescence emits predominantly blue light which is strongly absorbed by blood and tissue, it is important do ex vivo analysis of the organs as well to ensure bacterial colonies are not missed. Another use of lux-tagged bacteria has also been to detect biofilms and to develop cleaning methods against them, as well as to test the efficacy of hand sanitisers and disinfectants. As well as monitoring the growth and development of pathogenic bacteria, lux -tagged probiotic bacteria can also be monitored in foods that contain them as well as tracking the bacteria using in vivo imaging to understand their lifecycle and environment. 230,231

4.3 Mapping pollution in ecosystems

If the lux gene is expressed continuously, luciferase and luciferin will be formed continuously and the baseline light intensity would change on addition of the target analyte, depending on how well the bacterial cell survives. Alternatively, the lux gene can be controlled in an inducible manner wherein it would be fused to a promoter that is regulated by the compound of interest. In this case, the concentration of the compound can be quantitatively detected by measuring the bioluminescence intensity. 118 Previously bacterial bioluminescence sensors were reported to analyse a variety of analytes including zinc, bioavailable toluene and uranium. 234–236 Some recent examples in the development of bacterial bioluminescence biosensors to detect various analytes of ecotoxicology interest can be seen below ( Table 7 ). Like most assays, careful pre-treatment of the sample to eliminate interference causing agents is essential to get meaningful results.

The other common application of bioluminescence in ecotoxicology and pollutant monitoring is ATP quantification using the firefly bioluminescence ATP assay in both aquatic environments and bioaerosols in the atmosphere. ATP bioluminescence-based sensors have been used to determine the total ATP in water bodies including ocean environments and drinking water up to a detection limit of 1.1 × 10 −11 M. 244,245 This would include ATP from not just bacteria but also fungal cells and any parasitic protozoa. ATP bioluminescence-based sensors have also been used to detect the location and density of several air-borne bacteria in both artificially created and natural bioaerosols in indoor environments. 246 The biosensors reported have either used fabricated paper disks immobilised with luciferase/ D -luciferin or sensors microfluidic chips. 247,248 Air was vented into and bubbled into a bio-sampler bottle containing 20 mL of deionised water to capture any cells found in the air. This solution was then concentrated and heated to lyse the cells. This lysate was then dripped on the fabricated paper disks immobilised with luciferase/ D -luciferin. The fabricated paper disks with immobilised with luciferase/ D -luciferin were reported to have up to 10 times longer shelf-life compared to the liquid assay reagents when stored at room temperature. 247 Although this is a quick method to identify air-borne bacteria and their levels in studies where the identity of the bacterium is known i.e. artificially created bioaerosols, it is important to calibrate the assay effectively with known samples and use the ATP bioluminescence assay together with another assay to validate the results. 249–251

4.4 Culture and heritage – preservation of art work

4.5 sensing of ph, metal ions, reactive oxygen species (ros), enzymes, drug molecules, and membrane potential including in cellulo applications.

Caged D -luciferin probes have been used as sensors for enzyme activity, 262–269 small molecule sensors for molecules such as glycans, 270 hydrogen sulphide, 271 hypochlorous acid, 272 carbon monoxide 273,274 and hydrogen peroxide, 275 and sensors for metal ions such as copper, 276 iron, 277,278 and cobalt. 279 The benefit of bioluminescence is that no incident light is needed and so the signal to noise ratio is higher and therefore more accurate. Although a potential drawback of these bioluminescence-based probes compared to similar fluorescent probes is that the bioluminescent probes are administered in much higher concentrations in cell-based assays than fluorescent probes, and this might affect the physiological conditions of the cells. Nonetheless, several caged-luciferins have been successfully used for in vivo imaging of mice and there has been an excellent recent review covering the advances in this area. 280 Moreover, the first example of a bioluminescent probe that can measure mitochondrial membrane potential in a non-invasive manner in vivo has just been reported to be a caged luciferin probe that called a ‘mitochondria-activatable luciferin’ (MAL probe) ( Fig. 8 ). The MAL probe is uncaged by a bioorthogonal Staudinger reaction with an organic azide (Azido-TPP1), to release a functional luciferin, which will emit light in the presence of luciferase. The triphenylphosphonium (TPP) groups on both the organic azide and the caged luciferin directs both reagents to the mitochondria. The rate of uncaging and hence rate of formation of active luciferin is proportional to the combined changes in mitochondrial membrane potential and plasma membrane potential. 281

The other type of luciferin probes that have been reported are designed to use the Bioluminescent Enzyme-Induced Electron Transfer (BioLeT) process to modify the light output generated. Bioluminescent Enzyme-Induced Electron Transfer (BioLeT) is analogous to photoinduced electron transfer (PeT) which has often been incorporated in the design of fluorescent probes. 282,283 The design concept is that the singlet excited-state oxyluciferin species can be quenched by the electron transfer from the highest energy molecular orbital (HOMO) of an electron rich benzene moiety in close proximity. This was first reported in the design of a sensor for nitric oxide (NO), which is very dim in the absence of NO, due to BioLeT, but significantly brighter in the presence of NO, due to the absence of the electron-donating moiety ( Fig. 9 ). 284 This work also reported the successful use of this probe in vivo mice models. The authors have also reported another BioLet probe with turn-on luminescence that detect highly reactive oxygen species. 285

The Nano-lantern developed by Nagai et al. (Section 3.2) were also developed further in the same piece of work to detect ATP concentration. 170 A chimeric fusion protein of the Nano-lantern with a subunit of bacterial F o F 1 -ATP synthase led to the development of Nano-lantern (ATP1) which exhibited an increase in light output on the addition of ATP with a K d of 0.3 mM. This was then used to visualise ATP formation in chloroplasts. 170 A number of Nano-lantern based GECIs were also developed by genetically engineering the Nano-lantern probe with a calcium-sensing domain from an established fluorescent Ca 2+ sensor to form Nano-lantern (Ca 2+ ) which gave comparable output and sensitivity to the fluorescent, genetically encoded Ca 2+ sensor it was developed from. 170

Another important class of luciferase-based sensors are the luciferase-based indicators of drugs (LUCIDs) developed by Johnsson and co-workers. 288 These are semisynthetic bioluminescent sensor proteins that consist of three components: a receptor protein for the drug of interest covalently linked to a luciferase (Nluc), which is linked to a self-labelling protein such as SNAP-tag ( Fig. 10A ). The self-labelling protein was further linked to a synthetic molecule that consists of a fluorophore that can accept BRET from the luciferin–luciferase reaction and a ligand for the receptor protein. Binding of the protein with the ligand, lead to close proximity of the Nluc with the fluorophore and red-shifted emission due to BRET. In the presence of a drug molecule, this interaction is perturbed and hence a measure of the ratio of blue light/red light leads to a measure of drug concentration. LUCIDs were shown capable of detecting both small-molecule drugs and larger peptidic and macrocyclic drugs as well. The LUCIB and analytes were spotted on filter paper and the light output measured using a digital camera making them useful candidates for point-of-care diagnostics. Later Johnsson and co-workers reported the use of antibodies in place of the receptor protein to make the technology more easily accessible. 289

A number of BRET-based antibody sensors have also been reported. Merkx and co-workers reported a luminescent antibody sensing (LUMABS) technology to detect antibodies in blood plasma. 290 In these single protein sensors Nluc is connected to a green fluorescent protein mNeonGreen via a semiflexible linker and two antibody binding epitopes. A helper domain is found on each protein that keep both them in close contact to allow efficient BRET in the absence of an antibody ( Fig. 10B ). When an antibody binds to the sensing domain, the close proximity of the two light emitting proteins is disrupted leading to loss of BRET. A measure of this signal allows a ratiometric measure of antibody concentration. Initially this assay was optimised to a 384 well plate and the light output measured using a mobile phone. This technology has been further developed to enable identification of non-peptide epitopes, 291 optimised to use as a microfluidic paper-based analytical device, 292 and optimised further to require very small volumes of blood (∼5 μL) by depositing the biological machinery on cotton threads. 293

As discussed earlier, whole cell bioluminescent bacterial biosensors are widely used to detect heavy metal concentrations such as mercury, zinc and chromium in ecotoxicology studies. 294–296 For more details on this please refer to Section 4.3.

