In this study we have generated sequence resources for the poorly characterised genus Argulus (A. foliaceus, A. coregoni and A. siamensis) and for the Branchiura more generally. While there were two existing genomic datasets for Argulus species (A. siamensis and A. foliaceus), these were limited in terms of their coverage. The new transcriptomes comprise larger data sets for Argulus with 84,256,934 reads assembled into 66,940 contigs for A. foliaceus and a first transcriptome for A. coregoni, 10,840,092 reads, assembled to give 40,954 contigs.
As there is no reference genome for Argulus, the transcriptomes of both A. foliaceus and A. coregoni were assembled de novo using Trinity v2.1.1 software. The assembled transcriptomes are important for both genomic and proteomic studies [11], especially in terms of providing the complete ORF information for genes coding for proteins that may play important roles in biological processes and might therefore provide potentially suitable vaccine candidates [12] or targets for anti-parasiticides.
It was reported by Clark & Greenwood [31] that 50 % of crustacean transcripts from transcriptomic studies were orthologous genes to a model organism and most of these genes belong to biological processes involving metabolism, development, or regulation of a biological process. This concurs with the Argulus GO outcomes, as the predominant genes from the biological process analysis were involved in response to metabolic processes. This was a predictable result as shown from other organisms, due to the enormous number of GO terms that cover basic processes required to maintain a living organism [47]. One of the most important processes, and one that has some shared properties between invertebrates and vertebrates is the innate immune system [48]. Knowledge of crustacean immune and stress responses are key to understanding crustacean physiology [46]. Although, the annotation method applied in this study is not sufficient to provide detailed information about the immune pathways, the GO molecular functions associated with the Argulus transcripts highlighted specific processes that can also be involved in host-parasite interactions such as hydrolase, transferase and metallopeptidase activities.
Hydrolases have been identified from different parasite venoms which are known to include proteases, peptidases and glycosidase enzymes [34, 35]. As we have not only identified hydrolase transcripts here in Argulus tissues, but also recently in Argulus SEPs through proteomic analysis [11], the presence of hydrolases might reflect their contribution to host-parasite interactions in Argulus species. The identified metalloproteases from different arthropods are assumed to be related to different biological processes such as digestion, cuticle degradation and host immune suppression [47]. In general, the GO annotations from the three-species identified a high representation of genes involved in metabolism, protein binding and nucleotide binding. These are linked with high basal metabolic activity [51], and although the precise reason is not known, various studies on the effects of parasitism on infected mammals have reported that parasites increase the basal metabolic rate of the host [52]. The patterns of the GO analysis from three Argulus species were similar. Further characterisation of the transcripts involved in immune pathway functions and their potential contribution to successful parasitism can provide a valuable addition to understanding of Argulus biology and physiology.
The construction of a limited phylogenetic tree based on the top 100 shared genes for the four transcriptome datasets from Argulus, available from both this study and from previous data for other arthropods, confirms the position of the sequenced Argulus species with respect to the superclass Oligostraca, as previously suggested by Regier et al. [45]. These results suggest that A. coregoni is more closely related to A. foliaceus than to A. siamensis. This probably reflects the fact that A. foliaceus and A. coregoni are indigenous Argulus species in northern Europe and UK with closer and more recent evolutionary links than A. siamensis, an invasive species from Asia. Long association in similar environments is also reflected by similar host specificities, or lack of them, allowing them to infect such disparate species as salmonids e.g., rainbow trout (O. mykiss) and cyprinids e.g., common carp (C. carpio). This may also mean that they share more similar mechanisms for successful parasite-host interaction.
Comparative functional annotation of overlapping clusters among the Argulus species identified 6,674 shared gene clusters, suggesting continued conservation after speciation. Identification of conserved genes can help in the search for more universal control targets in these species, for other Argulus species and perhaps for Branchiuran parasites more widely.
