RESUMO
BACKGROUND: Control of the sea louse Caligus rogercresseyi in the Chilean salmonid industry is reliant on chemical treatments. Azamethiphos was introduced in 2013, although other organophosphates were previously used. In 2014, reduced sensitivity to azamethiphos was detected in the Los Lagos Region using bioassays. The main target of organophosphates is the enzyme acetylcholinesterase (AChE). Mutations in the AChE gene are the main cause of organophosphate resistance in arthropods, including other sea lice. In the present study, we aimed to characterize C. rogercresseyi AChE(s) gene(s) and to study the association between AChE variants and azamethiphos resistance in this sea louse species. METHODS: Samples of adult male and female C. rogercresseyi were collected in the Los Lagos Region in 2014. Twenty-four hour exposure bioassays with azamethiphos were performed to select sensitive and resistant lice. The full-length cDNA coding sequences encoding for two AChEs in C. rogercresseyi were molecularly characterized. One of the AChE genes was screened by direct sequencing in the azamethiphos-selected lice to search for variants. An additional louse sampling was performed before and after an azamethiphos treatment in the field in 2017 to validate the findings. RESULTS: The molecular analysis revealed two putative AChEs in C. rogercresseyi. In silico analysis and 3D modelling of the protein sequences identified both of them as invertebrate AChE type 1; they were named C. rogercresseyi AChE1a and 1b. AChE1a had the characteristics of the main synaptic AChE, while AChE1b lacked some of the important amino acids of a typical AChE. A missense change found in the main synaptic AChE (1a), F318F/V (F290 in Torpedo californica), was associated with survival of C. rogercresseyi at high azamethiphos concentrations (bioassays and field treatment). The amino acid change was located in the acyl pocket of the active-site gorge of the protein. CONCLUSIONS: The present study demonstrates the presence of two types of AChE1 genes in C. rogercresseyi. Although enzymatic assays are needed, AChE1a is most probably the main synaptic AChE. The function of AChE1b is unknown, but evidence points to a scavenger role. The AChE1a F/V318 variant is most probably involved in organophosphate resistance, and can be a good marker for resistance monitoring.
Assuntos
Acetilcolinesterase/genética , Antiparasitários/farmacologia , Copépodes/enzimologia , Doenças dos Peixes/parasitologia , Polimorfismo Genético/genética , Salmão/parasitologia , Sequência de Aminoácidos , Animais , Biomarcadores , Chile , Copépodes/efeitos dos fármacos , Copépodes/genética , Resistência a Medicamentos , Feminino , Masculino , Organotiofosfatos/farmacologia , Filogenia , Alinhamento de Sequência/veterináriaRESUMO
Cathepsins are proteases involved in the ability of parasites to overcome and/or modulate host defenses so as to complete their own lifecycle. However, the mechanisms underlying this ability of cathepsins are still poorly understood. One excellent model for identifying and exploring the molecular functions of cathepsins is the marine ectoparasitic copepod Caligus rogercresseyi that currently affects the Chilean salmon industry. Using high-throughput transcriptome sequencing, 56 cathepsin-like sequences were found distributed in five cysteine protease groups (B, F, L, Z, and S) as well as in an aspartic protease group (D). Ontogenic transcriptome analysis evidenced that L cathepsins were the most abundant during the lifecycle, while cathepsins B and K were mostly expressed in the larval stages and adult females, thus suggesting participation in the molting processes and embryonic development, respectively. Interestingly, a variety of cathepsins from groups Z, L, D, B, K, and S were upregulated in the infective stage of copepodid, corroborating the complexity of the processes involved in the parasitic success of this copepod. Putative functional roles of cathepsins were conjectured based on the differential expressions found and on roles previously described in other phylogenetically related species. Moreover, 140 single nucleotide polymorphisms (SNP) were identified in transcripts annotated for cysteine and aspartic proteases located into untranslated regions, or the coding region. This study reports for the first time the presence of cathepsin-like genes and differential expressions throughout a copepod lifecycle. The identification of cathepsins together with functional validations represents a valuable strategy for pinpointing target molecules that could be used in the development of new delousing drugs or vaccines against C. rogercresseyi.
Assuntos
Catepsinas/genética , Copépodes/enzimologia , Copépodes/patogenicidade , Doenças dos Peixes/parasitologia , Perfilação da Expressão Gênica , Salmão/parasitologia , Animais , Copépodes/genética , Copépodes/crescimento & desenvolvimento , Feminino , Masculino , Filogenia , Polimorfismo de Nucleotídeo Único , TranscriptomaRESUMO
Acute silver effects on whole-body ion regulation and Na(+),K(+)-ATPase activity were evaluated in the euryhaline copepod Acartia tonsa. Experiments were run at 20 degrees C, three different salinities (5, 15 and 30 ppt), in either the absence or the presence of food (diatom Thalassiosira weissflogii; 2 x 10(4)cells/mL). Standard static-renewal procedures were used. Copepods were acutely (48 h) exposed to silver (AgNO(3)) concentrations equivalent to the 48-h EC10 (dissolved Ag=3, 49, and 94 microg/L), 48-h EC30 (dissolved Ag=5, 71, and 125 microg/L) or 48-h EC50 (dissolved Ag=7, 83, and 173 microg/L) values in the absence of food or to the 48-h EC50 (dissolved Ag=35, 90, and 178 microg/L) values in the presence of food. These values were previously determined under the same experimental conditions at salinities 5, 15 and 30 ppt, respectively. Endpoints analyzed were whole-body ion concentrations (Na(+), Cl(-), and Mg(2+)) and Na(+),K(+)-ATPase activity. In starved copepods, lower whole-body Na(+) and Mg(2+) concentrations were observed in salinities 5 and 30 ppt, respectively. Also a higher whole-body Na(+),K(+)-ATPase activity was observed in all salinities tested. Data from fed copepods indicate that all these salinity effects were completely associated with starvation. Silver exposure induced a decrease in the whole-body Mg(2+) concentration in starved copepods in salinities 5 and 30 ppt and a Na(+),K(+)-ATPase inhibition in both starved and fed copepods in all salinities tested. Thus, food addition in the experimental media completely protected against silver effects on Mg(2+) concentration, but not against those on Na(+),K(+)-ATPase activity. In starved copepods, enzyme inhibition was dependent on silver concentration and a relationship between this parameter and mortality was observed in all salinities tested. Therefore, Na(+),K(+)-ATPase molecules seem to be a key site for acute silver toxicity in marine invertebrates, as reported for freshwater fish and crustaceans.