Paratransgenic manipulation of tsetsemiR275alters the physiological homeostasis of the fly’s midgut environment

Abstract Tsetse flies are vectors of parasitic African trypanosomes ( Trypanosoma spp.). Current disease control methods include fly-repelling pesticides, trapping flies, and chemotherapeutic treatment of infected people. Inhibiting tsetse’s ability to transmit trypanosomes by strengthening the fly’s natural barriers can serve as an alternative approach to reduce disease. The peritrophic matrix (PM) is a chitinous and proteinaceous barrier that lines tsetse’s midgut. It protects the epithelial cells from the gut lumen content such as food and invading trypanosomes, which have to overcome this physical barrier to establish an infection. Bloodstream form trypanosomes shed variant surface glycoproteins (VSG) into tsetse’s gut lumen early during the infection establishment. The VSG molecules are internalized by the fly’s PM-producing cardia, which results in a reduction in tsetse miR275 expression and a sequential molecular cascade that compromises the PM integrity. In the present study, we investigated the role(s) of miR275 in tsetse’s midgut physiology and trypanosome infection processes by developing a paratransgenic expression system. We used tsetse’s facultative bacterial endosymbiont Sodalis glossinidius to express tandem antagomir -275 repeats (or miR275 sponge) that constitutively reduce miR275 transcript abundance. This paratransgenic system successfully knocked down miR275 levels in the fly’s midgut, which consequently obstructed blood digestion and modulated infection outcomes with an entomopathogenic bacteria and with trypanosomes. RNA sequencing of cardia and midgut tissues from the paratransgenic tsetse confirmed that miR275 regulates processes related to the expression of PM-associated proteins and digestive enzymes as well as genes that encode abundant secretory proteins. Our study demonstrates that paratransgenesis can be employed to study microRNA-regulated pathways in arthropods housing symbiotic bacteria. Author Summary Tsetse flies transmit African trypanosomes, which are the parasites that cause sleeping sickness in human in sub-Saharan Africa. When tsetse ingests a blood meal containing trypanosomes, the expression level of a microRNA ( miR275 ) decreases in the fly’s gut. This process results in a series of events that interrupt the physiological homeostasis of the gut environment. To further understand the function of miR275 in tsetse fly, we genetically modified a tsetse’s native bacterial symbiont, reintroduced the genetically modified bacterium back into the fly, and successfully knocked down the miR275 expression in tsetse’s midgut. These ‘paratransgenic’ flies (which house genetically modified bacteria) presented impaired digestive processes and were highly susceptible to infection with trypanosomes. Lastly, we discovered that miR275 regulates tsetse secretory pathways. Our novel paratransgenic expression system can be applied to study the function of other microRNAs and how they regulate disease transmission in tsetse and other insect systems.