S. W. Feist

Histopathological biomarkers in estuarine fish species for the assessment of biological effects of contaminants

The increasing emphasis on the assessment and monitoring of estuarine ecosystems has highlighted the need to deploy appropriate biological indices for these locations. Fish diseases and histopathology, with a broad range of causes, are increasingly being used as indicators of environmental stress since they provide a definite biological end-point of historical exposure. This study reports on the histopathological alterations observed in selected organs and tissues of three species of estuarine fish (Platichthys flesus, Pomatoschistus minutus and Zoarces viviparus), captured from four British estuaries (the Tyne, Tees, Mersey and Alde), differently impacted by contaminants, including PAHs. A biannual sampling regime was used to identify the important seasonal variations that occur in terms of the observed biological effects. Inflammatory lesions and hepatocellular fibrillar inclusions attained their highest prevalence in P. flesus captured from the Tyne, Tees and Mersey. The presence of pre-neoplastic and neoplastic toxicopathic lesions was highest in P. flesus captured from these sites, when compared to fish from the Aide reference site. In particular, the prevalence of hepatic foci of cellular alteration (up to 43.3%) and hepatocellular adenoma (up to 10%) were highest in P. flesus captured from the Mersey estuary. Intersex (ovotestis) was only recorded in male P.flesus captured from the Mersey estuary (up to 8.3%) and from male Z. viviparous captured from the Tyne estuary (25%). Pathologies associated with the gill and the kidney were also most prevalent in fish captured from the Tyne, Tees and Mersey estuaries. This study has successfully applied histopathology to an estuarine monitoring program, both for the recording of toxicopathic lesions in the liver and other organs, and for the detection of the endpoint of endocrine disruption, intersex. As such, it provides a powerful integrative tool for the assessment of biological effects of contaminants in these environments.

The extent of oestrogenic contamination in the UK estuarine and marine environments — further surveys of flounder

In 1996, The Centre for Environment, Fisheries and Aquaculture Science (CEFAS) initiated a project to establish whether oestrogenic materials are present in UK estuarine and marine waters at biologically significant concentrations, and to investigate some of the possible effects which these may have in flounder (Platichthys flesus). Early results are published elsewhere; this paper describes the results of a second wider survey of vitellogenin and reproductive abnormalities in UK flounder. Vitellogenin levels in male blood plasma in the period from spring to winter 1997 were found to be significantly elevated (in comparison with a clean reference site on the Alde estuary) in at least one sample from most of the 11 estuaries investigated. The exceptions were the Tamar and the Dee where all fish appeared entirely normal. In broad terms, the degree of oestrogenic contamination as measured by male vitellogenesis in the various estuaries was ranked in the following descending order: Tees > Mersey > Tyne > Wear = Humber = Clyde = Southampton Water = Thames > Dee = Tamar. VTG concentrations in Tees, Mersey and Tyne male fish were extremely high (> 100,000 ng/ml), and often exceeded those normally found in sexually mature females. At most locations, ovotestis conditions in male flounder were entirely absent but 9% of male Mersey fish and 7% of male Tyne fish contained ovotestis. In a few cases, eggs were fully developed with yolk granules. Most testes did not show gross morphological abnormalities related to oestrogenic exposure, although one testis from a Mersey fish appeared to be almost entirely composed of eggs. Abnormal sex ratios were not seen in any estuary. The paper concludes by outlining a new research programme which will be addressing the biological significance of these observations.

SURVEYS OF PLASMA VITELLOGENIN AND INTERSEX IN MALE FLOUNDER (PLATICHTHYS FLESUS ) AS MEASURES OF ENDOCRINE DISRUPTION BY ESTROGENIC CONTAMINATION IN UNITED KINGDOM ESTUARIES: TEMPORAL TRENDS, 1996 TO 2001

Plasma vitellogenin (VTG) concentrations and the presence of the ovo-testis (intersex) condition have been recorded in male flounder (Platichthys flesus) captured from several United Kingdom (UK) estuaries since 1996 as part of the endocrine disruption in the Marine Environment (EDMAR) project and earlier programs. It has been confirmed that plasma VTG concentrations in male flounder have remained elevated in several UK estuaries (e.g., Tees, Mersey, and Tyne) throughout the period covered by this study. However, the time-series data indicate that plasma VTG, a measure of environmental estrogen contamination, has decreased in fish captured from several estuaries, especially those of the Tyne and Mersey. Shorter time-series data sets from the Forth and Clyde estuaries also suggest a decrease in estrogen contamination at these sites. Trends associated with specific point sources of estrogenic contamination show site-specific patterns. For instance, plasma VTG levels in male flounder captured near the Howdon sewage treatment outfall (Tyne) have shown a steady decline to near baseline levels in 2001, while the plasma of male fish captured at a site adjacent to the Dabholm Gut discharge in the Tees estuary have shown little evidence of a sustained decline. The occurrence of the intersex condition was detected at a low but consistent prevalence through the study period, with the majority of cases recorded in fish captured from the Tyne and Mersey estuaries. The data set does not allow conclusions to be drawn about any temporal trends associated with this condition. The significance of the findings and possible mitigating influences are discussed in terms of the impacts on wild fish and the role of effluent treatment in reducing these.

