*Article* **First Report of OvoA Gene in Marine Arthropods: A New Candidate Stress Biomarker in Copepods**

**Vittoria Roncalli 1,\*, Chiara Lauritano 2,† and Ylenia Carotenuto 1,†**


**Abstract:** Ovothiol is one of the most powerful antioxidants acting in marine organisms as a defense against oxidative stress during development and in response to environmental cues. The gene involved in the ovothiol biosynthesis, OvoA, is found in almost all metazoans, but open questions existed on its presence among arthropods. Here, using an in silico workflow, we report a single OvoA gene in marine arthropods including copepods, decapods, and amphipods. Phylogenetic analyses indicated that OvoA from marine arthropods separated from the other marine phyla (e.g., Porifera, Mollusca) and divided into two separate branches, suggesting a possible divergence through evolution. In the copepod *Calanus finmarchicus*, we suggest that OvoA has a defense role in oxidative stress as shown by its high expression in response to a toxic diet and during the copepodite stage, a developmental stage that includes significant morphological changes. Overall, the results of our study open possibilities for the use of OvoA as a biomarker of stress in copepods and possibly also for other marine holozooplankters. The finding of OvoA in copepods is also promising for the drug discovery field, suggesting the possibility of using copepods as a new source of bioactive compounds to be tested in the marine biotechnological sector.

**Keywords:** zooplankton; natural products; antioxidant; transcriptome mining

#### **1. Introduction**

Ovothiols are low molecular weight thiol-containing methylated amino acids with unique antioxidant properties that are broadly distributed among invertebrates, microalgae, protozoans, and bacteria [1]. Playing a key role in the maintenance of cellular redox homeostasis, ovothiols allow the organism to overcome environmental stress conditions. In marine organisms, ovothiols play a key role also during development as suggested by their antioxidant activity during oxidative stress at fertilization and larval development in the sea urchin [2], and during gametogenesis in the mollusc *Mytilus galloprovincialis* collected from polluted sites [3]. Ovothiols also act as a defense against the immune system of host cells during parasite infections [4,5], and as a protective compound in the mucus of Polychaeta [6]. These molecules have also been suggested as signaling molecules released in the urine of cephalopods [6], in pathways induced by light in microalgae [7,8], and as pheromones in marine worms and cone snails [9].

Recent studies showed new ovothiol bioactivities, highlighting interesting possible applications of this antioxidant in the pharmaceutical sector. Ovothiol A isolated from the sea urchin *Paracentrotus lividus* oocytes, reduced the cell viability of the human liver carcinoma cell line (Hep-G2) by activating autophagy [10]. Additional possible ovothiol A antiatherogenic activities have been found by cell-based assays suggesting its application for cardiovascular diseases associated with oxidative and inflammatory stress, as well as

**Citation:** Roncalli, V.; Lauritano, C.; Carotenuto, Y. First Report of OvoA Gene in Marine Arthropods: A New Candidate Stress Biomarker in Copepods. *Mar. Drugs* **2021**, *19*, 647. https://doi.org/10.3390/md19110647

Academic Editors: Angelo Fontana, Donatella Degl'Innocenti, Marzia Vasarri and Bill J. Baker

Received: 13 October 2021 Accepted: 19 November 2021 Published: 20 November 2021

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endothelial dysfunction [11]. In addition, in an in vivo study in mice ovothiol A showed activity against liver fibrosis progression [12].

The ovothiol biosynthetic pathway includes three enzymatic steps in which OvoA is the key enzyme with a bifunctional role. First, the OvoA enzyme, 5-histidylcysteine sulfoxide synthase, catalyzes the addition of the cysteine sulfur group into histidine to produce an intermediate; subsequently the intermediate is cleaved by sulfoxide b-lyase (OvoB) into thiohistidine that is finally methylated to ovothiol (π-N-methyl-5-thiohistidine) by OvoA. This final step is specific of the S-adenosylmethionine (SAM) methyltransferase domain situated in the C-terminal of the enzyme. OvoA also contains an N-terminal DNA damage-inducible (DinB) superfamily domain and a formylglycine-generating sulfatase (FGE-sulfatase) domain that contains the recognition/binding sites for the substrates (cysteine and histidine) [13] (Figure 1). The methyltransferases that can methylate the a-amino group of ovothiol A to form ovothiol B and C are not yet known.