The saliva of almost all studied blood feeding arthropods has been shown to contain a range of immunomodulating proteins. Specific components have active functions in altering the host immune response to help blood sucking [53] and exploiting aspects of these powerful substances can provide an effective way to control disease distribution [54]. A range of proteins / genes that have been previously characterised as being important immune mediators for blood-feeding insects and other haematophagous parasites, and identified recently by our group in SEPs of the parasitic crustaceans, L. salmonis and A. foliaceus [11, 12], were here discovered in Argulus spp. transcriptome for the first time and indeed for the Branchiura more widely. Proteins identified as trypsin, serpin, serine protease, cathepsin-L, aspartic protease, ferritin, cysteine protease, enolase, phospholipase, adenosine deaminase, apyrase, metalloprotease, thrombin inhibitor, and venom serine proteases in shared transcript clusters for the three Argulus species provide potential candidates for investigation of Argulus controls in the future.
Transcripts of a trypsin-like protease encoding 25 annotated proteins were detected in Argulus. Skin mucus of Atlantic salmon (Salmo salar L.) infected by sea lice L. salmonis contains trypsin-like proteases derived from the parasite, which suggests that the secretion of this protease during feeding is a mechanism to impair the host immune response [28, 29, 54–58]. Seven types of L. salmonis trypsin-like proteases have been inferred to play roles in digestion and other host-parasite interactions [59]. Valenzuela-Munoz et al. similarly identified the up-regulation of trypsin-1 gene in the sea louse Caligus rogercresseyi (Boxshall & Bravo, 2000) post-treatment with an organophosphate parasiticide, suggesting the gene to be associated with resistance / susceptibility to organophosphates [56], while Braden et al [60] found significant upregulation of trypsin-1 in L. salmonis after 2 days post infection of lice feeding on Atlantic salmon (S. salar). Serine protease inhibitors (serpins), identified for the first time here in Argulus, are recognised as the largest protein group in tick saliva and were suggested to play an important role during tick feeding by modulating host immunity [33, 61–65]. Serpins characterised from Ixodes scapularis saliva were found to inhibit thrombin and trypsin activity and lower platelet aggregation [66]. Serpins are also recognised from insects, for example salivary serpin from Aedes aegypti mosquitoes, serves as an active anti-coagulant due to its ability to inhibit Factor Xa, which is the activated form of the coagulation factor thrombo-kinase (Stark & James 1998). Serpins have also been noted to be differentially expressed in salivary glands of the mosquito Anopheles culicifacies, suggesting involvement in the blood feeding process [40]. We have previously identified serpins in A. foliaceus SEPs suggesting their role in facilitating parasitism and modulating host immune responses in argulids, and the presence of trypsins suggests their role in parasite-host interactions and digestion [11].
Transcripts for cathepsin-L identified from Argulus in the present study may signify a role for this protease in digestion and / or modulation of the host immune system. In Hysterothylacium aduncum (Rudolphi, 1802), a parasitic anisakid nematode of fish, cathepsin L was proposed to be a vital protein involved in digestion processes [68]. Cathepsin L was also identified as a potential anticoagulant in SEPs of adult Haemonchus contortus, a parasitic nematode of sheep and other ruminants [38]. Franta et al. suggested that this could provide a novel target for control of ticks and tick-borne pathogens [69]. Transcripts for cathepsin-L were also reported to be expressed by the copepodid life stages of sea lice, C. rogercresseyi [34, 70], and were upregulated in feeding adult L. salmonis [60] and showed activity in SEPs of pre-adult and adult L. salmonis [12, 71], suggesting a likely role in modulating the host immune system to facilitate attachment and feeding [71].
