Cleavage and gastrulation in the Kuruma shrimp Penaeus (Marsupenaeus) japonicus (Bate): A revised cell lineage and identification of a presumptive germ cell marker

A previous study suggested that mesendoderm (ME) cell arrest occurred at the 64‐cell stage and a ring of eight presumptive naupliar mesoderm cells or crown cells surrounded the blastopore in the Kuruma shrimp Penaeus (Marsupenaeus) japonicus. Since this varied from the pattern observed in other penaeoidean shrimp, cleavage and gastrulation was re‐examined in P. japonicus using the nucleic acid stain Sytox Green and confocal microscopy. In contrast to the earlier study, cleavage and gastrulation followed the pattern observed in other penaeoidean shrimp. The ME cells arrested at the 32‐cell stage, ingressed into the blastocoel, and resumed division after a three cell cycle delay. Nine naupliar mesoderm or crown cells surrounded the blastopore and their descendants invaginated during gastrulation. An intracellular body (ICB) was detected by Sytox Green and SYTO RNASelect staining to be segregated to one ME cell in P. japonicus, as described previously in Penaeus monodon. Staining of the ICB was eliminated by pre‐treatment with RNase but not DNase. The ICB was also found in two other penaeoidean shrimp, Penaeus vannamei (Family Penaeidae) and Sicyonia ingentis (Family Sicyoniidae). The results support the hypothesis that the ICB is a germ granule found in the Dendrobranchiata.     

Cleavage and gastrulation of the euphausiacean<Emphasis Type="Italic"> Meganyctiphanes norvegica</Emphasis> (Crustacea,Malacostraca)

According to the Articulata hypothesis the cleavage of arthropods must be derived from spiral cleavage. However, arthropods show a great variety of cleavage modes with a widespread occurrence of superficial cleavage. In the Malacostraca, holoblastic cleavage occurs in some taxa such as Amphipoda, Euphausiacea and Dendrobranchiata. In particular, the cleavage of euphausiaceans has been proposed to be a modified spiral cleavage. The cell lineage of early stages up to blastoderm formation of the euphausiacean Meganyctiphanes norvegica is reconstructed using recent methods of fluorescent staining. Only the oblique angle of the mitotic spindles during the transition from the 2- to the 4-cell stage resembles the spiral cleavage mode. At the 8-cell stage, four cells each form a pattern of two interlocking bands which is preserved until the 122-cell stage. One blastomere is delayed in division and shows an oblique division from the fourth cleavage on. It is the precursor cell of two enlarged and cleavage-arrested cells at the 32-cell stage. At the 62-cell stage, these two cells are surrounded by eight cells following a specific cell division pattern during the subsequent division cycles. The cleavage pattern of M. norvegica occurs in two mirror images. A comparative approach reveals distinct similarities between the early cleavage patterns of Euphausiacea and Dendrobranchiata which are suggested to be homologous. Furthermore, the relationships to non-malacostracan cleavage patterns are discussed. It is shown that the early cleavage pattern of M. norvegica does not offer an example of a spiral cleavage within arthropods.     

Cleavage and gastrulation of the dendrobranchiate shrimp Penaeus monodon (Crustacea,Malacostraca, Decapoda)

The cleavage pattern of the black tiger shrimp Penaeus monodon was analyzed from the first division until gastrulation. Observations were based on microscopy combined with the use of fluorescent dyes, histological techniques, and computer based three-dimensional reconstructions. Early cleavage is holoblastic and follows a stereotypic pattern, which largely corresponds to what is known from other dendrobranchiate decapods. However, for the first time in this group, we report the presence of an intracellular structure throughout early development. This intracellular body (icb) marks the lineage of one of the two enlarged and division-delayed mesendoderm cells that initiate gastrulation. The identity of the icb as a natural marker and putative determinant of the germ line and its implications on the establishment of the body axes are discussed. The icb as a landmark reveals that the same stereotypic cell division pattern can lead to different fates of individual cells. Hence, the results of this study permit an additional approach to study the relation between cell lineage pattern and the identity of cell lineages.     

