Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod (Gadus morhua) macrophages

The facultative intracellular bacterium Francisella noatunensis causes francisellosis in Atlantic cod (Gadus morhua), but little is known about its survival strategies or how these bacteria evade the host immune response. In this study we show intracellular localisation of F. noatunensis in cod macrophages using indirect immunofluorescence techniques and green fluorescent labelled bacteria. Transmission electron microscopy revealed that F. noatunensis was enclosed by a phagosomal membrane during the initial phase of infection. Bacteria were at a later stage of the infection found in large electron-lucent zones, apparently surrounded by a partially intact or disintegrated membrane. Immune electron microscopy demonstrated the release of bacterial derived vesicles from intracellular F. noatunensis, an event suspected of promoting phagosomal membrane degradation and allowing escape of the bacteria to cytoplasm. Studies of macrophages infected with F. noatunensis demonstrated a weak activation of the inflammatory response genes as measured by increased expression of the Interleukin (IL)-1β and IL-8. In comparison, a stronger induction of gene expression was found for the anti-inflammatory IL-10 indicating that the bacterium exhibits a role in down-regulating the inflammatory response. Expression of the p40 subunit of IL-12/IL-17 genes was highly induced during infection suggesting that F. noatunensis promotes T cell polarisation. The host macrophage responses studied here showed low ability to distinguish between live and inactivated bacteria, although other types of responses could be of importance for such discriminations. The immunoreactivity of F. noatunensis lipopolysaccharide (LPS) was very modest, in contrast to the strong capacity of Escherichia coli LPS to induce inflammatory responsive genes. These results suggest that F. noatunensis virulence mechanisms cover many strategies for intracellular survival in cod macrophages.     

Francisella noatunensis in Atlantic cod (Gadus morhua L.); waterborne transmission and immune responses

This is the first report that confirms waterborne transmission of francisellosis in Atlantic cod. To investigate the transmission of disease, particle reduced water was transferred from a tank with intraperitoneally infected cod to a tank with healthy cod. Waterborne transmission of Francisella noatunensis was confirmed in the effluent group using immunohistochemistry and real-time quantitative PCR (RT-qPCR). The bacteria were located inside the accumulated macrophage-like cells. Specific and high antibody responses against live and inactivated bacteria were observed. Oil adjuvant had no effect on the antibody responses against inactivated F.?noatunensis compared to saline formulation. The antigen epitope was a 20-25?kDa component of F.?noatunensis suggested to be lipopolysaccharide detected by Western blot, Sypro Ruby and Silver staining. Systemic immune reactions were investigated by measuring the expression of IFN-γ, IL-1β and IL-10 genes with RT-qPCR. After i.p. injection of live bacteria, a significant up-regulation of IFN-γ and IL-1β expression was observed from 15 to 60 days post infection in spleen and head kidney. In intestine, IFN-γ was significantly up-regulated after 30 days whereas rectum showed no significant differences in expression. Elevated expression of IL-10 was observed in all the organs tested but was only significantly up-regulated at 60 days post infection in intestine from i.p. infected fish. For the cohabitant group, IL-1β and IFN-γ was up-regulated in spleen whereas intestine and rectum showed a down-regulation after 60 days. IL-10 was up-regulated in intestine of cohabitant fish from day 30 to day 60. These results indicate that F.?noatunensis infection provokes both specific antibody responses and long term inflammatory responses in cod. The present study provides new knowledge about infection routes and shows that both humoral and cellular defence mechanisms are triggered by F.?noatunensis in cod.     

Francisella noatunensis subsp. noatunensis is the aetiological agent of visceral granulomatosis in wild Atlantic cod Gadus morhua

During the 1980s and 1990s wild-caught cod displaying visceral granulomatosis were sporadically identified from the southern North Sea. Presumptive diagnoses at the time included mycobacterial infection, although mycobacteria were never cultivated or observed histologically from these fish. Farmed cod in Norway displaying gross pathology similar to that identified previously in cod from the southern North Sea were recently discovered to be infected with the bacterium Francisella noatunensis subsp, noatunensis. Archived formalin-fixed paraffin-embedded tissues from the original North Sea cases were investigated for the presence of Mycobacterium spp. and Francisella spp. using real-time polymerase chain reaction, DNA sequencing and immunohistochemistry. Whilst no evidence of mycobacterial infection was found, F. noatunensis subsp. noatunensis was identified in association with pathological changes consistent with Francisella infections described from farmed cod in recent years. This study shows that francisellosis occurred in wild-caught cod in the southern North Sea in the 1980s and 1990s and demonstrates that this disease predates intensive aquaculture of cod.     

