Caligus rogercresseyi serine proteases: Transcriptomic analysis in response to delousing drugs treatments

    Caligus rogercresseyi serine proteases: Transcriptomic analysis in response to delousing drugs treatments Diego Valenzuela-Miranda, Cristian Gallardo-Esc´arate PII: DOI: Reference:

S0044-8486(16)30427-6 doi: 10.1016/j.aquaculture.2016.08.027 AQUA 632286

To appear in:

Aquaculture

Received date: Revised date: Accepted date:

4 February 2016 18 August 2016 19 August 2016

Please cite this article as: Valenzuela-Miranda, Diego, Gallardo-Esc´ arate, Cristian, Caligus rogercresseyi serine proteases: Transcriptomic analysis in response to delousing drugs treatments, Aquaculture (2016), doi: 10.1016/j.aquaculture.2016.08.027

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ACCEPTED MANUSCRIPT Caligus rogercresseyi serine proteases: Transcriptomic analysis in response to

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delousing drugs treatments

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Valenzuela-Miranda, Diego & Gallardo-Escárate, Cristian*

Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Sustainable

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Aquaculture Research (INCAR), Department of Oceanography, Biotechnology Center,

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University of Concepción, Concepción, Chile. *Corresponding author: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT Trypsin and chymotrypsin are the most abundant serine proteases in crustaceans, and these

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typically fulfill roles in dietary protein digestion and the immune response. However, recent

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evidence has demonstrated that serine proteases are also capable of catalyzing the degradation of non-protein molecules, such as pyrethroid insecticides. The present study identified trypsin and

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chymotrypsin in the salmon louse Caligus rogercresseyi and evaluated the transcriptional

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modulation of these after exposure to the delousing compounds deltamethrin and azamethiphos. High-throughput transcriptome sequencing identified 44 putative trypsin-like and 7 putative

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chymotrypsin-like transcripts in C. rogercresseyi that showed differentiated transcriptional modulation after drug exposure. Overall, deltamethrin exposure modulated the transcriptional

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response greater than azamethiphos. A sex-biased transcriptional response was evidenced for Cr-

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Tryp1-6-10-36 and 41 under exposure to either antiparasitic agent, with upregulation 100-fold

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greater than the control group. Together, these results suggest that trypsin-like transcripts in C. rogercresseyi might play a role in metabolizing delousing drugs. The identification of trypsinlike transcripts, together with functional validations, is a valuable strategy for pinpointing target molecules that could be used in developing new management strategies for C. rogercresseyi.

Keywords: Deltamethrin, Azamethiphos, Trypsin, Chymotrypsin, Transcriptomics, qPCR.

ACCEPTED MANUSCRIPT BACKGROUND The sea louse Caligus rogercresseyi is the most prevalent ectoparasite that affects the salmon

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industry in Chile (Carpio, Basabe, Acosta, Rodríguez, Mendoza, Lisperger, Zamorano, González,

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Rivas, Contreras, Haussmann, Figueroa, Osorio, Asencio, Mancilla, Ritchie, Borroto, Estrada, 2011). Mitigation strategies against sea lice are based on delousing compounds such as

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pyrethroids and organophosphates (Olsvik, Ørnsrud, Lunestad, Steine, Fredriksen, 2014).

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However, intensive use of these agents has exerted strong selective pressures, leading to loss of sensitivity to pesticides in some sea lice populations (Bravo, Silva, Monti, 2012; Helgesen,

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Bravo, Sevatdal, Mendoza, Horsberg, 2014). This effect has resulted in greater quantities of delousing compounds being used, translating into economic and environmental concerns for the

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Chilean aquaculture industry.

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The emergence of the insecticide resistance has been reported as the ability of the sea

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louse to modify its genetic, biochemical, and cellular machineries (Pedra, McIntyre, Scharf, Pittendrigh, 2004). In insects, the following models have been described for the development of resistance: penetration resistance (thicker cuticle for decreased entry of insecticides); behavioral resistance (knockdown resistance); metabolic resistance and target site modifications (Mamidala, Asela, Wijeratne, Kornacker, Sudhamalla, Rivera-Vega, Hoelmer, Meulia, Jones, Mittapalli, 2012). For metabolic resistance, the modification of enzymes such as cytochrome P450 oxidase, glutathione S transferase, and carboxylesterases plays a pivotal role in drug resistance (Adelman, Kilcullen, Koganemaru, Anderson, Anderson, Miller, 2011). Particularly, carboxylesterases possess a catalytic triad (serine, histidine, and glutamic acid) that can hydrolyze the ester bond of deltamethrin, thus destabilizing its structure and promoting its degradation (Moataza, Mohamed, Taha, 2008; S. Reissmann, Greiner, 1992; Wu, Tian, Wu, Langdon, Kurtis, Shen, Ma, Li, Gu, Hu, Zhu, 2004; Yang, Sun, Zhang, Qian, Sun, Ma, Sun, Hu, Tan, Wang, Zhu, 2008). Certain

ACCEPTED MANUSCRIPT enzymes with esterase activity, such as the serine proteases trypsin and chymotrypsin, have shown catalytic triads similar to those found in carboxylesterases (Moataza, Mohamed, Taha,

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2008; S. Reissmann, Greiner, 1992). This has led to a search for novel functions of serine

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proteases beyond the classical roles in protein digestion, coagulation, the immune response and signal transduction (Ramalho-Ortigão, Kamhawi, Rowton, Ribeiro, Valenzuela, 2003).

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The development of resistant traits are often associated as the result of a strong selective

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pressures, where certain individuals can overcome these adverse conditions and leave offspring (James, 1989 ). Over the last 20 years, around 450 arthropod species have presented pesticide-

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resistant traits (Soderlund, Bloomquist, 1990). In helminthes and mosquitoes, the main mechanism against antiparasitic compounds has been associated with a lost or reduced affinity

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between receptors and drugs (ffrench-Constant, Rocheleau, Steichen, Chalmers, 1993; Köhler,

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2001). Moreover, several transcripts related to proteolysis have been linked to insecticide