Penaeus monodon chitin-binding protein (PmCBP) is involved in white spot syndrome virus (WSSV) infection

White spot syndrome virus (WSSV) can cause the most serious viral disease of shrimp and has a wide host range among crustaceans. Although researches show a lot about its genome and structure, information concerning the mechanism of how WSSV infects' cells is lacking. In this study, some experiments were applied to confirm the biological meaning of the protein–protein interaction between WSSV envelope protein, VP53A, and Penaeus monodon chitin-binding protein (PmCBP). Immunofluorescent study indicated that PmCBP is located on the cell surface of host cells. PmCBP amounts of about 34 kDa can be detected in both P. monodon and Litopenaeus vannamei tissues by Western blotting. In the in vivo neutralization experiment, both rVP53A and rPmCBP that were produced by Esherichia coli can promote resp. a 40% and 20% survival rate of the shrimp which were challenged by WSSV. Furthermore, a yeast-two-hybrid result revealed that PmCBP could interact with at least 11 WSSV envelope proteins. Those findings suggest that PmCBP may be involved in WSSV infection.     

Effect of salinity and body weight on ecophysiological performance of the Pacific white shrimp (Litopenaeus vannamei)

Ecophysiological responses of Litopenaeus vannamei were evaluated as functions of environmental salinity and animal size. Growth rate, routine metabolic rate, limiting oxygen concentration, and marginal metabolic scope were determined for L. vannamei acclimated to, and tested at, salinities of 2, 10, and 28 ppt, all at 28 °C. Routine metabolic rate (RMR), estimated as oxygen-consumption rate per unit body weight for fasted, routinely-active shrimp, was independent of salinity but decreased with increasing shrimp weight. Limiting oxygen concentration for routine metabolism (LOCr) decreased with increased shrimp weight for the 10 and 28 ppt treatments, but not for the 2 ppt treatment. Marginal metabolic scope (MMS = RMR/LOCr) also decreased with increasing shrimp weight and was independent of salinity. Growth rate was significantly less at 2 ppt than at either 10 or 28 ppt, which gave similar growth rates.     

地衣芽孢杆菌对南美白对虾零水交换养殖系统细菌群落的影响

【目的】地衣芽孢杆菌（Bacillus licheniformis）对南美白对虾（Penaeus vannamei）免疫反应、抗病性和营养的影响已被广泛研究，但零水交换养殖系统下地衣芽孢杆菌对对虾肠道和养殖水环境微生物群落的影响尚不清楚。【方法】通过收集添加地衣芽孢杆菌在饲料或水中后，对虾肠道、池水和池低沉积物样品，通过16S rRNA基因测序和线性判别分析（linear discriminant analysis effect size，LEfSe）进行微生物分析。【结果】结果表明，添加地衣芽孢杆菌对对虾的生长影响较小。此外，添加方式的不同对对虾肠道菌群的影响较小。但添加地衣芽孢杆菌可以有效地改变对虾肠道微生物群落，并改善对虾免疫力。【结论】这些结果有助于全面了解在零水交换养殖系统中，通过饲料和水添加地衣芽孢杆菌后对虾肠道和环境的变化，从而为选择正确的益生菌以及如何添加益生菌维持对虾健康提供基础信息。     

欧文氏弧菌毒力蛋白PirAB的原核表达与纯化

对虾肝胰腺坏死病的爆发造成了对虾养殖产业的严重亏损。从上海地区凡纳滨对虾中分离出1株欧文氏弧菌(Vibrio owensii SH-14),该菌株可导致凡纳滨对虾死亡,且死虾出现对虾肝胰腺坏死病的典型症状。经PCR扩增欧文氏弧菌毒力蛋白PirA与PirB对应基因序列,并将其连接到表达质粒pET-21b(pirA)和pGEX-4t-1(pirB)。通过优化诱导表达和亲和层析纯化条件,最终获得大量高纯度的目的蛋白。经冷冻干燥保存为后期抗体合成以及进一步毒力效应研究提供参考。     

Cathepsin B from the white shrimp Litopenaeus vannamei: cDNA sequence analysis, tissues-specific expression and biological activity

Cathepsin B is a cystein proteinase scarcely studied in crustaceans. Its function has not been clearly described in shrimp species belonging to the sub-order Dendrobranchiata, which includes the white shrimp Litopenaeus vannamei and other species from the Penaeidae family. Studies on vertebrates suggest that these lysosomal enzymes intracellularly hydrolize protein, as other cystein proteinases. However, the expression of the gene encoding the shrimp cathepsin B in the midgut gland was affected by starvation in a similar way as other digestive proteinases which extracellularly hydrolyze food protein. In this study the white shrimp L. vannamei cathepsin B (LvCathB) cDNA was sequenced, and characterized. Its gene expression was evaluated in various shrimp tissues, and changes in the mRNA amounts were compared with those observed on other digestive proteinases from the midgut gland during starvation. By using qRT-PCR it was found that LvCathB is expressed in most shrimp tissues except in pleopods and eye stalk. Changes on LvCathB mRNA during starvation suggest that the enzyme participates during intracellular protein hydrolysis but also, after food ingestion, it participates in hydrolyzing food proteins extracellularly as confirmed by the high activity levels we found in the gastric juice and midgut gland of the white shrimp.     