Molecular and bioassay-based detection of Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis)

Toxoplasma gondii is associated with morbidity and mortality in a variety of marine mammals, including fatal meningoencephalitis in the southern sea otter (Enhydra lutris nereis). The source(s) of T. gondii infection and routes of transmission in the marine environment are unknown. We hypothesise that filter-feeding marine bivalve shellfish serve as paratenic hosts by assimilation and concentration of infective T. gondii oocysts and their subsequent predation by southern sea otters is a source of infection for these animals. We developed a TaqMan PCR assay for detection of T. gondii ssrRNA and evaluated its usefulness for the detection of T. gondii in experimentally exposed mussels (Mytilus galloprovincialis) under laboratory conditions. Toxoplasma gondii-specific ssrRNA was detected in mussels as long as 21 days post-exposure to T. gondii oocysts. Parasite ssrRNA was most often detected in digestive gland homogenate (31 of 35, i.e. 89%) compared with haemolymph or gill homogenates. Parasite infectivity was confirmed using a mouse bioassay. Infections were detected in mice inoculated with any one of the mussel sample preparations (haemolymph, gill, or digestive gland), but only digestive gland samples remained bioassay-positive for at least 3 days post-exposure. For each time point, the total proportion of mice inoculated with each of the different tissues from T. gondii-exposed mussels was similar to the proportion of exposed mussels from the same treatment groups that were positive via TaqMan PCR. The TaqMan PCR assay described here is now being tested in field sampling of free-living invertebrate prey species from high-risk coastal locations where T. gondii infections are prevalent in southern sea otters.     

Detection of bacteria originally isolated from Alexandrium spp. in the midgut diverticula of Mytilus edulis after water-borne exposure

Bacteria associated with toxic dinoflagellates have been implicated in the production of paralytic shellfish poisoning (PSP) toxins, but it has not been substantiated that bacteria are truly capable of autonomous PSP toxin synthesis or what role bacteria may play in shellfish toxification. In this study, different putatively PSP toxin producing bacteria originally isolated from toxic Alexandrium spp. were exposed to the blue mussel Mytilus edulis. To document that these bacteria accumulated in the digestive tract of the mussels, hybridization techniques that use rRNA targeted oligonuceotides for in situ identification of these bacteria were applied. The mussel hepatopancreas was dissected and paraffin and frozen sections were made. The dissected glands were hybridized with digoxigenin-labelled 16S rRNA oligonucleotide probes. Results demonstrate that mussels will readily uptake and accumulate these bacteria in the hepatopancreas. However, the mussels were not rendered toxic by the ingestion of the bacteria as determined by HPLC with UV detection for PSP toxins and determination of sodium channel blocking activity using the mouse neuroblastoma assay. Thus, although the role that bacteria play in mussel toxification remains unclear, methods are now available which will aid in further investigation of this relatively unexplored area.     

Experimental Transmission of Infectious Pancreatic Necrosis Virus from the Blue Mussel,Mytilus edulis,to Cohabitating Atlantic Salmon (Salmo salar) Smolts

Integrated multitrophic aquaculture (IMTA) reduces the environmental impacts of commercial aquaculture systems by combining the cultivation of fed species with extractive species. Shellfish play a critical role in IMTA systems by filter-feeding particulate-bound organic nutrients. As bioaccumulating organisms, shellfish may also increase disease risk on farms by serving as reservoirs for important finfish pathogens such as infectious pancreatic necrosis virus (IPNV). The ability of the blue mussel (Mytilus edulis) to bioaccumulate and transmit IPNV to naive Atlantic salmon (Salmo salar) smolts was investigated. To determine the ability of mussels to filter and accumulate viable IPNV, mussels were held in water containing log 4.6 50% tissue culture infective dose(s) (TCID₅₀) of the West Buxton strain of IPNV ml⁻¹. Viable IPNV was detected in the digestive glands (DGs) of IPNV-exposed mussels as early as 2 h postexposure. The viral load in mussel DG tissue significantly increased with time and reached log 5.35 ± 0.25 TCID₅₀ g of DG tissue⁻¹ after 120 h of exposure. IPNV titers never reached levels that were significantly greater than that in the water. Viable IPNV was detected in mussel feces out to 7 days postdepuration, and the virus persisted in DG tissues for at least 18 days of depuration. To determine whether IPNV can be transmitted from mussels to Atlantic salmon, IPNV-exposed mussels were cohabitated with naive Atlantic salmon smolts. Transmission of IPNV did occur from mussels to smolts at a low frequency. The results demonstrate that a nonenveloped virus, such as IPNV, can accumulate in mussels and be transferred to naive fish.     

