Replacement of fishmeal with a microbial single-cell protein induced enteropathy and poor growth outcomes in barramundi (Lates calcarifer) fry.

Fish meal (FM) replacement is essential for the sustainable expansion of aquaculture. This study focussed on the feasibility of replacing FM with a single-cell protein (SCP) derived from methanotrophic bacteria (Methylococcus capsulatus, Bath) in barramundi fry (Lates calcarifer). Three isonitrogenous and isoenergetic diets were formulated with 0%, 6.4% and 12.9% inclusion of the SCP, replacing FM by 0%, 25% and 50%. Barramundi fry (initial body weight 2.5 ± 0.1 g) were fed experimental diets for 21 days to assess growth performance, gut microbiome composition and gut histopathology. Our findings revealed that both levels of SCP inclusion induced detrimental effects in barramundi fry, including impaired growth and reduced survival compared with the control group (66.7% and 71.7% survival in diets replacing FM with SCP by 25% and 50%, respectively; p < .05). Both dietary treatments presented mild necrotizing enteritis with subepithelial oedema and accumulation of PAS positive, diastase resistant droplets within hepatocytes (ceroid hepatopathy) and pancreatic atrophy. Microbiome analysis revealed a marked shift in the gut microbial community with the expansion of potential opportunistic bacteria in the genus Aeromonas. Reduced overall performance in the highest inclusion level (50% SCP) was primarily associated with reduced feed intake, likely related to palatability issues, albeit pathological changes observed in gut and liver may also play a role. Our study highlights the importance of meticulous optimization of SCP inclusion levels in aquafeed formulations, and the need for species and life-stage specific assessments to ensure the health and welfare of fish in sustainable aquaculture practices.

Effects of replacing fishmeal with methanotroph (Methylococcus capsulatus, Bath) bacteria meal (FeedKind®) on growth and intestinal health status of juvenile largemouth bass (Micropterus salmoides).

A ten-week feeding trial evaluated the feasibility of methanotroph (Methylococcus capsulatus) bacteria meal (FeedKind®, FK) as a fishmeal substitute in largemouth bass (Micropterus salmoides) diets. Six isonitrogenous and isoenergetic diets with different inclusion levels of FK (0 (fishmeal group), 43, 86, 129, 172 and 215 g/kg) were formulated to replace 0, 50, 100, 150, 200 and 250 g/kg fishmeal, respectively. The results showed that FK inclusion level could reach 129 g/kg without significantly affecting growth or feed coefficient rate (P > 0.05), while growth performance was decreased and feed coefficient rate increased when FK inclusion levels exceeded 129 g/kg (P < 0.05). Increase in FK inclusion levels tended to reduce plasma total cholesterol and total triglyceride whilst plasma total protein, albumin, alanine aminotransferase and aspartate aminotransferase in FK treatment groups were unchanged compared with fishmeal group (P > 0.05). FK inclusion levels at 43 g/kg and 86 g/kg were not detrimental to intestinal morphology whilst it was unfavourable when FK inclusion levels exceeded 86 g/kg as the total length of intestinal wall thickness and villus height, villus height were obviously decreased compared with fishmeal group (P < 0.05). As regards to inflammatory cytokine genes, FK instead of fishmeal increased the expression levels of TLR2, RelA, TNF-α, IL-1ß, IL-10 and TGF-ß, 43 g/kg and 86 g/kg FK decreased the expression level of Caspase-3 (P < 0.05). In conclusion, 129 g/kg FK can replace 150 g/kg fishmeal without negative effects on the growth performance, and replacing 100 g/kg fishmeal with 86 g/kg FK is more beneficial to intestinal health.

Interlaboratory variability in screening for Bonamia ostreae, a protistan parasite of the European flat oyster Ostrea edulis.

The spread of the protozoan parasite Bonamia ostreae is of major concern to the European flat oyster Ostrea edulis industry. Many studies have looked at the sensitivity of individual methods available to screen for B. ostreae, but in this study, 3 separate laboratories examined 4 methods of diagnosis currently used routinely in laboratories: heart imprints, histology, polymerase chain reaction (PCR) and in situ hybridisation (ISH). The results were compared to estimate interlaboratory variability. Heart imprints and histology had the highest reproducibility amongst the 3 laboratories, with greatest agreement between detection of infected and uninfected individuals. PCR had the highest detection level in every laboratory. These positives were related to the presence of confirmed infections but also in unconfirmed infections, possibly due to the presence of traces of B. ostreae DNA in oysters where clinical infections were not observed. PCR, in combination with histology or ISH, provided the most reliable detection levels in every laboratory. Variation in results for PCR and ISH observed between laboratories may be due to the different protocols used by each laboratory for both methods. Overall, the findings from the 3 laboratories indicated that at least 2 methods, with fixed protocols, should be used for the accurate detection and determination of infection prevalence within a sample. This combination of methods would allow for a clearer and more precise diagnosis of B. ostreae, preventing further spread of the disease and providing more accurate detection levels and epidemiological information.

