Table1_Knockdown of Sec16 causes early lethality and defective deposition of the protein Rp30 in the eggshell of the vector Rhodnius prolixus.DOCX

In nearly every species of insect, embryonic development takes place outside of the mother’s body and is entirely dependent on the elements that the mother had previously stored within the eggs. It is well known that the follicle cells (FCs) synthesize the eggshell (chorion) components during the process of choriogenesis, the final step of oogenesis before fertilization. These cells have developed a specialization in the massive production of chorion proteins, which are essential for the protection and survival of the embryo. Here, we investigate the function of Sec16, a protein crucial for the endoplasmic reticulum (ER) to Golgi traffic, in the oocyte development in the insect Rhodnius prolixus. We discovered that Sec16 is strongly expressed in vitellogenic females’ ovaries, particularly in the choriogenic oocyte and it is mainly associated with the FCs. Silencing of Sec16 by RNAi caused a sharp decline in oviposition rates, F1 viability, and longevity in adult females. In the FCs, genes involved in the unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and autophagy were massively upregulated, whereas the mRNAs of Rp30 and Rp45—which code for the two major chorion proteins - were downregulated as a result of Sec16 silencing, indicating general proteostasis disturbance. As a result, the outer surface ultrastructure of Sec16-silenced chorions was altered, with decreased thickness, dityrosine crosslinking, sulfur signals, and lower amounts of the chorion protein Rp30. These findings collectively demonstrate the critical role Sec16 plays in the proper functioning of the FCs, which impacts the synthesis and deposition of particular components of the chorion as well as the overall reproduction of this vector.

在几乎所有昆虫物种中，胚胎发育均发生于母体外，且完全依赖于母体预先储存在卵内的物质。众所周知，卵泡细胞（follicle cells, FCs）会在卵壳发生（choriogenesis）过程中合成卵壳（chorion）组分——该过程是受精前卵子发生（oogenesis）的最后阶段。这类细胞特化形成了大规模合成卵壳蛋白的能力，而卵壳蛋白对于胚胎的保护与存活至关重要。本研究以红猎蝽（Rhodnius prolixus）为对象，探究Sec16蛋白在其卵母细胞发育中的功能：Sec16是介导内质网（endoplasmic reticulum, ER）至高尔基体运输的关键蛋白。研究发现，Sec16在卵黄生成期雌虫的卵巢中高表达，尤其在卵壳发生期的卵母细胞中，且主要定位于卵泡细胞。通过RNA干扰（RNAi）沉默Sec16后，雌成虫的产卵率、F1代存活率及寿命均显著下降。在卵泡细胞中，未折叠蛋白反应（unfolded protein response, UPR）、泛素-蛋白酶体系统（ubiquitin-proteasome system, UPS）及自噬相关基因均被显著上调；而编码两种主要卵壳蛋白的Rp30与Rp45的mRNA表达量则因Sec16沉默而下调，这表明蛋白稳态整体受到扰动。相应地，经Sec16沉默后的卵壳其外表超微结构发生改变：厚度降低、二酪氨酸交联（dityrosine crosslinking）水平下降、硫信号减弱，且卵壳蛋白Rp30的含量减少。综上，本研究结果证实了Sec16在卵泡细胞正常功能发挥中的关键作用，其可影响卵壳特定组分的合成与沉积，并最终影响该媒介昆虫的整体繁殖能力。