Attraction of Stomoxys sp. to various fruits and flowers in Mali.

The attraction of three Stomoxys species to 26 fruits and 26 flowers of different plant species was investigated in two different sites in Mali during 2008. Stomoxys niger bilineatus Grunberg (Diptera: Muscidae) was attracted to a wider spectrum of species, significantly attracted by four fruits and eight flowers compared with control traps, whereas S. sitiens Rondani (Diptera: Muscidae) was attracted to six fruits and seven flowers of different plants, and S. calcitrans L. (Diptera: Muscidae) was only attracted to one fruit and three flowers. Cold anthrone assays showed a significantly higher prevalence of sugar feeding amongst all three species at the lagoon site than at the site near Mopti. The rhythm of activity study shows temporally separated blood- and sugar-feeding periods for S. niger bilineatus and S. sitiens, but not for S. calcitrans. A comparison between blood and sugar feeding throughout the day shows that sugar feeding activity is as frequent as blood feeding activity. Because not much is known about the preferred sugar sources for Stomoxys species in their natural habitats, the present study provides valuable information regarding the attraction capability of several plants with possible future implication for Stomoxys control strategies.

Understanding attraction stimuli of the cat flea, Ctenocephalides felis, in non-chemical control methods.

Comparisons were conducted of flea catches of four commercially available flea traps in the laboratory and under field conditions, in both rural and urban locations. The results clearly showed the My Flea Trap™, which utilizes an intermittent light to attract fleas, to be far superior in trapping ability to the three continuous light traps; it caught up to 23 times as many fleas as the other traps. Altering the lighting mechanism to provide continuous rather than intermittent light significantly decreased the number of fleas captured. In addition, the use of a green filter significantly increased trapping efficiency, whereas the addition of a heat source had no apparent effect.

Control of Culex quinquefasciatus in a storm drain system in Florida using attractive toxic sugar baits.

Attractive toxic sugar baits (ATSBs) were used to control mosquitoes in the storm drains of a residential area on the outskirts of St Augustine, Florida. The drainage system was newly constructed and no mosquitoes were breeding inside it. The area covered by the storm drains was divided in half; 10 drains served as control drains and 16 drains served as experimental drains. The baits, which consisted of a mixture of brown sugar, fruit juice, green dye marker and boric acid, were presented at the entrances of the treated drains and exit traps were positioned over the drain openings and the connecting tubes leading to retention ponds. Similar baits with orange dye and without toxin were presented at the entrances of control drains. A total of 220 pupae of Culex quinquefasciatus (Diptera: Culicidae) were released in each control and toxin-treated drain, and the numbers of recovered mosquitoes were examined to determine the effectiveness of ATSBs in the storm drain system. An average of 178.2 mosquitoes exited each drain in the control area; 87.0% of these had fed on the baits and were stained orange, whereas 13.0% were unstained. In the toxin-treated drains, 83.7% of hatched females and 86.6% of hatched males were controlled by the baits.

Effective control of adult Culex pipiens by spraying an attractive toxic sugar bait solution in the vegetation near larval habitats.

This study evaluated the use of an insecticide-treated fruit juice bait against adult Culex pipiens s.l. L. from sewage ponds in Israel. The attractive toxic sugar bait (ATSB) solution (fruit juice, sugar, food dye, oral insecticide spinosad, and BaitStab, a mixture of slow-release substances and preservatives) was sprayed onto 10-15% of the surrounding vegetation of these ponds. The same bait solution, without insecticide, was sprayed onto vegetation at a similar site as a control. Mosquito abundance was monitored in treatment and control sites with six CDC light traps. Mosquitoes in the experimental sites decreased from approximately 125 to approximately eight per trap. Mosquito abundance in the control sites was fairly stable and averaged approximately 60 per trap during the study. Before starting the study parity of mosquitoes from the experimental and control sites averaged approximately 20% multiparous females. After bait/insecticide application, only approximately 3% of the females were multiparous in the treatment area.

Plant tissues: the frugal diet of mosquitoes in adverse conditions.

