Crustacean Hosts Research Articles

Two in one: A case study of two rhizocephalan species invading the nervous tissue of one host

AbstractCertain parasites can manipulate their hosts to enhance their own fitness and transmission success. Rhizocephalan barnacles are a notable example, inducing significant changes in their crustacean host's morphology, physiology, and behaviour. Until recently, it was believed that the spider crab Scyra aff. ferox is parasitized by a single species Sacculina pilosella in the Sea of Japan. However, previous molecular studies have revealed a complex of unrelated species, Sacculina pugettiae (fam. Sacculinidae) and Parasacculina pilosella (fam. Polyascidae). These rhizocephalans can even parasitize one host specimen simultaneously. Here, we investigated the interaction of S. pugettiae and P. pilosella with the nervous system of their common host. Species were identified based on receptacle morphology, with further validation through 18S gene phylogenetic analysis. The parasites interacted differently with the host nervous system: S. pugettiae had goblet‐shaped organs in the ganglion periphery, while P. pilosella lacked these structures, instead possessing numerous neuropil rootlets. In case of simultaneous infection, both goblet‐shaped organs and neuropil rootlets were present. Histochemistry revealed the presence of muscular rosettes in the rootlets of both species, suggesting a similar organization of the muscular system despite phylogenetic differences. Scanning electron microscopy confirmed the presence of host cell projections enveloping the rootlets. This study provides insights into the morphological features of rhizocephalan‐decapod interaction and highlights differences in their interaction with host nervous tissue between families. Our results also confirmed the loss of the goblet‐shaped organs in Polyascidae.

Exposure to fungal infection decreases eye size in the zooplankton, Daphnia

Abstract Immune responses can be energetically expensive and subject to trade-offs. Prior work on the freshwater zooplankton, Ceriodaphnia cornuta, demonstrated an association between eye size and infection, leading to questions about whether investment in eyes trades off against investment in immunity. We used the crustacean host, Daphnia dentifera, and its fungal parasite, Metschnikowia bicuspidata, to investigate the relationships between eye size, parasite resistance and infection. In the field, we found a negative correlation between size-corrected eye area (SCEA) and Metschnikowia infection, suggesting that either SCEA decreases infection (thereby indicating resistance) or that infection decreases SCEA. Controlled laboratory experiments reinforced the latter result: exposure to the fungal parasite decreased a host’s SCEA, regardless of the parasite dose or host genotype. We also uncovered significant plasticity in this trait—both host age and resource level increased SCEA. Identifying causality in physiological correlations is challenging. Our results suggest that negative associations between parasitism and energetically-expensive traits can arise through plasticity.

Contrasting alterations in brain chemistry in a crustacean intermediate host of two acanthocephalan parasites

The dynamic properties of neural systems throughout life can be hijacked by so-called manipulative parasites. This study investigated changes in the brain chemistry of the amphipod Gammarus fossarum in response to infection with two trophically-transmitted helminth parasites known to induce distinct behavioral alterations: the bird acanthocephalan Polymorphus minutus and the fish acanthocephalan Pomphorhynchus tereticollis. We quantified brain antioxidant capacity as a common marker of homeostasis and neuroprotection, and brain total protein, on 72 pools of six brains. We analyzed the concentration of serotonin (5HT), dopamine (DA) and tyramine in 52 pools of six brains, by using ultrafast high performance liquid chromatography with electrochemical detection (UHPLC-ECD). Brain total protein concentration scaled hypo-allometrically to dry body weight, and was increased in infected gammarids compared to uninfected ones. The brain of gammarids infected with P. minutus had significantly lower total antioxidant capacity relative to total proteins. Infection with P. tereticollis impacted DA level compared to uninfected ones, and in opposite direction between spring and summer. Brain 5HT level was higher in summer compared to spring independently of infection status, and was decreased by infection after correcting for brain total protein concentration estimated from dry whole-body weight. The potential implication of 5HT/DA balance in parasite manipulation, as a major modulator of the reward-punishment axis, is discussed. Taken together, these findings highlight the need to consider both brain homeostatic and/or structural changes (antioxidant and total protein content) together with neurotransmission balance and flexibility, in studies investigating the impact of parasites on brain and behavior.

Testing the radiation cascade in postglacial radiations of whitefish and their parasites: founder events and host ecology drive parasite evolution.

