The rodent filaria Litomosoides sigmodontis harbour Wolbachia, endosymbionts essential for worm embryogenesis, larval development and adult survival. To study the effect of tetracycline, which depletes Wolbachia, on the development of microfilariae (L1s, MF) to L3 in the intermediate host Ornithonyssus bacoti, and to observe the development of Wolbachia-depleted L3s in Mongolian gerbils (Meriones unguiculatus); microfilaremic gerbils were treated orally with tetracycline for 6 weeks (primary infected Tet) or untreated (primary Con). Treatment resulted in a significant reduction of Wolbachia per MF in primary Tet gerbils. Naïve mites then fed on the primary Tet and primary Con gerbils in the week after treatment ended, when MF levels were not significantly different, and used to infect new gerbils (secondary infected ) Tet, secondary Con) via natural infection. The infection rate from dissected mites was 9% and 54% (primary Tet and primary Con, respectively). After 3 months, worms were isolated from secondary gerbils. Significantly fewer female worms developed in secondary Tet gerbils. In contrast, there was no difference in the number of male worms that developed in secondary gerbils, resulting in a male biased sex-ratio. Although secondary Tet male worms had fewer Wolbachia than secondary Con males, development was not impaired. Female worms that developed from Wolbachia-depleted MF had Wolbachia levels equivalent to worms from secondary Con animals. Thus, tetracycline pre-treatment selected for female worms with high numbers of Wolbachia, whereas male worms had median Wolbachia levels significantly lower than secondary Con males. Therefore, female worms require a higher threshold of Wolbachia for their development. The worms analysed were only exposed to tetracycline as MF, ruling out direct effects of tetracycline during larval development in the mites or secondary gerbils, suggesting that the depletion of Wolbachia in MF was the cause of impaired larval development.