In the late 1880s, Raphaël Dubois was among the first to suggest bacteria could be responsible for the light emitted by some animals (Harvey, 1957). In the beginning of the twentieth century, Balthazar Osorio (1912) provided clear and convincing evidence of such symbiosis, when luminescent bacteria were described in high density within a dedicated fish gland, called the light organ (Balthazar Osorio was cited in Hickling, 1926). Since then, luminous bacterial symbiosis has been the subject of interest among the scientific community working on bioluminescence, to such an extent that, by the mid-twentieth century, luminescence of many organisms was thought to have bacterial origin. However, some of these assessments have been refuted later (Herring, 1977).

Bioluminescence ability is shared by about 8 % of all known fish species (Paitio et al., 2016). Amongst luminous fishes, bacterial luminescence is the rule for almost half of them (48 %) (Davis et al., 2016). To date, symbiotic bacteria are recognized as responsible for the luminescence of some fishes and squids (Davis et al., 2016; Haygood, 1993; Lindgren et al., 2012). Although forms of symbiotic luminescence have been suggested for some shark species or pyrosomes (tunicates) (Dunlap and Urbanczyk, 2013; Leisman et al., 1980), no evidence of luminous bacteria has been found so far (Claes and Mallefet, 2009; Renwart et al., 2014; Widder, 2002) and a recent study has definitely rejected a bacterial origin in the velvet belly lanternshark (Duchatelet et al., 2019). Concerning luminous squids, intrinsic bioluminescence is more common, and symbiotic light organs are known in only two families (Sepiolidae and Loliginidae) (Lindgren et al., 2012; Nishiguchi et al., 2004).

Symbiotic luminescence seems more common in benthic or coastal environments for fish and squid as well (Haygood, 1993; Lindgren et al., 2012; Paitio et al., 2016). Shallow-water fishes with luminous bacterial symbionts include flashlight fishes (Anomalopidae), ponyfishes (Leiognathidae) and pinecone fishes (Monocentridae) (Davis et al., 2016; Morin, 1983). For deep-sea fishes, anglerfishes (Ceratiodei) and cods (Moridae) are among the common examples of luminous-bacteria hosts.

Bacterial and intrinsic light organs are predominantly internal, ventrally located (Paitio et al., 2016). Many luminous organisms with ventral light organs likely use the emitted light to conceal themselves by counterillumination. This defensive strategy allows luminous species to match with the intensity, spectrum and angular distribution of the downwelling light, thus obliterating their silhouette and therefore avoiding dusk-active piscivorous predators (Claes et al., 2010; Johnsen et al., 2004; Warner et al., 1979). Amongst bacterial light symbioses, counterillumination has been demonstrated for the bobtail squid Euprymna scolopes (Jones and Nishiguchi, 2004) and some leiognathids fish (McFall-Ngai and Morin, 1991) and hypothesized for other bioluminescent fishes (Dunlap et al., 2009; McAllister, 1967). Less common but more striking, some organisms found in the families Monocentridae and Anomalopidae and numerous deep-sea anglerfishes belonging to the suborder Ceratoidei exhibit externally located light organs colonized by bacteria (Haygood, 1993). The external light organs of flashlight fish have been demonstrated to be used to illuminate the nearby environment and detect prey (Hellinger et al., 2017), or schooling behavior (Gruber et al., 2019), while the lure of female anglerfish is generally believed to be used for mate-finding purposes and prey attraction (Herring, 2007).

Like most symbiotic bacterial associations with animals, luminous bacteria are acquired from the surrounding environment by individuals, independently of their ancestry (i.e., horizontally transmitted) (Baker et al., 2019; Haygood, 1993; McFall-Ngai, 2014). One of the best-documented symbioses is the association of Aliivibrio fischeri with the bobtail squid Euprymna scolopes (Nyholm and McFall-Ngai, 2004; Ruby, 1996). Through the easy independent cultivation of both partners in the laboratory, this symbiosis has become a perfect model for studying the process of bacterial colonization into the light organ and understanding bacteria–animal interactions, broadly speaking (Mandel and Dunn, 2016; McFall-Ngai, 2014).

