Elsevier

Gene

Volume 536, Issue 1, 15 February 2014, Pages 1-8
Gene

Transcriptome analysis of the couch potato (CPO) protein reveals an expression pattern associated with early development in the salmon louse Caligus rogercresseyi

https://doi.org/10.1016/j.gene.2013.11.100Get rights and content

Highlights

  • We characterize a putative CPO gene from the salmon louse C. rogercresseyi.

  • We evidence CrCPO expression pattern associated with early development.

  • Eight SNPs located in the UTRs and one located in the coding region were detected.

Abstract

The couch potato (CPO) protein is a key biomolecule involved in regulating diapause through the RNA-binding process of the peripheral and central nervous systems in insects and also recently discovered in a few crustacean species. As such, ectoparasitic copepods are interesting model species that have no evidence of developmental arrest. The present study is the first to report on the cloning of a putative CPO gene from the salmon louse Caligus rogercresseyi (CrCPO), as identified by high-throughput transcriptome sequencing. In addition, the transcription expression in larvae and adults was evaluated using quantitative real-time PCR. The CrCPO cDNA sequence showed 3261 base pairs (bp), consisting of 713 bp of 5′ UTR, 1741 bp of 3′ UTR, and an open reading frame of 807 bp encoding for 268 amino acids. The highly conserved RNA binding regions RNP2 (LFVSGL) and RNP1 (SPVGFVTF), as well the dimerization site (LEF), were also found. Furthermore, eight single nucleotide polymorphisms located in the untranslated regions and one located in the coding region were detected. Gene transcription analysis revealed that CrCPO has ubiquitous expression across larval stages and in adult individuals, with the highest expression from nauplius to copepodid stages. The present study suggests a putative biological function of CrCPO associated with the development of the nervous system in salmon lice and contributes molecular evidence for candidate genes related to host–parasite interactions.

Introduction

The couch potato (CPO) protein was first isolated and cloned from Drosophila embryos, and its expression in neuronal precursors and their daughter cells evidences its pivotal role in the development and proper biological functioning of the peripheral and central nervous systems (Bellen et al., 1992, Glasscock and Tanouye, 2005). In addition to the expression of CPO in the nervous system, this protein is also expressed in larval ring glands, suggesting a biological function likely involved with neuroendocrine activity during larval development (Harvie et al., 1998). Regarding its molecular characteristics, CPO belongs to an RNA-binding protein family that has complex structural features such as a molecular size over 100 kb that encodes for at least three transcripts with alternative splicing, a lack of the AUG initiation codon, and the probable encoding of three different proteins (Bellen et al., 1992).

The CPO gene gains its name from several partial loss-of-function alleles that cause hypoactive behavior, such as abnormal geotaxis, phototaxis, and flight behavior, in adults of Drosophila, thus evidencing a mutant genotype in insects with life strategies that involve diapause (Saunders et al., 1989). Diapause is a classic adaptation to seasonality in arthropods, and its expression can result in an extreme extension of lifespan as well as an enhanced resistance to environmental challenges. So far, experiments using quantitative trait mapping, as carried out by Schmidt et al. (2008), have revealed the RNA-binding protein encoding CPO to be major genetic locus that modulates the diapause phenotype in Drosophila melanogaster. Furthermore, sequence analysis from natural populations demonstrated that variation for the diapause phenotype is caused by a single Lys/Ile substitution, and the polymorphisms also show geographic variation according to latitudinal cline (Tyukmaeva et al., 2011).