Detection of Bonamia exitiosa (Haplosporidia) in European flat oysters Ostrea edulis cultivated in mainland Britain.

Following a reported mortality event of European flat oysters Ostrea edulis in southwestern England in December 2010, a sample of 30 oysters was examined using histology and molecular techniques. Histological examination of the oysters revealed the presence of microcell stages in the haemocytes and connective tissues of 3 out of the 30 animals examined. One animal showing marked haemocyte infiltration of the connective tissues was considered to be infected with Bonamia ostreae based on the presence of small uninucleate microcells measuring approximately 1 to 1.5 µm in diameter. Two other oysters were considered by histology to be infected with B. exitiosa. Infected haemocytes contained up to 5 microcells, measuring approximately 2 to 3 µm in diameter with a central or subcentral nucleus. Rarely, larger plasmodia-like multinucleated stages were noted in the haemocyte cytoplasm characterised by its irregular shape and increased eosinophilic cytoplasm. Haemocyte infiltration of the connective tissues surrounding the digestive gland and the mantle was noted along with necrosis of the tissues associated with the infection. Molecular analysis of the infected animals confirmed the presence of B. exitiosa in the sample. This study describes the parasite from flat oysters cultured in the UK; subsequent targeted sampling has not detected the parasite in flat oyster populations at this or other sites within the UK.

Comparing the standardized amino acid digestibility of an alternative protein source with commercially available protein-based ingredients using the precision-fed cecectomized rooster assay.

Using single-cell-based proteins in pet foods is of interest, but little testing has been done. Therefore, our objective was to determine the amino acid (AA) digestibilities, assess protein quality of a novel microbial protein (MP) (FeedKind), and compare it with other protein-based ingredients using the precision-fed cecectomized rooster assay. Test ingredients included: MP, chicken meal (CM), corn gluten meal (CGM), pea protein (PP), and black soldier fly larvae. Thirty cecectomized roosters (n = 6/ingredient) were randomly assigned to test ingredients. After 24 h of feed withdrawal, roosters were tube-fed 15 g test ingredient and 15 g corn, and then excreta were collected for 48 h. Endogenous AA corrections were made using additional roosters. Digestible indispensable AA score (DIAAS)-like values were calculated to determine protein quality according to Association of American Feed Control Officials (AAFCO), The European Pet Food Industry Federation, and National Research Council reference values for growing and adult dogs and cats. Data were analyzed using the Mixed Models procedure of SAS 9.4, with P ≤ 0.05 being significant. All reactive lysine:total lysine ratios, an indicator of heat damage, were higher than 0.9, except for CM (0.86). Digestibility of indispensable and dispensable AA were >85% and >80% for MP, respectively, with indispensable AA digestibilities being >80% for all other ingredients. In general, CGM had the highest, while CM had the lowest AA digestibilities. Two exceptions were lysine and tryptophan. Lysine digestibility for MP was higher than that of all other ingredients, while tryptophan digestibility for MP was higher than that of CM, CGM, and PP. Threonine digestibility was highest for CGM and MP. Valine digestibility was highest for CGM, PP, and MP. DIAAS-like calculations identified limiting AA of each ingredient and depended on the reference used and life stage and species of animal. Using AAFCO guidelines, all DIAAS-like values for MP were >100 suggesting that it could be used as the sole source of protein in adult dog and cat diets; only methionine had DIAAS-like values <100 for growing kittens. For dogs, limiting AA was most commonly methionine, threonine, and tryptophan in the other protein sources. For cats, limiting AA was most commonly lysine and methionine. Lysine was severely limited in CGM across all life stages considered. Further research in dogs and cats is necessary, but our data suggest that the MP tested has high AA digestibilities and is a high-quality protein source that may be useful in pet foods.

