Numerical Taxonomy of Old World Phlebotominae (Diptera: Psychodidae): 2. Restatement of Classification upon Subgeneric Morphological Characters

Numerical analyses (correspondence analysis, ascending hierarchical classification, and cladistics) were done with morphological characters of adult phlebotomine sand flies. The resulting classification largely confirms that of classical taxonomy for supra-specific groups from the Old World, though the positions of some groups are adjusted. The taxa Spelaeophlebotomus Theodor 1948, Idiophlebotomus Quate & Fairchild 1961, Australophlebotomus Theodor 1948 and Chinius Leng 1987 are notably distinct from other Old World groups, particularly from the genus Phlebotomus Rondani & Berté 1840. Spelaeomyia Theodor 1948 and, in particular, Parvidens Theodor & Mesghali 1964 are clearly separate from Sergentomyia França & Parrot 1920.

Batflies Parasitic on Some Phyllostomid Bats in Southeastern Brazil: Parasitism Rates and Host-parasite Relationships

Ectoparasitic batflies were studied on 12 species of phyllostomid bats, by making 35 nightly collections of bats using mist nets at the "Panga" Ecological Reservation near Uberlândia, State of Minas Gerais, southeastern Brazil, from August 1989 to July 1990. Eleven species of Streblidae and one of Nycteribiidae were collected on 12 species of bats. Prevalence of ectoparasitic flies was lower than those reported by other authors for the New World and may be the result of the lack of caves in the study area, causing bats to roost in less favorable locations, forming smaller colonies. The fly, Trichobius joblingi Wenzel, was found on Carollia perspicillata (Linnaeus), showing preference for adult male bats. This could be explained by the predominance of males in the bat colonies, and by the fact that females rest in isolation during the reproductive period making them less exposed to the parasites. The streblid flies, Aspidoptera falcata Wenzel and Megistopoda proxima (Séguy), were found on Sturnira lilium (Geoffroy). A. falcata occurred mainly on young and adult females, whereas M. proxima did not show any preferences relative to the reproductive condition of the host. Ecological factors are important in determining differential numbers of parasites occurring on the different sexes, ages and reproductive state of the hosts.

Laboratory and Field Evaluation of Biodegradable Polyesters for Sustained Release of Isometamidium and Ethidium

An overview is presented of the results obtained with biodegradable sustained release devices (SRDs) containing a mixture of polymers and either isometamidium (ISMM) or ethidium. Under controlled laboratory conditions (monthly challenge with tsetse flies infected with Trypanosoma congolense) the protection period in SRD treated cattle could be extended by a factor 2.8 (for ethidium) up to 4.2 (for ISMM) as compared to animals treated intramuscularly with the same drugs. Using a competitive drug ELISA ISMM concentrations were detected up to 330 days after the implantation of the SRDs, whereas after i.m. injection the drug was no longer present three to four months post treatment. Two field trials carried out in Mali under heavy tsetse challenge showed that the cumulative infection rate was significantly lower in the ISMM-SRD implanted cattle than in those which received ISMM intramuscularly. Using ethidium SRD, however, contradictory results were obtained in field trials in Zambia and in Mali. The potential advantages and inconvenients of the use of SRDs are discussed and suggestions are made in order to further improve the currently available devices.

Review on the Molecular Tools for the Understanding of the Epidemiology of Animal Trypanosomosis in West Africa

The epidemiology of animal trypanosomosis around Bobo-Dioulasso (Burkina Faso, West Africa) benefited a lot in the last years from the progress of molecular tools. The two most used molecular techniques were the polymerase chain reaction for the diagnosis of the disease in cattle and the characterization of the trypanosomes in the host and the vector on one hand, and the microsatellite DNA polymorphism in tsetse flies to study the intraspecific genetic variability of the vector on the other hand. The results obtained in the Sideradougou area during a recent two year survey with these techniques, associated with many other georeferenced informations concerning vector and cattle distribution, natural environment, landuse, ground occupation, livestock management, were combined in a Geographical Information System. This new approach of a complex pathogenic system led to a better evaluation of the risk of trypanosome transmission.

Contribution to the sand fly fauna (Diptera: Phlebotominae) of Rio Grande do Sul, Brazil and Leishmania (Viannia) infections

American cutaneous leishmaniasis (ACL) is endemic in the State of Rio Grande do Sul, Brazil. As reports of nearby hospitals suggest, the Parque Estadual do Turvo seems to serve as a source for the disease. During three months from November 1996 to February 1997 we collected, in this park, 2,228 sandflies (10 Lutzomyia species and 2 6species). We applied the polymerase chain reaction to 920 females which belong to the following species: Lutzomyia migonei, Lu. pessoai, Lu. fischeri, Lu. misionensis, Lu. lanei, Lu. neivai, Lu. shannoni, and Lu. monticola, in an attempt to verify natural infection by Leishmania (Viannia), the causative agent of ACL. Le. (Viannia) infections were demonstrated by DNA amplification from two Lu. pessoai and one Lu. misionensis female. Lu. pessoai have been found with leptomonas in the gut believed to be Le. (V.) braziliensis in other endemic areas of northeastern and southeastern Brazil. However, Lu. misionensis has never been found carrying a natural infection of Le. (Viannia).

Quantitative phenetics and taxonomy of some phlebotomine taxa

Elucidating the evolution of Phlebotominae is important not only to revise their taxonomy, but also to help understand the origin of the genus Leishmania and its relationship with humans. Our study is a phenetic portrayal of this history based on the genetic relationships among some New Word and Old Word taxa. We used both multilocus enzyme electrophoresis and morphometry on 24 male specimens of the Old Word genus Phlebotomus (with three of its subgenera: Phlebotomus, Spelaeophlebotomus and Australophlebotomus), and on 67 male specimens of the three New World genera, Warileya, Brumptomyia and Lutzomyia, (with three subgenera of Lutzomyia: Lutzomyia, Oligodontomyia and Psychodopygus). Phenetic trees derived from both techniques were similar, but disclosed relationships that disagree with the present classification of sand flies. The need for a true evolutionary approach is stressed.

