*3.2.5 Ophiuroidea*

Most species that exhibit semi-intrinsic bioluminescence acquire their luciferin via predation, most notably on luminescent copepods or on their predators. However, it is also feasible that filter feeders will be able to acquire coelenterazine and other luciferins through their diet. One such example is seen in the ophiuroids or brittle stars where many species have been shown to emit light [81, 82]. One such example is the brittle star *A. filiformis*, whose bioluminescence has been studied from a biochemical perspective for the past decade. This species feeds on suspended organic matter by extending its arms into the water column [83, 84]. Each of its arms are covered with light-emitting cells called photocytes that have been shown to be dependent on coelenterazine as a source of luciferin [81, 84, 85]. Additionally, the enzyme involved in its luminescent reaction was shown to be homologous to *Renilla* luciferase, which is a coelenterate also thought to acquire its luciferin from its diet [81, 86].

A recent study monitored *A. filiformis* kept in an aquarium for several months whilst controlling its diet [82]. Over five months a depletion in *A. filiformis'* luminescence was observed when fed a coelenterazine-free diet, strongly suggesting it acquired components for luminescence through filter feeding [82]. This was validated as there was a quick recovery in its luminescent capabilities once the brittle star was fed coelenterazine supplemented food. This animal signifies that semiintrinsic luminescent systems are not simply found among tertiary consumers. This also supports the notion that numerous other filter and detrital feeding organisms that exhibit luminescence, acquire their substrates via their diet.

#### *3.2.6 Tunicata*

While it has not fully been confirmed yet, it is possible a large number of other filter feeding marine organism can acquire luminescent components from their diet. Within the chordates the subphylum Tunicata, comprises of a number of species shown to produce luminescence, although compared with other luminescent organisms these remain poorly studied. Within the tunicates, luminescence is well represented among the appendicularians with several species being confirmed to produce luminescence. One such example within this group is the larvacean *O. dioica*, which is a free-swimming tunicate that dwells in the photic zone of the ocean [87]. The animal has transparent body and a tadpole-like appearance throughout its life cycle, ranging in size from 0.5 to 1 mm. Light emission occurs as blue flashes of light from its body that can be induced by mechanical stimulation [88]. This animal has also been reported to emit light in the presence of coelenterazine, so it is possible that these are able to acquire coelenterazine from exogenous sources [87]. Larvaceans like *O. dioica* can secrete their luminescence as a mucus that will capture and collect particulate organic matter whilst the animals are filter feeding [89]. These secretions form luminescent "houses" or clusters of organic matter which can harbour all of the components for the bioluminescent reaction. On mechanical stimulation, these "houses" emit blue light showing that the components luminescence are all present in a way such that coelenterazine does not undergo autooxidation. This display of luminescence supports coelenterazine being utilised by this and other filter feeders for semi-intrinsic luminescence as stable luciferins can potentially be found in particulate organic matter that these organisms can feed on [87, 88, 90].

Another example of luminescence in tunicates is found in pyrosomes which are pelagic tunicates known for their sustained bright blue luminescence as well as their capacity to form sporadic and yet massive blooms such as those observed in this region [91]. There is currently a lack of consensus on the origin of luminescence in this species. A recent study has shown that light emission occurs in the presence of coelenterazine for the species *Pyrosoma atlanticum* [92]. Moreover, using transcriptomic analysis, an enzymatic sequence was identified as being similar to the luciferase found in the Cnidarian *Renilla reniformis* that uses coelenterazine as its light emitter. Subsequent expression of this gene showed that light emission occurred in the presence of coelenterazine strongly supporting that this is the luciferase involved in pyrosome bioluminescence [92]. Coelenterates and some echinoderms have been shown to utilise luciferases with a similar structure to *Renilla*, and a number of these are thought to acquire coelenterazine through their diets. Therefore, it is entirely feasible that pyrosomes such as this species attain coelenterazine through filter feeding, which may also occur for various other luminescent tunicates. However, it should also be noted that recent studies have identified and characterised potentially luminescent bacterial symbionts within *P. atlanticum* [93] which supports several previous studies on this system. Determining how this organism obtains its luminescence will rely on further confirmation what the source of light emission is in this tunicate.

#### *3.2.7 Mollusca*

Like previously mentioned phyla, some luminescent molluscs are able to acquire coelenterazine through their diet. This includes the clam *Pholas dactylus*, as well as several species of squid that have been shown to possess coelenterazine in their livers [94]. However, these animals do not use coelenterazine directly as their source of luciferin for bioluminescence. Instead, they use modified forms of this substrate, for example the firefly squid utilises a disulphate form of coelenterazine

#### **Figure 3.**

*Photograph of* Watasenia scintillans *taken under natural light (upper) and in a dark room (lower) showing the luminescent photophores along its body. Photographs taken by Yuichi Oba.*

in its luminescent reaction [95]. These produce a dim continuous blue bioluminescence from ventral photophores, as well as a bright blue flash of luminescence (470 nm) from light organs on its arm tips after being mechanically stimulated [96]. The flashing ability may be used as a means of intra-specific communication and recognition although this has not yet been defined. The enzymatic oxidation of coelenterazine disulphate [luciferin] in the presence of Mg2+ has led to emissions of blue light, however how or why obtained coelenterazine is modified remains undetermined [95, 97].

