This thesis is focused on the bloodmeal digestion of phlebotomine sand flies and its effects on Leishmania development within their midguts. In the first part, we studied various parameters of bloodmeal digestion in four sand fly species differing in susceptibility to Leishmania donovani to evaluate the effects on vector competence. Both proven vectors of L. donovani (Phlebotomus orientalis and P. argentipes) showed lower trypsin activity and slower formation of the peritrophic matrix (PM) than refractory species (P. papatasi and Sergentomyia schwetzi). Remarkably, P. orientalis and P. argentipes strikingly differed from each other in a time course of bloodmeal digestion. Phlebotomus orientalis females showed very slow bloodmeal digestion with low peaks of proteolytic activities and defecated around day five post bloodmeal. In contrast, P. argentipes females digested faster with a very high peak of chymotrypsin activity, their PM was present for only a short time and defecation was finished by day three post bloodmeal. We presume that the period between the degradation of the PM and defecation (i.e. time frame when Leishmania bind to the midgut to avoid expulsion with bloodmeal remnants), is one of crucial parameters affecting the establishment of Leishmania in the sand flies. In both natural...

Leishmania development in their vectors is closely connected with bloodmeal digestion. This thesis focuses on factors affecting bloodmeal digestion, egg development and Leishmania infection within the sand fly gut. First, we compared the effect of mammalian (rabbit) and avian (chicken) blood on digestion and eggs development in Phlebotomus duboscqi. Sand flies fed on chickens had twice lower protein concentrations in the midgut and significantly lower trypsin activity compared to those fed on rabbits. The highest differences in the trypsin activity were observed during first 24 hours post bloodmeal. In addition, females fed on chickens had slower eggs development and their eggs were 10 % smaller compared to those fed on rabbits. In the second part of the thesis we tested the effect of mosquito hormone TMOF on the trypsin activity and eggs development of Lutzomyia longipalpis. Rabbit blood with TMOF (28 mg/ml) was presented to the females via a membrane feeding system. Sand flies fed on blood with TMOF had 15 - 35 % less trypsin activity than control females fed on only rabbit blood. In addition, females fed on blood with TMOF had developed 30 % less eggs and their eggs were 12 - 24 % smaller compared to control group. However the effect of TMOF we observed was lower than that described previously...

Human Monocytic Ehrlichiosis (HME) is an emerging disease first described in 1987 and is transmitted by the bite of Amblyomma americanum. Over the past 10 years, the CDC has documented increasing ehrlichiosis case reports nationwide. Our study site is a golf-oriented retirement community located in the Cumberland Plateau of Tennessee. In 1993, four men at the study site had symptoms consistent with HME which prompted a CDC outbreak investigation and led community managers to mitigate ticks feeding on deer. The objectives of this study were to measure the efficacy of current tick mitigation attempts, to determine the level of infection and composition of tick-borne disease in the study area, and to assess which wildlife species are potentially acting as reservoirs for disease. Ticks were sampled in the community at eight sites of ‘4-poster’ acaricide applicator utilization and at seven untreated sites. Close to the ‘4-poster’ devices, larval, nymphal, and adult tick abundances were reduced by 90%, 68% and 49% respectively (larval p<0.001, nymphal p<0.001, adult p=0.005) relative to the untreated areas. We extracted DNA from A. americanum ticks collected at the treatment and non-treatment sites and tested for Ehrlichia spp. infections. Of 253 adult and nymphal A. americanum tested, we found 1.2% to be positive for Ehrlichia chaffeensis, 4.7% positive for Ehrlichia ewingii, and 1.6% positive for Panola Mountain Ehrlichia; in combination this prevalence is similar to that reported in other Ehrlichia-endemic areas of the eastern U.S.. We also performed blood meal analysis on DNA from A. americanum ticks and the results suggest that the most significant reservoir hosts for Ehrlichia spp. are white-tailed deer, turkeys, grey squirrels, and Passeriformes. We conclude that while the ‘4-poster’ acaricide applicators reduce the number of ticks close to treatment, at the density at which they are currently being used (8 applicators per 52.6 km2, average distance between applicators = 6.6km) they will have no large-scale effect on the community’s tick population. In order to accomplish area-wide reduction of A.americanum and Ehrlichia spp. in this locale, community managers should develop an integrated management strategy that utilizes other techniques in addition to ‘4-poster’ devices.

Phlebotomine sand flies (Diptera, Psychodidae) are small blood sucking insects which are only proven vectors of neglected tropical disease called leishmaniasis. Sand flies of the genus Phlebotomus act as vectors in the Old World and those of genus Lutzomyia are vectors in the New World. However, not all of them are capable of transmitting the disease. It is therefore crutial to conclusively determinate the species and have up-to-date knowledge of their natural occurence. Routine identification based on morphological characters is challenging due to intraspecific variability of these or their possible damage during the capture and preparation. In adition, correct assessment of distinctive cahracters, especially for females, is difficult and requires certain expertise. Thus, approaches of molecular taxonomy have been recently increasingly used for sand flies species determination. This thesis presents usage of DNA sequencing and MALDI-TOF protein profiling for routine determination of sand flies caught in southeastern Europe and adjacent regions, where we have lack of information of present sand flies species. Another part of this thesis focus on closer examination of relations between selected closely related species within species complexes. Key words: Phlebotomus, molacular identification, DNA...