*3.1.1 Cypridinid luciferin in the midshipman fish*

Several species of midshipman fish have been shown to utilise cypridinid luciferin as a substrate in their own luminescent reactions, despite showing no identifiable capability to synthesise their own luciferin [43]. A notable example of this has been observed consistently in the species *Porichthys notatus*, which can be found along the Pacific coast of the North American continent [44]. This species is characterised by an array of over 700 dermal photophores distributed along its head and body [45, 46]. Whilst light emission is restricted to specific organelle structures and can be stimulated mechanically, this is not sufficient to constitute a wholly intrinsic luminescent system. Moreover, non-luminescent individuals of the species have been identified when caught in the North Pacific off the coast of Oregon, where despite possessing the photophores in the same pattern, they did not exhibit luminescence [47]. This lack of luminescence was attributed to these animals not having a source of luciferin available from their diet at all of their life stages [48].

By adding small amounts of cypridinid luciferin to *P. notatus*, either by feeding them ostracods, or by intraperitoneal doses of as little as 6 μg of luciferin it was possible to induce luminescence [44]. This also was shown to be possible for completely non-luminescent individual midshipman fish and confirmed cross-reactivity of *P. notatus'* luciferase with cypridinid luciferin led to light emission [43]. It was identified that following consumption of ostracods, *P. notatus* is able to absorb the cypridinid luciferin through its gut. From here the substrate is believed to be able to bind non-specifically to erythrocytes in the blood plasma, possibly preventing autooxidation as it is transferred to the organelles of *P. notatus* where it can be oxidised in the presence of the luciferase enzyme to result in an emission of blue light [43, 49]. Light emission from the addition cypridinid luciferin to non-luminescent *P. notatus*, was indistinguishable from naturally luminescent Californian *P. notatus* [49].

The midshipman fish is a visually active nocturnal predator, that can utilise this acquired cypridinid luciferin to facilitate its hunting strategies. It has been speculated that the array of photophores on its body can mimic the light emission seen in euphausiid swarms, attracting unsuspecting prey [43, 50, 51]. This ability in combination with its highly evolved eyesight have allowed for it to be an effective nocturnal predator, feeding on both luminescent and non-luminescent organisms [52]. Cypridinid luciferin is not isolated to this species and has been found in several other luminescent coastal fishes including in the families, Pempheridae and Apogonidae [53]. Apogonids, or cardinalfishes are mostly reef dwelling with several species exhibiting visceral light organs that produce luminescence [54]. Similarly, Pempheridae commonly known as sweeper fishes, also have photophores along the length of their bodies and tend to be found in shallow marine and brackish waters [54]. It is likely that these species acquire their luminescence from ostracods, in a similar manner to the midshipman fish, though this is still to be confirmed experimentally.

## *3.1.2 Coelenterazine in Myctophid and Stomiid fishes*

Cypridinid luciferin accounts for the luminescence observed in only a few species of bony fish as well as within ostracods, meaning it does not encompass a large amount of the total luminescence in marine environments. The most ubiquitous luciferin found in marine organisms is coelenterazine with species across multiple

phyla utilising this as their substrate for light emission [12, 40]. Among the fishes, numerous species of Myctophidae and Stomiiformes have been shown to utilise coelenterazine for bioluminescence, which is obtained through their diet, either by predating directly on coelenterazine producing copepods such as *Metridia pacifica*, or indirectly by predating on the consumers of these copepods [55, 56].

Myctophids, commonly known as lanternfish, are one of the most widespread and abundant families of mesopelagic fish in the oceans. They are distributed globally, with over 250 species identified across 33 genera and 2 subfamilies [56, 57]. Lanternfish are taxonomically distinguished by specific patterns of luminescent photophores that have allowed for a diverse array of strategies for both prey detection and predator avoidance [58, 59]. Generally, Lanternfish have two kinds of photophores, one along the body with the other proximal to their eyes (**Figure 2**). These two sets of photophores are able to illuminate independently from one another allowing for a variety of ecological functions. Photophores arranged on the ventral surface produce a constant dim blue luminescent glow and can allow for counterillumination similar to other luminescent fishes, which would allow lanternfish to blend into the surrounding water column [56]. This would facilitate an ability to ambush prey as well as to hide from potential predators in the water column. These arrays of photophores form species specific patterns, which may allow for them to be used in intraspecific recognition [56, 60]. In addition to this array of photophores on the body, most lanternfish have one or more larger photophores on their head, usually positioned sub-orbitally or in the direct vicinity of their eyes [61]. Unlike the photophores on the ventral surface, these emit light in brief intermittent brilliant flashes. This is thought to allow either for predation by illuminating their prey, as well as being used to avoid predators by flashing and startling any larger organisms [56, 62]. Given that these suborbital photophores have sexual dimorphism, it is also possible that their main role is in communication within the species [56, 63].

Lanternfish feed predominantly on a variety of zooplankton including copepods such as *M. pacifica*, which would facilitate a source of coelenterazine luciferin for their luminescence, although it is difficult to assess this given the difficulties of maintaining deep sea fish such as myctophids in aquaria for sufficient amounts of time [55]. Lanternfishes are a major food source for a number of marine predators, including whales and dolphins. More importantly, they are also predated upon by squid and other larger lanternfishes, that also possess luminescence using coelenterazine or one of its derivatives [59]. Therefore, these potentially provide a key link in food webs by facilitating the transfer of coelenterazine from zooplankton to megafauna.