Single-cell-based proteins are of interest for use in pet foods, but little testing has been done. The objective of this experiment was to compare the amino acid (AA) digestibilities and protein quality of a novel microbial protein (MP) (FeedKind) with chicken meal (CM), corn gluten meal (CGM), pea protein (PP), and black soldier fly larvae ingredients using the precision-fed cecectomized rooster assay. Cecectomized roosters were tube-fed the test ingredients and excreta were collected. All reactive lysine:total lysine ratios, an indicator of heat damage, were higher than 0.9, except for CM. Digestibility of indispensable and dispensable AA were >85% and >80% for MP, respectively, with indispensable AA digestibilities being >80% for all other ingredients. In general, CGM had the highest, while CM had the lowest AA digestibilities. Lysine and tryptophan were exceptions, being highest for MP. Threonine and valine digestibilities were also high for MP. Digestible indispensable AA score-like values identified limiting AA of each ingredient. Limiting AA was most commonly methionine, threonine, and tryptophan for dogs and lysine and methionine for cats. Our data suggest that the MP tested has high AA digestibilities and is a high-quality protein source that may be useful in pet foods.

Parasites and pathogens of the endosymbiotic pea crab (Pinnotheres pisum) from blue mussels (Mytilus edulis) in England.

A histopathological survey of the commensal pea crab (Pinnotheres pisum) from the mantle cavities of blue mussels (Mytilus edulis) has been conducted. A total of 266 pea crabs from eight sites around the English coastline were examined. Of these, 82 were negative for any visible infections by histology. The remaining pea crabs were infected with an intranuclear bacilliform virus designated as P. pisum bacilliform virus (PpBV) in the hepatopancreatic epithelial cells, peritrichous ciliates on the gills, an intracytoplasmic microsporidian infection of the hepatopancreatocytes, a myophilic microsporidian infection, the gregarine Cephaloidophora fossor in the hepatopancreas, the entoniscid isopod Pinnotherion vermiforme, a low level nematode infection and an acanthocephalan cystacanth. Host reactions to infections were generally subdued. Results are discussed in relation to the endocommensal habitat of the pea crabs.

Histopathological survey of pathogens and commensals of white-clawed crayfish (Austropotamobius pallipes) in England and Wales.

A histopathological survey of white-clawed crayfish (Austropotamobius pallipes) from six rivers in England and Wales was conducted between November 2007 and September 2011. A. pallipes bacilliform virus (ApBV) was present in four samples; infected crayfish showed pathological responses ranging from mild hypertrophy of infected nuclei and emargination of chromatin through to loss of architecture of the hepatopancreatic cells. Crayfish were found to be hosts to two different ciliate species, mites, nematodes, digeneans and the microsporidian Thelohania contejeani. The variation in disease presence between populations highlights the importance of conducting appropriate surveys of native crayfish prior to movement of animals to refugia sites for relocation, restocking or breeding programmes.

A Natural Gas Fermentation Bacterial Meal (FeedKind®) as a Functional Alternative Ingredient for Fishmeal in Diet of Largemouth Bass, Micropterus salmoides.

A 10-week growth study was conducted to evaluate the effect of a natural gas fermentation bacterial meal (FeedKind®, FK) as a fishmeal (FM) alternative in largemouth bass (Micropterus salmoides) (48.0 ± 0.03 g). Four isonitrogenous and isoenergetic diets were formulated including one commercial control (C, 42% FM) and three experimental diets with gradient FK of 3% (FK3, 29%FM), 6% (FK6, 26%FM) and 9% (FK9, 23%FM), respectively. FK-fed groups showed significantly higher SR than that of C group. The WGR and SGR of fish fed FK3 and FK6 were significantly higher than those of FK9, but not statistical different from the C group. FK-fed groups showed higher apparent digestibility coefficients of dry matter and nutrients. Further, FK-fed groups increased the ratio of SOD/MDA in the plasma and liver, and the upregulation of intestinal Keap1 and downregulation of HIF1α was found in FK3. Furthermore, FK-fed groups showed higher microbial richness and diversity. Pearson correlation analysis found that antioxidant relevant biomarkers were negatively correlated with the relative abundance of certain potential beneficial bacteria. In conclusion, supplemented up to 3-6% FK replacing FM in a low FM diet of largemouth bass could increase growth, survival rate, antioxidant capacity, and improve gut microbiota.

Diseases of crayfish: a review.

A systematic review of parasites, pathogens and commensals of freshwater crayfish has been conducted. All major groups of disease causing agents have been covered including viruses, bacteria, fungi, protistans and metazoans. Most agents tend to cause limited problems for crayfish. Exceptions to this include fungi, bacteria and viruses. However, in many cases, these tend to be isolated reports in either a specific geographical location or in individual animals. The apparent absence of pathology associated with these agents in crayfish should not be taken to suggest that movements of crayfish to new geographical areas is necessarily acceptable. Several examples are given where seemingly healthy animals have been moved to new areas leading to mortality of other crayfish within the same area as a direct result of transmission of pathogens to naïve hosts. Some future research needs are proposed, including the need for pathogen characterisation and production of disease-free crayfish for aquaculture.