Knowledge of the mechanisms involved in the selection and the establishment of bacterial symbionts in the squid–Vibrio symbiosis have considerably improved over the last few decades. Harvest of the luminous symbionts from the bacterioplankton is driven by microbial recognition and molecular dialog (Kremer et al., 2013; Nyholm et al., 2000; Nyholm and McFall-Ngai, 2004; Pankey et al., 2017; Schwartzman and Ruby, 2016; Visick and Ruby, 2006). Moreover, bacterial colonization of host tissues induces the morphogenesis process of the light organ and appears to signal its further development and maturation (McFall-Ngai and Ruby, 1991; Montgomery and McFall-Ngai, 1998). The luminescence feature is essential for a correct morphogenesis process of the light organ and symbiont persistence inside (McFall-Ngai et al., 2012; Visick et al., 2000).

While the bobtail squid model provides a window to understand the establishment of such symbioses, this system cannot be systematically transferred to other bacterial luminous symbioses. Although less well-known, the other associations are no less important and many questions remain unsolved since they might be harder to study.

To date, 11 bacterial species are known to be involved in light organ symbioses (Table 1). In a light organ, the bacterial population is most of the time monospecific (Dunlap and Urbanczyk, 2013; Ruby, 1996).

Considering that fish and squid housing luminous bacteria are never found without symbionts in nature, the symbiosis appears obligatory for hosts (Haygood, 1993). In contrast, most symbiotic bacteria are viable outside the light organ, and thus are considered to be facultatively symbiotic. These facultative symbiotic bacteria are readily culturable under laboratory conditions, outside the host light organ. Exceptions have been highlighted for the luminous symbionts of two groups of fish, the flashlight fish and the deep-sea anglerfish (Dunlap and Kita-tsukamoto, 2006; Haygood and Distel, 1993). Indeed, despite the fact that the bacterial origin of the light was proved by microscopic observation and that genes from luminous bacteria were amplified (Haygood and Distel, 1993), bacterial cultivation has not yet been successful. Thanks to the emergence of genome sequencing, the complete genome of these symbionts has been reported in the last years. Analyses revealed a genome reduction in size by about 50 % and 80 % for anglerfish and flashlight fish symbionts, respectively, compared to facultative luminous symbionts or free-living relatives (Hendry et al., 2014, 2016, 2018). Genome reduction is a common trait shared by bacteria involved in obligatory symbiosis (Moran et al., 2009) and explains the inability of these symbionts to grow in laboratory cultures. Flashlight fish and anglerfish symbionts appear to be obligately dependent on their hosts for growth, as some metabolic capacities (e.g., genes necessary for amino acid synthesis) are absent in the genome.

Although light organs can differ in form, size or location according to the host (see Table 1), some structural and functional features are common for all of them. Luminous bacteria are densely packed within tubules which connect to the exterior of the light organ (Haygood, 1993; Nealson, 1979). The host provides nutrients and oxygen to the tubules through a highly vascularized system (Tebo et al., 1979). Bioluminescent bacteria emit light continuously in the light organ, as they do in laboratory cultures (Nealson and Hastings, 1979). However, the light intensity varies over time. As for self-luminescent fish, bacterial light organs have evolved with a multitude of adaptations of tissue, to serve as reflectors, diffusers, screens and light-conducting channels (Haygood, 1993; Munk et al., 1998). Such anatomical features assist in directing and enhancing light output (Sparks et al., 2005). In addition, the host can control the light diffusion through different mechanisms, which may be external lids, chromatophores, organ rotation, filters, occlusion with a shutter or muscle contraction (Hansen and Herring, 1977; Herring, 1977; Johnson and Rosenblatt, 1988). As an example, for counterillumination, controlling the intensity of light output gives the host a better camouflage, adapting its silhouette to environmental changes in light (Jones and Nishiguchi, 2004; McFall-Ngai and Morin, 1991). For intraspecies communication, it permits the production of sudden flashes or a specific signal/rhythm of light (e.g., schooling behavior, Gruber et al., 2019).

