Characterization of bacterial communities associating with larval development of Yesso Scallop (<Emphasis Type="Italic">Patinopecten yessoensisis</Emphasis> Jay, 1857) by high-throughput sequencing

Bacterial community presumably plays an essential role in inhibiting pathogen colonization and maintaining the health of scallop larvae, but limiting data are available for Yesso scallop (Patinopecten yessoensisis Jay, 1857) larval development stages. The aim of this study was to characterize and compare the bacterial communities associating with Yesso scallop larval development at fertilized egg S1, trochophora S2, D-shaped larvae S3, umbo larvae S4, and juvenile scallop S5 stages by Illumina high-throughput sequencing. Genomic DNA was extracted from the larvae and their associating bactera, and a gene segment covering V3-V4 region of 16S rRNA gene was amplified and sequenced using an Illumina Miseq sequencer. Overall, 106760 qualified sequences with an average length of 449 bp were obtained. Sequences were compared with those retrieved from 16S rRNA gene databases, and 4 phyla, 7 classes, 15 orders, 21 families, 31 genera were identified. Proteobacteria was predominant phylum, accounting for more than 99%, at all 5 larval development stages. At genus level, Pseudomonas was dominant at stages S1 (80.60%), S2 (87.77%) and S5 (68.71%), followed by Photobacterium (17.06%) and Aeromonas (1.64%) at stage S1, Serratia (6.94%), Stenotrophomonas (3.08%) and Acinetobacter (1.2%) at stage S2, Shewanella (25.95%) and Pseudoalteromonas (4.57%) at stage S5. Moreover, genus Pseudoalteromonas became dominant at stages S3 (44.85%) and S4 (56.02%), followed by Photobacterium (29.82%), Pseudomonas (11.86%), Aliivibrio (8.60%) and Shewanella (3.39%) at stage S3, Pseudomonas (18.16%), Aliivibrio (14.29%), Shewanella (4.11%), Psychromonas (4.04%) and Psychrobacter (1.81%) at stage S4. From the results, we concluded that the bacterial community changed significantly at different development stages of Yesso Scallop larvae.     

Developmental Dynamics of the Larval Muscle System of Bay Scallop (Argopecten irradians)

Though the larval development of bivalves has been extensively studied for commercial purposes,the dynamic development of larval muscle system remains largely unknown.In this study,we characterized the larval muscle system at different developmental stages(D-shaped veligers,umbo veligers and spats)in the bay scallop(Argopecten irradians)by phalloidin staining and under a confocal microscopy.The functional muscles are initially established at the early stage of veligers,which have four pairs of velar retractors and one anterior adductor.At the veliger stage,the velum and posterior retractor muscles are functionally important for velar contractility but undergo an irreversible shrink until they disappear at the end of the larval stage.During metamorphosis,three crucial modifications take place in the larval muscle system.The metamorphosis process involves the gradual degeneration of velum retractors,mantle margin development from an unfolded to a three-fold state,and remodeling of the adductor muscle system from dimyarian(two adductors)to monomyarian condition(one adductor)as in juveniles/adults.All retractor muscles are composed of striated muscle,but both anterior and posterior adductors have smooth and striated components.These findings highlight that the morphological changes at different stages are typical features of myogenesis in scallops.The present knowledge on the developmental dynamics of myogenesis in the bay scallop will not only improve our understanding of phenotypic diversity of larval myoanatomy in bivalves,but also provide useful information on the larval culture in hatcheries.     

Effect of zinc on the growth and development of larvae of bay scallopArgopecten Irradians

The 48-h straight-hinge larvae ofArgopecten irradians were exposed for 9 days to various concentrations of zinc in seawater. The growth rate of larvae in the 50 ppb Zn mediums was 77% that of the controls, and nearly zero in the 200 ppb Zn mediums. A progressive decrease in growth and increase in larval deformity and mortality was observed with increasing zinc concentrations from 50 to 200 ppb. 50 and 100 ppb Zn resulted in delayed development of eye spot and metamorphosis and reduced numbers of larvae at both stages. All larvae subjected to higher zinc concentrations died before attaining the eye-spot stage. The zinc concentrations which caused 50% reduction in growth (G C₅₀) and 50% mortality (LC₅₀) were 109 and 120 ppb respectively. Growing in the medium with 100 ppb Zn, the larvae fed withIsochrysis galbana cultured in a medium with 100 ppb Zn showed more suppressed growth and development than those fed with normal food. It is suggested that zinc contaminated food has strong influence on the bay scallop larvae. Contribution No. 1616 from the Institute of Oceanology, Academia Sinica.     

