Abstract
Investigation of proteins identified in the secretory and excretory products (SEPs) of the infectious copepodid stage of the salmon louse Lepeophtheirus salmonis
Background: The salmon louse (Lepeophtheirus salmonis (Krøyer, 1837)) is a caligid copepod ectoparasite of salmonids that feeds upon the blood, mucus, and skin of its hosts. While later life stages are known to secrete virulence factors that dampen host immune responses and facilitate feeding and survival, the functional protein components of secretory and excretory products (SEPs) produced by copepodids, the initial infectious stage of L. salmonis , remain uncharacterized.
Methodology: L. salmonis copepodids were hatched from egg strings and incubated at 10°C until 7 days post-hatch. Copepodid batches (n=4 replicates) were then incubated for three hours at 10°C to allow SEP production. Adult males and females (n=2 replicates) were incubated separately at densities of three or two lice mL⁻¹, respectively, under the same conditions for comparison. SEPs were filtered, precipitated, digested with trypsin, and subjected to LC-MS/MS analysis to determine protein composition. Proteins were identified by searching LC-MS/MS data against an L. salmonis database and further analyzed in silico with SignalP and InterPro.
Results: A total of 433 distinct L. salmonis proteins were detected across copepodid samples (mean 95.5 ± 146.74 (1 S.D.)), while adult samples contained 117 proteins (mean 56 ± 12.70). Investigation of signal peptides revealed 164 copepodid and 69 adult proteins were secretory in nature. Among adult samples, 31 secreted proteins were unique to females and 10 to males. Across treatments, adult and copepodid SEPs shared 21 secreted proteins, including 8 proteases, 2 protease inhibitors, and 2 uncharacterized proteins. Of these proteins with GO annotations for biological process and cellular component, 75% were involved in proteolysis and 50% were localized extracellularly. However, adult and copepodid secretory proteins differed considerably in composition and cellular localization, with distinct proteases, protease inhibitors, and proteins implicated in immune regulation. Notably, 67% of the 9 secreted proteins with GO terms found only in adults were extracellular, compared to 30.7% of 52 copepodid-specific secreted proteins. Additionally, copepodid and adult secretions contained 23 and 4 unique uncharacterized proteins, respectively.
Conclusions: Analysis of L. salmonis copepodid secretions reveals a diverse array of proteins putatively involved in the facilitation of parasitism. The characterization of these proteins provides novel insights into early host-parasite interactions and identifies potential drug and vaccine targets, warranting further investigation through in silico analysis and in situ hybridization.