The impact of oestrogenic and androgenic contamination on marine organisms in the United Kingdom: summary of the EDMAR programme

This paper summarises results of the EDMAR programme which is investigating oestrogenic and androgenic endocrine disruption in UK coastal waters. Most of the data concern fish. Four species (flounder, viviparous blenny and two sand gobies) are experiencing feminisation in industrialised estuaries. In males this includes vitellogenin (VTG) synthesis, ovotestis induction and/or feminised sexual characteristics. Although reproductive success may be impaired in some cases, implications for fish populations are still unclear. Suspected causative contaminants include natural oestrogenic substances and synthetic oestrogen mimics. The majority of the oestrogenic activity is adsorbed to sediments, and routes of exposure may include benthic food chain transfer. Some natural androgenic substances are also being discharged to estuaries, but their activity appears low.

Comparison of biomarker and pathological responses in flounder (Platichthys flesus L.) induced by ingested polycyclic aromatic hydrocarbon (PAH) contamination

This study investigated the response of biomarker measurements and histopathological indicators of polycyclic aromatic hydrocarbon (PAH) exposure in the flounder (Platichthys flesus L.). Flounder were fed food spiked with a mixture of four PAHs at an environmentally relevant range of concentrations for either one or six months. Ethoxyresorufin-O-deethylase (EROD) activity was elevated following 1 month exposure to PAH concentrations up to 50 mgkg(-1) in food. Bile metabolite concentrations were found to increase with PAH concentration, up to 500 mgkg(-1) PAH. By comparison, no DNA adducts were detected and there were no significant histopathological changes observed. After 6 months exposure, EROD levels were not elevated but bile metabolites showed a similar dose dependent relationship as in the 1 month experiment, while DNA adducts were only detected in the highest PAH exposure groups. No significant histopathological changes were observed. The results are discussed with respect to the implications for the use of these methods in environmental monitoring studies.

Emergence of cold water strawberry disease of rainbow trout Oncorynchus mykiss in England and Wales: outbreak investigations and transmission studies.

Cold water strawberry disease (CWSD), or red mark syndrome (RMS), is a severe dermatitis affecting the rainbow trout Oncorynchus mykiss. The condition, which presents as multifocal, raised lesions on the flanks of affected fish, was first diagnosed in Scotland in 2003 and has since spread to England and Wales. Results of field investigations indicated the condition had an infectious aetiology, with outbreaks in England linked to movements of live fish from affected sites in Scotland. Transmission trials confirmed these results, with 11 of 149 and 106 of 159 naive rainbow trout displaying CWSD-characteristic lesions 104 to 106 d after being cohabited with CWSD-affected fish from 2 farms (Farm B from England and Farm C from Wales, respectively). The condition apparently has a long latency, with the first characteristic lesions in the previously naive fish not definitively observed until 65 d (650 day-degrees) post-contact with affected fish. Affected fish from both outbreak investigations and the infection trial were examined for the presence of viruses, oomycetes, parasites and bacteria using a combination of techniques and methodologies (including culture-independent cloning of PCR-amplified bacterial 16S rRNA genes from lesions), with no potentially causative infectious agent consistently identified. The majority of the cloned phylotypes from both lesion and negative control skin samples were assigned to Acidovorax-like beta-Proteobacteria and Methylobacterium-like alpha-Proteobacteria.

