CRISPR/Cas9-mediated Serine protease 2 disruption induces male sterility in Spodoptera litura

Bi, Honglun; Xu, Xia; Li, Xiaowei; Wang, Yaohui; Zhou, Shutang; Huang, Yongping

doi:10.3389/fphys.2022.931824

Cited by 13 publications

(8 citation statements)

References 69 publications

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“…Trypsin-type serine proteases are known as components transferred from males to females and are crucial for fertility in diverse species [ 24 – 26 , 38 ]. Despite our previous work found that the mutation of Ser1 causes male sterility in B .…”

Section: Discussionmentioning

confidence: 99%

SPSL1 is essential for spermatophore formation and sperm activation in Spodoptera frugiperda

Qian,

Yang,

et al. 2023

PLoS Genet

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The reproductive process in various species has undergone evolutionary adaptations at both the physiological and molecular levels, playing a significant role in maintaining their populations. In lepidopteran insects, the spermatophore is a unique structure formed in the female reproductive system, in which sperm storage and activation take place. It is known that the formation of the spermatophore is regulated by seminal fluid proteins derived from males. However, studies investigating the genetic mechanisms behind spermatophore formation in lepidopterans have been limited. In this study, our focus was on SPSL1, a gene that encodes a trypsin-type seminal fluid protein in Spodoptera frugiperda, a pest species with global invasive tendencies. Our findings revealed that SPSL1 expression was predominantly observed in the male reproductive tracts, and the disruption of this gene resulted in male sterility. Surprisingly, fluorescence analysis indicated that the absence of SPSL1 did not affect spermatogenesis or sperm migration within the male reproductive system. However, when females mated with SPSL1-mutant males, several defects were observed. These included disruptions in spermatophore formation, sperm activation in the copulatory bursae, and sperm migration into the spermathecae. Additionally, mass spectrometry analysis highlighted reduced levels of energy-related metabolites, suggesting that SPSL1 plays an essential role in promoting hydrolysis reactions during copulation. Consequently, our study demonstrates that SPSL1 is crucial for male fertility due to its functions in spermatophore formation and sperm activation. This research provides valuable insights into the genetic factors underlying reproductive processes in lepidopteran insects and sheds light on potential strategies for controlling invasive pest populations.

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Section: Discussionmentioning

confidence: 99%

SPSL1 is essential for spermatophore formation and sperm activation in Spodoptera frugiperda

Qian,

Yang,

et al. 2023

PLoS Genet

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“…In melon fly, the suppression of ZcTSSK3 and ZcTSSK1 significantly decreases the sperm count and affects hatchability 13 . In lepidopteran insects, knockout of the different serine protease genes leads to male infertility in Spodoptera litura , Bombyx mori , and Plutella xylostella 14,15 . Collectively, these studies highlight the basic significance of several serine proteases in the regulation of male reproductive processes across different species, including insects.…”

Section: Introductionmentioning

confidence: 86%

Exploring the role of the ovary‐serine protease gene in the female fertility of the diamondback moth using CRISPR/Cas9

Asad,

Liao,

Chen

et al. 2024

Pest Management Science

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BACKGROUNDOogenesis is a complex pathway necessary for proper female reproduction in insects. Ovary‐serine protease (Osp) is a homologous gene of serine protease Nudel (SpNudel) and plays an essential role in the oogenesis and ovary development of Drosophila melanogaster. However, the function of Osp is not determined in Plutella xylostella, a highly destructive pest of cruciferous crops.RESULTSThe PxOsp gene comprised a 5883‐bp open‐reading frame that encoded a protein consisting of 1994 amino acids, which contained four conserved domains. PxOsp exhibited a high relative expression in adult females with a specific expression in the ovary. Through the utilization of CRISPR/Cas9 technology, homozygous mutants of PxOsp were generated. These homozygous mutant females produced fewer eggs (average of 56 eggs/female) than wildtype (WT) females (average of 97 eggs/female) when crossed with WT males, and these eggs failed to hatch. Conversely, mutant males produced normal progeny when crossed with WT females. The ovarioles in homozygous mutant females were significantly shorter (5.02 mm in length) and contained fewer eggs (average of 3 eggs/ovariole) than WT ovarioles (8.09 mm in length with an average of 8 eggs/ovariole). Moreover, eggs laid by homozygous mutant females were fragile, with irregular shapes and unable to maintain structural integrity due to eggshell ruptures. However, no significant differences were observed between WT and mutant individuals regarding developmental duration, pupal weight, and mating behavior.CONCLUSIONOur study suggested that PxOsp plays a vital role in female reproduction, particularly in ovary and egg development. Disrupting PxOsp results in a recessive female sterility while leaving the male reproductive capability unaffected. This report represents the first study of a haplosufficient gene responsible for female fertility in lepidopteran insects. Additionally, these findings emphasize PxOsp as a potential target for genetically‐based pest management of P. xylostella.This article is protected by copyright. All rights reserved.

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“…Over recent years, more biological approaches have been developed to effectively control S. litura 18 , 19 . Biopesticides are the biological agents that are used to control pests, and are derived from fungi, bacteria, viruses, plants, animals, and certain minerals.…”

Section: Introductionmentioning

confidence: 99%

Determination of internal controls for quantitative gene expression of Spodoptera litura under microbial pesticide stress

Wu,

Luo,

Zeng

et al. 2024

Sci Rep

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Quantitative real-time polymerase chain reaction (qRT-PCR) has become a commonly used method for the quantification of gene expression. However, accurate qRT-PCR analysis requires a valid internal reference for data normalization. To determine the valid reference characterized with low expression variability among Spodoptera litura samples after microbial pesticide treatments, nine housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), arginine kinase, ubiquitin C, actin-5C (ACT5C), actin, ribosomal protein S13 (RPS13), tubulin, acidic ribosomal protein P0 (RPLP0) and ubiquinol-cytochrome c reductase, were evaluated for their suitability using geNorm, Normfinder, BestKeeper, RefFinder and the comparative delta CT methods in this study. S. litura larvae after direct treatment (larvae were immersed in biopesticides), indirect treatment (larvae were fed with biopesticide immersed artificial diets) and comprehensive treatment (larvae were treated with the first two treatments in sequence), respectively with Metarhizium anisopliae, Empedobacter brevis and Bacillus thuringiensis, were investigated. The results indicated that the best sets of internal references were as follows: RPLP0 and ACT5C for direct treatment conditions; RPLP0 and RPS13 for indirect treatment conditions; RPS13 and GAPDH for comprehensive treatment conditions; RPS13 and RPLP0 for all the samples. These results provide valuable bases for further genetic researches in S. litura.

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CRISPR/Cas9-mediated Serine protease 2 disruption induces male sterility in Spodoptera litura

Cited by 13 publications

References 69 publications

SPSL1 is essential for spermatophore formation and sperm activation in Spodoptera frugiperda

SPSL1 is essential for spermatophore formation and sperm activation in Spodoptera frugiperda

Exploring the role of the ovary‐serine protease gene in the female fertility of the diamondback moth using CRISPR/Cas9

Determination of internal controls for quantitative gene expression of Spodoptera litura under microbial pesticide stress

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