Understanding Marine Mussel Adhesion

In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion.     

The Effects of Natural Hybridization on the Regulation of Doubly Uniparental Mtdna Inheritance in Blue Mussels (Mytilus Spp.)

Blue mussels in the Mytilus edulis species complex have a doubly uniparental mode of mtDNA inheritance with separate maternal and paternal mtDNA lineages. Female mussels inherit their mtDNA solely from their mother, while males inherit mtDNA from both parents. In the male gonad the paternal mtDNA is preferentially replicated so that only paternal mtDNA is transmitted from fathers to sons. Hybridization is common among differentiated blue mussel taxa; whenever it involves M. trossulus, doubly uniparental mtDNA inheritance is disrupted. We have found high frequencies of males without and females with paternal mtDNA among hybrid mussels produced by interspecific matings between M. galloprovincialis and M. trossulus. In contrast, hybridization between M. galloprovincialis and M. edulis does not affect doubly uniparental inheritance, indicating a difference in the divergence of the mechanisms regulating mtDNA inheritance among the three blue mussel taxa. Our data indicate a high frequency of disrupted mtDNA transmission in F(1) hybrids and suggest that two separate mechanisms, one regulating the transmission of paternal mtDNA to males and another inhibiting the establishment of paternal mtDNA in females, act to regulate doubly uniparental inheritance. We propose a model for the regulation of doubly uniparental inheritance that is consistent with these observations.     

New genotypes and factors associated with Cryptosporidium detection in mussels (Mytilus spp.) along the California coast

A 3 year study was conducted to evaluate mussels as bioindicators of faecal contamination in coastal ecosystems of California. Haemolymph samples from 4680 mussels (Mytilus spp.) were tested for Cryptosporidium genotypes using PCR amplification and DNA sequence analysis. Our hypotheses were that mussels collected from sites near livestock runoff or human sewage outflow would be more likely to contain the faecal pathogen Cryptosporidium than mussels collected distant to these sites, and that the prevalence would be greatest during the wet season when runoff into the nearshore marine environment was highest. To test these hypotheses, 156 batches of sentinel mussels were collected quarterly at nearshore marine sites considered at higher risk for exposure to livestock runoff, higher risk for exposure to human sewage, or lower risk for exposure to both faecal sources. Cryptosporidium genotypes detected in Haemolymph samples from individual mussels included Cryptosporidium parvum, Cryptosporidium felis, Cryptosporidium andersoni, and two novel Cryptosporidium spp. Factors significantly associated with detection of Cryptosporidium spp. in mussel batches were exposure to freshwater outflow and mussel collection within a week following a precipitation event. Detection of Cryptosporidium spp. was not associated with higher or lower risk status for exposure to livestock faeces or human sewage sources. This study showed that mussels can be used to monitor water quality in California and suggests that humans and animals ingesting faecal-contaminated water and shellfish may be exposed to both host-specific and anthropozoonotic Cryptosporidium genotypes of public health significance.     

Monitoring the survival of fish-pathogenic Francisella in water microcosms

In this report, the survival behaviour of fish pathogenic Francisella in water microcosms was investigated under laboratory conditions. Two isolates of Francisella noatunensis (NCIMB14265(T) and PQ 1106), from fish held in seawater and freshwater, were inoculated into natural (nonsterile) and sterile sea- and freshwater microcosms, respectively, and monitored under different temperature conditions (4, 8 and 12 °C) over a period of 60 days. The culturability of the strains was inversely related to the water temperature. Strain NCIMB14265(T) was found to survive longer in seawater than PQ 1106 held in freshwater at equivalent temperatures. The survival of both strains was higher in sterile than in nonsterile microcosms. These results were confirmed by quantitative PCR analysis targeting the succinate dehydrogenase (sdhA) gene. A cell viability assay coupled with FISH analyses showed that F. noatunensis cells enter a viable but not culturable (VBNC) state after a period in water. However, although metabolically active, the VBNC cells were not pathogenic to cod (Gadhus morhua) following an intraperitoneal challenge, under the conditions tested. The data presented contribute to a better understanding of the behaviour of F. noatunensis in natural seawater and freshwater environments, and show the need for further investigation of the role of VBNC cells in the environmental transmission of this pathogen.     