Sexing sand fly pupae (Diptera: Psychodidade: Phlebotominae)

We describe a technique to separate male and female pupae of sand flies. This has reduced the labour to separate flies after emergence and also allows the isolation of unmated adults for behavioural and physiological studies.

Further support for a palaearctic origin of Leishmania

The fossil record and systematics of murid rodents, reservoirs of zoonotic cutaneous leishmaniasis in the Palaearctic, Oriental, African, Nearctic and Neotropical, strongly support a Palaearctic origin of Leishmania. The fossil record and systematics of phlebotomine sand flies reinforce this idea. Interpretations of molecular data that place the origin of Leishmania in the Neotropical are inconsistent with the natural histories of reservoirs and vectors. The evolutionary pattern of New World rats (Sigmodontinae) indicates that they may be the most important reservoirs of zoonotic cutaneous leishmaniasis throughout their range.

Sand fly evolution and its relationship to Leishmania transmission

The evolutionary relationships of sand flies and Leishmania are discussed in this report, which draws distinctions between co-association, co-evolution and co-speciation (or co-cladogenesis). Examples focus on Phlebotomus vectors of Le. infantum and Le. major in the Mediterranean subregion.

Mosquitocidal bacterial toxins: diversity, mode of action and resistance phenomena

Bacteria active against dipteran larvae (mosquitoes and black flies) include a wide variety of Bacillus thuringiensis and B. sphaericus strains, as well as isolates of Brevibacillus laterosporus and Clostridium bifermentans. All display different spectra and levels of activity correlated with the nature of the toxins, mainly produced during the sporulation process. This paper describes the structure and mode of action of the main mosquitocidal toxins, in relationship with their potential use in mosquito and/or black fly larvae control. Investigations with laboratory and field colonies of mosquitoes that have become highly resistant to the B. sphaericus Bin toxin have shown that several mechanisms of resistance are involved, some affecting the toxin/receptor binding step, others unknown.

The use of bacterial larvicides in mosquito and black fly control programmes in Brazil

Bacillus spp. based larvides are increasingly replacing, with numerous advantages, chemical insecticides in programmes for controlling black fly and mosquito populations. Brazil was among the pioneers in adopting Bacillus thuringiensis israelensis (B.t.i) to control black flies. However, the major current mosquito control programme in Brazil, the Programme for Eradication of Aedes aegypti launched in 1997, only recently decided to replace temephos by B.t.i based larvicides, in the State of Rio de Janeiro. In the last decade, works developed by research groups in Brazilian institutions have generated a significant contribution to this subject through the isolation of B. sphaericus new strains, the development of new products and the implementation of field trials of Bacillus efficacy against mosquito species under different environmental conditions.

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Genetic variability among populations of Lutzomyia (Psathyromyia) shannoni (Dyar 1929) (Diptera: Psychodidae: Phlebotominae) in Colombia

Polyacrylamide gel electrophoresis was used to elucidate genetic variation at 13 isozyme loci among forest populations of Lutzomyia shannoni from three widely separated locations in Colombia: Palambí (Nariño Department), Cimitarra (Santander Department) and Chinácota (Norte de Santander Department). These samples were compared with a laboratory colony originating from the Magdalena Valley in Central Colombia. The mean heterozygosity ranged from 16 to 22%, with 2.1 to 2.6 alleles detected per locus. Nei's genetic distances among populations were low, ranging from 0.011 to 0.049. The estimated number of migrants (Nm=3.8) based on Wright's F-Statistic, F ST, indicated low levels of gene flow among Lu. shannoni forest populations. This low level of migration indicates that the spread of stomatitis virus occurs via infected host, not by infected insect. In the colony sample of 79 individuals, the Gpi locus was homozygotic (0.62/0.62) in all females and heterozygotic (0.62/0.72) in all males. Although this phenomenon is probably a consequence of colonization, it indicates that Gpi is linked to a sex determining locus.

Ootaxonomic investigation of five Lutzomyia species (Diptera, Psychodidae) from Venezuela

The eggshell fine structure of five sand fly species from Venezuela belonging to the genus Lutzomyia (L. migonei, L. ovallesi, L. absonodonta, L. gomezi and L. panamensis) was examined by scanning electron microscopy. The chorionic sculpturing of L. migonei, L. ovallesi, L. absonodonta and L. gomezi was characterized by series of columns arranged in palisade to form sinuous ridges. In inter-ridge areas, the basal layer was covered with fibrous material. The outer chorion of L. panamensis had a pattern known as "mountain- or volcano-like". The morphology of the posterior pole and aeropyle had a common structure in the five species, with some species-specific characters. The eggshell features of the five species are compared with those of other phlebotomine sand flies.

Effect of a second bloodmeal on the oesophagus colonization by Leishmania mexicana complex in Lutzomyia evansi (Diptera: Psychodidae)

Migration and colonization of the oesophagus by Leishmania mexicana parasites were enhanced after digestion of a second bloodmeal intake in Lutzomyia evansi. This event has epidemiological significance since it affects the infection susceptibility of this sand fly species, which is a proven vector of L. chagasi in Colombian and Venezuelan visceral leishmaniasis foci. Also, it may explain the host seeking behaviour displayed by some partially bloodfed flies found inside houses.