Another derivative found in several molluscs is dehydrocoelenterazine. This is an oxidised form of coelenterazine and was identified as the luciferin required in the luminescence of the clam *P. dactylus*, the purple back flying squid *Sthenoteuthis oualaniensis* and recently the Humboldt squid *Dosidicus gigas* [98]. In *D. gigas*, a blue bioluminescent light is emitted from an array of photophores on their body [39]. These structures are small, ovoid rice-like granules that are embedded in the muscle all over the squid on the mantle, fins, head, arms and tentacles [99]. It is entirely possible that this and other squids can obtain coelenterazine from lanternfishes which they are known to predate on. This coelenterazine may undergo an enzymatic oxidation to form dehydrocoelenterazine which is then utilised in its light emission (**Figure 3**).

#### **3.3 Non imidazopyrazinone substrates**

All examples of semi-intrinsic luminescence so far have involved either coelenterazine or cypridinid luciferin as the substrate. Dinoflagellate luciferin has also been shown be required by several heterotrophic organisms that appear to not be able to synthesise this luciferin. Dinoflagellates are unicellular organisms that account for the majority of bioluminescence observed in the surface ocean [100, 101]. The compounds involved with luminescence are regulated on a diurnal circadian rhythm, along with photosynthetic components. This means that dinoflagellates conduct primary production during the day and only produce bioluminescence at night, when this would be most effective. The structure of this luciferin was originally determined from *Pyrocystis lunula*. The compound is a linear tetrapyrrole which is very sensitive to non-enzymatic oxidation and is most likely to have derived from chlorophyll [102]. Within different species of dinoflagellates there is variation in the intensity and duration of light emission but in general light is emitted from organelles known as scintillons [101].

Dinoflagellate luciferin shows no similarities to other luciferins and is found in forms, one within dinoflagellates and another with two hydroxyl moieties in euphausiids (krill). This similarity suggests that there is some form of dietary link [102, 103]. Studies have shown luminescent euphausiids occurred in high densities which coincided with large populations of dinoflagellates during late spring [104]. Additionally, heterotrophic species of dinoflagellate, such as *Noctiluca scintillans* have been shown to feed on luminescent dinoflagellates such as *P. lunula*. When their diet was controlled in the lab to exclude luminescent dinoflagellates and all other phytoplankton, they were shown to lose their capacity to emit light [101]. Moreover, when fed other non-dinoflagellate phytoplankton, luminescence was maintained, suggesting that *N. scintillans* can synthesise the tetrapyrrole luciferin from chlorophyll [105]. These examples suggest other luciferins and their precursors may be taken up in the diets and utilised by consumers that already express the required luciferases for other non-imidazopyrazinone luciferins.

#### **4. "Kleptoprotein" luminescence**

A general consensus among semi-intrinsic luminescent systems is that the components of the light emission utilised by other organisms are the substrates rather than enzymes. As most of these animals acquire luminescence through their diets, any exogenous components would need to be able to withstand digestion and

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

potentially transport through the blood plasma to the luminogenic organs. Given this it seems unlikely that the enzymatic component of luminescence would be able to be obtained in this manner, as they would likely be denatured and completely broken-down during digestion [13].

However, a recent study on the *Parapriacanthus* fish, has shown that it is able to obtain both its luciferin and luciferase from its prey. Like midshipman fish, *Parapriacanthus ransonneti* predates on ostracods, which provide a source of cypridinid luciferin that is used in its light emission [13]. When *P. ransonneti* was fed on the ostracod *Cypridina noctiluca,* the luciferase identified from its light organs was identical to the luciferase of this species. When a different species of luminescent ostracod, *Vargula hilgendorfii* was identified in another individual fish, the identified luciferase was now the same as this ostracod, demonstrating the ability to specifically uptake luciferases from its diet to the fish's light organs [13]. Transcriptomic analysis of *P. ransonneti*, showed no transcripts corresponding to an ostracod-type luciferase, further highlighting that this was acquired via the diet (**Figure 4**).

This is the first reporting of this type of phenomenon in bioluminescence, and up until now it was assumed that any consumed luciferase enzyme would be broken down into amino acids or oligopeptides before being absorbed via the gut wall as nutrients [13]. However, the possibility of protein uptake without being fully broken down and retaining activity has been reported in several vertebrate immune systems. An example of this is seen in M cells within the mammalian intestinal epithelia as these have an important role in the immune system by transporting macromolecules and microbes into the cell via pinocytosis [106]. Similar examples of this have been observed in cyprinid fishes so it is feasible these or similar structures could facilitate the transfer of ostracod luciferase to the photophores of this animal [13].

