斑节对虾血淋巴低丰度蛋白双向电泳技术体系的优化

文章探索并优化了斑节对虾（Penaeus monodon）血淋巴双向电泳技术体系,对斑节对虾血淋巴总蛋白抽提方法进行优化,减小高丰度蛋白的影响;对双向电泳过程各步骤,如IPG胶条pH范围的选择、等电聚焦程序、上样量等进行了优化,得到高分辨率的2-DE图谱,用PDQuest软件进行了初步分析。结果表明,用PEG 6000能更快捷有效地去除血淋巴中的高丰度蛋白,增加低丰度蛋白的点数及浓度;在抽提蛋白过程中,利用预冷的80%丙酮洗涤,在一向电泳过程中设置100 V低压除盐,能更有效地去除蛋白中的盐分;采用18 cm、pH 3~10 NL 的中型胶条,能更好地显示低丰度蛋白点的分布;被动水化上样300 μg结合硝酸银染色方法,能有效提高双向电泳图谱中蛋白点的分离度和分辨率。该试验为斑节对虾及其他甲壳动物血淋巴蛋白质组学相关研究提供了稳定可重复的研究方法。     

Hollow sperm syndrome during spermatogenesis in the giant tiger shrimp Penaeus monodon (Fabricius 1798) from eastern Australia

During spermatogenesis, giant tiger shrimp (Penaeus monodon) from Queensland, eastern Australia had a high proportion of testicular spermatids that appeared ‘hollow’ because their nuclei were not visible with the haematoxylin and eosin stain. When examined by transmission electron microscopy, the nuclei of hollow spermatids contained highly decondensed chromatin, with large areas missing fibrillar chromatin. Together with hollow spermatids, testicular pale enlarged (PE) spermatids with weakly staining and marginated chromatin were observed. Degenerate‐eosinophilic‐clumped (DEC) spermatids that appeared as aggregated clumps were also present in testes tubules. Among 171 sub‐adult and adult P. monodon examined from several origins, 43% displayed evidence of hollow spermatids in the testes, 33% displayed PE spermatids and 15% displayed DEC spermatids. These abnormal sperm were also found at lower prevalence in the vas deferens and spermatophore. We propose ‘Hollow Sperm Syndrome (HSS)’ to describe this abnormal sperm condition as these morphological aberrations have yet to be described in penaeid shrimp. No specific cause of HSS was confirmed by examining either tank or pond cultured shrimp exposed to various stocking densities, temperatures, salinities, dietary and seasonal factors. Compared with wild broodstock, HSS occurred at higher prevalence and severity among sub‐adults originating from farms, research ponds and tanks. Further studies are required to establish what physiological, hormonal or metabolic processes may cause HSS and whether it compromises the fertility of male P. monodon.     

Effect of Sargassum wightii fucoidan on growth and disease resistance to Vibrio parahaemolyticus in Penaeus monodon post‐larvae

The polysaccharide – fucoidan was extracted from brown seaweed Sargassum wightii and its antibacterial activity was screened by agar well diffusion method. The maximum zone of inhibition observed was 15.66 mm in 20 mg mL^?1 concentration against Vibrio parahaemolyticus. The Minimum Inhibitory Concentration (MIC) of the fucoidan was 12 mg mL^?1 against V. parahaemolyticus. The fucoidan was then enriched with Artemia nauplii at four different concentrations such as 100, 200, 300 and 400 mg L^?1 for 12 h. The enriched Artemia nauplii were fed to Penaeus monodon post‐larvae for 20 days and the growth performance was assessed. The weight gain and SGR of the control group were 0.2432 g and 15.78%, respectively. But, in experimental groups fed with fucoidan enriched Artemia nauplii, the weight gain and SGR were increased and were respectively ranged from 0.2602 to 0.3161 g and from 16.11 to 17.05%. The P. monodon post‐larvae were challenged with V. parahaemolyticus for a period of 30 days showed a reduction in mortality percentage of experimental groups over the control group and it was ranged between 36.97 and 89.86%. During the challenge test, the V. parahaemolyticus load was also enumerated from the infected shrimp at every 10 day intervals. In the control group, the Vibrio load showed a linear increase in hepatopancreas and muscle tissues from 10th to 30th days of challenge test, whereas in the experimental groups, the Vibrio load established a declining trend with the advancement of challenged test.     

