**Abstract**

Light emission is widespread in the oceans, with over three quarters of all observed marine species exhibiting bioluminescence. Several organisms such as the copepod *Metridia pacifica* and the ostracod *Vargula hilgendorfii* have been proven to synthesise their luciferin and luciferase to facilitate light emission. However, many luminescent species lack the capability to do this and instead it is possible that they acquire some of the components for their luminescence through predation or filter feeding on organisms that produce luciferins or precursors to these molecules. This has resulted in many organisms using certain luciferins, such as coelenterazine, as their substrate without possessing a clear mechanism to synthesise these. This chapter will review several examples of these semi-intrinsic luminescent systems and how the substrates and enzymes can be obtained for these reactions. Moreover, it will look at why particular luciferins, such as coelenterazine, are more widespread and utilised in this manner compared to other substrates.

**Keywords:** Bioluminescence, Semi-Intrinsic, Luciferin, Coelenterazine, Imidazopyrazinone

### **1. Introduction**

Bioluminescence is a chemical process numerous organisms utilise to produce light. This reaction has been studied in a wide range of taxa, in terms of its chemistry, evolutionary history and purpose in ecology [1]. This ability to emit light via a chemical reaction can be found in a diverse range of phyla, ranging from simple unicellular bacteria and protists to more complex organisms such as cephalopods and elasmobranchs [1]. Generally, this is a chemical reaction that involves the oxidation of a luciferin compound in the presence of a luciferase enzyme. This produces an unstable intermediate (usually a cyclic peroxide) that breaks down to produce a compound generically called oxyluciferin and gives off a large amount of energy as light [2, 3].

This phenomenon has evolved independently at least 94 and potentially over 100 times [4] across both marine and terrestrial genera, and around 80% of bioluminescent genera occur in the oceans [5, 6]. In marine ecosystems, it is estimated that up to 95% of organisms that dwell below 200 m depth are able to emit light [7–9]. Given the widespread utilisation of this phenomenon, there are a diverse array of luminescent systems that exist with several different substrates and a wide variety of associated enzymes.

Unlike the enzymatic component of the reaction where individual species are capable of expressing unique enzymes, luciferins are more conserved, and the same structures can be found across multiple distinct phyla. As of now at least 10 natural

luciferins have been identified in terms of their chemical structure [4, 10]. Of those, the four main marine groups of luciferins are bacterial luciferin, tetrapyrrole used by dinoflagellates and krill, cypridinid luciferin used by several species of fish and ostracods and coelenterazine which is used by luminescent organisms in at least 9 different phyla [11].

Despite being a critical component for light emission, many marine organisms do not produce their own luciferins, and obtain these small organic compounds from their diet by grazing or predating on other luminescent organisms [1]. These species exhibit semi-intrinsic luminescence, as they still express their own luciferase enzymes, however they can obtain the substrates and potentially precursors to luciferin needed for luminescence through their diets [12]. Some have even shown the capacity to obtain the enzymatic component of the luminescent reaction through their diet as well [13]. With regards to this phenomenon the most notable examples of semi-intrinsic luminescence involve coelenterazine and cypridinid luciferin [14].

This chapter will review the prevalence of known semi-intrinsic luminescent systems and how these organisms have attained light emission. Moreover, it will look at why these reactions and predator–prey relationships have evolved over time and discuss why certain substrates are more commonly observed in semi-intrinsic luminescence.

#### **2. Sources of luminescence in semi-intrinsic systems**

Identifying the presence of luminescence in an organism is well established and involves identifying the luciferin and luciferase involved in the reaction and separating them. The basic technique for luciferin and luciferase separation, developed by Dubois [15, 16] is termed "hot-cold extract". In this method, two water extracts of luminogenic tissue are prepared [3, 16]. The use of cold extract allows to preserve the activity of the enzyme (luciferase), while the heated fraction destroys the proteins and yields the luciferin, and when both extracts are mixed together an *in vitro* luminescence is produced [3, 16]. Each extract can be purified to allow for the identification of the amino acid sequence corresponding to the luciferase and the chemical structure of the luciferin [3, 17].

However, this in of itself does not establish how the luminescent organism obtained these components. A possible method to identify this is by constructing the transcriptome of an organism to prove the luciferase enzyme was expressed and not obtained through diet [3, 18]. However, this is a lot more difficult when it comes to identifying whether an organism can synthesise its own luciferin, as very few biosynthetic pathways have been established.

Despite this, it has been shown by controlling the diet of a number of higher taxa that their luminescence is dependent on the consumption of particular organisms [12, 19]. Subsequently, it has been possible to identify several organisms at lower trophic levels that can produce their own luciferin, including the ostracod *Vargula hilgendorfii* [20] and the copepod *Metridia pacifica* [14], both shown in **Figure 1**.

