Sensitivity and specificity of real‐time PCR and bacteriological culture for francisellosis in farm‐raised Nile tilapia (Oreochromis niloticus L.)

Despite the worldwide occurrence of Francisella noatunensis subsp. orientalis (Fno) infection in farmed tilapia, sensitivity and specificity estimates of commonly used diagnostic tests have not been reported. This study aimed to estimate the sensitivity and specificity of bacteriological culture and qPCR to detect Fno infection. We tested 559 fish, sampled from four farms with different epidemiological scenarios: (i) healthy fish in a hatchery free of Fno; (ii) targeted sampling of diseased fish with suggestive external clinical signs of francisellosis during an outbreak; (iii) convenience sampling of diseased and clinically healthy fish during an outbreak; and (iv) sampling of healthy fish in a cage farm without a history of outbreaks, but with francisellosis reported in other farms in the same reservoir. The qPCR had higher median sensitivity (range, 48.8–99.5%) than culture (range, 1.6–74.4%). Culture had a substantially lower median sensitivity (1.6%) than qPCR (48.8%) to detect Fno in carrier tilapia (farm 4). Median specificity estimates for both tests were >99.2%. The qPCR is the superior test for use in surveillance and monitoring programmes for francisellosis in farmed Nile tilapia, but both tests have high sensitivity and specificity which make them fit for use in the diagnosis of Fno outbreaks.     

Donor effect on cultured pearl nacre development and shell matrix gene expression in Pinctada margaritifera reared in different field sites

Understanding the respective roles played by donor and recipient pearl oysters in pearl quality determination in relation to the environment is a challenge for the pearl industry. In most Pinctada species, pearl size is mainly related to recipient oyster growth performance but also relies to some extent on the biomineralisation activity of the pearl sac, a tissue that originates from the donor oyster mantle. We examined donor effect on pearl size in response to culture in the lagoons on Arutua and Apataki atolls. Overall, nacre weight and thickness were greater in Arutua than in Apataki, but sensitivity to the environment differed between donors. Some donors were associated with significantly heavier and thicker nacre in Arutua (I group), while others had similar results at the two sites (NI group). On average, up to 20% of the pearl size could be attributed to the donor but, in group I, donor effect was responsible for up to 36% of nacre weight determination. Additionally, a real‐time PCR expression study of eight matrix protein genes related to biomineralisation in the pearl sac showed that MSI60, pearlin and pif177 were significantly and positively correlated with nacre weight and thickness, with the latter two genes explaining the larger pearl size observed in Arutua. Donor oysters in P. margaritifera therefore play a key role in pearl size improvement, related to the role of the shell matrix protein genes. Understanding such contributions could help in the design of genetic selection plans for specific and adapted donor oyster lines.     

Infection dynamics of Bonamia exitiosa on intertidal Ostrea angasi farms

Bonamia spp. cause epizootics in oysters worldwide. In southern Australia, Bonamia exitiosa Hine, Cochennac and Berthe, 2001 threatens aquaculture of Ostrea angasi Sowerby, 1871. Bonamia spp. infections can display strong seasonality, but seasonal dynamics of B. exitiosa–O. angasi are unknown. Ostrea angasi naïve to B. exitiosa infection were stocked onto farms in three growing regions, and B. exitiosa was monitored seasonally for one year. Environmental parameters we measured did not correlate with B. exitiosa prevalence or infection intensities. Extreme temperatures suggest O. angasi culture systems need development. Bonamia exitiosa prevalence increased over time. After three months, O. angasi had B. exitiosa prevalence of 0.08–0.4, and after one year, the prevalence was 0.57–0.88. At some sites, O. angasi had >0.5 B. exitiosa prevalence in >6 months, but at other sites, >9 months passed before prevalence was >0.5. Bonamia exitiosa infection intensities were low with no seasonal pattern but were affected by the interaction of site, season and oyster meat:shell ratio. Understanding infection and initiating a breeding programme for resistance would provide benefits for O. angasi industry expansion.     

