Dual microphallid infections in shore crabs (Carcinus maenas): Site-level variation and phylogenetic insights in a globally invasive host

Dual microphallid infections in shore crabs (Carcinus maenas): Site-level variation and phylogenetic insights in a globally invasive host | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dual microphallid infections in shore crabs (Carcinus maenas): Site-level variation and phylogenetic insights in a globally invasive host Alexander T. Bedford, Grace Crocker, Andrew F. Rowley, Charlotte E. Davies This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7425079/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Feb, 2026 Read the published version in Parasitology Research → Version 1 posted 13 You are reading this latest preprint version Abstract Digenean trematodes commonly use marine crustaceans as intermediate hosts, yet their distribution and host-specific dynamics remain underexplored in temperate European systems. In this study, metacercarial stages of Microphallus similis and M. primas were identified in the hepatopancreas of Carcinus maenas collected from two distinct locations in South Wales, UK across a 4-month period. M. similis exhibited consistently high prevalence (~ 76%) at both sites, while M. primas occurred only as a co-infection, with lower prevalence (2–9%) and infection intensity. At Oxwich Bay, crab size and coloration were significantly associated with trematode infection, whereas no such associations were observed at Mumbles Head. Phylogenetic analysis confirmed species-level divergence between M. similis and M. primas . Overall parasite burden was significantly higher at Mumbles Head, with larger crabs harbouring more metacercariae. Additionally, metacercariae from Mumbles Head were significantly larger than those from Oxwich Bay, suggesting site-specific variation in parasite development. Carcinus maenas Microphallus similis Microphallus primas Digenean metacercariae Intermediate host Parasite burden Spatial variation Marine parasitology Host-parasite interactions Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The superfamily Microphalloidea (Ward, 1901; class Trematoda) is a group of ca . 170 genera of parasites often found associated with aquatic animals (Kostadinova and Pérez-del-Olmo, 2024 ). Many microphallids (family Microphallidae; Ward, 1901) including species of Microphallus and Maritrema , employ a three-host life cycle with the vertebrate definitive hosts (often birds or fish) producing eggs that infect the first intermediate host (usually molluscs) by the generation of ciliated miracidia. Within these latter hosts, daughter sporocysts or rediae are produced that are found in various tissues including the digestive gland, gonads and mantle. Cercarial development occurs in molluscan tissues and large numbers of free-swimming cercariae are released to infect the second intermediate host, usually a crustacean, where they become encysted (termed metacercariae) in the interstitial tissues in the hepatopancreas or less frequently in the haemal channels of the gills (Saville and Irwin 2005 ; Stentiford and Feist, 2008). When these hosts are predated upon by birds, the cycle is completed. Some digeneans, including microphallids, have evolved a truncated two host life cycle with a vertebrate definitive host and a single intermediate molluscan host. This simplified life cycle may give such digeneans an evolutionary advantage (Poulin and Cribb, 2002 ). The intertidal zone in rocky and sandy shores, and estuarine mud flats, can be productive hubs for these complex digenean life cycles with their abundant populations of resident littorinid snails, second intermediate hosts such as crabs and amphipods, and fish and sea bird populations, that predate on either molluscs (in truncated life cycles) or crustaceans in the typical three host life cycle (Poulin and Cribb, 2002 ). Parasites can have both direct (e.g. host survival) and indirect (e.g. habitat changes affecting other non-parasitised organisms) effects in such ecosystems (Mouritsen and Poulin, 2002 ). While few studies have examined levels of parasitism in all hosts in one location, Thieltges and co-workers comprehensively determined how parasites can affect food webs and population dynamics in the mud flats of the Wadden Sea in Europe (Thieltges et al., 2008 , 2013 , 2018 ). These studies revealed how parasites need to be incorporated into considerations of food webs both as prey and predators and shown how they are key players in shaping coastal ecosystems. Microphallus similis (Jägerskiöld, 1900) is a common microphallid parasite in intertidal and subtidal regions across the Northern hemisphere in Europe, Iceland and North America (Stunkard, 1957 ; James, 1968 ; Blakeslee et al., 2009 , 2015 , 2020a , b ; Barnard et al., 2020 ). Like other microphallids, it is a generalist parasite capable of using several definitive, first and second intermediate hosts. The definitive hosts include various species of gulls and waders (Stunkard, 1957 ). The first intermediate hosts comprise several species of periwinkles, Littorina saxatilis (James, 1969 ; Galaktionov and Bustnes, 1999 ; Galaktionov and Skirnisson, 2000 ; Bojko et al., 2017 ), L. obtusata (Stunkard, 1957 ; Galaktionov and Bustnes, 1999 ; Galaktionov and Skirnisson, 2000 ) L. mariae (Galaktionov and Skirnisson, 2000 ) L. littorea (Galaktionov and Bustnes, 1999 ) and L. arcana (Bojko et al., 2017 ). The second intermediate hosts have also been identified as the European shore crab (also referred to as the green crab), Carcinus maenas (Stunkard, 1957 ; Tkach et al., 2002; Galaktionov et al., 2012 ; Blakesee et al., 2015, 2020a,b; Ro et al., 2022 ), spider crabs, Hyas araneus (Galaktionov and Bustnes, 1999 ; Prokofiev et al., 2016 ) and edible (brown) crabs, Cancer pagurus (Crothers, 1966 ; Stentiford, 2008 ). C. maenas , is not only ecologically dominant in its native range but is also one of the world’s most invasive marine species, introduced to multiple continents including North America, South Africa, and Australia. Its role as an intermediate host for trematodes such as Microphallus similis and M. primas has implications not just for native parasite transmission, but also for parasite spillover, host-switching, and altered ecological dynamics in invaded regions. Despite this, few studies have examined infection dynamics in natural crab populations using molecular confirmation of parasite identity, particularly in the context of co-infection. Remarkably, both native and non-native (i.e. non-native invasive species) shore crabs have been found to harbour this parasite but levels of parasitism in North America seem to be lower than in those sites surveyed in the native range in Northern Europe (Blakeslee et al., 2009 ) probably because of ‘parasite escape’. This study aimed to examine the populations of digenean parasites in shore crabs found in two locations in South Wales U.K., namely Mumbles Head and Oxwich Bay. Both intertidal locations have large populations of C. maenas and periwinkles as potential first intermediate hosts. These sites were chosen as previous long term disease surveys have been carried out at both of these locations (e.g. Smith et al., 2015 ; Davies et al., 2019 , 2020a , b , 2022 ) but although encysted metacercariae were found in shore crabs via histology in our previous reports (e.g. Davies et al., 2022 ), they were neither quantified in terms of intensity of infection or identified using reliable (i.e. DNA sequence-based) methods. We also wished to investigate if crabs could be hosts for more than one species of digenean parasite as occurs in the first intermediate molluscan hosts such as periwinkles (James, 1969 ; Esch et al., 2001 ) and mudsnails (Field and Irwin, 1999 ). Materials and Methods Study area The study was conducted on spring tides at two rocky intertidal sites around Swansea, South Wales, UK: Oxwich Bay (51°33'11.4"N 4°09'03.7"W) and Mumbles Head (51° 34' 8.36"N, 3° 58' 35.93"W). Oxwich Bay, situated on the south shore of the Gower Peninsula primarily consists of sandy sediment, with subtidal and intertidal rocky reefs to the southwest (the collection site). Mumbles headland, situated on a rocky shore, is located to the south of Swansea Bay. Positioned within the Bristol Channel, both shores feature substantial tidal ranges (8.5m spring tides; 4.1m neap tides) (Collins et al., 1979). Oxwich Bay lies within multiple conservation designations, including a Special Area of Conservation (SAC) and Site of Special Scientific Interest (SSSI), whereas Mumbles Head, despite its proximity, falls outside formal marine protected area boundaries. Crab collection and storage Crab collection was conducted at both locations three times (April, June and August 2024) following an initial pilot study in February 2024 involving only 13 crabs. Approximately 30 shore crabs (see Supplementary Information, Table S1 for further details) were randomly sampled in the intertidal zone during each collection point. At Oxwich Bay, samples were collected exclusively using manual collection by boulder turning. At Mumbles Head, an additional collection method was employed: baited crab pots, which were deployed and immersed 24 hr prior to collection. Surveyed crabs were transported back in seaweed to an aquarium where they remained for > 48 hr. During this time crabs were fed ad libitum with mussels. Laboratory regime All crabs were processed within 48 hr post collection and placed on ice for a minimum of 20 min prior to examination. Biometric data were recorded for each crab. including sex, moult stage [inter-moult (hard) or post-moult (soft)], fouling (visible epibionts on the crab surface), external presence of potential diseases and causing agents ( Sacculina , pigment loss, shell disease), carapace width (mm), ventral carapace appearance (green, yellow, orange/red), limb loss or additional damage (e.g., exoskeletal fractures) and weight. Approximately 300 μl of haemolymph was extracted from each crab using a 23-gauge hypodermic needle fitted to a 1 ml syringe. Haemolymph appearance was deemed either ‘normal’ (clear to slightly cloudy) or milky in appearance as an initial indication of potential systemic infection. A small drop of haemolymph was then placed on to a microscope slide for screening for the presence of Hematodinium , haplosporidians, and fungi known to proliferate in this tissue (Davies et al., 2019, 2020a,b, 2022) using phase contrast optics of an Olympus BX41 microscope. Subsequently, crabs were sacrificed via placing at -18 °C for 30-45 min. The whole hepatopancreas was removed, weighed, and stored at -18 °C for later processing. Post storage, the hepatopancreas samples were left to thaw at RT for ca . 30 min. Each sample was disassociated in 4-5 ml of 3% NaCl and vacuum filtered through a sterile 150 µm pore size low density polyethylene cell strainer (pluriStrainer, Leipzig, Germany). Metacercarial cyst counts were performed on the filter using a binocular dissecting microscope. Metacercariae were manually removed from the filter, either individually or in small groups, placed in Eppendorf tubes, and stored at -18 ºC for later DNA extraction. A subsample of cysts (n = 134) was visualised using darkfield or brightfield optics of an Olympus BX41 microscope equipped with a digital camera. Cyst sizes were calculated using imageJ (Schindelin et al., 2012) by measuring height and width of the inner most cyst wall. DNA extraction, amplification, sequencing and phylogenetic analyses DNA was extracted from thawed cysts isolated from host tissue using a Qiagen Blood and Tissue Kit (Qiagen, Hilden, Germany) using adapted manufacturer’s instructions by extending the proteinase K digestion step to overnight. Extracted DNA was quantified using Qubit ® dsDNA Broad Sensitivity Assay Kit and a Qubit ® Fluorometer (ThermoFisher Scientific, Altrincham, UK). All PCR reactions were carried out in 25 μl total reaction volumes containing 12.5 µl of 2 x BioMix (New England Biolabs Inc., Ipswich, USA), 0.5 μl of each primer at a concentration of 10 μM (Eurofins, Ebersberg, Germany), 0.5 μl of nuclease-free water (Invitrogen™, Leicestershire, UK), 1 μl of genomic DNA ( ca . 