FicD genes in invertebrates: A tale of transposons, pathogenic and integrated viruses.

Many gene families are shared across the tree of life between distantly related species because of horizontal gene transfers (HGTs). However, the frequency of HGTs varies strongly between gene families and biotic realms suggesting differential selection pressures and functional bias. One gene family with a wide distribution are FIC-domain containing enzymes (FicDs). FicDs catalyze AMPylation, a post-translational protein modification consisting in the addition of adenosine monophosphate to accessible residues of target proteins. Beside the well-known conservation of FicDs in deuterostomes, we report the presence of a conserved FicD gene ortholog in a large number of protostomes and microbial eukaryotes. We also reported additional FicD gene copies in the genomes of some rotifers, parasitic worms and bivalves. A few dsDNA viruses of these invertebrates, including White spot syndrome virus, Cherax quadricarinatus iridovirus, Ostreid herpesvirus-1 and the beetle nudivirus, carry copies of FicDs, with phylogenetic analysis suggesting a common origin of these FicD copies and the duplicated FicDs of their invertebrate hosts. HGTs and gene duplications possibly mediated by endogenous viruses or genetic mobile elements seem to have contributed to the transfer of AMPylation ability from bacteria and eukaryotes to pathogenic viruses, where this pathway could have been hijacked to promote viral infection.

Environmental vibrio phage-bacteria interaction networks reflect the genetic structure of host populations.

Phages depend on their bacterial hosts to replicate. The habitat, density and genetic diversity of host populations are therefore key factors in phage ecology, but our ability to explore their biology depends on the isolation of a diverse and representative collection of phages from different sources. Here, we compared two populations of marine bacterial hosts and their phages collected during a time series sampling program in an oyster farm. The population of Vibrio crassostreae, a species associated specifically to oysters, was genetically structured into clades of near clonal strains, leading to the isolation of closely related phages forming large modules in phage-bacterial infection networks. For Vibrio chagasii, which blooms in the water column, a lower number of closely related hosts and a higher diversity of isolated phages resulted in small modules in the phage-bacterial infection network. Over time, phage load was correlated with V. chagasii abundance, indicating a role of host blooms in driving phage abundance. Genetic experiments further demonstrated that these phage blooms can generate epigenetic and genetic variability that can counteract host defence systems. These results highlight the importance of considering both the environmental dynamics and the genetic structure of the host when interpreting phage-bacteria networks.

Effect of predation risk on parasite transmission from first to second intermediate trematode hosts.

Predators can affect parasite-host interactions when directly preying on hosts or their parasites. However, predators may also have non-consumptive indirect effects on parasite-host interactions when hosts adjust their behaviour or physiology in response to predator presence. In this study, we examined how chemical cues from a predatory marine crab affect the transmission of a parasitic trematode from its first (periwinkle) to its second (mussel) intermediate host. Laboratory experiments revealed that chemical cues from crabs lead to a threefold increase in the release of trematode cercariae from periwinkles as a result of increased periwinkle activity. This positive effect on transmission was contrasted by a 10-fold reduction in cercarial infection rates in the second intermediate host when we experimentally exposed mussels to cercariae and predator cues. The low infection rates were caused by a substantial reduction in mussel filtration activity in the presence of predator cues, preventing cercariae from entering the mussels. To assess the combined net effect of both processes, we conducted a transmission experiment between infected periwinkles and uninfected mussels. Infection levels of mussels in the treatments with crab cues were sevenfold lower than in mussels without crab chemical cues. This suggests that predation risk effects on mussel susceptibility can counteract the elevated parasite release from first intermediate hosts, with negative net effects on parasite transmission. These experiments highlight that predation risk effects on parasite transmission can have opposing directions at different stages of the parasite's life cycle. Such complex non-consumptive predation risk effects on parasite transmission may constitute an important indirect mechanism affecting prevalence and distribution patterns of parasites in different hosts across their life cycle.

Phage-host coevolution in natural populations.

