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In this study, we demonstrate how stable carbon (C) and nitrogen (N) isotope analysis can be used to estimate the allocation of specific dietary ingredients, incorporated into four artificially formulated feeds supplemented with varying amounts of dried seaweed Ulva armoricana (0, 50, 150 and 200 g/kg), to the production of gonads by Tripneustes gratilla over a 20‐week growth trial. Results indicate that U. armoricana is an important dietary ingredient for gonad production, accounting for an average of 37.33 ± 1.03% of the isotopic signal recorded for gonads at the end of the growth trial. This is significantly more than fish meal and soya, which only contributed an average of 11.40 ± 1.81 and 3.80 ± 0.09%, respectively, of the isotopic signal of the gonads. Maize and wheat each account for almost 25% of all the dietary ingredients used to formulate the feeds, but their relative contribution to gonad production only averaged 8.55 ± 0.58 and 3.80 ± 0.05%, respectively, among the diets. This indicates that, at least for gonad development, these ingredients may not be required at such high levels. These findings support previous data on the importance of Ulva in abalone and urchin diets, and demonstrate the value of stable isotope analysis for assessing the contribution of specific dietary ingredients in new feed formulations, particularly where growth of specific tissues is being investigated.

A large dinoflagellate bloom in Walker Bay (South Africa) in January 2017 impacted 3 land-based abalone farms resulting in the death of several million animals. Satellite-derived images of Chl-a from the Ocean and Land Colour Imager (OLCI) on board the European Space Agency Sentinel-3 A showed bloom initiation in late December 2016 and dispersal in mid-February 2017. The bloom was dominated by two dinoflagellate species identified by light microscopy as Gonyaulax spinifera (Claparede & Lachmann) Diesing, 1866 and Lingulodinium polyedrum (Stein) Dodge, 1989. These morphologically based identifications were confirmed by phylogenetic analysis using partial sequences of the large subunit rDNA of both dinoflagellates. The appearance of yessotoxins (YTX) in abalone clearly coincided with increases in dinoflagellate concentrations. Yessotoxins in both the plankton and abalone were dominated by the two analogues homo-YTX and 45-hydroxy-YTX. The absence of toxins in a clonal culture of L. polyedrum implicated G. spinifera as the likely source of YTX. Toxin concentrations were found to be highest in the gills which showed the most significant pathology, including severe, generalized disruption of the gill epithelium characterized by degeneration and necrosis of epithelial cells accompanied by a modest inflammatory response. Some farms undertook pre-emptive or emergency harvesting to reduce financial losses.

The dusky kob Argyrosomus japonicus is an emerging aquaculture species in South Africa. To support industry growth, existing hatcheries frequently sell or donate excess eggs to neighbouring farms, raising concern over the potential transfer of infectious disease agents. Chemical disinfectants are frequently used to prevent transmission of diseases, but their application has shown large variations between finfish species and very little is known about the efficacy and impact of disinfectants on dusky kob eggs or larvae. A thorough understanding of the bacterial communities associated with specific fish species can inform the need for specific chemical agents and guide a more targeted approach for disease control and treatment. Therefore, this study used next generation sequencing (NGS, 16S rDNA gene) and downstream bioinformatics analysis to describe the bacterial microbiome associated with the eggs and rearing water of dusky kob. Four chemical disinfectants [chloramine-T (C-T), povo‑iodine (P I), hydrogen peroxide (H2O2) & formaldehyde (CH2O)], applied at different concentrations and times, were then tested for their ability to disinfect eggs and seawater. The impact of each disinfectant on early larval development was also assessed. A total of 11,605 individual bacterial operational transcriptional units (OTUs) were identified from the dusky kob egg (7352 OTUs) and hatchery water (4253 OTUs) samples. Non-metric multidimensional scaling (NMDS) analysis revealed distinct differences in the composition of the microbiota associated with the egg and seawater samples. A moderate level of bacterial diversity was observed in all samples, with the seawater samples dominated by Rhodobacteraceae (65%) and Flavobacteriaceae (14%); whereas egg samples were dominated by Pseudoalteromonadaceae (49%), Vibrionaceae (27%) and Colwelliaceae (8%). A classical univariate statistical comparison identified 6 differentially abundant OTUs at phylum level, 12 at family level and 19 at genus level; with members of the family Vibrionaceae found to be significantly (P = 0.0011) more abundant on dusky kob eggs samples. All tested chemical disinfectants were capable of reducing the bacterial load in seawater in a dose dependent manner, with the number of culturable bacteria on eggs decreasing significantly following exposure to P I and CH2O for five minutes (43% and 88% reduction in CFUs, respectively), compared with C-T and H2O2 (3% & 10%). Similarly, P I and CH2O significantly decreased the culturability of Vibrio species on eggs (79% & 75%), compared with C-T and H2O2 (12% & 14%). None of the disinfectants appeared to have any adverse impact on larval growth and morphology.