Sphaerothecum destruens pathology in cyprinids

Sphaerothecum destruens is a significant intracellular parasite of fish which has caused disease and mortalities in cultured north American Chinook salmon Oncorhynchus tshawytscha and Atlantic salmon Salmo salar. Several hosts for S. destruens have been identified within the Salmonidae family, and the histopathology of the infection can differ between hosts. Recently, S. destruens has been associated with the most invasive cyprinid species in Europe, topmouth gudgeon Pseudorasbora parva. Accurate disease identification based on thorough descriptions of clinical signs and histopathology in this new range of hosts is thus paramount to support further epizootiological studies. In this study, the associated histopathology of S. destruens infection is described along with its pathogenesis in the endangered cyprinid sunbleak Leucaspius delineatus. Histological examination of 100 L. delineatus in a wild population in the south of England revealed the presence of S. destruens infections, with a prevalence of 5% with S. destruens, suggesting an over-dispersed distribution within the L. delineatus sample. Clinical signs of the infection were absent, but histological examination revealed the presence of both disseminated and nodular lesions in several organs.

Plastic parasites: Extreme dimorphism creates a taxonomic conundrum in the phylum Microsporidia

In this paper, we combine field observations of highly statistically significant co-occurrence with histopathological, ultrastructural and molecular phylogenetic analyses, to provide evidence for extreme morphological plasticity in a microsporidium parasite infecting the musculature of marine crabs. The parasite appears to alternate between lineages that culminate in production of either bizarre needle-like spores in the peripheral sarcoplasm of heart and skeletal muscle fibres (reminiscent of Nadelspora canceri infecting Cancer magister) or alternatively, Ameson-like spores with pronounced surface projections, in the skeletal muscles (as for Ameson pulvis, previously described infecting Carcinus maenas). Both lineages occur in direct contact with the cytoplasm of host muscle cells and can exist simultaneously within the same cell. Pathological data appears to reveal a remarkable shift in morphology during pathogenic remodelling of host tissues. Sequence analysis of multiple clones derived from amplification of the ssrRNA gene from infected regions of the heart and skeletal muscles appear to confirm the genetic identity of the two lineages. Furthermore, derived ssrRNA gene sequences are more similar (>99%) to N. canceri than to the coparasite Ameson michaelis infecting Callinectes sapidus (93%). Although molecular phylogenetic data support transfer of A. pulvis into the genus Nadelspora, the expansion in the generic description required to include such widely divergent characteristics is so significant as to be unfeasible within the current taxonomic framework of the phylum Microsporidia. At present, it is preferable to propose that the parasite infecting C. maenas forms a clade with other morphologically diverse but phylogenetically and ecologically similar muscle-infecting microsporidians from marine crustacean hosts. Given the strong evidence for significant plasticity in morphology amongst members of the phylum Microsporidia, novel approaches to phylogeny, based predominantly upon the informed use of molecular sequence data, are now deemed a necessity.

Histopathological assessment of liver and gonad pathology in continental slope fish from the northeast Atlantic Ocean

The deep-sea environment is a sink for a wide variety of contaminants including heavy metals and organic compounds of anthropogenic origin. Life history traits of many deep-water fish species including longevity and high trophic position may predispose them to contaminant exposure and subsequent induction of pathological changes, including tumour formation. The lack of evidence for this hypothesis prompted this investigation in order to provide data on the presence of pathological changes in the liver and gonads of several deep-water fish species. Fish were obtained from the north east region of the Bay of Biscay (north east Atlantic Ocean) by trawling at depths between 700 and 1400 m. Liver and gonad samples were collected on board ship and fixed for histological processing and subsequent examination by light microscopy. Hepatocellular and nuclear pleomorphism and individual cases of ovotestis and foci of cellular alteration (FCA) were detected in black scabbardfish (Aphanopus carbo). Six cases of FCA were observed in orange roughy (Hoplostethus atlanticus) (n = 50) together with a single case of hepatocellular adenoma. A wide variety of inflammatory and degenerative lesions were found in all species examined. Deep-water fish display a range of pathologies similar to those seen in shelf-sea species used for international monitoring programmes including biological effects of contaminants. This study has confirmed the utility of health screening in deep-water fish for detecting evidence of prior exposure to contaminants and has also gained evidence of pathology potentially associated with exposure to algal toxins.

Comparative pathology in bivalves: Aetiological agents and disease processes

Comparative pathology as a scientific discipline studies animal diseases in relation to their aetiology, pathogenesis and prognosis. Among the main aspects of this discipline, regressive changes, host defense responses with pathological implications and progressive changes, represent the majority of the possible responses of cells and tissues to pathogens and exposure to chemicals. One of the most persistent issues in the field of invertebrate pathology is the variability in terminology and definition, which has led to confusion in scientific communication. The aim of this paper is to provide an overview of the pathological basis of bivalve disease (defensive, regressive and progressive phenomena) and contribute to the standardised terminology for bivalve molluscan disease in the context of comparative pathology.