Shigenobu Shuji, Yoda Shinichi, Ohsawa Sonoko, Suzuki Miyuzu
Laboratory of Evolutionary Genomics, Trans-Scale Biology Center, National Institute for Basic Biology, Okazaki, Japan.
Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Japan.
PLoS Genet. 2025 Jul 21;21(7):e1011557. doi: 10.1371/journal.pgen.1011557. eCollection 2025 Jul.
The production of overwintering eggs is a critical adaptation for winter survival among many insects. Melanization contributes to eggshell pigmentation and hardening, consequently enhancing resistance to environmental stress. The complex life cycle of the pea aphid (Acyrthosiphon pisum), a model hemipteran insect with remarkable reproductive capacity, involves cyclical parthenogenesis. It enables the production of black overwintering eggs that undergo obligate diapause to survive under unfavorable conditions. Laccase2 (Lac2) is essential for cuticle sclerotization and pigmentation in other insects. We hypothesized that Lac2 plays a critical role in aphid eggshell pigmentation and survival during diapause. To test the hypothesis, we used CRISPR/Cas9 ribonucleoprotein microinjections and a novel Direct Parental CRISPR (DIPA-CRISPR) method to knockout Lac2. In Lac2 knockout (KO) crispants (G0), pigment-less eggs correlated with induced indel rates. Additionally, eggshell pigmentation was completely lost in homozygous Lac2 knockouts, leading to embryonic lethality. Observation of late-stage embryos in KO diapause eggs suggested that lethality occurred during late embryogenesis or hatching. Furthermore, eggshell stiffness was significantly reduced in Lac2 KOs, highlighting the role of this gene in eggshell hardening. Moreover, fungal growth was observed in KO eggs. These findings reveal the essential roles of Lac2 in eggshell pigmentation, hardening, late embryonic development, hatching, and fungal protection, which are critical for pea aphid survival during overwintering diapause. This study also advances CRISPR/Cas9-mediated genome editing in pea aphids by addressing the challenges associated with their unique biology, including complex life cycles, obligatory diapause, bacterial endosymbiosis, inbreeding depression, and high nuclease activity. Our optimized protocol achieved efficient targeted mutagenesis and germline transmission, thereby generating stable KO lines. Additionally, we successfully applied DIPA-CRISPR to aphids by inducing mutations via adult oviparous female injections in fertilized eggs. These robust genome-editing protocols will facilitate functional studies in aphids, a key model for research on evolution, ecology, development, and agriculture.
产生越冬卵是许多昆虫适应冬季生存的关键方式。黑化作用有助于卵壳色素沉着和硬化，从而增强对环境压力的抵抗力。豌豆蚜（Acyrthosiphon pisum）是一种具有显著繁殖能力的半翅目昆虫，其复杂的生命周期涉及周期性孤雌生殖。这使得豌豆蚜能够产生黑色的越冬卵，这些卵会经历专性滞育以在不利条件下存活。漆酶2（Lac2）在其他昆虫的表皮硬化和色素沉着过程中至关重要。我们推测Lac2在蚜虫卵壳色素沉着和滞育期间的存活中起关键作用。为了验证这一假设，我们使用CRISPR/Cas9核糖核蛋白显微注射和一种新型的直接亲代CRISPR（DIPA-CRISPR）方法敲除Lac2。在Lac2基因敲除（KO）的突变体（G0）中，无色素的卵与诱导的插入缺失率相关。此外，纯合Lac2基因敲除个体的卵壳色素沉着完全丧失，导致胚胎致死。对处于滞育期的KO卵中的晚期胚胎进行观察表明，致死现象发生在胚胎发育后期或孵化期。此外，Lac2基因敲除个体的卵壳硬度显著降低，突出了该基因在卵壳硬化中的作用。此外，在KO卵中观察到了真菌生长。这些发现揭示了Lac2在卵壳色素沉着、硬化、胚胎后期发育、孵化以及真菌防护方面的重要作用，这些对于豌豆蚜在越冬滞育期间的生存至关重要。本研究还通过应对与豌豆蚜独特生物学特性相关的挑战，包括复杂的生命周期、专性滞育、细菌内共生、近亲繁殖衰退以及高核酸酶活性，推动了CRISPR/Cas9介导的豌豆蚜基因组编辑技术的发展。我们优化后的方案实现了高效的靶向诱变和种系传递，从而产生了稳定的KO品系。此外，我们通过对受精的卵进行成年产卵雌虫注射诱导突变，成功地将DIPA-CRISPR应用于蚜虫。这些强大的基因组编辑方案将有助于对蚜虫进行功能研究，蚜虫是进化、生态、发育和农业研究的关键模型。
