Transcriptome analysis of the couch potato (CPO) protein reveals an expression pattern associated with early development in the salmon louse Caligus rogercresseyi (original) (raw)
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Couch potato (CPO) is an RNA-binding protein involved in the regulation of nervous system development and adult diapause in insects. Within insects, this protein is highly conserved, yet it has not been identified in another large arthropod group, the Crustacea. Here, functional genomics was used to identify putative CPO homologs in the copepod Calanus finmarchicus, a planktonic crustacean that undergoes seasonal diapause. In silico mining of expressed sequence tag (EST) and 454 pyrosequencing data resulted in the identification of two full-length CPO proteins, each 205 amino acids long. The two C. finmarchicus CPOs (Calfi-CPO I and II) are identical in sequence with the exception of three amino acids, and are predicted to possess a single RNA recognition motif (RRM). Sequence comparison of the two Calfi-CPOs with those of insects shows high levels of amino acid conservation , particularly in their RRMs. Using the C. finmarchicus sequences as queries, ESTs encoding partial CPOs were identified from two other crustaceans, the parasitic copepod Lernaeocera branchialis and shrimp Penaeus monodon. Surprisingly, no convincing CPO-encoding transcripts were identified from crustacean species with very large (>100,000) EST datasets (e.g. Litopenaeus vannamei, Daphnia pulex and Lepeophtheirus salmonis), suggesting that CPO transcript/protein may be expressed at very low levels or absent in some crustaceans. RNA-Seq data suggested stage-specific expression of CPO in C. finmarchicus, with few transcripts present in eggs (which represent mixed embryonic stages) and adults, and high levels in nauplii and copepodites; stages exhibiting high CPO expression are consistent with a role for it in neuronal development.
PLoS ONE, 2014
Despite the economic and environmental impacts that sea lice infestations have on salmon farming worldwide, genomic data generated by high-throughput transcriptome sequencing for different developmental stages, sexes, and strains of sea lice is still limited or unknown. In this study, RNA-seq analysis was performed using de novo transcriptome assembly as a reference for evidenced transcriptional changes from six developmental stages of the salmon louse Caligus rogercresseyi. EST-datasets were generated from the nauplius I, nauplius II, copepodid and chalimus stages and from female and male adults using MiSeq Illumina sequencing. A total of 151,788,682 transcripts were yielded, which were assembled into 83,444 high quality contigs and subsequently annotated into roughly 24,000 genes based on known proteins. To identify differential transcription patterns among salmon louse stages, cluster analyses were performed using normalized gene expression values. Herein, four clusters were differentially expressed between nauplius I-II and copepodid stages (604 transcripts), five clusters between copepodid and chalimus stages (2,426 transcripts), and six clusters between female and male adults (2,478 transcripts). Gene ontology analysis revealed that the nauplius I-II, copepodid and chalimus stages are mainly annotated to aminoacid transfer/repair/breakdown, metabolism, molting cycle, and nervous system development. Additionally, genes showing differential transcription in female and male adults were highly related to cytoskeletal and contractile elements, reproduction, cell development, morphogenesis, and transcription-translation processes. The data presented in this study provides the most comprehensive transcriptome resource available for C. rogercresseyi, which should be used for future genomic studies linked to host-parasite interactions.
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 2014
The salmon louse Caligus rogercresseyi is the dominant ectoparasite species affecting the salmon aquaculture industry in the Southern hemisphere, and it is currently the main cause for economic losses in Chilean aquaculture. However, despite the great concern over Caligus infestations, genomic information on this louse is still scarce, even while the need to develop high-resolution molecular markers is growing. This study provides the first deep transcriptome survey to identify thousands of SNP markers from C. rogercresseyi, with a total of 69,466 SNPs identified using the MiSeq platform (Illumina®), 30,605 (52%) of which were found in contigs successfully annotated against known protein databases. Furthermore, in silico gene expression profiles associated with SNP variants were evaluated, and the results evidenced a wide array of genes that were down-and upregulated throughout the developmental stages of C. rogercresseyi. Interestingly, putative KEGG pathways involved in resistance to antiparasitic agents were also identified, where ten pathways were associated with the nervous system and one was related to ABC transporters. Taken together, this information could be highly useful for investigating the molecular underpinnings involved in the susceptibility or resistance of salmon lice to chemical treatments.