Paramarteilia canceri sp. n. (Cercozoa) in the European edible crab (Cancer pagurus) with a proposal for the revision of the order Paramyxida Chatton, 1911.

A new species, Paramarteilia canceri sp. n., is described using light and electron microscopy from the edible crab Cancer pagurus L. captured from the English Channel. No external symptoms were noted, although infected animals were typically lethargic and unresponsive to external stimuli. Organs of infected animals were shrunken and collapsed compared with apparently healthy individuals. Although the infection was systemic, marked host responses were only noted in the hepatopancreas where the parasite induced a pronounced haemocytic infiltration. Prevalence of infection throughout the study was 1.1%, with a maximum monthly prevalence of 3%. The intracellular parasite was typically 15 microm in length and composed of a primary cell containing up to three secondary cells derived by internal cleavage. Each secondary cell contains two bicellular spores. The parasite is readily differentiated from the other described paramyxean species by a combination of the number of secondary and tertiary cells. In light of this new discovery, a revision of the order Paramyxida Chatton, 1911 is proposed based upon comparison to the original descriptions of this parasite group in various species of invertebrate hosts. The proposed classification is based on the number of cells within the spores (tertiary cells), so that only three genera remain within the order, namely Marteilia Grizel, Comps, Bonami, Cousserans, Duthoit et Le Pennec, 1974, Paramarteilia Ginsburger-Vogel et Desportes, 1979 and Paramyxa Chatton, 1911. Subsequent discrimination of species is based on a combination of the number of secondary cells within the primary cell and the number of tertiary cells within secondary cells. It is proposed that the genus Marteilioides Comps, Park et Desportes, 1986 is suppressed and the type species of the genus, M. chungmuensis Comps, Park et Desportes, 1986, is transferred to Marteilia and that the other representative of the genus, M. branchialis Anderson et Lester, 1992, is transferred to Paramarteilia. Further, Paramyxoides Larsson et Køie, 2005 is considered as a junior synonym of Paramyxa and its type and only species, Paramyxoides nephtys Larsson et Køie, 2005, is transferred to Paramyxa.

Anisakis simplex sensu lato associated with red vent syndrome in wild adult Atlantic salmon Salmo salar in England and Wales.

Wild Atlantic salmon Salmo salar with swollen and/or bleeding vents (red vent syndrome) have been observed in rivers across England and Wales. Low numbers of salmon with this condition have previously been recorded, but reports have increased in 2006 and 2007. Affected and non-affected salmon were sampled from fish traps and their vent pathology was examined. Third stage larvae of the nematode Anisakis simplex sensu lato were observed in the tissue surrounding the vent in all affected fish. Histology of the vent showed gross lesions with an inflammatory response associated with non-encapsulated nematode worms in the connective tissue and skeletal muscle. The presence of the parasite in the unusual location of the vent areas, with associated haemorrhaging, is possibly a new emerging pathological association with the nematode A. simplex infection in Atlantic salmon. The reason for this unusual tissue tropism by larval A. simplex and the significance on already depleted salmon stocks are currently unknown.

Effect of microbial pathogens on the diversity of aquatic populations, notably in Europe.

The expansion of aquaculture and the demand for ornamental fish have resulted in the large-scale movements of aquatic animals and their pathogens. Here we review the most important non-native fish and shellfish pathogens in European waters and their global impacts on wild fish host populations. The role of theoretical models in the study of the impact of microbial pathogens is discussed, including its integration into risk assessments.

Myxobolus buckei sp. n. (Myxozoa), a new pathogenic parasite from the spinal column of three cyprinid fishes from the United Kingdom.