Aquatic organisms are constantly exposed to environmental stimuli or anthropogenic pollutants. As such, diapause is of critical importance for the life history of some crustaceans, including calanoid and cylopoid copepods (Hirche, 1996, Bron et al., 2011). For instance, a prolonged dormancy phase during the life cycle of Calanus finmarchicus is a strategy, which allows it to avoid the unfavorable environmental conditions typical of the upper ocean from late summer to early spring in the subarctic North Atlantic (Carmichael et al., 2013, Olsvik et al., 2013). Furthermore, the functional genomics approach has been used to identity putative CPO homologs in C. finmarchicus (Christie et al., 2013). Mining of 454 pyrosequencing datasets and bioinformatics analyses from published databases resulted in the identification of two full-length CPO proteins and two additional putative sequences in the parasitic copepod Lernaeocera branchialis and the shrimp Penaeus monodon. According to the authors, no convincing CPO-encoding transcripts were identified for crustacean species with very large datasets, such as Litopenaeus vannamei, Daphnia pulex, and Lepeophtheirus salmonis, suggesting that CPO transcription has a low expression or is absent in some crustacean species.

The salmon louse Caligus rogercresseyi is a marine ectoparasite that is the cause of high economic losses for the salmon aquaculture industry in Chile. This parasite produces significant physiological and pathological consequences for infected fish (Bartsch et al., 2013). For instance, heavy infections lead to the erosion of the epidermis, exposure of the dermis, and in severe cases, exposure of skeletal muscle (Skilbrei et al., 2013). However, subclinical and physiological effects, which include stress, increased plasma cortisol concentrations, and changes in blood glucose, electrolyte, and plasma sex-steroid concentrations, are the more common impacts of sea lice (Saksida et al., 2013). Salmon lice infestations have been managed by antiparasitic agents such as organophosphates (Jones et al., 1992, Roth et al., 1996), pyrethroids (Sevatdal and Horsberg, 2003), hydrogen peroxide (Bravo et al., 2010), and avermectins (Duston and Cusack, 2002, Bravo et al., 2008). However, the overexposure of parasites to chemical control agents tends to promote drug resistance in wild populations (ffrench-Constant et al., 2004). In order to address this concern and that of the scarce genomic knowledge on the molecular functions affected by anti-sea louse treatments, investigations of novel candidate genes that could be related to the invertebrate nervous system are required. In this context, CPO is of interest to the current study as there is evidence related to its functional role during early developmental stages in C. finmarchicus. Interestingly, the model species C. rogercresseyi differs in comparison to other sea lice in regard to its life cycle strategy and association with its host fish and in its lack of a diapause stage.

This study is the first to report a putative CPO gene from the salmon louse C. rogercresseyi, as identified by high-throughput transcriptome sequencing. Additionally, transcription expression was performed in copepodid and chalimus larval stages and in female and male adults so as to evaluate expression patterns associated to ontogenetic development.

Section snippets

Laboratory conditions for salmon lice culturing

Ovigerous specimens of C. rogercresseyi were collected from recently harvested fish at the AquaChile salmon processing plant in Puerto Montt, Chile. Individuals were transported back to the laboratory on ice, and their egg strings were then removed and placed in culture buckets supplied with seawater flow (12 °C) and gentle aeration. Eggs were allowed to hatch and develop until the infectious copepodid stage, at which point they were harvested for RNA extraction and cDNA library construction.

Molecular characterization of CPO cDNA from C. rogercresseyi

From the in silico analysis of high-throughput transcriptome sequencing and RACE technique, a complete cDNA sequence of a putative CPO gene from the sea louse C. rogercresseyi was obtained. The molecular characteristics showed a total length of 3261 base pairs (bp), which contained an open reading frame of 807 bp encoding for 268 amino acids. Untranslated regions were also identified with lengths of 713 bp and 1741 bp for 5′ UTR and 3′ UTR, respectively. This latter sequence showed a

Discussion

Copepods are a group of small aquatic crustaceans described as the most abundant metazoans on earth, and their life histories comprise planktonic and benthic cycles with important ecological links in the marine ecosystem (Humes, 1994, Hirche, 1996), including parasitic interactions with both farmed and wild marine finfish (Krkosek, 2010, Nowak et al., 2011). Members of the family Caligidae, and especially of genera Caligus and Lepeophtheirus, are the most important parasites of the salmon

Conflict of interest

The authors declare no conflict of interest in the current study.

Acknowledgments

This study was supported by the FONDAP (15110027) project granted by CONICYT-Chile.

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