A method for stable gene knock-down by RNA interference in larvae of the salmon louse (Lepeophtheirus salmonis)

Highlights

• Presentation of a method for RNAi in accurately staged Lepeophtheirus salmonis larvae.

• Significant RNAi mediated knock-down was only found in molting nauplius I larvae.

• Stable knock-down in the whole measurement period between 2 and 12 days.

• The method was tested and found to be robust in eight different genes.

Abstract

The salmon louse (Lepeophtheirus salmonis), an ectoparasitic copepod of salmonid fish, is a major threat to aquaculture in Norway, Ireland, Scotland and Canada. Due to rise in resistance against existing pesticides, development of novel drugs or vaccines is necessary. Posttranscriptional gene silencing by RNA interference (RNAi), when established in a high throughput system is a potential method for evaluation of molecular targets for new medical compounds or vaccine antigens. Successful use of RNAi has been reported in several stages of salmon lice. However, when we employed a previously described protocol for planktonic stages, no reproducible down-regulation of target genes was gained. In the present study, we describe a robust method for RNAi, where nauplius larvae are soaked in seawater added double stranded RNA (dsRNA). In order to test for when dsRNA may be introduced, and for the efficacy and duration of RNAi, we performed a series of experiments on accurately age determined larvae, ranging from the hatching egg to the copepodid with a salmon louse coatomer and a putative prostaglandin E synthase gene. Presumptive knock-down was monitored by real time PCR. Significant gene silencing was obtained only when nauplius I larvae were exposed to dsRNA during the period in which they molted to nauplius II. A knock down effect could be detected 2 days after soaking, and it remained stable until the last measurement, on day 12. Soaking nauplius I larvae, knock-down was verified for six additional genes with a putative role in molting. For one chitinase, a loss-of-function phenotype with abnormal swimming was obtained. Hence, RNAi, induced in the nauplius, may facilitate studies of the molecular biology of the louse, such as the function of specific genes in developmental processes and physiology, host recognition, host-parasite interaction, and, in extension, the engineering of novel medicines.

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.