Reasons for changes of hemocyte densities and the relationship between hemocyte density and high temperature resistance of Bombyx mori larvae LI Tian, LIU Hao-Yu, WANG Ge-Min, LI Ya-Teng, YU Hua-Xian, YAN Dong-Sheng, GUO Yu, ZHANG Tai-Yun, CHEN Ping
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【Aim】Hemocyte plays a leading role in insect hemolymph immunity. Analyzing the changes of hemocyte density of the silkworm, Bombyx mori larvae and their causes, and the relationship between hemocyte density and B. mori resistance, is an important part of hemocyte immune regulation and resistance breeding of silkworm.【Methods】Hemocyte density (hemocyte number) in 10 μL hemolymph of the silkworm variety Dazao at different instars (the day-1-4 4th instar, the day-1-8 5th instar and the wandering larval stage) was evaluated by a hemocytometer. The cross-sectional image area was estimated by ImageJ software to represent the relative size of hematopoietic organs. The numbers of hemocytes in the hemolymph of the day-1 5th instar larvae of different silkworm varieties including Jingsong, Haoyue, Liangguang 2, 932, Furong, 7532 and Xianghui were measured by a hemocytometer. The relative expression levels of hematopoietic regulation factor genes BmBCFI, BmUsh, BmGATA and BmLz in the hemolymph of the day-1 5th instar larvae of Haoyue and Liangguang 2 were detected by qRT-PCR. After the day-1 and -3 5th instar larvae of Dazao were infected by Escherichia coli for different time, the hemocyte numbers in the hemolymph were measured by a hemocytometer and the relative expression levels of the above hematopoietic regulation factor genes in the hemolymph were detected by qRT-PCR. After RNAi of BmBCFI, BmUsh and BmLz for different time, the relative expression levels of target genes in the hemolymph of the 5th instar larvae of Dazao were detected by qRT-PCR. At 6 h after the 5th instar larvae of Dazao were infected by E. coli following RNAi of BmBCFI, BmUsh and BmLz for 48 h, the relative expression levels of target genes and immune-related genes BmRelish, BmCactus, BmSpzl and BmPGRP-LE in the hemolymph were detected by qRT-PCR, and the numbers of different types of hemocytes in the hemolymph were counted by a hemocytometer. The correlations between the hemocyte number and viability of the 5th instar larvae at high temperature of 35℃ were analyzed for 12 practical silkworm varieties.【Results】The hemocyte numbers of Dazao gradually increased during the 4th instar larval stage, reached a peak at the day-1 5th instar larval stage, began to decrease at the day-2 5th instar larval stage, and then slightly increased during the wandering stage. The hematopoietic organs gradually grew during the early and middle 5th instar larval stages, reached a peak at the day-6 5th instar larval stage, then diminished significantly and finally disintegrated. The time and trend of changes in the size of hematopoietic organs were not consistent with those of the changes of the hemocyte numbers. The expression levels of hematopoietic regulation factor genes BmBCFI, BmUsh and BmLz in Liangguang 2 with higher hemocyte numbers were higher than those in Haoyue with lower hemocyte numbers. After the 5th instar larvae of Dazao were infected by E. coli, the expression levels of BmBCFI, BmUsh and BmLz were significantly up-regulated when hemocyte numbers reached a peak. The oenocytoid numbers significantly reduced after down-regulating BmLz or BmUsh via E. coli infection following RNAi, and the plasmatocyte number and the number of total hemocytes significantly decreased after down-regulating BmBCFI via E. coli infection following RNAi. The expression levels of BmRelish in the Imd pathway and BmCactus in the Toll pathway were both significantly down-regulated after down-regulating BmBCFI or BmUsh via E. coli infection following RNAi, implying that BmBCFI and BmUsh are related to the activation of NF-κB signaling pathway and the formation of immune factors such as antimicrobial peptides. The hemocyte numbers varied greatly among different silkworm varieties, and the summer-autumn varieties tended to have higher hemocyte numbers than spring varieties. There was a negative correlation between the hemocyte number and the death index of the 5th instar larvae of silkworm reared at high temperature of 35℃.【Conclusion】Expression level of hematopoietic regulation factor genes of B. mori can affect the hemocyte density. BmUsh and BmLz influence the oenocytoid density of B. mori, and BmBCFI is associated with the plasmatocyte formation. Moreover, BmBCFI and BmUsh are likely to be related to the activation of NF-κB signaling pathway in B. mori. The hemocyte density is positively correlated with the viability of B. mori larvae fed at high temperature.