4.6 Gene assays

One of the most common applications of Fluc and Rluc bioluminescence is their use as reporter genes for the study of gene expression in prokaryotic and eukaryotic cells and systems. 301–304 A notable recent and relevant example is the use of luciferase based assays to determine the infectivity of viruses such as the various coronaviruses in different host cell types. 305,306 Such an assay was also used to establish that SARS-Cov-2 coronavirus and a closely related RaTG13 coronavirus that was found in bats can both successful infect human cells to produce daughter viruses (Fig. 12) . 307 The interaction and binding of the spike glycoprotein in coronaviruses to the cell receptor Angiotensin-converting enzyme 2 (ACE2) in human cells is considered key in the entry of the viruses into human host cells. 308 Plasmids containing the genes for Fluc and the genes for the spike protein from the coronavirus strain of interest were co-transfected into host cells and incubated for 72 h. The pseudoviruses formed after this period were collected and these pseudoviruses would have the desired spike-protein on their surface and the genetic material encoding for Fluc inside of them. These pseudoviruses were then incubated with ACE-2 expressing human cells for 60 h. If the viruses are successfully able to infect the human cells, daughter viruses and Fluc would be produced. On addition of D -luciferin, the light output would be a measure of infectivity.

There has also been a report of the blue light from a Nanoluc-furimazine reaction being used to activate a photoactive LOV protein that in-turn uncages a transcription factor – so in essence the light is used to regulate gene expression, although this assay can also be used as a protein–protein interaction assay. 309 In their assay, protein A is linked to a light-activated LOV protein as well as a transcription factor through a protease cleavage site and protein B is linked to Nanoluc as well as a protease (tobacco etch virus protease TEVp). When proteins A and B come into contace with each other, and blue light is emitted from Nanoluc in the presence of Furimazine, this light activates the LOV protein, which changes conformation to present the protease cleavage site to the TEVp protease. The protease works on the protease cleavage site and releases the transcription factor, which then heads towards the nucleus for transcription. In the work, the transcription factor induces the transcription of the red fluorescent protein mCherry. The readout of the assay is the result of this transcription and hence the expression and fluorescence of mCherry ( Fig. 13 ). This is one of the few examples of light from the bioluminescent reaction being used to control an effector function.

4.7 Protein–protein interactions (PPIs)

BRET is based on the concept of Förster resonance energy transfer, which is a non-radiative energy transfer between two luminescent molecules, an excited state donor that transfers its energy to an acceptor which then emits light. The efficiency of the energy transfer is dependent on the distance between the donor and acceptor and their respective dipoles, which means that for efficient energy transfer to occur the molecules must be in close proximity to each other (1–10 nm), 313 and have the correct orientation. 314 In BRET the donor is a photoprotein such as Aequorin or a luciferin molecule such as coelenterazine or furimazine, while the acceptor is often the green fluorescent protein GFP, which emits green light. This circumvents the problems with Förster resonance energy transfer (FRET) with fluorophores, such as the need for an external light source, photobleaching of the donor fluorescent protein, simultaneous excitation of both the donor and acceptor molecules and autofluorescence in cells or animal models ( Fig. 14 ).

Previously, BRET based assays were well established to study various protein–protein interactions of interest such as oncology based targets including p53/hDM2 as well as G-coupled protein receptors both in vitro and in cellulo . 315,316 More recently the inhibition or stabitisation of other PPIs of interest has also been successfully detected using the NanoBRET technology in which BRET from the bright Nanoluc to an acceptor chromophore allows the determination of the proximity and orientation of two proteins of interest. Examples of such analysis include the localisation and conformation of viral HCV NS5A protein, 317 analysis of the interaction between the PRAS40 and hippo pathway, 318 analysis of the CD26-ADA-A 2A R trimeric complex in cells, 319 the use of miniG proteins as probes for GPCRs, 320 and analysis of the interaction between the H2 relaxin protein and the RXFP1 protein. 321 With the development of BRET acceptors that emit red light, NanoBRET technology has also been used to measure protein–ligand interactions in vivo mouse models of breast cancer to determine target engagement. 322 For a more detailed review on the developments in NanoBRET technology, please refer to the recently published review on the topic. 311 The vast majority of BRET systems use the Nanoluc/Furimazine combination as the energy donor as it is significantly brighter than D -luciferin/Fluc and is also much a smaller enzyme than Fluc, which makes tagging it on to proteins of interest easier and more useful as it is unlikely to disturb the protein's natural state. However, this is of limited use for in vivo imaging studies. Consequently, some efforts have been made towards red-shifted BRET systems. In this regard, Fluc enzyme mutants such as Ppy RE10 ( λ max 617 nm with D -luciferin) has been covalently labelled with nrIR fluorescent dyes such as Alexa-Fluor680. This resulted in BRET emission of λ max 705 nm with an acceptor to donor emission ratio of 34.0 (Fig. 15) . 323 Another interesting avenue has been BRET from a luciferase/luciferin combination to an appropriate quantum dot. Quantum dots are nanoparticles (diameters ∼ 2–10 nm) composed of a semiconducting material with diameters in the range of 2–10 nm. Due to their high surface-to-volume ratios they demonstrate a number of interesting properties such as fluorescence. For example, NanoLuc was covalently linked to a polymer-coated CdSe/ZnS core–shell quantum dot QD705 that emits at λ max 705 nm. BRET from the Nanoluc/Furimazine reaction to the quantum dot led to red-shifted emission at 705 nm and this was used to image a tumour in mouse. 324 Nonetheless, the toxicity of quantum dots is a cause of concern for many, particularly for in vivo applications, leading to research into more biocompatible quantum dots. 325

As brighter and red-shifted BRET systems are being engineered, 184 and tagging technology is improving as well, 326 it can be reasonably expected that increasing numbers of in vivo monitoring of protein–protein interactions and protein–ligand interactions will emerge in the near future.

Split luciferase assays are also widely used to detect and evaluate PPIs. 327 These have been based on various luciferases including the firefly luciferase, click-beetle luciferase, Gaussia luciferase and Renilla luciferases, which have all been used to sensitively monitor dynamic PPIs with close to real-time kinetics both in vitro and in vivo . 328,329 The luciferase is split into 2 portions with one consisting of the N-terminal and the other of the C-terminal domain. Each of these portions is tagged to proteins of interest. On addition of a ligand, the proteins of interest are brought together and both termini of the luciferase come in close proximity to each other, hence reconstituting the luciferase reporter function ( Fig. 16 ).