Reciprocal effects of adaptive radiations on the evolution of interspecific interactions, like parasitism, remain barely explored. We test whether the recent radiations of European whitefish (Coregonus spp.) across and within perialpine and subarctic lakes promote its parasite Proteocephalus fallax (Platyhelminthes: Cestoda) to undergo host repertoire expansion via opportunity and ecological fitting, or adaptive radiation by specialization. Using de novo genomic data, we examined P. fallax differentiation across lakes, within lakes across sympatric host species, and the contributions of host genetics versus host habitat use and trophic preferences. Whitefish intralake radiations prompted parasite host repertoire expansion in all lakes, whereas P. fallax differentiation remains incipient among sympatric fish hosts. Whitefish genetic differentiation per se did not explain the genetic differentiation among its parasite populations, ruling out codivergence with the host. Instead, incipient parasite differentiation was driven by whitefish phenotypic radiation in trophic preferences and habitat use in an arena of parasite opportunity and ecological fitting to utilize resources from emerging hosts. Whilst the whitefish radiation provides a substrate for the parasite to differentiate along the same water-depth ecological axis as Coregonus spp., the role of the intermediate hosts in parasite speciation may be overlooked. Parasite multiple-level ecological fitting to both fish and crustacean intermediate hosts resources may be responsible for parasite population substructure in Coregonus spp. We propose parasites' delayed arrival was key to the initial burst of postglacial intralake whitefish diversification, followed by opportunistic tapeworm host repertoire expansion and a delayed nonadaptive radiation cascade of incipient tapeworm differentiation. At the geographical scale, dispersal, founder events, and genetic drift following colonization of spatially heterogeneous landscapes drove strong parasite differentiation. We argue that these microevolutionary processes result in the mirroring of host-parasite phylogenies through phylogenetic tracking at macroevolutionary and geographical scales.

Leeches Baicalobdella torquata feed on hemolymph but have a low effect on the cellular immune response of amphipod Eulimnogammarus verrucosus from Lake Baikal.

Lake Baikal is one of the largest and oldest freshwater reservoirs on the planet with a huge endemic diversity of amphipods (Amphipoda, Crustacea). These crustaceans have various symbiotic relationships, including the rarely described phenomenon of leech parasitism on amphipods. It is known that leeches feeding on hemolymph of crustacean hosts can influence their physiology, especially under stressful conditions. Here we show that leeches Baicalobdella torquata (Grube, 1871) found on gills of Eulimnogammarus verrucosus (Gerstfeldt, 1858), one of the most abundant amphipods in the Baikal littoral zone, indeed feed on the hemolymph of their host. However, the leech infection had no effect on immune parameters such as hemocyte concentration or phenoloxidase activity and also did not affect glycogen content. The intensity of hemocyte reaction to foreign bodies in a primary culture was identical between leech-free and leech-infected animals. Artificial infection with leeches also had only a subtle effect on the course of a model microbial infection in terms of hemocyte concentration and composition. Despite we cannot fully exclude deleterious effects of the parasites, our study indicates a low influence of a few leeches on E. verrucosus and shows that leech-infected amphipods can be used at least for some types of ecophysiological experiments.

Shrimp classification for white spot syndrome detection through enhanced gated recurrent unit-based wild geese migration optimization algorithm.

The major dangerous viral infection for cultivated shrimps is WSSV. The virus is extremely dangerous, spreads swiftly, and may result in up to 100% mortality in 3-10days. The vast wrapped double stranded DNA virus known as WSSV describes a member of the Nimaviridae viral family's species Whispovirus. It impacts a variety of crustacean hosts but predominantly marine shrimp species that are raised for commercial purposes. The entire age groups are affected by the virus, which leads to widespread mortality. Mesodermal and ectodermal tissues, like the lymph nodes, gills, and cuticular epithelium, represents the centres of infection. Complete genome sequencing related to the WSSV strains from Thailand, China, and Taiwan has identified minute genetic variations amongst them. There exist conflicting findings on the causes of WSSV pathogenicity, which involve variations in the size associated with the genome, the count of tandem repeats, and the availability or lack of certain proteins. Hence, this paper plans to perform the shrimp classification for the WSSV on the basis of novel deep learning methodology. Initially, the data is gathered from the farms as well as internet sources. Next, the pre-processing of the gathered shrimp images is accomplished using the LBP technique. These pre-processed images undergo the segmentation process utilizing the TGVFCMS approach. The extraction of the features from these segmented images is performed by the PLDA technique. In the final step, the classification of the shrimp into healthy shrimp and WSSV affected shrimp is done by the EGRU, in which the parameter tuning is accomplished by the wild GMO algorithm with the consideration of accuracy maximization as the major objective function. Performance indicators for accuracy have been compared with those of various conventional methods, and the results show that the methodology is capable of accurately identifying the shrimp WSSV illness.

