投喂不同油脂饲料对中华绒螯蟹免疫、代谢及耐低氧性能的影响

为研究饲料中添加鱼油和豆油对中华绒螯蟹(Eriocheir sinensis)成蟹生长、免疫、代谢和耐低氧性能的影响,配制了添加不同比例鱼油和豆油的3种蟹用饲料,添加3%鱼油饲料组、3%豆油饲料组、3%鱼油和豆油混合组(1∶1,质量比),将其分别投喂中华绒螯蟹115 d后测量蟹体重、壳长和壳宽的变化,再将其放入溶解氧(dissolved oxygen,DO)为(9.06±0.06)mg/L和(2.57±0.44)mg/L的水体中,测定其免疫、代谢指标及耐低氧性能的变化。结果发现:投喂添加3种不同油脂饲料的中华绒螯蟹各组间体重无显著性差异;低氧胁迫对中华绒螯蟹代谢指标影响较大;添加鱼油和豆油混合油饲料组中华绒螯蟹血细胞密度、血蓝蛋白含量及超氧化物歧化酶、酸性和碱性磷酸酶、乳酸脱氢酶的活性都为最高,说明鱼油与豆油混合添加对中华绒螯蟹免疫和抗氧化能力有促进作用,并增加其耐低氧能力。     

CuSO4对中华绒螯蟹蜕皮、生长和存活的影响

通过向水中添加不同浓度的铜(Cu2 ),观察其对中华绒螯蟹(Eriocheir sinensis)Ⅰ期幼蟹(0.020±0.01g)和12月龄扣蟹(3.34±0.26 g)的毒性影响。Cu2 对Ⅰ期幼蟹24,48,72和96h的半致死浓度(LC50)分别为0.70,0.43,0.33和0.22 mg/L,而对12月龄扣蟹相应的LC50分别是18.20,10.23,9.12和8.51mg/L。中华绒螯蟹Ⅰ期幼蟹在0.00,0.01,0.02,0.03,0.05和0.08mg/L Cu2 的水环境中的蜕皮率、增重率和存活率的比较研究结果表明,虽然各浓度组存活率均高于50%,但其随着Cu2 浓度的增高而降低。增重率和蜕皮率的变化趋势与存活率相似。此外,研究了中华绒螯蟹12月龄扣蟹在0.00,0.01,0.05,0.10,0.50,1.00和2.50 mg/L Cu2 的水环境中蜕皮率、增重率和存活率的变化。结果显示,各组存活率均高于50%,除0.01mg/L处理组的存活率略高于对照组外,总的变化趋势是随着Cu2 浓度的增高而降低。增重率和蜕皮率随着Cu2 浓度的增高,总的变化趋势亦逐渐降低。相关性分析表明,中华绒螯蟹Ⅰ期幼蟹和12月龄扣蟹的生长、蜕皮和存活与水中添加Cu2 的浓度增加有极显著的负相关(P<0.01)。     

饥饿对中华绒螯蟹(Eriocheir sinensis)幼体发育的影响

对刚孵化的中华绒螯蟹第一期的蚤状幼体经不同时间的饥饿后再投喂,发现饥饿可以明显降低幼体的存活率和延长幼体的发育期。实验表明:对中华绒螯蟹第一期的蚤状幼体的饥饿时间(t)和发育期长(D)呈线性关系(D=4.6303+1.3226t r=0.970p<0.01)。对于中华绒螯蟹第一期的蚤状幼体,当起始饥饿时间超过了4d,再予以投饵,幼体均不能恢复正常的发育和蜕皮功能,得出中华绒螯蟹的不可恢复点(the point of no-return,PNR)大约为4d。通常以产生50%的幼体死亡的饥饿期即PNR₅₀,来表明幼体对饥饿的抵抗能力,实验得出中华绒螯蟹第一期的蚤状幼体的PNR₅₀大约为48h。     

