Herpes-like virus infections in hatchery-reared bivalve larvae in Europe: specific viral DNA detection by PCR

Periodic losses in oyster hatcheries are regularly reported in Europe. Herpes-like virus infections seem to play a key role. Polymerase chain reaction (PCR) was used to investigate the presence of herpes-like virus DNA in larval samples belonging to different bivalve species from different geographical origins. Seventeen samples of the 81 analysed appeared positive for the herpes-like virus DNA by PCR. These results confirm previous data indicating that herpes-like virus infections occur in commercial French hatcheries. Polymerase chain reaction positive results were also obtained for bivalve larval samples originating from Spain and the UK. The number of virus DNA positive samples depended on the primer pair used. The primer pair C2/C6 appears well adapted for herpes-like virus DNA detection because of processing ease and great sensitivity. Positive samples were observed in four bivalve species: Crassostrea gigas , Ostrea edulis , Ruditapes decussatus and Ruditapes philippinarum . Herpes-like virus DNA detection is reported in larval R. decussatus for the first time. Many samples in which viral DNA was detected by PCR correspond to larval batches presenting mortalities. Herpes-like viruses may be one of the causative agents of mortalities observed in bivalve hatcheries.     

Identification and counting of Pacific oyster Crassostrea gigas larvae by object detection using deep learning

Natural seedling collection is widely used in the culture of various bivalve species. For successful natural seedling collection, collectors must be installed when larvae appear in the water column at a stage immediately before attachment. Aquaculture farmers generally identify target larvae by morphological features through microscopic examination in a time- and labor-expensive exercise, which also requires a level of expertise to ensure accurate larval identification. We develop a deep-learning-based object-detection technique that ultimately might reduce the time and effort required to accurately identify and count Pacific oyster larvae, render their identification more consistent, and negate the need for expertise. Images of plankton net samples collected in Matsushima and Sendai bays, Japan, were taken using a new photographic device with a CMOS image sensor. Images of oyster larvae identified by an expert were used to create a library of labeled images to train a deep-learning model, which proved to be 82.4% accurate in precision, 90.8% in recall, and 86.4% in F-measure. A further method for estimating larval shell height from the rectangular shape of oyster larval images is also developed. The standardized mean difference in shell height between measurements and estimates is 3.3%. This deep-learning model has the potential to significantly reduce the time and effort required to identify oyster larvae in plankton samples, and thereby costs of this exercise.     

Comparison of Development and Larval Growth of Four Venerid Clams

The development and larval morphology of four venerid calms, Ruditapes philippinarum, Mactra veneriformis, Cyclina sinensis , and Meretrix lusoria , which cohabit the intertidal zone in western coastal Korea, were compared using laboratory culture techniques. At 87 μm, the fertilized eggs of C. sinensis and M. lusoria were the largest and at 53 μm, those of M. veneriformis were the smallest. D-shaped larvae of M. lusoriu were the largest and those of M. veneriformis were the smallest measuring at 135 μm and 89 μm, respectively. D-shaped larvae of R. philippinarum and M. lusoria had symmetrical shoulder angles and an elliptical ventral form, in contrast to the asymmetrical shoulder angles and round ventral forms of M. veneriformis and C. sinensis . In general, pediveliger larvae of all species in the study were yellow, but those of M. veneriforks and C sinenis were a more pronounced yellow. In between the early D-shaped and pediveliger stage, 7 and 17 d elapsed for M. lusonia and C. sinensis larvae, respectively. In the early larval stages for all species, the sheU length was longer than the height. However, shell length and height later became approximately the same size in all species except R. philippinarum , which exhibited a flat shape. These results indicate that for these four venerid clams, the different characteristics in larval growth and external morphology provide the evidence necessary for larval identification of natural seed production despite the fact that they spawn concurrently in the intertidal zone.     

