DNA methylase 1 influences temperature responses and development in the invasive pest Tuta absoluta.

DNA methylase 1 (Dnmt1) is an important regulatory factor associated with biochemical signals required for insect development. It responds to changes in the environment and triggers phenotypic plasticity. Meanwhile, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae)-a destructive invasive pest-can rapidly invade and adapt to different habitats; however, the role of Dnmt1 in this organism has not been elucidated. Accordingly, this study investigates the mechanism(s) underlying the rapid adaptation of Tuta absoluta to temperature stress. Potential regulatory genes were screened via RNAi (RNA interference), and the DNA methylase in Tuta absoluta was cloned by RACE (Rapid amplification of cDNA ends). TaDnmt1 was identified as a potential regulatory gene via bioinformatics; its expression was evaluated in response to temperature stress and during different development stages using real-time polymerase chain reaction. Results revealed that TaDnmt1 participates in hot/cold tolerance, temperature preference and larval development. The full-length cDNA sequence of TaDnmt1 is 3765 bp and encodes a 1254 kDa protein with typical Dnmt1 node-conserved structural features and six conserved DNA-binding active motifs. Moreover, TaDnmt1 expression is significantly altered by temperature stress treatments and within different development stages. Hence, TaDnmt1 likely contributes to temperature responses and organismal development. Furthermore, after treating with double-stranded RNA and exposing Tuta absoluta to 35°C heat shock or -12°C cold shock for 1 h, the survival rate significantly decreases; the preferred temperature is 2°C lower than that of the control group. In addition, the epidermal segments become enlarged and irregularly folded while the surface dries up. This results in a significant increase in larval mortality (57%) and a decrease in pupation (49.3%) and eclosion (50.9%) rates. Hence, TaDnmt1 contributes to temperature stress responses and temperature perception, as well as organismal growth and development, via DNA methylation regulation. These findings suggest that the rapid geographic expansion of T absoluta has been closely associated with TaDnmt1-mediated temperature tolerance. This study advances the research on 'thermos Dnmt' and provides a potential target for RNAi-driven regulation of Tuta absoluta.

Opsin mutants alter host plant selection by color vision in the nocturnal invasive pest Tuta absoluta.

In insects, vision is crucial in finding host plants, but its role in nocturnal insects is largely unknown. Vision involves responses to specific spectra of photon wavelengths and opsins plays an important role in this process. Long-wavelength sensitive opsin (LW opsin) and blue-sensitive opsin (BL opsin) are main visual opsin proteins and play important in behavior regulation.We used CRISPR/Cas9 technology to mutate the long-wavelength-sensitive and blue wavelength-sensitive genes and explored the role of vision in the nocturnal invasive pest Tuta absoluta. Light wave experiments revealed that LW2(-/-) and BL(-/-) mutants showed abnormal wavelength tropism. Both LW2 and BL mutations affected the preference of T. absoluta for the green environment. Mutations in LW2 and BL are necessary to inhibit visual attraction. The elimination of LW2 and BL affected the preference of leaf moths for green plants, and mutations in both induced a preference in moths for white plants. Behavioral changes resulting from LW2(-/-) and BL(-/-) mutants were not affected by sense of smell, further supporting the regulatory role of vision in insect behavior. To the best of our knowledge, this is the first study to reveal that vision, not smell, plays an important role in the host-seeking behavior of nocturnal insects at night, of which LW2 and BL opsins are key regulatory factors. These study findings will drive the development of the "vision-ecology" theory.

Bacterial Communities of the Internal Reproductive and Digestive Tracts of Virgin and Mated Tuta absoluta.

Microorganisms can affect host reproduction, defense, and immunity through sexual or opportunistic transmission; however, there are few studies on insect reproductive organs and intestinal bacterial communities and their effects on mating. Tuta absoluta is a worldwide quarantine pest that seriously threatens the production of Solanaceae crops, and the microbial community within tomato leafminers remains unclear. In this study, 16s rRNA sequencing was used to analyze bacterial communities related to the reproductive organs and intestinal tracts of tomato leafminers (the sample accession numbers are from CNS0856533 to CNS0856577). Different bacterial communities were found in the reproductive organs and intestinal tracts of females and males. Community ecological analysis revealed three potential signs of bacterial sexual transmission: (1) Mating increased the similarity between male and female sex organs and intestinal communities. (2) The bacteria carried by mated individuals were found in unmated individuals of the opposite sex but not in unmated individuals of the same sex. (3) The bacteria carried by unmated individuals were lost after mating. In addition, the abundances of bacterial communities carried by eggs were significantly higher than those of adult worms. Our results confirm that mating leads to the transfer of bacterial communities in the reproductive organs and gut of tomato leafminers, and suggest that this community strongly influences the reproductive process.

Krüppel-homologue 1 regulates the development of Tuta absoluta and its cascade regulation pattern in the juvenile hormone signalling pathway.

Tomato leaf miner, Tuta absoluta (Meyrick), is one of the most destructive quarantine pests globally. It has been confirmed that Krüppel-homologue 1 (kr-h1) plays a key role in the regulation of juvenile hormone (JH). However, it is unclear how kr-h1 regulates the synthesis of JH and its cascade regulation pattern in tomato leaf miner. Here, we obtained the six JH signalling genes (kr-h1, Methoprene-tolerant, Forkhead box O, Juvenile acid methyltransferase, Juvenile hormone esterase and Fatty acid synthase 2), and applied RNA interference to explore the role of kr-h1 and the seven genes (plus Vitellogenin) regulation relationship in T. absoluta. Bioinformatics analysis revealed the structural characteristics of kr-h1 protein and JH receptor Met, which contained eight C2H2 zinc finger structures and three typical domains of the bHLH-PAS family, respectively. The expression levels of Met and Vg were upregulated after RNAi of kr-h1 gene, while the gene levels of JHAMT and FAS2 were downregulated. Furthermore, topical application of JH analogue to second instar larvae could induce the expression of kr-h1 and inhibit the expression of Met. Our study reveals the mechanism by which kr-h1 regulates JH pathway genes, which could be applied to control the growth of tomato leaf miners.

