Silencing of prophenoloxidase (proPO) gene in freshwater prawn, Macrobrachium rosenbergii, makes them susceptible to white spot syndrome virus (WSSV).

Prophenoloxidase (proPO) is very important to protect the invertebrates from microbial infections. Our previous studies revealed that proPO was up-regulated in WSSV-injected Macrobrachium rosenbergii and is responsible for protecting M. rosenbergii from WSSV. In order to prove this mechanism, an attempt was made in the present study to silence the proPO gene in freshwater prawn by injection of dsRNA-proPO followed by WSSV challenge. Two partial fragments of proPO with the size of 251 and 331 bp were used to synthesize dsRNA using LITMUS38i vector and E. coli. The bacterially synthesized dsRNA-proPO was used to silence proPO gene to determine its involvement in developing resistance in prawn against WSSV. In proPO gene-silenced prawn, 100% mortality was observed after WSSV challenge whereas no mortality was observed in prawn injected with WSSV alone. The WSSV infection in gene-silenced prawn was confirmed by PCR, and its propagation was quantified by ELISA and real-time PCR at different time intervals. Real-time PCR assay revealed a significant reduction in the expression of proPO gene in WSSV-challenged proPO-silenced prawn when compared to normal prawn. Level of proPO was reduced significantly in the haemolymph of proPO-silenced prawn when compared to prawn injected with PBS.

Sensitivity to near-future CO2 conditions in marine crabs depends on their compensatory capacities for salinity change.

Marine crabs inhabit shallow coastal/estuarine habitats particularly sensitive to climate change, and yet we know very little about the diversity of their responses to environmental change. We report the effects of a rarely studied, but increasingly prevalent, combination of environmental factors, that of near-future pCO2 (~1000 µatm) and a physiologically relevant 20% reduction in salinity. We focused on two crab species with differing abilities to cope with natural salinity change, and revealed via physiological and molecular studies that salinity had an overriding effect on ion exchange in the osmoregulating shore crab, Carcinus maenas. This species was unaffected by elevated CO2, and was able to hyper-osmoregulate and maintain haemolymph pH homeostasis for at least one year. By contrast, the commercially important edible crab, Cancer pagurus, an osmoconformer, had limited ion-transporting capacities, which were unresponsive to dilute seawater. Elevated CO2 disrupted haemolymph pH homeostasis, but there was some respite in dilute seawater due to a salinity-induced metabolic alkalosis (increase in HCO3- at constant pCO2). Ultimately, Cancer pagurus was poorly equipped to compensate for change, and exposures were limited to 9 months. Failure to understand the full spectrum of species-related vulnerabilities could lead to erroneous predictions of the impacts of a changing marine climate.

Infestation of shore crab gills by a free-living mussel species.

Parasitic and commensal species can impact the structure and function of ecological communities and are typically highly specialized to overcome host defences. Here, we report multiple instances of a normally free-living species, the blue mussel Mytilus edulis Linnaeus, 1758, inhabiting the branchial chamber of the shore crab Carcinus maenas (Linnaeus, 1758) collected from widely separated geographical locations. A total of 127 C. maenas were examined from four locations in the English Channel, one location in the Irish Sea and two locations at the entrance of the Baltic Sea. The branchial chambers of three crabs (one from the English Channel and two from Gullmar Fjord, Sweden) were infested with mussels resembling the genus Mytilus. Sequencing at the Me15/16 locus on the polyphenolic adhesive protein gene confirmed the identity as M. edulis. Bivalve infestation always occurred in larger red male individuals. Up to 16 mussels, ranging from 2 to 11 mm in shell length, were found in each individual, either wedged between gill lamellae or attached to the branchial chamber inner wall. This is one of the first reports of a bivalve inhabiting crustacean gills and is an intriguing case of a normally free-living prey species infesting its predator.

Real-time PCR quantification of the in vitro effects of crustacean immunostimulants on gene expression in lobster (Homarus gammarus) granular haemocytes.

This paper presents data from an investigation of the mode of action of five different crustacean immunostimulants presented to European lobster (Homarus gammarus) granulocytes cultured in vitro. The experiments were designed to test whether or not the immunostimulants could cause the short-term up-regulation of genes coding for immune proteins without causing the cells to degranulate. Quantitative measurements of mRNA transcript abundance were made using real-time PCR and it was first necessary to isolate the complete gene sequences coding for the proteins prophenoloxidase (proPO), beta-1,3-glucan binding protein (betaGBP) and beta-actin (beta-act) in the lobster. These sequences were used to design TaqMan primer and fluorescent probe sets. The presented data indicated that the majority of the tested immunostimulants did not induce the up-regulation of immune-related gene expression in the granulocytes in isolation. Alternative modes of action, including the in vivo up-regulation of gene expression in haemopoetic tissues, are discussed.