In the pursuit of better pest-and vector-control strategies, attention returns to an old proven technology, the sterile insect technique (SIT) and related insect population-suppression methods. A major obstacle for any of these approaches that involves the release of sterile males is the separation of males from females during the mass rearing stage, in order to improve the cost-efficiency of these methods and to prevent the release of biting and disease-vectoring females. This review describes recent sex-sorting developments in dipteran flies with an emphasis on assessing the suitability of these methods for large-scale rearing of male vectors for mass release. Sexing Is an Obstacle in Genetic Pest-control ProgramsHighlights Sexing Diptera represents a major obstacle to operationalizing vector-control methods based on the mass release of males, such as the sterile insect technique or the incompatible insect technique.

RNA interference (RNAi) in insects is routinely used to ascertain gene function, but also has potential as a technology to control pest species. For some insects, such as beetles, ingestion of small quantities of double-stranded RNA (dsRNA) is able to knock down a targeted gene's expression. However, in other species, ingestion of dsRNA can be ineffective owing to the presence of nucleases within the gut, which degrade dsRNA before it reaches target cells. In this study, we observed that nucleases within the gut of the Queensland fruit fly (Bactrocera tryoni) rapidly degrade dsRNA and reduce RNAi efficacy. By complexing dsRNA with liposomes within the adult insect's diet, RNAi-mediated knockdown of a melanin synthesis gene, yellow, was improved significantly, resulting in strong RNAi phenotypes. RNAi efficiency was also enhanced by feeding both larvae and adults for several days on dsRNAs that targeted two different dsRNase gene transcripts. Co-delivery of both dsRNase-specific dsRNAs and yellow dsRNA resulted in almost complete knockdown of the yellow transcripts. These findings show that the use of liposomes or co-feeding of nuclease-specific dsRNAs significantly improves RNAi inhibition of gene expression in B. tryoni and could be a useful strategy to improve RNAi-based control in other insect species.

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RNA interference (RNAi) techniques are being developed for a range of pest insect control technologies, including the sterile insect technique (SIT) and double-stranded RNA (dsRNA)-based insecticides. In SIT applications, where >99% of the released males should be sterile to meet industry standards, the efficiency of RNAi will need to be improved for many insect species if this technology is to be adopted. Endogenous dsRNases can impede dsRNA delivery in some insects, and, here, we investigated whether dsRNases in the midgut could limit RNAi efficacy in the mosquito Aedes aegypti. Ten putative dsRNases were identified in the Ae. aegypti genome, with two highly expressed in the midguts of larvae. Using an ex vivo assay, we observed that dsRNA was rapidly degraded within the mosquito larva’s gut. Double-stranded RNA targeting these two dsRNases, when fed to the larvae, effectively reduced gut dsRNase activity. When these dsRNase-specific dsRNAs were co-delivered with dsRNA targeting a cyan fluorescent protein (CFP) reporter gene, greater knockdown of CFP fluorescence was observed. These results suggest that inhibiting dsRNase activity could enable the implementation of RNAi-based mosquito control methods.

Artemisinin partial resistance may facilitate selection of Plasmodium falciparum resistant to combination therapy partner drugs. We evaluated 99 P. falciparum isolates collected in 2021 from northern Uganda, where resistance-associated PfK13 C469Y and A675V mutations have emerged, and eastern Uganda, where these mutations are uncommon. With the ex vivo ring survival assay, isolates with the 469Y mutation (median survival 7.3% for mutant, 2.5% mixed, and 1.4% wild type) and/or mutations in Pfcoronin or falcipain-2a, had significantly greater survival; all isolates with survival >5% had mutations in at least one of these proteins. With ex vivo growth inhibition assays, susceptibility to lumefantrine (median IC50 14.6 vs. 6.9 nM, p < 0.0001) and dihydroartemisinin (2.3 vs. 1.5 nM, p = 0.003) was decreased in northern vs. eastern Uganda; 14/49 northern vs. 0/38 eastern isolates had lumefantrine IC50 > 20 nM (p = 0.0002). Targeted sequencing of 819 isolates from 2015–21 identified multiple polymorphisms associated with altered drug susceptibility, notably PfK13 469Y with decreased susceptibility to lumefantrine (p = 6 × 10−8) and PfCRT mutations with chloroquine resistance (p = 1 × 10−20). Our results raise concern regarding activity of artemether-lumefantrine, the first-line antimalarial in Uganda.

Background Artemisinin resistance mutations in Plasmodium falciparum kelch13 (Pfk13) have begun to emerge in Africa with Pfk13-R561H as the first reported, found in Rwanda in 2014, but limited sampling left questions about its early distribution and origin. Methods We genotyped P. falciparum positive dried blood spot (DBS) samples from a nationally representative 2014-15 Rwanda Demographic Health Surveys (DHS) HIV study. DBS were subsampled from DHS sampling clusters with &gt;15% P. falciparum prevalence as determined by rapid testing or microscopy done during the DHS study (n clusters = 67, n samples = 1873). Results We detected 476 parasitemias among 1873 residual blood spots from a 2014-15 Rwanda Demographic Health Survey. We sequenced 351 samples revealing 341/351 were wild type (97.03% weighted) and 4 samples (1.34% weighted) harbored R561H which were significantly spatially clustered. Other nonsynonymous mutations found were V555A (3), C532W (1), and G533A (1). Conclusions Our study better defines the early distribution of R561H in Rwanda. Previous studies only observed the mutation in Masaka as of 2014, but our study indicates its presence in higher-transmission regions in the southeast of the country at that time.

Recent developments in molecular biology and genomics have revolutionized biology and medicine mainly in the developed world. The application of next generation sequencing (NGS) and CRISPR-Cas tools is now poised to support endemic countries in the detection, monitoring and control of endemic diseases and future epidemics, as well as with emerging and re-emerging pathogens. Most low and middle income countries (LMICs) with the highest burden of infectious diseases still largely lack the capacity to generate and perform bioinformatic analysis of genomic data. These countries have also not deployed tools based on CRISPR-Cas technologies. For LMICs including Tanzania, it is critical to focus not only on the process of generation and analysis of data generated using such tools, but also on the utilization of the findings for policy and decision making. Here we discuss the promise and challenges of NGS and CRISPR-Cas in the context of malaria as Africa moves towards malaria elimination. These innovative tools are urgently needed to strengthen the current diagnostic and surveillance systems. We discuss ongoing efforts to deploy these tools for malaria detection and molecular surveillance highlighting potential opportunities presented by these innovative technologies as well as challenges in adopting them. Their deployment will also offer an opportunity to broadly build in-country capacity in pathogen genomics and bioinformatics, and to effectively engage with multiple stakeholders as well as policy makers, overcoming current workforce and infrastructure challenges. Overall, these ongoing initiatives will build the malaria molecular surveillance capacity of African researchers and their institutions, and allow them to generate genomics data and perform bioinformatics analysis in-country in order to provide critical information that will be used for real-time policy and decision-making to support malaria elimination on the continent.

Artemisinin resistance mutations in Plasmodium falciparum kelch13 (Pfk13) have begun to emerge in Africa. Pfk13-R561H was the first reported African mutation found in Rwanda in 2014, but limited sampling left questions about its early distribution and origin. We detected 476 parasitemias among 1873 residual blood spots from a 2014-15 Rwanda Demographic Health Survey. We sequenced 351 samples revealing 341/351 were wild type (97.03% weighted) and 4 samples (1.34% weighted) harbored R561H which were significantly spatially clustered. Our study better defines the early distribution of R561H in Rwanda and suggests that the origin may have involved higher-transmission regions.