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resistance, given that a balance between the degradation and synthesis of novel proteins is required to overcome drug exposure (Pedra, McIntyre, Scharf, Pittendrigh, 2004). In this context, an overexpression of transcripts associated with a peptidase has been reported in DDT-resistant strains of Drosophila melanogaster and Anopheles gambiae (Bonizzoni, Afrane, Dunn, Atieli, Zhou, Zhong, Li, Githeko, Yan, 2012; Pedra, McIntyre, Scharf, Pittendrigh, 2004). Likewise, strong proteolytic activity was observed in azamethiphos-resistant strains of Musca domestica (Ahmad, 1970; Vontas, Blass, Koutsos, David, Kafatos, Louis, Hemingway, Christophides, Ranson, 2005). Furthermore, a comparative study between deltamethrin-resistant and -susceptible strains of A. gambiae reported higher levels of transcripts with peptidase and trypsin domains, suggesting the participation of these enzymes in pesticide resistance (Bonizzoni, Afrane, Dunn, Atieli, Zhou, Zhong, Li, Githeko, Yan, 2012). It was later demonstrated that serine proteases from Culex pipens pallenses could directly catalyze the degradation of insecticides such as

ACCEPTED MANUSCRIPT deltamethrin, thus conferring resistance to this pesticide (Xiong, Fang, Chen, Yang, He, Zhou, Shen, Ma, Sun, Zhang, Zhu, 2014; Yang, Sun, Zhang, Qian, Sun, Ma, Sun, Hu, Tan, Wang, Zhu,

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2008). Despite this evidence, serine proteases in sea lice have been mainly associated with the

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inflammatory response in Atlantic salmon (Salmo salar) during infection (Firth, Johnson, Ross, 2000), but these have not yet been linked to the development of pesticide resistance in C.

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rogercresseyi (Helgesen, Bravo, Sevatdal, Mendoza, Horsberg, 2014).

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A key step in the management of pesticide resistance is identifying the underlying molecular mechanism for this phenomenon, such as genetic variations among individuals, but

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few genomic studies have examined the molecular underpinnings for drug resistance in C. rogercresseyi. However, high-throughput sequencing has revolutionized the field of functional

expression

analyses,

thus

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differential

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genomics in non-model organisms (Ekblom, Galindo, 2011), and it is a powerful tool for building

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foundation

towards

understanding

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ecophysiological adaptations (Mamidala, Asela, Wijeratne, Kornacker, Sudhamalla, RiveraVega, Hoelmer, Meulia, Jones, Mittapalli, 2012). For instance, RNA-Seq analysis has been used to show the modulation of the antioxidant system in C. rogercresseyi during exposure to deltamethrin (Chavez-Mardones, Gallardo-Escárate, 2014). Nevertheless, knowledge of drug response mechanisms and the proteins involved in such mechanisms is still limited. Considering all of the above, the present study conducted high-throughput transcriptome sequencing in adult C. rogercresseyi males and females exposed to either deltamethrin or azamethiphos in order to 1) identify sequences related to trypsin and chymotrypsin, and 2) assess the modulation of these after exposure to delousing pesticides against the sea louse C. rogercresseyi.

METHODS

ACCEPTED MANUSCRIPT Samples and bioassays with deltamethrin and azamethiphos Female and male specimens of C. rogercresseyi were collected from recently harvested fish at a

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salmon farm located in the X Region of Chile (41º40’48.5”S; 73º02’31.34”E¨). Deltamethrin

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(AlphaMax) was prepared via serial dilutions with seawater to four concentrations: 0, 1, 2 and 3 parts per billion (ppb). Stock solution of 1 ppm (parts per million) was also prepared for each

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bioassay by diluting 100 µl of deltamethrin in 999.9 ml of seawater. With respect to

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azamethiphos (Bayer), stock solutions of 333 ppm were diluted in methanol (5 mg azamethiphos + 15 ml ethanol) to four concentrations: 0, 1, 3 and 10 ppb. For each treatment, 30 individuals

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(fifteen females and fifteen males) were exposed to each delousing compound dilution in Petri plates containing 50 ml of seawater. Each experiment was carried out in triplicate, and the

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exposure period to either deltamethrin or azamethiphos was 40 and 30 min according the

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manufacturer’s instructions, respectively. During exposure, salmon lice were maintained at 12ºC.

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After 24 h, the organisms were fixed in RNAlater® RNA Stabilization Reagent (Ambion, USA) and stored at -80ºC until RNA extraction. The protocols for bioassays were performed according to the SEARCH Consortium (2006)

RNA isolation and Illumina sequencing According to the bioassays results, individuals exposed to 3 ppb of either deltamethrin or azamethiphos were selected for high-throughput transcriptome sequencing. For this purpose, total RNA pooled from 10 different individuals was considered for cDNA libraries construction using TruSeq RNA Sample Preparation kit v2 (Illumina, San Diego, CA, USA). Two biological replicates were sequenced by the MiSeq (Illumina) platform using sequencing runs of 2  250

ACCEPTED MANUSCRIPT paired-end reads at the Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary

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Center for Aquaculture Research (INCAR), University of Concepción, Chile.

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Identification of differentially expressed serine protease transcripts

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Molecular characterization of serine protease transcripts was conducted using the published 83,444 contigs yielded from de novo transcriptome assembly for C. rogercresseyi as a query

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sequence (Gallardo-Escarate, Valenzuela-Munoz, Nunez-Acuna, 2014). Contig annotation was

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performed using the CLC Genomics Workbench software (v7.1, CLC Bio, Denmark). For this, the nucleotide sequences were blasted against a non-redundant protein database (BLASTx) using

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a word size = 3, gap cost existence = 11, gap cost extension = 1, and a BLOSUM62 matrix.

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Sequences with an E-value < 1E-10 were considered. Furthermore, in silico protein analysis was conducted using the Geneious Pro bioinformatics software (v5.1.7, Aarhus Denmark). From pre-

predicted.

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selected contigs, open reading frames (ORFs) were identified, and protein sequences were

Sequence data analysis and phylogenetic relationships The trypsin- and chymotrypsin-like sequences from C. rogercresseyi were used to query all nonredundant protein sequences curated at the NCBI using the online program Protein Blast (Blastn and

BLASTx

algorithms

used

http://www.ncbi.nlm.nih.gov/).

