Hematophagy in the salmon louse (Lepeophtheirus salmonis) : Characterization of genes and proteins involved in parasite blood-feeding

The salmon louse (Lepeophtheirus salmonis) is an obligate, hematophagous ectoparasite infecting salmonid fish such as the Atlantic salmon (Salmo salar). The parasite is at present the number one threat to the Norwegian salmon farming industry. There is a high density of hosts in salmon farms, and due to the high mutation rate and fecundity of the parasite, the salmon louse is currently ahead of the development of effective pest controls. Resistance has been reported against several available chemotherapeutants, and non-therapeutic interventions are as per now not sufficient to treat salmon louse infestations. Particularly adult female lice have blood as a major dietary component. This is a high-risk, high-reward strategy which the parasite is well-adapted to. Blood is highly nutritious and constantly renewed in the vertebrate host, but also contains toxic, yet necessary, compounds such as iron and heme (iron protoporphyrin IX). The salmon louse is likely a heme auxotroph, as many homologous enzymes of the conserved heme biosynthetic pathway are not found within its genome. As such, the salmon louse is innately dependent on its host for iron and heme supply. Blood-feeding and subsequent iron and heme trafficking are thus essential to study in the salmon louse, and could potentially reveal candidate proteins for pest management by e.g. vaccination or nutritional deprivation. Ferritin is a well-conserved multi-subunit iron storage and distribution protein that can be either cytosolic or secreted. In the present study, several ferritin-encoding genes were identified in the salmon louse genome (LsFer1, 2, 3 and 4). Secreted heavy chain homolog (LsFer1) and secreted light chain homolog (LsFer2) transcripts were found to be expressed in the salmon louse intestine, where the precursor protein is probably loaded with iron intracellularly before it is secreted to the hemolymph. RNA interference-mediated knockdown of these two transcripts resulted in severe phenotype alterations for the adult female salmon louse. In both knockdown groups, it was unable to produce proper egg strings, and with an almost complete egg hatching failure. Histological sections of knockdown animal genital segments revealed that the developing oocytes lacked the structure and integrity seen in normal developing oocytes. Furthermore, the knockdown animals lacked the ability to fully engorge in salmon blood. Additionally, in wildtype lice, the transcript levels of LsFer1 and LsFer2 were decreasing during starvation, further indicating the importance of these genes in relation to the blood meal. Further, an investigation of a CD36-like protein named L. salmonis heme scavenger receptor class B (LsHSCARB) was performed. The transcript and protein were located to the salmon louse intestine. The receptor has an extracellular domain likely facing the intestinal lumen. The postulation was that the domain could scavenge host-derived heme. The domain was able to bind to heme in silico as demonstrated by ligand docking using bioinformatical tools. The receptor was also able to bind heme in vitro, as shown using a recombinantly expressed protein in a hemin-agarose pull down assay. During a starvation period, the transcript levels of LsHSCARB in the adult female salmon louse decreased steadily, suggesting that the receptor is down-regulated by the absence of a food source. RNAi mediated receptor ablation in vivo led to a decrease in absorbed heme levels in salmon louse tissues, and knockdown animals had shorter egg strings with a lower hatching success. Because having blood as a diet requires proper handling of blood-components, a hypothesis was that initiating in blood-feeding would to a shift in the salmon louse transcriptome. Results in this thesis indicate that the salmon louse normally starts feeding on blood in the mobile preadult I stage, but that those lice that were attached to the vascular fish gills were feeding on blood already in the chalimus I stage. The lice attached to the gills also developed at a slower pace than those lice elsewhere on the host. Chalimus larvae located on gills were therefore sampled for RNA-sequencing and subsequent gene expression analyses, and compared to chalimus larvae from host fins and general body surfaces, that were of similar instar age. Several transcripts were found differentially expressed in chalimus larvae on gills, among these ferritins, digestive enzymes, genes of unknown functions and genes with FNII domains, to mention some. These genes could be vital for blood-feeding in the salmon louse. Combined, these results stress the importance for the salmon louse to maintain proper ways of handling the blood meal, particularly the micronutrients iron and heme. This thesis builds on the little knowledge on iron and heme biology in L. salmonis from earlier, and provides a further understanding of salmon louse hematophagy. The results of this thesis could possibly be used to lay the groundwork for future methods of controlling the salmon louse infestations in the salmon aquaculture

Without a pinch of salt: effect of low salinity on eggs and nauplii of the salmon louse (Lepeophtheirus salmonis)