Coevolution between bacteriophages (phages) and their bacterial hosts occurs through changes in resistance and counter-resistance mechanisms. To assess phage-host evolution in wild populations, we isolated 195 Vibrio crassostreae strains and 243 vibriophages during a 5-month time series from an oyster farm and combined these isolates with existing V. crassostreae and phage isolates. Cross-infection studies of 81,926 host-phage pairs delineated a modular network where phages are best at infecting co-occurring hosts, indicating local adaptation. Successful propagation of phage is restricted by the ability to adsorb to closely related bacteria and further constrained by strain-specific defence systems. These defences are highly diverse and predominantly located on mobile genetic elements, and multiple defences are active within a single genome. We further show that epigenetic and genomic modifications enable phage to adapt to bacterial defences and alter host range. Our findings reveal that the evolution of bacterial defences and phage counter-defences is underpinned by frequent genetic exchanges with, and between, mobile genetic elements.

Windows of opportunity: Ocean warming shapes temperature-sensitive epigenetic reprogramming and gene expression across gametogenesis and embryogenesis in marine stickleback.

Rapid climate change is placing many marine species at risk of local extinction. Recent studies show that epigenetic mechanisms (e.g. DNA methylation, histone modifications) can facilitate both within and transgenerational plasticity to cope with changing environments. However, epigenetic reprogramming (erasure and re-establishment of epigenetic marks) during gamete and early embryo development may hinder transgenerational epigenetic inheritance. Most of our knowledge about reprogramming stems from mammals and model organisms, whereas the prevalence and extent of reprogramming among non-model species from wild populations is rarely investigated. Moreover, whether reprogramming dynamics are sensitive to changing environmental conditions is not well known, representing a key knowledge gap in the pursuit to identify mechanisms underlying links between parental exposure to changing climate patterns and environmentally adapted offspring phenotypes. Here, we investigated epigenetic reprogramming (DNA methylation/hydroxymethylation) and gene expression across gametogenesis and embryogenesis of marine stickleback (Gasterosteus aculeatus) under three ocean warming scenarios (ambient, +1.5 and +4°C). We found that parental acclimation to ocean warming led to dynamic and temperature-sensitive reprogramming throughout offspring development. Both global methylation/hydroxymethylation and expression of genes involved in epigenetic modifications were strongly and differentially affected by the increased warming scenarios. Comparing transcriptomic profiles from gonads, mature gametes and early embryonic stages showed sex-specific accumulation and temperature sensitivity of several epigenetic actors. DNA methyltransferase induction was primarily maternally inherited (suggesting maternal control of remethylation), whereas induction of several histone-modifying enzymes was shaped by both parents. Importantly, massive, temperature-specific changes to the epigenetic landscape occurred in blastula, a critical stage for successful embryo development, which could, thus, translate to substantial consequences for offspring phenotype resilience in warming environments. In summary, our study identified key stages during gamete and embryo development with temperature-sensitive reprogramming and epigenetic gene regulation, reflecting potential 'windows of opportunity' for adaptive epigenetic responses under future climate change.

Recent advances in bivalve-microbiota interactions for disease prevention in aquaculture.

In bivalves, no clear-cut functional role of microbiota has yet been identified, although many publications suggest that they could be involved in nutrition or immunity of their host. In the context of climate change, integrative approaches at the crossroads of disciplines have been developed to explore the environment-host-pathogen-microbiota system. Here, we attempt to synthesize work on (1) the current methodologies to analyse bivalve microbiota, (2) the comparison of microbiota between species, between host compartments and their surrounding habitat, (3) how the bivalve microbiota are governed by environmental factors and host genetics and (4) how host-associated microorganisms act as a buffer against pathogens and/or promote recovery, and could thereby play a role in the prevention of disease or mortalities.

Viral Decoys: The Only Two Herpesviruses Infecting Invertebrates Evolved Different Transcriptional Strategies to Deflect Post-Transcriptional Editing.