This example of a "kleptoprotein" form of luminescence where both the substrate and the enzyme are provided through the diet, provides an additional novel category of luminescent reactions, as of yet not considered. Moreover, this highlights the possibility that other luminescent species may utilise this capability to obtain active exogenous luciferase from their gut. Potentially, this may include several species of fishes that predate on ostracods, whose light organs are often connected to their digestive tracts. This research may suggest that semi-intrinsic and "kleptoprotein" luminescent behaviours may be more widespread than previously considered, with proteins associated with other biological processes potentially being able to be attained via diet as opposed to gene expression.

#### **Figure 4.**

*Ventral view of* Parapriacanthus ransonneti *taken in a dark room to capture the light emission from these body regions. Photos by Okinawa Commemorative National Government Park (Ocean Expo Park), Okinawa Churaumi Aquarium.*

### **5. Why semi-intrinsic luminescence occurs?**

Semi-intrinsic luminescence has been shown to exist in a number of organisms and is hypothesised to exist in several others. Cypridinid luciferin and dinoflagellate luciferin have been shown to be taken up by predators of ostracods and dinoflagellates respectively, notably several species of fishes, and euphausiid shrimp. However, the majority of semi-intrinsic luminescence, in addition to the majority of bioluminescence in the oceans involves using coelenterazine. Dietary uptake of coelenterazine has been shown in coelenterates, echinoderms, and decapod shrimp, while it is also strongly supported to be the source of luciferin in myctophid and stomiid fishes, chaetognaths, tunicates and several species of squid. Moreover, coelenterazine can be modified via oxidation or di-sulfonation, once it is taken up by species, allowing for a variety of different light reaction mechanisms to occur with this molecule. It is important to understand why some animals use semi-intrinsic luminescence, and the potential evolutionary origins of this, and how coelenterazine may spread across the food web and be the most common light emission system in the oceans. It is useful to consider whether this phenomenon along with "kleptoprotein" luminescence is a lot more widespread in other biological processes and systems.

There are two main groups of hypotheses on why bioluminescence evolved originally; one based around changes in the luciferin (substrate-centric hypothesis) [5, 107] and another that suggests changes occurred in what became the luciferase enzyme (enzyme-centric hypothesis) [108]. The first hypothesis suggests that the luciferin substrate evolved in order to protect organisms from reactive oxidative species (*e.g*., hydrogen peroxide) in the water column [108]. Luminescent animal migrated to deeper water to evade visual predators and at these depths there was no longer significant oxidative stress. Therefore, the active selection pressure switched to the luminescent, communicative properties of luciferins, leading to more specific adaptations to predation, survival, and communication [1].

The alternate hypothesis focuses on the enzyme luciferase and that these molecules were originally less specific oxygenase enzymes [108]. The oxygenase enzymes mutated as a result of animals migrating to deeper waters to either evade visual predators, or to predate on organisms that have migrated to deeper water [5]. The mutation in oxygenase enzymes associated with display functions would result in external luminescence being exhibited [109]. These display pigments would previously have been associated with warning colourations or patterns to both recognise species and attract potential mates. There is evidence for enzyme-based hypotheses in terms of enhancement of visual signals [5]. However, there is no biochemical or genetic evidence that would support this hypothesis, and the mutation of the luciferase enzyme alone would not explain the convergent evolution of the bioluminescent reaction in multiple phyla [1, 5].

Whether one or a combination of both hypotheses are more viable for the origins of luminescence, both allow for the possible co-evolution of predators and prey that may utilise the same source of luminescence. Convergent evolution caused by environmental factors may have allowed for the presence of various enzymes that were compatible with the same substrate resulting in coelenterazine being utilised by both animals that can synthesise it as well as their predators. Moreover, given the energetic costs associated with synthesising luciferins, it may simply be more efficient for some of these organisms to acquire exogenous sources instead.

Semi-intrinsic luminescent organisms, particularly those that harbour coelenterazine, have shown the potential spread and dispersal across the food web for not just luciferins, but other molecules that may be involved in biological processes. A major source of coelenterazine is found in the copepod *M. pacifica* which is grazed

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

upon by a variety of organisms including coelenterates, lanternfishes, euphausiids, and radiolarians. Additionally, these animals, particularly lanternfishes are predated upon by tertiary consumers such as squid, stomiid fishes and luminescent sharks [110]. The consumption of copepods by zooplankton and higher taxa can lead to particulate organic matter or marine snow forming and descending to the depths of the ocean. These aggregates will contain detritus, plankton and larvacean houses, meaning that it is highly likely for free-available coelenterazine to be present. The coelenterazine within this particulate organic matter can then be taken up by filter feeders such as echinoderms and tunicates, allowing for them to utilise coelenterazine in their luminescent displays.

In a number of these organisms, luciferin has been identified in a sulfonated form. The most notable example of this is in the firefly squid, however sulfonated luciferins have been identified in *V. hilgendorfii* and *Renilla reniformis* [3]. This form is more stable than free forms of coelenterazine, and it is possible this is a stored form of luciferin that may prevent auto-oxidation that can occur. This more stable form may prevent breakdown and oxidation of the substrate when it is in the water column or during digestion. Potentially, a lot of these semi-intrinsic luminescent organisms will obtain their luciferins in this form, and then have the capability to de-sulfonate the luciferin to make it available for luminescence.