马氏珠母贝肠道及其养殖水体可培养细菌群落结构

【目的】了解马氏珠母贝(Pinctada fucata martensii)肠道及其养殖水体可培养细菌的群落组成。【方法】采用2216E平板涂布法研究海区养殖马氏珠母贝肠道与养殖水体的可培养菌群种类及丰度。【结果与结论】马氏珠母贝肠道及其养殖水体的可培养细菌归属于2门(变形菌门和厚壁菌门)3纲7目10科23属56种。属水平上,肠道中以弧菌属(74.7%)和假交替单胞菌属(18.7%)为主;养殖水体中α-变形菌纲的FJ943236_g属(40.7%)丰度最大,弧菌属(16.7%)相对肠道丰度较低。样品共有菌属为弧菌属、假交替单胞菌属、发光杆菌属和芽孢杆菌属;肠道特有菌属为希瓦氏菌属和盐单胞菌属;养殖水体特有菌属主要为FJ943236_g、鲁杰氏菌属和Nautella。在种水平上,7个种为二者共有;马氏珠母贝肠道和养殖水体特异性菌种分别为18个和31个。虽然门水平上马氏珠母贝肠道中可培养细菌群落与其养殖水体中的细菌群落大致相似,但在属、种水平上二者差异明显。     

Effects of environmental oxygen deficiency on embryos and larvae of bay scallop,Argopecten irradians irradians

The experimental results showed that: 1) The embryonic development of bay scallop is inhibited at a dissolved oxygen range of 1.38–3.64×10⁻³ at 23°C, and completely blocked below the lower limit. 2) The tolerance of larvae to anoxia increased with larval sizes and was related to their oxygen debt. 3) The scallop larvae exhibited specific behavioral responses to oxygen deficiency, which finally led to velum disintegration and larval death. The possible relationship between environmental oxygen deficiency and the disease of disintegration of the larval velum is also discussed. In this study, considerable oxygen debt was found in bay scallop larvae, which was greater in small animals. Based on the works of previous authors, a new concept is proposed for the estimation of oxygen debt, namely, the compensatory rate of oxygen debt (CROD). This can be used in intra—or interspecific comparison of oxygen debt. The results can be helpful in the management of water quality and for the prevention of larval diseases encountered in scallop culture. Contribution No. 2167 from the Institute of Oceanology, Chinese Academy of Sciences.     

The potential of ocean acidification on suppressing larval development in the Pacific oyster Crassostrea gigas and blood cockle Area inflata Reeve

We evaluated the effect of pH on larval development in larval Pacific oyster(Crassostrea gigas) and blood cockle(Arca inflata Reeve).The larvae were reared at pH 8.2(control),7.9,7.6,or 7.3beginning 30 min or 24 h post fertilization.Exposure to lower pH during early embryonic development inhibited larval shell formation in both species.Compared with the control,larvae took longer to reach the D-veliger stage when reared under pH 7.6 and 7.3.Exposure to lower pH immediately after fertilization resulted in significantly delayed shell formation in the Pacific oyster larvae at pH 7.3 and blood cockle larvae at pH 7.6 and 7.3.However,when exposure was delayed until 24 h post fertilization,shell formation was only inhibited in blood cockle larvae reared at pH 7.3.Thus,the early embryonic stages were more sensitive to acidified conditions.Our results suggest that ocean acidification will have an adverse effect on embryonic development in bivalves.Although the effects appear subtle,they may accumulate and lead to subsequent issues during later larval development.     