#### **2.1 Cypridinid luciferin**

Cypridinid luciferin was the first marine luminescent substrate to be identified in terms of its chemical structure. This compound was first isolated and crystallised by Shimomura and colleagues [21, 22], and the structure was determined by Kishi et al. [23], allowing for the detailed study of the biochemistry of this reaction [1]. The ostracod *V. hilgendorfii* was shown to secrete a luminescent mucus when disturbed, emitting a bright blue light at a peak wavelength of 453–455 nm [24]. The *Semi-Intrinsic Luminescence in Marine Organisms DOI: http://dx.doi.org/10.5772/intechopen.99369*

#### **Figure 1.**

*Photographs of luminescent organisms known to synthesise their luciferins. The ostracod* Vargula hilgendorfii *(upper) synthesises cypridinid luciferin and the copepod* Metridia pacifica *(lower) synthesises coelenterazine. Photos taken by ken-ichi Onodera, and Yuichi Oba.*

luminescent cloud of mucus is emitted from specialised glands from two types of cell, one producing the luciferin and the other the luciferase [25].

Kato and colleagues [26, 27] showed that ostracod luciferin is synthesised from tryptophan, isoleucine, and arginine, via a currently unknown pathway. This was

observed by labelling the amino acid L-tryptophan with deuterium before feeding the ostracod *V*. *hilgendorfii* with this to confirm incorporation into the cypridinid luciferin [20]. *V*. *hilgendorfii* was shown to be the first example of a species that could use free amino acids to synthesise its imidazopyrazinone-type substrate, cypridinid luciferin. While this is used by several bioluminescent species, it makes up a small component of total systems in marine environments [20, 28].

#### **2.2 Coelenterazine**

The majority of luminescent organisms in marine environments with known or partially studied light emission systems utilise coelenterazine. Coelenterazine is an imidazopyrazinone compound (3,7-dihydroimidazopyrazin-3-one structure) that occurs exclusively in marine organisms in a wider range of phyla (at least nine) than any other luciferin [4]. These include radiolarians, ctenophores, cnidarians, molluscs, multiple arthropods, and some fish [29]. A large proportion of these organisms are assumed to have taken up this luciferin through their diet with only a few organisms shown to synthesise their own substrate [30–32]. The coelenterazine molecule was originally given its name due to the initial discovery of its presence in coelenterates, namely *A. victoria* and *Renilla reniformis* [33]. *A. victoria* is a hydrozoan jellyfish that emits a green light at 508 nm from a ring of photocytes on the peripheral regions of its umbrella [3]. Variants of this substrate exist in several species of squid either as a coelenterazine disulphate [34] or as dehydrocoelenterazine [35, 36].

Whilst coelenterazine has been found in a diverse array of phyla, a biosynthetic pathway and origin has not yet been determined for the majority of species, which are thought to obtain coelenterazine through their diet [12]. Coelenterazine has been shown to be synthesised in the deep-sea copepod, *Metridia pacifica,* via a similar mechanism to that observed for cypridinid luciferin in *Vargula hilgendorfii* wherein free amino acids are biosynthesised to form the coelenterazine luciferin [20, 26]. By labelling L-tyrosine and L-phenylalanine with deuterium it was proven that *M. pacifica* was able to incorporate these amino acids into its diet and that it was able to synthesise coelenterazine from two molecules of L-tyrosine and one molecule of L-phenylalanine [14]. Given that *M. pacifica* is at a lower trophic level it is likely to be predated upon by several higher taxa, many of which exhibit their own luminescent reactions [14, 37].

Recently it has been proposed that luminescent ctenophores are also able to produce their own luminescent components. The phylum Ctenophora or comb jellies are similar to the coelenterates in their morphology and apart from the family Pleurobrachiidae, all are presumed to be luminescent [38]. Ctenophores had previously been considered to be a source of coelenterazine synthesis in the oceans as there are reports of bioluminescence at early developmental stages [39]. When fed a coelenterazine-free non-luminescent diet, ctenophores were still shown to possess this substrate via mass spectrometry [40]. This recent study has implications that a number of other marine organisms, in addition to *M. pacifica* and Ctenophora, have the capacity to synthesise luciferin, which can provide a clear source of coelenterazine for a number of semi-intrinsic luminescent organisms.

#### **3. Semi-intrinsic luminescent systems**

#### **3.1 Luminescence in fish**

Most notable semi-intrinsic luminescence occurs in higher trophic levels such as among fishes. Several species have been shown to utilise the imidazopyrazinone type substrates cypridinid luciferin and coelenterazine in luminescent reactions [1, 3, 41], though they are shown to express their own luciferase enzymes [6]. Often these have evolved to harbour luminescence in specialised regions of the body that allow for particular behaviours and functions for luminescence [1, 42].
