Elimination of malaria depends on mastering transmission and understanding the biological basis of Plasmodium infection in the vector. The first mosquito organ to interact with the parasite is the midgut and its transcriptomic characterization during infection can reveal effective antiplasmodial responses able to limit the survival of the parasite. The vector response to Plasmodium vivax is not fully characterized, and its specificities when compared with other malaria parasites can be of fundamental interest for specific control measures. Experimental infections were performed using a membrane-feeding device. Three groups were used: P. vivax-blood-fed, blood-fed on inactivated gametocytes, and unfed mosquitoes. Twenty-four hours after feeding, the mosquitoes were dissected and the midgut collected for transcriptomic analysis using RNAseq. Nine cDNA libraries were generated and sequenced on an Illumina HiSeq2500. Readings were checked for quality control and analysed using the Trinity platform for de novo transcriptome assembly. Transcript quantification was performed and the transcriptome was functionally annotated. Differential expression gene analysis was carried out. The role of the identified mechanisms was further explored using functional approaches. Forty-nine genes were identified as being differentially expressed with P. vivax infection: 34 were upregulated and 15 were downregulated. Half of the P. vivax-related differentially expressed genes could be related to autophagy; therefore, the effect of the known inhibitor (wortmannin) and activator (spermidine) was tested on the infection outcome. Autophagic activation significantly reduced the intensity and prevalence of infection. This was associated with transcription alterations of the autophagy regulating genes Beclin, DRAM and Apg8. Our data indicate that P. vivax invasion of An. aquasalis midgut epithelium triggers an autophagic response and its activation reduces infection. This suggests a novel mechanism that mosquitoes can use to fight Plasmodium infection.

Haemagogus (Haemagogus) janthinomys (Dyar, 1921), the major neotropical vector of sylvatic yellow fever virus, is notoriously difficult to maintain in captivity. It has never been reared beyond an F₁ generation, and almost no experimental transmission studies have been performed with this species since the 1940s. Herein we describe installment hatching, artificial blood feeding, and forced-mating techniques that enabled us to produce small numbers of F₃ generation Hg. janthinomys eggs for the first time. A total of 62.8% (1562/2486) F₁ generation eggs hatched during ≤10 four-day cycles of immersion in a bamboo leaf infusion followed by partial drying. Hatching decreased to 20.1% (190/944) in the F₂ generation for eggs laid by mosquitoes copulated by forced mating. More than 85% (79/92) female F₂ mosquitoes fed on an artificial blood feeding system. While we were unable to maintain a laboratory colony of Hg. janthinomys past the F₃ generation, our methods provide a foundation for experimental transmission studies with this species in a laboratory setting, a critical capacity in a region with hyper-endemic transmission of dengue, Zika, and chikungunya viruses, all posing a risk of spillback into a sylvatic cycle.

Malaria remains a public health issue across the world. Different methods have been analyzed to achieve this disease’s elimination, such as the vector control of Anopheles spp. Control strategies include the use of different classes of insecticides, although the accelerated evolution of vectors resistant to them makes the development of alternative control methods necessary. Therefore, entomopathogenic fungi have been considered to be promising biopesticides, given that they are safe for human beings and the environment. This study aimed to evaluate the entomopathogenic activity of fungi collected in the Amazon Rainforest against adult female Anopheles aquasalis mosquitoes. Females were exposed to four different species of fungi and observed daily to evaluate their survival rate. Also, fungi species’ behavior was analyzed through scanning electron microscopy (SEM). Those exposed to Trichoderma harzianum and Penicillium citrinum had their survival rate reduced. SEM confirmed the development of fungi on the mosquitoes after 48 h. The findings suggest that the entomopathogenic potential of the fungi used in this study should be considered, given the reduction in the survival rate of Anopheles aquasalis mosquitoes.