To

determine

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identity/similarity of amino acids between sequences, a multiple alignment of trypsin- and chymotrypsin-like sequences in C. rogercresseyi and of publicly available proteins for other species was performed with the MUSCLE (Edgar, 2004) alignment algorithm included in Geneious Pro (v8.0.4, Biomatters, New Zealand). Later, a phylogenetic tree was constructed

ACCEPTED MANUSCRIPT based on a Blosum62 matrix, with a gap open penalty of 12, gap extension penalty of 3, ends-free global alignment, Jukes–Cantor genetic distance model, and the neighbor-joining method. The

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data were bootstrapped 1000 times using Chinchilla lanigera and Aedes aegyptii for

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chymotrypsin- and trypsin-like sequences, respectively, as out-groups in order to estimate the internal stability of each node. The structural motifs in proteins were analyzed with the SMART

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online tool (http://smart.embl-heidelberg.de/) (Letunic I, Copley RR, Schmidt S, Ciccarelli FD,

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T, 2004), and signal peptides within predicted proteins were sought using the SecretomeP (http://www.cbs.dtu.dk/services/

SecretomeP)

and

SignalP

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(http://www.cbs.dtu.dk/services/SignalP) software.

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Analysis of serine protease transcription in silico

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Different RNA-Seq analyses were performed for each library by mapping filtered reads against

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the putative serine protease transcripts. The considered parameters included a minimum read length fraction = 0.8, minimum read similarity fraction = 0.9, and unspecific read match limit = 10 in relation to the reference. Expression values were estimated as reads per kilobase of exon model (RPKM) and then normalized by totals, using state numbers in reads per 1,000,000. A proportion-based test was conducted to identify the differentially expressed genes among different transcripts, with false discovery rate-corrected P value < 0.05. The Manhattan distance metric was used for hierarchical cluster analysis, and genes were classified as differentially expressed if they exhibited a more than two-fold change between the two samples. Statistical significance was established at P < 0.05 based on the Kal's Z-test (Kal AJ, van Zonneveld AJ, Benes V, van den Berg M, MG, 1999) between pairs of females and males exposed to deltamethrin or azamethiphos.

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RT-qPCR validation

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To validate the expression profile obtained through RNA-seq analysis, quantitative real time PCR

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(qPCR) analysis was performed on seven candidate genes (6 trypsins and 1 chymotrypsin) selected from those most differentially expressed during deltamethrin and azamethiphos

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exposure. Here, consensus contigs were selected and used as a primer design template for the

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qPCR assay of relative expression, including the β tubulin gene as an endogenous control (Table 1) as previously validated (Gallardo-Escarate, Valenzuela-Munoz, Nunez-Acuna, Chavez-

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Mardones, Maldonado-Aguayo, 2014). Primer design was performed using the Primer3 tool included in Geneious Pro (v8.0.4, Biomatters, New Zealand). The qPCR runs were performed

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with StepOnePlus™ (Applied Biosystems, Life Technologies, USA) and cDNA was synthetized

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using Total RNA extracted from pools of individuals from both deltamethrin and azamethiphos

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bioassays using the Ribopure™ kit (Ambion®, Life Technologies™, USA) following the manufacturer's instructions. In addition, primers with amplification efficiency between 95-105% were considered for further analyses and the 2−ΔΔCT Method was used to determine the relative quantity (RQ) for each gene (Livak, Schmittgen, 2001). Three technical replicates were measured for each one of the control and challenged groups using the Maxima® SYBR Green/ROX qPCR Master Mix (Thermo Scientific, USA). The amplification conditions were as follows: 95°C for 10 min, 40 cycles at 95°C for 30 s, 60ºC for 30 s, and 72°C for 30 s. The data obtained were analyzed through the Kruskal-Wallis test with the Statistica software (v7.0, StatSoft, Inc.). Statistically significant differences were accepted with a P < 0.05. SNPs mining from trypsin-like genes Single nucleotide polymorphism (SNP) candidates were identified from the de novo assembly of female and male C. rogercresseyi exposed to deltamethrin or azamethiphos using the CLC

ACCEPTED MANUSCRIPT Genomics Workbench and quality-based variant detection. SNPs mining followed a neighborhood radius = 11, a maximum gap and mismatch count = 2, a minimum neighborhood

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quality = 15, a minimum coverage = 20, a minimum variant frequency (%) = 25.0, and a

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maximum expected alleles (ploidy) = 2. The frequencies of SNPs found in all contigs were

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calculated using the same software and were tabulated in Excel spreadsheets for analysis.

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RESULTS Identification of putative trypsin-like transcripts

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Based on the reference transcriptome available for C. rogercresseyi (Gallardo-Escarate, Valenzuela-Munoz, Nunez-Acuna, 2014), 44 putative trypsin-like and 7 putative chymotrypsin-

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like transcripts were identified. For use in future discussions, the trypsin and chymotrypsin-like

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sequences discovered in this study were denoted as Cr-Tryp 1-6-10-36-41-42 and Cr-Chym 1.

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Seven selected candidate genes with the greatest differential expressions to deltamethrin and azamethiphos exposure were selected for sequence analysis. Signal peptide prediction revealed a signal peptide in the amino terminal end, with a putative cleavage site, for Cr-Tryp 1-6-10-36-4142 and Cr-Chym1 (Table 2). Structural analysis through SMART showed that trypsin- and chymotrypsin-like sequences also contained the serine protease domain with signature and putative endopeptidase activities. Furthermore, BLASTp analysis established that the predicted amino acid sequences were homologous with trypsin in other crustaceans, including Lepeophtheirus salmonis and Caligus clemensi (Table 2). Conserved cysteine residues and a catalytic triad were also identified by multiple sequence alignments of serine protease sequences (Fig.1-2). Homology and phylogenetic analysis

ACCEPTED MANUSCRIPT BLASTx data showed that the deduced amino acid sequences shared high similarity with other crustacean species (Table 2). For instance, Cr-Tryp41 was 60% similar to trypsin-like from L.

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salmonis, whereas Cr-Tryp10 shared a 74% identity with trypsin-like from C. clemensi. The

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evolutionary relationships of serine proteases from C. rogercresseyi were evaluated after aligning the amino acid sequences. Subsequent phylogenetic analysis using the neighbor joining method

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with the Jukes-Cantor genetic distance model demonstrated well-sustained nodes in the

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consensus and topology trees, as shown by high bootstrap values. For trypsin-like sequences, phylogenetic analysis shows three well-supported clades (Fig. 3). The clade represented in red

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gathers different types of trypsin-2 like sequences, including the one previously described for the salmon lice L. salmonis. On the other hand, trypsin-1 sequences were grouped in two clades, a

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green one including sequences for anionic trypsins and a blue one grouping the remaining

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trypsin-1 like sequences described for C. rogercresseyi (Fig. 3) with bootstrap values around

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100%. On the other hand, analysis of chymotrypsin-like from C. rogercresseyi revealed high homology (75%) of Cr-Chym 1 with L. salmonis. Phylogenetic analysis revealed three supported clades, including a green one for chymotrypsin-C, a blue one for chymotrypsin-2 and a red one gathering sequences for chymotrypsin-like sequences described for different arthropods (Fig. 4).