Stomiiform fishes include four families comprising of Gonostomatidae (bristlemouths), Phosichthyidae (lightfishes), Sternoptychidae (hatchetfishes), and the Stomiidae (dragonfishes) [64]. Among the dragonfishes, all species identified within this group have been shown to be bioluminescent, harbouring their light emission within specialised arrays of photophores. Apart from the Arctic Ocean, Stomiidae fishes are distributed globally, residing in the mesopelagic zone of the ocean between 200 and 1000 m depth, with some species recorded to a depth more than 4000 m [64, 65]. Luminescence may well be derived from the coelenterazine in their diets, with several species showing cross reactivity with coelenterazine in a similar way to some lanternfish [3]. However, it has been difficult to determine whether these animals are capable of synthesising their own luciferin, given that it is not yet possible to collect and maintain stomiid fishes in aquaria for any length of time. Dragonfishes are predators, utilising their bioluminescent emissions both as lures and as means to illuminate prey in order to facilitate prey capture [64]. Most feed on squid, shrimps and other fishes including lanternfishes, which may facilitate a source for coelenterazine in a number of these species [64].

*Semi-Intrinsic Luminescence in Marine Organisms DOI: http://dx.doi.org/10.5772/intechopen.99369*

#### **Figure 2.**

*Photographs of* Diaphus *sp. captured from a lateral (upper) and ventral view (middle). Displaying the photophores that produce a blue luminescent light (lower). Photographs taken by Yuichi Oba.*

Support for a dietary origin for luciferin in a number of stomiids is supported by their ability to uptake other small molecules to utilise in light emission. An example of this is shown in several species of loose-jaw dragonfish (*Malacosteus* spp.), that have a rare ability to emit longer wavelengths of luminescence that is red in colour, as opposed to blue light which is more ubiquitous in the oceans [1]. *Malacosteus* can also detect red wavelengths of light using a distinct mechanism requiring derivatives of bacteriochlorophylls *c* and *d* that enhance its sensitivity to these longer wavelengths [66]. As vertebrates are unable to synthesise chlorophyll, *Malacosteus* could obtain this through a diet, predominantly of grazers such as copepods that will contain phytoplankton derived pigments in their guts [64]. This strongly supports the concept that other small organic compounds such as luciferins can be taken up by dragonfishes, as well as other Stomiiformes to utilise in their bioluminescent reactions.

#### **3.2 Other Coelenterazine utilising systems**

Semi-intrinsic luminescence is clearly present in several marine vertebrates that utilise either cypridinid luciferin or coelenterazine as their substrate. However, this alone does not account for the diverse array of marine phyla that use coelenterazine in their bioluminescent behaviours. Many organisms previously considered to synthesise coelenterazine have since been shown to obtain this through their diet, including in the cnidarians where this was first discovered.

#### *3.2.1 Cnidaria (Coelenterates)*

Bioluminescence within the phylum Cnidaria has been studied more than in any other marine invertebrate. Most notably the hydromedusa *A. victoria* which emit light via the enzymatic oxidation of coelenterazine in the presence of calcium [12]. Unlike most coelenterazine utilising organisms that emit blue light, in *A. victoria*, light emission is green due to a green fluorescent protein. This emits green light via resonance energy transfer from the aequorin photoprotein [67]. According to Shimomura [3], photoproteins can be distinguished from luciferases by two general means, not requiring molecular oxygen for light emission and being capable of emitting light proportional to the amount of protein present [68]. Isolated aequorin can appear to emit light only by adding Ca2+, and once the reaction is complete the protein does not appear to immediately be available for further reactions [69].

By controlling the diet of *A. victoria* in the lab it was possible to show that they are dependent on a dietary supply for their luciferin. When provided with an external source of luciferin to uptake after this, *A. victoria* was able to regain its luminescence [12]. The diet of *A. victoria* will consist of a variety of zooplankton, including luminescent copepods such as *M. longa* as well as luminescent ctenophores, which could provide a dietary source for their luminescence. Several other notable examples of luminescent coelenterates are presumed to obtain coelenterazine from their diet including the sea pansy, *Renilla* sp. and the sea cactus *Cavernularia obesa* [70, 71]. These anthozoans are found predominantly in tropical waters and may be able to obtain coelenterazine by feeding on suspended detrital matter that may contain the substrate.

#### *3.2.2 Crustacea*

Among the crustacea there is proven case of a fully intrinsic luminescent system in the copepod *Metridia pacifica*, and a probable case in the decapod shrimp *Systellaspis debilis* which appears to have the ability to synthesise the molecule from free amino acids [72]. Zooplanktonic species such as these potentially provide a source for a lot of the coelenterazine utilised in semi-intrinsic luminescent systems found in many marine organisms. However not all crustacea are able to perform this, and some such as the lophogastrid shrimp, *Neognathophausia ingens*, have been shown to require coelenterazine from their diet [31, 73].

These shrimp use bioluminescence to evade predators as they emit a brilliant blue cloud of luminescence when agitated that acts as a smoke screen [74]. Given that deep water visual predators have highly sensitive eyes, the bioluminescent ink cloud will have a much greater effect in startling nearby predators than the ink clouds produced by most cephalopods [75]. It is possible that producing this amount of luminescent material has a high energetic so it may be easier from an evolutionary perspective to obtain this through their diet instead of via an internal biosynthetic pathway.

#### *3.2.3 Radiolaria*

An assumption may be that as the majority of coelenterazine in the ocean is produced and utilised by eukaryotes, that organisms such as protists would synthesise their own source of luciferin rather than obtain it through their diets. However even protozoa such as several radiolarian species are not only capable of bioluminescence but obtain coelenterazine through their diet [1]. For example, bioluminescence has been found in several species of *Thalassicolla* and *Sphaerozoum* [29]. As protists they may appear to be unable to possess semi-intrinsic luminescence, however

these species are heterotrophic, and capable of consuming and digesting larger prey including zooplanktonic copepods [76]. As to the function of luminescence in these organisms it remains poorly understood, although given their dietary acquisition of luciferin, light emission may assist in prey attraction and capture [1].