果胶和木聚糖对中华绒螯蟹肠道菌群结构的影响

本研究旨在揭示果胶和木聚糖对中华绒螯蟹(Eriocheir sinensis)肠道菌群结构的影响。将果胶和麦麸(木聚糖源)分别以8%和30%的比例添加到饲料中制成试验饲料,将经过酶解的果胶和麦麸添加到饲料中制备成对照饲料,饲养初始体重为(5.9依0.4)g的中华绒螯蟹8周。养殖试验结束后,采用16S r DNA荧光定量PCR法分析测定了中华绒螯蟹肠道菌群结构。结果显示中华绒螯蟹肠道细菌主要由变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、梭杆菌门(Fusobacteria)的细菌组成。果胶组肠道菌群丰度指数Ace和Chao分别低于其对照组31%和39%,肠道菌群多样性指数Simpson比对照组高25%,而Shannon指数比对照组低48%。木聚糖组Ace和Chao指数均比果胶组高约25%,比其酶解对照组高约14%。木聚糖组的多样性指数与其酶解木聚糖组及果胶组的差异较小。上述结果表明,果胶和木聚糖均影响中华绒螯蟹肠道的菌群结构,但两者对肠道菌群丰度的影响不同。     

中华绒螯蟹幼体发育阶段对淀粉营养需要的研究

本实验研究了饲料中淀粉含量对中华绒螯蟹幼体生长发育与淀粉酶活力的影响。结果表明:饲料中不同淀粉含量对中华绒螯蟹幼体存活率、变态率和增重量的影响显著(P1、Z2、Z3、Z4)或下降(Z5、M)趋势,且饲料中淀粉含量对幼体淀粉酶活力变化有显著影响(P1、Z2、Z34、Z5、M的适宜淀粉需求量分别为18%、22%、18%、18%-22%、18%、14%-18%。       

从线粒体16S rDNA序列探讨绒螯蟹类的系统发生关系

测定了绒螯蟹类各物种的线粒体16SrDNA部分片段的序列,构建了NJ树、ML树和MP树。序列歧异数据比较和各系统发生树都支持新绒螯蟹属(Neoeriocheir)为一个独立的属。在3种系统发生树中,直额绒螯蟹(Eriocheir recta)都是绒螯蟹属(Eriocheir)所有其它成员的姐妹群,并且广东珠江1只直额绒螯蟹标本的16SrDNA部分序列与台湾产台湾绒螯蟹(Eriocheir formasa)的相应序列相同。这些结果不支持平绒螯蟹属(Platyeriocheir)是一个有效的属,并表明E．formosa是E．recta的同物异名。绒螯蟹属(Eriocheir)所有其它成员聚为一个单系的分支,支持中华绒螯蟹、合浦绒螯蟹与日本绒螯蟹属于同一个物种Eriocheir japonica。16SrDNA部分序列的比对表明,产于台湾的日本绒螯蟹的此段序列与合浦绒螯蟹的相同,产于崇明岛的和产于美国旧金山海湾的中华绒螯蟹的此段序列与中华绒螯蟹单元型B的序列相同。     

背角无齿蚌四种组织细胞原代培养的比较

以背角无齿蚌（Anodonta woodiana）血细胞（含颗粒细胞和透明细胞）、消化腺细胞（含上皮样细胞和圆形小细胞）、外套膜细胞（含上皮样细胞、透明细胞、圆形大细胞和圆形小细胞）、斧足细胞（含上皮样细胞、透明细胞、圆形大细胞和圆形透亮细胞）为材料,比较了上述各类细胞的原代培养特征以及不同条件下的细胞存活率,优化了细胞制备及细胞活性鉴定的方法。结果显示,上述各类组织细胞存活率受不同培养温度、培养时间和培养基种类的影响,其中,血细胞96 h在20℃下、培养基2中的存活率最高,为94.67%±0.47%;消化腺细胞48 h在20℃下、培养基3中的存活率最高,为93.67%±1.70%;外套膜细胞48h在15℃下、培养基1中的存活率最高,为93.67%±1.7%;斧足细胞48h在15℃下、培养基1的存活率最高,为94.33%±0.94%。相较于25℃,在20℃和15℃下,4种组织细胞96h的存活率更为理想。本研究旨在为背角无齿蚌组织细胞系的建立及贝类细胞水平毒理学的研究提供基础数据。     