**Figure 1.** Ovothiol A biosynthetic pathway. Schematic representation of Ovothiol A pathway which consists of three steps (1–3) catalyzed by two enzymes (in bold). (1) OvoA enzyme (5-histidylcysteine sulfoxide synthase) catalyzes the addition of the cysteine sulfur group into histidine to produce an intermediate (not shown); (2) The intermediate (not shown) is cleaved by sulfoxide β lyase (OvoB) into thiohistidine which is then (3) methylated by OvoA to ovothiol (π-N-methyl-5-thiohistidine). Chemical structures were downloaded from the National Center for Biotechnology Information (NCBI) PubChem database. Histidine: PubChem Identifier CID: 6274, https://pubchem.ncbi.nlm. nih.gov/compound/Histidine, accessed on the 3 November 2021; Cysteine: PubChem Identifier CID: 6419722, https://pubchem.ncbi.nlm.nih.gov/compound/Cysteine, accessed on the 3 November 2021; OvothiolA: PubChem Identifier CID: 130131 https://pubchem.ncbi.nlm.nih.gov/compound/ Ovothiol-A, accessed on the 3 November 2021.

Braunshausen and Seebeck [14] were the first to characterize the OvoA gene from the bacterium *Erwinia tasmaniensis* and the protozoan *Trypanosoma cruzi*. They also found homologous OvoA enzymes in more than 80 genomes ranging from proteobacteria to uniand multicellular eukaryotes [14]. From a phylogenetic point of view, OvoA has been reported in many metazoans including Porifera, Emichordata, and Placozoa. Through evolution, the gene was lost twice, once in the common ancestor of nematodes and arthropods and once in the ancestor of Osteichthyes fish [15]. In freshwater fish, ovothiol A has been identified in the metabolites of the lens and other tissues as well as in the eggs, suggesting that these organisms might not have the gene, but are able to acquire the metabolite through their diet [16,17]. In contrast, still little is known of arthropods; it has been suggested that the lack of OvoA in most terrestrial species (e.g., insects) could be related to a specific role of ovothiol in the transition from the aquatic to the terrestrial environment [1,15]. Recently, Brancaccio and coauthors [18] conducted a genomic and metagenomics data mining to investigate the distribution and diversification of the enzymes involved in ovothiol biosynthesis in bacteria. They observed a horizontal gene transfer event of OvoB from Bacteroidetes living in symbiosis with Hydrozoa and suggested that the evolution of ovothiol biosynthesis may have involved symbiosis processes [18]. Overall, from all these studies, it

is clear that ovothiol A is an important antioxidant as it is conserved in many metazoans; however, the studies highlight the need to better investigate the presence of OvoA gene in other phyla.

Gene expression changes of OvoA have been reported in *P. lividus* during development and when exposed to stress conditions. Relative expression of OvoA was high in eggs and decreased immediately after fertilization, remaining low in the early developmental stages (early and the swimming blastula) with a final significant increase in the last larval stage (pluteus) [2]. A significant increase in the expression of OvoA has also been reported in larvae exposed algae and to the metals Cd and Mn [2,19]. Overall, these results suggested that in *P. lividus* ovothiol may act as a protective compound against environmental stressors, and/or as a regulation factor during development.

The aim of this study was to explore the occurrence and diversity of the OvoA gene in marine arthropods. Since the OvoB gene has not been identified in metazoan genomes or transcriptomes, except hydrozoans [1,18], we focused our investigation on OvoA. Using a well-established in silico workflow we mined the new publicly available transcriptomic resources for copepods, expanding the searches also to malacostraca. Copepods are an important component in most trophic marine food webs [20–22]. As part of zooplankton, those tiny crustaceans live in highly variable environments and are constantly subject to natural and anthropogenic-related stressful conditions that might compromise their cellular redox homeostasis [23–27]. However, in these herbivorous consumers, still little is known on which genes are activated during detoxification and which genes are responsible of antioxidant production. Given the high antioxidant properties of ovothiol in many marine organisms, we decided to examine the occurrence of OvoA gene in copepods. Using an in silico workflow, we identified OvoA transcripts in copepods but also in other marine arthropods. The identified OvoA transcripts were used in a phylogenetic analysis to support their annotation and to investigate their relationship to other marine metazoans. Lastly, using previous RNA-Seq-based studies, the expression of OvoA across development and after feeding on toxic phytoplankton species was investigated in two crustacean copepods, to evaluate the potential role of ovothiol as protective antioxidant in these holozooplankters.

## **2. Results**
