Salmon louse labial gland enzymes: implications for host settlement and immune modulation.

Salmon louse (Lepeophtheirus salmonis) is a skin- and blood-feeding ectoparasite, infesting salmonids. While feeding, labial gland proteins from the salmon louse may be deposited on the Atlantic salmon (Salmo salar) skin. Previously characterized labial gland proteins are involved in anti-coagulation and may contribute to inhibiting Atlantic salmon from mounting a sufficient immune response against the ectoparasite. As labial gland proteins seem to be important in the host-parasite interaction, we have, therefore, identified and characterized ten enzymes localized to the labial gland. They are a large group of astacins named L. salmonis labial gland astacin 1-8 (LsLGA 1-8), one serine protease named L. salmonis labial gland serine protease 1 (LsLGSP1), and one apyrase named L. salmonis labial gland apyrase 1 (LsLGAp1). Protein domain predictions showed that LsLGA proteins all have N-terminal ShK domains, which may bind to potassium channels targeting the astacins to its substrate. LsLGA1 and -4 are, in addition, expressed in another gland type, whose secrete also meets the host-parasite interface. This suggests that LsLGA proteins may have an anti-microbial function and may prevent secondary infections in the wounds. LsLGAp1 is predicted to hydrolyze ATP or AMP and is, thereby, suggested to have an immune dampening function. In a knockdown study targeting LsLGSP1, a significant increase in IL-8 and MMP13 at the skin infestation site was seen under LsLGSP1 knockdown salmon louse compared to the control, suggesting that LsLGSP1 may have an anti-inflammatory effect. Moreover, most of the identified labial gland proteins are expressed in mature copepodids prior to host settlement, are not regulated by starvation, and are expressed at similar or higher levels in lice infesting the salmon louse-resistant pink salmon (Oncorhynchus gorbuscha). This study, thereby, emphasizes the importance of labial gland proteins for host settlement and their immune dampening function. This work can further contribute to anti-salmon louse treatment such as vaccine development, functional feed, or gene-edited salmon louse-resistant Atlantic salmon.

Small, charged proteins in salmon louse (Lepeophtheirus salmonis) secretions modulate Atlantic salmon (Salmo salar) immune responses and coagulation.

Little is known about glandular proteins secreted from the skin- and blood-feeding ectoparasite salmon louse (Lepeophtheirus salmonis). The labial gland has ducts extending into the oral cavity of the lice, and the present study aimed to identify novel genes expressed by this gland type and to investigate their role in modulation of host parameters at the lice feeding site. Five genes associated with labial gland function were identified and named Lepeophteirus salmonis labial gland protein (LsLGP) 1-4 and 1 like (LsLGP1L). All LsLGPs were predicted to be small charged secreted proteins not encoding any known protein domains. Functional studies revealed that LsLGP1 and/or LsLGP1L regulated the expression of other labial gland genes. Immune dampening functions were indicated for LsLGP2 and 3. Whereas LsLGP2 was expressed throughout the parasitic life cycle and found to dampen inflammatory cytokines, LsLGP3 displayed an increased expression in mobile stages and appeared to dampen adaptive immune responses. Expression of LsLGP4 coincided with moulting to the mobile pre-adult I stage where hematophagous feeding is initiated, and synthetic LsLGP4 decreased the clotting time of Atlantic salmon plasma. Results from the present study confirm that the salmon louse secretes immune modulating and anti-coagulative proteins with a potential application in new immune based anti-salmon louse treatments.

The potential for cleaner fish-driven evolution in the salmon louse Lepeophtheirus salmonis: Genetic or environmental control of pigmentation?

The parasitic salmon louse represents one of the biggest challenges to environmentally sustainable salmonid aquaculture across the globe. This species also displays a high evolutionary potential, as demonstrated by its rapid development of resistance to delousing chemicals. In response, farms now use a range of non-chemical delousing methods, including cleaner fish that eat lice from salmon. Anecdotal reports suggest that in regions where cleaner fish are extensively used on farms, lice have begun to appear less pigmented and therefore putatively less visible to cleaner fish. However, it remains an open question whether these observations reflect a plastic (environmental) or adaptive (genetic) response. To investigate this, we developed a pigment scoring system and conducted complimentary experiments which collectively demonstrate that, a) louse pigmentation is strongly influenced by environmental conditions, most likely light, and b) the presence of modest but significant differences in pigmentation between two strains of lice reared under identical conditions. Based on these data, we conclude that pigmentation in the salmon louse is strongly influenced by environmental conditions, yet there are also indications of underlying genetic control. Therefore, lice could display both plastic and adaptive responses to extensive cleaner fish usage where visual appearance is likely to influence survival of lice.

The effect of temperature on ability of Lepeophtheirus salmonis to infect and persist on Atlantic salmon.