In squid–Vibrio symbiosis, bacterial luminescence genes are regulated with a quorum-sensing system, a cell-density-dependent process. When the cell density reaches a certain level, autoinducers responsible for triggering the synthesis of the genes involved in light emission are accumulated in sufficient amounts, and light is emitted (Nealson et al., 1970; Verma and Miyashiro, 2013). Interestingly, A. fischeri produces a higher level of luminescence within the light organ than in laboratory cultures, despite a similarly high cell density (Boettcher and Ruby, 1990). Hence, Verma and Miyashiro (2013) suggested that the light organ environment offers specific conditions such as the levels of oxygen, iron or phosphate, to enhance bacterial light emission. Here again, while the control mechanisms of the squid–Vibrio symbiosis are well-understood, those of the other symbioses remain enigmatic and there are indications that they may vary. For example, the absence of the quorum-sensing-gene detection in anglerfish and flashlight fish symbionts suggests a constitutive light emission by the bacteria (Hendry et al., 2016, 2018).

For all symbioses, luminous symbionts, within the light organ, reach a very high density which reduces the oxygen availability, essential for the light reaction. Such oxygen limitation leads to a decrease in the specific luminescence activity (Boettcher et al., 1996). The bacterial population inside the light organ is regulated by the host, by coupling the restriction of the growth rate and the expulsion of symbionts. Growth repression is thought to reduce the energetic cost of the symbiosis to the host (Haygood et al., 1984; Ruby and Asato, 1993; Tebo et al., 1979). Additionally, since luminous bacteria are densely packed inside tubules communicating with the exterior of the light organ (Haygood, 1993), the cell number of symbionts is regulated by the regular expulsion of most of the bacterial population, followed by a period of regrowth of the remaining symbionts. Concerning the well-known squid–Vibrio symbiosis, its daily release is highly correlated with the diel pattern of the host behavior. Indeed, the bobtail squid expels 95 % of the luminous symbionts in the surrounding environment at dawn, the beginning of its inactive phase. The remaining 5 % of A. fischeri grow through the day and the highest concentration is reached at the end of afternoon, at the nocturnal active phase of the squid (Nyholm and McFall-Ngai, 2004; Ruby, 1996). Currently, with the exception of the squid–Vibrio symbiosis, accurate data on the symbiont release are still largely unknown. Indeed, the frequency of release may vary and occur more than once a day as has been shown for some flashlight and pinecone fishes (Haygood et al., 1984).

Regular expulsion of symbionts maintains favorable conditions in the light organ for the bacterial population, but it also seeds the environment with luminous symbionts for colonization of the next host generation. The consequence is a release of a huge quantity of bioluminescent bacteria in the seawater, inducing a major contribution to the ocean microbiome. To make it more concrete and provide an order of magnitude, two examples are proposed. Using laboratory experiments on different fishes (Monocentridae, Anomalopidae), Haygood et al. (1984) estimated a release of between 107 and 109 bioluminescent bacterial cells per day and per individual. Another study on the Hawaiian bobtail squid (E. scolopes) has estimated that the squid expels about 5×108 bioluminescent bacterial cells per day and per individual (Lee and Ruby, 1994). These discharges lead to a regular luminous-bacteria enrichment of the areas inhabited by these organisms.

Depending on the anatomical location of the light organ (see Table 1), luminous symbionts are released through pores or ducts into the surrounding seawater or into the digestive tract (Haygood, 1993; Nealson and Hastings, 1979). An enteric lifestyle has indeed been suggested for the luminous bacteria (Ruby and Morin, 1979; Nealson, 1979).

Qualitative and quantitative studies showed that the luminous bacteria are dynamic over time and space. Seasonal variations have been identified, in both abundance and predominant species (O'Brien and Sizemore, 1979; Ruby and Nealson, 1978; Yetinson and Shilo, 1979). A wide variability has been observed in species repartition over depth and between geographic areas (DeLuca, 2006; Gentile et al., 2009; Nealson and Hastings, 1979; Ramaiah and Chandramohan, 1992; Ruby et al., 1980). Horizontal, vertical and seasonal variations were presumed to reflect physiological preferences most of the time, and particularly temperature or salinity sensitivity (Orndorff and Colwell, 1980; Ramesh et al., 1990; Ruby and Nealson, 1978; Shilo and Yetinson, 1979; Yetinson and Shilo, 1979). Some works mentioned that symbiotic niches, such as light organs and enteric tracts, may serve to inoculate the planktonic population (Nealson et al., 1984; Nealson and Hastings, 1979; Ramesh et al., 1990; Ruby et al., 1980). To our knowledge, very few studies focused intensively on the contribution of species-specific symbiotic associations on the occurrence and distribution of luminous bacteria in the surrounding water. Amongst these rare studies, Lee and Ruby (1994) reported that the abundance of A. fischeri, the luminous symbiont of the Hawaiian squid E. scolopes was 24 to 30 times higher, in both water column and sediments, in areas inhabited by the squids than in similar locations where squids were not observed.