Nectonema horsehair worms (Nematomorpha) parasitic in the Tanner crab Chionoecetes bairdi, with a note on the relationship between host and parasite phylogeny.

Nectonema nematomorphs utilize marine crustacean hosts in their life cycle; 16 decapod and 1 isopod genera have been reported to date as host genera. This study reports the first case of Nectonema parasitic in the Tanner crab Chionoecetes bairdi, adding another known host genus. A single nematomorph juvenile was recovered from the body cavity of each of 2 ovigerous female crabs. A nucleotide sequence for the 18S rRNA gene (1854 bp) was determined from 1 Nectonema individual. The 18S sequence showed Kimura 2-parameter (K2P) distances of 10.0, 2.0, and 1.7% from 18S sequences from Nectonema sp. from an isopod host, N. agile, and N. munidae, respectively. In an 18S-based tree, the unknown species was the sister taxon to a clade comprising N. agile and N. munidae, both of which also utilize decapod hosts. The phylogenetic relationships among the 3 Nectonema species parasitic in decapods were not congruent with the phylogeny of the hosts, not supporting a hypothesis of nematomorph-host co-evolution.

Transmission Routes and a New Freshwater Crustacean Host for Infectious Precocity Virus (IPV)

Macrobrachium rosenbergii holds significant importance in aquaculture within tropical and subtropical regions globally. An infection called infectious precocity virus disease (IPVD), caused by infectious precocity virus (IPV), has emerged in M. rosenbergii in China and causes significant economic losses. The diseased M. rosenbergii presents with the characteristics of sexual precocity and slow growth. Elaborating the route of transmission and host range of IPV is necessary to prevent the disease. Transmission of IPV to healthy M. rosenbergii can occur through the ingestion of IPV-infected tissue, immersion of viral filtrate, cohabitation with infected M. rosenbergii, or water-borne transmission in contaminated environment. Results showed that fertilized eggs and different growth and development stages (larvae Ⅰ–Ⅵ, postlarvae, juveniles, and adult prawns) from infected M. rosenbergii were negative for IPV, indicating that vertical transmissions did not occur. The results of artificial infection experiments showed that M. nipponense tested positive for the presence of IPV, but not in fishes Carassius auratus, Pangasius bocourti, Micropterus salmoides, and Oreochromis mossambicus, and crabs Scylla paramamosain and Eriocheir sinensis. IPV could cause slow growth in M. nipponense and mainly infected the second pereiopod, brain, eyes, and gills of M. nipponense. M. nipponense was found to be a new freshwater crustacean host for IPV. From December 2019 to March 2022, 260 M. nipponense samples in four regions were collected, including 230 M. nipponense IPV-positive samples according to nested PCR and 40 M. nipponense IPV-negative samples according to nested PCR. These findings could aid in preventing and managing IPV infections in crustaceans.

Prevalence of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) in Farmed Procambarus clarkii of the Middle and Lower Reaches of the Yangtze River in China.

Procambarus clarkii is an important economic aquaculture species worldwide. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) infects numerous crustacean hosts, including P. clarkii. However, there have been few reports on the prevalence of IHHNV in P. clarkii. In this study, 200 farmed P. clarkii were collected from Anhui, Jiangsu, Zhejiang, Hunan, Hubei, and Sichuan provinces in China. PCR detection was employed per the protocol by the World Organization for Animal Health (WOAH) to identify and detect the presence of IHHNV. The positive rate of IHHNV in different provinces ranged from 16.7 to 56.7%, and the overall IHHNV-positive rate was 38.5%. IHHNV strains isolated in this study related closely to infectious IHHNV and split into two major distinct branches. Besides, the IHHNV strains shared a high homology (93.4-99.4%). These findings suggest that a high prevalence of IHHNV was established in farmed P. clarkii in the middle and lower reaches of the Yangtze River.