Before the development of Nanoluc, Gaussia luciferase was arguably the most suited for protein-fragment complementation assays (PCA) as it is ATP independent, can be located in the extra-cellular space, is shown to be brighter than Rluc as part of these assays and the N-terminal and C-terminal enzyme fragments are small in size. The first split Gaussia luciferase assay was reported by Michnick et al. In their work, they interrogated the FRB/FKBP protein–protein interaction, using rapamycin as a ligand to induce complex formation, and FK506 as a competitive inhibitor. The PPI dynamics were visualised both in vitro and in vivo . 330 Tao et al. reported the development of a split Gluc template which was adapted to visualise the protein–protein interaction of three different PPIs namely CaM/M13 with Ca 2+ ions behaving as the ligand and the interactions of the ligand binding domains of (LBD) of representative steroid hormone receptors such as androgen receptor (AR), glucocorticoid receptor (GR), and oestrogen receptor (ER) with various petide or small-molecule ligands. 331 Others have reported PCAs based on Gluc for visualisation of PPIs both in vitro and in vivo mouse models. 332 Recently, split Nano luciferase has been used to determine protein–protein interactions in plant cells, wherein the PPI between receptor kinase flagellin-sensitive 2 (FLS2) and plant receptor kinase BAK1 (BRI1-associated receptor kinase 1) was found to be induced by bacterial flg22 peptide through a split Nanoluc system. 333 Split luciferase assays have also been used to study viruses, 334,335 as well as to detect protein–protein aggregation in human cells. 336

4.8 High-throughput screening

For example, Tan and co-workers reported the development of high-throughput screening assay to identify inhibitors of coronaviruses. 337 In particular, they replaced the ns2 accessory gene in the human coronanavirus strain HCoV-OC43 with the gene for Renilla luciferase ( Rluc ) to form a functional reporter virus strain rOC43-ns2DelRluc whose pathogenicity was unaltered. This mutant virus strain was then used to infect cells, and Rluc was expressed in the infected cells during viral replication. On addition of coelenterazine, the light output was proportional to the Rluc levels in the cells, which are a measure of viral replication. On addition, of small-molecule compounds that inhibit viral replication, a significant reduction in light-output was observed. 338 This assay could be readily adapted in a 96-well format ( Fig. 17 ).

More recently, similar high-throughput assays have been developed for screening for antibiotics in aquatic samples, 237 screening of antibacterial dental adhesives against mutants of streptococcus, 339 and monitoring and inhibiting kinase activity. 340,341

Both Fluc and Rluc have been extensively used in high-throughput screening campaigns against large compound libraries in both biochemical assays and cell-based assays. Whilst these assays are extremely useful and still widely used, it is important to note that they have some limitations. For example, both Fluc and Rluc can be inhibited by various small molecules. For example, Fluc in particular suffers from competitive inhibition from compound classes that have similar chemical structures to that of its substrate D -luciferin including benzothiazoles, benzimidazoles, benoxazoles and biaryl oxadiazoles. This can lead to false-positives in inhibitory assays for these compounds. Moreover, some compounds can also lead to increased trasnscription or translation of the reporter enzyme leading to a false-negative result. A critical discussion on the use of bioluminesce based assays is covered in seminal reviews written by Inglese and co-workers. 342,343

4.9 In vivo imaging

For bioluminescence in vivo imaging the cells of interest are genetically modified to include the gene for luciferase production. These cells can then be injected and tracked in the body of the small mammal, when the respective luciferin is added. Whilst, eukaryotic cells are often genetically tagged with Fluc, Rluc or Nluc mutants, bacterial cells are genetically modified to incorporate the lux codon responsible for bacterial bioluminescence. The light output is then recorded using a cooled charge-coupled device camera ( Fig. 18 ). This allows the monitoring of in vivo processes in real time, without the need to sacrifice the animal.

Bioluminescence imaging has also been effectively used in imaging the development of infectious diseases both in vitro and in vivo . 347 Genetically modified bioluminescent pathogens, such as bacteria, parasites, viruses and fungi have been designed and monitored both in vivo and in vitro , in the presence and absence of therapies to test their effectiveness. The bioluminescent light output has been shown to correlate with the infection load.

Some notable examples include the first real time visualisation of the influenza virus in ferrets infected with A/California/04/2009 H1N1 virus (CA/09) encoding Nanoluc (Nluc) luciferase. 348 The replication and development of human coronavirus strain HCoV-OC43 in the central nervous system of live mice was achieved using an Rluc reporter and coelenterazine, 349 while another study reported the entry sites of encephalitis viruses in the central nervous system of mice, using the Fluc/ D -luciferin system. 350 An engineered firefly luciferase was also used with D -luciferin to monitor the development of a Candida albicans fungal infection real time in mice using in vivo imaging. 351

In vivo BLI is also the modality of choice when monitoring the development of parasitic infections that cause neglected tropical diseases such as those from Toxoplasma gondii , 352 Trypanosoma cruzi , 353,354 and Leishmania amazonesis . 355,356 The gene for firefly luciferase can be readily encoded into these parasites and as bioluminescent output from the D -luciferin/Fluc system is ATP dependent, only living parasites are selectively imaged, which would not necessarily be the case in fluorescence imaging. Moreover, as the imaging technique is non-invasive, the diseased mice can be kept alive and monitored over time whilst being administered different treatments.

Bacterial infections such as Klebsiella pneumoniae , Citrobacter rodentium and antibiotic resistance to them is also monitored using BLI; however the bacterial lux operon is often used for this. 357,358 The bacteria of interest are genetically encoded with the lux operon for bacterial bioluminescence and then injected into the mouse. The mouse is then treated with antibiotics and imaged over time to visualise the development of a disease. This technique has often been used to visualise the effectiveness of photodynamic therapy (PDT) on the treatment of bacterial infections such as surgical wounds, burns and lacerations as an alternative to treatment with antibiotics to combat antibiotic resistance. For example, dermal abrasions on mice infected with bioluminescent methicillin-resistant S. aureus (MRSA) were monitored while a treatment of PDT using a phthalocyanine derivative and toluidine blue with red light was administered by Hamblin and co-workers. 359

Although now BLI reporters can emit light up to a wavelength of 750 nm, there is still much to be desired in terms of brightness to achieve desirable outcomes in in vivo imaging. Moreover, research into fluorescent probes has demonstrated that light output in the NIR-II window (1000–1700 nm) has significantly better penetration through blood and tissue than light in the NIR-I window. 360 This is the next frontier in bioluminescence in vivo imaging.