Abalone Haliotis midae exhibiting typical clinical signs of tubercle mycosis were discovered in South African culture facilities in 2006, posing a significant threat to the industry. The fungus responsible for the outbreak was identified as a Peronosporomycete, Halioticida noduliformans. Currently, histopathology and gross observation are used to diagnose this disease, but these 2 methods are neither rapid nor sensitive enough to provide accurate and reliable diagnosis. Real-time quantitative PCR (qPCR) is a rapid and reliable method for the detection and quantification of a variety of pathogens, so therefore we aimed to develop a qPCR assay for species-specific detection and quantification of H. noduliformans. Effective extraction of H. noduliformans genomic DNA from laboratory grown cultures, as well as from spiked abalone tissues, was accomplished by grinding samples using a pellet pestle followed by heat lysis in the presence of Chelax-100 beads. A set of oligonucleotide primers was designed to specifically amplify H. noduliformans DNA in the large subunit (LSU) rRNA gene, and tested for cross-reactivity to DNA extracted from related and non-related fungi isolated from seaweeds, crustaceans and healthy abalone; no cross-amplification was detected. When performing PCR assays in an abalone tissue matrix, an environment designed to be a non-sterile simulation of environmental conditions, no amplification occurred in the negative controls. The qPCR assay sensitivity was determined to be approximately 0.28 pg of fungal DNA (~2.3 spores) in a 25 µl reaction volume. Our qPCR technique will be useful for monitoring and quantifying H. noduliformans for the surveillance and management of abalone tubercle mycosis in South Africa.

The use of probiotics in aquaculture is proving to be highly effective in improving disease resistance, nutrition and/or growth of cultured organisms. We have shown that Haliotis midae fed a probiotic-supplemented diet have an improved survival and growth rate compared to animals not fed probiotics. The growth rate of small (20 mm) and large (67mm) abalone was improved by 8% and 34%, respectively, in two separate eight-month farm growth trials. Furthermore, the number, and phagocytic activity, of circulating haemocytes was significantly higher (P

Heavy metals, such as copper, zinc and cadmium, represent some of the most common and serious pollutants in coastal estuaries. In the present study, we used a combination of linear and artificial neural network (ANN) modelling to detect and explore interactions among low-dose mixtures of these heavy metals and their impacts on fundamental physiological processes in tissues of the Eastern oyster, Crassostrea virginica. Animals were exposed to Cd (0.001-0.400 microM), Zn (0.001-3.059 microM) or Cu (0.002-0.787 microM), either alone or in combination for 1 to 27 days. We measured indicators of acid-base balance (hemolymph pH and total CO(2)), gas exchange (Po(2)), immunocompetence (total hemocyte counts, numbers of invasive bacteria), antioxidant status (glutathione, GSH), oxidative damage (lipid peroxidation; LPx), and metal accumulation in the gill and the hepatopancreas. Linear analysis showed that oxidative membrane damage from tissue accumulation of environmental metals was correlated with impaired acid-base balance in oysters. ANN analysis revealed interactions of metals with hemolymph acid-base chemistry in predicting oxidative damage that were not evident from linear analyses. These results highlight the usefulness of machine learning approaches, such as ANNs, for improving our ability to recognize and understand the effects of sub-acute exposure to contaminant mixtures.