Bioluminescent bacteria also seem to be the cause of the spectacular and still largely unexplained events, so-called milky seas (Lapota et al., 1988; Nealson and Hastings, 2006). Milky seas are characterized by an unusual brightness on the ocean surface and extend over such a large area that the light emitted is detectable from space (Miller et al., 2005). The light emission pattern of milky seas is continuous and homogeneous, which is consistent with light emission from bacteria and easily distinguished from blooms of dinoflagellates.

Outside of spatially restricted niches, such as light organ or gut environments, the role of the dispersed luminous cells in the marine environment was a matter of debate, and it was thus mentioned that non-symbiotic bacteria may have no ecological significance (Hastings and Greenberg, 1999; Nealson and Hastings, 1979). However, Herren et al. (2004) suggested that luminous bacteria are more often attached to particles than free-living, which was confirmed by Al Ali et al. (2010). Many bacteria, including bioluminescent bacteria (Ruby and Asato, 1993; Zhang et al., 2016), can develop swimming behavior to colonize the sinking organic material, therefore reaching a cell density 100 to 10 000 times higher than in the water column (up to 108 to 109 cells mL-1) (e.g., Ploug and Grossart, 2000).

Bacteria that glow on particles can attract macroorganisms. After being ingested, they will find a more favorable environment to live and grow in their gut (Andrews et al., 1984; Ruby and Morin, 1979). Actually, this is the preferred current hypothesis that supports a positive selection related to the dispersion and propagation of the bacteria. Indeed, luminous bacteria growing on particulate matter could produce enough light to be visible by other organisms. For bacterial species with light production under cell-density control (i.e., under quorum-sensing regulation), the high cell concentration reached on particles can allow the sufficient accumulation of the autoinducers, and thus the emission of light for attracting predators. For species for which light production is not subject to cell-density control (i.e., not under quorum-sensing regulation) (Tanet et al., 2019), to be able to produce light at a very low cell concentration could give them an advantage. Continuously glowing bioluminescent emissions are thought to attract predators (Nealson and Hastings, 1979). In the water column, the glowing bacteria aggregated on particles would lead to the detection, attraction, ingestion and decomposition of particles by larger organisms. Grazers would consume luminous matter at a higher rate than invisible particles. Being consumed and ending up in the gut, bacteria would benefit from a more suitable environment regarding the growth conditions and the nutrient accessibility. In the open ocean, and particularly in deep regions, where sparse nutrient supply prevails, nutrient-rich gut niches of the surrounding animals could appear as an oasis of life for bacteria. This dispersion hypothesis has also been strongly consolidated by field data where bacterial bioluminescence was observed in freshly egested fecal pellets and in materials collected from sediment traps (Andrews et al., 1984), as well as by laboratory experiments where glowing zooplankton were preferentially ingested by fishes (Zarubin et al., 2012).

The copiotrophic trait of luminous bacteria is another point supporting their particle-attached lifestyle. Bacterial population colonizing nutrient-rich environments (e.g., floating carcass, marine snow, fecal pellets or the gut tract of a marine eukaryote) are defined as copiotrophs, by opposition to the oligotrophs which are members of free-living microbial populations (Lauro et al., 2009). All luminous Vibrionaceae, except reduced genome symbionts, possess two chromosomes in their genome (Boyd et al., 2015; Zhang et al., 2016), with a high copy number of rRNA operons. Such genomic features, a large genome size and multiple rRNA operons, are considered to be an adaptation for a copiotrophic lifestyle (Klappenbach et al., 2000; Lauro et al., 2009). Copiotrophs are thought to have strong adaptability skills, permitting them to survive long enough between two nutrient-rich environments (Yooseph et al., 2010).