The highly versatile group of Herpesviruses cause disease in a wide range of hosts. In invertebrates, only two herpesviruses are known: the malacoherpesviruses HaHV-1 and OsHV-1 infecting gastropods and bivalves, respectively. To understand viral transcript architecture and diversity we first reconstructed full-length viral genomes of HaHV-1 infecting Haliotis diversicolor supertexta and OsHV-1 infecting Scapharca broughtonii by DNA-seq. We then used RNA-seq over the time-course of experimental infections to establish viral transcriptional dynamics, followed by PacBio long-read sequencing of full-length transcripts to untangle viral transcript architectures at two selected time points. Despite similarities in genome structure, in the number of genes and in the diverse transcriptomic architectures, we measured a ten-fold higher transcript variability in HaHV-1, with more extended antisense gene transcription. Transcriptional dynamics also appeared different, both in timing and expression trends. Both viruses were heavily affected by post-transcriptional modifications performed by ADAR1 affecting sense-antisense gene pairs forming dsRNAs. However, OsHV-1 concentrated these modifications in a few genomic hotspots, whereas HaHV-1 diluted ADAR1 impact by elongated and polycistronic transcripts distributed over its whole genome. These transcriptional strategies might thus provide alternative potential roles for sense-antisense transcription in viral transcriptomes to evade the host's immune response in different virus-host combinations.

Microbiome response differs among selected lines of Sydney rock oysters to ocean warming and acidification.

Oyster microbiomes are integral to healthy function and can be altered by climate change conditions. Genetic variation among oysters is known to influence the response of oysters to climate change and may ameliorate any adverse effects on oyster microbiome; however, this remains unstudied. Nine full-sibling selected breeding lines of the Sydney rock oyster (Saccostrea glomerata) were exposed to predicted warming (ambient = 24°C, elevated = 28°C) and ocean acidification (ambient pCO2 = 400, elevated pCO2 = 1000 µatm) for 4 weeks. The haemolymph bacterial microbiome was characterized using 16S rRNA (V3-V4) gene sequencing and varied among oyster lines in the control (ambient pCO2, 24°C) treatment. Microbiomes were also altered by climate change dependent on oyster lines. Bacterial α-diversity increased in response to elevated pCO2 in two selected lines, while bacterial ß-diversity was significantly altered by combinations of elevated pCO2 and temperature in four selected lines. Climate change treatments caused shifts in the abundance of multiple amplicon sequence variants driving change in the microbiome of some selected lines. We show that oyster genetic background may influence the Sydney rock oyster haemolymph microbiome under climate change and that future assisted evolution breeding programs to enhance resilience should consider the oyster microbiome.

Climate change alters the haemolymph microbiome of oysters.

The wellbeing of marine organisms is connected to their microbiome. Oysters are a vital food source and provide ecological services, yet little is known about how climate change such as ocean acidification and warming will affect their microbiome. We exposed the Sydney rock oyster, Saccostrea glomerata, to orthogonal combinations of temperature (24, 28 °C) and pCO2 (400 and 1000 µatm) for eight weeks and used amplicon sequencing of the 16S rRNA (V3-V4) gene to characterise the bacterial community in haemolymph. Overall, elevated pCO2 and temperature interacted to alter the microbiome of oysters, with a clear partitioning of treatments in CAP ordinations. Elevated pCO2 was the strongest driver of species diversity and richness and elevated temperature also increased species richness. Climate change, both ocean acidification and warming, will alter the microbiome of S. glomerata which may increase the susceptibility of oysters to disease.

Parallel analysis of miRNAs and mRNAs suggests distinct regulatory networks in Crassostrea gigas infected by Ostreid herpesvirus 1.