Fatty acid as markers to demonstrating trophic relationships among diatoms, rotifers and green-lipped mussels

Green-lipped mussels Perna viridis, collected from Peng Chau, Hong Kong were allotted into two treatment groups, each containing three experimental tanks. The first treatment group comprised of mussels fed with the diatom Thalassiosira pseudonana only, whereas the second treatment group contained mussels fed with the marine rotifer Brachionus plicatilis, which was in turn fed with diatom T. pseudonana. The mussels were fed two times each day over the experimental period of 14 days. On Days 4, 7 and 14, three mussels were collected from each tank of each treatment group and treated as a single replicate. Fatty acid profiles of diatoms, marine rotifers and the three organs (digestive gland, mantle margin and adductor muscle) of the two mussel groups were analyzed. Results showed that monosaturated fatty acid (MUFA) 16:1n7 was conserved along the food chain among diatoms, marine rotifers and green-lipped mussels. This suggested that 16:1n7 or the ratio of 16:1n7 to saturated fatty acid (SFA) 16:0 can be a trophic marker for diatom T. pseudonana and elevated amounts of 16:1n7 in mussels can reflect the dominance of diatoms in its diet. The present results also showed that rotifers could accumulate MUFA 18:1n7 and PUFA 20:4n6 which were transferred up to mussels, especially 18:1n7, as zooplankton have the ability to synthesize or actively accumulate certain fatty acids that they need for growth or reproduction. There was an increase in the amount of 18:1n7 in the digestive gland of mussels fed with rotifers but the level of this fatty acid remained unchanged in those fed with diatoms, further confirming that 18:1n7 can be used as a marker for the presence of rotifers in trophic relationship studies. The relatively faster responses in the digestive gland of mussels to acquire the fatty acid signatures from their food suggested that the fatty acid profiles in the digestive gland can be a good marker to show a short-term fluctuation of food conditions in the marine environment.     

Effects of Adult Dreissena polymorpha on Settling Juveniles and Associated Macroinvertebrates

The impact of Dreissena polymorpha settlement on recruitment of juvenile mussels and density of other macroinvertebrates was studied in field experiments using blank concrete blocks and tiles (control), blocks and tiles with attached empty zebra mussel shells, and blocks and tiles with attached living mussels. On blocks, dominant invertebrate taxa showed colonization patterns coinciding with increased habitat complexity owing to zebra mussel settlement or the biodeposition of faeces and pseudofaeces. Adult and especially juvenile zebra mussels preferred blocks with empty shells to blank blocks and blocks with living mussels; this might possibly be caused by a chemical cue that induces gregarious settlement. Lower recruitment on blocks with attached living mussels compared to blocks with only shells could be the consequence of ingestion of larvae by adult mussels and of competition for food. On tiles, the sediments deposited and the organic content of the sediment were investigated. Sedimentation was significantly higher on shell‐only and live‐mussel tiles compared to blank tiles. Organic matter differed significantly between blank and live‐mussel tiles.     

The effect of chlorpyrifos on protein content and acid DNase activity in the mussel,Mytilus galloprovincialis

We evaluated the effects of short-term exposure to an organophosphate pesticide chlorpyrifos on the digestive gland and gills of the mussel Mytilus galloprovincialis. We studied metabolic activity by quantifying protein content and physiological function responses using acid DNase activity. The increase in protein content was observed in both the target tissues of mussels exposed to 0.03 μg/L chlorpyrifos when compared with control mussels. The pattern of acid DNase activity in digestive gland and gills indicated a tissue-specific response, although the lowest concentration of chlorpyrifos caused changes in acid DNase activity in both tissues. In the digestive gland, the increase of acid DNase activity was observed in mussel exposed to 0.03 μg/L chlorpyrifos, followed by decrease up to 100 μg/L chlorpyrifos. Enzyme activity in the gills showed a dose response effect. The results support the use of acid DNase activity in the digestive gland as a sensitive response to an environmentally relevant range of pesticide concentrations. It may also indicate an effect on mussel physiological status.     