4.10 Disease therapy using the light from bioluminescence

Both the Fluc and Rluc systems have been used as genetically encoded sources of light for photosensitisers. For example, Theodossiou et al. reported that increased cell death was observed when both the photosensitiser Rose Bengal and D -luciferin were administered to a Fluc expressing cancer cell line, in the absence of ambient light. 361 This work was contested by a later report that reported that the increase in cell death was insignificant when non-toxic levels of photosensitiser and luciferin were used, and that the quantum yield of the light output from the D -luciferin/Fluc reaction was the limiting factor. 362 Another study by Lai et al. reported Rluc-immobilized quantum dots-655 (QD-Rluc8) for bioluminescence resonance energy transfer (BRET)-mediated PDT using the photosensitiser Foscan® to target cancer cells both in vitro and in vivo in mice. 363 Although the PDT did not completely eradicate the tumour, it significantly delayed tumour growth, and it was proposed that this method of low-light dosage, compared to the use of external light, may cause lower inflammation and unnecessary death of healthy tissue.

Yun and co-workers reported Rluc and rose-bengal conjugates that generate singlet oxygen by bioluminescence resonance energy transfer (BRET). In their work, they used bovine serum albumin (BSA) as a central backbone and conjugated Rluc and rose-bengal to the BSA to achieve the desired distance between Rluc and Rose Bengal which allowed both moieties to be functional, without quenching the emission from either of them ( Fig. 19 ). When coelenterazine was administered to this system, evidence of cytotoxicity and oxidative stress was observed in cells. 14,364 Unsurprisingly, the uptake of the large and bulky conjugate into cells was poor and optimisation is required on that front.

It is to be noted, that an improvement in the light output efficiency of the bioluminescent reaction, as well as the BRET efficiency between the coelenteramide and the photosensitiser would improve the generation of singlet oxygen.

An alternative approach that circumvents the need for cellular uptake of large and bulky biological conjugates could be to use a nanoparticle to bring the luciferase and photosensitiser in close proximity. This approach was taken by Wu et al. and in their work they encapsulated Rose-Bengal in biodegradable poly(lactic- co -glycolic acid) (PLGA) nanoparticles, which were then conjugated with Fluc. In the presence of D -luciferin, effective PDT and cancer cell death was observed both in vitro and in vivo in mice suffering from cancer of the liver. 365

Although increasing numbers of studies with PDT activated by BRET from bioluminescence are being reported, the scope of the work is somewhat limited by amongst other factors, the brightness of the luciferin/luciferase reaction. As brighter luciferin/luciferase pairs are developed, it is hoped that this area will also expand into new territories.

4.11 Effector applications

The earliest targets to be affected by bioluminescent light output were channelrhodopsins. Channelrhodopsins are light-sensitive ion channels that are naturally found in algae and are responsible for their movement in response to light i.e. phototaxis. 369,370 When expressed in neurons, rhodopsins allow light to control the state of the ion-channel by either opening it or closing it and hence the ability of the neuron to fire action potentials. 371 The channelrhodopsins absorb blue light (480 nm), so are ideal targets for Rluc , Gluc or Nluc . 372 The gene for channelrhodopsins can be encoded together with the gene for luciferase leading to a fusion protein consisting of a channelrhodopsins with a luciferase. This led to the development of ‘luminopsins’ which are now a class of optogenetic reporters, in which the addition of coelenterazine luciferin and subsequent light output controls the state of the ion-channel, and hence the ability of neurone cells to fire action potentials. 17,373–376 The use of bioluminescent light to control gene expression has been discussed in Section 4.6.

Bioluminescent light has also been used for novel photo-uncaging reactions by Winssinger and co-workers. In 2018, they reported a photo-uncaging using BRET from Nluc to a ruthenium photocatalyst to release pyridinium species. Further developments by this group in 2019 saw the first true ‘bioluminolysis’ wherein no photocatalyst was needed to uncage drugs and molecules of interest using BRET from Nanoluc-Halotag chimera protein (Hluc) to a coumarin photocage ( Fig. 20 ). 15,16

Photopharmacology including photodynamic therapy, photouncaging and photoisomerism is an area of growing interest in medicinal chemistry to develop and herness new therapies. 377 Bioluminescence has potential to be of great use here as a light source in vivo in close proximity to species of interest. The fact that luciferases are usually genetically encoded makes this a challenging endeavour in humans and complex on several fronts. However, with the advent of luciferase conjugated nanoparticles, this challenge in the delivery of the enzyme to its desired site of action might soon be getting addressed.

5. Blue-sky research and new horizons

5.1 glowing plants.

To circumvent the problem of delivery of D -luciferin to the cells of interest, it was thought that an autoluminescent plant would be of more use. To this end, genetically modified plants were produced, in which the bacterial lux operon would be expressed in the plastids, but this failed to produce sufficient light, partly due to the fact that bioluminescent bacteria emit blue light, whilst chlorophyll absorbs strongly in that region. Moreover, the expression of the bacterial bioluminescent system was found to be toxic to the plant. 115 Since the recent discovery of the fully genetically encoded bioluminesce pathway of fungal bioluminescence in 2018, 126 this year two different groups reported successfully creating genetically modified plants with the fungal bioluminescence pathway genetically encoded into them, making them autoluminescent. 128,382 This opens up a new avenue for BLI in plant research as it has moved BLI in plants from the laboratory Petri-dish to more real life examples grown in soil. Moreover, it also opens the exciting avenue of using such plants for ‘green’ lighting purposes in the future. Notably, in their work Yampolsky and Sarkisyan et al. also reported an autoluminescent mammalian cell line using the genes responsible for fungal bioluminescence. 128 However, no attempt was made to compare the brightness of the light output with that obtained from the bacterial ilux operon.

Apart from genetically encoded autoluminescent systems, a nanobionic light emitting plant has also been reported in the literature. In this system, Fluc was conjugated into silica nanoparticles (SNP-Luc) and D -luciferin was conjugated into poly(lactic- co -glycolic acid) (PLGA-LH 2 ) nanoparticles. Both types of nanoparticles were slowly able to release their cargo inside plant cells to allow the bioluminescence reaction to take place and to give the plant a yellow-green glow. 18 However, a pressurized bath infusion of nanoparticles was used to administer the mixture of nanoparticles to the plant, which makes this sort of a light emitting plant unsustainable.

5.2 Lifestyle

As the use of bioluminescent systems become more widespread, they have inspired both scientists and artists alike towards innovation and novel applications. There are a number of examples of artists that are inspired by and actively use bioluminescence in their work. Novel and rare art work known as ‘living art’ has been produced using some bioluminescent systems – namely bioluminescent bacteria and the bioluminescence from single-celled dinoflagellate. 384 The bacterial bioluminescence art work is done on Petri dishes and lasts up to 2 weeks, gradually dying off and depicting different aspects of the art as the light fades. Thus, these bioluminescent systems serve as tools for the artist's expression as well as starting points for science communication ( Fig. 21 ).

5.3 Limitations of bioluminescent systems

6. conclusions, conflicts of interest, acknowledgements, notes and references.

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In Coral Fossils, Searching for the First Glow of Bioluminescence

A new study resets the timing for the emergence of bioluminescence back to millions of years earlier than previously thought.

By Sam Jones

Bioluminescence is used throughout the animal kingdom, particularly in marine environments, to lure prey, startle predators and even act as camouflage in the surrounding light .

“We always say it’s light-limited in the deep sea, but there are a lot of organisms that produce their own light,” said Andrea Quattrini , a zoologist at the Smithsonian National Museum of Natural History in Washington.