Differential gene expression in response to fungal pathogen exposure in the aquatic invertebrate, Daphnia dentifera.

While vertebrate immune systems are appreciated for their complexity and adaptability, invertebrate immunity is often considered to be less complex. However, immune responses in many invertebrates likely involve sophisticated processes. Interactions between the crustacean host Daphnia dentifera and its fungal pathogen Metschnikowia bicuspidata provide an excellent model for exploring the mechanisms underlying crustacean immunity. To explore the genomic basis of immunity in Daphnia, we used RNA-sequencing technology to quantify differential gene expression between individuals of a single host genotype exposed or unexposed to M. bicuspidata over 24 h. Transcriptomic analyses showed that the number of differentially expressed genes between the control (unexposed) and experimental (exposed) groups increased over time. Gene ontology enrichment analysis revealed that differentially expressed genes were enriched for immune-related molecules and processes, such as cuticle development, prostaglandin, and defense response processes. Our findings provide a suite of immunologically relevant genes and suggest the presence of a rapidly upregulated immune response involving the cuticle in Daphnia. Studies involving gene expression responses to pathogen exposure shine a light on the processes occurring during the course of infection. By leveraging knowledge on the genetic basis for immunity, immune mechanisms can be more thoroughly understood to refine our understanding of disease spread within invertebrate populations.

A qPCR-based method to detect the eel parasitic nematode Anguillicola crassus in intermediate and final hosts.

Being able to systematically detect parasitic infection, even when no visual signs of infection are present, is crucial to the establishment of accurate conservation policies. The nematode Anguillicola crassus infects the swimbladder of anguillid species and is a potential threat for eel populations. In North America, naïve hosts such as the American eel Anguilla rostrata are affected by this infection. The accidental introduction of A. crassus following restocking programs may contribute to the actual decline of the American eel in Canada. We present a quantitative real time PCR-based method to detect A. crassus infection in final and intermediate hosts. We tested two protocols on samples from different geographical origins in Canada: 1) a general detection of A. crassus DNA in pools of young final hosts (glass eels) or crustacean intermediate hosts 2) a detection at the individual scale by analyzing swim bladders from elvers, or from adult yellow and silver eels. The DNA of A. crassus was detected in one pool of zooplankton (intermediate host) from the Richelieu River (Montérégie-Québec), as well as in individual swim bladders of 13 elvers from Grande and Petite Trinité rivers (Côte-Nord-Québec). We suggest that our qPCR approach could be used in a quantitative way to estimate the parasitic burden in individual swim bladders of elvers. Our method, which goes beyond most of previous developed protocols that restricted the diagnosis of A. crassus to the moment when it was fully established in its final host, should help to detect early A. crassus infection in nature.

QTL study reveals candidate genes underlying host resistance in a Red Queen model system.

Specific interactions of host and parasite genotypes can lead to balancing selection, maintaining genetic diversity within populations. In order to understand the drivers of such specific coevolution, it is necessary to identify the molecular underpinnings of these genotypic interactions. Here, we investigate the genetic basis of resistance in the crustacean host, Daphnia magna, to attachment and subsequent infection by the bacterial parasite, Pasteuria ramosa. We discover a single locus with Mendelian segregation (3:1 ratio) with resistance being dominant, which we call the F locus. We use QTL analysis and fine mapping to localize the F locus to a 28.8-kb region in the host genome, adjacent to a known resistance supergene. We compare the 28.8-kb region in the two QTL parents to identify differences between host genotypes that are resistant versus susceptible to attachment and infection by the parasite. We identify 13 genes in the region, from which we highlight eight biological candidates for the F locus, based on presence/absence polymorphisms and differential gene expression. The top candidates include a fucosyltransferase gene that is only present in one of the two QTL parents, as well as several Cladoceran-specific genes belonging to a large family that is represented in multiple locations of the host genome. Fucosyltransferases have been linked to resistance in previous studies of Daphnia-Pasteuria and other host-parasite systems, suggesting that P. ramosa spore attachment could be mediated by changes in glycan structures on D. magna cuticle proteins. The Cladoceran-specific candidate genes suggest a resistance strategy that relies on gene duplication. Our results add a new locus to a growing genetic model of resistance in the D. magna-P. ramosa system. The identified candidate genes will be used in future functional genetic studies, with the ultimate aim to test for cycles of allele frequencies in natural populations.

