Puparia are commonly found in tsetse fly larviposition sites during studies on larval ecology. This chitinous shell is representative of past or ongoing exploitation of these sites by tsetse flies. The morphological characteristics of the puparium are not sufficiently distinctive to allow identification of the species. This study explores the applicability of biomolecular techniques on empty puparia for tsetse fly species identification. Five techniques were compared for DNA extraction from tsetse fly puparia, 1/Chelex® 100 Resin, 2/CTAB, 3/Livak's protocol, 4/DEB + proteinase K and 5/QIAamp® DNA Mini kit, using two homogenisation methods (manual and automated). Using a combination of two primer pairs, Chelex, CTAB, and DEB + K proved the most efficient on fresh puparia with 90, 85, and 70% samples identified, respectively. Shifting from fresh to one- to nine-month-old puparia, the Chelex method gave the best result allowing species identification on puparia up to seven months old. The subsequent testing of the Chelex extraction protocol identified 152 (60%) of 252 field-collected puparia samples at species level. The results show that reliable genetic identification of tsetse flies species can be performed from empty puparia, what can prove of great interest for future ecological studies on larviposition sites. The Chelex technique was the most efficient for DNA extraction, though the age-limit of the samples stood at seven months, beyond which DNA degradation probably compromises the genetic analysis.

Differentiation of niche by means of resource partitioning facilitates coexistence of species with similar requirements. Here we analyse the association between different habitats (i.e. nest types) and two Diptera species of the poorly known Family Carnidae that coexist during their larval and pupal stage in the nests of troglodytic bird species. We also describe for the first time the puparium of Hemeromyia anthracina and Hemeromyia longirostris and offer morphometric data of the puparia of these two species and of Carnus hemapterus. Both the smaller size and the occurrence of well-developed spiracles allow easy discrimination of the puparium of C. hemapterus. The puparia of both Hemeromyia species is very similar and only differ in the distance between the small spiracles. Hemeromyia anthracina and C. hemapterus coexisted in nest boxes but the former species did not occur in natural sandy cavities where, in turn, C. hemapterus was highly prevalent. Carnus hemapterus prevalence did not differ between nest boxes and natural cavities but its abundance was higher in the first type of nest. This study shows clear associations of the two dipteran species with specific types of nests. Yet, some conditions are seemingly acceptable for both species.