Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
BMC Vet Res. 2013 Sep 30;9:189. doi: 10.1186/1746-6148-9-189.
Abdominal segment deformity disease (ASDD) of cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei causes economic loss of approximately 10% in affected specimens because of the unsightliness of distorted abdominal muscles. It is associated with the presence of viral-like particles seen by electron microscopy in the ventral nerve cords of affected shrimp. Thus, shotgun cloning was carried out to seek viral-like sequences in affected shrimp.
A new retrovirus-like element of 5052 bp (named abdominal segment deformity element or ASDE) was compiled by shotgun cloning and 3' and 5' RACE using RNA and DNA extracted from ventral nerve cords of ASDD shrimp. ASDE contained 7 putative open reading frames (ORF). One ORF (called the PENS sub-domain), had a deduced amino acid (aa) sequence homologous to the GIY-YIG endonuclease domain of penelope-like retrotransposons while two others were homologous to the reverse transcriptase (RT) and RNaseH domains of the pol gene of non-long terminal repeat (non-LTR) retrotransposons (called the NLRS sub-domain). No single amplicon of 5 kb containing both these elements was obtained by PCR or RT-PCR from ASDD shrimp. Subsequent analysis indicated that PENS and NLRS were not contiguous and that NLRS was a host genetic element. In situ hybridization using a dioxygenin-labeled NLRS probe revealed that NLRS gave positive reactions in abdominal-ganglion neurons of ASDD shrimp but not normal shrimp. Preliminary analysis indicated that long-term use of female broodstock after eyestalk ablation in the hatchery increased the intensity of RT-PCR amplicons for NLRS and also the prevalence of ASDD in mysis 3 offspring of the broodstock. The deformities persist upon further cultivation until shrimp harvest but do not increase in prevalence and do not affect growth or survival.
Our results suggested that NLRS is a shrimp genetic element associated with ASDD and that immediate preventative measures could include shorter-term use of broodstock after eyestalk ablation and/or discard of broodstock that give strong RT-PCR reactions for NLRS. In the longer term, it is recommended, if possible, that currently used, domesticated shrimp lines be selected for freedom from NLRS. The molecular tools developed in this work will facilitate the management and further study of ASDD.
养殖凡纳滨对虾（Litopenaeus vannamei）的腹部节段畸形病（ASDD）导致受影响标本的经济损失约为 10%，因为变形的腹部肌肉不美观。它与电子显微镜下在受影响虾的腹神经索中观察到的类似病毒颗粒有关。因此，进行了鸟枪法克隆以寻找受影响虾中的类似病毒序列。
通过鸟枪法克隆和使用从 ASDD 虾的腹神经索提取的 RNA 和 DNA 进行 3' 和 5'RACE，编译了一个新的逆转录病毒样元件，长 5052bp（命名为腹部节段畸形元件或 ASDE）。ASDE 包含 7 个推定的开放阅读框（ORF）。一个 ORF（称为 PENS 亚域）具有与 penelope 样逆转录转座子的 GIY-YIG 内切酶结构域同源的推导氨基酸（aa）序列，而另外两个与非长末端重复（non-LTR）逆转录转座子的 pol 基因的逆转录酶（RT）和 RNaseH 结构域同源（称为 NLRS 亚域）。通过 PCR 或 RT-PCR 从未受影响的虾中未获得包含这两个元件的 5kb 单一扩增子。随后的分析表明，PENS 和 NLRS 不连续，NLRS 是宿主遗传元件。使用二氧杂环素标记的 NLRS 探针进行原位杂交显示，NLRS 在 ASDD 虾的腹神经节神经元中产生阳性反应，但在正常虾中没有。初步分析表明，在孵化场中切除眼柄后长期使用雌亲虾增加了 NLRS 的 RT-PCR 扩增子的强度，并且在亲虾的无节幼体 3 代中增加了 ASDD 的流行率。在收获虾之前，畸形会持续存在，但不会增加流行率，也不会影响生长或存活。
我们的结果表明，NLRS 是与 ASDD 相关的虾遗传元件，立即采取的预防措施可能包括在切除眼柄后短期使用亲虾和/或丢弃对 NLRS 产生强烈 RT-PCR 反应的亲虾。从长远来看，如果可能的话，建议选择目前使用的、驯化的虾系，使其免受 NLRS 的影响。本工作中开发的分子工具将有助于 ASDD 的管理和进一步研究。