BACKGROUND: Since 2008, the aquaculture production of Crassostrea gigas was heavily affected by mass mortalities associated to Ostreid herpesvirus 1 (OsHV-1) microvariants worldwide. Transcriptomic studies revealed the major antiviral pathways of the oyster immune response while other findings suggested that also small non-coding RNAs (sncRNA) such as microRNAs might act as key regulators of the oyster response against OsHV-1. To explore the explicit connection between small non-coding and protein-coding transcripts, we performed paired whole transcriptome analysis of sncRNA and messenger RNA (mRNA) in six oysters selected for different intensities of OsHV-1 infection. RESULTS: The mRNA profiles of the naturally infected oysters were mostly governed by the transcriptional activity of OsHV-1, with several differentially expressed genes mapping to the interferon, toll, apoptosis, and pro-PO pathways. In contrast, miRNA profiles suggested more complex regulatory mechanisms, with 15 differentially expressed miRNAs (DE-miRNA) pointing to a possible modulation of the host response during OsHV-1 infection. We predicted 68 interactions between DE-miRNAs and oyster 3'-UTRs, but only few of them involved antiviral genes. The sncRNA reads assigned to OsHV-1 rather resembled mRNA degradation products, suggesting the absence of genuine viral miRNAs. CONCLUSIONS: We provided data describing the miRNAome during OsHV-1 infection in C. gigas. This information can be used to understand the role of miRNAs in healthy and diseased oysters, to identify new targets for functional studies and, eventually to disentangle cause and effect relationships during viral infections in marine mollusks.

Selection of Vibrio crassostreae relies on a plasmid expressing a type 6 secretion system cytotoxic for host immune cells.

Pacific oyster mortality syndrome affects juveniles of Crassostrea gigas oysters and threatens the sustainability of commercial and natural stocks of this species. Vibrio crassostreae (V. crassostreae) has been repeatedly isolated from diseased animals, and the majority of the strains have been demonstrated to be virulent for oysters. In this study, we showed that oyster farms exhibited a high prevalence of a virulence plasmid carried by V. crassostreae, while oysters, at an adult stage, were reservoirs of this virulent population. The pathogenicity of V. crassostreae depends on a novel transcriptional regulator, which activates the bidirectional promoter of a type 6 secretion system (T6SS) genes cluster. Both the T6SS and a second chromosomal virulence factor, r5.7, are necessary for virulence but act independently to cause haemocyte (oyster immune cell) cytotoxicity. A phylogenetically closely related T6SS was identified in V. aestuarianus and V. tapetis, which infect adult oysters and clams respectively. We propose that haemocyte cytotoxicity is a lethality trait shared by a broad range of mollusc pathogens, and we speculate that T6SS was involved in parallel evolution of pathogen for molluscs.

Molecular Targets for Coevolutionary Interactions Between Pacific Oyster Larvae and Their Sympatric Vibrios.

Bacteria of the Vibrio genus are the most predominant infectious agents threatening marine wildlife and aquaculture. Due to the large genetic diversity of these pathogens, the molecular determinants of Vibrio virulence are only poorly understood. Furthermore, studies tend to ignore co-evolutionary interactions between different host populations and their locally encountered Vibrio communities. Here, we explore the molecular targets of such co-evolutionary interactions by analyzing the genomes of nine Vibrio strains from the Splendidus-clade showing opposite virulence patterns towards two populations of Pacific oysters introduced into European Wadden Sea. By contrasting Vibrio phylogeny to their host specific virulence patterns, we could identify two core genome genes (OG1907 and OG 3159) that determine the genotype by genotype (G × G) interactions between oyster larvae and their sympatric Vibrio communities. Both genes show positive selection between locations targeting only few amino acid positions. Deletion of each gene led to a loss of the host specific virulence patterns while complementation with OG3159 alleles from both locations could recreate the wild type phenotypes matching the origin of the allele. This indicates that both genes can act as a genetic switch for Vibrio-oyster coevolution demonstrating that local adaptation in distinct Vibrio lineages can rely on only few genes independent of larger pathogenicity islands or plasmids.

Global invasion genetics of two parasitic copepods infecting marine bivalves.

