Amplification and Characterization of DDT Metabolizing Delta Class GST in Sand Fly, Phlebotomus argentipes (Diptera: Psychodidae) From Bihar, India.

Phlebotomus argentipes is an established vector for Visceral leishmaniasis prevalent in the Indian subcontinent. Insect Glutathione S-transferases (GST) enzyme plays a pivotal role in the metabolism of xenobiotics and chemical insecticides. We report herein the identification and characterization of a delta class GST from the sandfly, P. argentipes. The resulting clone (rParg-GSTδ) is successfully sequenced, which revealed 76.43% and 66.32% gene identity with GST from Phlebotomus papatasi (Scopoli; Diptera: Psychodidae) and Lutzomiya longipalpis (Lutz and Neiva; Diptera: Psychodidae), respectively. The identified rParg-GST amino acid Blast results revealed 82.6% homology to delta class GST of Phlebotomus papatasi and more than 50% homology to Lepidoptera which comprises butterflies and moths. The Phylogenetic analysis of Parg-GST with different classes of Insect GSTs further supported its classification as delta class. A functional recombinant Parg-GSTδ protein (rParg-GSTδ) was expressed in Escherichia coli (Migula; Enterobacterales: Enterobacteriaceae) cells in a soluble form, purified to homogeneity and found to be active against a substrate 1-chloro-2,4-dintrobenzene (CDNB) and lipid peroxidation by-product 4-Hydrxynonenal (4-HNE). Interestingly, rParg-GSTδ demonstrates high dehydrochlorination activity against dichlorodiphenyltrichloroethane (DDT) i.e., 16.27 nM/µg in high performance liquid chromatography (HPLC) assay. These results provide evidence of direct DDT metabolism property exhibited by P. argentipes GST and set the foundation to decipher the metabolic resistance mechanism in P. argentipes against insecticides.

Insecticide susceptibility of the sand fly leishmaniasis vector Phlebotomus argentipes in Sri Lanka.

BACKGROUND: Leishmania donovani-induced and sand fly-transmitted leishmaniasis is a growing health problem in Sri Lanka. Limited knowledge on biological and behavioral characteristics of probable vector Phlebotomus argentipes hinders disease control. Here, insecticide susceptibility patterns of P. argentipes were investigated with exploration of probable underlying resistance mechanisms. METHODS: Adult sand flies were collected using standard cattle baited net traps and CDC light traps from selected sites in four districts. Adult F1 progeny of P. argentipes were exposed to different concentrations of DDT, malathion, deltamethrin and propoxur using WHO susceptibility bioassay kits. Post-1-h knockdown and post-24-h mortality were recorded and analyzed. Metabolic enzyme activity and the sensitivity of the acetylcholinesterase target-site were determined by biochemical assays using wild-caught flies. Extracted fly DNA samples were tested for the presence of knockdown-resistance (kdr) type mutations. RESULTS: The LC100 values for DDT, malathion, propoxur and deltamethrin were 0.8-1.5%, 0.9-2.0%, 0.017-0.03% and 0.007% respectively. Insecticide-susceptibility levels were higher than the discriminating dosages established for Aedes mosquitoes, except for malathion. The lowest susceptibility levels (except for deltamethrin) were detected in the Mamadala population, whereas the highest levels were detected in the Mirigama population. The percentage of knocked-down sand flies was < 75% at any tested concentration, including those, which exhibited 100% mortality after 24 h. Elevated activity levels of glutathione S-transferase (3%, 7%, 12.5% and 14%) and esterase (2%, 5%, 5.5% and 6.5%) were detected in flies that originated from Mirigama, Pannala, Thalawa and Mamadala respectively, while monooxygenase quantities remained below the cut-off level. Ten to 34.5% of flies were heterozygous for acetylcholinesterases target-site insensitivity, associated with organophosphate and carbamate resistance. Pyrethroid-resistance-associated L1014F kdr-type mutation in the voltage gated sodium channel gene was detected in 30/53 flies. CONCLUSIONS: Populations of P. argentipes in Sri Lanka are largely susceptible to common insecticides, except for malathion (used extensively in the past for malaria control). Their insecticide susceptibility appears negatively associated with past malaria endemicity of the study sites, with signs of early insecticide tolerance. Presence of insecticide target site insensitivity in a notable proportion of flies and enhanced insecticide metabolizing enzyme activities imply potential future challenges for leishmaniasis control, with a call for urgent proactive measures for its containment.

Detection and functional characterization of sigma class GST in Phlebotomus argentipes and its role in stress tolerance and DDT resistance.