Phenotypic indicators for cultured pearl size improvement in the black‐lipped pearl oyster (Pinctada margaritifera): towards selection for the recipient growth performance

The black‐lipped pearl oyster, Pinctada margaritifera, is the most important farmed species in French Polynesia and the basis of the most valuable export industry. Mass production of black pearls relies on a surgical operation requiring tissue from a donor pearl oyster to be grafted, together with a nucleus made of shell, into the gonad of a recipient oyster. Improving pearl size through family selection remains one of the main challenges for future aquaculture development. This study analyses the relative contribution of donor and recipient oysters to pearl size. To this end, hatchery‐produced donor oysters of two batches, large and small (based on shell height), were used to supply grafts for recipients, which were then monitored individually for their growth performance by recording shell height, width, and thickness, and total live weight (flesh + shells) every 6 months (four biometric measurement times) over 20 months of culture. Pearls issued from the two batches of donors showed no significant differences in nacre weight or thickness. In contrast, recipient oyster shell height and total weight were increasingly positively correlated with these pearl size parameters over the culture period, becoming significant at 8 months post‐grafting. Potential therefore exists to use shell height and oyster weight as phenotypic indicators for selective breeding of recipient oysters with high growth performance to increase pearl size in P. margaritifera.     

Siltation increases the susceptibility of surface‐cultured eastern oysters (Crassostrea virginica) to parasitism by the mudworm Polydora websteri

Mudworm (Polydora websteri) parasitism is known to result in unsightly mud blisters in eastern oysters (Crassostrea virginica), resulting in reduced product quality and increased vulnerability to illness and environmental stress. While typically a concern only for bottom‐grown oysters, an abnormal severe outbreak of P. websteri in surface‐cultured oysters in New Brunswick, Canada, was reported in 2013, along with an anecdotally reported concurrent increase in siltation. Although heavier loads of silt are reported to increase P. websteri infestations in oysters, studies report mixed effects of siltation to this regard. Here, we report the results of a field experiment testing the effect of siltation on P. websteri recruitment in surface‐grown oysters at an aquaculture site in Richibucto, New Brunswick. Live oysters of similar size were deployed at the aquaculture site and were left to collect P. websteri recruits under one of two siltation treatments (high vs. low) for 50 days. Results suggested that P. websteri recruitment correlated with metrics of oyster size (shell weight, length, width, depth and surface area). When P. websteri counts were standardized for oyster shell morphometry, P. websteri recruitment was significantly higher in oysters exposed to the high siltation treatment, accumulating approximately 1.5× as many P. websteri as oysters exposed to the low siltation treatment. Our results suggest that higher amounts of siltation on surface‐cultured oysters can result in increased rates of P. websteri parasitism. Enhanced cleaning regimes may help to alleviate the impacts of P. websteri in surface‐grown oysters, although other mitigation strategies exist.     

Seasonal Fouling Stress on the Farmed Pearl Oyster,Pinctada fucata,from Southeastern Arabian Sea

Biofouling on the periostracum of pearl oysters and on the cages has been considered as a stress factor causing mortality of the farmed stock, reducing growth rates and also affecting pearl quality. In farming experiments using the pearl oyster, Pinctada fucata, at Kollam Bay (India), biofouling was found to be a problem. This study was conducted to understand the effect of fouling on the mortality of pearl oysters kept in suspended culture, to identify the main foulers, the seasonal variation in biofouling and species successions in the community with reference to abiotic factors. The average monthly mortality rate (MR) was estimated as 0.117 ± 0.002 and the monthly variations were significantly different (P < 0.01). The total fouling (0.163 ± 0.002 g/g oyster) and biofouling weights (0.166 ± 0.007 g/g oyster) were high in December when the fouling community was composed of several species and dominated by the ascidian, Didemnum sp. December was also the period when the MRs peaked indicating that this organism was the main cause of mortality in pearl farms in Kollam Bay. A clear seasonality in the fouling community (25 species belonging to nine phyla) has been observed in the present study. On the basis of this study, monthly cleaning of oysters is advocated except during December, January, and March when the cleaning should be fortnightly.     