The dazzling glow of bioluminescence is common in Octocorallia, also known as octocorals, a class of over 3,000 Anthozoa species including sea fans, sea pens and soft corals. The prevalence of bioluminescence in these sessile animals makes a lot of sense, Dr. Quattrini said: “They settle somewhere and they’re there.”

How long organisms have been able to emit light is at the center of recent research by Dr. Quattrini and colleagues. Their latest study, published Tuesday in the journal Proceedings of the Royal Society B , resets the timing for the emergence of bioluminescence back to about 540 million years ago, from the existing understanding that it appeared in small marine crustaceans 267 million years ago .

The researchers based their finding on recent octocoral evolutionary tree work, octocoral fossils and modeling to trace the ancestral past of the tiny organisms.

Bioluminescence is believed to have evolved nearly 100 times across history , caused by a simple chemical reaction, when a light-producing molecule called a luciferin reacts with an enzyme called luciferase.

“This ability to bioluminesce is giving these animals some type of survival or fitness advantage,” said Danielle DeLeo , the lead author on the study and a biologist affiliated with Florida International University and the Smithsonian National Museum of Natural History.

Dr. DeLeo was first captivated by the remarkable glow over a decade ago, while studying the impact of the 2010 Deepwater Horizon oil spill on deep sea communities. Descending a thousand meters below the surface in a submersible, she recalled, “you look out the window and all you see is bioluminescence.”

Now she studies bioluminescence in a range of invertebrates — including deep sea shrimp that spew bioluminescent vomit — and for years has been interested in when this basic yet stunning form of communication first emerged.

Setting the stage to answer that question, in 2022, Dr. Quattrini and her former adviser, Catherine McFadden at Harvey Mudd College in California, who is also an author on the new study, revised the octocoral tree of life based on new genetic data.

In the new study, the researchers incorporated dated octocoral fossils to determine when branches on the tree diverged.

They then collected data on the presence or absence of bioluminescence in as many of those species as possible, pulling from previous research as well as their continuing work. From there, they used a series of statistical models to work back in time, over hundreds of millions of years, to calculate the probability of bioluminescence for each common ancestor on the tree.

Every iteration of the analysis landed them at the same conclusion: that bioluminescence first popped up in the common ancestor of octocorals approximately 540 million years ago, around the beginning of the Cambrian era.

This was just before or at the start of the Cambrian explosion , when a huge number of new, more complex species arose.

“Light-sensing had already evolved by then,” but not quite in the form of eyes, said Elena Bollati , a marine biologist at the University of Copenhagen who was not involved in the study. “Predators weren’t really common before the Cambrian explosion either,” she added, “so this has interesting implications regarding the original function of bioluminescence.”

This work lends support to a longstanding theory that — because the chemical reaction underlying bioluminescence uses up oxygen — bioluminescence first evolved as a defense mechanism against the production of dangerous oxygen-containing molecules called free radicals. Glowing may have just been a byproduct but, as it turned out, a useful one.

“You can take care of oxygen and you can make light and you can perhaps deter predators or attract prey, and then you’re going to be successful into future generations,” Dr. Quattrini said.

Now the researchers are focused on a genetic test that, using just a small piece of tissue, will assess if an animal’s luciferase enzyme is functional, an indicator that it can bioluminesce. This technique will remove the need to collect an entire octocoral colony to physically test if it glows, said Dr. DeLeo, helping protect these creatures from the potential threats of oil drilling, fishing and other anthropogenic hazards.

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Bioluminescence first evolved in animals at least 540 million years ago, pushing back previous oldest dated example

by Smithsonian

Bioluminescence first evolved in animals at least 540 million years ago in a group of marine invertebrates called octocorals, according to the results of a new study from scientists with the Smithsonian's National Museum of Natural History.

The results, published April 23, in the Proceedings of the Royal Society B: Biological Sciences , push back the previous record for the luminous trait's oldest dated emergence in animals by nearly 300 million years, and could one day help scientists decode why the ability to produce light evolved in the first place.

Bioluminescence—the ability of living things to produce light via chemical reactions—has independently evolved at least 94 times in nature and is involved in a huge range of behaviors including camouflage, courtship, communication and hunting. Until now, the earliest dated origin of bioluminescence in animals was thought to be around 267 million years ago in small marine crustaceans called ostracods .

But for a trait that is literally illuminating, bioluminescence's origins have remained shadowy.

"Nobody quite knows why it first evolved in animals," said Andrea Quattrini, the museum's curator of corals and senior author on the study.

But for Quattrini and lead author Danielle DeLeo, a museum research associate and former postdoctoral fellow, to eventually tackle the larger question of why bioluminescence evolved, they needed to know when the ability first appeared in animals.

In search of the trait's earliest origins, the researchers decided to peer back into the evolutionary history of the octocorals, an evolutionarily ancient and frequently bioluminescent group of animals that includes soft corals, sea fans and sea pens.

Like hard corals, octocorals are tiny colonial polyps that secrete a framework that becomes their refuge, but unlike their stony relatives, that structure is usually soft. Octocorals that glow typically only do so when bumped or otherwise disturbed, leaving the precise function of their ability to produce light a bit mysterious.

"We wanted to figure out the timing of the origin of bioluminescence, and octocorals are one of the oldest groups of animals on the planet known to bioluminesce," DeLeo said. "So, the question was when did they develop this ability?"

Not coincidentally, Quattrini and Catherine McFadden with Harvey Mudd College had completed an extremely detailed, well-supported evolutionary tree of the octocorals in 2022 . Quattrini and her collaborators created this map of evolutionary relationships, or phylogeny, using genetic data from 185 species of octocorals.

With this evolutionary tree grounded in genetic evidence , DeLeo and Quattrini then situated two octocoral fossils of known ages within the tree according to their physical features. The scientists were able to use the fossils' ages and their respective positions in the octocoral evolutionary tree to date to figure out roughly when octocoral lineages split apart to become two or more branches.

Next, the team mapped out the branches of the phylogeny that featured living bioluminescent species.

With the evolutionary tree dated and the branches that contained luminous species labeled, the team then used a series of statistical techniques to perform an analysis called ancestral state reconstruction.

"If we know these species of octocorals living today are bioluminescent, we can use statistics to infer whether their ancestors were highly probable to be bioluminescent or not," Quattrini said. "The more living species with the shared trait, the higher the probability that as you move back in time that those ancestors likely had that trait as well."

The researchers used numerous different statistical methods for their ancestral state reconstruction, but all arrived at the same result: Some 540 million years ago, the common ancestor of all octocorals were very likely bioluminescent. That is 273 million years earlier than the glowing ostracod crustaceans that previously held the title of earliest evolution of bioluminescence in animals.

DeLeo and Quattrini said that the octocorals' thousands of living representatives and relatively high incidence of bioluminescence suggests the trait has played a role in the group's evolutionary success. While this further begs the question of what exactly octocorals are using bioluminescence for, the researchers said the fact that it has been retained for so long highlights how important this form of communication has become for their fitness and survival.

Now that the researchers know the common ancestor of all octocorals likely already had the ability to produce its own light, they are interested in a more thorough accounting of which of the group's more than 3,000 living species can still light up and which have lost the trait. This could help zero in on a set of ecological circumstances that correlate with the ability to bioluminesce and potentially illuminate its function.