脂肪酸对离体培养中华绒螯蟹肝胰腺PUFAs合成相关基因表达的影响

长期以来海水水生动物和淡水水生动物多不饱和脂肪酸合成能力一直是众多研究的重点。相比于淡水水生动物,海水水生动物更容易从食物中获得多不饱和脂肪酸,因此其合成多不饱和脂肪酸的能力低于淡水水生动物。中华绒螯蟹作为一种洄游型甲壳动物,其脂肪酸合成能力的研究显得更加重要。本研究通过中华绒螯蟹肝胰腺离体培养技术,利用不同的脂肪酸培养中华绒螯蟹肝胰腺组织,旨在探讨不同脂肪酸对中华绒螯蟹肝胰腺多不饱和脂肪酸合成相关基因表达的影响,为研究饲料中添加不同脂肪酸对中华绒螯蟹多不饱和脂肪酸合成的影响提供了一定的理论参考依据。本研究表明:油酸会显著增加中华绒螯蟹肝胰腺ELOVL6、fad6、和fad9基因的表达量;不同脂肪酸对fad6和fad9基因表达量的影响相似,且相比于EPA和DHA,亚麻酸和油酸会显著增加fad6和fad9基因的表达。通过本研究,我们得出EPA和DHA等多不饱和脂肪酸会抑制多不饱和脂肪酸合成的过程。进一步证实了海水水生动物多不饱和脂肪酸合成能力和海水中高含量的多不饱和脂肪酸之间的关系。     

基于稳定同位素的黄河三角洲盐碱地稻蟹种养系统中华绒螯蟹食物源分析

为探讨盐碱地稻蟹种养系统中华绒螯蟹食物源构成,2020年6—10月,在山东东营垦利区,采集稻田中华绒螯蟹及其所有可能食物来源样品,包括植物(伊乐藻、菹草、金鱼藻、浮萍、水稻茎叶、稻谷)、动物(底栖动物、浮游动物)、有机碎屑和人工饲料(配合饲料、玉米粕),并利用碳、氮稳定同位素(δ¹³C和δ¹⁵N)值进行分析,定量其在中华绒螯蟹食物组成中的贡献率。结果表明: 食物源样品δ¹³C值范围为-30.09‰～-11.24‰,δ¹⁵N值范围为0.03‰～12.78‰,不同食物源δ¹³C和δ¹⁵N值呈现明显差异。中华绒螯蟹肌肉中δ¹³C值变化范围为-24.61‰～-20.08‰,δ¹⁵N值变化范围为4.74‰～9.21‰,表明稻蟹种养系统中华绒螯蟹食物来源较丰富。养殖期间各食源贡献率为植物(46.7%～57.1%)>动物(21.5%～24.5%)>人工饲料(10.9%～21.3%)>有机碎屑(7.1%～7.9%)。可见,盐碱地稻田系统天然饵料基本能够满足中华绒螯蟹摄食需求,即使本试验投喂非动物性人工饵料,也未改变中华绒螯蟹主要食源贡献率。     

中华绒螯蟹呼肠孤病毒样病毒病研究

为了探讨中华绒螯蟹(Eriocheir sinensis)颤抖病的发生原因,从表现出颤抖症状的中华绒螯蟹中初步分离纯化出一种病毒,人工感染健康触,出现明显的阳性反应,并从人工感染的蟹的血液、肠道、性腺等组织中检测到病毒,表明分离到的病毒为中华绒螯蟹的病原 ,且对野生锯齿华溪蟹(Sesarnadehanni)也具感染性.电镜观察结果显示该病毒粒子呈球状,无囊膜,大小为55nm左右.病毒核酸在1%琼脂糖凝胶电泳中,电泳图谱呈现3/3/4/2型,总分子量约20kb.该核酸对DNaseⅠ不敏感,对RNase敏感,推测病毒核酸为dsRNA,从病毒形态大小、病毒核酸等特性初步确定该病毒为呼肠孤病毒样病毒(Reovirus-like Virus).该研究为探讨中华绒螯蟹颤抖病的发生原因及防治方法有很大的指导意义.     