Effects of different dietary lipid contents on growth and lipase activity of Eriocheir sinensis larvae

The effects of different dietary lipid content on the growth and lipase activity of Eriocheir sinensis larvae were studied in the paper. The results showed that the survival, metamorphic rate and weight gain of E. sinensis larvae at different stages of growth all varied significantly with lipid content (P<0.05). Further, the survival and metamorphosis rates were the highest during the larval phases Z₃ to Z₄, and the weight gain was the highest during the larval phases Z₅ to M. During the first 20 h after metamorphosis of every larval stage, the lipase activity increased over time at Z₁, Z₂, Z₃ and M and declined at Z₄ and Z₅, and was influenced significantly by lipid content (P<0.05). In addition, lipase activity at each larval stage began to respond to dietary lipid contents 4 h after the larvae were fed, and tended to be stable after 12 h. The diets with higher lipase activity and lower lipid content were selected to give the suitable recipe of lipid requirements at each larval stage. It was concluded that the suitable lipid requirements at Z₁, Z₂, Z₃, Z₄, Z₅ and M were 6%, 4%, 6%, 8%, 8% and 10%, respectively.     

Yield trait improvement of bay scallops following complete diallel crosses between different scallop stocks

This study describes the complete diaUel hybridization between newly introduced bay scallop stock （W） from Canada and local commercial stock （D） grown under laboratory conditions, in China. Larval survival and growth during all life stages （larvae, spat, and adult） were compared among hybrid （DW, WD） and purebred （DD, WW） populations. Significant heterosis was detected for survival during the larval stage （〉 1% of the mid-parent values）. The mean heterosis （Hm） varied in growth throughout the life span. More than 50% of the Hm values were positive and negative in the DW and WD groups, respectively. The influence of maternal effects and mating types （intrapopulation vs. interpopulation crosses） on growth for all life stages was not consistent. Larval survival did not differ significantly （P〉0.05） with maternal effect or mating type. In the harvest stage, shell length （SL）, shell height （SH）, shell width （SW）, and total weight （TW） were larger in the hybrid compared with the inbred groups. Positive Hm values were observed in SL （1.5%）, SW （5.8%）, and TW （12.3%）, and were more significant in the DW groups （6.1%, 4.5%, 6.8%, and 27.2%）. These results suggest that hybridization between two geographic populations is a good tool for improving bay scallop growth. However, unstable heterosis between the two populations requires further study.     

The potential of ocean acidification on suppressing larval development in the Pacific oyster Crassostrea gigas and blood cockle Arca inflata Reeve

We evaluated the effect of pH on larval development in larval Pacific oyster （Crassostrea gigas） and blood cockle （Arca inflata Reeve）. The larvae were reared at pH 8.2 （control）, 7.9, 7.6, or 7.3 beginning 30 min or 24 h post fertilization. Exposure to lower pH during early embryonic development inhibited larval shell formation in both species. Compared with the control, larvae took longer to reach the D-veliger stage when reared under pH 7.6 and 7.3. Exposure to lower pH immediately after fertilization resulted in significantly delayed shell formation in the Pacific oyster larvae at pH 7.3 and blood cockle larvae at pH 7.6 and 7.3. However, when exposure was delayed until 24 h post fertilization, shell formation was only inhibited in blood cockle larvae reared at pH 7.3. Thus, the early embryonic stages were more sensitive to acidified conditions. Our results suggest that ocean acidification will have an adverse effect on embryonic development in bivalves. Although the effects appear subtle, they may accumulate and lead to subsequent issues during later larval development.     

施氏獭蛤幼虫和稚贝发育及行为的研究

进行了施氏獭蛤(Lutraria sieboldii Reeve)的人工育苗、幼虫发育及行为观察实验。结果显示:在施氏獭蛤幼虫发育过程中,壳顶后期幼虫只有鳃丝及足出现,而没有眼点出现,发达的足和鳃丝是幼虫变态的标志;幼虫变态期明显的分为3～5d的爬行期和3～5d的附着期;爬行期幼虫有单条水管形成,附着期幼虫只有一条分叉的足丝,水管为单管型;底栖稚贝期开始形成两条水管,底栖稚贝早期的幼虫不具备潜沙能力,只进行爬行活动,随着水管的快速发育,幼虫逐渐具备潜沙能力,幼虫壳长达2500μm、水管长达6000μm以上时,最终具备和成体一样的完全潜沙能力。     

Characterization of Bacterial Community Associated with Four Organs of the Yesso Scallop (Patinopecten yessoensis) by High-Throughput Sequencing