RNA-Seq analysis of trypsin-like genes and RT-qPCR validation Trypsin-likes sequences from C. rogercresseyi displayed differential expression patterns during the lifecycle, and sequences such as Cr-Tryp 13-23-35-7-15-33 and 34 were mainly associated with early larval stages while others, such as Cr-Tryp 37-38-1-42-43-40-41 and 36, had higher transcript expression in the adult stages (Fig. 5). Under deltamethrin treatment, several serine proteases were associated with sex-dependent transcription activity (Fig. 6). Variants such as CrTryp 1-6 and 41 were differentially expressed in both males and females, reaching expressions up

ACCEPTED MANUSCRIPT to 100-fold more than the control group (Tables 3, 4). Interestingly, these variants were also upregulated after azamethiphos exposure (Fig. 7). However, only 9.5-fold changes were

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estimated as compared to the control group (Tables 5, 6). To verify these results, qPCR

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validations were performed, and the results revealed different expression patterns for both pesticides, with a higher expression levels in individuals exposed to deltamethrin (see Fig. 1S).

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Regarding individuals treated with deltamethrin, sex-dependent transcriptional responses were

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found. Thus, Cr-Tryp 1-10 and 41 variants were upregulated in males more than females exposed to 1, 2, and 3 ppb (Fig. 8A). On the other hand, individuals exposed to azamethiphos revealed

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lower transcriptional modulation. Herein, Cr-tryp10 was the unique transcript that displayed significant differences between sexes of sea lice (Fig. 8B). Interestingly, the relative expression

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of Cr-Tryp10 was enhanced with increasing azamethiphos concentrations (Fig. 1S). Finally, the

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Cr-Tryp 2-6-10-36-41-42 and Cr-Chym1 expression values obtained by qPCR were correlated

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with those obtained by RNA-Seq, supporting that the expression values of sexes and treatments were relatively constant (data not shown).

SNPs mining from Trypsin-like genes Among the different serine protease transcripts identified for C. rogercresseyi, 16 sequences evidenced the presence of SNPs mutations between males and females (Tables S1-4). Among these, several mutations were present exclusively in males or females. For instance, mutations in Cr-Tryp 38 and 32 were present exclusively in females, while SNPs in Cr-Chym 2, Cr-Chym 3, and Cr-Tryp29 were just present in males. In addition, putative SNPs were classified as transitions (A/G or C/T) or transversions (C/G or A/T). Cr-Tryp 39 showed five SNPs identified in the ORF region, as well as 5' and 3'UTR regions. In turn, Cr-Tryp 27 presented one SNP in the ORF region (Table S1).

ACCEPTED MANUSCRIPT DISCUSSION The transcriptome represents the complete gene expression profile of an organism under a

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defined condition. Previous reports have used high-throughput sequencing analysis to evaluate

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different developmental stages of the sea louse, from early larvae, such as the nauplius stage, to adult stages and these transcriptomes were used for comparative analyses (Gallardo-Escarate,

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Valenzuela-Munoz, Nunez-Acuna, 2014). Furthermore, transcriptomes obtained in the present

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study from females and males exposed to deltamethrin or azamethiphos were also assessed (Nunez-Acuna, Gallardo-Escarate, 2015; Valenzuela-Miranda, Nunez-Acuna, Valenzuela-

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Munoz, Asgari, Gallardo-Escarate, 2015; Valenzuela-Munoz, Sturm, Gallardo-Escarate, 2015). To assess the role of peptidases during the drug response in C. rogercresseyi, several serine

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protease transcripts were identified from the sea lice transcriptome. Through BLAST and

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SMART platforms, conserved domains were identified for 51 serine proteases corresponding to

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trypsin- and chymotrypsin-like sequences. The transcriptional modulation of these variants was assessed by RNA-Seq after exposure to deltamethrin and azamethiphos, and based on transcriptional modulation; seven serine proteases were selected for further analyses. Homology and phylogenetic analyses revealed a high degree of identity for serine proteases from C. rogercresseyi with trypsin and chymotrypsin from other crustaceans and insects. This result is consistent with previous reports where trypsin from L. salmonis indicated 46% of similarity with crustaceans and insects (Johnson, Ewart, Osborne, Delage, Ross, Murray, 2002). Additionally, signal peptide analysis was performed for the most regulated serine proteases after drug exposure. Thus, signature motifs associated to protein secretion through the classical pathway were identified in Cr-Tryp 1 and 29, meanwhile motifs related with protein secretion through the non-classical pathway were present in Cr-Tryp 36. These results evidenced that these serine proteases could synthetized and secreted by C. rogercresseyi cells and therefore

ACCEPTED MANUSCRIPT they could be acting at extracellular level. It has been described that several serine proteases from other crustaceans contributes to extracellular digestion (Rojo, Muhlia-Almazan, Saborowski,

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García-Carreño, 2010) and therefore this particular trypsin might have a potential role in the

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extracellular digestion of drugs in C. rogercresseyi. On the other hand, multiple protein alignment revealed the presence of eight conserved cysteines responsible for forming disulfide

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bonds and a catalytic triad that are characteristic of the S1 serine protease family (Carter, Wells,

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1988). However, differences in the signal peptide and catalytic triad among serine proteases suggest that these variants could be involved in different molecular processes. These differences

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have also been described in L. salmonis, in which seven different isoforms of trypsin-like proteins with variations in the signal peptide were reported (Johnson, Ewart, Osborne, Delage,

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Ross, Murray, 2002).

