Effects of azadirachtin on the biology of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) adult female, the main vector of American visceral leishmaniasis.

The effects of azadirachtin A added to the sucrose diet of the adult females on the mortality, oviposition, and hatching of the sand fly vector of American visceral leishmaniasis Lutzomyia longipalpis (Lutz & Neiva, 1912) were investigated. Concentrations of 0.1, 1.0, and 10.0 microg/mg of azadirachtin significantly increased insect mortality in comparison with control insects. The same dose also significantly reduced oviposition but not hatching. After a long development period, significantly fewer adult insects were obtained from eggs hatching by azadirachtin-treated females in a dose-response manner. These results indicate that azadirachtin is a potent sterilizer that could be used against the development of Lu. longipalpis populations and as a tool for studying physiological and biochemical processes in phlebotomine species.

In Vitro Inhibition of Leishmania Attachment to Sandfly Midguts and LL-5 Cells by Divalent Metal Chelators, Anti-gp63 and Phosphoglycans.

Leishmania braziliensis and Leishmania infantum are the causative agents of cutaneous and visceral leishmaniasis, respectively. Several aspects of the vector-parasite interaction involving gp63 and phosphoglycans have been individually assayed in different studies. However, their role under the same experimental conditions was not studied yet. Here, the roles of divalent metal chelators, anti-gp63 antibodies and purified type I phosphoglycans (PGs) were evaluated during in vitro parasite attachment to the midgut of the vector. Parasites were treated with divalent metal chelators or anti-gp63 antibodies prior to the interaction with Lutzomyia longipalpis/Lutzomyia intermedia midguts or sand fly LL-5 cells. In vitro binding system was used to examine the role of PG and gp63 in parallel. Treatment with divalent metal chelators reduced Le. infantum adhesion to the Lu. longipalpis midguts. The most effective compound (Phen) inhibited the binding in both vectors. Similar results were observed in the interaction between both Leishmania species and the cell line LL-5. Finally, parallel experiments using anti-gp63-treated parasites and PG-incubated midguts demonstrated that both approaches substantially inhibited attachment in the natural parasite-vector pairs Le. infantum/Lu. longipalpis and Le. braziliensis/Lu. intermedia. Our results suggest that gp63 and/or PG are involved in parasite attachment to the midgut of these important vectors.

Effects of azadirachtin on the development and mortality of Lutzomyia longipalpis larvae (Diptera: Psychodidae: Phlebotominae).

The effects of azadirachtin A added to the standard diet on the development, mortality, and metamorphosis of Lutzomyia longipalpis Lutz & Neiva, 1912 were studied. Concentrations of 0.1, 1.0, and 10.0 microg of azadirachtin/mg of diet significantly increased larval mortality in comparison with nontreated insects. Concentrations 0.1 and 1.0 microg blocked the molt of larvae, which remained as third instars until the end of the experiment. The 10 microg/mg concentration resulted in greater molt inhibition. In this group, all insects stopped their development as second instars. Simultaneous addition of ecdysone (1 microg/mg) to the standard diet containing azadirachtin counteracted the effects of azadirachtin on mortality and inhibition of ecdysis. These results indicate that azadirachtin is a potent growth inhibitor of L. longipalpis.

Effects of temperature and photoperiod on daily activity rhythms of Lutzomyia longipalpis (Diptera: Psychodidae).

BACKGROUND: Insect vectors have been established as models in Chronobiology for many decades, and recent studies have demonstrated a close relationship between the circadian clock machinery, daily rhythms of activity and vectorial capacity. Lutzomyia longipalpis, the primary vector of Leishmania (Leishmania) infantum in the New World, is reported to have crepuscular/nocturnal activity in the wild. However, most of these studies applied hourly CDC trap captures, which is a good indicative of L. longipalpis behaviour, but has limited accuracy due to the inability to record the daily activity of a single insect during consecutive days. In addition, very little is known about the activity pattern of L. longipalpis under seasonal variations of average temperature and day length in controlled laboratory conditions. METHODS: We recorded the locomotor activity of L. longipalpis males under different artificial regimes of temperature and photoperiod. First, in order to test the effects of temperature on the activity, sandflies were submitted to regimes of light/dark cycles similar to the equinox photoperiod (LD 12:12) combined with different constant temperatures (20°C, 25°C and 30°C). In addition, we recorded sandfly locomotor activity under a mild constant temperature (25°C with different day length regimes: 8 hours, 12 hours and 16 hours). RESULTS: L. longipalpis exhibited more activity at night, initiating dusk-related activity (onset time) at higher rather than lower temperatures. In parallel, changes of photoperiod affected anticipation as well as all the patterns of activity (onset, peak and offset time). However, under LD 16:08, sandflies presented the earliest values of maximum peak and offset times, contrary to other regimes. CONCLUSIONS: Herein, we showed that light and temperature modulate L. longipalpis behaviour under controlled laboratory conditions, suggesting that sandflies might use environmental information to sustain their crepuscular/nocturnal activity, as well as other important aspects as mating and host-seeking at appropriate times in different seasons. Our results depict previously unappreciated aspects of the L. longipalpis daily rhythms of activity that might have important epidemiological implications.

Comparative study of the phlebotomine sand fly species (Diptera: Psychodidae: Phlebotominae) of the genera Nyssomyia Barretto, 1962, Bichromomyia Artemiev, 1991, and Migonemyia Galati, 1995, vectors of American cutaneous leishmaniasis in Brazil.

Phlebotominae, a group of insects with great medical importance especially in Brazil, are responsible for transmitting causal agents of cutaneous and visceral leishmaniasis. In Brazil, the most important species of Leishmania Ross, 1903 are L. (Viannia) braziliensis Vianna, 1911, whose main vectors are Nyssomyia intermedia Lutz & Neiva, 1912, Ny. neivai Pin-to, 1926, Ny. whitmani Antunes & Coutinho, 1939 and Migonemyia migonei França, 1920; and L. (Leishmania) amazon-ensis Lainson & Shaw, 1972, for which Bichromomyia flaviscutellata Mangabeira, 1942 is the main vector. The present study sought to investigate the morphological as well as geometrical and linear morphometric characteristics of these five sand flies in an attempt to cluster these species. Our aim was to reveal some of the characters that might help identify these phlebotomine species and also be useful in future phylogenetic studies. Comparative analyses by linear and geometric morphometric characters allowed us to distinguish the genera of these sand flies and assess the taxonomic position of Ny. intermedia and Ny. neivai, the so-called "cryptic species". Significant differences were observed in several of the analyzed structures, including the centroid size of the wings and the ratio between the ejaculatory filament and its tip. Based on the linear morphometric analytical results, the size of the centroids of the wings and their shapes indicated that these three species of Nyssomyia are phenetically more similar to Mg. migonei (all vectors of L. (V.) braziliensis) than to Bi. flavis-cutellata (vector of L. (L.) amazonensis). These results are in agreement with the division of the genera Nyssomyia and Bichromomyia.