Analyses of Endocrine Regulation and Expression of Genes Related to the Molting Signaling Pathway in the Molting Cycle of Macrobrachium rosenbergii

doi:10.13560/j.cnki.biotech.bull.1985.2023-0169

Abstract

Abstract:

Molting is the important physiological process in Macrobrachium rosenbergii. This work is aimed to explore the endocrine regulation of M. rosenbergii and the expression patterns of related genes in the molting pathway, and to reveal the molecular regulatory pathways of M. rosenbergii molting. The activities of molt-related enzymes（glutamine synthetase, β-N acetylglucosaminidase and chitinase）and ecdyhormone contents in the molting cycle of hepatopancreas and hemolymph in M. rosenbergii were determined, and the expressions of Mr-ETHR, Mr-FTZ-F1, Mr-ECR, Mr-RXR, and Mr-MIH were detected by RT-qPCR. The results of enzymatic activity showed that glutamine synthetase was more active in hemolymphatic than in hepatopancreatic（P<0.05）. β-N acetylglucosaminidase had much higher pre-molting activity in hepatopancreatic and hemolymphatic than in the postmolt stage（P<0.05）. In the hepatopancreas, chitinase activity was the highest in the postmolt stage（P<0.05）. The content of ecdytropin in the hepatopancreatic and hemolymphatic was the lowest in the intermolt stage and the highest in the postmolt stage. The ORF length of Mr-ETHR gene was 1 173 bp, encoding 390 amino acids, and the sequence was relatively conserved. The ORF length of Mr-FTZ-F1 gene was 1 206 bp, encoding 401 amino acids. The Mr-ETHR, Mr-RXR and Mr-ECR expressed the highest in the postmolt stage, while Mr-FTZ-F1 was the highest in the premolt stage. The expressions of Mr-MIH reached the highest in the intermolt stage and the lowest in the postmolt stage. Cluster and correlation analyses showed that Mr-ETHR and Mr-RXR and Mr-ECR expression patterns were closely related and had a very significant positive correlation（r=0.7030, P<0.01; r=0.8680, P<0.01）, Mr-FTZ-F1 and Mr-MIH expression patterns were similar and positively correlated（r=0.6665, P<0.01）, while Mr-FTZ-F1 was negatively correlated with Mr-ECR and Mr-ETHR（r=-0.8339, P<0.01; r=-0.6275, P<0.01）. It was shown that the molting process of R. rosenbergii was regulated by glutamine synthetase, β-N-acetylaminoglucosidase and chitinase, and Mr-ETHR, Mr-RXR, Mr-ECR, Mr-FTZ-F1 and Mr-MIH were involved in regulating the molting process of M. rosenbergii, and Mr-ETHR, Mr-RXR and Mr-ECR were positively regulated in the molting signaling pathway, while Mr-FTZ-F1 and Mr-MIH were negatively regulated. Mr-ETHR, Mr-RXR and Mr-ECR play a positive regulatory role in the molt signaling pathway, while Mr-FTZ-F1 and Mr-MIH play a negative regulatory role. This study's results provided a foundation for the study of molting regulation mechanism in the downstream of the molting signaling pathway in crustaceans.

Key words: Macrobrachium rosenbergii, molting hormone, molting period, molting signaling pathways

DING Li, DU Ting-ting, TANG Qiong-ying, GAO Quan-xin, YI Shao-kui, YANG Guo-liang. Analyses of Endocrine Regulation and Expression of Genes Related to the Molting Signaling Pathway in the Molting Cycle of Macrobrachium rosenbergii[J]. Biotechnology Bulletin, 2023, 39(9): 300-310.

Figures/Tables 7

Fig. 1 Schematic diagram of the regulation process of molting in arthropods MIH receptor is G protein-coupled receptor. When MIH binds to the receptor, intracellular cAMP increases, activating Ca2+ channels on the cell membrane, and Ca2+ enters the cell and activates calmodulin（CaM）. CaM dephosphorylates nitric oxide synthase activates NOS, then NO is formed with arginine as a substrate, and then this activates guanylate cyclase type I（GC-I）, GC-I converts GTP to cGTP, and activates cGTP-dependent protein kinases in cells. The active PKA enters the nucleus to regulate the synthesis of ecdyhormone, and after 20E enters the nucleus, it binds to heterodimer（ECR/USP/RXR）to initiate early response genes, such as E75, E74 and Br-C, and then regulates early-late genes, such as HR3, HR4 and HR38, and finally these early and late response genes regulate late response genes, such as FTZ-F1, and finally complete molting. Endocrine Inka cells secrete ecdy-initiating hormone（ETH）acting on the ecdy-initiating hormone receptor（ETHR）, initiating molt

Table 1 Primer sequences used in the experiment

引物名称Primer name	引物序列Primer sequence（5'-3'）	产物长度Product length/bp	退火温度Annealing temperature/℃
FTZ-F1-F-1	CCTATTATGCTGCGGGAGT	649	55
FTZ-F1-R-1	CACCGTTGATGTGCTTGTA	649	55
FTZ-F1-F-2	TGTCCGTACTGTCGCTTTC	627	52
FTZ-F1-R-2	ACCAACTCCCGCAGCATA	627	52
FTZ-F1-F-3	CCTATTATGCTGCGGGAGT	595	55
FTZ-F1-R-3	GAAGCTCTGGCAGTAAATCC	595	55
ETHR-F-1	GTCCCGTTGCTGGTTCTT	535	55
ETHR-R-1	ATGACGCTGACCCTGTTG	535	55
ETHR-F-2	CGATGACGCTGACCCTGT	537	55
ETHR-R-2	AAGCCAAGGCGATAACAG	537	55
ETHR-F-3	ATAGTGCAGCCGCAGATT	635	53.5
ETHR-R-3	GGATCACCTGCACCAACGTA	635	53.5
RTFTZ-F1-F	GGATCACCTGCACCAACGTA	120	57
RTFTZ-F1-R	GGAAACGATCTGCGAACTGC	120	57
RTETHR-F	GTCCCGTTGCTGGTTCTT	165	55
RTETHR-R	GTCTCGCTCGCATTTGTG	165	55
RTMIH-F	AGCCCTGAGTGTCTGTCC	123	57
RTMIH-R	CCTTGCGTTGTCTGGTT	123	57
RTRXR-F	GATCGGCAGTCCCCTTTGAA	109	57
RTRXR-R	TTGGACACACTGGGAGAAGC	109	57
RTECR-F	AGAGCCGCATAAAGTGGAGA	134	57
RTECR-R	CTCAGGTCGGTCAGGATGTT	134	57
18S-F	TATACGCTAGTGGAGCTGGAA	313	60
18S-R	GGGGAGGTAGTGACGAAAAAT	313	60

Fig. 2 Dynamics in molting-related enzyme activity in M. rosenbergiiat at different molting cycles C: Intermolt stage. D: Premolt stage. E: Molt stage. AB: Postmolt stage. Different letters indicated significant differences（P<0.05）, The same below

Fig. 3 Dynamics of ecdyhormone content in hepatopancreas and hemolymph in M. rosenbergii at different molting stages

Fig. 4 Phylogenetic trees of Mr-ETHR（A）and Mr-FTZ-F1（B）based on NJ method

Fig. 5 Expressions of Mr-ETHR and Mr-FTZ-F1 in hepatopancreatic and hemolymphatic tissues at different molting stages

Fig. 6 Expressions of Mr-ETHR, Mr-FTZ-F1, Mr-RXR, Mr-ECR and Mr-MIH genes in M. rosenbergii at different molting cycles

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