Daily patterns in parasite processes: diel variation in fish louse transcriptomes.

Parasites, similar to all other organisms, time themselves to environmental cues using a molecular clock to generate and maintain rhythms. Chronotherapeutic (timed treatment) techniques based on such rhythms offer great potential for improving control of chronic, problematic parasites. Fish lice are a key disease threat in aquaculture, with current control insufficient. Assessing the rhythmicity of fish lice transcriptomes offers not only insight into the viability of chronotherapy, but the opportunity to identify new drug targets. Here, for the first known time in any crustacean parasite, diel changes in gene transcription are examined, revealing that approximately half of the Argulus foliaceus annotated transcriptome displays significant daily rhythmicity. We identified rhythmically transcribed putative clock genes including core clock/cycle and period/timeless pairs, alongside rhythms in feeding-associated genes and processes involving immune response, as well as fish louse drug targets. A substantial number of gene pathways showed peak transcription in hours immediately preceding onset of light, potentially in anticipation of peak host anti-parasite responses or in preparation for increased feeding activity. Genes related to immune haemocyte activity and chitin development were more highly transcribed 4 h post light onset, although inflammatory gene transcription was highest during dark periods. Our study provides an important resource for application of chronotherapy in fish lice; timed application could increase efficacy and/or reduce dose requirement, improving the current landscape of drug resistance and fish health while reducing the economic cost of infection.

Identification of proteins from the secretory/excretory products (SEPs) of the branchiuran ectoparasite Argulus foliaceus (Linnaeus, 1758) reveals unique secreted proteins amongst haematophagous ecdysozoa.

BACKGROUND: It is hypothesised that being a blood-feeding ectoparasite, Argulus foliaceus (Linnaeus, 1758), uses similar mechanisms for digestion and host immune evasion to those used by other haematophagous ecdysozoa, including caligid copepods (e.g. sea louse). We recently described and characterised glands associated with the feeding appendages of A. foliaceus using histological techniques. The work described in the present study is the first undertaken with the objective of identifying and partially characterising the components secreted from these glands using a proteomic approach. METHODS: Argulus foliaceus parasites were sampled from the skin of rainbow trout (Oncorhynchus mykiss), from Loch Fad on the Isle of Bute, Scotland, UK. The proteins from A. foliaceus secretory/excretory products (SEPs) were collected from the supernatant of artificial freshwater conditioned with active adult parasites (n = 5-9 per ml; n = 560 total). Proteins within the SEPs were identified and characterised using LC-ESI-MS/MS analysis. Data are available via ProteomeXchange with identifier PXD016226. RESULTS: Data mining of a protein database translated from an A. foliaceus dataset using ProteinScape allowed identification of 27 predicted protein sequences from the A. foliaceus SEPs, each protein matching the criteria of 2 peptides with at least 4 contiguous amino acids. Nine proteins had no matching sequence through OmicsBox (Blast2GO) analysis searches suggesting that Argulus spp. may additionally have unique proteins present in their SEPs. SignalP 5.0 software, identified 13 proteins with a signal sequence suggestive of signal peptides and supportive of secreted proteins being identified. Notably, the functional characteristics of identified A. foliaceus proteins/domains have also been described from the salivary glands and saliva of other blood-feeding arthropods such as ticks. Identified proteins included: transporters, peroxidases, metalloproteases, proteases and serine protease inhibitors which are known to play roles in parasite immune evasion/induction (e.g. astacin), immunomodulation (e.g. serpin) and digestion (e.g. trypsin). CONCLUSIONS: To our knowledge, the present study represents the first proteomic analysis undertaken for SEPs from any branchiuran fish louse. Here we reveal possible functional roles of A. foliaceus SEPs in digestion and immunomodulation, with a number of protein families shared with other haematophagous ectoparasites. A number of apparently unique secreted proteins were identified compared to other haematophagous ecdysozoa.

Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets.