We used Illumina high-throughput sequencing of PCR-amplified V3-V4 16 S rRNA gene regions to characterize bacterial communities associated with the adductor muscles, gills, gonads and intestines of the Yesso scallop(Patinopecten yessoensis) from waters around Zhangzidao, Dalian, China. Overall, 421,276 optimized reads were classified as 25 described bacterial phyla and 308 genera. Firmicutes, Proteobacteria, Tenericutes, Bacteroidetes, Chlamydiae and Spirochaetae accounted for > 97% of the total reads in the four organs. The bacterial 16 S rDNA sequences assigned to Firmicutes and Proteobacteria were abundant in the adductor muscles, gills and gonads; while reads from Tenericutes were dominant in the intestines, followed by those from Firmicutes, Chlamydiae, Proteobacteria and Bacteroidetes. At the genus level, the dominant genera in the adductor muscles, gills and gonads appeared to be Bacillus, Enterococcus and Lactococcus, whereas Mycoplasma was dominant in the intestines. The relative abundances of Bacillus, Enterococcus, Lactococcus, Alkaliphilus, Raoultella, Paenibacillus and Oceanobacillus were significantly lower in the intestine than in the other three organs. Cluster analysis and principal coordinates analysis of the operational taxonomy units profile revealed significant differences in the bacterial community structure between the intestine and the other three organs. Taken together, these results suggest that scallops have intestine-specific bacterial communities and the adductor muscles, gills and gonads harbor similar communities. The difference in the bacterial community between organs may relate to unique habitats, surroundings, diet and their respective physiological functions.     

Comparison of growth and survival of larvae among different shell color stocks of bay scallop Argopecten irradians irradians (Lamarck 1819)

In order to improve production and breed new broods of bay scallopArgopecten irradians irradians, different-colored orange, purple and white lines were established by two mating methods of self-fertilization and mass spawning at a commercial scallop hatchery in spring, 2002. And then larval growth and survival of different lines was compared to test whether there is a relationship between shell color and growth and survival at early developmental stage. Both growth and survival have no significant differences among different experimental larvae of self-fertilization or mass spawning. Results are as following in the order of orange, purple and white shell stock. For the self-fertilization, growth rates of larvae were 6.174, 6.412, and 6.599 μm/d, respectively. Survival rates of larvae at Day 3 were 74.41%, 76.86%, and 82.05%; Day 6 were 49.14%, 65.63%, and 52.79%; and Day 9 were 25.06%, 20.80%, and 26.47%, respectively. For the mass spawning, the growth rates were 7.836, 7.941, and 7.878 μm/d, respectively. Survival rates at Day 3 were 93.05%, 91.95%, and 92.50%; Day 6 were 79.17%, 78.05%, and 82.50%; and Day 9 were 34.72%, 36.67%, and 38.33%, respectively. The absence of any relationship between shell color and growth and survival at the larval stage may be resulted from their common genetic basis. This work was financially supported by Project of Scientific Innovation, Chinese Academy of Sciences (ZKCX2-211).     

Ontogenetic development in the morphology and behavior of loach （Misgurnus anguillicaudatus） during early life stages

Loach （Misgurnus anguillicaudatus） is a commercially important fish in China and an ideal aquaculture species. However, culturists experience high larval and juvenile mortality during mass production. To provide insight into ways to improve larviculture techniques, we describe the morphological characteristics and behavior of loach during the larval and early juvenile stages. Yolksae larvae ranged from 2.8 to 4.0 mm body length （BL） between days 0 to 4; preflexion larvae ranged from 3.6 to 5.5 mm BL between days 4 to 6; flexion larvae ranged from 4.8 to 8.1 mm BL between days 5 and 14; and post-flexion larvae ranged from 7. l to 15.7 mm BL between days 11 to 27; the minimum length and age of juveniles was 14.1 mm BL and 23 d, respectively. Loach are demersal from hatch through to the early juvenile stages. A suite of morphological characteristics （e.g., external gill filament and ventral mouth opening） and behavioral traits have developed to adapt to demersal living. We observed positive allometric growth in eye diameter, head length, head height, and pectoral fin length during the early larval stages, reflecting the priorities in the development of the organs essential for survival. Our results provide a basis for developing techniques to improve the survival of larval and juvenile loach during mass production.     