Journal of Fish Diseases, 2018
The salmon louse (Lepeophtheirus salmonis), an ectoparasitic copepod on salmonids, has become a major threat for the aquaculture industry. In search for new drugs and vaccines, transcriptome analysis is increasingly used to find differently regulated genes and pathways in response to treatment. However, the underlying gene expression changes going along with developmental processes could confound such analyses. The life cycle of L. salmonis consists of eight stages divided by moults. The developmental rate of salmon lice on the host is not uniform. Individual-and sex-related differences are found leading to individuals of unlike developmental status at same sampling time point after infection. In this study, we analyse L. salmonis from a time series by RNA sequencing applying a method of separating individuals of different instar age independent of sampling time point. Lice of four stages divided into up to four age groups within the stage were analysed in triplicate (total of 66 samples). Gene expression analysis shows that the method for sorting individuals was successful. Many genes show cyclic expression patterns over the moulting cycles. Overall gene expression differs more between lice of different age within the same stage than between lice of different stage but same instar age.
BMC Genomics, 2015
Background: Nuclear receptors have crucial roles in all metazoan animals as regulators of gene transcription. A wide range of studies have elucidated molecular and biological significance of nuclear receptors but there are still a large number of animals where the knowledge is very limited. In the present study we have identified an RXR type of nuclear receptor in the salmon louse (Lepeophtheirus salmonis) (i.e. LsRXR). RXR is one of the two partners of the Ecdysteroid receptor in arthropods, the receptor for the main molting hormone 20-hydroxyecdysone (E20) with a wide array of effects in arthropods. Results: Five different LsRXR transcripts were identified by RACE showing large differences in domain structure. The largest isoforms contained complete DNA binding domain (DBD) and ligand binding domain (LBD), whereas some variants had incomplete or no DBD. LsRXR is transcribed in several tissues in the salmon louse including ovary, subcuticular tissue, intestine and glands. By using Q-PCR it is evident that the LsRXR mRNA levels vary throughout the L. salmonis life cycle. We also show that the truncated LsRXR transcript comprise about 50% in all examined samples. We used RNAi to knock-down the transcription in adult reproducing female lice. This resulted in close to zero viable offspring. We also assessed the LsRXR RNAi effects using a L. salmonis microarray and saw significant effects on transcription in the female lice. Transcription of the major yolk proteins was strongly reduced by knock-down of LsRXR. Genes involved in lipid metabolism and transport were also down regulated. Furthermore, different types of growth processes were up regulated and many cuticle proteins were present in this group.
2011
Background: Lepeophtheirus salmonis is an ectoparasitic copepod feeding on skin, mucus and blood from salmonid hosts. Initial analysis of EST sequences from pre adult and adult stages of L. salmonis revealed a large proportion of novel transcripts. In order to link unknown transcripts to biological functions we have combined EST sequencing and microarray analysis to characterize female salmon louse transcriptomes during post molting maturation and egg production. Results: EST sequence analysis shows that 43% of the ESTs have no significant hits in GenBank. Sequenced ESTs assembled into 556 contigs and 1614 singletons and whenever homologous genes were identified no clear correlation with homologous genes from any specific animal group was evident. Sequence comparison of 27 L. salmonis proteins with homologous proteins in humans, zebrafish, insects and crustaceans revealed an almost identical sequence identity with all species. Microarray analysis of maturing female adult salmon lice revealed two major transcription patterns; up-regulation during the final molting followed by down regulation and female specific up regulation during post molting growth and egg production. For a third minor group of ESTs transcription decreased during molting from pre-adult II to immature adults. Genes regulated during molting typically gave hits with cuticula proteins whilst transcripts up regulated during post molting growth were female specific, including two vitellogenins. Conclusion: The copepod L.salmonis contains high a level of novel genes. Among analyzed L.salmonis proteins, sequence identities with homologous proteins in crustaceans are no higher than to homologous proteins in humans. Three distinct processes, molting, post molting growth and egg production correlate with transcriptional regulation of three groups of transcripts; two including genes related to growth, one including genes related to egg production. The function of the regulated transcripts is discussed in relation to post molting morphological changes in adult female salmon louse. There is clear evidence that transcription of the major yolk proteins is not induced before some of the post molting growth of abdomen and the genital segment has occurred. A hallmark for the observed growth is transcription of many putative cuticula proteins prior to the size increase.
