Introduction
A substantial factor that makes parasites a serious pest is their high reproductive ability. Therefore, understanding how parasites produce plentiful offspring and how their reproductive capability can be suppressed constitutes a major subject in pest control and management research. Sea lice are ectoparasitic copepods that cause significant economic losses in both wild and farmed marine finfish by feeding on the hosts’ mucus and blood, resulting in skin injuries and increased susceptibility to secondary infections. Among these parasites, Caligus rogercresseyi is the most important species affecting salmon aquaculture in the southern hemisphere. To mitigate the adverse impacts caused by this species, it is critical to identify the molecular mechanisms involved in its reproductive and developmental processes. Therefore, this research aims to achieve two goals: i) to identify the expression patterns and localization of selected genes putatively involved in germ cell differentiation and gonad development in C. rogercresseyi, and ii) to investigate the effects of RNAi-induced gene silencing of candidate targets on the reproductive output of this sea lice species.
Materials and Methods
We characterized a panel of gene homologs that are putatively involved in reproduction and gonad development in this species. RNA-seq and qPCR were used to profile the expression levels of these genes at different developmental stages and between sexes. To clarify the anatomy of the reproductive system and gonadal organization of this parasite, we performed histological and immunohistochemical assessments. We identified the localization of candidate reproductive genes, including Vasa , Ecdysone Receptor (EcR ), and Retinoid X Receptor (RXR ), using in situ hybridization (ISH) . We designed and synthesized dsRNA for the gene silencing of Vasa and RXR , and we adapted a method for subcuticular hemocelic microinjection of dsRNA for the first time in this species. Finally, we assessed the effects RNAi-induced gene silencing on the egg strings formation and embryo development and examined the ovarian organization of treated females using histology.
Results
The expression of a panel of 18 nuclear receptors (NRs) transcript sequences was identified using RNA-seq and RT-qPCR data. Higher relative expression was found in females compared to chalimus and male stages for Cr-RXR , Cr-EcR and hormone receptor 3 (Cr-HR3 ). Localization of Cr-RXR and Cr-EcR transcripts was examined using ISH , showing strong labeling in ovaries, oocytes, and intestine of sea lice. Furthermore, RNAi-induced gene silencing of Cr-RXR caused delayed egg string production, severely reduced fecundity, and generated abnormal gonad and embryo development (Bustos et. al 2023) (Fig. 1) . The Cr-Vasa gene expression patterns were assessed by qPCR, and the results showed a significantly higher relative expression level in adult females compared to copepodid , chalimus, and adult male stages. In situ hybridization revealed strong positive signal in male testes, but also in the intestine and cuticle, while in females, it was observed in the ovaries, oocytes, cuticle, intestine, and egg strings. RNAi-mediated gene silencing of Cr-Vasa impacted embryonic development and reproductive output in adult female lice. Females from the dsVasa-treated group displayed unusual phenotypes, including shorter egg strings with numerous extra-embryonic inclusions, irregularly shaped abnormal embryos, and aborted egg strings.
Discussion
The study expands the knowledge of NRs in parasitic copepods, examining their diversity, expression, and localization in C. rogercresseyi . The findings suggest a physiological role for NRs in C. rogercresseyi reproductive tract development, expanding our knowledge of genes involved in reproduction and development in sea lice. Cr-Vasa gene expression was significantly higher in adult females compared to copepodid , chalimus and adult male stages, indicating a potential role in female reproductive development. Tissue-specific localization of Cr-Vasa mRNA showed strong expression in the testes and ovaries of male and female lice, respectively, as well as in other tissues such as the cuticle and intestine. Finally, RNAi-mediated gene silencing of Cr-Vasa resulted in abnormal embryo development and impaired reproductive output, indicating the crucial role of Cr-Vasa in C. rogercresseyi embryonic and ovarian development.
Conclusions
This research provides insight into the molecular basis of reproduction in C. rogercresseyi, enhancing the value of this copepod as a novel model for molecular research. Furthermore, the functional characterization of specific genes involved in reproduction and development might be useful to identify potential targets for novel therapeutics approaches to control sea lice infestation in aquaculture.
References
Bustos P, Vidal-Pérez D, Schmitt P, Brown DI, and Farlora R. 2023. RNA interference-mediated silencing of R etinoid X Receptor causes reproductive failure in the sea lice Caligus rogercresseyi . Aquaculture 566, 739170 https://doi.org/10.1016/j.aquaculture.2022.739170
This study was supported by ANID-Chile FONDECYT 11150915 and FONDEF ID21I10276.