Microsomal triglyceride transfer protein in the ectoparasitic crustacean salmon louse (Lepeophtheirus salmonis)

Abstract

The salmon louse, Lepeophtheirus salmonis, is an endemic ectoparasite on salmonid fish that is challenging for the salmon farming industry and wild fish. Salmon lice produce high numbers of offspring, necessitating sequestration of large amounts of lipids into growing oocytes as a major energy source for larvae, most probably mediated by lipoproteins. The microsomal triglyceride transfer protein (MTP) is essential for the assembly of lipoproteins. Salmon lice have three L. salmonis MTP (LsMTP) transcript variants encoding two different protein isoforms, which are predicted to contain three β-sheets (N, C, and A) and a central helical domain, similar to MTPs from other species. In adult females, the LsMTPs are differently transcribed in the sub-cuticular tissues, the intestine, the ovary, and in the mature eggs. RNA interference-mediated knockdown of LsMTP in mature females gave offspring with significantly fewer neutral lipids in their yolk and only 10-30% survival. The present study suggests the importance of LsMTP in reproduction and lipid metabolism in adult female L. salmonis, a possible metabolic bottleneck that could be exploited for the development of new anti-parasitic treatment methods.

Keywords: Nile Red; Oil Red O; RNA interference; gene expression; lipid and lipoprotein metabolism; lipid transport; lipid transport proteins; lipoproteins; sea lice.

Fig. 1.

The organization of the LsMTP gene. A: Genetic structure of the three variants of LsMTP. LsMTP-A consisted of six exons with an initiator codon in exon 1. The 5′ UTR is represented with a white box. LsMTP-B was generated due to intron retention, with exon 1 as part of the 5′ UTR. LsMTP-C arose due to exon 1 skipping. Lipoprotein N-terminal domain (LpD-N) is shaded with gray. The positions of the fragments used for RNAi and in situ hybridization RNA probes (ISH) are also shown. Scale bar = 200 bp. B: Multiple nucleotide alignments of the 5′ UTR sequences of three LsMTP variants. The 5′ UTR nucleotide sequences of three variants are highlighted in gray. The arrowheads indicate the start codon (ATG). Lowercase letters represent the intron sequence. Gaps are displayed as dashed lines. C: N-terminal amino acid sequence alignment of three LsMTP variants. The predicted signal peptides for three variants are underlined. D: Overview table. The table lists the size of the variants, open reading frame (ORF), 5′ and 3′ UTRs, signal peptide, and LpD-N domain size in number of amino acids (AAs).

Fig. 2.

Structural analysis of LsMTP. A: Predicted secondary structures of LsMTP. LsMTP model consists of four functional domains: N-terminal β-sheet, central helical domain, C β-sheets, and A β-sheets. The ruler for amino-acid numbering is shown below. B: The tertiary structure of LsMTP was modeled using PHYRE protein structure prediction program. The left panel represents the full view of the ribbon structure of LsMTP protein, with cysteine residues (gray spheres) and residues of the salt bridges (cyan spheres). The right panel represents the zoom view of the interior of the N-terminal β-sheet (upper) and central helical domain (lower). Disulfide linkages have been formed between C101-C120 and C156-C182, whereas salt bridges have been formed between D96-R115-K117 and R473-E502. C, D: Multiple alignments of the conserved N-sheet and central helical domain of LsMTP with other MTPs. The conserved cysteine residues are shown with asterisks and residues of the salt bridges are highlighted with inverted triangles. E: Multiple alignment of the MTP-specific sequence. This region was present in salmon louse MTP and contained helix A. The black arrow below the sequence indicates the amino acid position (L734 in human MTP) important for the lipid transfer activity (37). Isoleucine (I653) was found at this position in salmon lice. The dotted line shows the helix as predicted by Jpred4 and PSSpred, which has not been described before in other MTPs.

Fig. 3.

In situ hybridization and RT-PCR analysis of LsMTP mRNAs in various tissues of an adult female salmon louse. A: Dorsal view of an adult female without egg-strings. The black dotted line indicates the area where sub-cuticular tissue is situated; a white straight dash-dot line represents the gut filled with blood. Asterisks (*) and hashtags (#) represent the positions of the ovaries and mature vitellogenic oocytes, respectively. B–E: Cross-sections of sub-cuticular tissue (B), intestine (C), ovaries (D), and vitellogenic oocytes (E) hybridized with antisense probes or sense probes (small inserts) as negative controls. F: RT-PCR analyses from cDNA templates of different tissues of adult female lice using LsMTP variant-specific primers. RT-PCR analysis of ef1α was carried out to determine the quantitative variations of LsMTP transcripts among samples. SQT, sub-cuticular tissue; IN, intestine; OV, ovaries; OO, oocytes. Scale bars = 1 mm (A), 200 μm (B, E), 100 μm (C, D).

Fig. 4.

Expression of the transcript levels of three different variants of LsMTP in various developmental stages of the salmon louse relative to transcript level in the copepodids. The insert shows the expression of the three variants in the copepodids. Naup I, nauplii I; Naup II, nauplii II; Cop, planktonic 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; YAD, young adult female. Error bars represent the SD (n = 5 for each stage).

Fig. 5.

Inhibition of LsMTP transcript by RNAi in adult female salmon lice. The expression level of LsMTP was quantified by Q-PCR in adult females injected with dsRNA in preadult (Fr 1) and young adult (Fr 2) females against control. The results represent the mean ± SEM of five biological replicates from each treatment group. Significant downregulation of LsMTP was found as compared with control (t-test, P < 0.05).

Fig. 6.

LsMTP transports maternal neutral lipids to developing embryos. Bright field (A–D) and confocal fluorescence (A′–D′) nauplii stained with Nile Red to visualize the lipid droplets. Accumulation of maternal neutral lipids was reduced in the nauplii of females injected with LsMTP dsRNA (C′–D′) as compared with nauplii produced from females injected with control dsRNA (A′–B′). Nauplii hatched from females injected with control dsRNA (E) and LsMTP dsRNA (F) were stained with a nonfluorescent dye (Oil Red O). Nauplii hatched from females injected with LsMTP dsRNA accumulate fewer neutral lipids (F), as compared with nauplii hatched from females treated with control dsRNA (E). G: Semi-quantification of neutral lipids with Oil Red O stain in the nauplii of females treated with control and LsMTP dsRNAs. Neutral lipids were reduced significantly (83%) in hatched nauplii of LsMTP dsRNA-treated females. Results are represented as the mean ± SD of nauplii (n = 25) hatched from seven independent replicates of control and LsMTP dsRNA-injected females. Scale bars = 100 μm (A/A′–D/D′), 500 μm (E, F).

Fig. 7.

Starvation reduces LsMTP transcript levels in adult female lice. Significant reduction of the expression level of LsMTP was found in starved animals, as compared with control animals (day 0) (t-test: P < 0.05). Refeeding (2 days) increased the expression of LsMTP as compared with starved and control (day 0) animals. Error bars represent the SD for each time point (n = 5).