Impact of RNA interference of acetylcholinesterase gene on the growth and survival of Pomacea canaliculata

doi:10.20199/j.issn.1672-2302.2024.06.005

Abstract

Abstract:

Objective To investigate the impact of RNA interference (RNAi) on the expression of the acetylcholinesterase (AChE) gene in Pomacea canaliculata and its effects on the growth and survival of the snail for potential application of RNAi technology in the control of this pest. Methods Double-stranded RNA (dsRNA) targeting the AChE gene of P. canaliculata was designed, and the corresponding plasmid was constructed and transformed into Escherichia coli to create engineered bacteria. The study included a blank control group, a negative control group, and experimental groups (F1, F2, F3, F4), with 10 snails in each group. The blank control group received no treatment, the negative control group was fed with E. coli carrying an empty vector, and the experimental groups F1, F2, F3, and F4 were fed with the engineered E. coli to express different AChE-dsRNAs. Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of AChE at 8, 12, and 24 hours post-treatment. The group showing the most significant change in AChE expression was selected for further feeding and injection tests to observe the survival and mortality of the snails. Results After feeding the snails with E. coli expressing AChE-dsRNA for 8 and 12 hours, group F2 showed a significant decrease in AChE expression (t=5.790, 10.110, P<0.01) compared to the negative control group, whereas groups F1, F3, and F4 did not show significant changes. After 24 hours of feeding, AChE expression was unaffected in the snails in each experimental group. Further feeding with E. coli expressing AChE-dsRNA-F2 for 30 days, the average weight of the snails in group F2 was increased by 16.9% [ranging from (0.560 9 ± 0.113 6) g to (0.655 9 ± 0.177 3) g], and the average weight of the snails in the negative control group was increased by 23.3%[ranging from (0.565 0 ± 0.131 7) g to (0.691 2 ± 0.156 8) g]. The difference in weight gain was statistically significant in both groups (F=2.720, P<0.05). Injection with E. coli expressing AChE-dsRNA-F2 led to a 30.0% mortality of the snails, compared to 13.3% in the control group, with significant difference between groups (χ²=4.910, P<0.05). Conclusion Feeding E. coli engineered to express AChE-dsRNA can effectively reduce the expression of the AChE gene in P. canaliculata and inhibit its weight gain, and injection with AChE-dsRNA-expressing E. coli may increase the mortality. Our findings suggest that RNAi technology may be a novel rationale in the control of P. canaliculata.

Key words: Pomacea canaliculata, RNA interference (RNAi), Acetylcholinesterase gene, Double-stranded RNA

CLC Number:

R184.3

ZHANG Jian, KAI Zhenpeng, CHENG Donghui, GUO Yunhai, ZHANG Yi. Impact of RNA interference of acetylcholinesterase gene on the growth and survival of Pomacea canaliculata[J]. Journal of Tropical Diseases and Parasitology, 2024, 22(6): 346-352.

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编号	引物	碱基对（bp）	基因编号
F1	F:5′ CACAGAATGGTCAGAGGGTC 3′ R:5′ TCCAGTCCACAATGTAGCG 3′	468	LOC112555322
F2	F:5′ CTGGCAGTGGAGAATGATGT 3′ R:5′ TGGAGTATGGCTCGGTTGA 3′	269	LOC112555322
F3	F:5′ GGCTGCTCGTCAGTATGTG 3′ R:5′ TTGTTTCTGTTGAGGTGGG 3′	366	LOC112567060
F4	F:5′ AATGGGTCCACGACAATG 3′ R:5′ TACGCCAATAATCACTTCC 3′	425	LOC112573528
F5	F:5′ AAGAGTGTCGGCGGTGTATC 3′ R:5′ CGTCTGTTGGTCTGGTGGTC 3′	324	LOC112567060

基因名称	引物
F1	F: 5′ ACAAGAATGGCCCGAGTACC 3′
F1	R :5′ ACGAGGCTGGGTAGGTAGAG 3′
F2	F: 5′ GTGTCGATGAACTACCGCCT 3′
F2	R :5′ ATTATGAGGACTGCCCCCGA 3′
F3	F: 5′ TCGTGCTCACCGTCTGTTAC 3′
F3	R :5′ CGAATCTCAGGTACGCCTGT 3′
F4	F: 5′ TGGGTCCACGACAATGTTGG 3′
F4	R :5′ TCAAGGGACCTCCACTCTCC 3′
内参基因RPL5	F: 5′ CGTATGCCAGAATTGAGGGT 3′
内参基因RPL5	R :5′ CAACATCCAAGTATGCACGG 3′

时间（d）	空白对照组（g）	阴性对照组（g）	F2组（g）
0	0.566 7±0.118 7	0.565 0±0.131 7	0.560 9±0.113 6
10	0.620 9±0.139 7	0.609 7±0.114 2	0.591 7±0.139 0
20	0.679 5±0.145 0	0.671 1±0.143 1	0.629 0±0.144 1
30	0.693 6±0.155 9	0.691 2±0.156 8	0.655 9±0.177 3