Infection with an acanthocephalan helminth reduces anxiety-like behaviour in crustacean host

Trophically transmitted heteroxenous parasites of diverse clades can decrease or reverse antipredator behaviours in their intermediate hosts, thereby increasing their chances of reaching their final hosts. Such behavioural alterations could result from compromised cognitive abilities affecting fear- or more generally stress-related neurophysiological pathways. We tested this hypothesis in a key model system in the study of parasitic manipulation, the fish acanthocephalan parasite Pomphorhynchus tereticollis and its intermediate crustacean host Gammarus fossarum, using the ‘threat of electric shock’ paradigm. We exposed uninfected and infected G. fossarum to chronic and/or acute electric shock programs at two different intensities (voltage), and then quantified their sheltering behaviour as a proxy for anxiety-like state. Infected gammarids did not express anxiety-like response to electric shocks, while uninfected gammarids hid more when exposed to acute treatments, and when exposed to the high intensity chronic treatment. Interestingly, the lack of response in infected gammarids depended on parasite developmental stage. Our results support the hypothesis that this acanthocephalan parasite impacts the general anxiety-like circuitry of their intermediate host. Further studies are needed to investigate whether it involves inappropriate processing of information, impaired integration, or altered activation of downstream pathways initiating behavioural action.

Molecular and morphological description of a novel microsporidian Inodosporus fujiokai n. sp. infecting both salmonid fish and freshwater prawns.

A new microsporidian disease of cultured rainbow trout Oncorhynchus mykiss has recently been confirmed in Japan, and the causative species was tentatively designated as Microsporidium sp. RBT-2021. Involvement of common prawn Palaemon paucidens in its transmission was suggested based on the previous feeding trials, although the microsporidian infection in P. paucidens was not confirmed. In this study, P. paucidens in Lake Biwa, Japan was investigated for microsporidian infection and 4 types of spores (types 1-4) were newly found. The nucleotide sequence of the small subunit ribosomal RNA gene was identical between type 1 and Microsporidium sp. RBT-2021, indicating they are conspecific. However, intriguingly, the spore morphology and the mode of development in fish and prawn were strikingly different. Morphological observations revealed type 1 in the prawn possesses characteristics of the genus Inodosporus Overstreet and Weidner, 1974, while Microsporidium sp. RBT-2021 in the trout exhibited the characteristics of the genus Kabatana Lom, Dyková and Tonguthai, 2000. In the phylogeny, type 1 was placed within a clade comprising Kabatana spp. and Inodosporus octosporus. Based on the morphological and molecular analyses, we describe Microsporidium sp. RBT-2021 as Inodosporus fujiokai n. sp. Together with the success of the previous prawn-feeding trials, this study strongly suggests I. fujiokai n. sp. has a multi-host life cycle utilizing fish and crustacean hosts and different modes of development in each host. Such polymorphic life cycle has barely been known among fish microsporidians. This study also suggests that the genus Kabatana is a junior synonym of the genus Inodosporus.

Narrower isotopic niche size in fish infected by the intestinal parasite Pomphorhynchus sp. compared to uninfected ones.

Examples of parasite-related effects on intermediate crustacean hosts are numerous but their ecological consequences on their vertebrate hosts are scarce. Here, we address the role of macroparasite infections on the trophic niche structure of definitive hosts and its potential physiological consequences using wild fish populations infected with an acantochephalan parasite Pomphorhynchus sp., a trophically transmitted intestinal worm. Infected and uninfected fish were sampled from six populations on the Marne River, France and the prevalence of intestinal parasites in the host populations ranged from 50% to 90%. Although the isotopic ratios (δ13 C and δ15 N) did not differ between infected and uninfected fish, we found a consistent pattern of isotopic niche size being considerably smaller in infected hosts when compared with noninfected ones. This was not explained by interindividual differences in intrinsic factors such as length/age or body condition between infected and uninfected fish. These results suggest a potential niche specialization of infected fish, which did not impair their energetic status.