Argulus spp. are economically important fish ectoparasites. The development of antiparasitic drugs is thus important and real time PCR is an indispensable tool in drug development. The analytical potential of RT-PCR depends upon accurate normalisation by the use of stable reference genes. Here, we identified stable reference genes of Argulus siamensis for validation of efficacy of drugs and drug targets. Seven candidate genes were evaluated by evaluating their expression under different states of Argulus using the RefFinder tool. The four algorithms together generated a comprehensive ranking with elongation factor-1 alpha (EF-1α) being the most stable and 18S ribosomal protein (18S) the least stable gene. Taking EF-1α and 18S genes as references, the effectiveness of six anti-parasitic compounds against Argulus was evaluated by studying their effect on the expression pattern of few ion channel genes; this was to understand their mode of action, besides validating the reference genes. EF-1α was found to be the most stable gene in the validation. Collectively, this study is the first report to validate the optimal reference genes of A. siamensis for normalisation, and the potential of the ion channel genes for evaluating effective drug targets in parasite control.

Parasitic sea louse infestations on wild sea trout: separating the roles of fish farms and temperature.

BACKGROUND: The causal relation between parasitic sea lice on fish farms and sea lice on wild fish is a controversial subject. A specific scientific debate has been whether the statistical association between infestation pressure (IP) from fish farms and the number of parasites observed on wild sea trout emerges purely because of a confounding and direct effect of temperature (T). METHODS: We studied the associations between louse infestation on wild sea trout, fish farm activity and temperature in an area that practices coordinated fallowing in Nordhordland, Norway. The data were sampled between 2009 and 2016. We used negative binomial models and mediation analysis to determine to what degree the effect of T is mediated through the IP from fish farms. RESULTS: The number of attached lice on sea trout increased with the T when the IP from fish farms was high but not when the IP was low. In addition, nearly all of the effect of rising T was indirect and mediated through the IP. Attached lice remained low when neighbouring farms were in the first year of the production cycle but rose substantially during the second year. In contrast to attached lice, mobile lice were generally seen in higher numbers at lower water temperatures. Temperature had an indirect positive effect on mobile louse counts by increasing the IP which, in turn, raised the sea trout louse counts. Mobile louse counts rose steadily during the year when neighbouring farms were in the first year of the production cycle and stayed high throughout the second year. CONCLUSIONS: The estimates of the IP effect on louse counts along with the clear biennial pattern emerging due to the production cycle of fish farms clearly indicate that fish farms play an important role in the epidemiology of sea lice on wild sea trout. Furthermore, the mediation analysis demonstrates that a large proportion of the effect of T on louse counts is mediated through IP.

Linking eggs and adults of Argulus spp. using mitochondrial DNA barcodes.

We have created barcode library for common Argulus spp. infecting Carassius auratus, which could also be used to identify premature forms of Argulus spp. even by non-professionals. Infected C. auratus was examined and purchased from ornamental fish-trading centers and the adult life stage of Argulus spp. was identified and DNA barcoded. The eggs of Argulus spp. were collected using bottle implants. The collected eggs are barcoded and precisely identified by matching with the adult sequences. Four species of adult Argulus spp. were identified, namely Argulus japonicus, Argulus indicus, Argulus siamensis, and Argulus foliaceus. Precise identification of egg samples was done by two different analyses, namely (i) BLAST analysis and (ii) phylogenetic clustering of adults and eggs. All egg samples including the control were precisely identified by BLAST analysis and the results are consistent with phylogenetic clustering of adult and egg's DNA barcodes. In order to establish the DNA barcode technology for the identification of all Argulus spp and its premature forms, the development of full-fledged barcode library that includes all species of this genus is very important for the benefit of ornamental fish industries.

Peptide discovery in the ectoparasitic crustacean Argulus siamensis: identification of the first neuropeptides from a member of the Branchiura.