Transcripts for aspartic protease 6 were also identified from Argulus in this study, this being a protein common to the three species examined. Aspartic proteases have been proposed to have a role during C. rogercresseyi feeding through haemoglobin proteolysis, where aspartic protease D was highly expressed in the chalimus and adult stages [34]. The salivary glands of blood feeding arthropods frequently contain anticoagulation inhibitors [32] and in this study we found evidence for the existence of the thrombin inhibitor rhodniin in all three Argulus species studied. It has previously been reported to prevent blood clotting by inhibiting thrombin [72] to facilitate blood acquisition. Apyrase is another immunomodulatory molecule, which we found in Argulus. This enzyme is believed to have anti-pain; anti-inflammatory and anti-haemostatic activity, thus potentially playing a role during feeding to prevent blood clotting and host behavioural responses [32]. Ribeiro et al. described the apyrase from R. prolixus to act as a platelet aggregation inhibitor [73]. A number of metalloproteases were also identified in Argulus, these being a common enzyme group in other blood-feeding arthropods as well, although it is important to note that metalloproteases also perform a wide range of other roles in arthropods more generally. Transcriptome analysis in conjunction with KEGG pathway analysis conducted by Sahoo et al. on A. siamensis resulted in the identification of proteases including serine proteases and metalloproteases which have been described to have antigenic properties in some ectoparasites [14]. Apyrase and metalloprotease found in tick salivary secretions were suggested to affect antiplatelet aggregation and coagulation activity [44] and may serve a similar function for processing blood meals in Argulus. Adenosine deaminase, which can act as a vasodilator and antiplatelet aggregation factor for arthropod feeding [32, 43] was also found in Argulus in the present study. By searching for adenosine deaminase using OrthoVenn BLAST feature, two contigs of adenosine deaminase transcripts resulted from Argulus species, adenosine deaminase and RNA-specific adenosine deaminase-1. In the sand fly Lutzomyia longipalpis [54] and the mosquito Aedes aegypti [74] adenosine deaminase can assist feeding and modulate host immune responses by metabolizing the purine 2- deoxyadenosine and adenosine to 2-deoxyinosine and inosine, correspondingly. The mechanism of immune evasion of salivary adenosine deaminase of Lu. longipalpis and Ae. Aegypti, is regulated by the removal of adenosine, a molecule associated with pain perception and mast cell degranulation. Then, the resulting product, inosine, functions as an immunomodulator by inhibiting the production of pro inflammatory cytokines [53].
The exact role of the cysteine protease transcript found here in all three Argulus species is not known but it was suggested to have roles in nutrition and / or host immune evasion in the nematode H. contortus (Rudolphi, 1803) Cobb, 1898 [38]. It was also suggested to have a critical function in the infection process of the trophont parasitic stages of the ciliate, Ichthyophthirius multifiliis and blood feeding parasitic copepod Phrixocephalus cincinnatus (C. B. Wilson, 1908) (Jousson et al., 2007; Perkins et al., 1997). Cysteine proteases have similarly been shown to play important roles in trematode parasites. Their roles include catalysing turnover of parasite peptides and growth factors and regulation of proteins, host tissue penetration and invasion, digestion and modulation of host defence systems [75, 76]. Phospholipases were also represented in the shared Argulus clusters, with 14 different clusters recognised in Swiss-Prot homology blasting. One of the most notable of these is phospholipase A2 (PLA2), a platelet-activating factor acetyl hydrolase, which is known to deactivate platelet-activating factor by Leishmania parasites [42] and known to facilitate blood feeding [77]. Transcripts for two types of ferritin were also found in Argulus in the present study, Ferritin-1 heavy chain and yolk ferritin, possibly involved in processing and storing iron after blood meals. Recently, ferritin was reported up-regulated in the salivary gland of the mosquito A. culicifaciens, after blood feeding [40], which prevents iron overload by assisting in iron homeostasis through iron transport and storage [78]. Hajdusek et al., working on Ixodes ricinus and Rhipicephalus annulatus (Say, 1821), illustrated the success of using the secreted form of ferritin 2 protein (FER2) as an antigen in an anti-tick vaccine [79]. Transcripts for enolase B, another protein suggested to be involved in blood digestion / feeding and defensive activity in ticks and other blood feeing parasites [80], were also found in Argulus spp. here as well as in sea lice, L. salmonis [70]. Verónica Díaz-Martín et al [81], reported that, enolase protein collected from the argasid tick Ornithodoros moubata saliva, known to bind with plasminogen, and hence may play a role in tick feeding by maintain blood fluidity. Investigations for both the cestode Echinococcus granulosus, (Batsch, 1786) [82] and the nematode Ascaris suum, (Goeze, 1782) have suggested enolase to be a promising vaccine candidate [41, 82].
Future work is needed to determine whether the identified compounds are secreted / expressed from Argulus feeding glands (i.e. pre-oral spine and proboscis glands), which we have recently characterised using histology and transmission electron microscopy[18]. This study substantially extends genomic resources for Argulus and is the first study to explore shared genes expressed across species, providing a preliminary list of potential feeding enhancers / immunomodulators / defensins, thus potential broad scope vaccine / drug targets.