Parasites, pathological conditions and mortality in QX-resistant and wild-caught Sydney rock oysters, Saccostrea glomerata

Differences in the survival of QX-resistant fifth generation (QXR₅) and wild-caught (Wild-Caught) Sydney rock oysters were assessed over the QX-disease risk period in the Pimpama River, SE Queensland. Cumulative mortality of Wild-Caught oysters (31.7%) was significantly greater than QXR₅ oysters (0.0%) over the 118 days of the experiment. PCR and histological results showed that Wild-Caught oyster did not die from QX disease. Histology revealed oysters were infected with disseminating hemocytic neoplasia, a Steinhausia-like infection, a Rickettsia-like organism infecting epithelial cells of the gill, digenean flukes encysted in the gonadal tissue and a gill response to an unknown toxin. The cause of mortality is attributed to disseminating hemocytic neoplasia.     

Microzooplankton as a food source for the Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>: seasonal variation in gut contents and food availability

In this study I investigated the seasonal occurrence of microzooplankton in the gut of the Pacific oyster Crassostrea gigas. Gut contents consisted of various microzooplankton, including tintinnid ciliates, dinoflagellates (Dinophysis spp.), copepods, copepod nauplii, bivalve larvae, and rotifers. Monthly variations in the abundance of protists (tintinnids and Dinophysis spp.) in the gut contents were significantly correlated with the variation in their abundances in the surrounding seawater, whereas this relationship was not significant with metazoans (copepods, copepod nauplii, and bivalve larvae). In laboratory experiments, oysters actively fed on ciliates. When oysters fed on ciliates labeled with a stable nitrogen isotope (¹⁵N), the ¹⁵N:total N ratio of the oyster tissues was significantly higher than that for oysters under a no-food condition, suggesting that oysters assimilate nitrogen of the ciliates into their body tissues. Based on these results, I conclude that microzooplankton forms an important food source for oysters, especially when other food sources such as phytoplankton are not abundant.     

Bayesian estimation of diagnostic sensitivity and specificity of a qPCR and a bacteriological culture method for Piscirickettsia salmonis in farmed Atlantic salmon (Salmo salar L.) in Chile

Early detection of piscirickettsiosis is an important purpose of government- and industry-based surveillance for the disease in Atlantic salmon farms in Chile. Real-time qPCRs are currently used for surveillance because bacterial isolation is inadequately sensitive or rapid enough for routine use. Since no perfect tests exist, we used Bayesian latent class models to estimate diagnostic sensitivity (DSe) and specificity (DSp) of qPCR and culture using separate two-test, single-population models for three farms (n = 148, 151, 44). Informative priors were used for DSp (culture (beta(999,1); qPCR (beta(98,2)), and flat priors (beta 1,1) for DSe and prevalence. Models were run for liver and kidney tissues combined and separately, based on the presence of selected gross-pathological signs. Across all models, qPCR DSe was 5- to 30-fold greater than for culture. Combined-tissue qPCR median DSe was highest in Farm 3 (sampled during P. salmonis outbreak (DSe = 97.6%)) versus Farm 1 (DSe = 85.6%) or Farm 2 (DSe = 83.5%), both sampled before clinical disease. Median DSe of qPCR was similar for liver and kidney, but higher when gross-pathological signs were evident at necropsy. High DSe and DSp and rapid turnaround-time indicate that the qPCR is fit for surveillance programmes and diagnosis during an outbreak. Targeted testing of salmon with gross-pathological signs can enhance DSe.     