烟台四十里湾浅海养殖生物及附着生物的化学组成、有机净生产量及其生态效应

对四十里湾养殖海区一些双壳贝类和藻类收获时的化学组成和有机净生产量进行了分析，不同双壳贝类软体有机碳含量差别不大，而N含量差异较大，栉孔扇贝软体N含量最高（12．36％），而牡蛎，毛坩相对较低（8％－9％），贻贝和菲律宾蛤仔贝壳N含量最高，为0．55％和0．56％，而栉孔扇贝壳则较低（0．1％），且贝壳，C，N，P在扇贝C，N，P总含量中所占的比例分别为6．2％，7．5％和6．9％，海带和石莼元素组成说明海区营养元素N的不足，海带的不同部位N含量差别很大，且在不同海区有较大变化，贻贝贝壳C，H，N和P含量在整个贻贝中占有相对大的比例，分别为30．4％，30．2％，31．8％，29．6％，菲律宾蛤仔，长牡蛎和中国蛤蜊等贝类贝壳中这些元素的相对含量也较高，布扇贝贝壳却较低，四十里湾海区每年因贝类和藻类的养殖，海区将至少减少850tN和78tP，其中贝壳的营养损失占据着相当比例，在海水交换受到一定限制，营养不很丰富，海区，养殖生物收获本身也会对海区的营养状况产生影响，根据贝壳N，P的含量，笔者认为在营养不很丰富的沿岸海区，贻贝，牡蛎最好不养或尽可能少养。     

Use of a probiotic for the culture of larvae of the Pacific oyster (Crassostrea gigas Thunberg)

Additions of bacteria (strain CA2) as a food supplement to xenic larval cultures of the oyster Crassostrea gigas consistently enhanced growth of larvae during different seasons of the year. Bacterial enhancement of larval growth occurred when either Isochrysis galbana (ISO), I. aff. galbana (T-ISO) or Pseudoisochrysis paradoxa (VA-12) were used as algal foods. Additions of CA2 bacteria at 10⁵ cells ml⁻¹ to cultures of algal-fed larvae increased larval growth, the proportion of larvae that set to produce spat, and the subsequent size of spat. A lower proportion of slow-growing larvae in populations receiving additions of CA2 bacteria compared with populations of larvae fed only algae, suggests a bacterial nutritional contribution to larval growth. Manipulation of bacterial populations present in bivalve larval cultures is a potentially useful strategy for the enhancement of oyster production.     

基于原位LAMP技术的牡蛎疱疹病毒(OsHV-1)易感宿主调查

牡蛎疱疹病毒(Ostreid herpesvirus 1, OsHV-1)给世界双壳贝类养殖业造成了严重的经济损失。10余种双壳贝类陆续被认定为易感宿主,仍有其他几种贝类仅有PCR核酸阳性数据,因确诊证据不足导致其易感性未得到充分评估。原位环介导等温核酸扩增(LAMP)检测技术相对传统原位杂交技术具有灵敏度高、方便快捷、可作为病原微生物感染证据的优点。为了在OsHV-1流行病学调查过程中实现病毒感染的快速检测和确诊,根据已报道的OsHV-1特异性LAMP检测引物,设计内引物,优化反应条件,建立了OsHV-1的原位LAMP检测方法。基于该方法对2019年以来采集的长牡蛎(Crassostrea gigas)、福建牡蛎(Crassostrea angulata)、栉孔扇贝(Chlamys farreri)、虾夷扇贝(Mizuhopecten yessoensis)、毛蚶(Scapharca subcrenata)和菲律宾蛤仔(Ruditapes philippinarum)样本进行检测。结果显示,毛蚶样本的OsHV-1原位LAMP检测结果呈阳性;其他几种贝类部分样本的实时定量PCR (qPCR)检测呈阳性,但原位LAMP检测呈阴性。对毛蚶样本的原位LAMP检测结果分析发现,病毒杂交信号主要分布在外套膜和肝胰腺等器官的结缔组织,推测感染的细胞为成纤维细胞和血淋巴细胞;在闭壳肌和斧足肌肉组织的肌细胞细胞核中也发现较多杂交信号。鳃丝内和周边偶现阳性信号,推测来自渗出的血淋巴细胞。基于原位LAMP技术的OsHV-1检测结果显示,毛蚶是OsHV-1的一种易感宿主,毛蚶结缔组织、肌肉组织和血淋巴细胞对该病毒有强亲嗜性。     

The first detection of okadaic acid and its derivatives in Korean bivalves by liquid chromatography-mass spectrometry

ABSTRACT:   Lipophilic toxin profiles associated with diarrhetic shellfish poisoning in Korean bivalves were investigated by liquid chromatography-mass spectrometry (LC-MS). Okadaic acid (OA), dinophysistoxins (DTX), pectenotoxins (PTX) and yessotoxin (YTX) in bivalves were quantified by LC-MS. Thirty-eight bivalve samples were collected from five different areas on the south coast of Korea during January to June 2006. Dinophysistoxin-1 (DTX1) and OA were found in some samples of both mussel Mytilus edulis and oyster Crassostrea gigas . The levels of DTX1 and OA found in the hepatopancreas of the samples were 0.04–0.23 µg/g and 0.03–0.08 µg/g, respectively. Dinophysistoxin-3 was found in the hepatopancreas of the short-necked clam Ruditapes philippinarum at a concentration of 0.06 µg/g. PTX and YTX were not detected from all of the samples examined. This is the first report of the unambiguous identification of OA and its derivatives, by LC-MS, in Korean bivalves.     