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Recent investigations suggest that organophosphorus insecticide resistance is conferred by

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two distinct genetic mechanisms. The first is based on an alteration of acetyl cholinesterase to an insensitive form through a mutation within its active site (Hemingway, 2000). The second mechanism meanwhile could be mediated by the esterase activity presented in this enzyme (Pan, Guo, Gao, 2009). In this context, the similarity between the catalytic triads present in serine proteases with those in carboxylesterases, together with the esterase activity shared between both enzymes, could also be related with the degradation of pesticides in C. rogercresseyi. Analysis of differentiated expression patterns for C. rogercresseyi at distinct developmental stages showed that some transcripts, such as Cr-Tryp 13-23-35-7 and 15, were more expressed in the larval stages, whereas Cr-Tryp 1-42-43-41 and 36 were more expressed in adults. Similar results have been found in L. salmonis, suggesting a possible function of the earlystage transcripts in regulating the yolk degradome (Skern-Mauritzen, Frost, Dalvin, Kvamme, Sommerset, Nilsen, 2009). In turn, expression values obtained by RNA-Seq and qPCR analyses

ACCEPTED MANUSCRIPT showed that serine proteases such as Cr-Tryp2 were positively regulated in females and males exposed to either deltamethrin or azamethiphos. Interestingly, high expression ratios were

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observed in transcripts such as Cr-trypsin2-9-10-14-18-19- 22-23-24-28-29 and 30, in which

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individuals exposed to deltamethrin evidenced expression values up to 80- and 2-fold more in females and males, respectively, as compared to the control group. Other transcripts, such as Cr-

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Tryp 15-35 and 40, were expressed only in females exposed to deltamethrin while transcripts Cr-

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Tryp 1-25-30-31 and Cr-Chym2 were expressed only in males. When individuals exposed to azamethiphos were analyzed for the same genes, only Cr-Tryp 2-10-14-15-18-19-24 and 25 were

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upregulated, but differences in expression against the control group were only present in males for Cr-Tryp 10-19 and 24 (fold change = 2.4,18.05, and 3.38, respectively) and not at all in males

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(Tables 5, 6).

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In general, the fold-change found in individuals exposed to azamethiphos (variation fold

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change 18- to 2-fold) was minor compared to those exposed to deltamethrin (variation fold change 80- to 2-fold). These differences could be explained by the frequency and time during which each drug has been used (James, 1989 ). For instance, the use of deltamethrin as a pesticide for sea lice has been more extensive than azamethiphos (Marín, Martin, Lewis, 2014), and it is consequently expected that this prolonged exposure has promoted the development of deltamethrin resistance more than azamethiphos resistance in C. rogercresseyi (Helgesen, Bravo, Sevatdal, Mendoza, Horsberg, 2014). Therefore, the strong selective pressure generated by the use of deltamethrin in sea lice management could explain the higher transcription values obtained in individuals exposed to deltamethrin as compared to those treated with azamethiphos. Besides the hypothesis that serine proteases could catalyze the degradation of pesticides, it has also been proposed that the role of these enzymes during the drug response is just a result of the energy requirements necessary to develop mitigation mechanisms (Pedra, McIntyre,

ACCEPTED MANUSCRIPT Scharf, Pittendrigh, 2004). However, the present results are not consistent with this postulation since high differences were obtained when comparing between treatments. If serine proteases

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expression pattern independent of the drug would be expected.

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were just associated with supplying the energy necessary for the drug response, a similar

For sex-dependent transcription values, RPKM values were generally higher in females

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than in males. These results are consistent with evidence that suggest that C. rogercresseyi

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females are less sensitive to deltamethrin than males (Helgesen, Bravo, Sevatdal, Mendoza, Horsberg, 2014). This differentiated resistance between sexes has also been reported in sea lice

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treated with emamectin (Bravo, 2010) and exposed to hydrogen peroxide, females being less susceptible to delousing compounds (Treasurer, Wadsworth, Grant, 2000). These differences

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have been attributed to the longer life cycle of females (Bravo, 2010), but loss of sensitivity in

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arthropods is closely related to the amount and frequency of pesticide application (Denholm,

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Devine, 2013). Despite that the abundance of serine protease transcripts increased with higher pesticide concentrations, this tendency was not maintained at the highest concentrations. This could be a result of the drug response mechanism having a high energetic cost, as has been demonstrated in the fly Lucilia cuprina (McKenzie, Whitten, Adena, 1982). Among genetic mutations, SNPs have been extensively studied since they can directly result in a phenotypic change (Liao, Lee, 2010). Considering this, SNPs were searched on different serine protease transcripts identified for C. rogercresseyi. As a result, 16 different SNP’s were associated to serine proteases. Analysis transcripts variants in females exposed to deltamethrin revealed that Cr-Tryp39 was upregulated up to 21-fold times more than the control group, while Cr-Tryp 27 was 2-fold. Similar results were shown for males exposed to deltamethrin, with a 9-fold change for Cr-Tryp 39 and a 3-fold change for Cr-Tryp 27. In addition, both females and males exposed to azamethiphos evidenced a 4-fold change for Cr-

ACCEPTED MANUSCRIPT Tryp 39 and a 2.8-fold change for Cr-tryp 27 as compared to the control group. The presence of SNP among the transcripts that evidenced a positive regulation after drug exposures, makes these

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mutations suitable candidates to assesses whether these variations can produce a functional

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variation among serine proteases. For instance, some mutations could be responsible for a change in the catalytic site of trypsin, and this alteration could therefore be associated with a change in

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the affinity of the enzyme with the drug. This relationship between SNP modification and

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phenotypic response has been previously shown in arthropods. For instance, a mutation within the ORF of cathepsin B is associated with resistance/susceptibility to the white spot virus that

MA

affects to Fenneropenaeus chinensis (Li, Meng, Kong, Luo, Luan, Cao, Liu, Pang, Shi, 2013). The present results are a promising step towards determining if the presence of polymorphisms in

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these sequences could be related with drug resistance in C. rogercresseyi through variations in

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the ORF or in regions that regulate mRNA stabilization, as observed in other species (Liaoa,

AC CE P

Leeb, 2010). However, further experiments are still required to fully discovery the role of serine proteases in the drug resistance mechanisms of sea lice.

CONCLUSIONS

This study identified the presence of 51 serine proteases, including 44 trypsin-like sequences and 7 chymotrypsin-like sequences, in C. rogercresseyi that were differentially expressed in individuals exposed to deltamethrin or azamethiphos. The putative role of trypsin and chymotrypsin in resistance to these pesticides was analyzed using RNAseq and qPCR. The results will accelerate efforts to understand the biochemical and genetic bases of insecticide resistance in C. rogercresseyi as well as aid in the implementation of new management strategies for sea lice control.