Isolation of novel psychrophilic bacteria from Arctic sea ice

The phylogenetic diversity of cuhurable psychrophilic bacteria associated with sea ice from the high latitude regions of Canadian Basin and Chukchi Sea, Arctic, was investigated. A total of 34 psychropilic strains were isolated using three methods of （ Ⅰ ） dilution plating （ at 4 ℃ ）, （ Ⅱ ） bath culturing （ at - 1 ℃ ） and dilution plating, and （ Ⅲ） cold shock （ -20 ℃ for 24 h）, bath culturing and dilution plating under aerobic conditions. Sea-ice samples were exposed to -20 ℃ for 24 h that might reduce the number of common microorganisms and encourage outgrowth of psyehrophilic strains. This process might be able to be introduced to isolation psychrophilic bacteria from other environmental samples in future study. 16S rDNA nearly full-length sequence analysis revealed that psychrophilic strains felled in two phylo- genetic divisions, γ-proteobacteria （in the genera ColweUia,Marinobaeter、Shewanella,Glaciecola,Marinomortas and Pseudoalteromortas ） and Cytophaga-Flexibacter-Baeteroides （ Flavobacterlum and Psyehroflexus）. Fifteen of bacterial isolates quite likely represented novel species （16S rDNA sequence similarity below 98% ）. One of strains （BSi20002） from Canadian Basin showed 100% sequence similarity to that of Marinobacter sp. ANT8277 isolated from the Antarctic Weddell sea ice, suggesting bacteria may have a bipolar distribution at the species level.     

Using iTRAQ-Based Quantitative Proteomics Analysis to Identify Differentially Expressed Proteins Related to Larval Development of Portunus trituberculatus

The swimming crab Portunus trituberculatus is an economically important marine crustacean species. Here isobaric tags for relative and absolute quantitation(iTRAQ) analysis were used to identify proteins that are differentially expressed during larval development of P. trituberculatus to elucidate the underlying mechanisms. In comparison with the first zoea stage(Z1), 3980 proteins were identified from 32789 peptides, which were matched with 115497 spectrums. A total of 241 proteins were screened with significantly differential expressions in all development stages. These 241 proteins are involved in various biological processes, such as cytoskeleton organization, protein synthesis, energy production and substance metabolism, physiological activities, and transport.Cluster analyses of the 241 differentially expressed proteins led to the generation of four protein clusters based on the overall similarity in protein expression patterns. Exactly 54, 70, 36, and 45 proteins clustered in profiles 10(0, 0, 0,-1, 0, 0), 15(0, 1, 0, 1, 0, 1), 18(0, 1, 2, 2, 1, 0), and 19(0, 1, 2, 3, 4, 5), respectively. Muscle development and exoskeleton renewal were important processes throughout the development stages. In addition, protein synthesis, degradation, and digestion actively occurred, especially at the Z4 stage. These results provide novel insights into the mechanisms underlying larval development of swimming crab and can assist in larval rearing.     

An integrative study of larval organogenesis of American shad <Emphasis Type="Italic">Alosa sapidissima</Emphasis> in histological aspects

We describe organogenesis at a histological level in American shad (Alosa sapidissima) larvae from 0 until 45 days after hatching (DAH). Larval development was divided into four stages based on the feeding mode, external morphological features, and structural changes in the organs: stage 1 (0–2 DAH), stage 2 (3–5 DAH), stage 3 (6–26 DAH) and stage 4 (27–45 DAH). At early stage 2 (3 DAH), American shad larvae developed the initial digestive and absorptive tissues, including the mouth and anal opening, buccopharyngeal cavity, oesophagus, incipient stomach, anterior and posterior intestine, differentiated hepatocytes, and exocrine pancreas. The digestive and absorptive capacity developed further in stages 2 to 3, at which time the pharyngeal teeth, taste buds, gut mucosa folds, differentiated stomach, and gastric glands could be observed. Four defined compartments were discernible in the heart at 4 DAH. From 3 to 13 DAH, the excretory systems started to develop, accompanied by urinary bladder opening, the appearance and development of primordial pronephros, and the proliferation and convolution of renal tubules. Primordial gills were detected at 2 DAH, the pseudobranch was visible at 6 DAH, and the filaments and lamellae proliferated rapidly during stage 3. The primordial swim bladder was first observed at 2 DAH and started to inflate at 9 DAH; from then on, it expanded constantly. The spleen was first observed at 8 DAH and the thymus was evident at 12 DAH. From stage 4 onwards, most organs essentially manifested an increase in size, number, and complexity of tissue structure.     