Sequence and polymerase chain reaction–restriction fragment length polymorphism analysis of the large subunit rRNA gene of bivalve: Simple and widely applicable technique for multiple species identification of bivalve larva

ABSTRACT:   One of the biggest and long-standing difficulties in investigation of larval ecology in the field is species-level identification. In the present study, we developed polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis based on the large subunit (LSU) rRNA gene (rDNA) D1/D2/D3 region for identification of multiple species of bivalve larvae using 14 species of bivalve collected from Maizuru Bay. The LSU rDNA D1/D2/D3 region of all analyzed species could be amplified by PCR using bvD1f/bvD3r primers, and RFLP analysis by Hae III digestion on the PCR products showed species-specific fragment patterns. Furthermore, this analysis applied to single bivalve larvae in Maizuru Bay revealed efficient amplification of the target region and the species-specific pattern from 80% of the larvae, 75% of which showed a pattern that matched a certain pattern of the adult bivalves. In addition, the analysis of inter- and intraspecies variation of the LSU rDNA D1/D2/D3 region using the sequence data of the genus Crassostrea from the DDBJ database showed high applicability of this RFLP analysis on closely related species. Because of the wide applicability and technical simplicity, this method can become the standard for the identification of bivalve larvae species once the sequence data of the LSU rDNA D1/D2/D3 region of many bivalve species have been accumulated.     

Two monoclonal antibodies for the recognition of Mytilus spp. larvae: studies on cultured larvae and tests on plankton samples

Monoclonal antibodies (mAbs) were generated against 2-day-old mussel larvae in an attempt to develop a rapid and rigorous method for the identification of mussel larvae in field plankton samples. Previously, we have shown that two of these mAbs recognised Galician Mytilus galloprovincialis obtained from monospecific cultures, but did not recognise the larvae of other bivalve species present in that area. To assess the possibility of using these mAbs in routine assays for measuring the abundance of mussel larvae in plankton, studies on cultured mussel larvae, at different stages of development, and tests on bivalve larvae from plankton samples were carried out. Initially, to see whether the two mAbs also recognise other mussel larval stages, they were tested against mussel larvae of different ages obtained from monospecific cultures. The results indicate that both antibodies stain all the stages tested, even 1-month-old postlarvae. In addition, we also demonstrate that these mAbs also recognise other forms of Mytilus. Both antibodies bind to M. galloprovincialis larvae from the Mediterranean Sea and M. edulis larvae. Finally, and more significantly, studies on field plankton samples were performed to confirm if both mAbs are really mussel-specific, and do not cross-react with larvae of any other bivalve species existing in the plankton. The results presented here clearly indicate that our two monoclonal antibodies specifically recognise the mussel larvae in field plankton samples from different geographical regions, but not the larvae of any other bivalve species. Thus, these monoclonal antibodies could be used for routine monitoring of mussel larvae in plankton samples from different sources.     

The potential for Chilean oysters, Tiostrea chilensis (Philippi, 1845), from two populations in northern New Zealand as a source of larvae for aquaculture

The use of the Chilean oyster. Tiostrea chilensis (Philippi. 1845), for aquaculture has been hampered by difficulty in obtaining commercial quantities of larvae due to the low fertility of the existing broodstock of this species taken from well-known populations in Chile and the South Island of New Zealand. The larval productions of two wild populations of this oyster in northern New Zealand were assessed as potential alternative sources of broodstock for aquaculture. The study found both populations produced larvae from young, small oysters, and that a much larger proportion of the population was brooding larvae each year than has been reported elsewhere. The mean fertility of one of these populations, at 71 056 ± 3701 (SE) larvae per oyster, was the highest reported for any population of this species. The significantly higher fertility of these populations makes them well suited for further development as broodstock for aquaculture.     