The salmon louse (Lepeophtheirus salmonis) is an ecologically and economically important parasite of salmonid fish. Temperature is a strong influencer of biological processes in salmon lice, with development rate increased at higher temperatures. The successful attachment of lice onto a host is also predicted to be influenced by temperature; however, the correlation of temperature with parasite survival is unknown. This study describes the effects of temperature on infection success, and survival on the host during development to the adult stage. To accurately describe infection dynamics with varying temperatures, infection success was recorded on Atlantic salmon (Salmo salar) between 2 and 10°C. Infection success ranged from 20% to 50% and was strongly correlated with temperature, with the highest success at 10°C. Parasite loss was monitored during development at eight temperatures with high loss of lice at 3 and 24°C, whilst no loss was recorded in the temperature range from 6 to 21°C. Sea temperatures thus have large effects on the outcome of salmon louse infections and should be taken into account in the management and risk assessment of this parasite. Improving understanding of the infection dynamics of salmon lice will facilitate epidemiological modelling efforts and efficiency of pest management strategies.

Identification of critical enzymes in the salmon louse chitin synthesis pathway as revealed by RNA interference-mediated abrogation of infectivity.

Treatment of infestation by the ectoparasite Lepeophtheirus salmonis relies on a small number of chemotherapeutant treatments that currently meet with limited success. Drugs targeting chitin synthesis have been largely successful against terrestrial parasites where the pathway is well characterised. However, a comparable approach against salmon lice has been, until recently, less successful, likely due to a poor understanding of the chitin synthesis pathway. Post-transcriptional silencing of genes by RNA interference (RNAi) is a powerful method for evaluation of protein function in non-model organisms and has been successfully applied to the salmon louse. In the present study, putative genes coding for enzymes involved in L. salmonis chitin synthesis were characterised after knockdown by RNAi. Nauplii I stage L. salmonis were exposed to double-stranded (ds) RNA specific for several putative non-redundant points in the pathway: glutamine: fructose-6-phosphate aminotransferase (LsGFAT), UDP-N-acetylglucosamine pyrophosphorylase (LsUAP), N-acetylglucosamine phosphate mutase (LsAGM), chitin synthase 1 (LsCHS1), and chitin synthase 2 (LsCHS2). Additionally, we targeted three putative chitin deacetylases (LsCDA4557, 5169 and 5956) by knockdown. Successful knockdown was determined after moulting to the copepodite stage by real-time quantitative PCR (RT-qPCR), while infectivity potential (the number of attached chalimus II compared with the initial number of larvae in the system) was measured after exposure to Atlantic salmon and subsequent development on their host. Compared with controls, infectivity potential was not compromised in dsAGM, dsCHS2, dsCDA4557, or dsCDA5169 groups. In contrast, there was a significant effect in the dsUAP-treated group. However, of most interest was the treatment with dsGFAT, dsCHS1, dsCHS1+2, and dsCDA5956, which resulted in complete abrogation of infectivity, despite apparent compensatory mechanisms in the chitin synthesis pathway as detected by qPCR. There appeared to be a common phenotypic effect in these groups, characterised by significant aberrations in appendage morphology and an inability to swim. Ultrastructurally, dsGFAT showed a significantly distorted procuticle without distinct exo/endocuticle and intermittent electron dense (i.e. chitin) inclusions, and together with dsUAP and dsCHS1, indicated delayed entry to the pre-moult phase.

Salmon louse rhabdoviruses: Impact on louse development and transcription of selected Atlantic salmon immune genes.

Recently, it has been shown that the salmon louse (Lepeophtheirus salmonis) is commonly infected by one or two vertically transmitted Lepeophtheirus salmonis rhabdoviruses (LsRVs). As shown in the present study, the viruses have limited effect on louse survival, developmental rate and fecundity. Since the LsRVs were confirmed to be present in the louse salivary glands, the salmon cutaneous immune response towards LsRV positive and negative lice was analyzed. In general, L. salmonis increased the expression of IL1ß, IL8 and IL4/13A at the attachment site, in addition to the non-specific cytotoxic cell receptor protein 1 (NCCRP-1). Interestingly, LsRV free lice induced a higher skin expression of IL1ß, IL8, and NCCRP-1 than the LsRV infected lice. The inflammatory response is important for louse clearance, and the present results suggest that the LsRVs can be beneficial for the louse by dampening inflammation. Further research is, however; needed to ascertain whether this is a direct modulatory effect of secreted virions, or if virus replication is altering the level of louse salivary gland proteins.

RNAi-mediated treatment of two vertically transmitted rhabdovirus infecting the salmon louse (Lepeophtheirus salmonis).

