TEMPERATURE ON DIV1 INFECTION IN MACROBRACHIUM ROSENBERGII AND TRANSCRIPTOMIC ANALYSIS
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摘要:
为探究温度对罗氏沼虾(Macrobrachium rosenbergii)感染十足目虹彩病毒1 (Decapod iridescent virus 1, DIV1)的影响及调控机制, 研究设置5组不同温度(26、28、30、32、34℃)对罗氏沼虾人工感染DIV1, 并统计其存活率, 结果显示温度34℃能够抑制罗氏沼虾体内的病毒复制, 减少死亡并延长其存活时间。对感染DIV1 24h和72h的罗氏沼虾肝胰腺、鳃及肌肉进行病毒载量测定, 结果表明感染DIV1的罗氏沼虾在72h内病毒迅速增殖, 但当水温升高至30℃及更高温度时其体内的病毒载量明显降低。此外, 采集罗氏沼虾不同温度下感染DIV1的肝胰腺进行转录组学分析, 结果表明共有8483个不同差异表达基因, 富集分析发现基因主要富集在花生四烯酸代谢(Arachidonic acid metabolism)、糖酵解/糖异生(Glycolysis / Gluconeogenesis)、α-亚油酸代谢(alpha-Linolenic acid metabolism)等与Warburg效应相关的代谢通路中, 推测这些基因和通路可能与病毒感染机制密切相关。对罗氏沼虾感染DIV1后的免疫基因CAT、Cu/ZnSOD、CTL、ACP的表达水平进行测定, 结果发现当温度为32℃时这些免疫基因的表达量显著高于其他温度, 表明高温能够促进罗氏沼虾的免疫基因表达量增加以抵御病毒入侵。研究通过分析不同温度对DIV1感染罗氏沼虾的影响, 初步揭示了温度对病毒复制的影响及调控机制, 为深入探究病毒感染的分子机制和开发抗病毒免疫技术奠定一定的基础。
Abstract:To investigate the effect and regulatory mechanism of temperature on Macrobrachium rosenbergii infected with Decapod iridescent virus 1 (DIV1), we set up five experimental groups at different temperatures (26, 28, 30, 32, and 34℃) for artificial DIV1 infection in this study, and calculated its survival rate. The results showed that the temperature of 34℃ significantly inhibited virus replication in M. rosenbergii, decreased mortality, and extended survival duration. The viral load in the hepatopancreas, gill, and muscle of M. rosenbergii infected with DIV1 was determined at 24h and 72h. The results found that the virus rapidly proliferated within 72hours, however, a notable reduction in the viral load decreased significantly when the water temperature increased to 30℃ or above. In addition, transcriptomic analysis was conducted on hepatopancreas of M. rosenbergii infected with DIV1 at different temperatures, and a total of 8483 differentially expressed genes were identified. Enrichment analysis revealed that these differentially expressed genes were mainly enriched in Arachidonic acid metabolism, Glycolysis/Gluconeogenesis, alpha-Linolenic acid metabolism and other metabolic pathways related to Warburg effect. It is speculated that these pathways and genes may be closely related to the mechanism of viral infection. Furthermore, the expression levels of immune genes CAT, Cu/ZnSOD, CTL, and ACP in M. rosenbergii infected with DIV1, revealing significantly higher expression at temperature of 32℃ compared to other temperatures. This indicates that high temperature can promote an increase in immune gene expression in M. rosenbergii to resist virus invasion. This study provides a comprehensive analysis of the temperature effects on DIV1 infection in M. rosenbergii, elucidating the effects and regulatory mechanism of temperature on virus replication. These findings lay a foundation for further exploration into the molecular mechanism of virus infection and the development of antiviral immune technology.
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图 1 罗氏沼虾在不同温度下感染DIV1后的存活率
Figure 1. Survival rate of M. rosenbergii after infection with DIV1 at different temperatures
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图 3 不同温度下感染DIV1 24h (A)和72h (B)罗氏沼虾的肝胰腺、鳃和肌肉组织中病毒载量的变化
不同字母表示组间差异显著(P<0.05)
Figure 3. Changes of viral load in hepatopancreas, gill, and muscle tissues of M. rosenbergii infected with DIV1 at different temperatures for 24h (A) and 72h (B)
Different letters indicate significant differences in groups (P<0.05)
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图 4 样本间基因表达的Pearson相关系数
颜色代表相关系数; 绿色. 高相关; 白色. 中等相关; 棕色. 低相关
Figure 4. Pearson correlation coefficient of gene expression in samples
The color represents the correlation coefficient; green represents high correlation, white represents medium correlation, brown represents low correlation
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图 5 C-vs.-T28 (A)、C- vs.-T32 (B)、C- vs.-T34 (C)差异表达基因的GO富集分析
Figure 5. GO enrichment analysis of differentially expressed genes in C- vs.-T28 (A), C- vs.-T32 (B), C- vs.-T34 (C)
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图 6 不同温度C- vs.-T28 (A)、C- vs.-T32 (B)、C- vs.-T34 (C)差异表达基因的KEGG富集分析
Figure 6. KEGG enrichment analysis of differentially expressed genes at different temperatures C-vs-T28 (A), C-vs-T32 (B), C-vs-T34 (C)
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图 7 罗氏沼虾感染DIV1后免疫基因的表达水平
**表示各组间有显著性差异(P<0.05)
Figure 7. The expression levels of immune genes in M. rosenbergii after infection with DIV1