The salmon louse is an economically important parasite on Atlantic salmon and poses a major threat to aquaculture. Several treatment methods have lost their effect due to resistance development in the lice. A rather new method for combatting sea lice is freshwater treatment where the various life stages of lice are differently affected by this treatment. In this study, we analyzed the effect of freshwater on the egg strings. A 3-h treatment with freshwater had a detrimental effect on the egg strings. First, the water penetrated the string, widening it, then entering the eggs and enlarging them. Finally, the ordered structure of the egg strings collapsed, and no alive animals hatched. Shorter treatments had a lower effectivity, and treatments with brackish water also showed milder effects. The egg strings were found to have a protective effect against low salinities, as hatched nauplii died rapidly under conditions that embryos survived. We also found that embryos react to low salinity on a molecular level by changing gene expression of several genes, when incubated in brackish water. Additionally, the hatching of embryos treated with brackish water was delayed in comparison to seawater controls.publishedVersio

The transcriptomic response of adult salmon lice (Lepeophtheirus salmonis) to reduced salinity

Salmon lice (Lepeophtheirus salmonis) are marine parasitic copepods living on salmonids and are challenging for salmon aquaculture. One of several treatment methods is the application of freshwater to the fish which can lead to lice loss. However, lab experiments have shown that salmon lice, acclimated to seawater, are capable of surviving for several weeks in freshwater, when attached to a host. If not attached to a host, they die within a few hours in freshwater but can survive a longer time in brackish water. The molecular mechanisms involved in the adaptation to low salinity of the louse have not been identified yet. In this study we incubated salmon lice, being attached to a host, or detached, in seawater, brackish water and freshwater for 4 h and 1 d, sampled the animals and used RNA-Seq to identify genes involved in these mechanisms. Freshwater incubation led to a much stronger regulatory response than brackish water and a longer incubation time gave a stronger effect than a short incubation. Among the most interesting genes, upregulated in low salinity water are in addition to several transporters, several enzymes involved in amino acid metabolism and especially in the proline biosynthesis. A strong upregulation of these enzymes might lead to an accumulation of proline which is known to be used as an osmolyte in other species. While the RNA-Seq experiment was performed with female samples, qPCR showed that at least 10 genes regulated in females, were also regulated in males.publishedVersio

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

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.publishedVersio

Heavy and light chain homologs of ferritin are essential for blood-feeding and egg production of the ectoparasitic copepod Lepeophtheirus salmonis

The salmon louse, Lepeophtheirus salmonis, is a hematophagous ectoparasite of salmonid fish. Due to its blood-feeding activity, the louse is exposed to great amounts of iron, which is an essential, yet potentially toxic mineral. The major known iron storage protein is ferritin, which the salmon louse encodes four genes of (LsFer1-4). Two of the ferritins are predicted to be secreted. These are one of the heavy chain homologs (LsFer1) and the light chain homolog (LsFer2). Here, we perform functional studies and characterize the two secreted ferritins. Our results show that knocking down LsFer1 and LsFer2 both negatively affect the parasite’s physiology, as it is not able to properly feed and reproduce. In a starvation experiment, the transcript levels of both LsFer1 and LsFer2 decrease during the starvation period. Combined, these results demonstrate the importance of these genes for the normal parasite biology, and they could thus potentially be targets for pest management.publishedVersio

Hematophagy in the salmon louse (Lepeophtheirus salmonis) : Characterization of genes and proteins involved in parasite blood-feeding