The plant tissue feeding of Anopheles sergentii Theobald, An. claviger Meigen, Culex pipiens Forskål and Aedes caspius Pallas (Diptera: Culicidae) was investigated. The relative frequency of plant feeding was compared to the general intake of sugar in different seasons and in different habitats in Israel. The study shows that mosquitoes feed on sugar-poor plant tissue in the lack of better options. Optimal conditions were in the sugar-rich En Fesha oasis, where plant tissue was found in only 0.3% of An. sergentii females, and, except for the winter, about 80% of females were sugar positive and the gut content was equivalent to 72.3 +/- 1.6 microg sucrose. By contrast, in the dry season in the desert, guts of 46.3% An. sergentii females contained plant tissue residues, only 46.0% were sugar positive, and the gut-content was less than a tenth, the equivalent of 5.6 +/- 0.9 microg sucrose. Age grouping of contemporary, desert An. sergentii showed that the growing population consisted mostly of newly emerged adults, as 80.2% of the females were nulliparous and only 12.1% of them survived one gonotrophic cycle.

Linkage between susceptibility of Phlebotomus papatasi to Leishmania major and hunger tolerance.

Phlebotomus papatasi sand flies, the vectors of Leishmania major, differ genetically in their susceptibility to the pathogens. The costs of infection appear to be so great that selection against Leishmania-susceptible flies could presumably occur, unless susceptibility is compensated for by some advantage. Foci of P. papatasi-transmitted L. major are mainly found in arid habitats where seasonal stress of dehydration and heat reduces the quantity of sugar in plant leaves. The sand flies feed on these leaves and with the lack of essential sugar only a few survive long enough to deposit eggs and transmit Leishmania. This association suggested that susceptibility to L. major infection may be linked with advantageous tolerance of sugar deprivation. Here we show that desert sand flies, provided with excess sugar, became progressively resistant to infection. Selection for survival under sugar-poor conditions increased the susceptibility of parent and first-generation offspring by more than 2-fold. The leaves of plants, on which flies naturally feed, contain more sugar in irrigated than in arid habitats. About 85% of first-generation flies colonized from a desert habitat retain experimental Leishmania infection compared to 25 % of offspring of flies from irrigated sites.

Hunger tolerance and Leishmania in sandflies.

The sandfly Phlebotomus papatasi transmits Leishmania major, the agent of cutaneous leishmaniasis, in desert and savannah regions of the Old World, where seasonal stress of dehydration and heat reduces the quantity of sugar in plant leaves. Without essential sugar, only a few flies that feed on leaves can survive for long enough to deposit eggs and transmit Leishmania. Accordingly, selection for hunger tolerance may also select for pathogen susceptibility in flies. Here we provide evidence of a link between these advantageous and costly properties by testing the susceptibility of flies selected by sugar deprivation and of flies from irrigated and arid habitats.

Sand fly feeding on noxious plants: a potential method for the control of leishmaniasis.

The sand fly Phlebotomus papatasi transmits Leishmania major, which causes cutaneous leishmaniasis, in vast regions of the Old World. In addition to blood, the sand flies feed on plants. In a study of this diet, we observed that one night of feeding on branches of Solanum jasminoides, Ricinus communis, or Bougainvillea glabra drastically shortened the life span of the sand flies. Flowering B. glabra attracted P. papatasi in the field. Nevertheless, in the region endemic for L. major in yards abounding with vector sand flies, the number of P. papatasi trapped near hedges of B. glabra was eight times less (62 versus 502 flies trapped) than in the control sites. The results imply that B. glabra affords local protection against sand fly bites and decreases the risk of leishmaniasis. We suggest that this and other ornamental plants that are harmful to sand flies can be used as a tool for this purpose.

Phlebotomus papatasi and Leishmania major parasites express alpha-amylase and alpha-glucosidase.

