Department of Biochemistry and Molecular Biology, Egerton University, Njoro Campus, PO Box 536-20115, Egerton, Kenya.
Department of Biochemistry, Microbiology and Biotechnology, School of Pure and Applied Sciences, Kenyatta University, Ruiru Campus, PO Box 43844-00100, Nairobi, Kenya.
Parasit Vectors. 2021 Jan 2;14(1):1. doi: 10.1186/s13071-020-04505-4.
Insect growth regulators (IGRs) can control insect vector populations by disrupting growth and development in juvenile stages of the vectors. We previously identified and described the curry tree (Murraya koenigii (L.) Spreng) phytochemical leaf extract composition (neplanocin A, 3-(1-naphthyl)-L-alanine, lumiflavine, terezine C, agelaspongin and murrayazolinol), which disrupted growth and development in Anopheles gambiae sensu stricto mosquito larvae by inducing morphogenetic abnormalities, reducing locomotion and delaying pupation in the mosquito. Here, we attempted to establish the transcriptional process in the larvae that underpins these phenotypes in the mosquito.
We first exposed third-fourth instar larvae of the mosquito to the leaf extract and consequently the inherent phytochemicals (and corresponding non-exposed controls) in two independent biological replicates. We collected the larvae for our experiments sampled 24 h before peak pupation, which was 7 and 18 days post-exposure for controls and exposed larvae, respectively. The differences in duration to peak pupation were due to extract-induced growth delay in the larvae. The two study groups (exposed vs control) were consequently not age-matched. We then sequentially (i) isolated RNA (whole larvae) from each replicate treatment, (ii) sequenced the RNA on Illumina HiSeq platform, (iii) performed differential bioinformatics analyses between libraries (exposed vs control) and (iv) independently validated the transcriptome expression profiles through RT-qPCR.
Our analyses revealed significant induction of transcripts predominantly associated with hard cuticular proteins, juvenile hormone esterases, immunity and detoxification in the larvae samples exposed to the extract relative to the non-exposed control samples. Our analysis also revealed alteration of pathways functionally associated with putrescine metabolism and structural constituents of the cuticle in the extract-exposed larvae relative to the non-exposed control, putatively linked to the exoskeleton and immune response in the larvae. The extract-exposed larvae also appeared to have suppressed pathways functionally associated with molting, cell division and growth in the larvae. However, given the age mismatch between the extract-exposed and non-exposed larvae, we can attribute the modulation of innate immune, detoxification, cuticular and associated transcripts and pathways we observed to effects of age differences among the larvae samples (exposed vs control) and to exposures of the larvae to the extract.
The exposure treatment appears to disrupt cuticular development, immune response and oxidative stress pathways in Anopheles gambiae s.s larvae. These pathways can potentially be targeted in development of more efficacious curry tree phytochemical-based IGRs against An. gambiae s.s mosquito larvae.
昆虫生长调节剂（IGRs）可以通过破坏媒介昆虫幼虫阶段的生长和发育来控制昆虫媒介种群。我们之前已经鉴定并描述了咖喱树（Murraya koenigii（L.）Spreng）植物化学叶提取物的组成（neplanocin A、3-（1-萘基）-L-丙氨酸、光黄素、terezine C、agelaspongin 和 murrayazolinol），它通过诱导形态发生异常、减少运动并延迟蛹化来破坏按蚊幼虫的生长和发育。在这里，我们试图确定这些表型在蚊子幼虫中所依赖的转录过程。
我们首先将蚊子的三到四龄幼虫暴露于叶提取物中，然后将其暴露于两种独立的生物重复中的固有植物化学物质（和相应的未暴露对照）。我们在实验中收集幼虫，在暴露幼虫的峰值蛹化前 24 小时采样，对照幼虫分别为 7 天和 18 天。峰值蛹化时间的差异是由于提取物诱导幼虫生长延迟。因此，两个研究组（暴露组与对照组）的年龄不匹配。然后，我们依次进行：（i）从每个重复处理中分离 RNA（整个幼虫），（ii）在 Illumina HiSeq 平台上对 RNA 进行测序，（iii）对文库（暴露组与对照组）之间的差异进行生物信息学分析，以及（iv）通过 RT-qPCR 独立验证转录组表达谱。
我们的分析表明，与未暴露的对照样本相比，暴露于提取物的幼虫样本中，与硬壳蛋白、保幼激素酯酶、免疫和解毒相关的转录物主要被显著诱导。我们的分析还表明，在暴露于提取物的幼虫中，与精胺代谢和表皮结构成分相关的途径发生改变，这可能与幼虫的外骨骼和免疫反应有关。提取物暴露的幼虫似乎还抑制了与蜕皮、细胞分裂和生长相关的途径，这些途径在幼虫中发挥功能。然而，鉴于暴露于提取物的幼虫与未暴露的幼虫之间存在年龄不匹配，我们可以将我们观察到的固有免疫、解毒、表皮和相关转录物和途径的调节归因于幼虫样本（暴露组与对照组）之间年龄差异的影响以及幼虫暴露于提取物。
暴露处理似乎破坏了按蚊 s.s 幼虫的表皮发育、免疫反应和氧化应激途径。这些途径可能成为开发更有效的咖喱树植物化学 IGRs 对抗按蚊 s.s 幼虫的潜在靶点。