Invasive species, and especially invasive parasites, represent excellent models to study ecological and evolutionary mechanisms in the wild. To understand these processes, it is crucial to obtain more knowledge on the native range, invasion routes and invasion history of invasive parasites. We investigated the consecutive invasions of two parasitic copepods (Mytilicola intestinalis and Mytilicola orientalis) by combining an extensive literature survey covering the reported putative native regions and the present-day invaded regions with a global phylogeography of both species. The population genetic analyses based on partial COI sequences revealed significant population differentiation for M. orientalis within the native region in Japan, while introduced populations in North America and Europe could not be distinguished from the native ones. Thus, M. orientalis' invasion history resembles the genetic structure and recent spread of its principal host, the Pacific oyster, Crassostrea gigas, while M. intestinalis lacks population genetic structure and has an overall low genetic diversity. Therefore, the native origin of M. intestinalis remains unclear. With this study, we demonstrate that even highly related and biologically similar invasive species can differ in their invasion genetics. From this, we conclude that extrapolating invasion genetics dynamics from related invasive taxa may not always be possible.

An Evolutionary Perspective of Dopachrome Tautomerase Enzymes in Metazoans.

Melanin plays a pivotal role in the cellular processes of several metazoans. The final step of the enzymically-regulated melanin biogenesis is the conversion of dopachrome into dihydroxyindoles, a reaction catalyzed by a class of enzymes called dopachrome tautomerases. We traced dopachrometautomerase (DCT) and dopachrome converting enzyme (DCE) genes throughout metazoans and we could show that only one class is present in most of the phyla. While DCTs are typically found in deuterostomes, DCEs are present in several protostome phyla, including arthropods and mollusks. The respective DCEs belong to the yellow gene family, previously reported to be taxonomically restricted to insects, bacteria and fungi. Mining genomic and transcriptomic data of metazoans, we updated the distribution of DCE/yellow genes, demonstrating their presence and active expression in most of the lophotrochozoan phyla as well as in copepods (Crustacea). We have traced one intronless DCE/yellow gene through most of the analyzed lophotrochozoan genomes and we could show that it was subjected to genomic diversification in some species, while it is conserved in other species. DCE/yellow was expressed in most phyla, although it showed tissue specific expression patterns. In the parasitic copepod Mytilicolaintestinalis DCE/yellow even belonged to the 100 most expressed genes. Both tissue specificity and high expression suggests that diverse functions of this gene family also evolved in other phyla apart from insects.

How invasive oysters can affect parasite infection patterns in native mussels on a large spatial scale.

There are surprisingly few field studies on the role of invasive species on parasite infection patterns in native hosts. We investigated the role of invasive Pacific oysters (Magallana gigas) in determining parasite infection levels in native blue mussels (Mytilus edulis) in relation to other environmental and biotic factors. Using hierarchical field sampling covering three spatial scales along a large intertidal ecosystem (European Wadden Sea), we found strong spatial differences in infection levels of five parasite species associated with mussels and oysters. We applied mixed models to analyse the associations between parasite prevalence and abundance in mussels and oysters, and 12 biological and environmental factors. For each parasite-host relationship, an optimal model (either a null, one-factor or two-factor model) was selected based on AIC scores. We found that the density of invasive oysters contributed to three of the 12 models. Other biological factors such as host size (six models), and the density of target or alternative host species (five models) contributed more frequently to the best models. Furthermore, for parasite species infecting both mussels and oysters, parasite population densities were higher in native mussels, attributed to the higher densities of mussels. Our results indicate that invasive species can affect parasite infection patterns in native species in the field, but that their relative contribution may be further mediated by other biological and environmental parameters. These results stress the usefulness of large-scale field studies for detailed assessments of the mechanisms underlying the impacts of invasive species on native host communities.

Within-generation and transgenerational plasticity of mate choice in oceanic stickleback under climate change.