盐度对菊黄东方鲀受精卵孵化和仔鱼生长的影响

比较在0、5、10、15、20、25、30(自然海水)、35、40、45、50等11个盐度梯度下,菊黄东方鲀(Takiugu flavidus)的受精卵发育和仔鱼生长情况,测定仔鱼的不投饵存活系数(SAI).结果表明:1)菊黄东方鲀受精卵在盐度0~50范围内均可孵化,最适盐度20~25.盐度(x)与孵化时间(y)呈正相关关系,表达式为:y=0.345 x+141.1,R2=0.956.2)实验盐度范围内,菊黄东方鲀仔鱼SAI值为1.55~30.10.3)在盐度5~45条件下,仔鱼皆可存活,存活率为3%~27%.其中,在10~30盐度下存活率较高,为20%~27%.经15 d的培育,仔鱼平均全长为6.28~7.34 mm.低盐度对菊黄东方鲀仔鱼生长更有利,在15~20盐度条件下,仔鱼生长较好,最终平均全长为7.30~7.34 mm.4)根据SAI值和生长状况,仔鱼生长和存活的适宜生长盐度为10~30,最适盐度范围为15~20.     

Distribution and elimination of Norfloxacin in Fenneropenaeus chinensis larvae

This study examined the distribution and elimination of Norfloxacin(NFLX) in Fenneropenaeus chinensis ovary and egg and newly hatched larvae.Mature parental shrimp were exposed to 4 or 10 mg L 1NFLX for 2 or 5 d.Ovary and eggs of the shrimp were sampled after spawning in order to detect NFLX residue using high-performance liquid chromatography(HPLC).Results showed that NFLX residue accumulated in F.chinensis eggs after the parental exposure,with the highest residue detected in ovary.To examine the fate of NFLX residue in larvae,we further determined the concentration of NFLX residue in F.chinensis eggs and larvae at 4 different developmental stages after 24-h exposure.From the newly metamorphosed larvae(0 h post-metamorphosis,h.p.m),samples were taken at different time intervals to 72 h.p.m.HPLC assay showed that the concentrations of NFLX residue in zoea exposed to 4 and 10 mg L 1NFLX were the highest at 1.5 h,i.e.,0.332 and 0.454 μg g 1,respectively.At the two NFLX exposure levels,the elimination time of half NFLX(half life) in nauplius was 45.36 and 49.85 h,respectively,followed by that in zoea(31.68 and 33.13 h),mysis larvae(42.24 and 47.28 h) and postlarvae(24.48 and 30.96 h).Both NFLX exposure levels had a germicidal effect.The distribution and elimination of NFLX residue in F.chinensis tissue,eggs and larvae correlated well with the drug exposure level.The disappearance of NFLX residue coincided with the larval growth,and the half-life of NFLX decreased with the larval development.     

Changes of Protein and Lipid Contents,Amino Acid and Fatty Acid Compositions in Eggs and Yolk-Sac Larvae of American Shad(Alosa sapidissima)

To investigate the changes of the biochemical composition of American shad(Alosa sapidissima) eggs and larvae at embryonic and early larval stages, samples were collected at different development stages from artificial fertilization to the end of yolk absorption including 2 h, 12 h and 30 h after fertilization and newly hatched larvae including 1 and 3 days after hatching. The composition of lipid, fatty acids, protein and amino acids were analyzed. The content of total protein exhibited a decreasing trend during embryogenesis and larval development, and a significant reduction was detected after hatching(P 0.05). The total lipid content remained relative stable. A significant reduction was detected in almost all amino acids after hatching except for glycine(P 0.05), while a significant decrease was found in the content of cysteine, proline, tyrosine, valine, isoleucine, leucine and phenylalanine during the yolk-sac phase(P 0.05). On the other hand, all the groups of fatty acids remained stable during the period of embryogenesis. But after hatching, a significant decrease was found in the content of C18:2n-6, C18:3n-6, SFA and ratio of EPA/ARA(P 0.05), while a significant increase was found in the content of C18:3n-3, C20:4n-6, C22:6n-3 and ratio of n-3/n-6(P 0.05). In conclusion, the combined data suggested that American shad utilizes the protein content as preferential energy substrates during embryonic and early larval developments with some specificity in the consumption of different amino acids.