Integrative omics analysis highlights the immunomodulatory effects of the parasitic dinoflagellate Hematodinium on crustacean hemocytes

Parasitic dinoflagellates in genus Hematodinium have caused substantial economic losses to multiple commercially valuable marine crustaceans around the world. Recent efforts to better understand the life cycle and biology of the parasite have improved our understanding of the disease ecology. However, studies on the host-parasite interaction, especially how Hematodinium parasites evade the host immune response are lacking. To address this shortfall, we used the comprehensive omics approaches (miRNA transcriptomics, iTRAQ-based proteomics) to get insights into the host-parasite interaction between hemocytes from Portunus trituberculatus and Hematodinium perezi in the present study. The parasitic dinoflagellate H. perezi remodeled the miRNome and proteome of hemocytes from challenged hosts, modulated the host immune response at both post-transcriptional and translational levels and caused post-transcriptional regulation to the host immune response. Multiple important cellular and humoral immune-related pathways (ex. Apoptosis, Endocytosis, ECM-receptor interaction, proPO activation pathway, Toll-like signaling pathway, Jak-STAT signaling pathway) were significantly affected by Hematodinium parasites. Through modulation of the host miRNome, the host immune responses of nodulation, proPO activation and antimicrobial peptides were significantly suppressed. Cellular homeostasis was imbalanced via post-transcriptional dysregulation of the phagosome and peroxisome pathways. Cellular structure and communication was seriously impacted by post-transcriptional downregulation of ECM-receptor interaction and focal adhesion pathways. In conclusion, H. perezi parasites could trigger striking changes in the miRNome and proteome of crustacean hemocytes, and this parasite exhibited multifaceted immunomodulatory effects and potential immune-suppressive mechanisms in crustacean hosts.

Inhibiting viral replication and prolonging survival of hosts by attenuating stress responses to viral infection

Viruses are obligate intracellular parasites relying on host cells to obtain biosynthetic precursors and energy to successfully infect the host. The metabolic profile of the host cell is known to be altered in response to viral infection to satisfy the resources demanded during viral replication. Previous data of ours showed that white spot syndrome virus (WSSV) elicited in a crustacean host (Procambarus clarkii) a rapid and long-lasting release of crustacean hyperglycemic hormone (CHH), a well-known carbohydrate-regulating and stress response-mediating endocrine hormone. Therefore, the WSSV-enhanced release of CHH could be responsible at least in part for the metabolic alterations in the WSSV-challenged host. To investigate the possible metabolic roles of CHH in the host-parasite interaction, we studied whether silencing CHH gene expression could inhibit WSSV propagation in tissues and reduce the mortality of the WSSV-infected animals. Data presented in this study showed that CHH gene silencing indeed resists the WSSV infection. Injection of CHH dsRNA at the dosage of 140 μg/g BW caused significant decreases of viral copy number in tissues of WSSV-infected host, particularly showing a pronounced effect in the endodermal tissues (including hepatopancreas and gastrolith disk). Furthermore, results from the cumulative mortality showed that the treatment of CHH dsRNA delayed death from WSSV. Injection of CHH dsRNA at the dosages of 70, 17, and 10 μg/ g BW significantly extended the mean survival time. Together, this study concludes that the silencing of the CHH gene does have an inhibitory effect on the replication of the white spot syndrome virus and can assist the host to mitigate the invasion of WSSV, through attenuating CHH-mediated stress responses.

Characterization of Two Novel Toti-Like Viruses Co-infecting the Atlantic Blue Crab, Callinectes sapidus, in Its Northern Range of the United States.

