A non-invasive method for sexing all castes of Prorhinotermes flavus (Blattodea: Psammotermitidae) using external...

Sex identification in termites is crucial for understanding caste development, colony organization, and reproductive strategies. Traditional methods, such as dissection and staining, are invasive and may damage specimens. In this study, a non-invasive method was developed to identify the sex of Prorhinotermes flavus (Bugnion and Popoff). The sternite morphology of all six castes, alate, neotenic, nymph, pseudergate, soldier, and larva, could be categorized into two groups. Micro-CT analyses revealed sex-specific gonadal structures in alates, neotenics, and nymphs, and their corresponding sex-specific sternite morphology was confirmed. Rearing trials were conducted to observe pseudergates molting into neotenics and soldiers, and larvae molting into pseudergates. Since the sex-specific sternite morphology of neotenics is known, we are able to confirm the sex of pseudergates, and consequently, that of soldiers and larvae. Our findings show that male alates and neotenics possess a visible ninth abdominal sternite with distinct styli. In female alates and neotenics, the seventh sternite completely covers the eighth and ninth sternites, and the styli are absent. In male pseudergates, soldiers, and nymphs, the ninth sternite with styli is also clearly visible, while in females, the seventh sternite partially covers the eighth and ninth sternites. Male larvae can be identified by the presence of bristles on the posterior margin of the eighth abdominal sternite, whereas in female larvae, these bristles are absent. This study presents the first comprehensive, non-invasive method for sexing all castes of P. flavus, enabling more precise research on caste development and social interactions in termite colonies.