Fish guts could also act as an enrichment vessel for the growth of luminous bacteria, and thus enhance their propagation (Nealson and Hastings, 1979; Ramesh and Venugopalan, 1988). When expelled with feces, enteric luminous bacteria can be easily isolated from the fresh fecal material. This fecal luminescence increased in intensity over a matter of hours, proving that luminous bacteria survive the digestive process and can proliferate on such organic material (Ruby and Morin, 1979). Hence, fish feces appear to be an important source of viable luminous bacteria in the marine environment and could affect both the distribution and the species composition of luminous populations. The luminescence of fecal particles has been reported numerous times and is always associated with luminous bacteria, due to the observation of continuous light emission or direct isolation (Andrews et al., 1984; Ramesh et al., 1990; Raymond and DeVries, 1976; Ruby and Morin, 1979; Zarubin et al., 2012).

In comparison with free-living luminous bacteria, few studies have focused on bioluminescence of marine snow and fecal pellets. Yet, observations on materials collected from sediment traps revealed light emission in 70 % of all samples, with two distinct patterns of light kinetics, probably due to the presence of different luminescent organisms (Andrews et al., 1984). Surface-sample (above 60 m depth) analyses reported that more than 90 % of the luminous-aggregate samples exhibited bacterial luminescence (Orzech and Nealson, 1984). Another study (between 2 and 17 m depth) also reported a large part of luminous marine snow, but more likely due to dinoflagellates (Herren et al., 2004).

Information relative to luminous bacteria in sediment is also limited. It is known that bioluminescent bacteria can be isolated from sediment samples (Ramesh et al., 1990), but rare data exist about their distribution or abundance. In some sediment samples, occurrence of luminous bacteria among total heterotrophic bacteria could reach up to 70 %, with seasonal variations (Ramesh et al., 1989), although less pronounced than in the water column (O'Brien and Sizemore, 1979). The main sources of luminous bacteria in sediments are likely the glowing sinking marine snow and benthic or demersal hosts, harboring symbiotic light organs with regular discharges.

More recently, sediment resuspension events (Durrieu de Madron et al., 2017) were correlated with newly formed deep-water events and deep-sea bioluminescent events recorded in the NW Mediterranean Sea (Martini et al., 2014; Tamburini et al., 2013a). Since the presence of active luminous bacteria has been demonstrated on the site (Martini et al., 2016), it has been hypothesized that resuspended luminescent bacteria present in sediment can be part of these luminescence events (Durrieu de Madron et al., 2017). Additionally, dense water formation, conveying particulate organic matter, could further increase luminous-bacteria proliferation and activity (Tamburini et al., 2013a).

Based on the ecological versatility of the bacterial bioluminescence reviewed above, we propose reconsidering the classical view of the fate of organic matter in the oceans. Figure 1 represents the guideline of the bioluminescence shunt hypothesis of the biological carbon pump.

Bioluminescent bacterial emissions are continuous over time and such a characteristic is thought to attract predators. Indeed, the light from bioluminescence contrasts well against the dim or dark background of the ocean depths. In the bathypelagic zone (1000–4000 m), where no daylight remains, bioluminescent emissions are considered the major visual stimulus (Warrant and Locket, 2004; Widder, 2002). For these reasons, symbiotic associations in light organs have been selected as an advantage for hosts (fish or squid). Luminous bacterial symbionts are successively acquired by juveniles and released into the seawater to control population concentration (Fig. 1, step 1). As indicated previously, the release of bioluminescent bacteria from light organs and fecal pellets could represent a huge quantity of bioluminescent bacteria in the water column. On dead organisms, luminous bacteria present in the gut of the host could initiate rapid propagation and decomposition of the host body and result in the formation of luminous debris in the marine environment. Based on the increase in light emission observed on dead marine animals, Wada et al. (1995) argue that, upon the death of the host, enteric luminous bacteria may have an important saprophytic lifestyle.

Recent studies underlined that fish vision is very-well-adapted to the detection and location of point-source bioluminescence (de Busserolles and Marshall, 2017; Mark et al., 2018; Musilova et al., 2019; Paitio et al., 2016; Warrant and Locket, 2004). Although less intensively documented than fishes, the crustacean (copepods, amphipods, isopods, etc.) visual system is also reported to have a sensitivity shift to bluer wavelengths, which aids their bioluminescence detection (Cohen and Forward, 2002; Frank et al., 2012; Marshall et al., 1999; Nishida et al., 2002). In the laboratory, Land et al. (1995) demonstrated that amphipods were attracted to a blue-light-emitting diode. Unfortunately, and despite these statements, rare studies have investigated the effect of bioluminescence on the ingestion rates of predators (Fig. 1, step 2). To our knowledge, the only one known is from Zarubin et al. (2012), who demonstrated that zooplankton is attracted to luminous particles and feeds on the luminous-bacteria-rich organic matter. Because of the ingestion of the luminous bacteria, the zooplankton itself starts to glow. Then, Zarubin et al. (2012) experimentally measured the 8-times-higher ingestion rate of glowing zooplankton by fishes, compared to non-luminous zooplankton.