The salmon louse (Lepeophtheirus salmonis) is an obligate, hematophagous ectoparasite infecting salmonid fish such as the Atlantic salmon (Salmo salar). The parasite is at present the number one threat to the Norwegian salmon farming industry. There is a high density of hosts in salmon farms, and due to the high mutation rate and fecundity of the parasite, the salmon louse is currently ahead of the development of effective pest controls. Resistance has been reported against several available chemotherapeutants, and non-therapeutic interventions are as per now not sufficient to treat salmon louse infestations. Particularly adult female lice have blood as a major dietary component. This is a high-risk, high-reward strategy which the parasite is well-adapted to. Blood is highly nutritious and constantly renewed in the vertebrate host, but also contains toxic, yet necessary, compounds such as iron and heme (iron protoporphyrin IX). The salmon louse is likely a heme auxotroph, as many homologous enzymes of the conserved heme biosynthetic pathway are not found within its genome. As such, the salmon louse is innately dependent on its host for iron and heme supply. Blood-feeding and subsequent iron and heme trafficking are thus essential to study in the salmon louse, and could potentially reveal candidate proteins for pest management by e.g. vaccination or nutritional deprivation. Ferritin is a well-conserved multi-subunit iron storage and distribution protein that can be either cytosolic or secreted. In the present study, several ferritin-encoding genes were identified in the salmon louse genome (LsFer1, 2, 3 and 4). Secreted heavy chain homolog (LsFer1) and secreted light chain homolog (LsFer2) transcripts were found to be expressed in the salmon louse intestine, where the precursor protein is probably loaded with iron intracellularly before it is secreted to the hemolymph. RNA interference-mediated knockdown of these two transcripts resulted in severe phenotype alterations for the adult female salmon louse. In both knockdown groups, it was unable to produce proper egg strings, and with an almost complete egg hatching failure. Histological sections of knockdown animal genital segments revealed that the developing oocytes lacked the structure and integrity seen in normal developing oocytes. Furthermore, the knockdown animals lacked the ability to fully engorge in salmon blood. Additionally, in wildtype lice, the transcript levels of LsFer1 and LsFer2 were decreasing during starvation, further indicating the importance of these genes in relation to the blood meal. Further, an investigation of a CD36-like protein named L. salmonis heme scavenger receptor class B (LsHSCARB) was performed. The transcript and protein were located to the salmon louse intestine. The receptor has an extracellular domain likely facing the intestinal lumen. The postulation was that the domain could scavenge host-derived heme. The domain was able to bind to heme in silico as demonstrated by ligand docking using bioinformatical tools. The receptor was also able to bind heme in vitro, as shown using a recombinantly expressed protein in a hemin-agarose pull down assay. During a starvation period, the transcript levels of LsHSCARB in the adult female salmon louse decreased steadily, suggesting that the receptor is down-regulated by the absence of a food source. RNAi mediated receptor ablation in vivo led to a decrease in absorbed heme levels in salmon louse tissues, and knockdown animals had shorter egg strings with a lower hatching success. Because having blood as a diet requires proper handling of blood-components, a hypothesis was that initiating in blood-feeding would to a shift in the salmon louse transcriptome. Results in this thesis indicate that the salmon louse normally starts feeding on blood in the mobile preadult I stage, but that those lice that were attached to the vascular fish gills were feeding on blood already in the chalimus I stage. The lice attached to the gills also developed at a slower pace than those lice elsewhere on the host. Chalimus larvae located on gills were therefore sampled for RNA-sequencing and subsequent gene expression analyses, and compared to chalimus larvae from host fins and general body surfaces, that were of similar instar age. Several transcripts were found differentially expressed in chalimus larvae on gills, among these ferritins, digestive enzymes, genes of unknown functions and genes with FNII domains, to mention some. These genes could be vital for blood-feeding in the salmon louse. Combined, these results stress the importance for the salmon louse to maintain proper ways of handling the blood meal, particularly the micronutrients iron and heme. This thesis builds on the little knowledge on iron and heme biology in L. salmonis from earlier, and provides a further understanding of salmon louse hematophagy. The results of this thesis could possibly be used to lay the groundwork for future methods of controlling the salmon louse infestations in the salmon aquaculture

The transcriptomic response of adult salmon lice (Lepeophtheirus salmonis) to reduced salinity

Salmon lice (Lepeophtheirus salmonis) are marine parasitic copepods living on salmonids and are challenging for salmon aquaculture. One of several treatment methods is the application of freshwater to the fish which can lead to lice loss. However, lab experiments have shown that salmon lice, acclimated to seawater, are capable of surviving for several weeks in freshwater, when attached to a host. If not attached to a host, they die within a few hours in freshwater but can survive a longer time in brackish water. The molecular mechanisms involved in the adaptation to low salinity of the louse have not been identified yet. In this study we incubated salmon lice, being attached to a host, or detached, in seawater, brackish water and freshwater for 4 h and 1 d, sampled the animals and used RNA-Seq to identify genes involved in these mechanisms. Freshwater incubation led to a much stronger regulatory response than brackish water and a longer incubation time gave a stronger effect than a short incubation. Among the most interesting genes, upregulated in low salinity water are in addition to several transporters, several enzymes involved in amino acid metabolism and especially in the proline biosynthesis. A strong upregulation of these enzymes might lead to an accumulation of proline which is known to be used as an osmolyte in other species. While the RNA-Seq experiment was performed with female samples, qPCR showed that at least 10 genes regulated in females, were also regulated in males

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

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

A scavenger receptor B (CD36)-like protein is a potential mediator of intestinal heme absorption in the hematophagous ectoparasite Lepeophtheirus salmonis

Abstract Intestinal absorption of heme has remained enigmatic for years, even though heme provides the most bioavailable form of iron. The salmon louse, Lepeophtheirus salmonis, is a heme auxotrophic ectoparasite feeding on large quantities of blood from its host, the salmon. Here we show that a scavenging CD36-like receptor is a potential mediator of heme absorption in the intestine of the salmon louse. The receptor was characterized by a heme binding assay using recombinantly expressed protein, in situ hybridization and immunohistochemistry, as well as functional knockdown studies in the louse. A computational structural model of the receptor predicted a binding pocket for heme, as also supported by in silico docking. The mRNA and protein were expressed exclusively in the intestine of the louse. Further, knocking down the transcript resulted in lower heme levels in the adult female louse, production of shorter egg strings, and an overall lower hatching success of the eggs. Finally, starving the lice caused the transcript expression of the receptor to decrease. To our knowledge, this is the first time a CD36-like protein has been suggested to be an intestinal heme receptor