Recent advances in high-throughput sequencing have facilitated the generation of large transcriptomic datasets for an ever-growing number of crustaceans, one being the carp louse Argulus siamensis. This and other members of the subclass Branchiura are obligate fish ectoparasites, and as such, are a major concern for commercial aquaculture. Using the extant transcriptome shotgun assembly (TSA) sequences for A. siamensis, 27 transcripts encoding putative neuropeptide precursors were identified, and their pre/preprohormones deduced and characterized using a well-established bioinformatics workflow. The structures of 105 distinct peptides were predicted from the deduced proteins, including isoforms of adipokinetic hormone (AKH), allatostatin A, allatostatin B, allatostatin C, allatotropin, bursicon α, bursicon ß, crustacean cardioactive peptide (CCAP), diuretic hormone 31, diuretic hormone 44, eclosion hormone, myosuppressin, neuroparsin, neuropeptide Y, orcokinin, pigment dispersing hormone, proctolin, short neuropeptide F, SIFamide, sulfakinin and tachykinin-related peptide. While several of the predicted peptides are known from other crustacean and/or insect species, e.g. RYLPT, a broadly conserved arthropod proctolin isoform, and PFCNAFTGCamide (disulfide bridging between the two cysteines), the stereotypical crustacean CCAP, the vast majority of them are described here for the first time, e.g. pQVNFSTKWamide, a new AKH/red pigment concentrating hormone superfamily member, pQEGLDHMFMRFamide, a novel myosuppressin, and SYKSKPPFNGSIFamide, a new member of the SIFamide family. As the peptides presented here are the only ones thus far described from A. siamensis, or for that matter, any branchiuran, they represent a new resource to begin investigations of peptidergic control of physiology and behavior in this and other related aquacultural pests.

De novo whole transcriptome analysis of the fish louse, Argulus siamensis: first molecular insights into characterization of Toll downstream signalling molecules of crustaceans.

Argulus siamensis is a major ectoparasitic pathogen of freshwater fish capable of causing substantial economic loss. None of the available control measures have been able to address the problem of argulosis resourcefully. To combat this pathogen effectively, it is necessary to have a comprehensive understanding of its life processes with information on various genes involved. The transcriptome studies can generate introductory information about genes participating in physiological processes of the parasite which could be targeted for their control. In this study, the transcriptome sequencing of A. siamensis was performed on Illumina HiSeq 2000 platform which generated 75,126,957 high quality reads. A total of 46,352 transcript contigs were assembled with average length of 1211bp and N50 length of 2302bp. In total, 19,290 CDS including 184 novel CDS and 59,019 open reading frames (ORFs) were identified from the assembled contigs. Gene ontology and Kyoto Encylopedia of Genes and Genomes pathway analysis were performed to classify contigs into their functional categories and regulation pathways. Additionally, 1171 simple sequence repeats were identified from the assembled contigs. Further, twelve contigs with high similarity with downstream molecules of the mammalian toll like receptor (TLR) pathway were validated by their inductive expressions in response to lipopolysaccharide (LPS) of Gram negative bacteria, Escherichia coli and Gram positive bacteria, Staphylococcus aureus. The transcriptome of an ectoparasite A. siamensis was sequenced, assembled, annotated, and the downstream signalling molecules of Toll pathway characterized. The transcriptome data generated will facilitate studies on functional genomics that will subsequently be applied for vaccine development and other control strategies against the parasite.

A bridge between original and novel states: ontogeny and function of "suction discs" in the Branchiura (Crustacea).

The emergence of novel structures in the course of evolution faces an explanatory problem, leaving the gap from the ancestral structures difficult to bridge. This difficulty is caused by the lack of intermediate stages. Branchiurans are ectoparasitic crustaceans which use a pair of "suction discs" to attach to their host. These structures are modified first maxillae. During ontogeny, the first maxillae transform from a normal cephalic appendage to the specialized suction disc. However, supposedly ancestral branchiurans lack the suction discs in the adults and the first maxilla remains a normal appendage throughout. We describe the muscular arrangements in the developing first maxillae in Argulus coregoni. The suction discs originate as a fusion of the first and second podomeres. The sucker muscles of the suction discs are homologous to the muscles that insert in the second podomere at the early larval stages. The developmental process of the suction disc can be seen as a "recapitulation" of the evolutionary process. We thus show how the first maxilla can maintain not just the biological role but also a functional continuity during the evolution of the novel structure. From this example it is obvious that the intermediate stages of the emerging novelty, if present in the ontogeny, can help solve at least some of the enigmatic appearances of novel structures.