Alpha-amylase and alpha-glucosidase activities were found in homogenates of young, unfed male and female Phlebotomus papatasi and in gut and salivary gland preparations. A significant increase in both enzyme activities in females and of alpha-amylase in males was recorded for flies that had fed overnight on a plant (Capparis spinosa). After plant feeding, alpha-amylase activity was relatively lower in female salivary glands and higher in guts, while in the males the activity in the salivary glands had increased. Alpha-glucosidase activity increased in guts of both sexes and in the salivary glands of the females. In addition, alpha-amylase activity was found in preparations of Leishmania major and L. infantum promastigotes, but not in those of L. donovani or L. tropica. Alpha-glucosidase activity was present in promastigote preparations of L. major, L. infantum, L. donovani, L. braziliensis, Crithidia fasciculata and Herpetomonas muscarum. It was lacking in similar preparations of L. tropica, Sauroleishmania agamae or Leptomonas seymouri. The growth rate of L. major promastigotes in medium supplemented with starch or with glucose was similar and it was significantly higher than in glucose poor medium. In this study, we demonstrate that P. papatasi and L. major possess the enzymes for hydrolyzing starch grains that are included in the plant tissue-diet of the sand flies.

The biological function of sand fly and Leishmania glycosidases.

This is a summary of the recent work on some glycosidases of sand flies and their Leishmania parasites. Glycosidases catalyze the hydrolysis of complex sugar subunits of polysaccharides into simple sugars. Leishmania major parasites secrete chitinase and N-acetylglucosaminase, which enables them to survive in the gut of the sand fly and are important in facilitating their transmission by the phlebotomine sand fly Phlebotomus papatasi. These enzymes are found in a wide range of trypanosomatids and the gene locus is highly conserved. The sand flies feed on plants and the ingested tissues may contain cellulose particles that the sand flies are unable to digest. Cellulolytic enzymes are secreted by L. major promastigotes and this may help to break down cellulose in infected flies and sustain their growth. Starch is a main photosynthesis product that is stored in leaves. Starch grains have been found in the midguts of field caught sand flies and alpha-amylase, the specific enzyme for starch, has been found in the salivary glands and other organs of Lutzomyia longipalpis and P. papatasi. Alpha-amylase and alpha-glucosidase are expressed by L. major promastigotes and alpha-glucosidase is secreted by several trypanosomatid genera, but not by all those examined. Primers originally designed to amplify P. papatasi amylase DNA sequences, by polymerase chain reaction (PCR), also amplified DNA from all Old World Leishmania species, indicating that the gene is highly conserved between sand flies and these parasites.

Photosynthesis modulates the plant feeding of Phlebotomus papatasi (Diptera: Psychodidae).

Phlebotomus papatasi (Scopoli), the vector of Leishmania major (Yakimoff & Schokhor), feeds on plants in desert habitats in the Jordan Valley. At the end of the dry summer, the life span of sand flies is short and the amount of sugars in their guts is small. In this season the plants are under the stress of heat and dehydration. This stress arrests the photosynthesis and decreases the amounts of the main end products, sucrose and starch. We presumed that the paucity of sugars in the sand fly plant tissue diet resulted from the arrest of photosynthesis. To test this assumption, we compared the feeding of sand flies on branches of Capparis spinosa (L.) that had been kept for 24 h in darkness and on branches cut after a normal day of photosynthesis. In darkness, the branches had lost more than half of their sugar content. Afterward they were fed upon overnight by 45.2% of female and 14.3% of male sand flies. A higher proportion of 81.0% females and 38.7% males fed on branches from natural conditions and these fed flies were significantly heavier. Laboratory experiments also showed that plant tissue meals of P. papatasi often include starch grains. Such grains were found also in 50% of field-caught males and females. The nutritive potential of plant tissues was demonstrated by the 33-d median survival of P. papatasi series that had been maintained on fresh C. spinosa branches and water.

Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors.