Glowing bacteria have been observed attached to particles of organic matter, marine snow and fecal pellets (Fig. 1, from symbionts in guts in step 1 and through predation in step 2) sinking into the deep ocean. Thus, while sinking into the deep, these glowing bacteria living on organic carbon particles (marine snow, fecal pellets, etc.) would lead to the detection, attraction, ingestion and decomposition of particles by larger organisms. Consumers would ingest luminous matter at a higher rate than invisible particles and consequently will increase luminous-microorganism dispersion by the egestion of fecal pellets. Bioluminescent sinking material should accelerate the consumption of organic matter by attracting grazing organisms. Interestingly, bacteria associated with animal guts are thought to be particularly adapted to high hydrostatic pressure (Deming et al., 1981; Ohwada et al., 1980; ZoBell and Morita, 1957). Indeed, certain bioluminescent bacteria resist high hydrostatic pressure (Brown et al., 1942), and some of them have a higher growth rate and emit more light than at atmospheric pressure (Martini et al., 2013). Such piezotolerance, or piezophile lifestyle, is undoubtedly an advantage for luminous bacteria attached to particles that are exposed to pressure variations during the sinking-particle fluxes (Tamburini et al., 2013b). The addition of these bioluminescent tags on particles has two indirect impacts (Fig. 1, steps 2 and 3). First, due to aggregate fragmentation by sloppy feeding and coprorhexy, fast-sinking particles are transformed into slow-sinking or suspended particles. Fragmentation has been shown to be the primary process controlling the sequestration of sinking organic carbon (Briggs et al., 2020). The second possibility is that organic matter ingestion leads to aggregation by repackaging, and the egested pellets of higher density are fast-sinking particles. Filter-feeder plankton, without visual detection and food selection by light, will also passively contribute to such aggregation or fragmentation of particles. For these organisms, bioluminescence can even have a negative effect since they can be identified by the luminous material filtered. Additionally, the consumption of organic material colonized by bioluminescent bacteria increases their dispersal rate provided by migrating zooplankton and even more so by actively swimming fish, following the conveyor-belt hypothesis (Grossart et al., 2010) (Fig. 1, step 4). After being ingested, bacteria (including luminous ones), attached to the particles consumed by zooplankton and fish, stay in their digestive tract. At night, these organisms migrate in the upper part of the water column and release feces in niches and at depth that, eventually, would not have been otherwise colonized by luminous bacteria. This dispersion, due to the expelling of luminous feces, is several orders of magnitude greater than that of waterborne free bacteria. Zooming on the carbon fluxes at the level of a gravitational sinking particle (Fig. 2), the bioluminescence shunt hypothesis implies that the bacterial glow of this particle increases the distance of visual detection. Such a distance can be up to several tens of meters according to Warrant and Locket (2004) and probably depends on the bioluminescent bacterial concentration and the visual perception of the organisms.

Sediment resuspension is another process implying the consumption of luminous bacteria by higher trophic levels (Fig. 1, step 5). This potentially re-inseminates bacteria into the bioluminescence loop through the consumption by epi-benthic organisms.

Considering this bioluminescence shunt hypothesis, all the processes described above show that bioluminescence affects the biological gravitational carbon pump (Boyd et al., 2019), either by increasing the carbon sequestration into the deep ocean or by slowing down the sinking rate of particles and consequently increasing their degradation and the remineralization rate. Bioluminescence and especially luminous bacteria may therefore influence the export and sequestration of biogenic carbon in the deep oceans (either positively or negatively). A better quantification of these processes and impacts in the biological carbon pump is a requirement in future studies.