50 – 200 ng/μl) and performed on a T100 PCR thermal cycler (BioRad Laboratories Inc., Watford, UK). Universal digenean primers derived from Tkach et al. (2003) and Galaktionov et al. (2012) were employed. These were: forward primer LSU-5 (5’-TAG GTC GAC CCG CTG AAY TTA AGC A-3’) and reverse primer LSU – 1500R (5’-GCT ATC CTG AGG GAA ACT TCG-3’). The cycling conditions were as follows: 3 min denaturation hold at 94 ºC; 35 cycles of 30 sec at 94 ºC, 30 sec at 60 ºC, 1 min at 72 ºC; and a final phase at 72 ºC for 5 min. Five microlitres of post PCR product was mixed with 1 μl of 6x DNA loading dye (ThermoFisher Scientific, Altrincham, UK) and loaded on to a 2% agarose/TAE gel stained with GreenSafe premium nucleic acid stain (NZYTech, Lisboa, Portugal). Gels were run for 45-60 min at 60 v with a 1 Kb Plus DNA Ladder (New England Biolabs, Hitchin, UK). Gel imaging was completed using a Molecular Imager® Gel Doc TM XR System (BioRad Laboratories Inc., Watford, UK). Positive samples were purified using ExoSAP-ITTM Express Fast High-Throughput PCR product clean-up (ThermoFisher Scientific, Altrincham, UK) for target sequencing (5:2; 4 min at 37°C, 1 min at 80°C). Amplicons were sent for DNA Sanger sequencing using both forward and reverse primers, synthesised by Eurofins (Ebersberg, Germany). Forward and reverse sequences were trimmed to remove primers, and consensus sequences were constructed using the CAP contig assembly extension in BioEdit software (Hall et al., 1999). All resulting sequences were subjected to the bioinformatic tool for similarity search BLAST (Camacho et al., 2009) to confirm identification and deposited in the GenBank database under the accession numbers PQ314574 – PQ314578, PQ314583 - PQ314592, and PQ314598- PQ314610 (See Supplementary Information, Table S2) Multiple sequence alignments were performed in CLUSTAL X v.2 (Larkin et al., 2007). Following quality control, sequences were combined with reference sequences to create two phylograms Alignments were analysed for the best fitting model using the IQ-TREE server, according to Bayesian information criterion (Kalyaanamoorthy et al . , 2017; Nguyen et al . , 2015; Hoang et al . , 2018; Trifinopoulos et al . , 2016). The final phylograms were constructed using Maximum Likelihood process with 1000 bootstrap replicates and annotated in iTOL (Letunic and Bork, 2019). Reference sequences comparing the same genetic region (locus) of both M. similis and M. primas obtained from a variety of hosts, were sourced from GenBank at NCBI (Benson et al., 2017). Statistical analyses Binomial logistic regression models with Logit link functions (following Bernoulli distributions) were used (MASS library) to examine the impact of specific predictors on the likelihood of detecting digenean trematodes in sampled crab populations and to examine the impact of specific predictors on metacercarial cyst load within the sampled crab population. All logistic models were run in RStudio v. 2024.04.2+764 using R v.3.6.0. Initially, all potential predictor variables were included in what are termed full models. Non-significant predictors were then sequentially eliminated using the drop1 function to develop reduced models with greater predictive accuracy. The drop1 function tests each reduced model, lacking the least significant predictor, against the original full model. If a reduced model showed a statistically significant difference (using a Chi-square test for binomial responses to evaluate differences in residual sum of squares), the excluded predictor was permanently omitted. This stepwise refinement continued until a final, optimized model was established. The full models included the input variables: month (April, June, August), carapace width (continuous number), sex (male or female), colour (green, yellow, red), pigment loss (0 or 1), haemolymph opacity (clear or milky, 0 or 1), fouling (presence of epibionts, 0 or 1). The initial model also included location (Mumbles or Oxwich) before being separated for further analysis (Table S3, Supplementary Information). To examine the impact of specific predictors on metacercarial cyst load within the sampled crab population, a Generalized Linear Model (GLM) with a Negative Binomial function was utilized (MASS library). This model was selected due normality tests indicating that the data presented with severe overdispersion (variance > mean). Prior to analysis, the dataset was refined using the subset function to exclude crabs with zero cysts. The input variables included in the model were as follows: location (Mumbles, Oxwich), month (April, June, August), carapace width (continuous number), sex (male or female), haemolymph opacity (clear or milky, 0 or 1), fouling (presence of epibionts, 0 or 1), and pigment loss (0 or 1). A Mann-Whitney test was conducted to determine if there was a statistically significant difference in the average cyst size between the two sample locations. Normality of the dataset was assessed using a Shapiro-Wilk test. This analysis was restricted to metacercariae with a spherical morphology only. The measurements of non-spherical metacercarial cysts were reported as height and width only. Graphics and statistical analyses were performed using GraphPad Prism v10.0.0 for Mac OS X and RStudio (v.2024.04.02+764) on R (v.3.6.0). Results General observations and cyst morphology In total, 163 crabs were sampled between April-August, 75 from Mumbles and 88 from Oxwich. An additional 13 crabs were also surveyed in February but the data from these are not included in this report. Biometric data including size range and sex ratios, as detailed in the Materials and Methods section, are shown in Supplementary Information Table S1. Examination of hepatopancreas samples under a binocular microscope revealed that ~76% were infected with trematode metacercariae. Cysts examined from the February (initial trial) and April-August (main trial) samples were spherical and all of similar size and general morphology (Figure 1A,C). Two morphologically distinct types of metacercariae were found from June onwards, with the original spherical forms as seen in February and April samples and an additional, asymmetrical form, found in only 9.3% of infected crabs from Mumbles and 2.3% from Oxwich (Figure 1B,D). Notably, these distinctive asymmetrical metacercariae were mostly observed in crabs with low intensity infections and always appeared as joint infections with the spherical form of metacercariae. Finally, there was no evidence of any cellular host response, specifically involving melanisation of the cysts, in any of the metacercariae examined. The diameter of 134 encysted metacercariae was measured. Among these, 120 from Mumbles (n = 86) and Oxwich (n = 34) were identified as spherical metacercariae consistent with the morphology described for Microphallus similis (Stunkard, 1957; Figure 1A,C). Normality of the dataset was assessed using a Shapiro–Wilk test, which indicated that the data were not normally distributed (P < 0.05), and therefore a Mann–Whitney U test was conducted to determine whether there was a statistically significant difference in the size of M. similis metacercariae between the two sample locations (Mumbles and Oxwich). This test indicated that there was a significant difference in cyst sizes between location (U = 1126, n 1 = 87, n 2 = 35, P = 0.0244), with significantly larger metacercariae at Mumbles than at Oxwich (292 ± 40 µm vs . 272 ± 23 µm, respectively, mean ± SD; Figure 2). The remaining measured metacercariae were identified by their oval (asymmetrical) morphology synonymous with descriptions of M. primas (Pina et al., 2011; Figure 1B,D). Due to a small number of metacercariae recorded, no statistical analysis was performed on cyst with oval-shaped morphology. General population measurements (n = 14), however, for metacercarial sizes showed mean dimensions (height vs . width) of 407 ± 16 by 313 ± 17 µm, respectively. Haemolymph screening using phase contrast microscopy identified Hematodinium infections in ca . 10% of crabs, with infection severity ranging from low (levels L1 and L2) to high (levels L3 and L4) (see Smith et al., 2015 and Davies et al., 2019 for a description of the severity levels). Additionally, 1.2% of crabs were found to have a Haplosporidian carcini infection (Davies et al., 2020a), and 1.8% were diagnosed with an unidentified fungal infection (Similar to Davies et al., 2020b). No crabs were found with externa of Sacculina carcini and while some crabs were found with shell disease lesions (see Supplementary Information Table S1) these were all superficial in nature. General and site-specific population observations of the prevalence of crab infestation Model 1, a binomial logistic regression model which combined data from both locations and used the presence of trematode parasites as the response variable, showed that crab size (carapace width) was the only factor associated with the presence of parasites (Table 1, Model 1). In terms of size, larger crabs were significantly ( P = 0.00286) more likely to present with trematode infections compared to parasite-free crabs (41 ± 11 vs . 33 ± 11 mm, respectively, mean ± SD). The prevalence of trematode infections in crabs sampled from Mumbles and Oxwich was 85% and 67%, respectively. The drop1 function revealed that despite an apparent lower infection presence at Oxwich, location was not an associated factor with trematode presence ( P = 0.092). Of the male crabs, 69% were parasitised whereas 83% of females presented with trematode metacercariae. Although this may suggest a higher prevalence of infection in female crabs, the combined model showed that sex was not a significant factor ( P = 0.18082) associated with trematode presence (Table 1, Model 1). To further explore the possible relationship between external factors and the presence of trematode parasites, the data were separated and analysed between the two locations (i.e. Mumbles vs . Oxwich). In Mumbles, 85% of surveyed crabs presented with trematode metacercariae. Model 2, which used the presence of trematode infection at this site as the response variable, indicated that no factors were associated with infection. In terms of crab size, the average carapace width of infected crabs from Mumbles was 43 ± 10 mm (range 19 – 61 mm). Although the combined model (Model 1) identified crabs of a larger size as a factor associated with trematode presence, subsequent analysis using the drop1 function in Model 2 deemed that crab size was not statistically significant for inclusion in the final (reduced) model (Table 1, Models 1 - 2). The drop1 function indicated a potentially lower presence of infection during August, however, the significant threshold ( P = 0.05) was not achieved (Table 1, Model 2). Additionally, sex, fouling (presence of epibionts), pigment loss, haemolymph opacity, and limb loss did not have a significant effect on parasitisation (data not shown). Of the crabs surveyed from Oxwich, 67% had metacercariae in the hepatopancreas. Using the presence of trematode infections at Oxwich as the response variable (Model 3), revealed that carapace width and colouration on the ventral surface were significant factors associated with trematode presence (Table 1, Model 3). In terms of size, larger crabs were significantly more likely ( P = 0.00062) to be infected with trematode parasites compared to parasite-free crabs (mean ± SD: 38.7 ± 10.5 vs . 29.9 ± 8.0 mm, respectively). Of the crabs surveyed from Oxwich, the colour distribution of infected individuals was as follows: 42% green, 7% yellow, and 51% red. Notably, shore crabs with a yellow ventral carapace coloration were significantly less likely to host trematode parasites than the other two colour morphs ( P = 0.02931; Table 1, Model 3). Table 1. Binomial logistic regression models (reduced from full models) testing the effects of biometric and environmental predictor variables on the overall presence of trematode parasites in the population. Models separated by location: Model 1, total population; Model 2, Mumbles; Model 3, Oxwich. Model Predictor Variable Estimate (slope) SE P-value Model 1 TremPres ~ Location + Sex + Carapace Width df = 159 AIC: 167.77 Location (Oxwich) - 0.71784 0.42619 0.09212 . Sex (Male) -0.55100 0.41173 0.18082 Carapace Width 0.05918 0.01984 0.00286 ** Model 2 TremPres ~ Month + Colour + Hemo.col df = 69 AIC: 69.402 Month (August) -2.3506 -1.714 0.08653 . Month (June) -0.2323 -0.275 0.78343 Colour (Red) -2.1355 -1.596 0.11047 Colour (Yellow) 15.3151 0.007 0.99426 HemoCol (Milky) 0.8920 0.962 0.33594 Model 3 TremPres ~ Carapace.Width + Colour df = 84 AIC: 94.678 Carapace Width 0.14027 0.04098 0.00062*** Colour (Red) 0.76112 0.59952 0.20424 Colour (Yellow) -2.53315 1.16239 0.02931* *Statistically significant * P ≤0.05, ** P ≤0.01,*** P ≤0.001 Abbreviation: SE, standard error Parasite intensity (i.e., loads ) There was a large range of the number of metacercariae/crab at both locations. In Mumbles this ranged from 1 – 265 cysts/crab (0.4 – 377 cysts/g wet weight hepatopancreas) while in Oxwich the equivalent data were 1 – 176 parasites/crab (0.3 – 86 cysts/g wet weight hepatopancreas). Model 4 shown in Table 2 combined data from both locations and used parasite count (cysts/crab) as the response variable. This model showed that location and carapace width were the only factors that had a significant effect on parasite load (Table 2). In terms of size, the model showed that larger crabs were significantly more likely to harbour a greater number of parasites (Table 2). Crabs sampled from Oxwich Bay had a significantly ( P = 0.009) lower parasite count compared to those in Mumbles Head (25 ± 40 vs . 41 ± 51 cysts/crab, respectively; mean ± SD). In terms of month, there was no significant difference in the number of parasite cysts per crab across sampling months (April, June, August) (31 ± 47 vs . 42 ± 49 vs . 26 ± 43, respectively; mean ± SD, Table 2, Model 4). Model 4 showed no significant difference ( P = 0.3) in the number of parasite cysts per crab when comparing males and female (29 ± 49 vs . 37 ± 44 parasites/crab, respectively; mean ± SD). Furthermore, haemolymph opacity, which can be an early indication of systematic infection, had no association ( P = 0.5956) with a change in parasite load (33 ± 47 vs . 