Evaluation of potential of lupin meal as an alternative to fish meal in juvenile Penaeus monodon diets

An indoor feeding trial in a flow-through marine water system was performed to evaluate the feasibility of using dehulled lupin Lupinus albus seed meal as a protein source to replace fish meal in diets for the juvenile Penaeus monodon. Five isonitrogenous (40% crude protein) diets formulated by replacing 0, 25, 50, 75, and 100% of fish meal protein by lupin meal protein were fed to shrimp (mean initial weight of 4.32 ± 0.23 g) three times daily at a feeding allowance of 5% body weight per day for 42 days. Shrimp fed diets with 0, 25, 50 and 75% replacement had similar (P > 0.05) weight gain, dry matter feed intake, feed conversion ratio (FCR), protein efficiency ratio (PER), and apparent net protein utilization (ANPU). Shrimp fed the highest dietary inclusion level of lupin meal (100% replacement) had significantly (P < 0.05) lower responses for all the above parameters than shrimp fed all other diets. Survival was high (87–100%) and similar for all diets. Apparent dry matter digestibility (ADMD) of diets with 25, 50, 75 and 100% replacement of fish meal with lupin meal was similar (75.6–76.6%) and significantly (P < 0.05) higher than that of diet with 0% replacement (73.9%). Diets containing the two highest lupin inclusion levels (75 and 100% replacement) had significantly (P < 0.05) better apparent protein digestibility (APD) than those containing the two lowest lupin meal inclusion levels (0 and 25% replacement). There were no significant differences (P > 0.05) in whole-body composition (dry matter, protein, lipid and ash) of shrimp fed on the various diets. Pellet water stability was inversely related to level of lupin meal inclusion. It was found, in this study, that up to 75% protein of fish meal can be replaced with the protein of dehulled lupin seed meal in diets for juvenile P. monodon. The diet with total replacement of fish meal containing 40% lupin meal was utilized very poorly by the shrimp.     

In vitro assembly of Penaeus monodon densovirus (PmDNV)‐like particles produced in a prokaryote expression system

Viral diseases are a significant problem in the shrimp aquaculture industry as outbreaks can cause significant mortality and economic loss. While it has been shown that triggering the shrimp RNA interference pathway through dsRNA is a potentially viable treatment pathway, this approach is hampered by the lack of a suitable delivery mechanism. Virus‐like particles (VLPs), which are structurally similar to native viruses but lack the genetic material, could possibly be developed as a delivery vehicle. To generate a candidate VLP, the Penaeus monodon densovirus (PmDNV) capsid protein was cloned with an added histidine tag and expressed in an E. coli expression system. While the protein was expressed in inclusion bodies, the recombinant PmDNV capsid protein could be dissolved and subsequently purified by nickel affinity column chromatography. The formation of VLP from this purified rPmDNV capsid protein was investigated by transmission electron microscopy, and PmDNV‐VLPs were observed that looked similar to the native PmDNV virion. Our results suggest that the PmDNV‐like particle could be promisingly applied towards vaccination and that this PmDNV‐like particle can potentially serve as a system for delivery of nucleic acids to trigger innate immunity in shrimp.     

草虾感染草虾杆状病毒（MBV）后肝胰腺上皮细胞病理变化的电镜观察

本文用改良的电镜固定液和新包埋剂制备超薄切片，在电镜下观察健康和感染ＭＢＶ的肝胰腺上皮细胞的超微病理变化，结果发现感染细胞的核质中有１个或多个包涵体和大量病毒粒子。随着包涵体和ＭＢＶ粒子增多，核染色质凝聚，核膜局部扩张，部份破裂，甚至完全消失，直至整个核崩解，使得ＭＢＶ和包涵体游离出来。胞质水肿，内质网髓鞘样变性，滑面内质网增生，脂滴增多，线粒体绒团样变性和并发细菌混合感染。作者认为核内包涵体和病     

蛋白水平对不同斑节对虾家系生长与消化酶活性的影响

为探究饲料蛋白水平与不同斑节对虾(Penaeus monodon)家系生长和消化酶之间的关系,研究了3种蛋白水平(34%、38%、42%)的饲料对16个斑节对虾家系的生长性状以及胃和肝脏消化酶活性的影响。经过56 d的养殖实验,结果表明:(1)随着蛋白水平的升高,所有斑节对虾家系的特定生长率均有所增加,F1、F2、F4、F7、F12、F13、F15等7个家系的特定生长率在34%、38%、42%3种蛋白水平组未见显著性差异(P>0.05),其余家系在3种或者其中的2种饲料之间有显著性差异(P<0.05);(2)斑节对虾特定生长率在家系和饲料蛋白质水平这两个因素间交互作用不显著(P=0.974);(3)所有斑节对虾家系的蛋白酶活性均随着饲料蛋白含量的增加而增加,淀粉酶活性有所下降,但规律不明显;(4)13个斑节对虾家系的肝脏蛋白酶活性、14个家系的胃蛋白酶活性均与家系特定生长率有着极显著的相关性(P<0.01),淀粉酶活性和特定生长率的相关性远小于蛋白酶活性。     

Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression

Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immune-related genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus-cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture.