Transmission of leishmaniasis is effected by a specific developmental stage, the metacyclic promastigote. The precursors of metacyclic promastigotes were a distinct subpopulation of parasites, identified for the first time as a new stage in the life-cycle and named leptomonad promastigotes. Microdissection of infected sandflies into 4 midgut regions and foregut allowed precursor-product relationships to be established for amastigote-procyclic promastigote, procyclic-nectomonad promastigote, nectomonad-leptomonad promastigote and leptomonad-metacyclic promastigote developmental switches. Metacyclic promastigotes occurred mainly in the thoracic midgut and cardia, coincident with the accumulation of a promastigote secretory gel (PSG) plug in these anterior regions. The gel-like plug was isolated from flies with mature infections and found to contain predominantly leptomonad promastigotes. The PSG plug also contained the majority (75 %) of the total metacyclic promastigote population in the sandflies, which were concentrated at the anterior pole. The PSG plug was found to be the main site of metacyclogenesis, and acted as a reservoir of leptomonad promastigotes from which metacyclic forms differentiated and migrated forward to promote the infective potential of the fly. The PSG plug occluded and distorted the midgut, forcing the stomodeal valve open and affecting the feeding success of the sandflies, such that they experienced difficulty in taking a full meal. Collectively, these data support the role of the PSG in the transmission of leishmaniasis, by conditioning the midgut environment for metacyclogenesis and altering the feeding ability of infected sandflies.Key words : Leishmania, sandfly, metacyclic promastigote, promastigote secretory gel (PSG), transmission. Parasites of the genus Leishmania are protozoa that occur within the phagolysosomes of the mammalian macrophages as non-flagellated amastigotes, and as motile flagellated promastigotes in phlebotomine sandflies. Infection of the sandfly begins after taking a bloodmeal from an infected host. Ingested amastigotes transform within the digesting bloodmeal and undergo development as a number of promastigote morphological forms (Killick-Kendrick, 1979). Upon engorgement the bloodmeal is encased by a peritrophic matrix, a chitinous sac formed from secretions of the midgut epithelium. Therein amastigotes transform into ovoid procyclic and long slender nectomonad forms, which initiate the growth of the infection. Towards the end of digestion of the bloodmeal the parasites escape from the peritrophic matrix to avoid expulsion during defecation, and further resist this process by attaching to the midgut microvillar border (Pimenta et al. 1992 ; Sacks et al. 1994). Subsequently, colonization of the anterior * Corresponding author : Tel : j44 151 708 9393. Fax : j44 151 708 9007. E-mail : m.e.rogers!liverpool.ac.uk regions of the midgut is accompanied by transformation to a variety of morphological forms, which include broad attached haptomonad pr...

Sand flies are the exclusive vectors of the protozoan parasite Leishmania1, but the mechanism of transmission by fly bite has not been determined nor incorporated into experimental models of infection. In sand flies with mature Leishmania infections the anterior midgut is blocked by a gel of parasite origin, the promastigote secretory gel (PSG)2,3. Here, we analyse for the first time the inocula from Leishmania mexicana infected Lutzomyia longipalpis sand flies. This revealed the size of the infectious dose, the underlying mechanism of parasite delivery by regurgitation, and the novel contribution made to infection by filamentous proteophosphoglycan (fPPG), a component of PSG found to accompany the parasites during transmission. Collectively, these results have important implications for understanding the relationship between parasite and its vector, the pathology of cutaneous leishmaniasis in humans and also the development of effective vaccines and drugs. These findings emphasise that to fully understand transmission of vector-borne diseases the interaction between all three participants must be considered.Leishmaniasis is a parasitic disease that currently infects some 12 million people worldwide, causing severe morbidity and mortality4. Infection is initiated by distinct life cycle stages, metacyclic promastigotes, that are introduced into the skin by fly bite along with sand fly saliva5-7. Leishmania are known to express various "virulence factors" in the sand fly, which may facilitate transmission to and infection of the mammalian host8-12. However, despite these discoveries our knowledge of parasite molecules that facilitate sand fly transmission is still limited. Furthermore, a number of key issues of transmission remain unresolved, such as the true infective dose, the mechanism of parasite delivery and the biological consequences of these upon infection. Significantly, in all Leishmania-vector combinations examined to date a gel-like plug, the parasite-derived PSG, blocks the anterior parts of the midgut coincident with the accumulation of metacyclic promastigotes2,3. An important structural component of PSG is fPPG, an unusual mucin-like glycoprotein unique to Leishmania13,14. Here we address these issues regarding transmission and reveal a novel contribution made by L. mexicana PSG to the infection process.Correspondence and requests for materials should be addressed to P. A.B. (pbates@liv.ac.uk To begin to understand the nature of the infective inoculum, the number and composition of L. mexicana parasites delivered during transmission was determined. A membrane feeding system was adapted to collect parasites egested by infected sand flies, revealing an average of 1086 parasites delivered per bite, highly enriched in metacyclic promastigotes (86-98%) ( Table 1). The only previous investigations quantitating egested parasites have been made using microcapillary forced feeding15,16. When this method was employed on L. mexicanainfected sand flies an average of 105 promastigotes were collected per...