First Report on CRISPR/Cas9-Based Genome Editing in the Destructive Invasive Pest Tuta Absoluta (Meyrick) (Lepidoptera: Gelechiidae).

The tomato leaf miner Tuta absoluta (Meyrick) is one of the world's most destructive pests of tomato, and because of its severe economic impacts, as well as the development of pesticide resistance, the species has been intensively studied, especially in regard to the identification of targets for T. absoluta control. However, functional genomic studies of T. absoluta have been constrained by a lack of effective genetic tools. Therefore, the aim of the present study was to develop a CRISPR/Cas9 zygote microinjection protocol for generating heritable mutations in T. absoluta, using the ommochrome synthesis gene cinnabar as an easily evaluated target gene. The injection of fertilised eggs with Cas9 protein and four sgRNAs, which targeted cinnabar exon 3, resulted in a mutagenesis rate of 31.9% for eggs reaching adulthood, and cinnabar mutagenesis resulted in either red or mosaic eye colour phenotypes. As such, this study is the first to report a complete and detailed CRISPR/Cas9 workflow for the efficient genome editing of the globally important invasive pest T. absoluta. The application of this robust genome-editing tool to T. absoluta will greatly facilitate the discovery of suitable RNAi control targets and the subsequent development of novel control strategies.

Chromatin-Remodelling ATPases ISWI and BRM Are Essential for Reproduction in the Destructive Pest Tuta absoluta.

The tomato leaf miner (Tuta absoluta) is one of the top 20 plant pests worldwide. We cloned and identified the chromatin-remodelling ATPase genes ISWI and BRM by RACE and bioinformatic analysis, respectively; used RT-qPCR to examine their expression patterns during different life cycle stages; and elucidated their roles in insect reproduction using double-stranded RNA injections. The full-length cDNA of TaISWI was 3428 bp and it encoded a 1025-aa polypeptide. The partial-length cDNA of TaBRM was 3457 bp and it encoded a 1030-aa polypeptide. TaISWI and TaBRM were upregulated at the egg stage. Injection of TaISWI or TaBRM dsRNA at the late pupa stage significantly inhibited adult ovary development and reduced fecundity, hatchability, and longevity in the adult females. To the best of our knowledge, the present study was the first to perform molecular characterisations of two chromatin-remodelling ATPase genes and clarify their roles in T. absoluta fecundity. Chromatin-remodelling ATPases are potential RNAi targets for the control of T. absoluta and other insect pests. The present study was also the first to demonstrate the feasibility of reproductive inhibitory RNAi as a putative approach for the suppression of T. absoluta and other Lepidopteran insect populations.

Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions.

Tuta absoluta is one of the most significant invasive pests affecting tomato plants worldwide. RT-qPCR has emerged as one of the most sensitive and accurate methods for detecting gene expression data. The screening of stable internal reference genes is the most critical step for studying the molecular mechanisms of environmental adaptability. The stable reference genes expressed in T. absoluta under specific experimental conditions have not yet been clarified. In this study, seven candidate reference genes (RPL27, RPS13, RPS15, EF1-α, TUB, TBP, and ß-actin) and their optimal numbers were evaluated under biotic (developmental stages and adult tissues) and abiotic (insecticide, temperature, and plant VOC) conditions using four software programs. Our results identified the following reference genes and numbers as optimal: three genes (EF1-α, RPS13, and RPL27) for different developmental stages (egg, larva, pupa, unmated adult), two genes (RPS13 and TBP) for adult tissues (antenna, head, thorax, abdomen, leg), two genes (TBP and RPS13) for insecticides (Bacillus thuringiensis, chlorpyrifos, abamectin-aminomethyl, and chlorantraniliprole), two genes (RPL27 and TUB) for temperature-induced stresses (0, 25, and 40 °C), and two genes (RPS13 and TUB) for VOC-induced stresses (nonanal, α-phellandrene, and tomato leaves). Our results provide a reference for selecting appropriate reference genes for further study of the functional genes of T. absoluta under different experimental conditions.

Molecular Characterization of TRPA Subfamily Genes and Function in Temperature Preference in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae).

To reveal the mechanism of temperature preference in Tuta absoluta, one of the top 20 plant pests in the world, we cloned and identified TaTRPA1, TaPain, and TaPyx genes by RACE and bioinformatic analysis, and clarified their expression profiles during different development stages using real-time PCR, and revealed their function in preference temperature by RNAi. The full-length cDNA of TaPain was 3136 bp, with a 2865-bp open reading frame encoding a 259.89-kDa protein; and the partial length cDNA of TaPyx was 2326-bp, with a 2025-bp open reading frame encoding a 193.16-kDa protein. In addition, the expression of TaTRPA1 and TaPyx was significantly lower in larvae than other stages, and it was significantly higher in pupae and newly emerging males for TaPain. After feeding target double-stranded RNA (dsRNA), the preferred temperature decreased 2 °C more than the control group. In conclusion, the results firstly indicated the molecular characterization of TRPA subfamily genes and their key role in temperature perception in T. absoluta, and the study will help us to understand the temperature-sensing mechanism in the pest, and will provide some basis for study of other Lepidoptera insects' temperature preference. Moreover, it is of great significance in enriching the research progress of "thermos TRP".