* * means significant difference in groups (P<0.05)
表 1 罗氏沼虾感染DIV1的转录组测序数据(过滤后)
Table 1 Transcriptome analysis data of hepatopancreas of M. rosenbergii infected with DIV1 (after filtering)
Sample Raw read number Trimmed read number Trimmed bases Raw Q20 rate (%) Raw Q30 rate (%) Useful read (%) C1 40656152 40270600 6071846179 97.97 93.98 99.05 C2 45844954 45362930 6839330599 97.82 93.66 98.95 C3 41687698 41320418 6229797577 98.09 94.29 99.12 T26-1 50214336 49673918 7484682542 98.13 94.42 98.92 T26-2 52973998 52477322 7911017426 98.38 95.04 99.06 T26-3 48144434 47595962 7173781641 98.08 94.32 98.86 T28-1 51755682 51295874 7733108510 98.32 94.85 99.11 T28-2 57000770 56368302 8499234370 98.12 94.38 98.89 T28-3 47886244 47449286 7153565373 98.18 94.39 99.09 T30-1 53964458 53491966 8062804860 98.45 95.21 99.12 T30-2 45954522 45543808 6865548233 98.31 94.81 99.11 T30-3 52823954 52240446 7873885256 98.18 94.6 98.9 T32-1 53489536 53001530 7991157162 98.36 94.95 99.09 T32-2 56704528 56141238 8461995874 98.32 94.91 99.01 T32-3 54654938 54195174 8171456679 98.45 95.17 99.16 T34-1 50771510 50347114 7589411546 98.46 95.18 99.16 T34-2 49617766 49172430 7411627603 98.45 95.24 99.10 T34-3 49058398 48590290 7324474436 98.30 94.83 99.05 
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表 2 转录组测序数据与参考基因组的比对结果
Table 2 The alignment results with transcriptome analysis data and the reference genome
Sample Clean reads Total mapped
(%)Multiple mapped
(%)Uniquely
mapped (%)Mapped to
gene (%)Mapped to
InterGene (%)Mappe to
exon (%)C1 40270600 95.25 15.48 84.52 81.91 18.09 91.33 C2 45362930 95.69 15.57 84.43 81.78 18.22 91.46 C3 41320418 95.47 15.28 84.72 83.02 16.98 91.56 T26-1 49673918 94.19 14.86 85.14 68.91 31.09 87.51 T26-2 52477322 95.51 14.69 85.31 77.01 22.99 88.87 T26-3 47595962 95.23 16.36 83.64 73.20 26.80 86.50 T28-1 51295874 95.61 15.09 84.91 77.57 22.43 88.77 T28-2 56368302 95.70 17.06 82.94 77.30 22.70 89.94 T28-3 47449286 96.09 16.52 83.48 81.28 18.72 90.47 T30-1 53491966 95.36 13.22 86.78 81.49 18.51 90.10 T30-2 45543808 95.28 13.89 86.11 79.79 20.21 90.35 T30-3 52240446 95.07 20.53 79.47 72.44 27.56 88.57 T32-1 53001530 95.58 12.10 87.90 80.67 19.33 91.18 T32-2 56141238 95.92 20.38 79.62 79.01 20.99 90.01 T32-3 54195174 95.60 11.92 88.08 82.18 17.82 91.41 T34-1 50347114 95.47 11.43 88.57 81.52 18.48 90.96 T34-2 49172430 94.74 11.30 88.70 78.30 21.70 91.07 T34-3 48590290 95.00 13.84 86.16 80.25 19.75 91.06
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表 3 不同温度感染组的富集通路及基因(部分)
Table 3 Enrichment pathways and genes in the infected groups at different temperatures.(Part)
Sample Up Down Total Enrichment pathway Enrichment gene C-vs-T26 499 787 1,286 Pentose and glucuronate interconversions DER、RDH、
Bco、HPSE、
art、CYP2、
ACP、Gba、
NAGA、PSAP、
CROT、Crot、
PEX、CROTRetinol metabolism Glycosaminoglycan degradation Ascorbate and aldarate metabolism Biosynthesis of unsaturated fatty acids Arachidonic acid metabolism Lysosome Peroxisome PPAR signaling pathway C-vs-T28 404 519 923 Retinol metabolism RDH、Bco、
CYP、art、
Cyp、UGP、
Gale、CAT、
SOD、Ctl、
Aga、Tspan4、
PicotBiosynthesis of unsaturated fatty acids Arachidonic acid metabolism Amino sugar and nucleotide sugar metabolism alpha-Linolenic acid metabolism Peroxisome Lysosome PPAR signaling pathway C-vs-T30 816 998 1,814 Amino sugar and nucleotide sugar metabolism PGM、Uap、
GALE、UGP、
Ctl、ANPEP、
Gclc、Casp、
GPX、SOD、
CYP、Pla、
RPS、RPLRetinol metabolism Glutathione metabolism Fructose and mannose metabolism Arachidonic acid metabolism Starch and sucrose metabolism Biosynthesis of unsaturated fatty acids Lysosome Peroxisome Ribosome C-vs-T32 1,148 1243 2,391 Ribosome AMY、TSPO、
Lrp、SLC、
CPA、PRCP、
Acp、PCK、
GMPPB、Ctl、
Gfus、PyK、Carbohydrate digestion and absorption Cholesterol metabolism Protein digestion and absorption PPAR signaling pathway Adipocytokine signaling pathway Amino sugar and nucleotide sugar metabolism Glycolysis / Gluconeogenesis Starch and sucrose metabolism Retinol metabolism C-vs-T34 1,105 964 2,069 Ribosome biogenesis in eukaryotes Rpp、UTP、
POP、REXO、
NOB、POLR、
RPI、HPSE、
Galns、LIPF、
TSPO、Tace、
NotchRNA polymerase RNA degradation Amino sugar and nucleotide sugar metabolism Arachidonic acid metabolism Glycosaminoglycan degradation Cytosolic DNA-sensing pathway Cholesterol metabolism Lysosome Notch signaling pathway
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