Comparison of PCR assays to detect Toxoplasma gondii oocysts in green-lipped mussels (Perna canaliculus).

Toxoplasma gondii is recognised as an important pathogen in the marine environment, with oocysts carried to coastal waters in overland runoff. Currently, there are no standardised methods to detect T. gondii directly in seawater to assess the extent of marine ecosystem contamination, but filter-feeding shellfish may serve as biosentinels. A variety of PCR-based methods have been used to confirm presence of T. gondii DNA in marine shellfish; however, systematic investigations comparing molecular methods are scarce. The primary objective of this study was to evaluate analytical sensitivity and specificity of two nested-PCR (nPCR) assays targeting dhps and B1 genes and two real-time (qPCR) assays targeting the B1 gene and a 529-bp repetitive element (rep529), for detection of T. gondii. These assays were subsequently validated for T. gondii detection in green-lipped mussel (Perna canaliculus) haemolymph using oocyst spiking experiments. All assays could reliably detect 50 oocysts spiked into mussel haemolymph. The lowest limit of detection was 5 oocysts using qPCR assays, with the rep529 primers performing best, with good correlation between oocyst concentrations and Cq values, and acceptable efficiency. Assay specificity was evaluated by testing DNA from closely related protozoans, Hammondia hammondi, Neospora caninum, and Sarcocystis spp. Both nPCR assays were specific to T. gondii. Both qPCR assays cross-reacted with Sarcocystis spp. DNA, and the rep529 primers also cross-reacted with N. caninum DNA. These studies suggest that the rep529 qPCR assay may be preferable for future mussel studies, but direct sequencing is required for definitive confirmation of T. gondii DNA detection.

Metabolic and immunological responses of male and female new Zealand Greenshell™ mussels (Perna canaliculus) infected with Vibrio sp.

Massive mortalities due to pathogens are routinely reported in bivalve cultivation that have significant economic consequences for the global aquaculture industry. However, host-pathogen interactions and infection mechanisms that mediate these interactions are poorly understood. In addition, gender-specific immunological responses have been reported for some species, but the reasons for such differences have not been elucidated. In this study, we used a GC/MS-based metabolomics platform and flow cytometry approach to characterize metabolic and immunological responses in haemolymph of male and female mussels (Perna canaliculus) experimentally infected with Vibrio sp. Sex-based differences in immunological responses were identified, with male mussels displaying higher mortality, oxidative stress and apoptosis after pathogen exposure. However, central metabolic processes appeared to be similar between sexes at 24 h post injection with Vibrio sp. DO1. Significant alterations in relative levels of 37 metabolites were detected between infected and uninfected mussels. These metabolites are involved in major perturbations on the host's innate immune system. In addition, there were alterations of seven metabolites in profiles of mussels sampled on the second day and mussels that survived six days after exposure. These metabolites include itaconic acid, isoleucine, phenylalanine, creatinine, malonic acid, glutaric acid and hydroxyproline. Among these, itaconic acid has the potential to be an important biomarker for Vibrio sp. DO1 infection. These findings provide new insights on the mechanistic relationship between a bivalve host and a pathogenic bacterium and highlight the need to consider host sex as a biological variable in future immunological studies.

First report of Toxoplasma gondii sporulated oocysts and Giardia duodenalis in commercial green-lipped mussels (Perna canaliculus) in New Zealand.

Pollution of marine ecosystems with the protozoan parasites Toxoplasma gondii, Cryptosporidium spp. and Giardia duodenalis can be studied using bivalve shellfish as biosentinels. Although evidence suggests that these parasites are present in New Zealand coastal waters, the extent of protozoal pollution has not been investigated. This study used optimised molecular methods to detect the presence of Cryptosporidium spp., G. duodenalis and T. gondii in commercially sourced green-lipped mussel (Perna canaliculus), an endemic species found throughout coastal New Zealand. A nested polymerase chain reaction was validated for detection of T. gondii DNA and applied to 104 commercially sourced mussels. Thirteen mussels were positive for T. gondii DNA with an estimated true prevalence of 16.4% using Bayesian statistics, and the presence of T. gondii in mussels was significantly associated with collection during the summer compared with that in the winter (P = 0.003). Consumption of contaminated shellfish may also pose a health risk for humans and marine wildlife. As only sporulated T. gondii oocysts can be infectious, a reverse transcriptase-polymerase chain reaction was used to confirm presence of a sporozoite-specific marker (SporoSAG), detected in four mussels. G. duodenalis assemblage B, known to be pathogenic in humans, was also discovered in 1% mussels, tested by polymerase chain reaction (n = 90). Cryptosporidium spp. was not detected in the sampled mussel haemolymph. Results suggest that New Zealand may have high levels of coastal contamination with T. gondii, particularly in summer months, and that naturally exposed mussels can ingest and retain sporulated oocysts, further establishing shellfish consumption as a health concern.

Impact of marine pollution in green mussel Perna viridis from four coastal sites in Karachi, Pakistan, North Arabian Sea: histopathological observations.