ACCEPTED MANUSCRIPT AUTHOR’S INFORMATION Dr. Cristian Gallardo-Escárate. E-mail: [email protected]

PT

Laboratory of Biotechnology and Aquatic Genomics

RI

Interdisciplinary Center for Aquaculture Research (INCAR)

SC

University of Concepción, Concepción, Chile

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ACKNOWLEDGMENTS

This work was supported by FONDAP (15110027), FONDECYT (1150077) awarded by

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CONICYT-Chile and CONICYT-PCHA/Doctorado Nacional/2015-21150728 grant

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REFERENCES

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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. Multiple-alignment from conserved domain of trypsin-like transcripts. Putative

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residues in the catalytic triad are shown in blue, and conserved cysteines are shown in

RI

magenta. Multiple alignment of Cr-trypsin-like transcripts from C. rogercresseyi and

SC

other trypsin-like members from Drosophila grimshawi (GH22866), Drosophila mojavensis (GI21243), Drosophila simulans (GD15412), Musca domestica

(KDR22111),

Panulirus

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(XP_005175140), Locusta migratoria (DAA64573), Zootermopsis nevadensis argus

(ADB66713),

Fenneropenaeus

chinensis

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(ACQ45454), Marsupenaeus japonicus (ACE80257), Litopenaeus vannamei (CAA75311), Lepeophtheirus salmonis (ADD38129), and C. rogercresseyi

alignment.

TE

D

(ACO11163), (ACO10347), (BT077108). Gaps are indicated by dashes to improve

AC CE P

Figure 2. Multiple-alignment from conserved domain of chymotrypsin-like transcripts. Putative residues in the catalytic triad H, D, and S are shown in blue, conserved cysteines are shown in red, and the putative residues involved in the substrate binding pockets of chymotrypsin are shown in yellow. Multiple alignment of chymotrypsin-like transcripts from C. rogercresseyi and other species, including Marsupenaeus japonicus (BAI49929), chymotrypsin-like elastase from Lepeophtheirus salmonis (ADD38646), putative chymotrypsin from Lutsomyia longipalpis (ABV60309) (ABV60292), chymotrypsin 1 from Tenebrio molitor (ABC88746), chymotrypsin B from Zootermopsis nevadensis (KDR14900), chymotrypsin-like elastase from Chinchilla lanigera (XP_005409930), chymotrypsin C from C. rogercresseyi (ACO10196),

chymotrypsin-like

from

Daphnia

pulex

(EFX79600)

and

ACCEPTED MANUSCRIPT chymotrypsin-like from Bombus patiens (XP_003492130). Gaps are indicated by dashes to improve alignment.

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Figure 3. Phylogenetic relationships among trypsin-like sequences of C. rogercresseyi and

RI

sequences reported from several species. Relationships with Zootermopsis nevadensis (KDR22111.1), C. rogercresseyi (BT077108.1), (ACO10347.1), Takifugu rubripes Panulirus

argus

(ADB66713.1),

SC

(XP_003979274.1),

Locusta

migratoria

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(DAA64573.1) and Lepeophtheirus salmonis (ADD38129.1). The sequences grouped in different clades are demarcated in distinct colors. The tree was constructed using

MA

the Neighbor-joining method and bootstrapping (1,000 iterations). The support values at the nodes are shown. Ciprinus carpio (AEZ68786) was used as an out-group.

D

Figure 4. Phylogenetic relationships among chymotrypsin sequences of C. rogercresseyi and

TE

sequences reported from several species. Relationships with Lepeophtheirus salmonis

AC CE P

(ADD24483), Lutzomyia longipalpis (ABV60291), (ABV60301), Poecilia reticulate (XP_008400098), Bombus impatiens (XP_003488148), Daphnia pulex (EFX79589), Marsupenaeus japonicus (BAI49929), Tenebrio molitor (ABC88746) and C. rogercresseyi (ACO10196). The sequences grouped in different clades are demarcated in distinct colors. The tree was constructed using Neighbor-joining method and bootstrapping (1,000 iterations). Chinchilla lanigera (XP_005409930) was used as an out-group. Figure 5. Heatmap of Cr-Tryp and Cr-Chym from different developmental stages of C. rogercresseyi. Transcript abundance is represented as RPKM values. Color scales show relative transcript expression.

ACCEPTED MANUSCRIPT Figure 6. Heatmap of trypsin-like transcripts from control and deltamethrin-exposed C. rogercresseyi females and males. Transcript abundance is represented as RPKM

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values. Color scales show relative transcript expression.

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Figure 7. Heatmaps of trypsin-like transcripts from control and azamethiphos-exposed C. rogercresseyi females and males. Transcript abundance is represented as RPKM

SC

values. Color scales show relative transcript expression.

NU

Figure 8. Temporal expression of Cr-tryp1-6-10-36-41-42 and Cr-chym1. Corresponding transcripts associated with female and male C. rogercresseyi exposed to A)

MA

Deltamethrin; and B) Azamethiphos. Data are presented as fold change expression values of different trypsin- and chymotrypsin-like transcripts. The relative expression

D

levels were normalized with β tubulin as an endogenous control. Data are presented

AC CE P

TE

as the mean expression ± standard deviation.

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure1

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SC

RI

PT

ACCEPTED MANUSCRIPT

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TE

D

MA

Figure 2

AC CE P

TE

D

MA

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SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 3

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 4

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 5

AC CE P

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D

MA

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SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 6

AC CE P

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D

MA

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SC

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PT

ACCEPTED MANUSCRIPT

Figure 7

Figure 8

r-

C

hy

m

1

42 C

r-

C

m

1

yp 42 hy

rTr

yp 41

*

PT

RI

SC

NU

4.0

C

yp

C

rTr

yp 36

*

rTr

41

C

rTr

MA

yp 10

6

1

3.0

C

yp

36

C

D

yp

yp

1.0

rTr

yp

10

Tr

Tr

rTr

r-

r-

2.0

C

rTr

yp

C

C

C

0.0

C

rTr

0.4

TE

B

C

ry p6

ry p1

0.3

AC CE P

5.0

rT

rT

Fold change

A

C

C

Fold change

ACCEPTED MANUSCRIPT

*

*

0.2

Female Male

0.1

0.0

AC CE P

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D

MA

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT List of tables Table 1. Sequence of the oligonucleotide primers used in RT-qPCR assays. Sequence (5’-3’)

Cr-Tryp-41-F

CAGACCCAATGAAATCAAG

Cr-Tryp-41-R

ATAGAGTTCGTGGTAGATG

Cr-Tryp-36-F

CTTTGAATTCCTCGTCCTT

Cr-Tryp-36-R

CAAAAACCAACTACGATGTC

Cr-Tryp-1-F

CATGATGAAGACCATTTCTG

Cr-Tryp-1-R

AAAGGAGACTTGGAAAGG

Cr-Tryp-6-F

TGTTCAGGTTGTCTCTGA

Cr-Tryp-6-R

GTTTCCTTGGACAAGTGG

Cr-Tryp-10-F

CACTTGTCCAAGGAAACA

Cr-Tryp-10-R

GATCCAGTCAAGGAACTC

Cr-Chym-1-F

AAGGAACTTGGTGACCTTTC

MA

NU

SC

RI

PT

Primers

Cr-Chym-1-R

CACTTGCCCAAGGAAACA

Cr-Tryp-42-F

CAACTTCAGACCAAATGAC

Cr-Tryp-42-R

CATAGAGTTCGTGGTAGAT

TE

AC CE P

Cr-β-tubulin-R

D

Cr-β-tubulin-F

TTTGTTGTGTGAGCTCTGGG GCTGATCTCCGAAAACTTGC

ACCEPTED MANUSCRIPT Table 2. BLASTx analyses for trypsin-like transcripts from C. rogercresseyi against the NCBI, non-redundant protein database that include domain and signal peptide analyses.