To this end, DeLeo said she and some of her co-authors are working on creating a genetic test to determine if an octocoral species has functional copies of the genes underlying luciferase, an enzyme involved in bioluminescence. For species of unknown luminosity, such a test would enable researchers to get an answer one way or the other more rapidly and more easily.

Aside from shedding light on the origins of bioluminescence, this study also offers evolutionary context and insight that can inform monitoring and management of these corals today. Octocorals are threatened by climate change and resource-extraction activities, particularly fishing, oil and gas extraction and spills, and more recently by marine mineral mining.

This research supports the museum's Ocean Science Center , which aims to advance and share knowledge of the ocean with the world. DeLeo and Quattrini said there is still much more to learn before scientists can understand why the ability to produce light first evolved, and though their results place its origins deep in evolutionary time, the possibility remains that future studies will discover that bioluminescence is even more ancient.

This study includes authors affiliated with Florida International University, the Monterey Bay Aquarium Research Institute, Nagoya University, Harvey Mudd College and University of California, Santa Cruz.

Journal information: Proceedings of the Royal Society B

Provided by Smithsonian

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What were the oldest animals to glow? A new study offers a clue.

Hundreds of plants, fungi, and animals can do it. Now scientists think bioluminescence may have evolved 540 million years ago in Earth’s ancient oceans.

From fireflies to glow worms, algae to squid, a dazzling array of organisms can perform an act of magic: they can generate their own light through a process known as bioluminescence. And it isn’t just an aesthetic wonder. It has evolved independently at least 100 times in nature and has dozens of diverse uses, from luring prey to freaking out predators to winking at a potential mate.  

But when did life first develop the ability to glow in the dark? For decades, scientists believed the oldest example of animal bioluminescence could be found in a diminutive marine crustacean known as an   ostracod , one that lived 267 million years ago and could light itself up. But a new study, published today in the   Proceedings of the Royal Society B , winds the clock on bioluminescence way, way back.

By studying an oft-bioluminescent group of deep-sea critters named octocorals, scientists have concluded that they shared a primeval light-bearing ancestor that lived 540 million years ago. This creature would have emerged during the Cambrian Explosion, a period in Earth’s history of seemingly supercharged evolutionary activity that saw many of the major animal groups we know today appear for the first time.

“That was a very exciting and pleasant surprise,” says   Danielle DeLeo , a deep-sea biologist at Florida International University and the study’s lead author.

“Bioluminescence, and light signaling in general, could be one of the oldest forms of communication that we have evidence of, which was not what we were originally expecting.”

In other words, the seas and oceans of the world are generally dark places. But almost as long as complex animals have existed, so too have there been lights flickering in the darkness.

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Traps, beacons, and klaxons.

Bioluminescence is a   cold light chemical reaction, one that requires the presence of luciferin—a light-making compound. Some lifeforms make luciferin themselves, while others absorb it from symbiotic organisms or by ingesting it. Some animals even give luciferin-containing bacteria or algae a comfortable abode in their bodies. But regardless of how luciferin is obtained, it is then combined with a catalyst (commonly luciferase) to generate luminescence, and different hues are emitted depending on how the luciferin molecules are arranged.

While a variety of land life forms have bioluminescence, by far the most biological fireworks can be found in the ocean:   three-quarters of marine animals are able to light themselves up in some way, and there’s almost no limit to their creativity.  

“It’s so diverse and variable,” says DeLeo. In some cases, bioluminescence can announce an animal’s quest for a paramour. Predators with rumbling stomachs can use the process to blind and stun their dinner, or draw gullible prey into their maws, or to act as a searchlight to spy a swimming snack.  

Bioluminescence is also used for defenses, including camouflage (making an animal’s underbelly glow so it blends in with the iridescent waves at the sea surface, for example) and as a decoy (detaching a luminous body part to distract a voracious predator, perhaps).  

Some deep-sea crustaceans even employ a flamboyantly guttural method of protection. “They have this bioluminescent vomit that they spew out when they’re startled,” says DeLeo.

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Was this prehistoric ‘killer shrimp’ as fierce as it looked?

These creatures are otherworldly. They destroy coral. And they're hard to kill.

Octocorals can also shine in the dark. Although superficially similar to the stony-housed polyp colonies that make up coral reefs many are familiar with, these   wiggly animals have a soft structure, along with a few other morphological quirks.  

And the purpose of their bioluminescence is debated. Although these immobile   inhabitants of the deep may sometimes use their light to lure in some tasty invertebrates to supplement their diet, they glow most noticeably when they are prodded—perhaps to startle a hungry predator.

“It’s what we call the burglar alarm hypothesis,” says   Jon Copley , a marine ecologist at the University of Southampton who wasn’t involved with the new work. “Bioluminescence is used to make a commotion, one that attracts the attention of potential predators of the predator.”

A bright, ancient time

Debates about purpose aside, DeLeo and her colleagues wanted to use octocorals to try something ambitious: find the earliest ancestor that could bioluminesce.  

A recent, detailed octocoral   evolutionary tree using genetic data from almost 200 species gave them that chance. First, they placed additional octocoral fossils with known ages on that tree to better illuminate how various lineages are related. They also mapped out the tree branches that featured living bioluminescent species. Then, they used statistical analyses to work out how probable it was that various ancestors were bioluminescent.

Ultimately, the team rewound the clock by 540 million years—back to the time of the common ancestor of octocorals, a creature that was almost certainly capable of self-illumination.  

“We did think there was a good chance the age of the most recent common ancestor was going to be hundreds of millions of years old. We didn’t realize quite how old!” says DeLeo.

That bioluminescence could be traced back to the Cambrian Explosion is an elegant finding. “It is the time that we knew that eyes were taking off,” says Copley, referring to animals that evolved the ability to detect light. It makes sense that bioluminescence would emerge around the same time. “I don’t think it’s a coincidence at all.”

But that primordial glowing was probably not used for today’s burglar alarm-like purpose. “We think this light production was more of a secondary byproduct,” says DeLeo—an inadvertent brilliance triggered by another biochemical reaction. But over time, the bioluminescent reactions “were kept because they started serving this really important function of communication, or light signaling.”

It's possible that the origins of bioluminescence may go back even further than the Cambrian. Perhaps, due to a paucity of fossils older than this period, scientists may never conclusively find out when this underwater starlight first appeared. But thanks to that initial ignition, a cornucopia of lifeforms today can light up their surroundings—giving researchers countless opportunities to study this remarkable ability.

“There’s so much more to discover,” says DeLeo.

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Bioluminescence-activated deep-tissue photodynamic therapy of cancer

Affiliations.