Rhabdoviruses are a family of enveloped negative-sense single-stranded RNA viruses infecting a variety of hosts. Recently, two vertically transmitted salmon louse (Lepeophtheirus salmonis) rhabdoviruses (LsRV) have been identified. The prevalence of these viruses was measured along the Norwegian coast and found to be close to 100%, and with the present lack of suitable cell lines to propagate these viruses, it is challenging to obtain material to study their host impact and infection routes. Thus, virus free lice strains were established from virus infected lice carrying one or both LsRVs by treating them with N protein dsRNA twice during development. The viral replication of the N protein was specifically down-regulated following introduction of virus-specific dsRNA, and virus-free lice strains were maintained for several generations. A preliminary study on infection routes suggested that the LsRV-No9 is maternally transmitted, and that the virus transmits from males to females horizontally. The ability to produce virus free strains allows for further studies on transmission modes and how these viruses influences on the L.salmonis interaction with its salmonid host. Moreover, this study provides a general fundament for future studies on how vertically transmitted rhabdoviruses influence the biology of their arthropod hosts.

Exocrine glands of Lepeophtheirus salmonis (Copepoda: Caligidae): Distribution, developmental appearance, and site of secretion.

Exocrine glands of blood-feeding parasitic copepods are believed to be important in host immune response modulation and inhibition of host blood coagulation, but also in the production of substances for integument lubrication and antifouling. In this study, we aimed to characterize the distribution of different types of salmon louse (Lepeophtheirus salmonis) exocrine glands and their site of secretion. The developmental appearance of each gland type was mapped and genes specifically expressed by glands were identified. Three types of tegumental (teg 1-3) glands and one labial gland type were found. The first glands to appear during development were teg 1 and teg 2 glands. They have ducts extending both dorsally and ventrally suggested to be important in lubricating the integument. Teg 1 glands were found to express two astacin metallopeptidases and a gene with fibronectin II domains, while teg 2 glands express a heme peroxidase. The labial glands were first identified in planktonic copepodids, with reservoirs that allows for storage of glandular products. The last gland type to appear during development was named teg 3 and was not seen before the preadult I stage when the lice become more virulent. Teg 3 glands have ducts ending ventrally at the host-parasite contact area, and may secrete substances important for the salmon lice virulence. Salmon lice teg 3 and labial glands are thus likely to be especially important in the host-parasite interaction. Proteins secreted from the salmon louse glands to its salmonid host skin or blood represents a potential interface where the host immune system can meet and elicit effective responses to sea lice antigens. The present study thus represents a fundamental basis for further functional studies and identification of possible vaccine candidates. J. Morphol. 277:1616-1630, 2016. © 2016 Wiley Periodicals, Inc.

Instar growth and molt increments in Lepeophtheirus salmonis (Copepoda: Caligidae) chalimus larvae.

The salmon louse (Lepeophtheirus salmonis) is an ectoparasitic copepod causing severe problems to the fish farming industry and to wild salmonids. Morphologically, all stages in the life cycle of L. salmonis have been described in detail based on successive samples from host populations. However, the rate of development differs between males and females as well as between individuals. It has therefore been difficult to observe development within stages, and this has led to a longstanding misinterpretation of the number of chalimus stages. Here samples of chalimi obtained for 12 consecutive days were observed daily in incubators. Chalimus 1 was able to molt in incubators only when fully grown and close to molting, whereas chalimus 2 was able to molt at about 60% of total instar growth. Total length instar growth was about 35% in both chalimus 1 and chalimus 2 and about equal among males and females; the cephalothorax increased by about 12% and the posterior body by about 80%. Instar growth was probably the main factor that led to the former belief that L. salmonis had four chalimus stages. Relative total length increase at molting was at the same order of magnitude as instar growth, but total length of females increased significantly more than that of males at molting. Consequently, a sexual size dimorphism was established upon molting to chalimus 2 and males were about 10% smaller than females. While growth by molting was mainly caused by cephalothorax increase, instar growth was mainly due to increase of the posterior body. The cephalothorax/total length ratio decreased from beginning to end of the instar phase suggesting that it may be used as an instar age marker. Male and female chalimus 2 can almost uniquely be identified by cephalothorax length. Chalimus 1 lasted between 5 and 6 days for males and between 6 and 7 days for females at 10°C. Chalimus 2 males lasted between 6 and 7 days and females between 7 and 8 days.

The Salmon Louse Lepeophtheirus salmonis (Copepoda: Caligidae) life cycle has only two Chalimus stages.

