Resolving the identity of commercially cultivated Ulva (Ulvaceae, Chlorophyta) in integrated seaweed-abalone aquaculture farms in South Africa.

Species of Ulva have a wide range of commercial applications and are increasingly being recognized as promising candidates for integrated aquaculture. In South Africa, Ulva has been commercially cultivated in integrated seaweed-abalone aquaculture farms since 2002, with more than 2000 tonnes of biomass cultivated per annum in land-based paddle raceways. However, the identity of the species of Ulva grown on these farms remains uncertain. We therefore characterized samples of Ulva cultivated in five integrated multi-trophic aquaculture farms (IMTA) across a wide geographical range and compared them with foliose Ulva specimens from neighboring seashores. The molecular markers employed for this study were the chloroplast-encoded Ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL), the Internal Transcribed Spacer (ITS) of the nuclear, and the chloroplast elongation factor tufA. All currently cultivated specimens of Ulva were molecularly resolved as a single species, U. lacinulata. The same species has been cultivated for over a decade, although a few specimens of two other species were also present in early South African IMTA systems. The name Ulva uncialis is adopted for the Ulva "Species A" by Fort et al. (2021), Molecular Ecology Resources, 22, 86) significantly extending the distribution range for this species. A comparison with wild Ulva on seashores close to the farms resulted in five new distribution records for South Africa (U. lacinulata, U. ohnoi, U. australis, U. stenophylloides, and U. aragoënsis), the first report of a foliose form of U. compressa in the region, and one new distribution record for Namibia (U. australis). This study reiterates the need for DNA confirmation, especially when identifying morphologically simple macroalgae with potential commercial applications.

Effects of chronic hypercapnia and elevated temperature on the immune response of the spiny lobster, Jasus lalandii.

The West Coast rock lobster (WCRL), Jasus lalandii, inhabits highly variable environments frequented by upwelling events, episodes of hypercapnia and large temperature variations. Coupled with the predicted threat of ocean acidification and temperature change for the coming centuries, the immune response in this crustacean will most likely be affected. We therefore tested the hypothesis that chronic exposure to hypercapnia and elevated seawater temperature will alter immune function of the WCRL. The chronic effects of four combinations of two stressors (seawater pCO2 and temperature) on the total number of circulating haemocytes (THC) as well as on the lobsters' ability to clear (inactivate) an injected dose of Vibrio anguillarum from haemolymph circulation were assessed. Juvenile lobsters were held in normocapnic (pH 8.01) or hypercapnic (pH 7.34) conditions at two temperatures (15.6 and 18.9 °C) for 48 weeks (n = 30 lobster per treatment), after which a subsample of lobsters (n = 8/treatment), all at a similar moult stage, were selected from each treatment for the immune challenge. Baseline levels of haemocytes (THC ml-1) and bacteria (CFU ml-1) in their haemolymph were quantified 24 h prior to bacterial challenge. Lobsters were then challenged by injecting 4 × 104V. anguillarum per g body weight directly into the cardiac region of each lobster and circulating haemocyte and culturable bacteria were measured at 20 min post challenge. No significant differences in THC ml-1 (p < 0.05) were observed between any of the treatment groups prior to the bacterial challenge. However lobsters chronically exposed to a combination of hypercapnia and low temperature had significantly higher (p < 0.05) THCs post-challenge in comparison with lobsters chronically exposed to hypercapnia and high temperature. A significant interactive effect was recorded between temperature and pH for the post-challenge THC data (two-way ANOVA, p = 0.0025). Lobster were very efficient at rendering an injected dose of bacteria non-culturable, with more than 83% of the theoretical challenge dose (∼1.7 × 105Vibrio ml-1 haemolymph) inactivated within the first 10 min following injection. Although differences in the inactivation of V. anguillarum were observed between treatment groups, none of these differences were significant. Clearance efficiency was in the following order: Hypercapnia/low temperature > normocapnia/high temperature > normocapnia/low temperature > hypercapnia/high temperature. This study demonstrated that despite chronic exposure to combinations of reduced seawater pH and high temperature, the WCRL was still capable of rapidly rendering an injected dose of bacteria non-culturable.