• 1 1. Graduate School of Nanoscience and Technology (WCU), Korea Advanced Institute of Science and Technology, 291 Daehak-Ro, Yusong-Gu, Daejon 305-701, Korea ; 2. Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-Ro, Yusong-Gu, Daejon 305-701, Korea ; 3. Department of Oncology, Asan Medical Center, Univ. Ulsan College of Medicine, Seoul , Korea.
• 2 1. Graduate School of Nanoscience and Technology (WCU), Korea Advanced Institute of Science and Technology, 291 Daehak-Ro, Yusong-Gu, Daejon 305-701, Korea.
• 3 4. Department of Pharmacology, Wonkwang Institute of Dental Research, School of Dentistry, Wonkwang University, Iksan, Chonbuk, 570-749, Korea ; 5. Wellman Center for Photomedicine, Massachusetts General Hospital, 65 Landsdowne St. UP-525, Cambridge, MA 02139, USA.
• 4 2. Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-Ro, Yusong-Gu, Daejon 305-701, Korea.
• 5 6. Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 790-784, Korea.
• 6 1. Graduate School of Nanoscience and Technology (WCU), Korea Advanced Institute of Science and Technology, 291 Daehak-Ro, Yusong-Gu, Daejon 305-701, Korea ; 2. Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-Ro, Yusong-Gu, Daejon 305-701, Korea.
• 7 1. Graduate School of Nanoscience and Technology (WCU), Korea Advanced Institute of Science and Technology, 291 Daehak-Ro, Yusong-Gu, Daejon 305-701, Korea ; 5. Wellman Center for Photomedicine, Massachusetts General Hospital, 65 Landsdowne St. UP-525, Cambridge, MA 02139, USA ; 7. Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, 40 Blossom St. Boston, MA 02114, USA.
• PMID: 26000054
• PMCID: PMC4440439
• DOI: 10.7150/thno.11520

Optical energy can trigger a variety of photochemical processes useful for therapies. Owing to the shallow penetration of light in tissues, however, the clinical applications of light-activated therapies have been limited. Bioluminescence resonant energy transfer (BRET) may provide a new way of inducing photochemical activation. Here, we show that efficient bioluminescence energy-induced photodynamic therapy (PDT) of macroscopic tumors and metastases in deep tissue. For monolayer cell culture in vitro incubated with Chlorin e6, BRET energy of about 1 nJ per cell generated as strong cytotoxicity as red laser light irradiation at 2.2 mW/cm(2) for 180 s. Regional delivery of bioluminescence agents via draining lymphatic vessels killed tumor cells spread to the sentinel and secondary lymph nodes, reduced distant metastases in the lung and improved animal survival. Our results show the promising potential of novel bioluminescence-activated PDT.

Keywords: Bioluminescence; Cancer; Photobiology; Photodynamic Therapy; Photomedicine; Photosensitizers; Resonance Energy Transfer.

Publication types

• Research Support, N.I.H., Extramural
• Research Support, Non-U.S. Gov't
• Chlorophyllides
• Disease Models, Animal
• Luminescence*
• Mice, Inbred C57BL
• Neoplasms / drug therapy*
• Photochemotherapy / methods*
• Porphyrins / metabolism*
• Radiation-Sensitizing Agents / metabolism*
• Radiation-Sensitizing Agents
• phytochlorin

Grants and funding

• P41 EB015903/EB/NIBIB NIH HHS/United States
• U54 CA143837/CA/NCI NIH HHS/United States
• P41EB015903/EB/NIBIB NIH HHS/United States
• U54CA143837/CA/NCI NIH HHS/United States

Impacts of 2020 Red Tide Event Highlighted in New Study

Collaborative paper documents extreme water conditions that led to fish die-offs.

• Brittany Hook - [email protected]

Published Date

Topics covered:.

• Bioluminescence
• Marine Biology

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Article content.

In the spring of 2020, a historic red tide event occurred in waters off Southern California. Driven by a bloom of the dinoflagellate Lingulodinium polyedra , this event brought nighttime displays of bioluminescence to beaches along the coast, from Baja California to Santa Barbara. While the bloom gained international attention for its stunning visual displays, it also had significant negative impacts, including mass mortality among fish and other marine organisms.

Marine scientists from UC San Diego’s Scripps Institution of Oceanography, National Oceanic and Atmospheric Administration (NOAA), and other organizations seized the opportunity to study the unprecedented event and its impacts on marine life, both in the wild and in aquariums. In a recent study, this multi-institutional team of 34 researchers identified deteriorating water quality — notably prolonged low oxygen conditions — as a significant factor in the mass mortality event. Their findings were published in the journal Elementa, and featured in a special issue focused on the 2020 red tide.

“Red tides are notoriously difficult to predict and study, so t he 2020 event presented a unique opportunity for the marine science community to collaborate and use existing infrastructure to figure out what was causing these die-offs,” said lead author Zachary Skelton, a marine biologist under contract at NOAA’s Southwest Fisheries Science Center (SWFSC) and recent graduate of Scripps Oceanography. “Our paper provides valuable insights into how low- or no-oxygen conditions can harm certain marine species, particularly those inhabiting estuaries and shallow coastal areas.”

The study was informed by data from existing long-term observation programs, alongside reports from community members, field surveys, and case studies from local aquariums that use local seawater to sustain their systems. The researchers found a correlation between extreme changes in water conditions, notably dissolved oxygen and pH levels, and the dynamics of the red tide bloom.

Sensor data along the coastline revealed extreme hypoxic (low oxygen) and hyperoxic (excess oxygen) conditions, especially in estuaries and near-shore coastal environments. These conditions impacted the health of numerous marine species, leading to the documented deaths of thousands of fishes and invertebrates, as well as several seabirds. Case studies from four local aquariums — including Birch Aquarium at Scripps, as well as two experimental aquariums at Scripps Oceanography and one at NOAA SWFSC — indicated that factors beyond large changes in oxygen and pH levels might have contributed to the observed mortality.

Since the red tide coincided with the early stages of the COVID-19 pandemic, the team had to obtain special permission from UC San Diego and other local institutions and authorities to conduct fieldwork. Once these permissions were secured, Skelton, along with Ben Frable, collection manager of marine vertebrates at Scripps Oceanography, visited multiple beaches and estuaries around San Diego to count, photograph, and measure the dead fish and invertebrates.

Their observations and community reports — shared via social media, email, and the citizen science platform iNaturalist — revealed mass die-offs of fish and invertebrates between late April and mid-May 2020, following sharp drops in oxygen levels. Fish die-offs were disproportionately reported in the La Jolla area (including La Jolla Cove, La Jolla Shores Beach, and Scripps Beach), the Encinitas area, and Agua Hedionda Lagoon in Carlsbad. Many of these locations are enclosed or geographically sheltered areas, which limits mixing and makes it easier for blooms to concentrate.

The red tide harmed many different species, but nearshore and bottom-dwelling species fared the worst. “Many of the species that live on or near the seafloor tend to be less mobile,” said Skelton. “These creatures often stick to their preferred habitats, even while experiencing stressful conditions.”

For instance, the spotted sand bass, usually found in shallow, sandy environments, suffered the highest reported mortality among fish species, with hundreds washing up on Encinitas beaches and at Agua Hedionda Lagoon. Similarly, hundreds of California bubble snails were among the most affected invertebrates. Divers also reported deaths of species like topsmelt, round stingray, sarcastic fringehead, sanddabs, rockfish, and thornback guitarfish, which were observed underwater but did not wash ashore. The authors noted that this red tide event did not seem to harm marine mammals or cause more reported strandings than usual.