Augmentation of the food supply for oyster larvae via bacterivorous flagellates: possible implications for larval breeding and oyster pond management

Abstract. Examination of the protist plankton in the Espevikpollen oyster pond indicates that the size fraction available to the pelagic larvae of Ostrea edulis L. includes a substantial amount of heterotrophic flagellates. As these kind of flagellates have been shown to be of good food value to veliger larvae of a related species ( Crassostrea gigas Thunberg), the heterotrophic flagellates may be of equal importance as the phototrophic flagellates to the veligers. Therefore, the loop via bacterial production and heterotrophic flagellates may play the same role for Ostrea veligers under pond conditions as this loop plays for the veligers of Crassostrea in certain Japanese aquaculture systems.     

肾上腺素对岩牡蛎幼虫变态的诱导

为了得到壳型规则、大小均一的单体牡蛎,本研究采用肾上腺素对岩牡蛎单体苗种生产的诱导条件进行研究,阐明了岩牡蛎单体苗种的最适诱导浓度、诱导时间和诱导密度。结果显示,肾上腺素能显著诱导岩牡蛎幼虫的不固着变态,最适诱导浓度为5×10–5 mol/L,最适诱导时间为1 h,提高诱导浓度和延长诱导时间导致岩牡蛎幼虫的不固着变态率、稚贝壳高和存活率显著降低;肾上腺素对低于8个/mL幼虫密度的诱导效果差异不显著,但稚贝壳高和存活率在8个/mL的培育密度下显著低于0.5～4个/mL,研究表明利用肾上腺素诱导岩牡蛎单体时,可大批量处理眼点幼虫,但稚贝充气培养的最适培养密度应不高于4个/mL。     

Image analysis: a new method for estimating triploidy in commercial bivalves

Abstract. For determination of ploidy levels in bivalves, three techniques are now routinely used: karyological determination, microfluorimetry and flow cytometry. This paper proposes an alternative technique which is based on determination of the optical density (OD) of stained nuclei using image analysis. A karyological and image analysis comparative experiment conducted on juveniles of Crassostrea gigas (Thunberg) and Ruditapes philippinarum (Adam & Reeve) is described and highlights the advantages of the image analysis technique. Initially developed on C. gigas and R. philippinarum , the image analysis method was easily extended to other bivalve species and to different developmental stages, as demonstrated by two experiments conducted on Ostrea edulis (L.). First, percentage of triploids was consistent from the D-larval to the adult stage of development, despite the use of different preparation techniques depending on the stage examined. Second, a rise in ploidy percentage demonstrated a differential mortality between diploid and triploid animals. Image analysis techniques are cheaper than flow cytometry but more expensive than microfluorimetry. However, image analysis equipment can also be used for algae, eggs and spermatozoa counting, and other purposes such as fluorescence measurements, analysis of autoradiographs and electrophorcsis gels, and measurement of immuno-histochemical reactions.     

太平洋牡蛎与葡萄牙牡蛎两亚种间杂交及其早期阶段生长与存活的杂种优势

利用山东青岛养殖的太平洋牡蛎(N)与汕头本地养殖的葡萄牙牡蛎(S)两个近缘种为亲本,采用正交设计建立了杂种组NS(N♂×S♀)和SN(S♂×N♀)与纯种组NN(N♂×N♀)和SS(S♂×S♀)4个不同的遗传组合,通过比较不同阶段(幼虫期、稚贝期、养成阶段)的生长和存活数据,研究了牡蛎近缘种间的杂种优势,目的为改良牡蛎的生产性状。结果表明,这两个近缘种之间杂交能够产生显著的杂种优势,杂交后代的生长与存活两个表型性状都得到改良。杂交组比近交组生长得快,杂种优势在幼虫期为37.44%,稚贝期为42.47%。杂交组也比近交组存活率高,8日龄幼虫存活率的杂种优势为76.80%、14日龄幼虫存活率的杂种优势可达107.70%,60、90和105日龄稚贝存活率的杂种优势分别为17.30%、15.62%和9.08%。研究表明,通过太平洋牡蛎和葡萄牙牡蛎两个近缘种间的杂交有望解决牡蛎养殖产业存在的育苗难、存活率低、生长慢、个体小等问题。     

Toxicity to marine bivalves of culture supernatant fluids of the bivalve-pathogenic Vibrio strain NCMB 1338 and other marine vibrios

Abstract. Culture supernatant fluids from twelve Vibrio strains were toxic to spat of the European and Pacific oysters, Ostrea edulis L., and Crassostrea gigas L. At least three factors were present in culture supernates of one of the most virulent strains (NCMB 1338). The first was a heat-labile protein of molecular weight approximately 39000 which was lethal to oyster spat. This toxin was released during the stationary phase of culture and 72-h culture supernates contained 23·0LC₅₀ml when assayed against 2–3 mm Crassostrea gigas spat for 24 h at 20°C. Under the same assay conditions, 7-mm C. gigas spat were six times less sensitive to the toxin. The second factor was a relatively heat-stable ciliostatic toxin of molecular weight < 5000 and the third a proteinase of molecular weight 30000 which degraded gill segments of Mytilus edulis L. In addition, a haemolysin of molecular weight 20000 was produced. All these factors could be involved in the pathogenesis of 'vibriosis' in oyster larvae.     