凡纳滨对虾繁育系统菌群结构及优势菌遗传多样性分析北大核心

【目的】探讨凡纳滨对虾(Litopenaeus vannamei)工厂化繁育系统内发生细菌性玻化症(shrimp postlarva bacterial vitrified syndrome,BVS)时期可培养微生物的菌群特征以及优势病原菌的遗传多样性。【方法】采用细菌体外培养方法结合基因测序技术对不同育苗阶段的亲虾、受精卵、无节幼体、蚤状幼体、糠虾幼体、仔虾,及其育苗池水和饵料内可培养细菌菌群的组成与结构特征进行研究,并通过多位点序列分析(multilocus sequence analysis,MLSA)方法解析病原菌的遗传多样性。【结果】系统内分离纯化的526株具有典型形态差异和群落优势的细菌分属于4门5纲16目24科38属113种。在纲水平上γ-变形菌纲(Gammaproteobacteria)丰度最高,共453株,占总分离株的86.1%;在属水平上弧菌属(Vibrio)丰度最高,共369株,占总分离株的70.2%;在种水平上,溶藻弧菌(Vibrio alginolyticus)为最优势种,共112株,占总分离株的21.3%,并且分布于整个繁育系统,在饵料中具有最高丰度。多元关联分析表明,随着对虾幼体的发育,饵料对幼体体内可培养细菌的菌群结构影响逐渐增加。对112株潜在溶藻弧菌的MLSA分析表明,其中100株菌株进一步确认为溶藻弧菌。进一步利用MLSA构建系统发育树分析其遗传多样性发现,100株溶藻弧菌分为9个簇,分离自同类样品的菌株广泛分布在不同的簇中。【结论】在BVS发生时期,凡纳滨对虾工厂化繁育系统中具有丰富的可培养微生物种类。对虾幼体发育过程中,饵料对幼体体内可培养细菌的菌群结构具有重要影响。溶藻弧菌为凡纳滨对虾工厂化繁育系统中的优势弧菌,分布于整个繁育系统,且具有丰富的遗传多样性。本研究为解析对虾繁育系统可培养微生物演替规律提供了数据支撑,也为对虾苗期病原防控和健康养殖提供了科学依据。     

凡纳滨对虾肠道红杆菌科细菌富集碳源筛选及其定向分离

【目的】红杆菌科(Rhodobacteraceae)细菌为凡纳滨对虾肠道微生物的优势类群,在健康对虾肠道中具有较高的相对丰度,是指示对虾健康的关键类群,探究对虾肠道红杆菌科细菌定向富集和分离方法,可为对虾养殖益生菌菌剂的研发提供基础。【方法】利用16S rRNA基因高通量测序技术研究不同碳源添加对凡纳滨对虾肠道中红杆菌科细菌的富集作用,筛选对红杆菌科细菌有显著富集作用的碳源;利用纯培养技术从红杆菌科细菌富集的样品中定向分离红杆菌科细菌,并对其进行鉴定和遗传多样性分析。【结果】添加短链脂肪酸(乙酸、丙酸、丁酸、戊酸)和碳酸氢钠对红杆菌科细菌有显著富集作用,主要富集到Cribrihabitans、Tritonibacter、Rhodovulum、Ruegeria、Sagittula和Thalassobius属相关菌株;对红杆菌科细菌相对丰度最高的样品进行稀释涂布培养,共分离纯化出303株细菌,分属于2门12科,其中红杆菌科细菌为主导类群共119株,主要包括Tritonibacter (90株)、Phaeobacter (25株)、Sulfitobacter (1株)、Ruegeria (1...     