Effects of pH, temperature, and osmolarity on the morphology and survival rate of primary hemocyte cultures from the Mitten Crab, Eriocheir sinensis

Hemocytes are the main immune defense cells in crustacean, and its in vitro culture can be a useful tool for the study of host and pathogen interaction. In the present study, the primary hemocyte culture of Chinese mitten crab (Eriocheir sinensis), including mixed and single hemocyte, was set up for the first time. In this study, different pH (6.4, 6.8, 7.2, 7.6, and 8.0), temperature (26, 28, and 30°C), and osmolarity (500, 700, 900, 1,100, and 1,300 mOsm kg^?1) values were tested. Moreover, the effects of two types of medium (1× L-15 and 3× L-15) with the same osmolarity on hemocyte culture were evaluated. After incubation at different culture conditions, the morphological changes (degranulation, lysis, shrinkage, and detachment) and survival rate of hemocytes were taken into account in order to evaluate the culture condition effect. Our results showed that the total hemocyte counts of Chinese mitten crab were about 2.5?×?10⁷ cells ml^?1, and three subpopulations of hemocytes were distinguished as granulocytes (43.46?±?4.98%), semigranulocytes (31.04?±1.95%), and hyalinocytes (25.50?±4.89%). The optimal culture condition for primary hemocytes of Chinese mitten crab was 3× L-15 medium, 1,100 mOsm kg^?1, pH 6.8 at 28°C. Hemocytes at optimal culture condition could retain a better morphology and higher survival rate: hemocytes retained a survival rate >60% after 5 d and >40% after 7 d. Furthermore, the hemocyte subpopulations were isolated by Percoll step gradient centrifugation and cultured in optimized hemocyte culture conditions. The results showed that hyalinocytes and semigranulocytes could maintain a survival rate of >50% after 15 d, while granulocytes only retained a survival rate of 26% after 5 d.     

Development of primary cell culture from <Emphasis Type="Italic">Scylla serrata</Emphasis>

This paper reports for the first time, the Primary cell culture of hepatopancreas from edible crab Scylla serrata using crab saline, L-15 (Leibovitz), 1 × L-15 + crab saline, 2 × L-15 + crab saline, 3 × L-15 and citrate buffer without any serum. We could isolate and maintain E (Embryonalzellen), F (Fibrenzellen), B (Blasenzellen), R (Restzellen) and G (Granular cells). Upon seeding the hepatopancreatic E, F, B, and R cells showed different survival pattern over time than granular cells. A modified L-15 (3×) medium supported the best survival of hepatopancreatic E, F B, and R cells in in-vitro culture. However granular cells could be maintained for 184 days with L-15 (1×) + crab saline. Fetal bovine serum was not effective additive and hampered cell viability in present study.     

Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 g l⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 μg ml⁻¹ chloramphenicol, 100 μg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2′-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT₅₀/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC₅₀. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.     

Characterization of cell clusters in larval hemolymph of the cabbage armyworm Mamestra brassicae and their role in maintenance of hemocyte populations

Maintenance of hemocyte populations is critical for both development and immune responses. In insects, the maintenance of hemocyte populations is regulated by mitotic division of circulating hemocytes and by discharge from hematopoietic organs. We found cell clusters in the hemolymph of Mamestra brassicae larvae that are composed of small, spherical cells. Microscopic observations revealed that the cells in these clusters are similar to immature or precursor cells present in hematopoietic organs. The results of bromodeoxyuridine (BrdU) incorporation experiments demonstrate that these cells are mitotically active. Furthermore, these cells maintain their immature state and proliferate until late in the last larval instar. The results of in vitro experiments showed that most of the cells changed their morphology to one consistent with plasmatocytes or granulocytes, and that the change was promoted by addition of larval hemolymph to the culture medium, in particular when hemolymph was collected at a prepupal stage. Taken together, our results suggested that cells in clusters may be an additional source of hemocytes during larval development.     