Plasticity, both within and across generations, can shape sexual traits involved in mate choice and reproductive success, and thus direct measures of fitness. Especially, transgenerational plasticity (TGP), where parental environment influences offspring plasticity in future environments, could compensate for otherwise negative effects of environmental change on offspring sexual traits. We conducted a mate choice experiment using stickleback ( Gasterosteus aculeatus) with different thermal histories (ambient 17°C or elevated 21°C) within and across generations under simulated ocean warming using outdoor mesocosms. Parentage analysis of egg clutches revealed that maternal developmental temperature and reproductive (mesocosm) environment affected egg size, with females that developed at 17°C laying smaller eggs in 21°C mesocosms, likely owing to metabolic costs at elevated temperature. Paternal developmental temperature interacted with the reproductive environment to influence mating success, particularly under simulated ocean warming, with males that developed at 21°C showing lower overall mating success compared with 17°C males, but higher mating success in 21°C mesocosms. Furthermore, mating success of males was influenced by the interaction between F1 developmental temperature and F0 parent acclimation temperature, demonstrating the potential role of both TGP and within-generation plasticity in shaping traits involved in sexual selection and mate choice, potentially facilitating rapid responses to environmental change. This article is part of the theme issue 'The role of plasticity in phenotypic adaptation to rapid environmental change'.

Infection by invasive parasites increases susceptibility of native hosts to secondary infection via modulation of cellular immunity.

Parasite spillover from invasive aliens to native species increases the risk of disease emergence within native biota-either by direct harm to the new host or by indirect effects like increased risks of secondary infection. One example for such a detrimental effect is the parasitic copepod Mytilicola intestinalis that infected blue mussels Mytilus edulis after being introduced into the North Sea in the early 20th century. Since 1949, the parasite was blamed for multiple mass mortalities of infested blue mussels but evidence for a direct causal involvement of M. intestinalis remained circumstantial. Here, we now examine the potential effects of primary infections by the invasive parasite on the susceptibility to secondary infections with virulent bacteria (Vibrio spp.) in a full factorial infection experiment combining parasite infection (control vs. infected) with different Vibrio infection treatments (control, bath challenge, injection) in environmental conditions that either favoured the host (ambient temperature) or the bacterium (elevated temperature). The influence of primary and secondary infections on cellular immunity (phagocytosis) and Vibrio load in the haemolymph was used to correlate these results to host survival. Our results suggest that the rate of secondary Vibrio infection is increased due to lower efficiency of the cellular immune response. As a consequence, the failure of clearing Vibrio from the haemolymph might increase mortality of mussels infected by M. intestinalis. This demonstrates that indirect effects of parasite invasions can outweigh direct effects of the infection highlighting the need for a more integrative approach to understand and predict the consequences of parasite invasions.

Pandemic GII.4 Sydney and Epidemic GII.17 Kawasaki308 Noroviruses Display Distinct Specificities for Histo-Blood Group Antigens Leading to Different Transmission Vector Dynamics in Pacific Oysters.

Noroviruses are the major cause of foodborne outbreaks of acute gastroenteritis, which are often linked to raw oyster consumption. Previous studies have suggested histo-blood group antigens (HBGA)-like structures in the oyster tissues as ligands for norovirus binding and persistence. To better understand how oysters function as vectors for the most common human noroviruses, we first tested the ability of the norovirus strains GI.1 West Chester, the pandemic GII.4 Sydney, and the epidemic GII.17 Kawasaki308 strains to interact with oyster tissues. Secondly, we explored how the HBGA preferences of these strains can affect their persistence in oyster tissues. We found limited HBGA expression in oyster tissues. HBGAs of A and H type 1 were present in the digestive tissues and palps of the Pacific oyster Crassostrea gigas, while the gills and mantle lacked any HBGA structures. By using Virus-like particles (VLPs), which are antigenically and morphologically similar to native virions, we were able to demonstrate that VLPs of GI.1 West Chester norovirus reacted with the digestive tissues and palps. Despite of the lack of HBGA expression in mantle, dominant GII.4 Sydney strain readily bound to all the oyster tissues, including the digestive tissues, gills, palps, and mantle. In contrast, no binding of the epidemic GII.17 Kawasaki308 VLPs to any of the investigated oyster tissues was observed. In synthetic HBGA and saliva-binding assays, GI.1 reacted with A type, H type, and Leb (Lewis b) HBGAs. GII.4 Sydney VLPs showed a broad binding pattern and interacted with various HBGA types. Compared to GI.1 and GII.4 VLPs, the GII.17 Kawasaki308 VLPs only weakly associated with long-chain saccharides containing A type, B type, H type, and Leb blood group epitopes. Our findings indicate that GI.1 and GII.4 noroviruses are likely to be concentrated in oysters, by binding to HBGA-like glycans, and therefore potentially leading to increased long term transmission. In regards to the GII.17 Kawasaki308 strain, we suggest that oysters can only function as short term transmission vector in periods of high environmental virus concentrations.