Evolutionary modification of T-brain (tbr) expression patterns in sand dollar

The sand dollars are a group of irregular echinoids that diverged from other regular sea urchins approximately 200 million years ago. We isolated two orthologs of T-brain (tbr), Smtbr and Pjtbr, from the indirect developing sand dollar Scaphechinus mirabilis and the direct developing sand dollar Peronella japonica, respectively. The expression patterns of Smtbr and Pjtbr during early development were examined by whole mount in situ hybridization. The expression of Smtbr was first detected in micromere descendants in early blastula stage, similar to tbr expression in regular sea urchins. However, unlike in regular sea urchin, Smtbr expression in middle blastula stage was detected in micromere-descendent cells and a subset of macromere-descendant cells. At gastrula stage, expression of Smtbr was detected in part of the archenteron as well as primary mesenchyme cells. A similar pattern of tbr expression was observed in early Peronella embryos. A comparison of tbr expression patterns between sand dollars and other echinoderm species suggested that broader expression in the endomesoderm is an ancestral character of echinoderms. In addition to the endomesoderm, Pjtbr expression was detected in the apical organ, the animal-most part of the ectoderm.     

高盐胁迫对凡纳滨对虾消化及免疫相关酶活力的影响

为探讨高盐对凡纳滨对虾(Litopenaeus vannamei)消化及免疫相关酶活力的影响,实验设置了30、40、50、60共4个盐度梯度。对虾体长(7.84±0.68)cm,养殖密度333尾/m~3,每个梯度设3个平行,实验周期30d。取血淋巴、肌肉、肝胰腺等组织,检测其超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、碱性磷酸酶(AKP)和酸性磷酸酶(ACP)及蛋白酶、脂肪酶、淀粉酶活力。结果表明,盐度显著影响凡纳滨对虾肝胰脏中胃蛋白酶、脂肪酶、淀粉酶的活力(P0.05);随着盐度增加,消化相关酶活力均不断下降,处理间差异显著(P0.05);盐度对凡纳滨对虾不同组织的免疫指标产生影响,表现为随着盐度升高,血淋巴中,AKP活力逐渐升高,ACP、CAT和SOD活力均表现为先升高后降低;肌肉中,AKP、ACP和SOD活力呈现先升高后降低的变化趋势;肝胰脏中,AKP活力呈现先降低后升高再降低的变化趋势,ACP活力高盐处理间差异不显著(P0.05),CAT活力先降低后升高,SOD活力盐度40后逐渐降低。实验结果初步说明,高盐显著影响凡纳滨对虾的消化及免疫相关酶活力,且盐度对不同组织中免疫酶活力影响存在一定的组织特异性,50以上的高盐胁迫对对虾消化和免疫相关酶活力的影响尤为显著。     

循环水养殖系统中凡纳滨对虾肠道微生物对三种复合益生菌制剂的响应

【目的】在循环养殖系统中应用不同的复合益生菌制剂,探讨凡纳滨对虾肠道菌群结构特征及免疫水平发生的变化。【方法】30 d养殖周期结束后,通过平板计数法分析肠道细菌总数和弧菌总数;基于高通量测序技术分析肠道样品V3+V4区菌群特征;采用实时荧光定量PCR技术分析免疫相关因子TLR1和Dorsal基因表达水平,阐述益生菌制剂应用的意义。【结果】益生菌制剂的应用降低了凡纳滨对虾肠道中细菌总数,抑制弧菌的生长,间接预防疾病的发生。不同益生菌制剂从不同程度上优化了凡纳滨对虾肠道菌群结构,提高高质量序列和有效OTU数量,Chao1、Simpson、Shannon指数显示了丰富度和多样性变化,复合益生菌制剂3效果较好。同时,菌群结构得到优化,其中益生菌制剂1组对拟杆菌门含量百分比产生显著影响。Toll受体TLR1和Toll通路中的Dorsal基因m RNA表达受到益生菌制剂的影响,1、3组促进了TLR 1表达,1、2、3组都促进了Dorsal基因表达。【结论】在循环水养殖系统中添加益生菌制剂可优化凡纳滨对虾肠道菌群特征,提高免疫水平,为病害防控和健康养殖提供理论依据。     

