BACKGROUND: Bactrocera dorsalis (Hendel), a very destructive insect pest of many fruits and vegetables, is widespread in many Asian countries. To facilitate control of this pest, it is essential to investigate its genetics and gene function using targeted gene disruption.RESULTS: Here, we describe successful targeted mutagenesis of the white and transformer genes in B. dorsalis through use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. Co-injection of the white sgRNA and Cas9 mRNA into B. dorsalis embryos caused eye color change, and the white mutations in the germline were heritable. CRISPR-mediated knockout of the sex determination gene transformer (tra) in B. dorsalis resulted in a male-biased sex ratio and adult flies with abnormal outer and interior reproductive organs. Small indels and substitutions were induced by CRIRPR for both genes.CONCLUSION: Our data demonstrate that somatic and germline genome engineering of the pest B. dorsalis can be performed efficiently using the CRISPR/Cas9 system, opening the door to the use of the CRISPR-mediated method for functional annotations of genes in B. dorsalis and for its population control using, for example, such as gene drive.

transformer (tra) is a switch gene of sex determination in many insects, particularly in Dipterans. However, the sex determination pathway in Bactrocera cucurbitae (Coquillett), a very destructive pest on earth, remains largely uncharacterized. In this study, we have isolated and characterized one female-specific and two male-specific transcripts of the tra gene (Bcutra) of B. cucurbitae. The genomic structure of Bcutra has been determined and the presence of multiple conserved Transformer (TRA)/TRA-2 binding sites in Bcutra has been found. BcuTRA is highly conservative with its homologues in other tephritid fruit flies. Gene expression analysis of Bcutra at different developmental stages demonstrates that the female transcript of Bcutra appears earlier than the male counterparts, indicating that the maternal TRA is inherited in eggs and might play a role in the regulation of TRA expression. The conservation of protein sequence and sex-specific splicing of Bcutra and its expression patterns during development suggest that Bcutra is probably the master gene of sex determination of B. cucurbitae. Isolation of Bcutra will facilitate the development of a genetic sexing strain for its biological control.

PurposeWe aimed to investigate the prevalence and risk factors of filamentous fungi (FF) carriage in human immunodeficiency virus (HIV)-infected patients in Guangdong province, along with its subsequent incidence of invasive fungal disease (IFD).MethodsSeven hundred and sixteen HIV-infected individuals from the outpatient clinic and 293 sex-matched healthy controls were recruited prospectively from May 1 to August 31, 2017. Fungi were isolated from oropharyngeal and nasopharyngeal swabs, then identified by morphological and molecular biological techniques. Logistic regression analysis was used to identify risk factors of pathogenic FF carriage. Pathogenic FF carriers were followed up through the end of 2019.ResultsOf the 716 included HIV-infected patients, 602 (84.1%) were male, the median age was 34 (27–42) years, and the median CD4+ count was 385 (254–542) cells/μl. Pathogenic FF were isolated in 119 (16.6%) cases with HIV infection and 40 (13.7%) healthy controls. Mucorales were found in 3 HIV-infected individuals and Talaromyces marneffei in 2 HIV-infected individuals, but not in healthy controls. History of cured opportunistic infections (OIs; OR, 1.97; 95% CI, 1.23–3.13, p = 0.004), and smoking (OR, 1.55; 95%CI, 1.03–2.32, p = 0.035) were independent risk factors of pathogenic FF carriage in HIV-infected individuals. A total of 119 pathogenic FF carriers with HIV infection were followed. During follow-up, 119 (100%) cases received antiretroviral therapy (ART) for at least 28 months, 107 (90%) cases had CD4+ counts>200 cells/μl, and none developed IFD.DiscussionPathogenic FF carriage is common in HIV-infected individuals but may not develop IFD in those who achieved immune reconstitution. Smoking and cured OIs history increase the risk of pathogenic FF carriage. Smoking abstinence and ART adherence are especially important for these patients.