Cr-Tryp6

Cr-Tryp1

Cr-Tryp42

2e-47 2e-37 2e-37 2e-37 2e-65 7e-34 8e-34 9e-34 2e-77 2e-44 1e-43 2e-43 1e-64 1e-47 3e-47 4e-47 3e-66 1e-56 2e-55 8e-55 1e-40 7e-34 4e-33 6e-33 2e-56 2e-46 2e-46 2e-46

SC

RI

88% 75% 75% 75% 97% 59% 60% 59% 99% 60% 59% 59% 83% 74% 73% 73% 78% 71% 62% 61% 81% 68% 70% 72% 81% 72% 73% 72

E-value

PT

Identity

NU

Cr-Tryp10

ACO11532 AAL71878 AAL71877 AAP55756 ACO11532 AAL71879 AAL71880 CAH61089 ACO11532 AAL71879 AAL71880 AAP55755 ACO11532 ACO14916 AAL71879 AAL71877 ACO11532 ACO14916 AAL71879 AAL71880 ACO11532 ACO11627 CAH61091 AAL71877 ACO11532 CAH61089 AAL71880 AAL71879

MA

Cr-Tryp36

Caligus rogercresseyi Lepeophtheirus salmonis Lepeophtheirus salmonis Lepeophtheirus salmonis Caligus rogercresseyi Lepeophtheirus salmonis Lepeophtheirus salmonis Lepeophtheirus salmonis Caligus rogercresseyi Lepeophtheirus salmonis Lepeophtheirus salmonis Lepeophtheirus salmonis Caligus rogercresseyi Caligus clemensi Lepeophtheirus salmonis Lepeophtheirus salmonis Caligus rogercresseyi Caligus clemensi Lepeophtheirus salmonis Lepeophtheirus salmonis Caligus rogercresseyi Caligus rogercresseyi Lepeophtheirus salmonis Lepeophtheirus salmonis Caligus rogercresseyi Lepeophtheirus salmonis Lepeophtheirus salmonis Lepeophtheirus salmonis

D

Cr-Tryp41

Trypsin 1 Trypsin type 4 Trypsin type 3 Trypsin type 1a Trypsin-1 Trypsin type 5 Trypsin type 6 Putative trypsin Trypsin 1 Trypsin type 5 Trypsin type 6 Trypsin 1c Trypsin-1 Trypsin-1 Trypsin Type 5 Trypsin type 3 Trypsin-1 Trypsin-1 Trypsin Type 5 Trypsin type 6 Trypsin-1 Putative trypsin Trypsin type 6 Trypsin type 5 Trypsin-1 Putative trypsin Trypsin type 6 Trypsin type 5

Accession number

Species

TE

Cr-Chym1

Description

AC CE P

ID

Domain Trypsin like serine protease

Signal Peptide (Cleavage site position) Secreted (26-27)

Trypsin like serine protease

Non Secreted

Trypsin like serine protease

Secreted by nonclassical pathway

Trypsin like serine protease

Non Secreted

Trypsin like serine protease

Non Secreted

Trypsin like serine protease

Secreted (27-28)

Trypsin like serine protease

Non Secreted

ACCEPTED MANUSCRIPT Table 3. Genes showing differential transcription expressions between C. rogercresseyi female controls and females exposed to deltamethrin. ID

Kal's Z-test foldchange

Unique gene reads control

P-value

RPKM control

Unique gene reads exposed

RPKM exposed

0

5

47.28

0

4

Cr-tryp24

36.57

0

7

Cr-tryp22

27.41

0

11

Cr-tryp18

7.30

0

12

Cr-tryp10

7.23

0

16

Cr-tryp30

7.03

0

Cr-tryp2

6.88

0

Cr-tryp9

5.91

0

Cr-tryp14

2.58

0

Cr-tryp35

2.55

0

Cr-tryp23

2.13

0

Cr-tryp19

2.11

Cr-tryp41 Cr-tryp15

236.98

137

19296.41

346.07

121

16471.45

478.89

116

17631.78

781.20

169

21555.55

346.85

27

2550.31

3275.15

81

23854.50

RI

80.89

Cr-tryp29

228.22

14

1615.88

8372.06

295

57988.53

53

5438.69

312

32398.40

83

970.59

61

2525.30

389

3334.88

283

8588.99

175

305.05

106

654.13

0

0

238.57

0

506.75

2.07

0

1586

102539.17

1768

213735.26

2.02

0

1

811.26

0

1656.92

Cr-chym2

-2.22

0

27

1053.97

2

476.33

Cr-tryp42

-2.24

0

1552

204093.82

88

91376.30

Cr-tryp4

-2.45

0

903

23108.99

97

9483.15

Cr-tryp17

-2.55

0

9

943.19

2

371.00

Cr-tryp12

-4.40

0

57

4178.21

6

954.29

MA

NU

6

56

TE

SC

Cr-tryp28

PT

Upregulated

AC CE P

D

Downregulated

Table 4. Genes showing differential transcription expression between C. rogercresseyi male controls and males exposed to deltamethrin. ID