Development of Leishmania parasites in the digestive tract of their sandfly vectors involves several morphological transformations from the intracellular mammalian amastigote via a succession of free and gut wall-attached promastigote stages to the infective metacyclic promastigotes. At the foregut midgut transition of Leishmania-infected sandflies a gel-like plug of unknown origin and composition is formed, which contains high numbers of parasites, that occludes the gut lumen and which may be responsible for the often observed inability of infected sandflies to draw blood. This "blocked fly" phenotype has been linked to efficient transmission of infectious metacyclic promastigotes from the vector to the mammalian host. We show by immunofluorescence and immunoelectron microscopy on two Leishmania/sandfly vector combinations (Leishmania mexicana/Lutzomyia longipalpis and L. major/Phlebotomus papatasi) that the gel-like mass is formed mainly by a parasite-derived mucin-like filamentous proteophosphoglycan (fPPG) whereas the Leishmania polymeric secreted acid phosphatase (SAP) is not a major component of this plug. fPPG forms a dense three-dimensional network of filaments which engulf the promastigote cell bodies in a gel-like mass. We propose that the continuous secretion of fPPG by promastigotes in the sandfly gut, that causes plug formation, is an important factor for the efficient transmission to the mammalian host.

Sugar meals and longevity of the sandfly Phlebotomus papatasi in an arid focus of Leishmania major in the Jordan Valley.

The sugar diet and life-span of Phlebotomus papatasi were studied in a typical zoonotic focus of Leishmania major in an arid area of the Jordan Valley during 1996-1997. Plant-tissue residues (cellulose particles) were identified in the stained guts of 23% of P. papatasi and significant amounts of sugar were found in the gut of 16%. Feeding on different plants was demonstrated by using their branches, suffused with cellulose stain, as baits in the field. Ingested, stained cellulose was detected in 10% of the sandflies (6% of males, 12.5% of females) caught near bait-branches of common local plants, mostly Chenopodiaceae. The similar rates of plant and sugar feeding, with the observed absence of aphids (ruling out the availability of honeydew), implied that the sugar meals of sandflies were obtained directly from plants. The relative paucity of sugar meals in P. papatasi coincided with a short life-span, evaluated by daily growth lines in the cuticle. The age of the oldest females was estimated to be 8 days, and 6 days for males. Under local conditions, the first gonotrophic cycle can be completed in 6 days and the usual transmission of L. major is apparently afterwards, when females ingest blood to initiate another cycle. Only about 9% of P. papatasi females survived > 6 days.

Lectins and toxins in the plant diet of Phlebotomus papatasi (Diptera: Psychodidae) can kill Leishmania major promastigotes in the sandfly and in culture.

Leishmania major promastigotes are agglutinated and die in their vector, Phlebotomus papatasi, after the sandflies feed on some plants that are found in their natural habitat. In in-vitro assays, extracts of Ricinus communis (Euphorbiaceae), Capparis spinosa (Capparaceae), Prosopis farcta (Mimosaceae) and Tamarix nilotica (Tamaricaceae) agglutinated and killed the parasites. This activity could be inhibited by specific carbohydrates, indicating that it was the result of various lectins in the extracts. An extract of Solanum luteum (Solanaceae) lysed the promastigotes under similar conditions and this cytotoxicity was not abated by the sugars tested. High mortality of promastigotes occurred in infected flies after they ingested an extract of R. communis, even when the extract fed to the flies had been pre-mixed with glucose, a carbohydrate that inhibited the agglutination caused by such an extract in vitro. The results indicate that the lectins and toxins found in the vegetation in L. major foci may decrease the transmission of the parasite.

Resistance of Phlebotomus papatasi to infection with Leishmania donovani is modulated by components of the infective bloodmeal.

The circumstances which permit the establishment of Leishmania infections in sandflies were investigated by altering the growth conditions for L. donovani parasites in the unsuitable vector Phlebotomus papatasi. Only 5.0% of the sandflies harboured a few parasites 3 days after feeding on promastigotes in defibrinated blood. Heparinized blood or the addition of trypsin inhibitor to the meals allowed persistence of infections (day 6) in 9.9% and 25.8% of the flies respectively. Meals of erythrocytes, saline and amastigotes produced 44.4% fly infection on day 6, while similar promastigote-initiated infections remained in 70.3% of the flies. Proteolytic activities in the guts of sandflies fed on the above meals without parasites, were the highest after defibrinated bloodmeals. Erythrocytes with saline decreased the maximal alkaline protease level from 20.8 U to 13.5 U/fly; that of trypsin from 3.9 U to 1.8 U/fly and that of the aminopeptidase from 5.5 U to 3.9 U/fly. After meals of heparinized blood, the maximal alkaline protease activity (12.0 U/fly) was also much lower than after defibrinated blood-feeding. The different diets which resulted in comparatively low enzymatic activities, including blood with trypsin inhibitor, also promoted the survival of infections. This implies that the proteolytic activity in the sandfly gut modulates the vector susceptibility.