Several Glutathione S-transferases (GSTs) enzymes, in insects, have previously been implicated in resistance developed against DDT and other insecticides. The GST enzyme particularly sigma class have important physiological role in detoxification of lipid peroxidation by-products in insects. Phlebotomus argentipes has been intensely exposed to DDT over years due to Indoor Residual Spray (IRS) programme for Kala-azar elimination in Bihar, India. However, in P. argentipes, role of GSTs in DDT resistance have not been elucidated. Here, sigma class GST of P. argentipes (Parg-GSTσ) was successfully cloned, expressed and purified by affinity chromatography. The recombinant Parg-GSTσ was found to be highly active towards cumene hydroperoxide and 4-HNE having specific activity 92.47 & 203.92 µM/min/mg of protein, respectively and exhibited low activity towards universal substrate CDNB i.e., 8.75 µM/min/mg of protein. RT-PCR and immunoblot analysis showed at least 2 and 1.8 fold overexpression of Parg-GSTσ in the single exposed and non exposed DDT resistant P. argentipes as compared to susceptible, implicating Parg-GSTσ also involved in DDT resistance probably by imparting enhanced stress tolerance. The DDT, H2O2 and temperature induction assays demonstrated stress-dependent induction of Parg-GSTσ expression indicating its important role in oxidative stress redressal.

Isoenzymatic characterization of Phlebotomus ariasi and P. perniciosus of canine leishmaniasis foci from Eastern Pyrenean regions and comparison with other populations from Europe.

An entomological survey was carried out in 2007 in two Pyrenean counties of Lleida province (north-eastern Spain), where cases of autochthonous canine leishmaniasis have been recently reported. Phlebotomus ariasi and P. perniciosus, vectors of Leishmania infantum in the Mediterranean area, were captured. The aim of the present study was to compare these phlebotomine populations with others captured in known leishmaniasis foci in Europe. Populations of these species were studied by analysing the polymorphism of seven enzymatic systems (HK, PGI, PGM, MDH, 6PGD, FUM and ACO) and compared with other specimens from endemic regions of France, Italy, Malta, Portugal and Spain captured in other campaigns, and also with previously published results. Phlebotomus ariasi was more polymorphic than P. perniciosus. Only the ACO locus had diagnostic alleles, but some other alleles show high characteristic frequencies for each species. The neighbour-joining trees separated two population groups in both species. On the basis of the isoenzyme study results, sand fly populations of the Pyrenean region in Lleida province are closely related to those of other nearby leishmaniasis endemic regions in France and Spain.

Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut.

BACKGROUND: In vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment. METHODS: In silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays. RESULTS: We identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to uninfected. Interestingly, during the early transition stage, substantial killing was observed in ISP2 knocked down (ISP2KD) parasites compared to wild type (WT), whereas ISP1 knocked down (ISP1KD) parasites remained viable. Therefore, our study clearly indicates that LdISP2 is a more effective inhibitor of serine peptidases than LdISP1. CONCLUSION: Our results suggest that the lack of ISP2 is detrimental to the parasites during the early transition from amastigotes to promastigotes. Moreover, the results of the present study demonstrated for the first time that LdISP2 has an important role in the inhibition of peptidases and promoting L. donovani survival inside the Phlebotomus argentipes midgut.

Effects of specific antisera targeting peritrophic matrix-associated proteins in the sand fly vector Phlebotomus papatasi.

In many hematophagous insects, the peritrophic matrix (PM) is formed soon after a blood meal (PBM) to compartmentalize the food bolus. The PM is an important component of vector competence, functioning as a barrier to the development of many pathogens including parasites of the genus Leishmania transmitted by sand flies. PM morphology and permeability are associated with the proteins that are part of the PM scaffolding, including several peritrophins, and chitin fibers. Here, we assessed the effects of specific antisera targeting proteins thought to be an integral part of the PM scaffolding and its process of maturation and degradation. Phlebotomus papatasi sand flies were fed with red blood cells reconstituted with antisera targeting the chitinase PpChit1, and the peritrophin PpPer2. Sand fly midguts were dissected at different time points and processed for light microscopy (LM), confocal and transmission electron (TEM) microscopies (24, 42-46, 48 and 72h PBM), scanning electron (SEM) (48h PBM) and atomic force (AFM) (30h PBM) microscopies. TEM and WGA-FITC staining indicate PM degradation was significantly delayed following feeding of flies on anti-PpChit1. AFM analysis at 30h PBM point to an increase in roughness' amplitude of the PM of flies that fed on either anti-PpChit1 or anti-PpPer2. Collective, our data suggest that antibodies targeting PM-associated proteins affects the kinetics of PM maturation, delaying its degradation and disruption and are potential targets on transmission-blocking vaccines strategies.

DNA barcoding for identification of sand fly species (Diptera: Psychodidae) from leishmaniasis-endemic areas of Peru.

Phlebotomine sand flies are the only proven vectors of leishmaniases, a group of human and animal diseases. Accurate knowledge of sand fly species identification is essential in understanding the epidemiology of leishmaniasis and vector control in endemic areas. Classical identification of sand fly species based on morphological characteristics often remains difficult and requires taxonomic expertise. Here, we generated DNA barcodes of the cytochrome c oxidase subunit 1 (COI) gene using 159 adult specimens morphologically identified to be 19 species of sand flies, belonging to 6 subgenera/species groups circulating in Peru, including the vector species. Neighbor-joining (NJ) analysis based on Kimura 2-Parameter genetic distances formed non-overlapping clusters for all species. The levels of intraspecific genetic divergence ranged from 0 to 5.96%, whereas interspecific genetic divergence among different species ranged from 8.39 to 19.08%. The generated COI barcodes could discriminate between all the sand fly taxa. Besides its success in separating known species, we found that DNA barcoding is useful in revealing population differentiation and cryptic diversity, and thus promises to be a valuable tool for epidemiological studies of leishmaniasis.

Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant.

BACKGROUND: Phlebotomus papatasi vectors zoonotic cutaneous leishmaniasis. Previous expression of recombinant P. papatasi acetylcholinesterase (PpAChE1) revealed 85% amino acid sequence identity to mosquito AChE and identified synthetic carbamates that effectively inhibited PpAChE1 with improved specificity for arthropod AChEs compared to mammalian AChEs. We hypothesized that the G119S mutation causing high level resistance to organophosphate insecticides in mosquitoes may occur in PpAChE1 and may reduce sensitivity to inhibition. We report construction, expression, and biochemical properties of rPpAChE1 containing the G119S orthologous mutation. METHODS: Targeted mutagenesis introduced the G119S orthologous substitution in PpAChE1 cDNA. Recombinant PpAChE1 enzymes containing or lacking the G119S mutation were expressed in the baculoviral system. Biochemical assays were conducted to determine altered catalytic properties and inhibitor sensitivity resulting from the G119S substitution. A molecular homology model was constructed to examine the modeled structural interference with docking of inhibitors of different classes. Genetic tests were conducted to determine if the G119S orthologous codon existed in polymorphic form in a laboratory colony of P. papatasi. RESULTS: Recombinant PpAChE1 containing the G119S substitution exhibited altered biochemical properties, and reduced inhibition by compounds that bind to the acylation site on the enzyme (with the exception of eserine). Less resistance was directed against bivalent or peripheral site inhibitors, in good agreement with modeled inhibitor docking. Eserine appeared to be a special case capable of inhibition in the absence of covalent binding at the acylation site. Genetic tests did not detect the G119S mutation in a laboratory colony of P. papatasi but did reveal that the G119S codon existed in polymorphic form (GGA + GGC). CONCLUSIONS: The finding of G119S codon polymorphism in a laboratory colony of P. papatasi suggests that a single nucleotide transversion (GGC → AGC) may readily occur, causing rapid development of resistance to organophosphate and phenyl-substituted carbamate insecticides under strong selection. Careful management of pesticide use in IPM programs is important to prevent or mitigate development and fixation of the G119S mutation in susceptible pest populations. Availability of recombinant AChEs enables identification of novel inhibitory ligands with improved efficacy and specificity for AChEs of arthropod pests.

Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi.

Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties.

BACKGROUND: Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. METHODS: A P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3'-5'-RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman's assay in microplates. RESULTS: A 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for L. longipalpis. Recombinant P. papatasi AChE1 was expressed in the baculovirus system and characterized as an insect acetylcholinesterase with substrate preference for acetylthiocholine and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine, BW284c51, malaoxon, and paraoxon, and was insensitive to the butyrylcholinesterase inhibitors ethopropazine and iso-OMPA. CONCLUSIONS: Results presented here enable the screening and identification of PpAChE mutations resulting in the genotype for insensitive PpAChE. Use of the recombinant P. papatasi AChE1 will facilitate rapid in vitro screening to identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of inhibition.

The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies.

BACKGROUND: Parasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. Leishmania development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female Phlebotomus perniciosus and compared the transcript expression profiles. RESULTS: A total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (PperPer1), two chymotrypsin-like proteins (PperChym1 and PperChym2), a putative trypsin (PperTryp3) and four putative microvillar proteins (PperMVP1, 2, 4 and 5). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (PperTryp1 and PperTryp2), a chymotrypsin (PperChym3) and a microvillar protein (PperMVP3). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in Leishmania infantum-infected and uninfected sand flies, which identified the L. infantum-induced down regulation of PperTryp3 at 24 hours post-blood meal. CONCLUSION: This midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of P. perniciosus, an important vector of visceral leishmaniasis in the Old World. Through the comparative analysis of the libraries we identified molecules differentially expressed during blood meal digestion. Additionally, this study provides a detailed comparison to transcripts of other sand flies. Moreover, our analysis of putative trypsins demonstrated that L. infantum infection can reduce the transcript abundance of trypsin PperTryp3 in the midgut of P. perniciosus.