37 ± 46 parasites/crab, clear vs . milky, respectively; mean ± SD, Table 2). However, the numbers of crabs with cloudy haemolymph were probably too small for meaningful analyses. Table 2. Generalised linear model with a negative binomial function testing the effect of biometric and environmental predictor variables on digenean trematode metacercarial cyst load in the total population of sampled C. maenas Model Predictor Variable Estimate (slope) SE P-value Model 4 Parasite count ~ Location + Month + Sex + Carapace Width + HemoCol + Pigment Loss + Fouling df = 116 AIC = 1082.5 Location (Oxwich) -0.79164 0.23512 0.00076 *** Month (August) -0.46575 0.27428 0.08949 . Month (June) -0.22969 0.26869 0.39263 Sex (Male) -0.44703 0.22956 0.05150 . Carapace Width 0.03817 0.01204 0.00153 ** HemoCol (Milky) -0.37159 0.46040 0.41960 Pigment Loss 0.02777 0.27760 0.92031 Fouling 0.81158 0.34743 0.01949 * *Statistically significant *P≤0.05, **P≤0.01,***P≤0.001 Abbreviation: SE, standard error Parasite identification and phylogenetic analysis Of the 123 trematode-infected crabs, a total of 26 samples from Mumbles (n = 15) and Oxwich (n =11) were successfully amplified and sequenced using the LSU-F/LSU-1500 oligonucleotides (Table S4, Figs S1-8, Supplementary Information). Of these sequences, 95.7% shared high similarity (>98.0% cover and identity) with M. similis from the slaty-backed gull ( Larus schistisagus ) from the Sea of Okhotsk, Russia (GenBank: HM584136 - HM584138). The remaining sequences derived from metacercariae from crabs harbouring the less common asymmetrical cysts also shared high similarity (>94% cover and identity) with those for M. primas from the mudsnail ( Peringia ulvae ). Following quality control, sequences were combined with reference sequences to create two phylograms (Figures 3 and 4). Figure 3 separated M. similis and M. primas into individual clades. Notably, most of the M. similis sequences formed a robust clade. In contrast, three M. primas sequences from August were unified in a single clade, while the one sequence from June displayed possible phylogenetic separation. Figure 4 delved deeper into the placement of the M. primas sequences from this study and the top 50 reference nucleotide sequences retrieved from GenBank. It shows the M. primas sequences clearly positioned in a clade alongside other M. primas , including samples from C. maenas in Portugal (Pina et al., 2011) and Hydrobia ulvae (a potential primary host) in the U.K. (Tkach et al., 2003). Discussion This study has shown that 76% crabs overall are subject to parasitisation by the microphallid, M. similis . It has also demonstrated that a smaller number of crabs from both sites were also infested with M. primas but at a much lower level (9% vs . 2% Mumbles vs . Oxwich). This latter parasite was never found to infect crabs that were free from M. similis . Furthermore, crabs were only found to harbour this parasite in the samples collected in the summer months of June and August. Finally, cyst sizes of M. similis differed significantly between Mumbles and Oxwich with those larger in the former site. Microphallus similis has been shown to utilise shore crabs as the second intermediate host in a variety of locations including South Wales (James, 1969) and Belfast Lough, Northern Ireland (Galaktionov et al., 2012) in the United Kingdom and in Northeast USA (Barnard et al., 2022) and Newfoundland, Canada (Blakeslee et al., 2020). Levels of parasitisation can vary dramatically from site to site but prevalences as high as 100% have been reported (see Blakeslee et al., 2009 for a summary of infection prevalences). Descriptions of the presence of M. primas in shore crabs are less frequent. Pina et al. (2011) used a morphological and molecular approach to identify this parasite in shore crabs from the Aviero estuary in Portugal. Davies et al. (2022) also identified two distinct forms of trematode metacercariae within the hepatopancreas of shore crabs from Mumbles Head using a histopathological approach. Although molecular techniques were not employed for species identification, the morphology of an additional morphotype of encysted metacercaria found on only one occasion in their year-long study closely resembles that of M. primas , as described in this study. This finding not only suggests the previous presence of M. primas in shore crabs from Swansea, U.K., but also demonstrates that co-infections with these two species of Microphallus are not unique at these locations. The prior potential identification of M. primas in 2019-2020 (Davies et al., 2022) also suggests that it is not a more recent introduction to the Swansea coastline. Finally, Stentiford and Feist (2008) using a histological approach concluded that the metacercariae found in the gills and hepatopancreas of shore crabs belonged to M. primas only although the morphology of some rounded forms in their illustrations is reminiscent of M. similis seen in our study – perhaps suggesting the presence of dual infections. The weakness of histology as an approach to identify metacercariae in <10 µm sections is that the plane of section does not always lend itself to interpretation of the 3D shape where forms can appear as spherical or ovoid depending on this aspect. Despite both digenean species M. similis and M. primas having been possibly identified in shore crabs across U.K. coastlines (e.g., Stentiford and Feist, 2005; Davies et al., 2022), research focusing on seasonal variations in prevalence within this secondary intermediate host is lacking. Based on the preliminary findings of this study, M. similis maintains a high level of presence within shore crab populations from Mumbles and Oxwich throughout the sampling period from late winter to summer. In contrast, M. primas only appeared and at a low prevalence from June onwards. These potential temporal changes in parasite presence may be due to factors including: (1). the migration of a population of crabs harbouring both M. similis and M. primas into the intertidal area between June and August, (2). an insufficient number of molluscan first intermediate host species to facilitate M. primas transmission in February and April, and/or (3). species variation in cercarial release which may also be influenced by environmental factors such as temperature. These possibilities are discussed below. The presence of M. primas at both Mumbles and Oxwich sites beginning in June may also be related to species-specific variations in cercarial release, potentially driven by environmental cues such as temperature, salinity and illumination. McCarthy et al. (2002) noted distinct patterns in the release and behaviour of cercariae among species; specifically, M. similis produced fewer but larger and more robustly swimming cercariae that gravitated towards darker waters, unlike other microphallid species. These observations suggest that factors such as light and the specific characteristics of cercariae release and movement could play critical roles in the successful localization of a viable host. It has been well documented that temperature also has a significant effect on the release and survival of trematode cercariae, with the release of many trematode species increasing with temperature (e.g., Poulin, 2006; Koprivnikar and Poulin, 2009a,b; Díaz-Morales et al., 2022). This suggests that the sudden appearance of M. primas could have been the direct cause of increasing summer temperatures in the intertidal zone. However, without a long-term comparative study of M. similis and M. primas , it remains uncertain whether these mechanisms are solely responsible for the inconsistent presence of M. primas , and that it is most likely to be influenced by a combination of host-parasite and environmental factors. The disparity between the levels of prevalence of M. similis and M. primas is also possibly associated with the habitat preference and population density of their respective first intermediate hosts. M. similis is known to have numerous first intermediate hosts within the genus Littorina, some of which have been reported at both Mumbles and Oxwich (Dixon and Pollard, 1985; Warwick et al., 1990). However, the only documented first intermediate host of M. primas is the mudsnail ( Peringia ulvae ). Although P. ulvae has been observed along coastlines within the Bristol Channel (Pinnion et al., 2007; authors personal observations), it is primarily located on their preferred habitats of mudflats and estuaries (i.e. not rocky shore areas such as those studied here). Stentiford and Feist (2005) noted a high prevalence of M. primas in the hepatopancreas and gills of shore crabs sampled from Southampton Water and Alde estuaries, U.K. Similarly, Pina et al. (2011), reported high a prevalence of M. primas in the hepatopancreas of shore crabs from the Aveiro Estuary in Portugal. This suggests the low prevalence of M. primas in this present study may be linked to the choice of survey site, in that intertidal rocky shores do not support a sufficient population of the established first intermediate hosts to facilitate the high levels of parasite transmission as observed in some estuarine environments. The size of encysted metacercariae of M. similis varies within species, often showing intraspecific variation due to different infection times and/or competition for space and resources within the host (Saldanha et al., 2009). Consistent with previous studies on M. similis (McCarthy et al., 2002) and M. primas (Saville and Irwin, 2005), the sizes of trematode metacercariae observed in this present study agree with these previous findings. Fredensborg and Poulin (2005) highlighted that the size of metacercariae is density-dependent; notably, higher intensity trematode infections are typically correlated with smaller-sized metacercariae. Based on these previous findings, it would be expected that shore crabs from Mumbles, given their higher infection intensities, should be more likely to host smaller M. similis metacercariae. However, contrary to these expectations, metacercariae isolated from crabs at Mumbles were significantly larger than those from hosts located at Oxwich Bay. An alternate explanation to our findings is that the cysts in Oxwich are less advanced in their development within the hepatopancreas than those in Mumbles and hence the observed size difference. In the current study, host size was found to be a significant factor in affecting the prevalence of digenean parasites but only at Oxwich Bay. This finding is consistent with previous research (e.g., Blakeslee et al., 2015; Koehler and Poulin, 2010; Koga, 2008), which suggests that larger, and therefore older, crabs, due to their longer exposure time, tend to accumulate more parasites and thus show higher parasitisation rates. However, while larger crabs naturally have more time to become parasitised, it is possible that host chemical cues (both attractive and inhibitory) may also be involved. Combes et al. (1994) and Haas (1994) have reviewed the significance of host chemical cues in cercariae host-finding behaviour in trematodes but information on the chemical nature and action of these is limited to a few trematodes such as schistosomes rather than those including M. similis and M. primas using crustaceans as second intermediate hosts. Given that larger C. maenas could emit a greater concentration of chemical cues due to their increased surface area of the gills and general body surface, they may attract more cercariae, thereby experiencing higher levels of parasite presence. Indeed, experimental studies of non-native shore crabs incubated with known numbers of cercariae from M. similis found that the largest crabs accrued the most metacercariae over a 5-day experimental period (Blakeslee et al., 2015). The absence of this finding in shore crabs from Mumbles could be caused by disparities in the two datasets. For instance, it is possible that the disparities in size of the crab populations sampled from Mumbles and Oxwich could contribute to the apparent lack of an observable relationship. The observed site-level variation in infection intensity and metacercarial size may be influenced by ecological and environmental differences between Oxwich Bay and Mumbles Head. These include differences in substrate type, hydrodynamic exposure, molluscan host availability, and crab microhabitats. For example, Mumbles Head's rocky intertidal structure may support higher densities of Littorina spp., facilitating sustained trematode transmission. Overall, we have demonstrated that shore crabs can host dual infestations as second intermediate hosts for at least two species of microphallids, and to our knowledge, this is the first such observation in crabs. The potential consequences of this infestation to the crabs surveyed is, however, unclear although Blakeslee et al. (2015) and Ro et al. (2022) reported only limited effects in terms of their behaviour of non-native C. maenas that could impact on their likelihood of predation by the definitive hosts. hese findings have implications beyond the surveyed Welsh sites. As C. maenas continues to expand its invasive range, understanding its parasite community structure, including potential co-infections, is critical for predicting ecological impact and managing disease risk. Studies have shown altered susceptibility, reduced parasite load, or novel host-switching in invaded regions (Blakeslee et al., 2009, 2020), raising the importance of monitoring parasite assemblages in both native and non-native populations. Molecularly confirmed records of species such as M. primas in C. maenas help clarify host–parasite relationships across the species' global range. Declarations Supplementary information . The supplementary information for this article can be found online. Acknowledgements . The authors are grateful to the Biosciences Technical Team in Swansea University (Hilary Williams, Jessica Minett, Jessica Bevan and Matthew Watkins) for their assistance. Author contributions . AFR and CED conceived and designed the study; all authors were involved in field and laboratory-based work; ATB, AFR and CED wrote the initial manuscript; all authors approved the final version of the manuscript at submission. Conflict of interest . The authors declare no conflicts of interest. Ethical standards . All institutional (Swansea University) guidelines and ethical approval for the collection, maintenance and euthanasia of crustaceans were followed. Research Ethics Approval Number: 2 2024 8411 7714. Funding No funding was received for this research. Clinical trial number Not applicable. Consent to Participate Not applicable. Consent to Publish Not applicable. Data Availability The datasets generated during the current study are available from the corresponding author. 