Knowledge of the effects that Thelazia callipaeda (Spirurida, Thelaziidae) infection has on the survival of its vector Phortica variegata (Drosophilidae, Steganinae) is scarce. The present study aimed to: (a) assess the rate of infection between experimentally infected (EI) and not experimentally infected (NEI) flies and (b) determine how T. callipaeda infection may affect the survival of P. variegata. In addition, fat composition was evaluated in flies that died during overwintering. Molecular analysis showed that T. callipaeda prevalence in flies that died before experimental infection, plus those from the NEI group, is 0.75% (i.e. 11 out of 1462 individuals). The EI group showed a significantly higher positivity to T. callipaeda (i.e. 51 out of 682 individuals; 7.48%) compared with the NEI group (i.e. 9 out of 750 individuals; 1.2%). Thelazia callipaeda DNA was detected until 147 days after experimental infection. This demonstrates that larvae of this eyeworm may survive in the fly for a significant period of the winter. Fat composition analysis showed that flies produced more unsaturated than saturated fatty acids during diapause, probably because unsaturated fatty acids remain in a liquid state at lower temperatures, providing anti-freeze properties to survive winter.