Host gill attachment causes blood-feeding by the salmon louse (Lepeophtheirus salmonis) chalimus larvae and alters parasite development and transcriptome

Abstract

Background: Blood-feeding is a common strategy among parasitizing arthropods, including the ectoparasitic salmon louse (Lepeophtheirus salmonis), feeding off its salmon host's skin and blood. Blood is rich in nutrients, among these iron and heme. These are essential molecules for the louse, yet their oxidative properties render them toxic to cells if not handled appropriately. Blood-feeding might therefore alter parasite gene expression.

Methods: We infected Atlantic salmon with salmon louse copepodids and sampled the lice in two different experiments at day 10 and 18 post-infestation. Parasite development and presence of host blood in their intestines were determined. Lice of similar instar age sampled from body parts with differential access to blood, namely from gills versus lice from skin epidermis, were analysed for gene expression by RNA-sequencing in samples taken at day 10 for both experiments and at day 18 for one of the experiments.

Results: We found that lice started feeding on blood when becoming mobile preadults if sitting on the fish body; however, they may initiate blood-feeding at the chalimus I stage if attached to gills. Lice attached to gills develop at a slower rate. By differential expression analysis, we found 355 transcripts elevated in lice sampled from gills and 202 transcripts elevated in lice sampled from skin consistent in all samplings. Genes annotated with "peptidase activity" were among the ones elevated in lice sampled from gills, while in the other group genes annotated with "phosphorylation" and "phosphatase" were pervasive. Transcripts elevated in lice sampled from gills were often genes relatively highly expressed in the louse intestine compared with other tissues, while this was not the case for transcripts elevated in lice sampled from skin. In both groups, more than half of the transcripts were from genes more highly expressed after attachment.

Conclusions: Gill settlement results in an alteration in gene expression and a premature onset of blood-feeding likely causes the parasite to develop at a slower pace.

Keywords: Blood-feeding; Ectoparasite; Gills; Hematophagy; RNA-sequencing; Salmon louse.

Fig. 1

Distribution of different stages and instar ages of lice. Stages and different instar ages of lice sampled at 10 days post-infestation (dpi) and at 18 dpi sampled from the fish body, fins and gills in both experiments (Experiment (Exp) 1 and Experiment 2) are shown. Number of fish sampled: n = 20 (Experiment 1, 10 dpi), n = 18 (Experiment 2, 10 dpi), n = 37 (Experiment 1, 18 dpi) and n = 34 (Experiment 2, 18 dpi). Lice instar ages were defined on photographs as described in the main text. Transcriptome sequencing was performed for lice from Experiment 1 10 dpi, Experiment 2 10 dpi (both chalimus I old) and Experiment 2 18 dpi (chalimus II old) marked with *

Fig. 2

Photographs of salmon lice with (right) and without (left) a blood-filled intestine. a Chalimus I larvae sampled 10 days post-infestation. b Chalimus II larvae sampled 18 days post-infestation. c Preadult I lice on frontal filament sampled 18 days post-infestation. The lice with blood-filled intestine were sampled from the gills and the others were sampled from the skin of their host. Scale-bars: 1 mm

Fig. 3

Overall differences in gene expression between different groups of lice. a Correspondence analysis (CA) plot showing the overall gene expression of the samples analysed in this study (pink and turquoise dots) in comparison with other chalimus I and chalimus II larvae divided into various instar ages taken from Eichner et al. [27]. b MA plots for DESeq2 comparing the different conditions (lice sampled from gills versus lice sampled from skin at 10 days post-infestation (dpi) and 18 dpi, respectively) in the samplings: Experiment (Exp) 1, sampled at 10 dpi, Experiment 2 sampled at 10 dpi and Experiment 2 sampled at 18 dpi. The average binary logarithm of the expression across all samples is shown on the x-axis and the binary logarithm of fold change is shown on the y-axis. Red dots indicate differentially expressed genes (DESeq2, Padj < 0.05) while grey dots are not differentially expressed between the two groups. c Principal components analysis (PCA) plots of the same data

Fig. 4

Scaled Venn diagrams for differentially expressed genes found in lice from skin and from gills. The Venn diagrams are showing the number of differentially expressed genes (DESeq2; Padj < 0.05) between chalimus larvae sampled from gills versus those sampled from skin, for Experiment (Exp) 1, sampled at 10 days post-infestation (dpi), Experiment 2, sampled at 10 dpi and Experiment 2 sampled at 18 dpi. The numbers of genes elevated in chalimus larvae sampled from gills in the different samplings are shown in (a) and the numbers of genes elevated in chalimus larvae sampled from fish skin in the different samplings are shown in (b). Representative GO terms for each group are given in the table in the bottom panel. A full list of enriched GO terms is shown in Additional file 15: Table S9 which are summarized as Tree maps in Additional file 1: Figure S1

Fig. 5

GOslim annotations of genes differently expressed in lice sampled from skin and gills. GO slim categories as well as enrichment of genes elevated in lice sampled from gills (a) and genes elevated in lice sampled from skin (b) in all three sampling points are shown (DESeq2; Padj < 0.05). Columns indicate the number of genes in each GOslim category and black dots show the enrichment of genes in that category in relation to number of genes of the specific category in the whole dataset

Fig. 6

Expression of genes with special domains in different tissues and stages. The expression profiles of genes found differentially expressed in all three sampling points (DESeq2; Padj < 0.05) with trypsin, other peptidase and FNII domains in various tissues and stages (LiceBase [60] and from the time series study (average values of biological parallels) by Eichner et al. [27]) are shown in hierarchical clusters. A blue stippled line is separating planktonic and parasitic stages. Key: Stable IDs with font in blue, domains other than trypsin predicted as well; font in green, predicted M13 peptidases; font in red, predicted Astacin peptidases