Calliphorid Hygrosensation Perception and the Effect of Humidity on Oviposition Behavior

Abstract

Forensic entomology relies on the predictable development of insects under specific environmental conditions to estimate the postmortem interval. However, the accuracy of these estimates can be affected by the time between death and insect colonization, the pre-appearance interval. In cases where insect evidence is absent, it is often unclear whether colonization has yet to occur or if environmental conditions, such as humidity, have inhibited oviposition. While the effects of temperature on blow fly behavior are well established, humidity remains less understood. This research examined the oviposition behavior of Phormia regina (Diptera: Calliphoridae) under varying humidity levels across different seasons, using populations collected from Indiana and Arizona. Results indicate that humidity alone did not significantly impact oviposition timing or egg output, although temperature and season were significant drivers of both outcomes (both p <0.001). Wild-caught flies were more sensitive to 70 % rh than lab-adapted flies, laying significantly more eggs (p = 0.04). Additionally, females reared under low humidity conditions laid the majority of their eggs in the evening, suggesting that developmental or maternal effects may influence oviposition behavior. Flies native to Arizona showed delayed oviposition at high humidity (85 %) compared to low (30 %) (p < 0.001), while Indiana flies produced more eggs across conditions. To explore the physiological mechanisms underlying humidity perception in P. regina, we analyzed the expression of odorant-binding protein 59a and ionotropic receptor 40a, genes associated with hygrosensation in Drosophila. Ir40a expression differed significantly by location (p = 0.003) and sex (p = 0.0007), with higher levels in males and in Arizona flies, but showed no response to humidity. In contrast, Obp59a expression differed significantly by both location (p = 0.031) and humidity (p = 0.046), with approximately 2.5-fold higher expression in flies from Arizona and those reared under low humidity conditions. Together, these patterns suggest distinct roles of these genes in humidity sensing. Collectively, these findings collectively demonstrate how humidity, temperature and geographic origin influence Phormia regina oviposition behavior and gene expression related to hygrosensation, providing critical insights to refine the accuracy and reliability of entomological evidence in postmortem interval estimation.