Functional characterization of codCath, the mature cathelicidin antimicrobial peptide from Atlantic cod (Gadus morhua)

Cathelicidins are among the best characterized antimicrobial peptides and have been shown to have an important role in mammalian innate immunity. We recently isolated a novel mature cathelicidin peptide (codCath) from Atlantic cod and in the present study we functionally characterized codCath. The peptide demonstrated salt sensitivity with abrogation of activity at physiological salt concentrations. In low ionic strength medium we found activity against marine and non-marine Gram-negative bacteria with an average MIC of 10 μM, weak activity against a Gram-positive bacterium (MIC 80 μM), and pronounced antifungal activity (MIC 2.5 μM). The results suggest the kinetics and mode of action of codCath to be fast killing accompanied by pronounced cell lysis. Extracellular products (ECPs) of three marine bacteria caused breakdown of the peptide into smaller fragments and the cleaved peptide lost its antibacterial activity. Proteolysis of the peptide on the other hand was abolished by prior heat-treatment of the ECPs, suggesting a protease involvement. We observed no cytotoxicity of the peptide in fish cells up to a concentration of 40 μM and the selectivity of activity was confirmed with bacterial and mammalian membrane mimetics. We conclude that the potent broad-spectrum activity of codCath hints at a role of the peptide in cod immune defense.     

Changes of Blue Mussels Mytilus edulis L. Lipid Composition Under Cadmium and Copper Toxic Effect

The lipid and fatty acid composition of the blue mussels Mytilus edulis L. gills and digestive glands was evaluated after 24 and 72 h of cadmium (Cd) and copper (Cu) exposure. Mussels were exposed to different cadmium (10, 100, and 500 μg/L) and copper (5, 50, and 250 μg/L) concentrations. Similar stress response of predominant membrane phospholipids level as well as polyenoic and non-methylene interrupted (NMI) fatty acids content was observed in mussel gills under both cadmium and copper effects. Increased NMI fatty acids level after 24 h, the metal ions treatment suggests that these acids contribute to the protective response to the membrane oxidative stress caused by accumulation of the metals. The content of cholesterol, some minor membrane phospholipids, and storage lipids (triacylglycerols, TAG) in the mussels’ organs alter significantly under the cadmium and copper effect. A two-step response at the digestive glands TAG level depends on the duration of the cadmium and copper treatments (24 and 72 h) on the mussels. The results demonstrate that Cd and Cu impact has adverse effects on gills and digestive glands lipid and fatty acids composition. The type of observed effects varies with the nature and concentration of the metal ions and depends on the role of the metals in the mussels’ life activity.     

Genome characterisation of the genus Francisella reveals insight into similar evolutionary paths in pathogens of mammals and fish

ABSTRACT: BACKGROUND: Prior to this study, relatively few strains of Francisella had been genome-sequenced. Previously published Francisella genome sequences were largely restricted to the zoonotic agent F. tularensis. Only limited data were available for other members of the Francisella genus, including F. philomiragia, an opportunistic pathogen of humans, F. noatunensis, a serious pathogen of farmed fish, and other less well described endosymbiotic species. RESULTS: We determined the phylogenetic relationships of all known Francisella species, including some for which the phylogenetic positions were previously uncertain. The genus Francisella could be divided into two main genetic clades: one included F. tularensis, F. novicida, F. hispaniensis and Wolbachia persica, and another included F. philomiragia and F. noatunensis. Some Francisella species were found to have significant recombination frequencies. However, the fish pathogen F. noatunensis subsp. noatunensis was an exception due to it exhibiting a highly clonal population structure similar to the human pathogen F. tularensis. CONCLUSIONS: The genus Francisella can be divided into two main genetic clades occupying both terrestrial and marine habitats. However, our analyses suggest that the ancestral Francisella species originated in a marine habitat. The observed genome to genome variation in gene content and IS elements of different species supports the view that similar evolutionary paths of host adaptation developed independently in F. tularensis (infecting mammals) and F. noatunensis subsp. noatunensis (infecting fish).     

Biological control of streptococcal infection in Nile tilapia Oreochromis niloticus (Linnaeus, 1758) using filter‐feeding bivalve mussel Pilsbryoconcha exilis (Lea, 1838)