Two epizootic Perkinsus spp. events in commercial oyster farms at Santa Catarina,Brazil

Perkinsus spp. have been detected in various bivalve species from north‐east Brazil. Santa Catarina is a South Brasil state with the highest national oyster production. Considering the pathogenicity of some Perkinsus spp., a study was carried out to survey perkinsosis in two oyster species cultured in this State, the mangrove oyster Crassostrea gasar and the Pacific oyster Crassostrea gigas. Sampling involved eight sites along the state coast, and oyster sampling was collected during the period between January 2013 and December 2014. For the detection of Perkinsus, Ray's fluid thioglycollate medium (RFTM) and histology were used, and for the identification of the species, PCR and DNA sequencing were used. Perkinsus spp. was found by RFTM in C. gigas and C. gasar from São Francisco do Sul. This pathology was also detected in C. gasar from Balneário Barra do Sul both, by RFTM and histology. Perkinsus marinus was identified in C. gigas and C. gasar from São Francisco do Sul and Perkinsus beihaiensis in C. gasar from Balneário Barra do Sul. This is the first report of P. marinus in C. gigas from South America. Results of this preliminary study suggest that both oyster species tolerate the species of Perkinsus identified, without suffering heavy lesions.     

The effects of stressors on the dynamics of Bonamia exitiosus Hine, Cochennec-Laureau & Berthe, infections in flat oysters Ostrea chilensis (Philippi)

Oysters, Ostrea chilensis, infected with Bonamia exitiosus were held under stressful conditions for 14 days, and then under normal conditions for 3.5 months, to determine the affects of stress on B.exitiosus infection levels. The stressors used were (1) air exposure for 8 h daily; (2) hot water (25–26 °C), or (3) cold water (7 °C) for 1 h daily; (4) hyposaline (15‰), or (5) hypersaline (39–40‰) water; (6) starvation in filtered sea water; and (7) vigorous stirring four times a day. A control tank held oysters in static sea water, which was changed daily. Oysters were also (8) kept in a trough among heavily infected oysters (trough exposure), or (9) in a trough without other oysters (control), for 4 months. Oysters in hyposaline conditions all died within 3 weeks, but this was apparently unrelated to Bonamia infection. Otherwise, cumulative mortalities were highest in trough exposure, cold, hypersaline and hot treatments. There was a significant difference in prevalence between treatments, and the stir, hot, cold, hypersalinity and trough exposure treatments had significantly higher intensities of infection than controls. The mean intensity of Bonamia infection was significantly higher among female and spent oysters than in male and hermaphrodite oysters.     

Technical and economic feasibility of integrating seahorse culture in shrimp/oyster farms

Integrated multi‐trophic aquaculture systems (IMTA) are building ecosystems designed to produce aquatic organisms with less environmental impact. We have developed a simple system and a management strategy for introducing seahorse (Hippocampus spp.) culture in shrimp/oyster farms to increase economic sustainability. The system includes broodstock, nursery and grow‐out phases of Hippocampus reidi G. culture. We performed a test in a 42.4‐ha marine shrimp/oyster farm, which showed that this system is technically feasible. Then, we performed a partial budget analysis including cost‐return, cash flow and financial feasibility analysis. More than 12 000 marketable size seahorses may be produced in 402 m² annually. Initial investment was only US$ 21 103.00. The benefit–cost ratio was ~US$ 20.00 for each US$ 1.00 spent, internal rate of return was 131% and payback period <2 years. In conclusion, the introduction of seahorse in shrimp/oyster IMTA system is technically feasible, profitable, resilient and shows high liquidity. This system may be easily established in developing countries and has potential to provide seahorses to supply different markets (live specimens for the marine aquarium trade and dried specimens for traditional Chinese medicine and the curio trade). In addition, it can indirectly contribute to preserve natural populations.     

Effects of temperature and photoperiod on the conditioning of the flat oyster (Ostrea edulis L.) in autumn