Myxobolus buckei sp. n. is described from the spinal column of Leuciscus cephalus (L.), Rutilus rutilus (L.) and Abramis brama (L.) from freshwater rivers in the North of England. The plasmodia develop within the remnants of the embryonic notochord in the intervertebral spaces. The spores are large, measuring (in microm) 14.0 +/- 0.7 x 11.5 +/- 0.6 (mean +/- SD), smooth, round to ellipsoid in valvular view with several sutural edge markings. The polar capsules are pyriform and of equal size, measuring 7.5 +/- 0.5 x 4.2 +/- 0.2 (mean +/- SD), with 11-12 turns of the polar filament arranged perpendicularly to the longitudinal axis of the polar capsule. The parasite has a large intercapsular appendix and large iodinophorous vacuole. The parasite can be differentiated from all known species of Myxobolus Bütschli, 1882 by a combination of the morphological characters defined. Infected fish show marked longitudinal compression of the body compared to uninfected individuals of the same year class, a feature which is pathognomonic for the disease. Histologically, host responses ranged from mild hypertrophy of the zygapophyseal process and expansion of the intervertebral membrane to complete hypertrophy and fusion of the vertebrae. Prominent notochord is present in the intervertebral spaces of infected fish and sporogony of the parasite leads to a vigorous focal inflammatory response involving proliferating fibroblast and osteogenic cells. The parasite causes a radial expansion of the centra and extensive dorsal and ventral outgrowths of the vertebrae leading to compression of the spinal cord and blood vessels running through the neural and haemal spines respectively. The parasite is considered highly pathogenic to juvenile cyprinids.

Mikrocytids are a broadly distributed and divergent radiation of parasites in aquatic invertebrates.

Microcell parasites have independently evolved in several eukaryotic lineages and are increasingly recognized as important and emerging pathogens of diverse hosts, including species of economic importance subject to international legislation concerning the trading of aquatic animals [1-3]. The microcell Mikrocytos mackini causes Denman Island disease of oysters and represents one of the most genetically divergent eukaryotes known. Mikrocytos has remained an isolated lineage with a limited distribution. We investigated two emerging diseases of juvenile crabs and oysters from the UK using massively parallel sequencing and targeted primer approaches to reveal that their causative agents are highly divergent lineages related to M. mackini (Paramikrocytos canceri n. gen. et n. sp. and M. mimicus sp. nov., respectively). We demonstrate a major new globally distributed parasite radiation (Mikrocytida ord. nov.) with phylogenetic affinities to the commercially important haplosporidian parasites of invertebrates. Mikrocytids have eluded detection because of their small size, intracellular habit, and extreme sequence divergence. P. canceri was frequently detected in a range of shoreline invertebrates, demonstrating that these newly recognized parasites are in fact common, diverse, and widespread and should be considered when assessing the risks of aquaculture activities, invasive species spread, and movements of ballast water and sediments with associated invertebrates.

The British river of the future: how climate change and human activity might affect two contrasting river ecosystems in England.

The possible effects of changing climate on a southern and a north-eastern English river (the Thames and the Yorkshire Ouse, respectively) were examined in relation to water and ecological quality throughout the food web. The CLASSIC hydrological model, driven by output from the Hadley Centre climate model (HadCM3), based on IPCC low and high CO(2) emission scenarios for 2080 were used as the basis for the analysis. Compared to current conditions, the CLASSIC model predicted lower flows for both rivers, in all seasons except winter. Such an outcome would lead to longer residence times (by up to a month in the Thames), with nutrient, organic and biological contaminant concentrations elevated by 70-100% pro-rata, assuming sewage treatment effectiveness remains unchanged. Greater opportunities for phytoplankton growth will arise, and this may be significant in the Thames. Warmer winters and milder springs will favour riverine birds and increase the recruitment of many coarse fish species. However, warm, slow-flowing, shallower water would increase the incidence of fish diseases. These changing conditions would make southern UK rivers in general a less favourable habitat for some species of fish, such as the Atlantic salmon (Salmo salar). Accidental or deliberate, introductions of alien macrophytes and fish may change the range of species in the rivers. In some areas, it is possible that a concurrence of different pressures may give rise to the temporary loss of ecosystem services, such as providing acceptable quality water for humans and industry. An increasing demand for water in southern England due to an expanding population, a possibly reduced flow due to climate change, together with the Water Framework Directive obligation to maintain water quality, will put extreme pressure on river ecosystems, such as the Thames.

Gyrodactylus quadratidigitus n. sp. (Monogenea: Gyrodactylidae), a parasite of the leopard-spotted goby Thorogobius ephippiatus (Lowe) from the south-western coast of the UK.

Gyrodactylus quadratidigitus n. sp. (Monogenea, Gyrodactylidae) is described from the gills, fins and skin of Thorogobius ephippiatus (Lowe) (leopard-spotted goby) from Portland Castle Bay, Dorset, UK. G. quadratidigitus n. sp. most closely resembles the species of Gyrodactylus previously described by Geets from Pomatoschistus microps (Krøyer) in the approximate shape and size of the attachment hooks but can be readily distinguished from other species of the genus by the distinctive square shape of the toe region of the marginal hook sickle, the anteriorly positioned cirrus bulb and the unusually short intestinal crura which extend only as far as the level of the testes.