Chitin synthesis in the salmon louse Lepeophtheirus salmonis

The salmon louse Lepeophtheirus salmonis is a parasitic copepod that lives and feeds on Atlantic salmon (Salmo salmar). At present, this parasite is the number one threat to the Norwegian aquaculture. The density of salmons living together in aquaculture sea cages gives the salmon lice optimal conditions for infecting and reproducing. Many delousing methods, both non-chemical and chemical, are available for the treatments of salmon lice. In salmon aquaculture, four benzoylureas-derived chitin synthesis inhibitors (CSI) are used: Hexaflumuron, diflubenzuron, teflubenzuron, and lufenuron. In-feed treatments based on diflubenzuron or teflubenzuron are used by Norwegian farmers, while hexaflumuron- and lufenuron-based treatments are known to be available in Chile. Despite many options for treating lice, current treatments are not optimal because 1) resistance can develop, 2) there are environmental issues associated with chemical treatments, and 2) non-chemical treatments can inflict stress-related damage on the fish. Therefore, new treatments are needed. By investigating the molecular biology of L. salmonis, it is hoped that new candidates can be discovered. Salmon lice have a rigid exoskeleton around the body, which must be removed for future growth to occur. During the development, the salmon louse molt to get to the next life stage. Molt, or ecdysis, is an essential process in all arthropods, and the salmon louse molts eight times to reach the adult life stage. Chitin is one of the main components of the exoskeleton, and the chitin synthesis pathway is very conserved in chitin-synthesizing organisms. The key chitin synthesis transcripts (LsGFAT, LsGNA1, LsAGM, LsUAP, and LsCHS1/2) were cloned and their sequences were analyzed to understand chitin synthesis in L. salmonis better. The results showed that L. salmonis has enzymes similar to other arthropods and these are most similar to other crustaceans' sequences, especially other copepods. The expression patterns of these chitin synthesis genes and chitinases (LsChi1, LsChi2, and LsChi4) were analyzed during the synthesis of a new exoskeleton. The formation of a new exoskeleton was analyzed in pre-adult I males. LsGFAT, LsCHS1, LsCHS2, LsChi1, and LsChi2 changed significantly during the synthesis of a new exoskeleton. The expression patterns of these genes were also analyzed in the nauplii stages. LsGFAT, LsCHS1, LsCHS2, and LsChi2 increased significantly from postmolt to premolt in nauplii II larvae, while in nauplius I, no molting-related expression patterns were observed. CSIs interfere with the chitin formation, but their exact mode of action is not known. Arthropods treated with CSIs have arrested molt, incomplete exoskeleton structure, and fertility reductions in adult females. Chitin synthesis related transcripts have been shown to be little affected by CSIs treatments, and different CSIs have been shown to require different concentrations to have the same effect on the same species. To find out if L. salmonis would react similarly to CSIs, four bioassays using hexaflumuron, diflubenzuron, lufenuron, and teflubenzuron, were performed on the nauplius I stage of L. salmonis. Bioassays revealed that the effective concentration needed to affect half of the animals tested was lowest for hexaflumuron and highest for diflubenzuron. The four chemicals had similar modes of action on the phenotype (molting arrest and abnormal copepodid with bloated appearances). The CSIs had negligible or no effects on the chitin synthesis (LsGFAT, LsGNA1, LsAGM, LsUAP, and LsCHS1/2) and chitinases (LsChi1, LsChi2, and LsChi4) transcripts. In the insects, Culex pipiens and Plutella xylostella, and in a gene-modified Drosophila melanogaster, the molecular mode of action of CSIs has been shown to target chitin synthase (CHS), the last enzyme in the chitin synthesis pathway. LsCHS1 and LsCHS2 were shown to have similar functions to insect CHS1 and CHS2, respectively. LsCHS1 was the main enzyme synthesizing chitin into the exoskeleton, and it also has a role in the development of oocytes. LsCHS2 was found to have a role in digestion and perhaps also in offspring formation. In summary, we demonstrated that the key chitin synthesis enzymes in L. salmonis are similar to homologous enzymes in other crustaceans. CSIs, do not affect key chitin synthesis or chitinase transcripts, and the phenotypes obtained in CSIs-treated larvae resemble phenotypes obtained after LsCHS1 and LsCHS2 knockdowns. This study revealed that the mode of action (here defined as functional or anatomical changes on the phenotype) of CSIs in L. salmonis larvae is similar to CSI action against other arthropods. This study contributes to the fundamental knowledge of the chitin synthesis pathway.

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