Kal's Z-test fold-change

P-value

Unique gene reads control

RPKM control

Unique gene reads exposed

RPKM exposed

Upregulated Cr-tryp29

44.08

0

3

260.32

120

11836.70

Cr-tryp14

11.67

0

111

1464.63

593

17641.57

Cr-tryp24

10.55

0

12

945.63

85

10294.81

Cr-tryp28

9.96

0

10

1114.18

114

11455.16

Cr-tryp31

9.58

0

23

2338.71

33

23119.86

Cr-tryp22

8.44

0

19

2100.00

183

18296.01

Cr-tryp18

7.21

0

6

301.05

33

2239.97

Cr-tryp25

4.37

0

4

169.88

5

766.06

ACCEPTED MANUSCRIPT 4.32

0

44

8282.95

250

36964.30

Cr-tryp10

3.63

0

27

7209.33

125

27045.22

Cr-tryp9

3.20

0

77

9116.65

413

30133.70

Cr-tryp23

2.52

0

198

389.44

229

1015.54

Cr-tryp30

2.38

0

10

404.66

12

995.31

Cr-chym2

2.24

0

58

2682.15

79

6218.45

Cr-tryp1

2.13

0

484

181223.76

1009

398837.04

Cr-tryp19

2.00

0

0

323.03

0

667.63

Cr-tryp39

-2.27

0

1699

Cr-tryp42

-2.31

0

1834

Cr-tryp5

-2.33

0

1552

Cr-tryp37

-2.39

0

1344

Cr-tryp38

-2.47

0

2426

Cr-chym3

-3.85

0

Cr-chym6

-4.11

0

Cr-tryp4

-4.23

0

Cr-chym5

-4.82

0

Cr-chym4

-5.26

0

Cr-tryp17

-6.86

0

PT

Cr-tryp2

69336.36

537

31415.57

232069.26

109

103577.06

38198.18

345

16845.49

38570.51

295

16630.08

79837.53

449

33260.16

118

2641.45

14

706.58

8

230.49

1

57.74

1038

31105.25

123

7577.62

95

2634.26

9

562.67

90

2845.22

8

557.82

6

886.88

2

133.30

MA

NU

SC

RI

Downregulated

TE

D

Table 5. Genes showing differential transcription expression between C. rogercresseyi female controls and females exposed to azamethiphos.

Upregulated Cr-tryp19

Kal's Z-test fold-change

AC CE P

ID

Unique gene reads control

P-value

RPKM control

Unique gene reads exposed

RPKM exposed

18.05

0

0

238.57

0

4317.81

12.15

0

1

249.20

0

3035.77

8.68

0

1

811.26

0

7058.96

5.21

0

12

346.85

3

1810.84

5.07

0

56

8372.06

23

42547.02

4.72

0

23

11402.24

2

54022.00

4.35

0

83

970.59

16

4232.85

3.38

0

7

478.89

0

1625.11

Cr-tryp23

3.35

0

175

305.054

26

1025.34

Cr-tryp26

3.01

0

15

322.54

2

972.93

Cr-tryp35

2.95

0

389

3334.88

51

9891.34

Cr-chym2

2.88

0

27

1053.97

5

3043.95

Cr-tryp31

2.75

0

116

11319.97

4

31260.29

Cr-tryp27

2.50

0

2

352.28

1

885.54

Cr-tryp10

2.40

0

16

3275.15

1

7895.31

Cr-tryp25

2.05

0

4

276.02

0

567.69

Cr-tryp34

-2.32

0

932

14879.62

19

6415.11

Cr-tryp20

-2.32

0

105

1266.37

2

545.70

Cr-tryp38

-2.74

0

2908

84702.92

47

30960.38

Cr-tryp4

-4.65

0

903

23108.99

9

4974.73

Cr-tryp42

-4.83

0

1552

204093.82

5

42320.85

Cr-tryp16 Cr-tryp15 Cr-tryp18 Cr-tryp2 Cr-tryp3 Cr-tryp14 Cr-tryp24

Downregulated

PT

ACCEPTED MANUSCRIPT

ID

Kal's Z-test fold-change

P-value

Unique gene reads control

0

Cr-tryp18

8.83

0

Cr-tryp15

7.06

0

Cr-tryp14

6.02

0

Cr-tryp31

5.71

0

Cr-tryp23

4.20

0

Cr-tryp2

4.06

0

Cr-tryp17

2.94

0

Cr-tryp1

2.31

0

Cr-tryp3

2.20

Cr-tryp25

2.10

RPKM exposed

237.93

0

2483.34

6

301.05

7

2688.31

1

880.18

0

6287.70

111

1464.63

53

8920.97 13514.43

23

2338.71

5

198

389.44

66

1656.00

44

8282.95

39

34056.16

886.88

2

2639.82

181223.76

160

423913.56

0

7

4041.60

0

9022.43

4.4409E-16

4

169.88

0

361.19

TE

6 484

0

1834

232069.26

23

75635.91

Cr-tryp11

AC CE P

Downregulated

Unique gene reads exposed

0

MA

10.32

D

Cr-tryp16

RPKM control

NU

Upregulated

SC

RI

Table 6. Genes showing differential transcription expression between C. rogercresseyi male controls and males exposed to azamethiphos.

-3.67

0

371

5039.54

8

1386.24

Cr-tryp10

-4.71

0

27

7209.33

0

1545.64

Cr-tryp22

-6.41

0

19

2100.00

1

330.72

Cr-tryp5 Cr-tryp12 Cr-tryp20 Cr-tryp43 Cr-chym5 Cr-tryp4 Cr-tryp42

-2.03

0

1552

38198.18

61

19013.22

-2.17

0

48

4167.02

5

1940.02

-2.21

0

168

2286.26

6

1041.59

-2.47

0

915

228974.73

31

93347.27

-2.50

0

95

2634.26

3

1061.17

-2.95

0

1038

31105.25

28

10646.39

-3.10

ACCEPTED MANUSCRIPT Statement of relevance The current manuscript describes the variety of trypsin and chemotrypsins in the sea lice Caligus

PT

rogercresseyi. Our findings suggest that trypsin-like transcripts in C. rogercresseyi might play a

RI

role in metabolizing delousing drugs. The identification of trypsin-like transcripts, is a valuable

AC CE P

TE

D

MA

NU

SC

strategy for developing new management strategies against C. rogercresseyi.

ACCEPTED MANUSCRIPT Highlights 1. The development of resistance of sea lice to delousing drugs is one of the main treats for Atlantic salmon aquaculture in Chile.

RI

PT

2. Trypsin had been recently related to drug resistance in mosquitos and therefore they could be and interesting target to enhance delousing strategies.

SC

3. We were able to identify multiple transcripts of serine proteases that had strong regulation after the exposure to delousing drugs.

AC CE P

TE

D

MA

NU

4. Developing strategies against serine proteases could improve the effectiveness of currently used drug delousing treatment.