Molecular characterization of the lipophorin receptor in the crustacean ectoparasite Lepeophtheirus salmonis

Abstract

The Salmon louse (Lepeophtheirus salmonis) is a marine ectoparasite of salmonid fish in the Northern Hemisphere and considered as a major challenge in aquaculture and a threat to wild populations of salmonids. Adult female lice produce a large number of lipid-rich eggs, however, the mechanism of maternal lipid transport into developing eggs during salmon louse reproduction has not been described. In the present study, a full-length L. salmonis lipophorin receptor (LsLpR) consisting of 16 exons was obtained by RACE and RT-PCR. The predicted ORF was 952 amino acids and structural analysis showed five functional domains that are similar to LpR of insects and decapods. Phylogenetic analysis placed the LsLpR together with LpRs from decapods and insects. Expression analysis revealed that the relative abundance of LsLpR transcripts was highest in the larvae and adult female lice. In adult females, the LsLpR transcripts and protein were found in the ovary and vitellogenic oocytes whereas, in larvae, the LsLpR transcripts were found in the neuronal somata of the brain and the intestine. Oil Red O stain results revealed that storage of neutral lipids was found in vitellogenic oocytes and ovaries of adult females, and in the yolk of larvae. Moreover, RNA interference (RNAi) was conducted to demonstrate the function of LsLpR in reproduction and lipid metabolism in L. salmonis. In larvae, the transcription of LsLpR was decreased by 44-54% while in an experiment LsLpR knockdown female lice produced 72% less offspring than control lice.

Fig 1. Exon-intron organization and structural analysis of LsLpR.

(A) LsLpR gene is composed of 16 exons separated by 15 introns and spanning a genomic region of 115.13 kbp. (B) Domains organization of LsLpR with other members of LDLR family. (C) Modelled structure of extracellular domains of LsLpR using PHYRE protein structure prediction program. Cysteine residues are coloured green, yellow residues provide pocket for calcium ion and bound calcium ions are shown as cyan spheres. (D) Single repeat from ligand binding domain shows the three disulphide bonds (C1-C3, C2-C5 and C4-C6). (E) Top view of β–propeller domain with five F/YWXD motifs.

Fig 2. Phylogenetic tree of selected lipoprotein receptors from vertebrates and invertebrates.

The tree was generated using Bayesian methods. LpR of L. salmonis (LsLpR) is shown in red. The yolk receptor (RME2) of the nematode (C. elegans) was used as an out-group. The nodes are labelled with posterior probabilities and for clarity only values < 100 are shown. The scale bar represents 0.4 amino acid substitutions per site.

Fig 3. Expression analysis of the LsLpR in various developmental stages of the salmon louse.

Expression levels of LsLpR in chalimus I was set as 1. Error bars represent the standard deviation (n = 5 samples for each stage). Abbreviations: Naup I, Nauplii I: Naup II, Nauplii II: Cop, free-living copepodids: Cha I, Chalimus I: Cha II, Chalimus II: Pad I M, Preadult I male: Pad I F, Preadult I female: Pad II M, preadult II male: Pad II F, Preadult II female.

Fig 4. Staining of neutral lipids in salmon lice.

Detection of neutral lipids by Oil Red O stain. Adult male (A) and adult female (B). Storage of lipids was detected mainly in mature eggs (II) but also in the ovary (I), of adult female lice. Maternally deposited lipids were found as droplets in the yolk of hatching nauplii (C). A reduction in lipid reserves was noted in copepodids of 7 dph (D) compared to newly hatched nauplii and no lipid droplets were found in copepodids (E) after 10 days of their hatching. Scale bars = (A, B, BII, C-E) 1 mm, (BI) 200 μm. Abbreviations: CM, cement gland; ME, mature eggs; IME, immature eggs.

Fig 5. Localization of LsLpR mRNA and protein in the salmon lice.

(A), (D) and (E) in situ hybridization. (A) Localization of the LsLpR transcripts in the intestine (In) and neuronal somata of the brain (Br) of copepodid. (B) Parallel slide of the copepodid stained with hematoxylin and erythrosine. (C) Dorsal view of an adult female without egg-strings. The asterisks (*) and hashtags (#) indicate the positions of the ovaries in the cephalothorax and mature vitellogenic oocytes in the genital segment of adult female louse respectively. (D) Localization of the LsLpR mRNA in the lumen of the ovarian tubules. (E) Localization of the LsLpR mRNA in the vitellogenic oocytes in the genital segment. No stain was seen in slides (small inserts) hybridized with sense RNA probe. (F) and (G) immunofluorescence with anti LpR. (F) Distribution of LsLpR protein was found in elongated structures, at the inner side of the tubular membrane (white arrow) together with the nuclei of the oocytes (nuclei were stained blue with DAPI). (G) Distribution of the LsLpR protein in the outer membrane of the vitellogenic oocytes. Scale bars indicate (A-B, E) 200 μm, (C) 1 mm, (D and G) 100 μm, (F) 50 μm.

Fig 6. Effect of RNAi on LsLpR transcript and lipid levels in copepodids.

(A) Relative Expression of LsLpR in the copepodids (7 dph) after knock downed in nauplius larva. Error bars show standard deviation. Asterisk represents significant difference (independent-samples T-test, p < 0.05) in mRNA levels of LsLpR between the control group (n = 5) and the knock-down group (n = 5). (B-D) Detection of neutral lipids by Oil Red O stain. Lipid contents in the copepodids hatched from LsLpR (fragments 1 + 3) (B) and control dsRNAs treated nauplii (C). Semi-quantification of total neutral lipids with Oil Red O stain in copepodids (n = 5, each replicate contains 25 animals) developed from nauplii treated with control and LsLpR dsRNAs (fragments 1 + 3) (D). No significant difference (independent-samples T-test, p > 0.05) was found between control group and LsLpR dsRNA-treated group. Scale bars = (B-C) 1 mm.

Fig 7. Treatment with dsRNA against LsLpR.

(A) Relative expression of LsLpR in the adult females after injection of dsRNA in pre-adult females (30–32 days post injection). (B) Relative expression of LsLpR after injection of dsRNA (fragment 1 + 3) in adult females and measured at days 5, 10 and 15 (post injection). Expression PCR was carried out on 5 female lice from control and knock-down group at each time point. (C) Relative expression of LsLpR in copepodids (n = 5 × 20) after knock down (fragments 1 + 3) in nauplii I, assayed before the infection of a host and in adult female lice at the time of termination of the experiment. Error bars show standard deviation and P-values for independent-samples T-test analysis are shown, expression levels of LsLpR in control versus LsLpR dsRNA-treated group.