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(A). Typical morphology of \u003cem\u003eM. similis\u003c/em\u003e metacercaria with characteristic spherical shape. (B). Morphology of asymmetrical \u003cem\u003eM. primas\u003c/em\u003e metcercaria. (C). Crab with large numbers of encysted \u003cem\u003eM. similis\u003c/em\u003e matacercariae clumped together in the interstitial tissue of the hepatopancreas. (D). Crab with dual infestations with \u003cem\u003eM. similis\u003c/em\u003e (left part of figure) and \u003cem\u003eM. primas\u003c/em\u003e (\u003csub\u003e*\u003c/sub\u003e) metcercariae. Scale bars = 100 µm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7425079/v1/1992acb226ab70bfe23a3582.png"},{"id":90993363,"identity":"b99f392d-3c7c-442b-8798-e315d8344aaa","added_by":"auto","created_at":"2025-09-10 11:56:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27886,"visible":true,"origin":"","legend":"\u003cp\u003eSize comparison (µm) of spherical trematode metacercariae extracted from the hepatopancreas of \u003cem\u003eCarcinus maenas\u003c/em\u003e sampled from two locations: Mumbles and Oxwich Bay. Boxplots display the median, interquartile range, and whiskers extend to the minimum and maximum observed values.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7425079/v1/e80f0e8651f211ca1a2709e9.png"},{"id":90993523,"identity":"25821d72-2d58-4a35-a9a5-c56c4eb69cc2","added_by":"auto","created_at":"2025-09-10 12:04:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":521785,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogram of the partial 28S rRNA gene region from trematode-infected crabs from this study and reference nucleotide sequences for microphallid species from various hosts retrieved from GenBank. Both \u003cem\u003eMicrophallus similis\u003c/em\u003e and \u003cem\u003eM. primas \u003c/em\u003esequences from this study are in \u003cstrong\u003ebold font\u003c/strong\u003e and are clearly in two, robust clades (highlighted pink and green, respectively). All clades with 99-100% bootstrap support are annotated with (l) at the node. Tree based on the evolutionary model TPM3u+F+G4, according to Bayesian information criterion from the IQ-TREE server (Nguyen et al. 2015; Trifinopoulos et al. 2016). The final tree was constructed using ML process with 1000 bootstrap replicates and annotated in iTOL (Letunic and Bork, 2019). Log-likelihood of the tree: -7449.0899 (s.e. 205.2217). Akaike information criterion (AIC) score: 15260.1798. Bayesian information criterion (BIC) score: 16301.8962.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7425079/v1/6650e273ec5ceabd4c46e5de.png"},{"id":90993367,"identity":"4386bc5f-ccb7-4f65-a285-79c5fd671d36","added_by":"auto","created_at":"2025-09-10 11:56:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":335253,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogram of the partial 28S rRNA gene region from \u003cem\u003eMicrophallus primas\u003c/em\u003e-infected crabs from this study and top 50 reference nucleotide sequences retrieved from GenBank. Sequences from this study are in \u003cstrong\u003ebold font\u003c/strong\u003e and are clearly in a robust clade (highlighted green) with other \u003cem\u003eM. primas\u003c/em\u003e. All clades with 92-100% bootstrap support are annotated with (l) at the node. Tree based on the evolutionary model GTR+F+I+G4, according to Bayesian information criterion from the IQ-TREE server (Nguyen et al. 2015; Trifinopoulos et al. 2016). The final tree was constructed using ML process with 1000 bootstrap replicates and annotated in iTOL (Letunic and Bork, 2019). Log-likelihood of the tree: -37716.7428 (s.e. 295.1992). Akaike information criterion (AIC) score: 75647.4857. Bayesian information criterion (BIC) score: 76411.5487.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7425079/v1/e3cff46903c1e8148123363f.png"},{"id":103251488,"identity":"5af35547-f818-41fa-a797-99070ca415d5","added_by":"auto","created_at":"2026-02-23 16:09:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5370408,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7425079/v1/aa6c2625-29cf-4889-bf93-b12cd429bf47.pdf"},{"id":90993531,"identity":"a964dc5d-6bae-4483-967c-e75dbd479e65","added_by":"auto","created_at":"2025-09-10 12:04:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14933962,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7425079/v1/622d955155fe2d0eb38a84ec.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dual microphallid infections in shore crabs (Carcinus maenas): Site-level variation and phylogenetic insights in a globally invasive host","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe superfamily Microphalloidea (Ward, 1901; class Trematoda) is a group of \u003cem\u003eca\u003c/em\u003e. 170 genera of parasites often found associated with aquatic animals (Kostadinova and P\u0026eacute;rez-del-Olmo, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Many microphallids (family Microphallidae; Ward, 1901) including species of \u003cem\u003eMicrophallus\u003c/em\u003e and \u003cem\u003eMaritrema\u003c/em\u003e, employ a three-host life cycle with the vertebrate definitive hosts (often birds or fish) producing eggs that infect the first intermediate host (usually molluscs) by the generation of ciliated miracidia. Within these latter hosts, daughter sporocysts or rediae are produced that are found in various tissues including the digestive gland, gonads and mantle. Cercarial development occurs in molluscan tissues and large numbers of free-swimming cercariae are released to infect the second intermediate host, usually a crustacean, where they become encysted (termed metacercariae) in the interstitial tissues in the hepatopancreas or less frequently in the haemal channels of the gills (Saville and Irwin \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Stentiford and Feist, 2008). When these hosts are predated upon by birds, the cycle is completed. Some digeneans, including microphallids, have evolved a truncated two host life cycle with a vertebrate definitive host and a single intermediate molluscan host. This simplified life cycle may give such digeneans an evolutionary advantage (Poulin and Cribb, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe intertidal zone in rocky and sandy shores, and estuarine mud flats, can be productive hubs for these complex digenean life cycles with their abundant populations of resident littorinid snails, second intermediate hosts such as crabs and amphipods, and fish and sea bird populations, that predate on either molluscs (in truncated life cycles) or crustaceans in the typical three host life cycle (Poulin and Cribb, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Parasites can have both direct (e.g. host survival) and indirect (e.g. habitat changes affecting other non-parasitised organisms) effects in such ecosystems (Mouritsen and Poulin, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). While few studies have examined levels of parasitism in all hosts in one location, Thieltges and co-workers comprehensively determined how parasites can affect food webs and population dynamics in the mud flats of the Wadden Sea in Europe (Thieltges et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These studies revealed how parasites need to be incorporated into considerations of food webs both as prey and predators and shown how they are key players in shaping coastal ecosystems.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMicrophallus similis\u003c/em\u003e (J\u0026auml;gerski\u0026ouml;ld, 1900) is a common microphallid parasite in intertidal and subtidal regions across the Northern hemisphere in Europe, Iceland and North America (Stunkard, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1957\u003c/span\u003e; James, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1968\u003c/span\u003e; Blakeslee et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Barnard et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Like other microphallids, it is a generalist parasite capable of using several definitive, first and second intermediate hosts. The definitive hosts include various species of gulls and waders (Stunkard, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1957\u003c/span\u003e). The first intermediate hosts comprise several species of periwinkles, \u003cem\u003eLittorina saxatilis\u003c/em\u003e (James, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Galaktionov and Bustnes, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Galaktionov and Skirnisson, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Bojko et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), L. \u003cem\u003eobtusata\u003c/em\u003e (Stunkard, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1957\u003c/span\u003e; Galaktionov and Bustnes, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Galaktionov and Skirnisson, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) \u003cem\u003eL. mariae\u003c/em\u003e (Galaktionov and Skirnisson, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) \u003cem\u003eL. littorea\u003c/em\u003e (Galaktionov and Bustnes, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and \u003cem\u003eL. arcana\u003c/em\u003e (Bojko et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The second intermediate hosts have also been identified as the European shore crab (also referred to as the green crab), \u003cem\u003eCarcinus maenas\u003c/em\u003e (Stunkard, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1957\u003c/span\u003e; Tkach et al., 2002; Galaktionov et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Blakesee et al., 2015, 2020a,b; Ro et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), spider crabs, \u003cem\u003eHyas araneus\u003c/em\u003e (Galaktionov and Bustnes, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Prokofiev et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and edible (brown) crabs, \u003cem\u003eCancer pagurus\u003c/em\u003e (Crothers, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1966\u003c/span\u003e; Stentiford, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). \u003cem\u003eC. maenas\u003c/em\u003e, is not only ecologically dominant in its native range but is also one of the world\u0026rsquo;s most invasive marine species, introduced to multiple continents including North America, South Africa, and Australia. Its role as an intermediate host for trematodes such as \u003cem\u003eMicrophallus similis\u003c/em\u003e and \u003cem\u003eM. primas\u003c/em\u003e has implications not just for native parasite transmission, but also for parasite spillover, host-switching, and altered ecological dynamics in invaded regions. Despite this, few studies have examined infection dynamics in natural crab populations using molecular confirmation of parasite identity, particularly in the context of co-infection. Remarkably, both native and non-native (i.e. non-native invasive species) shore crabs have been found to harbour this parasite but levels of parasitism in North America seem to be lower than in those sites surveyed in the native range in Northern Europe (Blakeslee et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) probably because of \u0026lsquo;parasite escape\u0026rsquo;.\u003c/p\u003e\u003cp\u003eThis study aimed to examine the populations of digenean parasites in shore crabs found in two locations in South Wales U.K., namely Mumbles Head and Oxwich Bay. Both intertidal locations have large populations of \u003cem\u003eC. maenas\u003c/em\u003e and periwinkles as potential first intermediate hosts. These sites were chosen as previous long term disease surveys have been carried out at both of these locations (e.g. Smith et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Davies et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003eb\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) but although encysted metacercariae were found in shore crabs via histology in our previous reports (e.g. Davies et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), they were neither quantified in terms of intensity of infection or identified using reliable (i.e. DNA sequence-based) methods. We also wished to investigate if crabs could be hosts for more than one species of digenean parasite as occurs in the first intermediate molluscan hosts such as periwinkles (James, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Esch et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and mudsnails (Field and Irwin, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eStudy area \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted on spring tides at two rocky intertidal sites around Swansea, South Wales, UK: Oxwich Bay (51\u0026deg;33\u0026apos;11.4\u0026quot;N 4\u0026deg;09\u0026apos;03.7\u0026quot;W) and Mumbles Head (51\u0026deg; 34\u0026apos; 8.36\u0026quot;N, 3\u0026deg; 58\u0026apos; 35.93\u0026quot;W). Oxwich Bay, situated on the south shore of the Gower Peninsula primarily consists of sandy sediment, with subtidal and intertidal rocky reefs to the southwest (the collection site). Mumbles headland, situated on a rocky shore, is located to the south of Swansea Bay. Positioned within the Bristol Channel, both shores feature substantial tidal ranges (8.5m spring tides; 4.1m neap tides) (Collins et al., 1979). Oxwich Bay lies within multiple conservation designations, including a Special Area of Conservation (SAC) and Site of Special Scientific Interest (SSSI), whereas Mumbles Head, despite its proximity, falls outside formal marine protected area boundaries. \u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eCrab collection and storage\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCrab collection was conducted at both locations three times (April, June and August 2024) following an initial pilot study in February 2024 involving only 13 crabs. Approximately 30 shore crabs (see Supplementary Information, Table S1 for further details) were randomly sampled in the intertidal zone during each collection point. At Oxwich Bay, samples were collected exclusively using manual collection by boulder turning. At Mumbles Head, an additional collection method was employed: baited crab pots, which were deployed and immersed 24 hr prior to collection. Surveyed crabs were transported back in seaweed to an aquarium where they remained for \u0026gt; 48 hr. During this time crabs were fed \u003cem\u003ead libitum\u003c/em\u003e with mussels.