The production of the flat oyster Ostrea edulis (L.) natural spat in Europe has decreased almost by 60% in the past ten years. Thus, the importance of the production of oyster spat in hatcheries is evident. One of the critical steps in hatchery production is broodstock conditioning, especially difficult in autumn, when gonadal development is in resting period. Conditioning is influence by temperature, photoperiod and nutrition. In this work, the effects of two temperature and three photoperiod regimes on the conditioning of O. edulis were studied for three years by stereological analyses and registering number and dates of spawning and larval yield. Temperature had a positive effect on the gonadal development of O. edulis during conditioning. The percentages of germinal cells in oysters conditioned with a gradient of temperature (14–18°C) were double compared to oysters conditioned at 15°C. Oysters conditioned with longer photoperiods showed higher percentages of germinal cells. There was no interaction between temperature and photoperiod. Spawning was observed in the oysters treated with daylight (8–16 h) ten weeks from the beginning of conditioning. Flat oysters conditioned with 8 h and 8–12 h of daylight delayed the first spawning for a month. Total larval production was higher in the oysters treated with the longest daylight gradient. Gonadal and gametogenic development was a non‐synchronic process and the spawning extended for around two months. A protocol for flat oyster broodstock conditioning in autumn by using both a gradient of temperature (14–18°C) and daylight (8–16 h) is proposed.     

Pearl grafting: Tracking the biological origin of nuclei by straightforward immunological methods

French Polynesia is renowned for the production of Tahitian black pearl. These gems are obtained by grafting a nucleus into the gonad of a receiving oyster together with a graft, i.e. a small section of mantle tissue of a donor oyster. This procedure initiates the formation of a pearl sack around the nucleus, and subsequently, the deposition of concentric layers of nacre. The nucleus plays a key‐role in pearl formation and its characteristics influence markedly the quality of the final product. As it is manufactured from mollusc shells, it contains a small percentage of organics. In the present paper, we used a set of biochemical techniques to characterize and compare the organic matrices from two types of nuclei that are currently used in French Polynesia: that from the freshwater mussel Amblema sp., and that from the pearl oyster Pinctada sp. To this end, we extracted the matrices from nuclei and performed FT‐IR, monodimensional electrophoresis, and enzyme‐linked immuno‐sorbent assay (ELISA). Our data show that the matrix associated with Amblema nuclei has a very different biochemical signature from that of Pinctada nuclei, a fact that may explain the improved tolerance of grafted oysters to nuclei of Pinctada origin. In the absence of complex physical methods of investigation, simple immunological techniques and FT‐IR performed on the extracted organic matrix are extremely reliable and effective for discriminating nuclei from these two sources. We assert that such techniques can be used as a diagnostic test to track unambiguously the biological origin of nuclei to avoid fraud.     

The capacity of oysters to regulate energy metabolism‐related processes may be key to their resilience against ocean acidification

Bivalve molluscs, such as oysters, are threatened by shifts in seawater chemistry resulting from climate change. However, a few species and populations within a species stand out for their capacity to cope with the impacts of climate change‐associated stressors. Understanding the intracellular basis of such differential responses can contribute to the development of strategies to minimise the pervasive effects of a changing ocean on marine organisms. In this study, we explored the intracellular responses to ocean acidification in two genetically distinct populations of Sydney rock oysters (Saccostrea glomerata). Selectively bred and wild type oysters exhibited markedly different mitochondrial integrities (mitochondrial membrane potential) and levels of reactive oxygen species (ROS) in their hemocytes under CO₂ stress. Analysis of these cellular parameters after 4 and 15 days of exposure to elevated CO₂ indicated that the onset of intracellular responses occurred earlier in the selectively bred oysters when compared to the wild type population. This may be due to an inherent capacity for increased intracellular energy production or adaptive energy reallocation in the selectively bred population. The differences observed in mitochondrial integrity and in ROS formation between oyster breeding lines reveal candidate biological processes that may underlie resilience or susceptibility to ocean acidification. Such processes can be targeted in breeding programs aiming to mitigate the impacts of climate change on threatened species.     

Broodstock conditioning of the Portuguese oyster (Crassostrea angulata,Lamarck, 1819): influence of different diets