Cryptic invasion of a parasitic copepod: Compromised identification when morphologically similar invaders co-occur in invaded ecosystems.

Despite their frequent occurrence and strong impacts on native biota, biological invasions can long remain undetected. One reason for this is that an invasive species can be morphologically similar to either native species or introduced species previously established in the same region, and thus be subject to mistaken identification. One recent case involves congeneric invasive parasites, copepods that now infect bivalve hosts along European Atlantic coasts, after having been introduced independently first from the Mediterranean Sea (Mytilicola intestinalis Steuer, 1902) and later from Japan (Mytilicola orientalis Mori, 1935). At least one report on M. intestinalis may have actually concerned M. orientalis, and M. orientalis thus qualifies as a "cryptic invader". Because these two parasitic copepods are morphologically similar, knowledge about their distribution, impact and interactions depends crucially on reliable species identification. In this study, we evaluated the reliability of morphological identification of these two species in parts of their invasive range in Europe (Dutch Delta and Wadden Sea) in comparison with molecular methods of well-established accuracy based on COI gene sequences and ITS1 restriction fragment length polymorphism. Based on seven easily measured or scored macro-morphological variables that were recorded for 182 individual copepods isolated from blue mussels (Mytilus edulis Linnaeus, 1758), principal component analysis showed two relatively distinct but overlapping morphological species groups for females, but no clear separation in males. Discriminant function analysis showed that the females can be discriminated reasonably well based on some of the morphological characteristics (identification error rate of 7%) while males cannot (error rate of 25%). The direction of the dorsolateral thoracic protuberances was identified as the most important trait for species discrimination, but among the morphological features checked, none could flawlessly discriminate between both species. We recommend the use of molecular techniques in future studies of invasive Mytilicola to reliably discriminate between the species. The morphological similarity of these two invaders suggests a more general problem of cryptic invasions and compromised identification of parasites in invaded ecosystems. This problem should be borne in mind whenever invasive parasites are investigated.

Dual transcriptomics reveals co-evolutionary mechanisms of intestinal parasite infections in blue mussels Mytilus edulis.

On theoretical grounds, antagonistic co-evolution between hosts and their parasites should be a widespread phenomenon but only received little empirical support so far. Consequently, the underlying molecular mechanisms and evolutionary steps remain elusive, especially in nonmodel systems. Here, we utilized the natural history of invasive parasites to document the molecular underpinnings of co-evolutionary trajectories. We applied a dual-species transcriptomics approach to experimental cross-infections of blue mussel Mytilus edulis hosts and their invasive parasitic copepods Mytilicola intestinalis from two invasion fronts in the Wadden Sea. We identified differentially regulated genes from an experimental infection contrast for hosts (infected vs. control) and a sympatry contrast (sympatric vs. allopatric combinations) for both hosts and parasites. The damage incurred by Mytilicola infection and the following immune response of the host were mainly reflected in cell division processes, wound healing, apoptosis and the production of reactive oxygen species (ROS). Furthermore, the functional coupling of host and parasite sympatry contrasts revealed the concerted regulation of chitin digestion by a Chitotriosidase 1 homolog in hosts with several cuticle proteins in the parasite. Together with the coupled regulation of ROS producers and antagonists, these genes represent candidates that mediate the different evolutionary trajectories within the parasite's invasion. The host-parasite combination-specific coupling of these effector mechanisms suggests that underlying recognition mechanisms create specificity and local adaptation. In this way, our study demonstrates the use of invasive species' natural history to elucidate molecular mechanisms of host-parasite co-evolution in the wild.