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eLaboratory regime \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll crabs were processed within 48 hr post collection and placed on ice for a minimum of 20 min prior to examination. Biometric data were recorded for each crab. including sex, moult stage [inter-moult (hard) or post-moult (soft)], fouling (visible epibionts on the crab surface), external presence of potential diseases and causing agents (\u003cem\u003eSacculina\u003c/em\u003e, pigment loss, shell disease), carapace width (mm), ventral carapace appearance (green, yellow, orange/red), limb loss or additional damage (e.g., exoskeletal fractures) and weight. Approximately 300 \u0026mu;l of haemolymph was extracted from each crab using a 23-gauge hypodermic needle fitted to a 1 ml syringe. Haemolymph appearance was deemed either \u0026lsquo;normal\u0026rsquo; (clear to slightly cloudy) or milky in appearance as an initial indication of potential systemic infection. A small drop of haemolymph was then placed on to a microscope slide for screening for the presence of \u003cem\u003eHematodinium\u003c/em\u003e, haplosporidians, and fungi known to proliferate in this tissue (Davies et al., 2019, 2020a,b, 2022) using phase contrast optics of an Olympus BX41 microscope. Subsequently, crabs were sacrificed via placing at -18 \u0026deg;C for 30-45 min. The whole hepatopancreas was removed, weighed, and stored at -18 \u0026deg;C for later processing. \u003c/p\u003e\n\u003cp\u003ePost storage, the hepatopancreas samples were left to thaw at RT for \u003cem\u003eca\u003c/em\u003e. 30 min. Each sample was disassociated in 4-5 ml of 3% NaCl and vacuum filtered through a sterile 150 \u0026micro;m pore size low density polyethylene cell strainer (pluriStrainer, Leipzig, Germany). Metacercarial cyst counts were performed on the filter using a binocular dissecting microscope. Metacercariae were manually removed from the filter, either individually or in small groups, placed in Eppendorf tubes, and stored at -18 \u0026ordm;C for later DNA extraction. A subsample of cysts (n = 134) was visualised using darkfield or brightfield optics of an Olympus BX41 microscope equipped with a digital camera. Cyst sizes were calculated using imageJ (Schindelin et al., 2012) by measuring height and width of the inner most cyst wall. \u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eDNA extraction, amplification, sequencing and phylogenetic analyses\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from thawed cysts isolated from host tissue using a Qiagen Blood and Tissue Kit (Qiagen, Hilden, Germany) using adapted manufacturer\u0026rsquo;s instructions by extending the proteinase K digestion step to overnight. Extracted DNA was quantified using Qubit\u003csup\u003e\u0026reg;\u003c/sup\u003e dsDNA Broad Sensitivity Assay Kit and a Qubit\u003csup\u003e\u0026reg;\u003c/sup\u003e Fluorometer (ThermoFisher Scientific, Altrincham, UK). \u003c/p\u003e\n\u003cp\u003eAll PCR reactions were carried out in 25 \u0026mu;l total reaction volumes containing 12.5 \u0026micro;l of 2 x BioMix (New England Biolabs Inc., Ipswich, USA), 0.5 \u0026mu;l of each primer at a concentration of 10 \u0026mu;M (Eurofins, Ebersberg, Germany), 0.5 \u0026mu;l of nuclease-free water (Invitrogen\u0026trade;, Leicestershire, UK), 1 \u0026mu;l of genomic DNA (\u003cem\u003eca\u003c/em\u003e. 50 \u0026ndash; 200 ng/\u0026mu;l) and performed on a T100 PCR thermal cycler (BioRad Laboratories Inc., Watford, UK). Universal digenean primers derived from Tkach et al. (2003) and Galaktionov et al. (2012) were employed. These were: forward primer LSU-5 (5\u0026rsquo;-TAG GTC GAC CCG CTG AAY TTA AGC A-3\u0026rsquo;) and reverse primer LSU \u0026ndash; 1500R (5\u0026rsquo;-GCT ATC CTG AGG GAA ACT TCG-3\u0026rsquo;). The cycling conditions were as follows: 3 min denaturation hold at 94 \u0026ordm;C; 35 cycles of 30 sec at 94 \u0026ordm;C, 30 sec at 60 \u0026ordm;C, 1 min at 72 \u0026ordm;C; and a final phase at 72 \u0026ordm;C for 5 min. \u003c/p\u003e\n\u003cp\u003eFive microlitres of post PCR product was mixed with 1 \u0026mu;l of 6x DNA loading dye (ThermoFisher Scientific, Altrincham, UK) and loaded on to a 2% agarose/TAE gel stained with GreenSafe premium nucleic acid stain (NZYTech, Lisboa, Portugal). Gels were run for 45-60 min at 60 v with a 1 Kb Plus DNA Ladder (New England Biolabs, Hitchin, UK). Gel imaging was completed using a Molecular Imager\u0026reg; Gel Doc\u003csup\u003eTM\u003c/sup\u003e XR System (BioRad Laboratories Inc., Watford, UK). Positive samples were purified using ExoSAP-ITTM Express Fast High-Throughput PCR product clean-up (ThermoFisher Scientific, Altrincham, UK) for target sequencing (5:2; 4 min at 37\u0026deg;C, 1 min at 80\u0026deg;C). Amplicons were sent for DNA Sanger sequencing using both forward and reverse primers, synthesised by Eurofins (Ebersberg, Germany). \u003c/p\u003e\n\u003cp\u003eForward and reverse sequences were trimmed to remove primers, and consensus sequences were constructed using the CAP contig assembly extension in BioEdit software (Hall et al., 1999). All resulting sequences were subjected to the bioinformatic tool for similarity search BLAST (Camacho et al., 2009) to confirm identification and deposited in the GenBank database under the accession numbers PQ314574 \u0026ndash; PQ314578, PQ314583 - PQ314592, and PQ314598- PQ314610 (See Supplementary Information, Table S2) \u003c/p\u003e\n\u003cp\u003eMultiple sequence alignments were performed in CLUSTAL X v.2 (Larkin et al., 2007). Following quality control, sequences were combined with reference sequences to create two phylograms Alignments were analysed for the best fitting model using the IQ-TREE server, according to Bayesian information criterion (Kalyaanamoorthy et al\u003cem\u003e.\u003c/em\u003e, 2017; Nguyen et al\u003cem\u003e.\u003c/em\u003e, 2015; Hoang et al\u003cem\u003e.\u003c/em\u003e, 2018; Trifinopoulos et al\u003cem\u003e.\u003c/em\u003e, 2016). The final phylograms were constructed using Maximum Likelihood process with 1000 bootstrap replicates and annotated in iTOL (Letunic and Bork, 2019). Reference sequences comparing the same genetic region (locus) of both \u003cem\u003eM. similis\u003c/em\u003e and \u003cem\u003eM. primas\u003c/em\u003e obtained from a variety of hosts, were sourced from GenBank at NCBI (Benson et al., 2017).\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eStatistical analyses\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBinomial logistic regression models with Logit link functions (following Bernoulli distributions) were used (MASS library) to examine the impact of specific predictors on the likelihood of detecting digenean trematodes in sampled crab populations and to examine the impact of specific predictors on metacercarial cyst load within the sampled crab population. All logistic models were run in RStudio v. 2024.04.2+764 using R v.3.6.0. Initially, all potential predictor variables were included in what are termed full models. Non-significant predictors were then sequentially eliminated using the drop1 function to develop reduced models with greater predictive accuracy. The drop1 function tests each reduced model, lacking the least significant predictor, against the original full model. If a reduced model showed a statistically significant difference (using a Chi-square test for binomial responses to evaluate differences in residual sum of squares), the excluded predictor was permanently omitted. This stepwise refinement continued until a final, optimized model was established. The full models included the input variables: month (April, June, August), carapace width (continuous number), sex (male or female), colour (green, yellow, red), pigment loss (0 or 1), haemolymph opacity (clear or milky, 0 or 1), fouling (presence of epibionts, 0 or 1). The initial model also included location (Mumbles or Oxwich) before being separated for further analysis (Table S3, Supplementary Information). \u003c/p\u003e\n\u003cp\u003eTo examine the impact of specific predictors on metacercarial cyst load within the sampled crab population, a Generalized Linear Model (GLM) with a Negative Binomial function was utilized (MASS library). This model was selected due normality tests indicating that the data presented with severe overdispersion (variance \u0026gt; mean). Prior to analysis, the dataset was refined using the subset function to exclude crabs with zero cysts. The input variables included in the model were as follows: location (Mumbles, Oxwich), month (April, June, August), carapace width (continuous number), sex (male or female), haemolymph opacity (clear or milky, 0 or 1), fouling (presence of epibionts, 0 or 1), and pigment loss (0 or 1).\u003c/p\u003e\n\u003cp\u003eA Mann-Whitney test was conducted to determine if there was a statistically significant difference in the average cyst size between the two sample locations. Normality of the dataset was assessed using a Shapiro-Wilk test. This analysis was restricted to metacercariae with a spherical morphology only. The measurements of non-spherical metacercarial cysts were reported as height and width only. Graphics and statistical analyses were performed using GraphPad Prism v10.0.0 for Mac OS X and RStudio (v.2024.04.02+764) on R (v.3.6.0).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eGeneral observations and cyst morphology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn total, 163 crabs were sampled between April-August, 75 from Mumbles and 88 from Oxwich. An additional 13 crabs were also surveyed in February but the data from these are not included in this report. Biometric data including size range and sex ratios, as detailed in the Materials and Methods section, are shown in Supplementary Information Table S1. Examination of hepatopancreas samples under a binocular microscope revealed that ~76% were infected with trematode metacercariae. Cysts examined from the February (initial trial) and April-August (main trial) samples were spherical and all of similar size and general morphology (Figure 1A,C). Two morphologically distinct types of metacercariae were found from June onwards, with the original spherical forms as seen in February and April samples and an additional, asymmetrical form, found in only 9.3% of infected crabs from Mumbles and 2.3% from Oxwich (Figure 1B,D). Notably, these distinctive asymmetrical metacercariae were mostly observed in crabs with low intensity infections and always appeared as joint infections with the spherical form of metacercariae. Finally, there was no evidence of any cellular host response, specifically involving melanisation of the cysts, in any of the metacercariae examined.\u003c/p\u003e\n\u003cp\u003eThe diameter of 134 encysted metacercariae was measured. Among these, 120 from Mumbles (n = 86) and Oxwich (n = 34) were identified as spherical metacercariae consistent with the morphology described for \u003cem\u003eMicrophallus similis\u003c/em\u003e (Stunkard, 1957; Figure 1A,C). Normality of the dataset was assessed using a Shapiro\u0026ndash;Wilk test, which indicated that the data were not normally distributed (P \u0026lt; 0.05), and therefore a Mann\u0026ndash;Whitney U test was conducted to determine whether there was a statistically significant difference in the size of \u003cem\u003eM. similis\u003c/em\u003e metacercariae between the two sample locations (Mumbles and Oxwich). This test indicated that there was a significant difference in cyst sizes between location (U = 1126, n\u003csub\u003e1\u003c/sub\u003e = 87, n\u003csub\u003e2\u003c/sub\u003e = 35, \u003cem\u003eP\u003c/em\u003e = 0.0244), with significantly larger metacercariae at Mumbles than at Oxwich (292 \u0026plusmn; 40 \u0026micro;m \u003cem\u003evs\u003c/em\u003e. 272 \u0026plusmn; 23 \u0026micro;m, respectively, mean \u0026plusmn; SD; Figure 2). The remaining measured metacercariae were identified by their oval (asymmetrical) morphology synonymous with descriptions of \u003cem\u003eM. primas\u003c/em\u003e (Pina et al., 2011; Figure 1B,D). Due to a small number of metacercariae recorded, no statistical analysis was performed on cyst with oval-shaped morphology. General population measurements (n = 14), however, for metacercarial sizes showed mean dimensions (height \u003cem\u003evs\u003c/em\u003e. width) of 407 \u0026plusmn; 16 by 313 \u0026plusmn; 17 \u0026micro;m, respectively.\u003c/p\u003e\n\u003cp\u003eHaemolymph screening using phase contrast microscopy identified \u003cem\u003eHematodinium\u003c/em\u003e infections in \u003cem\u003eca\u003c/em\u003e. 10% of crabs, with infection severity ranging from low (levels L1 and L2) to high (levels L3 and L4) (see Smith et al., 2015 and Davies et al., 2019 for a description of the severity levels). Additionally, 1.2% of crabs were found to have a \u003cem\u003eHaplosporidian carcini\u003c/em\u003e infection (Davies et al., 2020a), and 1.8% were diagnosed with an unidentified fungal infection (Similar to Davies et al., 2020b). No crabs were found with externa of \u003cem\u003eSacculina carcini\u003c/em\u003e and while some crabs were found with shell disease lesions (see Supplementary Information Table S1) these were all superficial in nature. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeneral and site-specific population observations of the prevalence of crab infestation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eModel 1, a binomial logistic regression model which combined data from both locations and used the presence of trematode parasites as the response variable, showed that crab size (carapace width) was the only factor associated with the presence of parasites (Table 1, Model 1). In terms of size, larger crabs were significantly (\u003cem\u003eP\u003c/em\u003e = 0.00286) more likely to present with trematode infections compared to parasite-free crabs (41 \u0026plusmn; 11 \u003cem\u003evs\u003c/em\u003e. 33 \u0026plusmn; 11 mm, respectively, mean \u0026plusmn; SD). The prevalence of trematode infections in crabs sampled from Mumbles and Oxwich was 85% and 67%, respectively. The drop1 function revealed that despite an apparent lower infection presence at Oxwich, location was not an associated factor with trematode presence (\u003cem\u003eP\u003c/em\u003e = 0.092). Of the male crabs, 69% were parasitised whereas 83% of females presented with trematode metacercariae. Although this may suggest a higher prevalence of infection in female crabs, the combined model showed that sex was not a significant factor (\u003cem\u003eP\u003c/em\u003e = 0.18082) associated with trematode presence (Table 1, Model 1).\u003c/p\u003e\n\u003cp\u003eTo further explore the possible relationship between external factors and the presence of trematode parasites, the data were separated and analysed between the two locations (i.e. Mumbles \u003cem\u003evs\u003c/em\u003e. Oxwich). In Mumbles, 85% of surveyed crabs presented with trematode metacercariae. Model 2, which used the presence of trematode infection at this site as the response variable, indicated that no factors were associated with infection. In terms of crab size, the average carapace width of infected crabs from Mumbles was 43 \u0026plusmn; 10 mm (range 19 \u0026ndash; 61 mm). Although the combined model (Model 1) identified crabs of a larger size as a factor associated with trematode presence, subsequent analysis using the drop1 function in Model 2 deemed that crab size was not statistically significant for inclusion in the final (reduced) model (Table 1, Models 1 - 2). The drop1 function indicated a potentially lower presence of infection during August, however, the significant threshold (\u003cem\u003eP\u003c/em\u003e = 0.05) was not achieved (Table 1, Model 2). Additionally, sex, fouling (presence of epibionts), pigment loss, haemolymph opacity, and limb loss did not have a significant effect on parasitisation (data not shown).\u003c/p\u003e\n\u003cp\u003eOf the crabs surveyed from Oxwich, 67% had metacercariae in the hepatopancreas. Using the presence of trematode infections at Oxwich as the response variable (Model 3), revealed that carapace width and colouration on the ventral surface were significant factors associated with trematode presence (Table 1, Model 3). In terms of size, larger crabs were significantly more likely (\u003cem\u003eP\u003c/em\u003e = 0.00062) to be infected with trematode parasites compared to parasite-free crabs (mean \u0026plusmn; SD: 38.7 \u0026plusmn; 10.5 \u003cem\u003evs\u003c/em\u003e. 29.9 \u0026plusmn; 8.0 mm, respectively). Of the crabs surveyed from Oxwich, the colour distribution of infected individuals was as follows: 42% green, 7% yellow, and 51% red. Notably, shore crabs with a yellow ventral carapace coloration were significantly less likely to host trematode parasites than the other two colour morphs (\u003cem\u003eP\u003c/em\u003e = 0.02931; Table 1, Model 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eBinomial logistic regression models (reduced from full models) testing the effects of biometric and environmental predictor variables on the overall presence of trematode parasites in the population. Models separated by location: Model 1, total\u003c/p\u003e\n\u003cp\u003epopulation; Model 2, Mumbles; Model 3, Oxwich.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eModel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePredictor Variable\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEstimate (slope)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eModel 1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTremPres ~ Location + Sex + Carapace Width\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003edf\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e= 159\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAIC: 167.77\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eLocation (Oxwich)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e- 0.71784\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.42619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.09212 \u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eSex (Male)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e-0.55100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.41173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.18082\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eCarapace Width\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0.05918\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.01984\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.00286 \u003cstrong\u003e**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eModel 2\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTremPres ~ Month + Colour + Hemo.col\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003edf\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e= 69\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAIC: 69.402\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eMonth (August)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e-2.3506\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-1.714\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.08653 \u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eMonth (June)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e-0.2323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-0.275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.78343\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eColour (Red)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e-2.1355\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-1.596\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.11047\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eColour (Yellow)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e15.3151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.99426\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eHemoCol (Milky)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0.8920\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.962\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.33594\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eModel 3\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTremPres ~ Carapace.Width + Colour\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003edf\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;= 84\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAIC: 94.678\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eCarapace Width\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0.14027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.04098\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.00062***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eColour (Red)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0.76112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.59952\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.20424\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eColour (Yellow)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e-2.53315\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.16239\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.02931*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Statistically significant *\u003cem\u003eP\u003c/em\u003e\u0026le;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026le;0.01,***\u003cem\u003eP\u003c/em\u003e\u0026le;0.001\u003c/p\u003e\n\u003cp\u003eAbbreviation: SE, standard error\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eParasite intensity (i.e., loads\u003c/em\u003e)\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThere was a large range of the number of metacercariae/crab at both locations. In Mumbles this ranged from 1 \u0026ndash; 265 cysts/crab (0.4 \u0026ndash; 377 cysts/g wet weight hepatopancreas) while in Oxwich the equivalent data were 1 \u0026ndash; 176 parasites/crab (0.3 \u0026ndash; 86 cysts/g wet weight hepatopancreas). Model 4 shown in Table 2 combined data from both locations and used parasite count (cysts/crab) as the response variable. This model showed that location and carapace width were the only factors that had a significant effect on parasite load (Table 2). In terms of size, the model showed that larger crabs were significantly more likely to harbour a greater number of parasites (Table 2). Crabs sampled from Oxwich Bay had a significantly (\u003cem\u003eP\u003c/em\u003e = 0.009) lower parasite count compared to those in Mumbles Head (25 \u0026plusmn; 40 \u003cem\u003evs\u003c/em\u003e. 41 \u0026plusmn; 51 cysts/crab, respectively; mean \u0026plusmn; SD). In terms of month, there was no significant difference in the number of parasite cysts per crab across sampling months (April, June, August) (31 \u0026plusmn; 47 \u003cem\u003evs\u003c/em\u003e. 42 \u0026plusmn; 49 \u003cem\u003evs\u003c/em\u003e. 26 \u0026plusmn; 43, respectively; mean \u0026plusmn; SD, Table 2, Model 4). Model 4 showed no significant difference (\u003cem\u003eP\u003c/em\u003e = 0.3) in the number of parasite cysts per crab when comparing males and female (29 \u0026plusmn; 49 \u003cem\u003evs\u003c/em\u003e. 37 \u0026plusmn; 44 parasites/crab, respectively; mean \u0026plusmn; SD). Furthermore, haemolymph opacity, which can be an early indication of systematic infection, had no association (\u003cem\u003eP\u003c/em\u003e = 0.5956) with a change in parasite load (33 \u0026plusmn; 47 \u003cem\u003evs\u003c/em\u003e. 37 \u0026plusmn; 46 parasites/crab, clear \u003cem\u003evs\u003c/em\u003e. milky, respectively; mean \u0026plusmn; SD, Table 2). However, the numbers of crabs with cloudy haemolymph were probably too small for meaningful analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eGeneralised linear model with a negative binomial function testing the effect of biometric and environmental predictor variables on digenean trematode metacercarial cyst load in the total population of sampled \u003cem\u003eC. maenas\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"631\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eModel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePredictor Variable\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEstimate (slope)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"9\" valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eModel 4\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eParasite count ~ \u0026nbsp;Location + Month + Sex + Carapace Width + HemoCol + Pigment Loss + Fouling\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003edf\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;= 116\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAIC = 1082.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eLocation (Oxwich)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e-0.79164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.23512 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.00076 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eMonth (August)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e-0.46575 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.27428 \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.08949 . \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eMonth (June)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e-0.22969 \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.26869 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.39263 \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eSex (Male)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e-0.44703 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.22956 \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.05150 . \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eCarapace Width\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.03817 \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.01204 \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.00153 **\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eHemoCol (Milky)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e-0.37159 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.46040 \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.41960 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003ePigment Loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.02777 \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.27760 \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.92031 \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eFouling\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.81158 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.34743 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.01949 *\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Statistically significant *P\u0026le;0.05, **P\u0026le;0.01,***P\u0026le;0.001\u003c/p\u003e\n\u003cp\u003eAbbreviation: SE, standard error\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eParasite identification and phylogenetic analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOf the 123 trematode-infected crabs, a total of 26 samples from Mumbles (n = 15) and Oxwich (n =11) were successfully amplified and sequenced using the LSU-F/LSU-1500 oligonucleotides (Table S4, Figs S1-8, Supplementary Information). Of these sequences, 95.7% shared high similarity (\u0026gt;98.0% cover and identity) with \u003cem\u003eM. similis\u003c/em\u003e from the slaty-backed gull (\u003cem\u003eLarus schistisagus\u003c/em\u003e) from the Sea of Okhotsk, Russia (GenBank: HM584136 - HM584138). The remaining sequences derived from metacercariae from crabs harbouring the less common asymmetrical cysts also shared high similarity (\u0026gt;94% cover and identity) with those for \u003cem\u003eM. primas\u003c/em\u003e from the mudsnail (\u003cem\u003ePeringia ulvae\u003c/em\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing quality control, sequences were combined with reference sequences to create two phylograms (Figures 3 and 4). Figure 3 separated \u003cem\u003eM. similis\u003c/em\u003e and \u003cem\u003eM. primas\u003c/em\u003e into individual clades. Notably, most of the \u003cem\u003eM. similis\u003c/em\u003e sequences formed a robust clade. In contrast, three \u003cem\u003eM. primas\u003c/em\u003e sequences from August were unified in a single clade, while the one sequence from June displayed possible phylogenetic separation. Figure 4 delved deeper into the placement of the \u003cem\u003eM. primas\u003c/em\u003e sequences from this study and the top 50 reference nucleotide sequences retrieved from GenBank. It shows the \u003cem\u003eM. primas\u003c/em\u003e sequences clearly positioned in a clade alongside other \u003cem\u003eM. primas\u003c/em\u003e, including samples from \u003cem\u003eC. maenas\u0026nbsp;\u003c/em\u003ein Portugal (Pina et al., 2011) and \u003cem\u003eHydrobia ulvae\u0026nbsp;\u003c/em\u003e(a potential primary host) in the U.K. (Tkach et al., 2003).