The Portuguese oyster Crassostrea angulata shows great potential in oyster farming. The conservation of pure populations of this species is important for production diversification and biodiversity preservation. In this way, the zootechnological development for seed hatchery production is extremely important. Broodstock conditioning is a key step in the process of rearing bivalves in a hatchery. Many factors regulate the reproductive cycle, being food one of the most important ones. To evaluate the effect of different diets on C. angulata reproductive performance, broodstock were conditioned with different food regimes formulated fundamentally by flagellates (Diet 1 – Pavlova lutheri and Isochrysis galbana clone T‐ISO; Diet 2 – P. lutheri, T‐ISO and Skeletonema costatum) and constituted fundamentally by diatoms (Diet 3 – S. costatum and Chaetoceros calcitrans; Diet 4 – P. lutheri, S. costatum and C. calcitrans). During conditioning, samples of oysters were collected to evaluate condition index, gonadal development and biochemical composition. At the end of the conditioning period, oysters were induced to spawn to evaluate reproductive output (fecundity, fertilization rate and D‐larvae development). The diets had an impact on the gametogenesis process, energy storage and reproductive output performance, being the best results those obtained in broodstock fed with the diatoms‐predominant diets. However, those fed with diets majority flagellates had an unsuccessful performance. Holistic approaches incorporating all results in this study reveal and reinforce the idea that the diatom species used presented the nutritional requirements to C. angulata broodstock, being essential in the conditioning phase.     

3种螺对养殖鲍外壳附着牡蛎的防除作用

为研究养殖皱纹盘鲍(Haliotis discus hannai Ino)外壳附着牡蛎的防除方法,对比了疣荔枝螺(Thais clavigera Kuster)、润泽角口螺(Ceratostoma rorifluum)和甲虫螺(Cantharus cecillei)对鲍外壳表面牡蛎的防除效果。以桑沟湾筏式养殖鲍为研究对象,自7月中旬开始,每隔15 d左右分别投放3种螺到鲍养殖笼中,共投放6次,以始终未投放螺的养殖笼为对照组。结果显示,实验结束时甲虫螺组中鲍壳上平均存活牡蛎数目为(0.04±0.04)个/只,疣荔枝螺组和润泽角口螺组疣荔枝螺组分别为(2.49±0.91)个/只和(2.21±1.05)个/只,对照组为(3.33±0.46)个/只。投放3种螺组中鲍壳上存活牡蛎数均显著显著低于对照组(P<0.05),甲虫螺也显著低于其他2种螺(P<0.05)。投螺时间也会影响螺防除牡蛎污损的效果,投放时间过晚会导致螺捕食牡蛎后有明显的壳残留,牡蛎的壳高达到0.5 cm左右再投放螺即可保证防除效果。另外甲虫螺组鲍壳上死亡牡蛎残留壳长度为(0.73±0.27) cm,显著低于疣荔枝螺组...     

Insights on the association between somatic aneuploidy and ostreid herpesvirus 1 detection in the oysters Crassostrea gigas,C. angulata and their F1 hybrids

Cytogenetic abnormalities associated with viral infections, including from viruses of the Herpesvirales order, have been reported in vertebrate species. Ostreid herpesvirus 1 (OsHV‐1) has been detected worldwide during mortality outbreaks of the Pacific oyster Crassostrea gigas. On the other hand, a high proportion of aneuploid cells in somatic tissues have been observed in C. gigas. In this study, we analysed the putative association between aneuploidy levels and the detection of OsHV‐1 in gills of C. gigas, the Portuguese oyster C. angulata and their F1 hybrids cultured in Ria Formosa (Portugal). OsHV‐1 was detected by PCR in 5.4% of the total of oysters analysed (n = 111) namely in 11.1%, 8.0% and 1.7% of C. gigas, C. angulata and F1 hybrid respectively. Sequencing analysis of a viral fragment amplified with the C2/C6 primer pair revealed a high similarity with the OsHV‐1 reference type. Moreover, in situ hybridization confirmed the presence of OsHV‐1 in gill tissue. Oysters where OsHV‐1 was detected had a significantly higher mean percentage of aneuploid cells (25%) than the ones where the virus was not detected (18%). However, the overall low percentage of positive samples contrasted with the high mean percentage of aneuploidy observed, with 50% of the oysters analysed showing a percentage of aneuploid cells between 20% and 30%. We hypothesize that somatic aneuploidy may adversely affect oysters making them more prone to OsHV‐1 infection, but the virus is unlikely to be the cause of somatic aneuploidy.