凡纳滨对虾生物絮团养殖系统中不同生境细菌群落特征

【目的】凡纳滨对虾生物絮团养殖系统(biofloc-based culture system, BFS)是一种基于培育和调控微生物群落的新型生态养殖模式。然而,目前对于BFS在不同生境中的微生物群落特征及其构建过程还不清楚。【方法】采用16S rRNA基因测序技术探究BFS在3种不同生境(水体、絮团和对虾肠道)的细菌群落组成;通过溯源分析和中性模型等方法,探究不同生境细菌群落的特征及其构建过程。【结果】3种生境的微生物群落多样性和组成具有显著性差异,絮团和对虾肠道的群落结构和组成最为相似,溯源结果显示对虾肠道有98.76%的细菌类群来自絮团,仅有0.83%的细菌类群来自水体;3种生境共有的细菌主要为鲁杰氏菌(Ruegeria),在水体、絮团和对虾肠道中的丰度分别为1.72%、7.34%和6.00%,水体中特有的扩增子变异序列(amplicon sequence variants, ASV)数量为89个,主要属于海茎状菌(Maricaulis)和欧文威克斯菌(Owenweeksia),絮团中有56个,主要为莱茵海默氏菌(Rheinheimera),而对虾肠道中仅有10个,主要属于玫瑰杆菌(Roseobacter);中性模型结果表明,水体、絮团和对虾肠道细菌群落构建均符合中性模型,表明3种生境中细菌群落构建均受中性过程主导。【结论】在BFS系统中,不同生境的微生物群落具有显著差异,对虾肠道细菌主要来自生物絮团,而3种生境的细菌群落构建过程由中性过程主导。这些结果为调控生物絮团养殖系统中微生物群落提供了理论依据。     

Histopathology in Pacific oyster (Crassostrea gigas) spat caused by the dinoflagellate Prorocentrum rhathymum

The recognition of an apparent association between seasonal oyster spat mortalities (up to 40%) and high Prorocentrum rhathymum density in the Little Swanport Estuary, Tasmania, prompted further experimental investigation into the toxicity by this dinoflagellate. Standard brine shrimp, haemolysis assays and intraperitoneal mouse bioassays revealed fast acting toxins in methanol but not aqueous extracts of P. rhathymum, with mice dying in less than 20 min. Oyster bioassays involved feeding spat (4 mm shell width) for 21 consecutive days on a diet of cultured P. rhathymum at simulated bloom densities (10⁴ cells ml⁻¹). No oyster mortality was observed, however, histopathological signs of thin, dilated gut tubules and sloughing of gut cells resembled those seen in affected field samples. In contrast to field samples, gill pathology was also observed in experimental exposure oysters.     

Spermatozoa ultrastructure of the pink shrimp Farfantepenaeus paulensis (Decapoda: Dendrobranchiata)

Braga, A.L., Nakayama, C.L., Suita de Castro, L.A. and Wasielesky, W. 2011. Spermatozoa ultrastructure of the pink shrimp Farfantepenaeus paulensis (Decapoda: Dendrobranchiata). —Acta Zoologica (Stockholm) 00 : 1–6. The spermatozoa ultrastructure of the pink shrimp Farfantepenaeus paulensis was investigated in this morphological study. Spermatophores and spermatozoa were analyzed by electron microscopy. The pink shrimp spermatophore is divided into two regions: the appendage and the spermatophore main body, where spermatozoa are grouped in a spermatic mass. Pink shrimp spermatozoa are unistellate and are composed of main body and single spike. The spermatozoa body comprises a perinuclear cytoplasmic band, nucleus, acrosomal cap, and subacrosomal region. The spermatozoa cell mean total length was 10.71 μm, the mean body diameter was 5.56 μm, and the mean spike length and diameter were 5.15 μm and 0.85 μm, respectively.     

Purification and Comparison of β-1,3-Glucan Binding Protein From White Shrimp (Penaeus vannamei)

A β-glucan binding protein (BGBP) was identified in both white (Penaeus vannamei) and blue shrimp (P. stylirostris) plasma. White shrimp BGBP was purified by affinity chromatography using immobilized laminarin, and its molecular and biological properties were described. White shrimp BGBP is a monomeric protein with a molecular mass of 100 kDa, similar to those described for other crustacean BGBPs. White and blue shrimp BGBPs can be detected with antisera against crayfish BGBP and brown shrimp BGBP. Both amino acid composition and N-terminal sequence are markedly similar to brown shrimp (P. californiensis) and crayfish (Pacifastacus leniusculus) BGBP, indicating that this recognition protein is present in freshwater and marine crustaceans.