Devastating farmed abalone mortalities attributed to yessotoxin-producing dinoflagellates.

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 (Claparède & 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.

Development and preliminary evaluation of a real-time PCR assay for Halioticida noduliformans in abalone tissues.

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.

Modelling interactions of acid-base balance and respiratory status in the toxicity of metal mixtures in the American oyster Crassostrea virginica.

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.

Differential localization and bacteriostasis of Vibrio campbellii among tissues of the Eastern oyster, Crassostrea virginica.

In bivalve mollusks the roles of individual tissues in antimicrobial defense remain unclear. In this study, Crassostrea virginica were injected in the adductor muscle with 10(5) live Vibrio campbellii. Major tissues were dissected at 10, 30, 60 or 120 min postinjection (PI); in each tissue undegraded (intact) bacteria were quantified by real-time PCR and culturable bacteria were enumerated by selective plating. At 10 min PI, accumulation of bacteria varied among tissues from approximately 2.4 x 10(3) (labial palps, digestive gland) to 24.2 x 10(3) (gonads) intact Vibrio g(-1). Neither distribution nor accumulation of intact bacteria changed with time except in the hemolymph. In most tissues, more than 80% of intact bacteria were culturable at 10 min PI and culturability decreased with time. In contrast, only 19% of intact bacteria in gonadal tissue could be cultured at 10 min PI, pointing to a major role for the gonadal tissues in antibacterial defense of molluscs.

Clearance of Vibrio campbellii injected into the hemolymph of Callinectes sapidus, the Atlantic blue crab: the effects of prior exposure to bacteria and environmental hypoxia.

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)<0.06 kPa; pH 7.8-8.0) or hypercapnic hypoxic (HH; P O2=4 kPa; CO(2)=1.8 kPa; pH 6.9-7.2) seawater received an injection (pre-challenge dose) of 1 x 10(5)Vibrio campbellii g(-1) crab. Control animals were injected with an equivalent dose of HEPES-buffered saline (1 microl g(-1) crab). At 2h or 24h after the pre-challenge injection, both Vibrio and saline-pre-challenged animals were injected with a dose of live V. campbellii (1 x 10(5)g(-1) crab). This second injection will be referred to as a second injection or challenge injection. Degradation in or physical removal of intact bacteria from hemolymph was quantified using real-time PCR; bacteriostasis was quantified as the percentage of intact bacteria that could not be recovered by selective plating. We demonstrated that bacteriostasis occurs in the hemolymph of blue crabs. Furthermore, blue crabs that received a challenge injection 2h after a pre-challenge dose of V. campbellii cleared culturable bacteria from their hemolymph more rapidly when compared to animals that received a pre-challenge dose of saline. This enhanced clearance of culturable bacteria was associated with an increase in antibacterial activity in the cell-free hemolymph. However, the enhanced clearance of culturable bacteria disappeared when the time interval between the pre-challenge and challenge dose was extended to 24h and when crabs were held in HH seawater throughout the experiment. Neither the time interval between the pre-challenge and the challenge dose nor exposure to HH altered the pattern of intact bacterial clearance in blue crabs. These results demonstrate that prior exposure to bacteria does not increase the susceptibility of C. sapidus to a second, sublethal dose of V. campbellii. In fact, a recent exposure to V. campbellii enhances the ability of blue crabs to render bacteria non-culturable and the immune mechanisms/effectors responsible for this are short lived and appear to be sensitive to low dissolved oxygen and high carbon dioxide concentrations in the environment.

Effects of hypercapnic hypoxia on inactivation and elimination of Vibrio campbellii in the Eastern oyster, Crassostrea virginica.

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 degrees 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 ( approximately 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.

Colonization of the gastrointestinal tract of the farmed South African abalone Haliotis midae by the probionts Vibrio midae SY9, Cryptococcus sp. SS1, and Debaryomyces hansenii AY1.

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 10(6) to 10(7) 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.