Each year the salmon louse (Lepeophtheirussalmonis Krøyer, 1838) causes multi-million dollar commercial losses to the salmon farming industry world-wide, and strict lice control regimes have been put in place to reduce the release of salmon louse larvae from aquaculture facilities into the environment. For half a century, the Lepeophtheirus life cycle has been regarded as the only copepod life cycle including 8 post-nauplius instars as confirmed in four different species, including L. salmonis. Here we prove that the accepted life cycle of the salmon louse is wrong. By observations of chalimus larvae molting in incubators and by morphometric cluster analysis, we show that there are only two chalimus instars: chalimus 1 (comprising the former chalimus I and II stages which are not separated by a molt) and chalimus 2 (the former chalimus III and IV stages which are not separated by a molt). Consequently the salmon louse life cycle has only six post-nauplius instars, as in other genera of caligid sea lice and copepods in general. These findings are of fundamental importance in experimental studies as well as for interpretation of salmon louse biology and for control and management of this economically important parasite.

Individual fish tank arrays in studies of Lepeophtheirus salmonis and lice loss variability.

In studies of the salmon louse Lepeophtheirus salmonis (Krøyer, 1837), experimental design is complicated by a highly variable and unpredictable lice loss among common experimental tanks and a substantial rate of host transfer within tanks. When fish hosting L. salmonis are maintained in individual tanks, unspecific effects such as host transfer, louse predation by cohabitant hosts and agonistic host interactions are excluded. This study suggests that it is possible to maintain Atlantic salmon Salmo salar infected with L. salmonis in an array of small, single fish tanks and, by doing so, provide an experimental system in which the loss of motile pre-adult and adult stages of L. salmonis is predictable. Here, lice can be collected shortly after detachment for detailed studies or to provide mortality curves of lice from individual fish. This represents an experimental approach improving precision in studies of L. salmonis, such as drug and vaccine efficacy assays, RNA interference (RNAi) studies and host-parasite interactions. The natural loss of pre-adult/adult L. salmonis from the system was higher for males than females. The loss of females appeared to be a process somewhat selective against large individuals. Inherent qualities of the host appeared to be of little significance in explaining the variability in loss of preadult/adult lice.

Establishment and characterisation of salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) laboratory strains.

The salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) is an ectoparasitic copepod which represents a major pathogen of wild and farmed salmonid fishes in the marine environment. In order to facilitate research on this ecologically and economically important parasite, a hatchery and culturing system permitting the closure of the life-cycle of L. salmonis in the laboratory was developed. Here, the hatchery system, breeding practices, and selected louse strains that have been maintained in culture in the period 2002-2009 are presented. The hatchery and culture protocol gave rise to predictable hatching of larvae and infections of host fish, permitting the cultivation of specific strains of L. salmonis for 22 generations. Both in- and out-bred lice and mutant colour strains have been established, and some of these strains were characterised by microsatellite DNA markers confirming their pedigree. No evidence of inbreeding depression, fitness or morphological changes was observed in any of the strains cultured. It is suggested that the culturing system, and the strains produced represent a significant resource for future research on this parasite.

Functional characterisation of the maternal yolk-associated protein (LsYAP) utilising systemic RNA interference in the salmon louse (Lepeophtheirus salmonis) (Crustacea: Copepoda).

The salmon louse (Lepeophtheirus salmonis) is an important pathogen in salmon aquaculture and a serious threat to wild populations of salmon. Knowledge of its basic biological processes such as reproduction is crucial for the control of this parasite and can facilitate development of a vaccine. Here, a novel yolk-associated protein, LsYAP, was characterised. Quantitative PCR and in situ analysis demonstrated that transcription of LsYAP takes place in the subcuticular tissue of adult females in the reproductive phase. LsYAP protein is transported and deposited in the developing eggs in the genital segment, where further processing takes place. The sequence characteristics, histological localisation and transcript regulation suggest that LsYAP is a yolk-associated protein. In addition, the use of RNA interference is, to our knowledge, demonstrated for the first time in a copepod. Treatment of adult females with double-stranded RNA led to lethality and deformations of offspring only. This result confirms that the LsYAP protein is produced in adult females but is utilised by the offspring.

Molecular characterization and classification of a clip domain containing peptidase from the ectoparasite Lepeophtheirus salmonis (Copepoda, Crustacea).

Clip domain containing serine peptidases (CSPs) include one or more N-terminal clip domain(s) and a C-terminal serine peptidase domain that shares traits with both chymotrypsin and trypsin. CSPs are found in arthropods and are involved in embryonic patterning, immune responses and blood clotting. Among crustaceans only one CSP, which activates prophenoloxidase in crayfish, have previously been reported. We here present LsCSP1, the first CSP found in copepods. LsCSP1 is expressed in the subcuticular tissue and the transcription appears to be upregulated during development. In conjunction with previous studies of CSPs, this study suggests that LsCSP1 may play a role in the immune responses of L. salmonis. Phylogenetic and structural analyses indicate that the CSPs and catalytically inactive CSP homologs (CSPHs) constitute a monophyletic lineage.