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study has shown that 76% crabs overall are subject to parasitisation by the microphallid, \u003cem\u003eM. similis\u003c/em\u003e. It has also demonstrated that a smaller number of crabs from both sites were also infested with \u003cem\u003eM. primas\u003c/em\u003e but at a much lower level (9% \u003cem\u003evs\u003c/em\u003e. 2% Mumbles \u003cem\u003evs\u003c/em\u003e. Oxwich). This latter parasite was never found to infect crabs that were free from \u003cem\u003eM. similis\u003c/em\u003e. Furthermore, crabs were only found to harbour this parasite in the samples collected in the summer months of June and August. Finally, cyst sizes of \u003cem\u003eM. similis\u003c/em\u003e differed significantly between Mumbles and Oxwich with those larger in the former site.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicrophallus similis\u003c/em\u003e has been shown to utilise shore crabs as the second intermediate host in a variety of locations including South Wales (James, 1969) and Belfast Lough, Northern Ireland (Galaktionov et al., 2012) in the United Kingdom and in Northeast USA (Barnard et al., 2022) and Newfoundland, Canada (Blakeslee et al., 2020). Levels of parasitisation can vary dramatically from site to site but prevalences as high as 100% have been reported (see Blakeslee et al., 2009 for a summary of infection prevalences). Descriptions of the presence of \u003cem\u003eM. primas\u003c/em\u003e in shore crabs are less frequent. Pina et al. (2011) used a morphological and molecular approach to identify this parasite in shore crabs from the Aviero estuary in Portugal. Davies et al. (2022) also identified two distinct forms of trematode metacercariae within the hepatopancreas of shore crabs from Mumbles Head using a histopathological approach. Although molecular techniques were not employed for species identification, the morphology of an additional morphotype of encysted metacercaria found on only one occasion in their year-long study closely resembles that of \u003cem\u003eM. primas\u003c/em\u003e, as described in this study. This finding not only suggests the previous presence of \u003cem\u003eM. primas\u003c/em\u003e in shore crabs from Swansea, U.K., but also demonstrates that co-infections with these two species of \u003cem\u003eMicrophallus\u003c/em\u003e are not unique at these locations. The prior potential identification of \u003cem\u003eM. primas\u003c/em\u003e in 2019-2020 (Davies et al., 2022) also suggests that it is not a more recent introduction to the Swansea coastline. Finally, Stentiford and Feist (2008) using a histological approach concluded that the metacercariae found in the gills and hepatopancreas of shore crabs belonged to \u003cem\u003eM. primas\u003c/em\u003e only although the morphology of some rounded forms in their illustrations is reminiscent of \u003cem\u003eM. similis\u003c/em\u003e seen in our study \u0026ndash; perhaps suggesting the presence of dual infections. The weakness of histology as an approach to identify metacercariae in \u0026lt;10 \u0026micro;m sections is that the plane of section does not always lend itself to interpretation of the 3D shape where forms can appear as spherical or ovoid depending on this aspect.\u003c/p\u003e\n\u003cp\u003eDespite both digenean species \u003cem\u003eM. similis\u003c/em\u003e and \u003cem\u003eM. primas\u003c/em\u003e having been possibly identified in shore crabs across U.K. coastlines (e.g., Stentiford and Feist, 2005; Davies et al., 2022), research focusing on seasonal variations in prevalence within this secondary intermediate host is lacking. Based on the preliminary findings of this study, \u003cem\u003eM. similis\u003c/em\u003e maintains a high level of presence within shore crab populations from Mumbles and Oxwich throughout the sampling period from late winter to summer. In contrast, \u003cem\u003eM. primas\u003c/em\u003e only appeared and at a low prevalence from June onwards. These potential temporal changes in parasite presence may be due to factors including: (1). the migration of a population of crabs harbouring both \u003cem\u003eM. similis\u003c/em\u003e and \u003cem\u003eM. primas\u003c/em\u003e into the intertidal area between June and August, (2). an insufficient number of molluscan first intermediate host species to facilitate \u003cem\u003eM. primas\u003c/em\u003e transmission in February and April, and/or (3). species variation in cercarial release which may also be influenced by environmental factors such as temperature. These possibilities are discussed below.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe presence of \u003cem\u003eM. primas\u003c/em\u003e at both Mumbles and Oxwich sites beginning in June may also be related to species-specific variations in cercarial release, potentially driven by environmental cues such as temperature, salinity and illumination. McCarthy et al. (2002) noted distinct patterns in the release and behaviour of cercariae among species; specifically, \u003cem\u003eM. similis\u003c/em\u003e produced fewer but larger and more robustly swimming cercariae that gravitated towards darker waters, unlike other microphallid species. These observations suggest that factors such as light and the specific characteristics of cercariae release and movement could play critical roles in the successful localization of a viable host. It has been well documented that temperature also has a significant effect on the release and survival of trematode cercariae, with the release of many trematode species increasing with temperature (e.g., Poulin, 2006; Koprivnikar and Poulin, 2009a,b; D\u0026iacute;az-Morales et al., 2022). This suggests that the sudden appearance of \u003cem\u003eM. primas\u003c/em\u003e could have been the direct cause of increasing summer temperatures in the intertidal zone. However, without a long-term comparative study of \u003cem\u003eM. similis\u003c/em\u003e and \u003cem\u003eM. primas\u003c/em\u003e, it remains uncertain whether these mechanisms are solely responsible for the inconsistent presence of \u003cem\u003eM. primas\u003c/em\u003e, and that it is most likely to be influenced by a combination of host-parasite and environmental factors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe disparity between the levels of prevalence of \u003cem\u003eM. similis\u003c/em\u003e and \u003cem\u003eM. primas\u003c/em\u003e is also possibly associated with the habitat preference and population density of their respective first intermediate hosts. \u003cem\u003eM. similis\u003c/em\u003e is known to have numerous first intermediate hosts within the genus \u003cem\u003eLittorina,\u003c/em\u003e some of which have been reported at both Mumbles and Oxwich (Dixon and Pollard, 1985; Warwick et al., 1990). However, the only documented first intermediate host of \u003cem\u003eM. primas\u003c/em\u003e is the mudsnail (\u003cem\u003ePeringia ulvae\u003c/em\u003e). Although \u003cem\u003eP. ulvae\u003c/em\u003e has been observed along coastlines within the Bristol Channel (Pinnion et al., 2007; authors personal observations), it is primarily located on their preferred habitats of mudflats and estuaries (i.e. not rocky shore areas such as those studied here). Stentiford and Feist (2005) noted a high prevalence of \u003cem\u003eM. primas\u003c/em\u003e in the hepatopancreas and gills of shore crabs sampled from Southampton Water and Alde estuaries, U.K. Similarly, Pina et al. (2011), reported high a prevalence of \u003cem\u003eM. primas\u003c/em\u003e in the hepatopancreas of shore crabs from the Aveiro Estuary in Portugal. This suggests the low prevalence of \u003cem\u003eM. primas\u003c/em\u003e in this present study may be linked to the choice of survey site, in that intertidal rocky shores do not support a sufficient population of the established first intermediate hosts to facilitate the high levels of parasite transmission as observed in some estuarine environments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe size of encysted metacercariae of \u003cem\u003eM. similis\u003c/em\u003e varies within species, often showing intraspecific variation due to different infection times and/or competition for space and resources within the host (Saldanha et al., 2009). Consistent with previous studies on \u003cem\u003eM. similis\u003c/em\u003e (McCarthy et al., 2002) and \u003cem\u003eM. primas\u003c/em\u003e (Saville and Irwin, 2005), the sizes of trematode metacercariae observed in this present study agree with these previous findings. Fredensborg and Poulin (2005) highlighted that the size of metacercariae is density-dependent; notably, higher intensity trematode infections are typically correlated with smaller-sized metacercariae. Based on these previous findings, it would be expected that shore crabs from Mumbles, given their higher infection intensities, should be more likely to host smaller \u003cem\u003eM. similis\u003c/em\u003e metacercariae. However, contrary to these expectations, metacercariae isolated from crabs at Mumbles were significantly larger than those from hosts located at Oxwich Bay. An alternate explanation to our findings is that the cysts in Oxwich are less advanced in their development within the hepatopancreas than those in Mumbles and hence the observed size difference.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the current study, host size was found to be a significant factor in affecting the prevalence of digenean parasites but only at Oxwich Bay. This finding is consistent with previous research (e.g., Blakeslee et al., 2015; Koehler and Poulin, 2010; Koga, 2008), which suggests that larger, and therefore older, crabs, due to their longer exposure time, tend to accumulate more parasites and thus show higher parasitisation rates. However, while larger crabs naturally have more time to become parasitised, it is possible that host chemical cues (both attractive and inhibitory) may also be involved. Combes et al. (1994) and Haas (1994) have reviewed the significance of host chemical cues in cercariae host-finding behaviour in trematodes but information on the chemical nature and action of these is limited to a few trematodes such as schistosomes rather than those including \u003cem\u003eM. similis\u003c/em\u003e and \u003cem\u003eM. primas\u003c/em\u003e using crustaceans as second intermediate hosts. Given that larger \u003cem\u003eC. maenas\u003c/em\u003e could emit a greater concentration of chemical cues due to their increased surface area of the gills and general body surface, they may attract more cercariae, thereby experiencing higher levels of parasite presence. Indeed, experimental studies of non-native shore crabs incubated with known numbers of cercariae from \u003cem\u003eM. similis\u003c/em\u003e found that the largest crabs accrued the most metacercariae over a 5-day experimental period (Blakeslee et al., 2015). The absence of this finding in shore crabs from Mumbles could be caused by disparities in the two datasets. For instance, it is possible that the disparities in size of the crab populations sampled from Mumbles and Oxwich could contribute to the apparent lack of an observable relationship. The observed site-level variation in infection intensity and metacercarial size may be influenced by ecological and environmental differences between Oxwich Bay and Mumbles Head. These include differences in substrate type, hydrodynamic exposure, molluscan host availability, and crab microhabitats. For example, Mumbles Head\u0026apos;s rocky intertidal structure may support higher densities of \u003cem\u003eLittorina\u003c/em\u003e spp., facilitating sustained trematode transmission.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, we have demonstrated that shore crabs can host dual infestations as second intermediate hosts for at least two species of microphallids, and to our knowledge, this is the first such observation in crabs. The potential consequences of this infestation to the crabs surveyed is, however, unclear although Blakeslee et al. (2015) and Ro et al. (2022) reported only limited effects in terms of their behaviour of non-native \u003cem\u003eC. maenas\u003c/em\u003e that could impact on their likelihood of predation by the definitive hosts. hese findings have implications beyond the surveyed Welsh sites. As \u003cem\u003eC. maenas\u003c/em\u003e continues to expand its invasive range, understanding its parasite community structure, including potential co-infections, is critical for predicting ecological impact and managing disease risk. Studies have shown altered susceptibility, reduced parasite load, or novel host-switching in invaded regions (Blakeslee et al., 2009, 2020), raising the importance of monitoring parasite assemblages in both native and non-native populations. Molecularly confirmed records of species such as \u003cem\u003eM. primas\u003c/em\u003e in \u003cem\u003eC. maenas\u003c/em\u003e help clarify host\u0026ndash;parasite relationships across the species\u0026apos; global range.