While olfactory neurons of silk moths are well known for their exquisite sensitivity to sex pheromone odorants, molecular mechanisms underlying this sensitivity are poorly understood. In searching for proteins that might support olfactory mechanisms, we characterized the protein profile of olfactory neuron receptor membranes of the wild silk moth Antheraea polyphemus. We have purified and cloned a prominent 67-kDa protein which we have named Snmp-1 (sensory neuron membrane protein-1). Northern blot analysis suggests that Snmp-1 is uniquely expressed in antennal tissue; in situ hybridization and immunocytochemical analyses show that Snmp-1 is expressed in olfactory neurons and that the protein is localized to the cilia, dendrites, and somata but not the axons. Snmp-1 mRNA expression increases significantly 1-2 days before the end of adult development, coincident with the functional maturation of the olfactory system. Sequence analysis suggests Snmp-1 is homologous with the CD36 protein family, a phylogenetically diverse family of receptor-like membrane proteins. CD36 family proteins are characterized as having two transmembrane domains and interacting with proteinaceous ligands; Snmp-1 is the first member of this family identified in nervous tissue. These findings argue that Snmp-1 has an important role in olfaction; possible roles of Snmp-1 in odorant detection are discussed.The antennae of silk moths are well known for their exquisite sensitivity to pheremonal odorants (1-3). Early reports demonstrated that the males of the wild silk moth Samia cynthia could locate a sex pheromone source over 2 miles away within several hours of their release (4). Studies of the silk moth Bombyx mori suggested that a single pheromone molecule was sufficient to activate olfactory neurons in the antenna (5). In insects, odors are detected by sensilla, small hair-like structures arrayed along the antennae. The sensilla are hollow, fluid-filled cuticular structures that contain the receptor cilia of olfactory neurons. Small holes penetrate through the wall of a sensillum, permitting entry of odor molecules; odorant-binding proteins are then thought to transport the odor molecules through the fluid-filled lumen to receptor proteins in the receptor membranes of the olfactory neurons (3, 6 -9).In searching for proteins that might support olfactory mechanisms, we characterized the protein profile of olfactory neuron receptor membranes of the wild silk moth Antheraea polyphemus. The morphology of the A. polyphemus antenna permits the relatively easy isolation of olfactory sensilla in a manner yielding olfactory receptor cilia as the only cellular component (10). This preparation is free of other parts of the olfactory neurons as well as of nonneuronal cells of the antenna, and it was previously used to identify a pheromonebinding membrane protein using a radiolabeled photoaffinity analog of the A. polyphemus sex pheromone (10). This protein co-migrated with bovine serum albumin on SDS gels (around 67 kDa) and appeared to be uniquely exp...

SNMP1-Apol is an antennal-specific protein of the wild silk moth Antheraea polyphemus; the protein is abundantly expressed and localized to the receptor membranes of sex-pheromone specific olfactory sensory neurons (OSNs). SNMP1-Apol is thought to function in odor detection based on its olfactory-specific expression, localization within OSNs, developmental time of expression, and apparent homology to the CD36 family of membrane-bound receptor proteins. In the current study, SNMP1-Apol homologues were identified from the moths Bombyx mori, Heliothis virescens, and Manduca sexta. These species posses antennal mRNAs encoding proteins with amino acid sequence identities ranging from 75-80%; these proteins are collectively designated SNMP1. A second M. sexta SNMP homologue, previously identified and partially sequenced [Robertson et al.: Insect Mol Biol 8:501-518, 1999] was fully sequenced and characterized. The encoded protein shares only 26-27% sequence identity with the SNMP1 proteins, and is thus designated SNMP2-Msex. The SNMP sequences were used to identify 14 and four possible homologues in Drosophila melanogaster and Caenorhabditis elegans genome databases, respectively; thus, greatly expanding CD36 family membership among the invertebrate lineages. Despite their sequence difference, SNMP1-Msex and SNMP2-Msex expression is localized to OSNs and occurs simultaneously with the onset of olfactory function. These findings suggest that SNMPs play a central role in odor detection in insects, and that the CD36 gene family is widely represented among animal phyla. The SNMPs are the only identified neuronal members of the CD36 family, and as such expand the activities of this gene family into roles influencing brain function and behavioral action.