The Eastern oyster, Crassostrea virginica , inhabits shallow coastal waters that frequently experience periods of low dissolved oxygen (hypoxia) and elevated CO 2 (hypercapnia) levels. Bacteria are extremely abundant in these environments and accumulate in large numbers in filter-feeding oysters, which can act as passive carriers of human pathogens. Although hypercapnic hypoxia (HH) can affect certain specific immune mechanisms, its direct effect on the inactivation, degradation and elimination of bacteria in oysters is unknown. This research was conducted to determine whether exposure to HH reduces the ability of C. virginica to inactivate and eliminate Vibrio campbellii following its injection into the adductor muscle. Oysters were held in fully air-saturated (normoxic; partial O 2 pressure [P O2 ] = 20.7 kPa, CO 2 < 0.06 kPa, pH 7.8 to 8.0) or HH (P O2 = 4 kPa, CO 2 = 1.8 kPa, pH 6.5 to 6.8) seawater at 25°C for 4 h before being injected in the adductor muscle with 10 5 live Vibrio campbellii bacteria and remained under these conditions for the remainder of the experiment (up to 24 h postinjection). Real-time PCR was used to quantify the number of intact V. campbellii bacteria, while selective plating was used to quantify the number of injected bacteria remaining culturable in whole-oyster tissues, seawater, and feces/pseudofeces at 0, 1, 4, and 24 h postinjection. We found that oysters maintained under normoxic conditions were very efficient at inactivating and degrading large numbers of injected bacteria within their tissues. Moreover, a small percentage (∼10%) of injected bacteria were passed into the surrounding seawater, while less than 1% were recovered in the feces/pseudofeces. In contrast, HH increased the percentage of culturable bacteria recovered from the tissues of oysters, suggesting an overall decrease in bacteriostasis. We suggest that poor water quality may increase the risk that oysters will harbor and transmit bacterial pathogens hazardous to human and ecosystem health.

The Atlantic blue crab, Callinectes sapidus (Rathbun), lives in a bacteria-rich environment that experiences daily fluctuations in water quality. In the present study, we tested the hypothesis that crustaceans with prior or ongoing exposure to bacteria in their hemolymph have an increased susceptibility to subsequent infections, and that acute exposure to low dissolved oxygen (hypoxia) and elevated carbon dioxide levels (hypercapnia) may further confound the ability of blue crabs to counter a subsequent infection. Adult male blue crabs held in well-aerated (normoxic; P O2=20.7 kPA; CO(2)

Viable cell counts and/or in situ hybridization were used to determine whether the probionts Vibrio midae SY9, Cryptococcus sp. SS1, and Debaryomyces hansenii AY1 can colonize the gastrointestinal tract of the South African abalone Haliotis midae. The number of culturable probiotic cells reisolated from H. midae fed probiotic-supplemented feed for 3 weeks ranged from 106 to 107 cfu/g gut material. A significant decrease (P < 0.05) in probiont numbers 2 days after feeding the probiotic-supplemented feed had been halted correlated with a significant decrease (P < 0.05) in intestinal protease and amylase activity. There was a positive correlation between Cryptococcus sp. SS1 and amylase activity (r2 = 0.681) and V. midae SY9.8 and protease activity (r2 = 0.711) in the H. midae intestine. Although culturable probionts were isolated from abalone that had not been fed probiotic-supplemented feed for a 2-week period, the drop in the number of probiotic cells colonizing the abalone digestive tract 2 days after feeding with the probiotic-supplemented feed had been halted indicates that farmed abalone should be fed probiotic-supplemented feed at least every second day for maximum benefit.