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Background: Usutu virus (USUV) is an emerging arbovirus transmitted predominantly by mosquitoes of the genus Culex, although several other genera, including Aedes, Culiseta, Mansonia, Anopheles, and Coquillettidia, have also been implicated as potential vectors. The virus was originally found in sub-Saharan Africa but is increasingly detected in Europe. While human infections are often asymptomatic, USUV has been linked to neuroinvasive disease. In temperate regions, where mosquito activity is seasonal, mechanisms enabling virus persistence are poorly understood. Vertical transmission has been proposed as a route for interepidemic maintenance, but experimental evidence remains limited.

Objective: To determine whether USUV can be vertically transmitted between mosquito generations and assess the effect of temperature on the vertical transmission rate in Cx. pipiens bioform molestus.

Methods: Laboratory-reared Cx. pipiens bioform molestus were provided with a bloodmeal infected with USUV and maintained at either 27°C or 23.5°C. Infected females (F0) were allowed to oviposit, and two successive generations (F1 and F2) were reared. Individuals from all life stages were screened for USUV RNA using RT-qPCR. Infection prevalence and relative viral RNA quantification were compared between temperature conditions and generations.

Results: At 27°C, the vertical transmission rate was high, with 94% (n = 30) of F₁ and 100% (n = 11) of F₂ adult mosquitoes testing positive, although USUV RNA was also detected in immature stages (eggs, larvae, and pupae). At 23.5°C, only one F₁ adult mosquito (1.25%) tested positive, with no infection detected in larvae or other immature stages and no F₂ infection was detected. Temperature had a significant effect on vertical transmission rate (p < 0.001), while differences between generations were not statistically significant.

Conclusion: These findings demonstrate that vertical transmission of USUV in Cx. pipiens bioform molestus occurs at significantly higher rates at 27°C (94–100%) compared to 23.5°C (1.25%), suggesting temperature-dependent mechanisms specifically affecting vertical inheritance. While temperature effects on viral acquisition and dissemination are well-established, our experimental design specifically tests vertical transmission by examininginfection in F₁ and F₂ offspring that had no direct exposure to the virus, allowing us to distinguish vertical inheritance from horizontal transmissionprocesses.