Queen Conch (<em>Strombus gigas</em>) Testis Regresses during the Reproductive Season at Nearshore Sites in the Florida Keys

BackgroundQueen conch (Strombus gigas) reproduction is inhibited in nearshore areas of the Florida Keys, relative to the offshore environment where conchs reproduce successfully. Nearshore reproductive failure is possibly a result of exposure to environmental factors, including heavy metals, which are likely to accumulate close to shore. Metals such as Cu and Zn are detrimental to reproduction in many mollusks. Methodology/Principal FindingsHistology shows gonadal atrophy in nearshore conchs as compared to reproductively healthy offshore conchs. In order to determine molecular mechanisms leading to tissue changes and reproductive failure, a microarray was developed. A normalized cDNA library for queen conch was constructed and sequenced using the 454 Life Sciences GS-FLX pyrosequencer, producing 27,723 assembled contigs and 7,740 annotated transcript sequences. The resulting sequences were used to design the microarray. Microarray analysis of conch testis indicated differential regulation of 255 genes (p<0.01) in nearshore conch, relative to offshore. Changes in expression for three of four transcripts of interest were confirmed using real-time reverse transcription polymerase chain reaction. Gene Ontology enrichment analysis indicated changes in biological processes: respiratory chain (GO:0015992), spermatogenesis (GO:0007283), small GTPase-mediated signal transduction (GO:0007264), and others. Inductively coupled plasma-mass spectrometry analysis indicated that Zn and possibly Cu were elevated in some nearshore conch tissues. Conclusions/SignificanceCongruence between testis histology and microarray data suggests that nearshore conch testes regress during the reproductive season, while offshore conch testes develop normally. Possible mechanisms underlying the testis regression observed in queen conch in the nearshore Florida Keys include a disruption of small GTPase (Ras)-mediated signaling in testis development. Additionally, elevated tissue levels of Cu (34.77 ng/mg in testis) and Zn (831.85 ng/mg in digestive gland, 83.96 ng/mg in testis) nearshore are similar to reported levels resulting in reproductive inhibition in other gastropods, indicating that these metals possibly contribute to NS conch reproductive failure.背景：相较于繁殖成功率较高的远岸环境，佛罗里达礁岛群近岸海域的大凤螺（Queen conch，学名*Strombus gigas*）繁殖活动受到显著抑制。近岸个体的繁殖失败可能源于暴露于近岸易富集的各类环境因子，其中包括重金属。铜（Cu）、锌（Zn）等重金属对诸多软体动物的繁殖均具有毒害作用。 材料与方法/主要结果：组织学检测显示，相较于繁殖机能健康的远岸大凤螺，近岸个体的性腺出现萎缩。为阐明引发组织病变与繁殖失败的分子机制，本研究构建了基因芯片（microarray）。研究人员利用454生命科学GS-FLX焦磷酸测序平台（454 Life Sciences GS-FLX pyrosequencer），对大凤螺的标准化cDNA文库进行构建与测序，最终获得27723条组装重叠群（contigs）与7740条注释转录本序列。所得序列被用于该基因芯片的探针设计。针对大凤螺睾丸的基因芯片分析显示，相较于远岸个体，近岸个体共有255个基因呈现差异表达（p<0.01）。研究通过实时逆转录聚合酶链式反应（real-time reverse transcription polymerase chain reaction）验证了4个目标转录本中3个的表达变化。基因本体（Gene Ontology, GO）富集分析显示，多个生物学过程出现显著变化：包括呼吸链（GO:0015992）、精子发生（GO:0007283）、小GTP酶介导的信号转导（GO:0007264）等。电感耦合等离子体质谱（Inductively coupled plasma-mass spectrometry）分析显示，部分近岸大凤螺组织内的锌（Zn）以及可能的铜（Cu）水平出现升高。 结论与意义：睾丸组织学结果与基因芯片数据的一致性表明，在繁殖季，近岸大凤螺的睾丸会发生退化，而远岸个体的睾丸则可正常发育。佛罗里达礁岛群近岸大凤螺睾丸退化的潜在机制之一，可能是睾丸发育过程中小GTP酶（Ras）介导的信号通路遭到破坏。此外，近岸个体组织内升高的铜（睾丸中为34.77 ng/mg）与锌（消化腺中为831.85 ng/mg，睾丸中为83.96 ng/mg）水平，与此前报道的可引发其他腹足类动物繁殖抑制的重金属浓度相近，提示这两类重金属可能是导致近岸大凤螺繁殖失败的诱因。