Expression of immune-related genes in rohu Labeo rohita (Hamilton) by experimental freshwater lice Argulus siamensis (Wilson) infection.

The crustacean ectoparasite, Argulus poses one of the major threats to carp culture due to absence of any suitable control measure. The study was undertaken to determine the expression of immune-related genes in three major immunocompetent organs viz., kidney, skin and liver of rohu (Labeo rohita) during experimental freshwater lice Argulus siamensis infection. Results showed that the expression of TLR 22-like, lysozyme G and ß2-microglobulin genes in kidney was significantly (P ≤ 0.05) down-regulated in lice-infected fish. On the other hand, no significant difference (P>0.05) in CXCa, lysozyme C, TNFα and complement component 3 (C3) expression was found between uninfected control and different degrees of lice infected fish. In the skin, the expression of TLR 22-like and TNFα genes were significantly up-regulated whereas that of C3 was significantly (P ≤ 0.05) down-regulated in lice-infected fish with respect to control fish. The expression of CXCa, lysozyme C and transferrin was not detected in the skin samples of fish. In the liver, the expression of CXCa, lysozyme G, ß2-microglobulin and transferrin was significantly (P≤0.05) up-regulated in lice-infected fish with respect to control fish whereas expression of C3 was significantly (P ≤ 0.05) down-regulated in lice-infected fish. The expression of TLR 22-like, lysozyme C, TNFα genes was not detected in the liver samples of fish. This study indicates that majority of the genes showed down-regulation in kidney tissue whereas up-regulation in liver and skin tissues except C3 in Argulus-infected fish. We show that infection with this parasite irrespective of intensity can also result in immune gene expression changes in tissues situated away from the site of parasite attachment and feeding. The information obtained here could be useful towards understanding the susceptibility of rohu to argulosis and mechanisms involved in protection of rohu to ectoparasitic infections, which is causing immense economic losses to freshwater aquaculture sector.

Analysis of genetic variability within Argulus japonicus from representatives of Africa, Middle East, and Asia revealed by sequences of three mitochondrial DNA genes.

This study investigated the genetic variability within fish louse Argulus japonicus (Crustacea: Branchiura) from Africa, Middle East, and Asia by polymerase chain reaction in three mitochondrial DNA (mtDNA) regions, namely, cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4). Six different sequences from a portion of the cox1 gene (pcox1) and a portion of the nad1 and nad4 genes (pnad1 and pnad4) for ten adult specimens from infected fish in China, Egypt, and Syria were amplified separately from individual and the amplicons were subjected to direct sequencing. A + T percentages were 68.8-69% for pcox1, 77.1-77.6% for pnad1, and 60.4-60.9% for pnad4. Among all the collected parasites, A. japonicus sequence variations were 0.0-1.9% for cox1, 0.0-2.3% for nad1, and 0.0-0.8% for nad4. In rivers, sequence variations among all individuals were 0.4-0.8% for cox1, 1.0-2.3% for nad1, and 0.4-0.8% for nad4, while sequence variations among all the collected parasites in fish farms were 0.6-1.9% for cox1, 0.0-1.7% for nad1, and 0.2-0.6% for nad4. The nad1 was the most variable gene among selected markers, while nad4 was a more conserved gene than cox1. All isolates of A. japonicus were sister to Argulus americanus in phylogenetic tree and they grouped together in one sub-clade, while isolates from China and Egypt fish farms were closely clustered together. However, moderate genetic drift and slight mutation could be observed among A. japonicus individuals. These findings demonstrated the convenience and attributes of the three selected mtDNA sequences for population genetic studies of A. japonicus where nad1 is a new and reliable marker to detect the sequence variation among A. japonicus individuals.