Abstract: Objective　To examine the impact of extreme temperature and drought stress on the survival of Bulinus globosus, so as to provide the theoretical evidence for the genomic research of Bulinus in absence of reference genes. Methods‌　B. globosus snail samples were collected from Kiwani Shehia in Pemba Island, Zanzibar, Tanzania, and offspring snails were obtained through laboratory breeding and reproduction. A total of 120 10⁃week⁃old B. globosus snails from the same generation were selected and randomly assigned into four groups, including the high⁃temperature drought (HD) group, normal temperature drought (D) group, low⁃temperature drought (LD) group, and the control (C) group, of 30 snails in each group. Snails in HD, D, and LD groups were placed in beakers containing dry soil at the bottom and subsequently housed in climate chambers at 35, 26 ℃, and 10 ℃, respectively, while snails in Group C were maintained in 500 mL petri dishes containing dechlorinated tap water at 26 ℃. Following 3 days of breeding, living snails in each group were collected, and soft tissues were dissected and isolated. Total RNA was extracted from snail soft tissues for library construction, followed by high⁃throughput sequencing on the Illumina HiSeq 4000 sequencing system. De novo transcriptome assembly was performed using the Trinity software, and the longest transcripts were selected as unigenes. Gene functional annotations of unigenes were conducted using the Diamond software against Gene Ontology (GO) knowledgebase, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, NCBI non⁃redundant (NR) protein sequences database, Protein Family (Pfam) database, and UniProtKB/Swiss⁃Prot (Swiss⁃Prot) knowledgebase. GO and KEGG enrichment analyses of differentially expressed genes (DEGs) were performed using the topGO and clusterProfiler software, respectively. In addition, four relevant genes were selected for validation using a real⁃time quantitative PCR (qRT⁃PCR) assay to verify the reliability of transcriptome sequencing results. Results　Following 3 days of breeding, there were 7, 20, 28, and 30 survival B. globosus snails in HD, LD, D, and C groups, with corresponding survival rates of 23.33% (7/30), 66.67% (20/30), 93.33% (28/30), and 100.00% (30/30), respectively ([χ2]  = 52.72, P < 0.001). De novo transcriptome assembly generated 176 942 unigenes, with annotation rates of 0.98%, 13.49%, 26.46%, 12.48%, and 14.39% against GO knowledgebase, KEGG pathway database, NR protein sequences database, Pfam database, and Swiss⁃Prot knowledgebase, respectively. There were 33 up⁃regulated and 72 down⁃regulated genes in Group D, 483 up⁃regulated and 815 down⁃regulated genes in Group HD, and 245 up⁃regulated and 172 down⁃regulated genes in Group LD relative to in Group C. Following removal of overlapping genes across groups and unmatched genes, 11 candidate genes were identified. GO and KEGG analyses revealed 3 heat shock protein (HSP)⁃related DEGs in these 11 candidate genes, which were annotated as HSP12.2, HSP70, and HSP20 genes and were all significantly up⁃regulated in each treatment group. Three immune⁃ and nervous system⁃related DEGs were identified, and were all significantly down⁃regulated in each treatment group, which were involved in the neural cell adhesion molecule L1⁃like protein pathway, fibrinogen binding protein pathway, and leukocyte elastase inhibitor⁃like protein pathway. qRT⁃PCR assay quantified that the expression trends of four genes related to temperature and drought stress across different treatment groups were highly consistent with transcriptome sequencing data. Conclusion　The survival rate of B. globosus significantly reduces under combined stresses of extreme temperature and drought, possibly due to an imbalance in its cellular homeostasis regulatory system.

Key words: Bulinus globosus, Schistosoma haematobium, De novo transcriptome, Temperature stress, Drought stress, Combined stress

摘要： 目的　探究极端温度和干旱胁迫对球形小泡螺生存能力的影响，为无参考基因条件下小泡螺基因组学研究提供理论依据。方法　自桑给巴尔奔巴岛Kiwani Shehia地区现场采集球形小泡螺样本，经实验室饲养、繁殖获取子代螺。选取120只来自同一代的10 周龄小泡螺作为实验螺，随机分配至高温干旱组（HD组）、常温干旱组（D组）、低温干旱组（LD组）和对照组（C组），每组30只螺。将HD组、D组、LD组小泡螺均置于底部为干燥土壤的烧杯中，随后分别置于35、26 ℃和10 ℃的气候培养箱中；C组小泡螺置于装有26 ℃去氯自来水的 500 mL培养皿中。饲养3 d后收集各组活螺，解剖分离软体组织，提取样本总 RNA并构建相应基因库，随后在Illumina HiSeq 4000 测序平台进行高通量测序。采用Trinity 软件进行从头转录组组装并选取最长的转录本作为单基因簇。使用DIAMOND软件对单基因簇进行基因功能注释，用于基因功能标注的数据库包括基因本体论（Gene Ontology，GO）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）、NCBI非冗余蛋白序列数据库（NCBI non⁃redundant protein sequences database，NR）、蛋白质家族数据库（Protein Family Database，Pfam）和瑞士⁃Prot蛋白序列数据库（UniProtKB/Swiss⁃Prot，Swiss⁃Prot）。分别使用topGO 软件和clusterProfiler软件对差异表达基因（differentially expressed genes，DEGs）进行GO、KEGG分析。另外选择4个相关基因进行实时荧光定量PCR（real⁃time quantitative PCR，qRT⁃PCR）验证，以确定转录组测序结果的可靠性。结果　饲养3 d后，HD组、LD组、D组和C组分别有7、20、28只和30只小泡螺存活，各组存活率分别为 23.33%（7/30）、66.67%（20/30）、93.33%（28/30）和 100.00%（30/30），差异有统计学意义（[χ2] = 52.72，P < 0.001）。通过无参转录组测序装配了176 942个单基因簇，在上述5个数据库中的标注率分别为0.98%、13.49%、26.46%、12.48%和14.39%。基因表达水平分析结果显示，与C组相比，D组共有33个基因上调、 72个基因下调，HD组共有483个基因上调、815个基因下调， LD组共有245个基因上调、172个基因下调。去除各组重叠基因及不匹配基因后，共确定了11个候选基因。GO和KEGG分析结果显示，上述11个DEGs中，热休克蛋白（heat shock protein，HSP）相关DEGs有3个，分别注释为HSP12.2、HSP70、HSP20基因，在各处理组中的表达均显著上调；免疫和神经系统相关DEGs有3个，在各处理组中均显著下调，涉及神经细胞黏附分子L1样蛋白通路、纤维蛋白原结合蛋白通路和白细胞弹性蛋白酶抑制剂样蛋白‌通路。qRT⁃PCR检测结果显示，4个与温度及干旱胁迫相关的基因表达水平在不同处理组中的表达趋势与转录组测序结果高度一致。结论　极端温度和干旱联合胁迫下球形小泡螺存活率降低，可能与其细胞内稳态调节系统失衡有关。

关键词: 球形小泡螺, 埃及血吸虫, 无参转录组, 温度胁迫, 干旱胁迫, 联合胁迫