Bacillus sphaericus inhibits hatching of phlebotomine sand fly eggs.

The effect of Bacillus sphaericus, at various concentrations, on hatching of phlebotomine sand fly eggs was examined using laboratory bioassays. Aqueous suspensions of B. sphaericus, strain 2362, inhibited hatching of eggs of Phlebotomus duboscqi and Sergentomyia schwetzi by 95% at concentrations as low as 0.05 and 0.11 mg/cm2, respectively. In contrast, B. sphaericus did not affect the ability of pupae to emerge as adults.

Species-specific detection of Leishmania in sandflies using an enzyme-linked immunosorbent assay.

A direct enzyme-linked immunosorbent assay (ELISA) using species-specific monoclonal antibodies was developed for the detection and identification of Leishmania in sandflies. A titration of mock-infected Phlebotomus papatasi showed that fewer than 2000 L. major promastigotes could be detected. The percentage of infected P. papatasi collected in the field, as determined by dissection, was compared to that revealed by the ELISA. Both methods gave similar results, irrespective of whether the flies were caught by sticky papers or light-traps. The percentage of infected flies determined by either method was also similar in experimentally infected colony reared sandflies. The ELISA can be carried out using multiple species-specific antibodies, and is as accurate as identification of infected sandflies by microscopical examination. The technique should be useful for identifying sandfly species involved in transmitting different species of Leishmania, and for rapid assessment of leishmanial infection rates in endemic regions.

Phlebotomine sand fly control using bait-fed adults to carry the larvicide Bacillus sphaericus to the larval habitat.

Sugar meals of plant origin are an important component of the sand fly diet. We show that sugar solution baits have potential as vehicles for phlebotomine sand fly control. In the laboratory, adult Phlebotomus duboscqi Neveu-Lemaire and Sergentomyia schwetzi (Adler, Theodor, and Parrot) that have consumed an aqueous sucrose solution containing Bacillus sphaericus Neide toxins and are subsequently eaten by larvae produce significant larval death (P < 0.01). In the field, when vegetation near animal burrows and eroded termite mounds was sprayed with sucrose solution with or without incorporation of the larval toxicant B. sphaericus, 40% of female sand flies fed in situ. Dispersing B. sphaericus-carrier sand flies caused significant larval mortality (P < 0.01) in resting and breeding sites in animal burrows 10-30 m from the sprayed vegetation for 2-12 wk posttreatment. Also, adult sand fly populations breeding and resting inside animal burrows were significantly reduced (P < 0.01) following direct application of the sucrose/B. sphaericus solution to the burrow entrances. This control effect lasted 4-10 wk post-treatment. The effect was not seen for sand fly populations breeding and resting inside eroded termite mounds. This approach may be useful for the application of biological control agents against phlebotomine sand flies in biotypes where larvae and adults use the same habitats.

Cellulase activity of Leishmania major in the sandfly vector and in culture.

The secretion of cellulose-degrading enzymes by Leishmania promastigotes in culture and in the sandfly vector was demonstrated. Two types of activity of cellulase enzyme-complexes were measured: endoglucanases, which randomly cleave cellulose chains and cellobioydrolases, which remove cellobiose from the nonreducing end of the molecule. The assays demonstrated that enzymes with these activities were secreted into the culture medium by Leishmania major, L. donovani, and L. braziliensis. These activities were also found in cultures of Sauroleishmania agamae, Leptomonas seymouri, Herpetomonas muscarum, Crithidia fasciculata and Trypanosoma brucei brucei that had a relatively low endoglucanase activity. Both endoglucanase and cellobiohydrolase activities were found in the gut of L. major-infected Phlebotomus papatasi, while gut preparations of uninfected sandflies had only cellobiohydrolase activity. The similar growth of L. major parasites in medium supplemented with either cellulose or glucose suggests these parasites can utilize cellulose.