Functional importance of groups I and II chitinases, CHT5 and CHT10, in turnover of chitinous cuticle during embryo hatching and post-embryonic molting in the red flour beetle, Tribolium castaneum

IF 3.2 2区 农林科学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Insect Biochemistry and Molecular Biology Pub Date : 2024-01-29 DOI:10.1016/j.ibmb.2024.104087
Myeongjin Kim , Mi Young Noh , Seulgi Mun , Subbaratnam Muthukrishnan , Karl J. Kramer , Yasuyuki Arakane
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Abstract

Chitinases (CHT) comprise a large gene family in insects and have been classified into at least eleven subgroups. Many studies involving RNA interference (RNAi) have demonstrated that depletion of group I (CHT5s) and group II (CHT10s) CHT transcripts causes lethal molting arrest in several insect species including the red flour beetle, Tribolium castaneum, presumably due to failure of degradation of chitin in their old cuticle. In this study we investigated the functions of CHT5 and CHT10 in turnover of chitinous cuticle in T. castaneum during embryonic and post-embryonic molting stages. RNAi and transmission electron microscopic (TEM) analyses indicate that CHT10 is required for cuticular chitin degradation at each molting period analyzed, while CHT5 is essential for pupal-adult molting only. We further analyzed the functions of these genes during embryogenesis in T. castaneum. Real-time qPCR analysis revealed that peak expression of CHT10 occurred prior to that of CHT5 during embryonic development as has been observed at post-embryonic molting periods in several other insect species. With immunogold-labeling TEM analysis using a fluorescein isothiocyanate-conjugated chitin-binding domain protein (FITC-CBD) probe, chitin was detected in the serosal cuticle but not in any other regions of the eggshell including the chorion and vitelline membrane layers. Injection of double-stranded RNA (dsRNA) for CHT5 (dsCHT5), CHT10 (dsCHT10) or their co-injection (dsCHT5/10) into mature adult females had no effect on their fecundity and the resulting embryos developed normally inside the egg. There were no obvious differences in the morphology of the outer chorion, inner chorion and vitelline membrane among eggs from these dsRNA-treated females. However, unlike dsCHT5 eggs, dsCHT10 and dsCHT5/10 eggs exhibited failure of turnover of the serosal cuticle in which the horizontal chitinous laminae remained intact, resulting in lethal embryo hatching defects. These results indicate that group I CHT5 is essential for pupal-adult molting, whereas group II CHT10 plays an essential role in cuticular chitin degradation in T. castaneum during both embryonic hatching and all of the post-embryonic molts. CHT10 can serve in place of CHT5 in chitin degradation, except during the pupal-adult molt when both enzymes are indispensable to complete eclosion.

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I 组和 II 组几丁质酶(CHT5 和 CHT10)在红面粉甲虫胚胎孵化和胚后蜕皮期间几丁质角质层更替中的功能重要性
几丁质酶(CHT)是昆虫中一个庞大的基因家族,至少分为 11 个亚群。许多涉及 RNA 干扰(RNAi)的研究表明,在包括红粉甲虫(Tribolium castaneum)在内的一些昆虫物种中,第一组(CHT5s)和第二组(CHT10s)CHT 转录本的耗竭会导致蜕皮停止,这可能是由于其旧角质层中的几丁质降解失败所致。在这项研究中,我们研究了 CHT5 和 CHT10 在蓖麻金龟子胚胎和胚后蜕皮阶段几丁质角质层周转中的功能。RNAi 和透射电子显微镜(TEM)分析表明,CHT10 在所分析的每个蜕皮期都对角质层几丁质降解是必需的,而 CHT5 仅对蛹-成虫蜕皮是必需的。我们进一步分析了这些基因在 T. castaneum 胚胎发生过程中的功能。实时 qPCR 分析表明,在胚胎发育过程中,CHT10 的表达峰值先于 CHT5,这在其他几种昆虫的胚后蜕皮期也有观察到。通过使用异硫氰酸荧光素结合几丁质结合域蛋白(FITC-CBD)探针进行免疫金标记 TEM 分析,在血清角质层中检测到了几丁质,但在蛋壳的任何其他区域(包括绒毛膜和卵黄膜层)均未检测到几丁质。将 CHT5 和 CHT10 的双链 RNA(dsRNA)或它们的共同注射(dsCHT5/10)注入成熟的成年雌虫体内对其繁殖力没有影响,所产生的胚胎在卵内发育正常。经dsRNA处理的雌性卵子的外绒毛膜、内绒毛膜和卵黄膜形态没有明显差异。然而,与dsCHT5卵不同的是,dsCHT10和dsCHT5/10卵表现出浆膜角质层周转失败,其中水平壳质层保持完整,导致胚胎孵化缺陷致死。这些结果表明,第 I 组 CHT5 对蛹到成虫的蜕皮至关重要,而第 II 组 CHT10 则在 T. castaneum 胚胎孵化和胚胎后期的所有蜕皮过程中对角质层几丁质降解起着至关重要的作用。CHT10 可代替 CHT5 进行几丁质降解,但在蛹-成虫蜕皮过程中除外,因为这两种酶都是完成蜕皮不可或缺的。
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来源期刊
CiteScore
7.40
自引率
5.30%
发文量
105
审稿时长
40 days
期刊介绍: This international journal publishes original contributions and mini-reviews in the fields of insect biochemistry and insect molecular biology. Main areas of interest are neurochemistry, hormone and pheromone biochemistry, enzymes and metabolism, hormone action and gene regulation, gene characterization and structure, pharmacology, immunology and cell and tissue culture. Papers on the biochemistry and molecular biology of other groups of arthropods are published if of general interest to the readership. Technique papers will be considered for publication if they significantly advance the field of insect biochemistry and molecular biology in the opinion of the Editors and Editorial Board.
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Foreword. Identification of a vital transcription factor of the alanine aminotransferase in the brown planthopper and its upstream regulatory pathways. Binding properties of olfactory proteins to host volatiles, free fatty acids and cuticular hydrocarbons in the termite Reticulitermes aculabialis. Nutrition influences immunity: Diet and host-parasite interactions Notch signaling regulates limb regeneration through Hes1 and HeyL in the Chinese mitten crab
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