Selection of a standard culture medium for primary culture of <Emphasis Type="Italic">Limulus polyphemus</Emphasis> Amebocytes

Summary This study provides information relevant to future research aimed at producing Limulus Amebocyte Lysate (LAL) in vitro, which would potentially reduce the need to harvest and bleed horseshoe crabs as in the current methods of LAL production. To address the need for primary culture of horseshoe crab amebocytes, this study tested the effects of a variety of standard insect cell culture media on amebocyte morphology and viability after 7 d of maintenance. Amerbocyte morphology was least altered from in vivo form in Grace’s Modified Insect Medium, with no observed degranulation of cells, as compared to the other media tested. There were significant differences in amebocyte viability among the six insect cell culture media tested. Grace’s Modified Insect Medium sustained viability of 77.2±5.1% (mean ± standard deviation) of amebocytes, followed distantly by Grace’s Insect Medium with 35.1±8.7% amebocyte viability. Results indicate that Grace’s Modified Insect Medium with horseshoe crab serum supplementation was the best candidate of the six media tested for future medium optimization for Limulus amebocyte requirements.     

Heat-inactivated coelomic fluid of the earthworm Perionyx excavatus is a possible alternative source for fetal bovine serum in animal cell culture

Fetal Bovine Serum (FBS) is used as a major supplement in culturing animal cells under in vitro conditions. Due to ethical concern, high cost, biosafety, and geographical as well as batchwise result variations, it is important to reduce or replace the use of FBS in animal cell culture. The major objective of this work is to evaluate the feasibility of heat-inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a possible alternative for FBS in animal cell culture experiments. The coelomic fluid (CF) was extruded from the earthworm using electric shock method and used for the experiments. Electric shock method is a simple non-invasive technique, which has no harmful effect on earthworms. Mouse primary fibroblast and HeLa cell lines were used in this study. Among HI-CF, autoclaved CF and crude CF, the supplement of medium with HI-CF shows positive results. The processed HI-CF (90°C for 5 min) at 10% supplement in cell culture medium promote maximum cell growth but cells need the initial support of FBS for the attachment to the culture flask. Microscopic observation and immunofluorescence assay with actin and lamin A confirm that the cellular and molecular morphology of the cells is maintained intact. The HI-CF of earthworm, P. excavatus has shown better cellular viability when compared with FBS and making it possible as an alternative supplement to minimize the use of FBS.     

An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

Hemolymph analysis and evaluation of newly formulated media for culture of shrimp cells (Penaeus stylirostris)

Summary Creation of a shirmp cell line has been an elusive goal. This failure may be due to the composition of the cell culture medium, which may be inadequate to support primary cultured cells. Shrimp hemolymph should contain the nutritional components needed to support cell growth and division. We report here the comprehensive biochemical analysis of hemolymph from the blue shrimp,Penaeus stylirostris (Litopenaeus stylirostris) (see Holthuis, L. B. Shrimps, and prawns of the world, in: FAO species catalog. Vol. 1. Rome: Food and Agriculture Organization of the United Nations; 1980), for free amino acids (FAAs), carbohydrates, electrolytes, metals, pH, and osmolality. Levels of hemolymph components were compared to 2×L-15 with 20% fetal bovine serum, a commonly used culture medium for crustacean cells. The FAAs, taurine and proline, and the metals, strontium and zinc, were significantly higher in hemolymph than in the 2×L-15 medium. In contrast, other FAAs were up to 50 times higher in the 2×L-15 medium than in the hemolymph. To mimic more closely the hemolymph composition, we created two new media based on either the 0.2×L-15 or the M199 medium. We compared the microscopic appearance of cells cultured in these media and evaluated deoxyribonucleic acid (DNA) and protein synthesis by³H-thymidine uptake and³⁵S-methionine uptake assays. The ovary cells ofP. stylirostris cultured in either of the new media formed monolayers, while the cells cultured in 2×L-15 medium did not. Despite these differences, there was no evidence of sustained DNA or protein synthesis with any of the media. Future studies to establish a shrimp cell line should focus on analysis of the cell cycle and on overcoming the molecular blocks to cell division.