Since bivalve mussels are able to graze heavily on bacteria, in this paper it is hypothesized that when mussels are cultured with fish, the filtering efficiency of the mussels will keep the bacterial population below a certain threshold and thus assist in reducing the risk of bacterial disease outbreaks. The ability of the filter‐feeding bivalve mussel Pilsbryoconcha exilis to control Streptococcus agalactiae was tested in a laboratory‐scale tilapia culture system. Juvenile Nile tilapia (Oreochromis niloticus), the bivalve mussel as well as the bacteria were cultured at different combinations using four treatments: treatment‐1: mussel and bacteria but no fish, treatment‐2: tilapia and mussel but no bacteria, treatment‐3: tilapia and bacteria but no mussel, and treatment‐4: tilapia, mussels, and bacteria. All treatments were run in three replicates; stocking rates were 10 tilapia juveniles; five mussels; and about 3.5 × 10⁵ colony forming units (CFU) ml^?1 of bacteria in 50‐L aquaria with 40‐L volume. The mussel reduced the bacterial population by 83.6–87.1% in a 3‐week period whereas in the absence of the mussel, the bacterial counts increased by 31.5%. Oresence of the mussel also resulted in significantly higher growth and lower mortality of tilapia juveniles than when the mussel was absent. The results of this experiment suggest that the freshwater mussel P. exilis could control the population of S. agalactiae in a laboratory‐scale tilapia culture system. Future studies should focus on the dynamic interactions among fish, mussels, and bacteria as well as on how input such as feed and other organic materials affect these interactions.     

Differential pattern of Cu/Zn superoxide dismutase isoforms in relation to tidal spatio-temporal changes in the blue mussel Mytilus edulis

Inducible antioxidant defences in marine organisms such as mussel bivalves are commonly used as biomarkers of pollutant-induced oxidative stress and their variations proposed as one of the biological effect measurements for assessment of contamination impact in aquatic environments. Among them, the copper/zinc superoxide dismutases (Cu/Zn-SODs) are metalloenzymes which play a key role in the protection of cells in case of oxidative stress. In order to observe possible variations of an antioxidant response in relation to tidal oscillations, the copper/zinc superoxide dismutase activity (Cu/Zn-SOD) was characterized in the digestive gland and gills of blue mussels sampled at high and low shore throughout the tidal cycle. Determination of SOD activity was performed on gels after isoelectro-focusing, allowing the revelation of three isoforms. In both tissues, high-shore mussels exhibited a higher level of total SOD activity than low-shore mussels. During emersion, a decrease of total SOD activity appeared in digestive gland for both groups. In high-shore mussels, the less acidic form contributed to 75% of the total activity, the second one to 20% and the more acidic one to 5% in both tissues before air exposure. During emersion, the relative contribution of the three isoforms to the total activity was markedly changed with a significant decrease in intensity of the first isoform and parallel increases in the two other ones. After re-immersion a progressive recovery of proportions of SOD isoforms was observed. In low-shore mussels, the relative contribution of the three isoforms to the total SOD activity showed similar changes. The observed variations could correspond to changes in the redox status of the mussels during tidal oscillations.     

Histopathology of the digestive gland of the bivalve mollusk Crenomytilus grayanus (Dunker, 1853) from southwestern Peter the Great Bay, Sea of Japan

The histomorphology of the digestive gland of the bivalve mollusk Crenomytilus grayanus from Sivuchya Bay, which is located in the southwest of Peter the Great Bay and subjected to the effect of polluted waters of Tumannaya River, was studied. Pathological changes of the digestive tubules, channels, and connective tissue of the gland were recorded in all the mussels studied. The epithelium of the tubules and channels was characteristic with erosive disturbances and by heavy vacuolization of digestive cells; connective tissue of the gland was specified by cells with lipofuscin (granulocytomes) and by foci of cells necrosis and lysis. Nervous fibers running in the gland were swollen in some mollusks. Strongly basophilic spherical formations, presumably one of the development stages of a parasitic plasmodium, were found in the granulocytomes and among vesicular cells of connective tissue of all the mussels. It was concluded that pathological changes in digestive gland of Gray’s mussel might be caused by chronic pollution of the bay and by parasitic invasion.     

The Escherichia coli Content of Mytilus edulis from Analysis of Whole Tissue or Digestive Tract

The dissected digestive tract of edible mussels from two sites was examined for Escherichia coli and the counts compared with those from the total tissue of parallel mussel samples. The method using 4 x 1 ml pour plates was preferred to the 1 x 4 ml pour plate or to the 2 x 0.2 ml spread plates. Counts from the digestive tract were up to sixfold higher than parallel counts using total tissue. Digestive tract inoculum also produced amplified Esch. coli detection when lightly polluted mussels were examined by the semi-quantitative percentage-clean technique. It was concluded that dissection of the bacteriologically rich digestive tract significantly increases the sensitivity of Esch. coli detection and has application in environmental assay procedures using mussels or other filter-feeding molluscs. Significant differences in the frequency of Esch. coli biotypes from the two mussel locations could be related to small differences in the pollution regimes at these two sites.