Lipid transporters and receptor in salmon louse (Lepeophtheirus salmonis) : Effect of RNAi Knockdown on oogenesis, embryonal development and larval maturation

Abstract

The salmon louse, Lepeophtheirus salmonis, is a marine ectoparasite of salmonids in the Northern Hemisphere. At present, salmon louse infestation is considered as one of the biggest challenges in the salmon farming industry, causing huge economic losses, and also considered a threat to wild populations of salmonids. Control of salmon lice on farmed salmon has mainly depended on the use of chemotherapeutants. However, over the past few years, the salmon louse has developed resistance against most available chemicals. As a consequence, non-chemical treatment methods such as cleaner fish have been introduced in salmon farming, but the production, health and welfare in the cleaner fish have been challenging. It is, therefore, evident that new treatment methods are needed to control this parasite. For this purpose, further understanding of the biology of this parasite is crucial to identify new principles or drug targets.

Lipids are an important source of energy for the growth and reproduction of animals. Other functions include their role in cellular signalling and as structural components in the cell membranes. In oviparous animals, females deposit lipids to maturing eggs to be utilized during embryogenesis and larval development. Transport of lipids through the circulation of animals to developing oocytes is facilitated by lipoproteins, which consist of lipids and protein components known as apolipoproteins. Lipoproteins carry lipids from the site of synthesis or storage to the site of utilization/storage while lipoprotein receptors facilitate uptake of lipoproteins. Previous studies in vertebrates and some insects showed that maturation of these lipoproteins is under the control of another protein known as microsomal triglyceride transfer protein (MTP). Female salmon lice produce large numbers of lipid-enriched eggs throughout its life span. Similar to other oviparous animals, female louse accumulates a large amount of lipids in developing eggs during vitellogenesis. In female salmon lice, transport of maternal lipids to growing oocytes of female lice has not been addressed before. Presence of genes encoding MTP, apolipoproteins (apoLps) and lipophorin receptor (LpR) may suggest a similar mechanism of lipid metabolism/transport as found in other organisms.

Lipoproteins require for extracellular transport of lipids to different tissues of animals and assembly, as well as secretion of these lipoproteins depend upon MTP. In oviparous species, female supply enough lipids to oocytes to secure successful embryogenesis and early larval development. It is likely that female salmon lice use similar lipoprotein based mechanism to supply maternal lipids to growing oocytes. Therefore it is important to study the role of MTP in the supply of lipids to growing oocytes. Three transcript variants of MTP were found in the salmon louse and all variants transcribed differently in different tissues of an adult female. Functional studies conducted through RNAi induced transcript knock down confirmed that female lice produce offspring with very low lipid contents and survival rate of 10-30% compare to control group animals. The present study suggests that MTP has an important function in reproduction and lipid metabolism in salmon louse and may be considered in the development of a new anti-parasitic treatment method.

Protein components of lipoproteins, apoLPs, are essential in the transport of lipids to different tissues of animals through their interaction with cell surface lipoprotein receptors. Similar to other oviparous animals, it is possible that female salmon louse use lipoproteins for the transport of maternal lipids to growing oocytes where apoLps of lipoproteins bind with lipoprotein receptors and release lipids to the oocytes. In salmon lice, two apoLps encoding genes (LsLp1 and LsLp2) were identified. Expression of both genes was found in the intestine and sub-cuticular tissue of adult female louse. RNAi mediated-knockdown of both genes in female louse confirmed significant reduction of transcripts levels. Female lice injected with LsLp1 double-stranded RNA produced short egg-strings as well as significantly fewer offspring compared to control lice. Knockdown of LsLp2 did not show any effect on the eggstring production and numbers of offspring compared with control lice. Functional studies were conducted through RNAi suggested that LsLp1 play an important role in reproduction of female lice.

Previous studies in different organisms show that members of low-density lipoprotein receptor (LDLR) superfamily mediate the endocytosis of lipoproteins. In salmon louse genome database, single gene homologous to insect lipophorin receptor was identified and named as L. salmonis lipophorin receptor (LsLpR). The LsLpR consists of 16 exons and encodes a protein of 952 amino acids. Structural analysis showed that the predicted structure of LsLpR contains five functional domains similar to LpR of insects. Phylogenetic analysis placed LsLpR together with LpR of insects. The highest abundance of LsLR transcripts was found in copepodids and adult females. In adult females, receptor transcripts and proteins were found in the ovary and vitellogenic oocytes. While in larvae, the LsLpR transcripts were found in the neuronal somata of the brain and in the intestine. Possible functions of LsLpR in reproduction and lipid metabolism were investigated through RNA interference. Knockdown in larvae decreased the transcription of LsLpR by 44-54%, and knockdown of LsLpR in adult female lice reduced the number of offspring by 72% compared with control lice.