Insect Pest Control Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Friedensstrasse 1, Seibersdorf, 2444, Austria.
Institute for Insect Biotechnology, Department of Insect Biotechnology in Plant Protection, Justus-Liebig-University Gießen, Winchesterstr. 2, Gießen, 35394, Germany.
BMC Biotechnol. 2024 Feb 1;24(1):7. doi: 10.1186/s12896-024-00832-x.
The Mediterranean fruit fly, Ceratitis capitata, is a significant agricultural pest managed through area-wide integrated pest management (AW-IPM) including a sterile insect technique (SIT) component. Male-only releases increase the efficiency and cost-effectiveness of SIT programs, which can be achieved through the development of genetic sexing strains (GSS). The most successful GSS developed to date is the C. capitata VIENNA 8 GSS, constructed using classical genetic approaches and an irradiation-induced translocation with two selectable markers: the white pupae (wp) and temperature-sensitive lethal (tsl) genes. However, currently used methods for selecting suitable markers and inducing translocations are stochastic and non-specific, resulting in a laborious and time-consuming process. Recent efforts have focused on identifying the gene(s) and the causal mutation(s) for suitable phenotypes, such as wp and tsl, which could be used as selectable markers for developing a generic approach for constructing GSS. The wp gene was recently identified, and efforts have been initiated to identify the tsl gene. This study investigates Ceratitis capitata deep orange (Ccdor) as a tsl candidate gene and its potential to induce tsl phenotypes.
An integrated approach based on cytogenetics, genomics, bioinformatics, and gene editing was used to characterize the Ccdor. Its location was confirmed on the right arm of chromosome 5 in the putative tsl genomic region. Knock-out of Ccdor using CRISPR/Cas9-NHEJ and targeting the fourth exon resulted in lethality at mid- and late-pupal stage, while the successful application of CRISPR HDR introducing a point mutation on the sixth exon resulted in the establishment of the desired strain and two additional strains (dor 12del and dor 51dup), all of them expressing tsl phenotypes and presenting no (or minimal) fitness cost when reared at 25 °C. One of the strains exhibited complete lethality when embryos were exposed at 36 °C.
Gene editing of the deep orange gene in Ceratitis capitata resulted in the establishment of temperature-sensitive lethal mutant strains. The induced mutations did not significantly affect the rearing efficiency of the strains. As deep orange is a highly conserved gene, these data suggest that it can be considered a target for the development of tsl mutations which could potentially be used to develop novel genetic sexing strains in insect pests and disease vectors.
地中海实蝇（Ceratitis capitata）是一种重要的农业害虫，通过包括不育昆虫技术（SIT）在内的全区域综合虫害管理（AW-IPM）进行管理。仅雄性释放可以提高 SIT 计划的效率和成本效益，这可以通过开发遗传性别鉴定品系（GSS）来实现。迄今为止，最成功的 GSS 是使用经典遗传方法构建的地中海实蝇 VIENNA 8 GSS，以及通过辐照诱导带有两个可选择标记的易位：白蛹（wp）和温度敏感致死（tsl）基因。然而，目前用于选择合适标记和诱导易位的方法是随机的和非特异性的，导致这一过程既费力又耗时。最近的努力集中在鉴定合适表型（如 wp 和 tsl）的基因和因果突变上，这些表型可以用作开发构建 GSS 的通用方法的可选择标记。wp 基因最近被鉴定出来，并且已经开始努力鉴定 tsl 基因。本研究调查了地中海实蝇深橙色（Ccdor）作为 tsl 候选基因及其诱导 tsl 表型的潜力。
使用细胞遗传学、基因组学、生物信息学和基因编辑的综合方法对 Ccdor 进行了表征。其位置在假定的 tsl 基因组区域的 5 号染色体右臂上被证实。使用 CRISPR/Cas9-NHEJ 敲除 Ccdor 并靶向第四外显子导致中晚期蛹期死亡，而成功应用 CRISPR HDR 在外显子第六位引入点突变导致所需菌株和另外两个菌株（dor 12del 和 dor 51dup）的建立，所有这些菌株都表现出 tsl 表型，并且在 25°C 下饲养时没有（或最小）的适应成本。其中一个菌株在胚胎暴露于 36°C 时表现出完全致死性。
地中海实蝇深橙色基因的基因编辑导致了温度敏感致死突变菌株的建立。诱导的突变并没有显著影响菌株的饲养效率。由于深橙色是一个高度保守的基因，这些数据表明它可以被视为 tsl 突变的目标，这可能有助于开发昆虫害虫和疾病媒介的新型遗传性别鉴定品系。