5种经济贝类在天津大港滩涂的养殖适应性研究

通过养殖试验,分析5种主要经济贝类的生长、存活及其养殖区的底质、生物饵料,探讨其在天津大港滩涂的养殖适应性。结果表明,5—8月份,贝类生长较慢,死亡率较高,9—11月份则相反;试验区夏季底质浮泥层较厚,海水中饵料生物缺乏,春秋季节则相反;该滩涂适于养殖长牡蛎、毛蚶,较适于养殖菲律宾蛤仔、青蛤,不适于养殖缢蛏。     

Transmission of lymphocystis disease virus to cultured gilthead seabream,Sparus aurata L., larvae

The transmission of lymphocystis disease virus (LCDV) to gilthead seabream, Sparus aurata L., larvae was investigated using fertilized eggs from a farm with previous reports of lymphocystis disease. LCDV genome was detected by PCR‐hybridization in blood samples from 17.5% of the asymptomatic gilthead seabream broodstock analysed. Using the same methodology, eggs spawned from these animals were LCDV positive, as well as larvae hatched from them. The presence of infective viral particles was confirmed by cytopathic effects development on SAF‐1 cells. Whole‐mount in situ hybridization (ISH) and immunohistochemistry (IHC) showed the presence of LCDV in the epidermis of larvae hatched from LCDV‐positive eggs. When fertilized eggs were disinfected with iodine, no viral DNA was detected either in eggs (analysed by PCR‐hybridization) or in larvae (PCR‐hybridization and ISH). These results suggest the vertical transmission of LCDV, the virus being transmitted on the egg surface. Larvae hatched from disinfected eggs remain LCDV negative during the endotrophic phase, as showed by PCR‐hybridization, ISH and IHC. After feeding on LCDV‐positive rotifers, viral antigens were observed in the digestive tract, which suggests that viral entry could be achieved via the alimentary canal, and that rotifers can act as a vector in LCDV transmission to gilthead seabream larvae.     

捕后干露对3种贝类活品品质的影响

为明确经济养殖贝类捕后早期干露耐受性,选取活力良好的菲律宾蛤仔、长牡蛎和虾夷扇贝在4℃条件下进行8 d的无水贮藏,以闭壳肌p H、糖原、腺苷三磷酸(ATP)关联化合物、磷酸精氨酸的含量和奥品脱氢酶活性为指标,研究3种贝类的品质变化规律.试验结果表明,无水贮藏期间,菲律宾蛤仔pH无明显变化(P<0.05),而长牡蛎和虾夷...     

盐度骤降对近江牡蛎和长牡蛎能量收支的影响

采用实验生态学方法,研究了盐度骤降(盐度分别为10、20,自然海水为对照组)对近江牡蛎(Crassostreaariakensis)和长牡蛎(Crassastreagigas)生理代谢的影响.结果显示,盐度骤降对长牡蛎和近江牡蛎的耗氧率(OR)、排氨率(NR)、排粪率(FER)均有显著影响(P＜0.05),且2种牡蛎对...     

广东沿海贝类4种重金属含量分析和评价

根据2007年6～9月对广东沿海4种贝类养殖区90个样品的检测结果,研究了贝类体内重金属的含量和累积,对贝类的质量安全进行评价。结果表明：（1）菲律宾蛤仔Ruditapes philippinarum体内含量大小顺序为铜（Cu）〉镉（Cd）〉铬（Cr）〉铅（Pb）,太平洋牡蛎Crassostreagigas和近江牡蛎C．rivularis的大小顺序都是Cu〉Cd〉Pb〉Cr,而翡翠贻贝Pernaviridis的大小顺序则是Cu〉Pb〉Cd〉Cr;（2）菲律宾蛤仔、太平洋牡蛎和翡翠贻贝未受重金属污染,但部分近江牡蛎体中Cd和Cu含量已超过中国农业行业标准“无公害食品：水产品有毒有害物质限量”标准,应该引起重视。