A method for stable gene knock-down by RNA interference in larvae of the salmon louse (Lepeophtheirus salmonis)
Graphical abstract
Introduction
The salmon louse (Lepeophtheirus salmonis) is an ectoparasitic copepod of salmonid fish, where it feeds on mucus, skin, and blood. It has a complex life cycle that includes eight developmental stages separated by molts (Hamre et al., 2013, Johnson and Albright, 1991, Schram et al., 1993). The initial stages (i.e. nauplius I and II) are planktonic, whereas the following copepodid stage detects and infects the host, on which five successive molts takes place before the louse reaches the sexually mature adult stage.
Dense infestations of salmon lice currently constitute a major challenge in Atlantic salmon aquaculture in Norway, Ireland, Scotland and Canada (Torrissen et al., 2013). Over the last 30 years, a small number of pesticides that target neuronal function (i.e. organophosphates, pyrethroides, avermectins) and enzymes (i.e. chitinase inhibitors) have been utilized to control the parasite. Due to rise in resistance, however, the efficacies of these compounds are becoming increasingly limited. Thus, to control the parasite, and, in extension, to secure current production and accommodate further growth in the aquaculture of salmonids, development of novel drugs and/or vaccines is a necessity. Here, high throughput experimental facilities and procedures to evaluate molecular targets for pesticides or vaccines, for instance by employing post transcriptional gene silencing through RNA interference (RNAi), are warranted. In addition to developing pharmaceuticals, loss-of-function RNAi screens may also facilitate studies of the molecular biology of the louse, such as the function of specific genes in developmental processes, host recognition, host-parasite interactions and physiology at different stages.
RNAi is a molecular technique by which double stranded RNA (dsRNA) inhibits gene expression by triggering degradation of gene-specific transcripts; to achieve this, the dsRNA has to be introduced into the organism and taken up by its cells. The method was initially established in the nematode worm Caenorhabditis elegans, where dsRNA was administrated by microinjection (Fire et al., 1998). Since then injection has also been employed for other species, for instance zebra fish Danio rerio (Wargelius et al., 1999). Furthermore, administration has been achieved by incubating – soaking – the organism in an aqueous solution of dsRNA. Such a method has been developed for C. elegans (Kuroyanagi et al., 2000), Caligus rogercresseyi (Carpio et al., 2011), and other species such as the sea anemone Aiptasia pallida (Dunn et al., 2007). Moreover, feeding with bacteria that express dsRNA has been utilized with success in C. elegans (Timmons and Fire, 1998) and, among others, the sponge Tethya wilhelma (Rivera et al., 2011). For the salmon louse, depending on the developmental stage, dsRNA has been administered either by soaking, or by microinjection: for preadult and adult stages RNAi is attained after microinjection. For instance, egg development has been abolished after microinjection of dsRNA for the egg yolk protein LsYAP (Dalvin et al., 2009). In newly hatched nauplius I and copepodids, RNAi by soaking in seawater containing dsRNA has been performed to silence a gene encoding a putative prostaglandin E synthase (Campbell et al., 2009). The experiment, however, was hampered by high cumulative mortality, and when employing the described protocol, we were unable to obtain knock-down of target genes.
Thus, the aim of the present study was to outline a sound method for RNAi in planktonic stages of the salmon louse. Our hypothesis was that the hatching or molting process might facilitate uptake of dsRNA, as during that processes the larvae have to absorb water to swell. By conducting a series of experiments on accurately aged larvae, with soaking at hatching, during molts, as well as at instar stages (i.e. between molts), we have determined when dsRNA may best be introduced. In addition, we have assessed the duration of gene knock-downs, confirmed a significant effect of RNAi on expression of eight genes and, for one of these, observed a loss-of-function phenotype.
