Symbiont-generated vitamin B6 is required to maintain proline homeostasis and fecundity in the tsetse fly

The tsetse fly obligate symbiont, Wigglesworthia glossinidae, produces a range of B vitamins, including pyridoxal phosphate (vitamin B6).  Tsetse utilizes proline instead of trehalose as a circulating nutrient source.  Proline biosynthesis from alanine involves alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B6) as a co-factor.  Here, we determined if production of vitamin B6 by Wigglesworthia is critical for the maintenance of proline homeostasis, specifically during the lactation period of tsetse’s viviparous reproductive cycle.  Expression of agat and host and symbiont genes involved in vitamin B6 synthesis increase in lactating flies.  Removal of symbionts via antibiotic treatment to generate aposymbiotic flies resulted in hypoprolinemia in lactating females and decreased fecundity. The resulting hypoprolinemia in aposymbiotic flies can be partially recovered by providing a diet supplemented with Wigglesworthia or yeast extracts.  Knockdown of agat in wild type flies reduced proline to similar levels as observed in aposymbiotic females.  Aposymbiotic flies treated with agat siRNA failed to recover their proline levels when their blood diet was supplemented with B vitamins or microbial extracts.  In conclusion, the presence of Wigglesworthia in tsetse is critical for the maintenance of proline hemostasis through the generation of specific B vitamins.