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e. The supplementary information for this article can be found online.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e. The authors are grateful to the Biosciences Technical Team in Swansea University (Hilary Williams, Jessica Minett, Jessica Bevan and Matthew Watkins) for their assistance. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e. AFR and CED conceived and designed the study; all authors were involved in field and laboratory-based work; ATB, AFR and CED wrote the initial manuscript; all authors approved the final version of the manuscript at submission.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e. The authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical standards\u003c/strong\u003e. All institutional (Swansea University) guidelines and ethical approval for the collection, maintenance and euthanasia of crustaceans were followed. Research Ethics Approval Number: 2 2024 8411 7714.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available from the corresponding author. Sequences generated are deposited in the GenBank database under the accession numbers PQ314574 \u0026ndash; PQ314578, PQ314583 - PQ314592, and PQ314598- PQ314610. All gel photos are in Supplementary Information (Table S4, Figs S1-8).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBarnard RB, Moore CS, Keogh CL, Blakeslee AMH (2020) If you encyst: evidence of parasite escape and host-switching among three co-occurring crabs. \u003cem\u003eMar Ecol Prog Ser\u003c/em\u003e \u003cstrong\u003e697\u003c/strong\u003e:67\u0026ndash;80. https://doi.org/10.3354/meps14143\u003c/li\u003e\n \u003cli\u003eBlakeslee AMH, Keogh CL, Byers JE, Kuris AM, Lafferty KD, Torchin ME (2009) Differential escape from parasites by two competing introduced crabs. \u003cem\u003eMar Ecol Prog Ser\u003c/em\u003e \u003cstrong\u003e393\u003c/strong\u003e:83\u0026ndash;96. https://doi.org/10.3354/meps08225\u003c/li\u003e\n \u003cli\u003eBlakeslee AMH, Keogh CL, Fowler AE, Griffen BD (2015) Assessing the effects of trematode infection on invasive green crabs in Eastern North America. \u003cem\u003ePLoS ONE\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e:e0128674. https://doi.org/10.1371/journal.pone.0128674\u003c/li\u003e\n \u003cli\u003eBlakeslee AMH, Barnard RB, Matheson K, McKenzie CH (2020a) Host-switching among crabs: species introduction results in a new target host for native parasites. \u003cem\u003eMar Ecol Prog Ser\u003c/em\u003e \u003cstrong\u003e636\u003c/strong\u003e:91\u0026ndash;106. https://doi.org/10.3354/meps13214\u003c/li\u003e\n \u003cli\u003eBlakeslee AMH, Ruocchio M, Moore CS, Keogh CL (2020b) Altered susceptibility to trematode infection in native versus introduced populations of the European green crab. \u003cem\u003eAquat Invasions\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e:177\u0026ndash;195. https://doi.org/10.3391/ai\u003c/li\u003e\n \u003cli\u003eBojko J, Grahame JW, Dunn AM (2017) Periwinkles and parasites: the occurrence and phenotypic effects of parasites in \u003cem\u003eLittorina saxatilis\u003c/em\u003e and \u003cem\u003eL. arcana\u003c/em\u003e in northeastern England. \u003cem\u003eJ Molluscan Stud\u003c/em\u003e \u003cstrong\u003e83\u003c/strong\u003e:69\u0026ndash;78. https://doi.org/10.1093/mollus/eyw047\u003c/li\u003e\n \u003cli\u003eCamacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e:421. https://doi.org/10.1186/1471-2105-10-421\u003c/li\u003e\n \u003cli\u003eCollins M, Ferentinos G, Banner FT (1979) The hydrodynamics and sedimentology of a high (tidal and wave) energy embayment (Swansea Bay, Northern Bristol Channel). \u003cem\u003eEstuar Coast Mar Sci\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e:49\u0026ndash;74\u003c/li\u003e\n \u003cli\u003eCombes C, Fournier A, Mon\u0026eacute; 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Report to the Department of Trade and Industry, U.K.\u003c/li\u003e\n \u003cli\u003ePoulin R (2006) Global warming and temperature-mediated increases in cercarial emergence in trematode parasites. \u003cem\u003eParasitology\u003c/em\u003e \u003cstrong\u003e132\u003c/strong\u003e:143\u0026ndash;151. https://doi.org/10.1017/S0031182005008693\u003c/li\u003e\n \u003cli\u003ePoulin R, Cribb TH (2002) Trematode life cycles: short is sweet? \u003cem\u003eTrends Parasitol\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e:176\u0026ndash;183. https://doi.org/10.1016/s1471-4922(02)02262-6\u003c/li\u003e\n \u003cli\u003eProkofiev VV, Kirill V, Galaktionov V, Levakin IA (2016) Patterns of parasite transmission in polar seas: Daily rhythms of cercarial emergence from intertidal snails. \u003cem\u003eJ Sea Res\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e:85\u0026ndash;98. https://doi.org/10.1016/j.seares.2015.07.007\u003c/li\u003e\n \u003cli\u003eRo H, Fowler AE, Wood CL, Blakeslee AMH (2022) Trematode parasites have minimal effect on the behavior of invasive green crabs. \u003cem\u003eAquat Invasions\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e:238\u0026ndash;258. https://doi.org/10.3391/ai.2022.17.2.07\u003c/li\u003e\n \u003cli\u003eSaldanha I, Leung TLF, Poulin R (2009) Causes of intraspecific variation in body size among trematode metacercariae. \u003cem\u003eJ Helminthol\u003c/em\u003e \u003cstrong\u003e83\u003c/strong\u003e:289\u0026ndash;293. https://doi.org/10.1017/S0022149X09224175\u003c/li\u003e\n \u003cli\u003eSaville DH, Irwin SWB (1991) In ovo cultivation of \u003cem\u003eMicrophallus primas\u003c/em\u003e (Trematoda: Microphallidae) metacercariae to ovigerous adults and the establishment of the life-cycle in the laboratory. \u003cem\u003eParasitology\u003c/em\u003e \u003cstrong\u003e103\u003c/strong\u003e:479\u0026ndash;484. https://doi.org/10.1017/s0031182000060005\u003c/li\u003e\n \u003cli\u003eSaville DH, Irwin SWB (2005) A study of the mechanisms by which the cercariae of \u003cem\u003eMicrophallus primas\u003c/em\u003e (Jag, 1909) Stunkard, 1957 penetrate the shore crab, \u003cem\u003eCarcinus maenas\u003c/em\u003e (L). \u003cem\u003eParasitology\u003c/em\u003e \u003cstrong\u003e131\u003c/strong\u003e:521\u0026ndash;529. https://doi.org/10.1017/S0031182005008048\u003c/li\u003e\n \u003cli\u003eSmith AL, Hirschle L, Vogan CL, Rowley AF (2015) Parasitisation of juvenile edible crabs (\u003cem\u003eCancer pagurus\u003c/em\u003e) by the dinoflagellate, \u003cem\u003eHematodinium\u003c/em\u003e sp.: pathobiology, seasonality and its potential effects on commercial fisheries. \u003cem\u003eParasitology\u003c/em\u003e \u003cstrong\u003e142\u003c/strong\u003e:428\u0026ndash;438. https://doi.org/10.1017/S0031182014001255\u003c/li\u003e\n \u003cli\u003eStentiford GD (2008) Diseases of the European edible crab (\u003cem\u003eCancer pagurus\u003c/em\u003e): a review. \u003cem\u003eICES J Mar Sci\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e:1578\u0026ndash;1592. https://doi.org/10.1093/icesjms/fsn134\u003c/li\u003e\n \u003cli\u003eStentiford GD, Feist SW (2005) A histopathological survey of shore crab (\u003cem\u003eCarcinus maenas\u003c/em\u003e) and brown shrimp (\u003cem\u003eCrangon crangon\u003c/em\u003e) from six estuaries in the United Kingdom. \u003cem\u003eJ Invertebr Pathol\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e:136\u0026ndash;146. https://doi.org/10.1016/j.jip.2005.01.006\u003c/li\u003e\n \u003cli\u003eStunkard HW (1957) The morphology and life-history of the digenetic trematode, \u003cem\u003eMicrophallus similis\u003c/em\u003e (J\u0026auml;gerski\u0026ouml;ld, 1900) Baer, 1943. \u003cem\u003eBiol Bull\u003c/em\u003e \u003cstrong\u003e112\u003c/strong\u003e:254\u0026ndash;266\u003c/li\u003e\n \u003cli\u003eThieltges DW, Hussel B, Hermann J, Jensen KT, Krakau M, Taraschewski H, Reise K (2008) Parasites in the northern Wadden Sea: a conservative ecosystem component over 4 decades. \u003cem\u003eHelgol Mar Res\u003c/em\u003e \u003cstrong\u003e62\u003c/strong\u003e:37\u0026ndash;47. https://doi.org/10.1007/s10152-007-0091-6\u003c/li\u003e\n \u003cli\u003eThieltges DW, Engelsma MY, Wendling CC, Wegner KM (2013) Parasites in the Wadden Sea food web. \u003cem\u003eJ Sea Res\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e:122\u0026ndash;133. https://doi.org/10.1016/j.seares.2012.06.002\u003c/li\u003e\n \u003cli\u003eThieltges DW, Mouritsen KN, Poulin R (2018) Ecology of parasites in mudflat ecosystems. In: Beninger PG (ed) \u003cem\u003eMudflat Ecology\u003c/em\u003e. Springer, Cham, pp 213\u0026ndash;242\u003c/li\u003e\n \u003cli\u003eTkach VV, Littlewood DTJ, Olson PD, Kinsella JM, Swuderski Z (2003) Molecular phylogenetic analysis of the Microphalloidea Ward, 1901 (Trematoda: Digenea). \u003cem\u003eSyst Parasitol\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e:1\u0026ndash;15. https://doi.org/10.1023/a:1025546001611\u003c/li\u003e\n \u003cli\u003eTrifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ (2016) W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e:W232\u0026ndash;W235. https://doi.org/10.1093/nar/gkw256\u003c/li\u003e\n \u003cli\u003eWarwick T, Knight AJ, Ward RD (1990) Hybridisation in the \u003cem\u003eLittorina saxatilis\u003c/em\u003e species complex (Prosobranchia: Mollusca). \u003cem\u003eHydrobiologia\u003c/em\u003e \u003cstrong\u003e193\u003c/strong\u003e:109\u0026ndash;116. https://doi.org/10.1007/BF00028070\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Carcinus maenas, Microphallus similis, Microphallus primas, Digenean metacercariae, Intermediate host, Parasite burden, Spatial variation, Marine parasitology, Host-parasite interactions","lastPublishedDoi":"10.21203/rs.3.rs-7425079/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7425079/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDigenean trematodes commonly use marine crustaceans as intermediate hosts, yet their distribution and host-specific dynamics remain underexplored in temperate European systems. In this study, metacercarial stages of \u003cem\u003eMicrophallus similis\u003c/em\u003e and \u003cem\u003eM. primas\u003c/em\u003e were identified in the hepatopancreas of \u003cem\u003eCarcinus maenas\u003c/em\u003e collected from two distinct locations in South Wales, UK across a 4-month period. \u003cem\u003eM. similis\u003c/em\u003e exhibited consistently high prevalence (~\u0026thinsp;76%) at both sites, while \u003cem\u003eM. primas\u003c/em\u003e occurred only as a co-infection, with lower prevalence (2\u0026ndash;9%) and infection intensity. At Oxwich Bay, crab size and coloration were significantly associated with trematode infection, whereas no such associations were observed at Mumbles Head. Phylogenetic analysis confirmed species-level divergence between \u003cem\u003eM. similis\u003c/em\u003e and \u003cem\u003eM. primas\u003c/em\u003e. Overall parasite burden was significantly higher at Mumbles Head, with larger crabs harbouring more metacercariae. Additionally, metacercariae from Mumbles Head were significantly larger than those from Oxwich Bay, suggesting site-specific variation in parasite development.\u003c/p\u003e","manuscriptTitle":"Dual microphallid infections in shore crabs (Carcinus maenas): Site-level variation and phylogenetic insights in a globally invasive host","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-10 11:56:23","doi":"10.21203/rs.3.rs-7425079/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-24T11:49:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T07:43:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-17T00:30:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-14T12:45:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-13T09:41:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"238858300491077418991120318334629471020","date":"2025-09-06T22:50:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"237935403694283924840753938209913788473","date":"2025-09-06T06:44:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69639138218924054408837269461072294923","date":"2025-09-04T14:26:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44698805499239178315739320490009602992","date":"2025-09-03T23:16:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-03T18:58:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-28T11:56:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-28T11:38:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasitology Research","date":"2025-08-21T10:14:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8d57147e-f9d0-4a80-a048-7ae984434be2","owner":[],"postedDate":"September 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:05:48+00:00","versionOfRecord":{"articleIdentity":"rs-7425079","link":"https://doi.org/10.1007/s00436-025-08614-w","journal":{"identity":"parasitology-research","isVorOnly":false,"title":"Parasitology Research"},"publishedOn":"2026-02-21 15:59:49","publishedOnDateReadable":"February 21st, 2026"},"versionCreatedAt":"2025-09-10 11:56:23","video":"","vorDoi":"10.1007/s00436-025-08614-w","vorDoiUrl":"https://doi.org/10.1007/s00436-025-08614-w","workflowStages":[]},"version":"v1","identity":"rs-7425079","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7425079","identity":"rs-7425079","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}