In the existing literature, to estimate the diversity and the distribution of bioluminescent bacteria, studies were based on a restricted number of sampling methods and instruments. These methods focused either on environmental samplings where bacteria are present or on organisms with associated bacteria.

First, vertical samplings in the water column were performed using sterile-bag samplers (Ruby et al., 1980), or later using Niskin bottles (mounted on rosette profilers, Fig. 1c) (Al Ali et al., 2010; Gentile et al., 2009; Kita-Tsukamoto et al., 2006; Martini et al., 2016; Yetinson and Shilo, 1979). This approach is commonly set up in oceanography but relies on relatively small volumes of water (up to 20 L). Furthermore, it does not fully capture the heterogeneity of the ecosystem since it provides one discreet sample over restricted time and space. Other instruments dedicated to the acquisition of sediment sampling are the multiple-core samplers, deployed onto the seafloor (Kita-Tsukamoto et al., 2006). For particulate organic carbon and fecal pellets, in order to describe the diversity of associated luminous bacteria, sediment traps (Fig. 1, item e) have been occasionally deployed from the surface down to the deep ocean (Andrews et al., 1984). Using these, fresh luminous material has been collected between 30 and 1900 m depth.

To study the presence of luminous symbionts in guts and light organs, larger organisms are caught. The most common way to catch deep-sea animals is the deployment of trawls and more generally nets (Fig. 1a–b). They are well-adapted to sample squid (Zamborsky and Nishiguchi, 2011) or fish, like the anglerfish (Freed et al., 2019). One particularity of these methods is that the sampling covers a large section of the water column and combines everything into one catch with a limited precision about depth layers. SCUBA diving is another method to gently select these large animals (Zamborsky and Nishiguchi, 2011). It has also been used to catch fecal pellets and sinking particles (Orzech and Nealson, 1984). Obviously, SCUBA diving has a strong depth limitation (generally above 50 m depth). It can be more efficient at night for some migrating species and has a restricted sampling size of organisms and number of samples carried back to the ship.

Once environmental samples or material from an organism's light organ have been acquired, the objective is either to describe the taxonomy and diversity of luminous bacteria or to quantify them. To do so, earlier studies have filtered seawater samples through a polycarbonate filter with a pore size of 0.2 µm to retain bacteria. The filter is then placed with the bacterial side up on growth medium in Petri dishes (Kita-Tsukamoto et al., 2006; Ruby et al., 1980). For symbiotic bacteria, light organs or guts are aseptically dissected shortly after death, and the content is homogenized before culture or microscopic observations (Dunlap, 1984). After hours of incubation, the total colony-forming units is observed; the luminous colonies can, then, be enumerated and selected for taxonomic investigation.

Further investigations of symbiotic associations, in relation to the surrounding environment, would require a reliable taxonomy of luminous bacteria and robust knowledge on species-specific symbiotic associations. As an example, Photobacterium phosphoreum was thought to be the specific symbiont of light organs of numerous deep-sea fish (Hendrie et al., 1970; Ruby et al., 1980; Ruby and Morin, 1978), before a phylogenetic analysis showed distinct evolutionary lineages in the P. phosphoreum clade according to the colonized habitat. This resolution revealed that all the P. phosphoreum symbionts isolated from light organs should actually be identified as P. kishitanii (Ast and Dunlap, 2005).

Since these first investigations on luminous bacteria in symbioses or in the environment, there has been a huge improvement in technology and molecular-biology techniques. To better evaluate the role of bioluminescence and luminous bacteria in the biological carbon pump, further studies have to follow an efficient strategy. Such a strategy will focus on quantifying this functional trait and how it impacts the transfer of organic carbon between trophic levels, as well as its sequestration into the deep ocean. This approach can be divided into several key points: (1) the assessment of the global importance of bioluminescence in the oceans, (2) the pursuit of investigations about the quantification and diversity of luminous bacteria and their variability between ecosystems (free-living in the water column, on sinking particles and fecal pellets, or in sediments), (3) the quantification of luminous bacterial release into the surrounding environment and the potential impact of diel vertical migration of zooplankton and fish, and (4) the quantification of the transfer rate of bacteria attached to glowing particles to zooplankton and the quantification of the effects on organic matter decomposition, sinking rate and fluxes, in comparison to non-glowing particles. In this review, future perspectives to allow major advances on these specific key points are proposed based on recently developed technologies.