Agri Gene
Small RNA sequencing in the copepod ectoparasite Caligus rogercresseyi was conducted to evidence putative roles of non-coding RNAs during the sea louse ontogeny. Here, differentially expressed miRNAs and mRNAs for each developmental stage were analyzed in parallel with bioinformatic gene target predictions. Based on sequence analysis, C. rogercresseyi miRNome comprises 673 conserved miRNAs, including precursors, 5′ and 3′ isomiRs. The conserved miRNAs include 40 families found in twelve different arthropods species. The results also showed that C. rogercresseyi miRNome exhibit stage-specific expression patterns, with miRNA-996-4 and miRNA-124 displaying sex-biased expression. Target prediction of these miRNAs identifies possible silencing mechanism of sex-related genes. Furthermore, bantam isomiRs were highly transcribed during the infective stage of copepodid and target prediction using differentially expressed genes in Atlantic salmon infested with sea lice, suggests a putative role of these miRNAs in the host-pathogen interaction. This is the first study reporting a miRNA repertoire in a marine copepod ectoparasite that affects the salmon aquaculture worldwide.
Frontiers in Marine Science
Phenylalanine hydroxylase (PAH) is a crucial enzyme involved in tyrosine biosynthesis, having roles in neurological and physiological processes. The purpose of PAH has received little attention in crustaceans despite extensive investigations in other arthropods. Here, we characterize the PAH gene for the first time in the parasite Lepeophtheirus salmonis, a copepod that is responsible for huge economic losses in salmonid fish farming. Phylogenetic and sequence analyses confirmed that LsPAH is closely related to the metazoan PAH with conserved ACT regulatory and catalytic domains. Temporal expression patterns revealed that LsPAH is expressed throughout all developmental stages peaking during the copepodite stages, suggesting an essential role in developmental physiology. We used RNAi to knockdown LsPAH expression in the nauplius I stage to study developmental function during the larval stages. PAH knockdown impaired larval development, molting and swimming ability with severe morphol...
International Journal for Parasitology, 2009
The salmon louse (Lepeophtheirus salmonis) is an important pathogen in salmon aquaculture and a serious threat to wild populations of salmon. Knowledge of its basic biological processes such as reproduction is crucial for the control of this parasite and can facilitate development of a vaccine. Here, a novel yolk-associated protein, LsYAP, was characterised. Quantitative PCR and in situ analysis demonstrated that transcription of LsYAP takes place in the subcuticular tissue of adult females in the reproductive phase. LsYAP protein is transported and deposited in the developing eggs in the genital segment, where further processing takes place. The sequence characteristics, histological localisation and transcript regulation suggest that LsYAP is a yolk-associated protein. In addition, the use of RNA interference is, to our knowledge, demonstrated for the first time in a copepod. Treatment of adult females with double-stranded RNA led to lethality and deformations of offspring only. This result confirms that the LsYAP protein is produced in adult females but is utilised by the offspring.
Gene expression in five salmon louse (Lepeophtheirus salmonis, Krøyer 1837) tissues
Marine Genomics, 2014
The Atlantic salmon, Salmo salar L, is an important species both for traditional fishery and fish farming. Many Atlantic salmon stocks have been declining and a suspected main contributor to this decline is the salmon louse (Lepeophtheirus salmonis); a parasitic copepod living off the salmonid hosts epidermal tissues and blood. Contributing to the growing body of knowledge on the molecular biology of the salmon louse we have utilized a microarray containing 11,100 salmon louse genes to study the gene expression patterns in selected tissues. This approach has yielded information about potential functions of the transcripts and tissues. Microarray analyses were preformed on subcuticular and frontal (neuronal and gland enriched tissue) tissues, as well as gut, ovary and testes of adult lice. Tissue specific transcriptomes were evident, allowing us to address main traits of functional partitioning between tissues and providing valuable insight into the biology of the louse. The results furthermore represent an important tool and resource for further experiments.