Section snippets
Salmon lice
A laboratory strain of salmon lice (Hamre et al., 2009) was maintained on farmed Atlantic salmon (Salmo salar) that were hand fed on a commercial diet and reared in sea water with a salinity of 34.5 ppt and a temperature of 10 °C. Egg-strings, nauplii and copepodids were kept in seawater from the same supply. Nauplii were obtained from hatching egg-string pairs kept in single wells in a flow through system (Hamre et al., 2009). All experimental procedures were performed in accordance with
Normal gene expression patterns
Expression patterns of the investigated genes are shown in Fig. 2. For all genes, there was significant regulation of mRNA levels between specific stages. LsETHR, LsPAD1, LsChi2 and LsCP1 displayed a high degree of regulation. LsPGES2, LsChi2, LsPAD1 and LsCP1 showed their highest level of relative expression during the nauplius II stage, while the expression of LsETHR reached distinct peak in copepodids. Some of the genes, such as, LsETHR, LsPAD1 and LsCP1 exhibited a very low relative
Discussion
In crustaceans, molting of the exoskeleton is initiated by deposition of a new intermolt cuticle underneath the old by the epidermal epithelium, concomitant with an increase in epidermal secretion of enzymes, such as chitinases and carboxypeptidases, which break down the old cuticle (Buchholz, 1989, Ote et al., 2005, Rocha et al., 2012, Sui et al., 2009, Watanabe et al., 1997). Finally, ecdysis is triggered by a motoric behavioral program that results in detachment and shedding of the old
Funding
This research has been funded by the Research Council Norway, SFI-Sea Lice Research Centre, Grant No. 203513.
Acknowledgment
We are grateful to Heidi Kongshaug and Lars Hamre for excellent help in the laboratory.
References (31)
- et al.
Juvenile hormone and insulin suppress lipolysis between periods of lactation during tsetse fly pregnancy
Mol. Cell. Endocrinol.
(2013) - et al.
Novel gene isolated from Caligus rogercresseyi: a promising target for vaccine development against sea lice
Vaccine
(2011) - et al.
Functional characterisation of the maternal yolk-associated protein (LsYAP) utilising systemic RNA interference in the salmon louse (Lepeophtheirus salmonis) (Crustacea: Copepoda)
Int. J. Parasitol.
(2009) - et al.
Validation of reference genes for transcription profiling in the salmon louse, Lepeophtheirus salmonis, by quantitative real-time PCR
Vet. Parasitol.
(2003) - et al.
Establishment and characterisation of salmon louse (Lepeophtheirus salmonis (Kroyer 1837)) laboratory strains
Parasitol. Int.
(2009) - et al.
SPK-1, a C-elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development
Mech. Dev.
(2000) - et al.
Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori
Comp. Biochem. Physiol. B
(2005) - et al.
Cuticular chitin synthase and chitinase mRNA of whiteleg shrimp Litopenaeus vannamei during the molting cycle
Aquaculture
(2012) - et al.
RNA interference mediated knockdown of the KDEL receptor and COPB2 inhibits digestion and reproduction in the parasitic copepod Lepeophtheirus salmonis
Comp. Biochem. Physiol. B: Biochem. Mol. Biol.
(2014) - et al.
Double-stranded RNA induces specific developmental defects in zebrafish embryos
Biochem. Biophys. Res. Commun.
(1999)
Molecular characterization and function of a p38 MAPK gene from Litopenaeus vannamei
Fish Shellfish Immunol.
Ultrastructure of the cuticle of the chalimus larva of the salmon louse Lepeophtheirus salmonis (Kroyer, 1837) (Copepoda: Caligidae)
Contrib. Zoolog.
Molt cycle and seasonal activities of chitinolytic enzymes in the integument and digestive-tract of the antarctic krill, euphausia-superba
Polar Biol.
RNA-interference methods for gene-knockdown in the sea louse, Lepeophtheirus salmonis: studies on a putative prostaglandin E synthase
Parasitology
Knockdown of actin and caspase gene expression by RNA interference in the symbiotic anemone Aiptasia pallida
Biol. Bull.
Cited by (42)
Identification of critical enzymes in the salmon louse chitin synthesis pathway as revealed by RNA interference-mediated abrogation of infectivity
2020, International Journal for ParasitologyChitin synthesis and degradation in Lepeophtheirus salmonis: Molecular characterization and gene expression profile during synthesis of a new exoskeleton
2019, Comparative Biochemistry and Physiology -Part A : Molecular and Integrative PhysiologyCitation Excerpt :LsChi2, on the contrary, exhibited an expression pattern correlated to molt with increasing level of transcripts from early to late premolt stages. This observation is similar to the expression of LsChi2 reported in nauplia II larvae (Eichner et al., 2014), which showed a significant increase in the transcriptional level from intermolt to premolt lice. Also, chitinases from Penaeus japonicas, L. vannamei, Euphausia superba, and B. mori are up-regulated during premolt stages (Abehsera et al., 2015; Buchholz, 1989; Rocha et al., 2012; Watanabe and Kono, 1997; Zhuo et al., 2014).
RNAi-directed knockdown in the cnidarian fish blood parasite Sphaerospora molnari
2024, Scientific Reports