Pathological changes are regarded as a standard technique to monitor the effects of pollutants in marine animals. Histopathological examination of the population of green mussel Perna viridis (L.) from four sites in Pakistan, namely, Manora Channel, Rehri Creek, Sandspit Backwaters and Bhanbore was conducted. The first three sites are on the Karachi coast, whereas the fourth one, Bhanbore is situated outside Karachi, and is considered to be less polluted. Two types of parasites, Rickettsia-like organisms and metacestode were found in the mussels studied. In the present study, we observed various pathological lesions, such as inflammatory responses, granulocytomas, lipofuscin pigments, vacuolation in the digestive gland and gonads, lamellar fusion and dilated hemolymphatic sinus in the gills of P. viridis. These observations indicate the extent of environmental pollution in the studied areas. Although, Bhanbore is considered to be relatively less polluted compared to other three sites, the present results have revealed that the waters of Bhanbore are also polluted as evidenced by the pathological changes observed in the mussels collected from there.

Mate locating and access behaviour of the parasitic pea crab, Nepinnotheres novaezelandiae, an important parasite of the mussel Perna canaliculus.

Pea crabs are globally ubiquitous symbionts in the marine environment that cause serious economic impact in the aquaculture production of several major bivalve species. However, little is known about their host-parasite interactions, especially the mating behaviour of these parasites that could prove useful for controlling their infestation in aquaculture. In this study, the mate location behaviour of male New Zealand pea crabs, Nepinnotheres novaezelandiae (Filhol, 1885), was observed when dwelling in its preferred host, the commercially important green-lipped mussel, Perna canaliculus. Given the cryptic behaviour of the male crabs, a novel trapping system was developed to determine whether male crabs would exit their mussel hosts in response to an upstream female crab. The presence of receptive female crabs placed upstream successfully attracted 60% of male crabs from their host over 24 h. Observations of the nocturnal mate-finding behaviour of male crabs were made in darkness using infrared video recordings. Males spent on average 49 min on empty hosts and never left a mussel containing a female conspecific once found, spending 200 min on average to gain entry to the mussel. Male crabs were often observed stroking the mantle edge of the mussel whilst attempting to gain entry, successfully increasing mussel valve gape during entry from 3.7 to 5.5 mm. A pheromone-based mate location system is likely used by this crab to greatly reduce the risks associated with the location of females.

Recruitment of the parasitic pea crab Nepinnotheres novaezelandiae into green-lipped mussels Perna canaliculus.

Pea crab species are globally ubiquitous parasites of marine bivalves including several major aquaculture species. However, little is known about the environmental factors that affect their recruitment into aquacultured mussels. The effect of depth and distance from shore on the recruitment of the parasitic pea crab Nepinnotheres novaezelandiae into New Zealand green-lipped mussels Perna canaliculus was examined with a field experiment. The incidence of pea crab infection of mussels over 295 d was nearly double when deployed at 5-10 m depth (1.97%) compared to 20-30 m depth (0.96%), although it was not significantly different due to the overall low period prevalence in the experimental population. The sex ratio of crabs recovered was significantly skewed towards females with a ratio of 1:14 (χ = 11.3, p < 0.001). Infection with pea crabs was found to significantly reduce final mussel shell height on average by 28% (21.0 mm) over 295 d (Mann-Whitney U = 6.0, p < 0.0001). This study confirms that parasitism of green-lipped mussels by pea crabs has a significant impact on the growth of the mussels and suggests that the incidence of pea crabs will be higher in shallower water and when mussels are in closer proximity to the shore. With no control methods available for preventing pea crab infection, these results suggest that moving mussel farms offshore has the potential to reduce the incidence of pea crabs in mussels and warrants larger-scale assessment.

Biological characteristics of parasitic Nepinnotheres novaezelandiae within a Perna canaliculus farm.

Pea crabs are commercially significant parasites in the aquaculture production of bivalves in many parts of the world. However, there is scant information available on the biology of these important parasites in aquaculture. The population structure, sex ratio, and breeding status were determined for the pea crab Nepinnotheres novaezelandiae residing in a typical green-lipped mussel Perna canaliculus farm in New Zealand. Of the 324 crabs randomly sampled, there were significantly more female (82.4%) than male (17.6%) crabs found. The vast majority of crabs (87.0%) were sexually mature and of these, females comprised 86.4% and males 13.6%. However, the sex ratio of immature crabs was relatively even, suggesting that male crabs may have higher mortality while searching for mates. Crab size was highly variable, indicating that recruitment to mussels in the farm was continuous. Carapace width ranged from 4.00 to 11.5 mm, with males tending to be smaller with a mean (±SE) carapace width of 6.31 ± 0.16 mm versus females with a carapace width of 8.03 ± 0.06 mm. The crabs did not show any preference for parasitizing mussels of different sexes. Despite the fact that the mussels were only 10 mo old, most female crabs were sexually mature (Stage V) and 89.3% were gravid. The mean (±SE) clutch size was 2592 ± 579 and clutch size was directly associated with female carapace width. The mussel farm pea crab population was estimated at 126390 ± 14144 individuals, including 93000 gravid females carrying a total of over 241 million eggs. Overall, the results show that pea crabs rapidly colonize farmed mussels and mature quickly to establish a significant breeding population within the mussel farm, with larval output capable of infecting nearby mussel farms as well as wild populations of bivalves.