Once it was established that a mass mortality event was underway, the team wanted to find out which aspect of the red tide bloom had the biggest impact on marine organisms. Red tides occur when there is a rapid increase or “bloom” in populations of marine algae, typically dinoflagellates or other species of phytoplankton. During the daytime, these organisms photosynthesize by taking up carbon dioxide and producing oxygen. At night, the collective bloom and community respiration draw up the surrounding oxygen while producing carbon dioxide, ultimately causing changes in seawater chemistry across the day/night cycles. At peak bloom, conditions are often hyperoxic for longer periods due to the increased amount of oxygen in the water. As the bloom starts to decline — either due to the natural lifecycle of these organisms or depletion of nearby nutrients — oxygen levels are reduced, often resulting in prolonged hypoxic conditions.

Study co-author and biological oceanographer Lillian McCormick analyzed data from 11 oceanographic sensor packages deployed on moorings off the coast of San Diego. These sensors were deployed at various depths, ranging from 1.5 meters (5 feet) to approximately 100 meters (328 feet), collecting data across estuaries, shallow nearshore environments, and farther offshore.

Shallower waters, particularly within estuaries and lagoons, bore the brunt of the red tide impacts, experiencing prolonged periods of hypoxia. Agua Hedionda Lagoon and Los Peñasquitos Lagoon were the hardest hit, enduring episodes of extreme hypoxia and even anoxia, where there was no oxygen. Construction near Los Peñasquitos Lagoon during the peak bloom led to the complete closure of the lagoon to the open ocean, which likely exacerbated the situation.

"The bloom was heavily concentrated within those estuaries, and likely worsened at one location by its complete closure for a number of weeks," said McCormick, a postdoctoral researcher at California Sea Grant and Scripps Oceanography. "These enclosed spaces don’t see as much flushing or movement of the water as we typically see in deeper, open ocean waters.”

The authors were surprised to find that the Scripps Pier sensor, located at a depth of 4 meters (13 feet), recorded intermittent hypoxia between April 30 and May 16, 2020, for a total of 82 hours. “You often see fish die-offs happening in estuaries, or in warm water areas like Florida,” said Skelton. “But open-ocean mortality associated with these hypoxic events is pretty rare in California, so the Scripps sensor provided some unique insight into that event.”

In addition to field observations, the authors analyzed water quality and mortality rates at four local aquarium facilities, each with different levels of filtration and supplemental oxygenation. As part of this research, members of the Tresguerres Lab at Scripps Oceanography examined the morphological changes in the gills of captive-held Pacific sardines in response to the red tide. Led by marine physiologists Garfield Kwan and Martín Tresguerres, the team employed microscopy and biochemistry techniques to examine the ionocyte, a specialized cell responsible for ion-transport akin to kidneys in mammals. Their findings revealed that sardines increased ion-transport to maintain nominal conditions during red tide exposure.

“This finding is notable because red tide events are difficult to predict and cannot be timed, so experimental data such as ours are very rare,” said Kwan, who is now a postdoctoral fellow at UC Davis. “We were fortunate to have a pre-exposure comparison of sardines due to the long-term sampling nature of the original experiment.”

McCormick and Skelton praised the collaborative efforts of all the authors, emphasizing the significant role each played in making the study possible. “It was a large community effort, with everyone contributing their existing data and working together to deepen our understanding of the issue,” said McCormick, “even when we were limited by the pandemic.”

View the published study to access the full list of 34 co-authors and their affiliated funding sources.

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Research on RNA editing illuminates possible lifesaving treatments for genetic diseases

A team at Montana State University published research that shows how RNA, the close chemical cousin to DNA, can be edited using CRISPRs. The work reveals a new process in human cells that has potential for treating a wide variety of genetic diseases.

Postdoctoral researchers Artem Nemudryi and Anna Nemudraia conducted the research alongside Blake Wiedenheft, professor in the Department of Microbiology and Cell Biology in MSU's College of Agriculture. The paper, titled "Repair of CRISPR-guided RNA breaks enables site-specific RNA excision in human cells," was published online in the journal Science and constitutes the latest advance in the team's ongoing exploration of CRISPR applications for programmable genetic engineering.

CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is a type of immune system that bacteria use to recognize and fight off viruses. Wiedenheft, one of the nation's leading CRISPR researchers, said that the system has been used for years to cut and edit DNA, but that applying similar technology to RNA is unprecedented. DNA editing uses a CRISPR-associated protein called Cas9, while editing RNA requires the use of a different CRISPR system, called type-III.

"In our previous work, we used type-III CRISPRs to edit viral RNA in a test tube," said Nemudryi. "But we wondered, can we program manipulation of RNA in a living human cell?"

To explore that question, the team programmed type-III CRISPR proteins to cut RNA containing a mutation that causes cystic fibrosis, restoring cell function.

"We were confident that we could use these CRISPR systems to cut RNA in a programmable manner, but we were all surprised when we sequenced the RNA and realized that the cell had stitched the RNA back together in a way that removed the mutation," said Wiedenheft.

Nemudryi noted that RNA is transient within the cell; it is constantly being destroyed and replaced.

"The general belief is that there's not much point in repairing RNA," he said. "We speculated that RNA would be repaired in living human cells, and it turned out to be true."

Wiedenheft has mentored the two postdoctoral researchers since their arrival at MSU nearly six years ago, and said that the impact of their scientific contributions will lead to significant and continued advancements.

"The work done by Artem and Anna suggests that RNA repair might be a fundamental aspect of biology and that harnessing this activity may lead to new lifesaving cures," said Wiedenheft. "Artem and Anna are two of the most brilliant scientists I have ever encountered, and I'm confident that their work is going to have a lasting impact on humanity."

RNA editing has important applications in the search for treatments of genetic diseases, Nemudryi said. RNA is a temporary copy of a cell's DNA, which serves as a template. Manipulating the template by editing DNA could cause unwanted and potentially irreversible collateral changes, but because RNA is a temporary copy, he said, edits made are essentially reversible and carry far less risk.

"People used Cas9 to break DNA and study how cells repair these breaks. Then, based on these patterns, they improved Cas9 editors," said Nemudraia. "Here, we hope the same will happen with RNA editing. We created a tool that allows us to study how the cells repair their RNA, and we hope to use this knowledge to make RNA editors more efficient."

In the new publication, the team shows that a mutation causing cystic fibrosis can be successfully removed from the RNA. But this is only one of thousands of known mutations that cause disease. The question of how many of them could be addressed with this new RNA editing technology will guide future work for Nemudryi and Nemudraia as they finish their postdoctoral training at MSU and prepare for faculty positions at the University of Florida this fall. Both credited Wiedenheft as a life-changing mentor.

"Blake taught us not to be afraid of testing any ideas," said Nemudraia. "As a scientist, you should be brave and not be afraid to fail. RNA editing and repair is the terra incognita. It's scary but also exciting. You feel you're working on the edge of science, pushing the limits to where nobody has been before."

• Human Biology
• Cystic Fibrosis
• CRISPR Gene Editing
• Biochemistry Research
• Organic Chemistry
• Biochemistry
• Forensic Research
• Double blind
• Origin of life
• Genetic code
• Molecular biology
• Human parainfluenza viruses

Story Source:

Materials provided by Montana State University . Original written by Reagan Cotton. Note: Content may be edited for style and length.

Journal Reference :

• Anna Nemudraia, Artem Nemudryi, Blake Wiedenheft. Repair of CRISPR-guided RNA breaks enables site-specific RNA excision in human cells . Science , 2024; DOI: 10.1126/science.adk5518

Cite This Page :

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