The factors that influence parasite associated bacterial microbial diversity and the geographic distributions of bacteria are not fully understood. In an effort to gain a deeper understanding of the relationship between the bacterial diversity of Ctenocephalides fleas and host species and the external environment, we conducted a metagenetic analysis of 107 flea samples collected from 8 distinct sampling sites in South Africa. Pooled DNA samples mostly comprising of 2 or 3 individuals sampled from the same host, and belonging to the same genetic cluster, were sequenced using the Ion PGM™ Hi-Q™ Kit and the Ion 316™ Chip v2. Differences were detected in the microbiome compositions between Ctenocephalides felis, Ctenocephalides canis and Ctenocephalides connatus. Although based on a small sample, C. connatus occurring on wildlife harboured a higher bacterial richness when compared to C. felis on domestic animals. Intraspecific differences in the microbial OTU diversity were detected within C. f. felis that occurred on domestic cats and dogs. Different genetic lineages of C. f. felis were similar in microbial compositions but some differences exist in the presence or absence of rare bacteria. Rickettsia and Bartonella OTU's identified in South African cat fleas differ from those identified in the USA and Australia. Intraspecific microbial compositions also differ across geographic sampling sites. Generalized dissimilarity modelling showed that temperature and humidity are potentially important environmental factors explaining the pattern obtained.

A high degree of specialization between host and parasite is a well-known outcome of a long history of coevolution, and it is strikingly illustrated in a coordination of their life cycles. In some cases, the arms race ensued at the establishment of a symbiotic relationship results in the adoption of manipulative strategies by the parasite. We have already learned that Steinina ctenocephali, a gregarine living in the alimentary canal of cat flea, Ctenocephalides felis follows its phenology and metamorphosis. Despite these findings the outcome of their symbiotic partnership (mutualist, parasitic or commensal) remains unclear. To address this important question, we measured life history parameters of the flea in the presence of varying infection intensities of gregarine oocysts in laboratory conditions. We found that neither the emergence nor survival rate of fleas was affected by harbouring the gregarines. More surprisingly, our results show that flea larvae infected with gregarines developed faster and emerged earlier than the control group. This gregarine therefore joins the selected group of protists that can modify physiological host traits and provides not only new model taxa to be explored in an evolutionary scenario, but also potential development of control strategies of cat flea.

The cat flea, Ctenocephalides felis, is a major pest species on companion animals thus of significant importance to the animal health industry. The aim of this study was to develop sampling and storage protocols and identify stable reference genes for gene expression studies to fully utilize the growing body of molecular knowledge of C. felis. RNA integrity was assessed in adult and larvae samples, which were either pierced or not pierced and stored in RNAlater at ambient temperature. RNA quality was maintained best in pierced samples, with negligible degradation evident after 10 days. RNA quality from non-pierced samples was poor within 3 days. Ten candidate reference genes were evaluated for their stability across four group comparisons (developmental stages, genders, feeding statuses and insecticide-treatment statuses). Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), 60S ribosomal protein L19 (RPL19) and elongation factor-1α (Ef) were ranked highly in all stability comparisons, thus are recommended as reference genes under similar conditions. Employing just two of these three stable reference genes was sufficient for accurate normalization. Our results make a significant contribution to the future of gene expression studies in C. felis, describing validated sample preparation procedures and reference genes for use in this common pest.

To investigate larval development of Acanthocheilonema reconditum in the cat flea Ctenocephalides felis, fleas were fed through an artificial feeding system with dog blood containing different concentrations of microfilariae (i.e. low, group L = 250; medium, group M = 500; high, group H = 1500 microfilariae per mL) or no microfilariae (group C). Fleas were sampled at 12 different time-points throughout the study period (D1–D28) and A. reconditum was detected by dissection, PCR and histology. Of 2105 fleas fed with infected dog blood, 891 (38·7%) died during the study before being sampled whilst the remaining (n = 1214) were examined for A. reconditum. Upon dissection, first-stage larvae (L1) were identified after 2 days post infection (D2), second-stage (L2) at D13 and infective third-stage larvae (L3) at D15. Eighteen (30%) of 60 pools of fleas molecularly examined tested positive. Histologically, L2 were detected at D13 in the sub-cuticle region embedded in the back muscle of one female flea. This study provides original data on larval development of A. reconditum in C. felis and reports on the usefulness of the artificial feeding system.

Aqueous neem seed kernels extracts (NSKE) were used to treat flea-infested goats in Morogoro, Tanzania to determine their efficacy for flea control. Mean population densities of the insects were significantly lower in the treated goats compared to their untreated counterparts (P < 0.05). It was concluded that NSKE has potential in controlling livestock fleas, but further studies are needed to determine the active principals against fleas and their mode of action.

Fourth instar larvae of laboratory-reared, Ctenocephalides felts and Xenopsylla. brasiliensis were exposed to 50% neem seed kernel extract (NSKE) for periods varying from 30 min to 24 hr. Mortality of larvae was observed from exposure periods of 2 hr and above in both species. At 24 hr exposure, 95.3% and 94.7% of C.felis and X. brasiliensis respectively were killed. LT50s were approximately 6 and 10.5 hr for C.felis and X. brasiliensis respectively. It was generally concluded that neem seed kernel extract was potentially larvicidal against the two flea species and that it could be effectively used for controlling the insects by properly dusting the latter's breeding sites with appropriate concentrations of the extract.

Ctenocephalides felis adults obtained from young goats belonging to the Faculty of Veterinary Medicine (SUA) were reared in captivity and the offsprings used for testing against permethrin. A total of 1260 fleas were exposed to 0.005% permethrin dust (0.5% a.i. diluted with fine sand) for periods varying from 30 min to 24 hr. Additionally, white rats infested with 2400 fleas were exposed to a similar mixture for 24 hr.

In the first set of tests, 100% mortality was obtained after a 2-hr exposure of the insects to the insecticide. Similarly, in the second set of tests, all the fleas were found dead at the end of 24-hr exposure.

It was generally concluded that permethrin is very effective against this flea species and that it could be used for controlling the ectoparasite in livestock, animal barns and residential houses in the area. It was finally recommended that similar tests with other flea species in the country, especially in plague endemic areas were desirable.