Reitmayer, C. M. | Search bioRxiv

bioRxiv2023

A Zika virus-responsive sensor-effector system in Aedes aegypti

Zika virus (ZIKV) is a recently re-emerged flavivirus transmitted primarily through the bite of an infected mosquito, Aedes aegypti being the main vector. ZIKV infection is associated with a range of adverse effects; infection during pregnancy can lead to foetal abnormalities, including microcephaly. Lacking a licensed vaccine, or specific therapeutics, control of ZIKV transmission focuses on vector control. However, in most transmission settings, current methods are insufficient to successfully control ZIKV, or other similarly-transmitted arboviruses such as dengue and chikungunya viruses. This has stimulated interest in genetics-based methods, either to reduce the number of mosquitoes ("population suppression"), or to make mosquitoes less able to transmit ("population modification"). Here, we describe a method to selectively eliminate infected mosquitoes, using a virus sensor inserted into the mosquito genome and coupled to a quorum-counting lethal effector. In mosquitoes, ZIKV normally establishes persistent, lifelong infection; survival of these infected mosquitoes is crucial to transmission potential. Correspondingly, removal of infected mosquitoes can reduce vectorial capacity of a mosquito population, i.e. ability to transmit. Since relatively few mosquitoes become infected, typically <2%, engineered hypersensitivity to ZIKV would have only a modest population-level fitness cost, and lower still if transmission were successfully reduced by such means.

bioengineering↗

bioRxiv2022

Fake it to break it: mimicking superinfection exclusion disrupts alphavirus infection and transmission in the yellow fever mosquito Aedes aegypti

Multiple viruses cause a phenomenon termed superinfection exclusion whereby a currently infected cell is resistant to secondary infection by the same or a closely related virus. In alphaviruses, this process is thought to be mediated, at least in part, by the viral protease (nsP2) which is responsible for processing the non-structural polyproteins (P123 and P1234) into individual proteins (nsP1-nsP4), forming the viral replication complex. Taking a synthetic-biology approach, we mimicked this naturally occurring phenomenon by generating a superinfection exclusion-like state in Aedes aegypti mosquitoes, rendering them refractory to alphavirus infection. By artificially expressing Sindbis virus (SINV) and chikungunya virus (CHIKV) nsP2 in mosquito cells and transgenic mosquitoes, we demonstrated a reduction in both SINV and CHIKV viral replication rates in cells following viral infection as well as reduced infection prevalence, viral titres and transmission potential in mosquitoes.

microbiology↗

bioRxiv2022

Small silencing RNAs expressed from W-linked retrocopies of Masculinizer target the male-determining gene PxyMasc during female sex determination in the Diamondback moth Plutella xylostella

The Lepidoptera are an insect order of cultural, economic and environmental importance, representing c. 10% of all described living species. Yet, for all but one of these species (silkmoth, Bombyx mori) the molecular genetics of how sexual fate is determined remains unknown. We investigated this in the diamondback moth (DBM - Plutella xylostella), a globally important, highly invasive and economically damaging pest of cruciferous crops. Our previous work uncovered a regulator of male sex determination in DBM - PxyMasc, a homologue of B. mori Masculinizer - which although initially expressed in embryos of both sexes, is then reduced in female embryos, leading to female-specific splicing of doublesex. Here, through sequencing small RNA libraries generated from early embryos and sexed larval pools, we identified a variety of small silencing RNAs (predominantly piRNAs) complementary to PxyMasc, whose temporal expression correlated with the reduction in PxyMasc transcript observed previously in females. Analysis of these small RNAs showed that they are expressed from tandemly-arranged, multi-copy arrays found exclusively on the W (female-specific) chromosome, which we term Pxyfem. Analysis of the Pxyfem sequences showed that they are partial cDNAs of PxyMasc mRNA transcripts, likely integrated into transposable element graveyards by the non-canonical action of retrotransposons (retrocopies), and that their apparent similarity to B. mori feminizer more probably represents convergent evolution. Our study helps elucidate the sex determination cascade in this globally important pest and highlights the shortcuts which retrotransposition events can facilitate in the evolution of complex molecular cascades, including sex determination. Significance statementUncovering the mechanisms which species have evolved to determine sex is of fundamental interest and provides avenues for pest management through genetic manipulation of these pathways. In insects, much of what is known regarding sex determination is concentrated within the Diptera and Hymenoptera, despite other orders (e.g. Lepidoptera) being of great ecological and economic importance. Here, using small RNA sequencing of embryonic and early larval samples, we uncover an RNAi-based sex determination system which silences the male determining gene PxyMasc in the Diamondback moth (Plutella xylostella) - a global pest of cruciferous crops. We track production of these silencing RNAs back to the W-chromosome where they are expressed from partial cDNA copies of PxyMasc. Our analysis suggests these are PxyMasc retrocopies, integrated via the non-canonical action of LTR retrotransposons and that similarities between this system and the feminizer system in Bombyx mori likely represent convergent evolution.

developmental biology↗