The advancement of high throughput sequencing has greatly facilitated the exploration of viruses that infect marine hosts. For example, a number of putative virus genomes belonging to the Totiviridae family have been described in crustacean hosts. However, there has been no characterization of the most newly discovered putative viruses beyond description of their genomes. In this study, two novel double-stranded RNA (dsRNA) virus genomes were discovered in the Atlantic blue crab (Callinectes sapidus) and further investigated. Sequencing of both virus genomes revealed that they each encode RNA dependent RNA polymerase proteins (RdRps) with similarities to toti-like viruses. The viruses were tentatively named Callinectes sapidus toti-like virus 1 (CsTLV1) and Callinectes sapidus toti-like virus 2 (CsTLV2). Both genomes have typical elements required for −1 ribosomal frameshifting, which may induce the expression of an encoded ORF1–ORF2 (gag-pol) fusion protein. Phylogenetic analyses of CsTLV1 and CsTLV2 RdRp amino acid sequences suggested that they are members of two new genera in the family Totiviridae. The CsTLV1 and CsTLV2 genomes were detected in muscle, gill, and hepatopancreas of blue crabs by real-time reverse transcription quantitative PCR (RT-qPCR). The presence of ~40 nm totivirus-like viral particles in all three tissues was verified by transmission electron microscopy, and pathology associated with CsTLV1 and CsTLV2 infections were observed by histology. PCR assays showed the prevalence and geographic range of these viruses, to be restricted to the northeast United States sites sampled. The two virus genomes co-occurred in almost all cases, with the CsTLV2 genome being found on its own in 8.5% cases, and the CsTLV1 genome not yet found on its own. To our knowledge, this is the first report of toti-like viruses in C. sapidus. The information reported here provides the knowledge and tools to investigate transmission and potential pathogenicity of these viruses.

‘Candidatus Mellornella promiscua’ n. gen. n. sp. (Alphaproteobacteria: Rickettsiales: Anaplasmataceae): An intracytoplasmic, hepatopancreatic, pathogen of the flatback mud crab, Eurypanopeus depressus

Bacterial pathogens are a long-standing threat to the longevity and survival of crustacean hosts. Their presence and continuing emergence require close monitoring to understand their impact on fished, cultured, and wild crustacean populations. We describe a new bacterial pathogen belonging to the Anaplasmataceae family (Alphaproteobacteria: Rickettsiales), providing pathological, ultrastructural, phylogenetic, and genomic evidence to determine a candidate genus and species (‘Candidatus Mellornella promiscua’). This bacterium was found to infect the mud crab, Eurypanopeus depressus, on the North Carolina coastline (USA) at a prevalence of 10.8%.‘Candidatus Mellornella promiscua’ was often observed in co-infection with the rhizocephalan barnacle, Loxothylacus panopaei. The bacterium was only found in the hepatopancreas of the mud crab host, causing cytoplasmic hypertrophy, tubule necrosis, large plaques within the cytoplasm of the host cell, and an abundance of sex-pili. The circular genome of the bacterium is 1,013,119 bp and encodes 939 genes in total. Phylogenetically, the new bacterium branches within the Anaplasmataceae. The genome is dissimilar from other described bacteria, with 16S gene similarity observed at a maximum of 85.3% to a Wolbachia endosymbiont.We explore this novel bacterial pathogen using genomic, phylogenetic, ultrastructural, and pathological methods, discussing these results in light of current bacterial taxonomy, similarity to other bacterial pathogens, and the potential impact upon the surrounding disease ecology of the host and benthic ecosystem.

Hematodinium sp. infection does not drive collateral disease contraction in a crustacean host.

Host, pathogen, and environment are determinants of the disease triangle, the latter being a key driver of disease outcomes and persistence within a community. The dinoflagellate genus Hematodinium is detrimental to crustaceans globally - considered to suppress the innate defences of hosts, making them more susceptible to co-infections. Evidence supporting immune suppression is largely anecdotal and sourced from diffuse accounts of compromised decapods. We used a population of shore crabs (Carcinus maenas), where Hematodinium sp. is endemic, to determine the extent of collateral infections across two distinct environments (open-water, semi-closed dock). Using a multi-resource approach (PCR, histology, haematology, population genetics, eDNA), we identified 162 Hematodinium-positive crabs and size/sex-matched these to 162 Hematodinium-free crabs out of 1191 analysed. Crabs were interrogated for known additional disease-causing agents; haplosporidians, microsporidians, mikrocytids, Vibrio spp., fungi, Sacculina, trematodes, and haemolymph bacterial loads. We found no significant differences in occurrence, severity, or composition of collateral infections between Hematodinium-positive and Hematodinium-free crabs at either site, but crucially, we recorded site-restricted blends of pathogens. We found no gross signs of host cell immune reactivity towards Hematodinium in the presence or absence of other pathogens. We contend Hematodinium sp. is not the proximal driver of co-infections in shore crabs, which suggests an evolutionary drive towards latency in this environmentally plastic host.