Targeting the midgut secreted PpChit1 reduces Leishmania major development in its natural vector, the sand fly Phlebotomus papatasi.

BACKGROUND: During its developmental cycle within the sand fly vector, Leishmania must survive an early proteolytic attack, escape the peritrophic matrix, and then adhere to the midgut epithelia in order to prevent excretion with remnants of the blood meal. These three steps are critical for the establishment of an infection within the vector and are linked to interactions controlling species-specific vector competence. PpChit1 is a midgut-specific chitinase from Phlebotomus papatasi presumably involved in maturation and degradation of the peritrophic matrix. Sand fly midgut chitinases, such as PpChit1, whether acting independently or in a synergistic manner with Leishmania-secreted chitinase, possibly play a role in the Leishmania escape from the endoperitrophic space. Thus, we predicted that silencing of sand fly chitinase will lead to reduction or elimination of Leishmania within the gut of the sand fly vector. METHODOLOGY/PRINCIPAL FINDINGS: We used injection of dsRNA to induce knock down of PpChit1 transcripts (dsPpChit1) and assessed the effect on protein levels post blood meal (PBM) and on Leishmania major development within P. papatasi. Injection of dsPpChit1 led to a significant reduction of PpChit1 transcripts from 24 hours to 96 hours PBM. More importantly, dsPpChit1 led to a significant reduction in protein levels and in the number of Le. major present in the midgut of infected P. papatasi following a infective blood meal. CONCLUSION/SIGNIFICANCE: Our data supports targeting PpChit1 as a potential transmission blocking vaccine candidate against leishmaniasis.

Functional characterization of a salivary apyrase from the sand fly, Phlebotomus duboscqi, a vector of Leishmania major.

Two transcripts coding for proteins homologous to apyrases were identified by massive sequencing of a Phlebotomus (P.) duboscqi salivary gland cDNA library. The sequence analysis revealed that the amino acids important for enzymatic activity including nucleotidase activity and the binding of calcium and nucleotides were well conserved in these molecules. A recombinant P. duboscqi salivary apyrase was expressed in Escherichia coli and purified. The resulting protein efficiently hydrolyzed ADP and ATP, but not AMP, GDP, CDP or UDP, in a calcium-dependent manner. Further, the recombinant protein inhibited ADP- and collagen-induced platelet aggregation. The results indicated that this salivary protein plays an important role in the blood-feeding process in P. duboscqi. Its unique enzymatic activity makes the salivary apyrase an attractive candidate as a therapeutic agent for the treatment of thrombotic pathologies as well as a reagent for a wide variety of research purposes.

Hyaluronidase of bloodsucking insects and its enhancing effect on leishmania infection in mice.

BACKGROUND: Salivary hyaluronidases have been described in a few bloodsucking arthropods. However, very little is known about the presence of this enzyme in various bloodsucking insects and no data are available on its effect on transmitted microorganisms. Here, we studied hyaluronidase activity in thirteen bloodsucking insects belonging to four different orders. In addition, we assessed the effect of hyaluronidase coinoculation on the outcome of Leishmania major infection in BALB/c mice. PRINCIPAL FINDINGS: High hyaluronidase activity was detected in several Diptera tested, namely deer fly Chrysops viduatus, blackflies Odagmia ornata and Eusimilium latipes, mosquito Culex quinquefasciatus, biting midge Culicoides kibunensis and sand fly Phlebotomus papatasi. Lower activity was detected in cat flea Ctenocephalides felis. No activity was found in kissing bug Rhodnius prolixus, mosquitoes Anopheles stephensi and Aedes aegypti, tse-tse fly Glossina fuscipes, stable fly Stomoxys calcitrans and human louse Pediculus humanus. Hyaluronidases of different insects vary substantially in their molecular weight, the structure of the molecule and the sensitivity to reducing conditions or sodium dodecyl sulphate. Hyaluronidase exacerbates skin lesions caused by Leishmania major; more severe lesions developed in mice where L. major promastigotes were coinjected with hyaluronidase. CONCLUSIONS: High hyaluronidase activities seem to be essential for insects with pool-feeding mode, where they facilitate the enlargement of the feeding lesion and serve as a spreading factor for other pharmacologically active compounds present in saliva. As this enzyme is present in all Phlebotomus and Lutzomyia species studied to date, it seems to be one of the factors responsible for enhancing activity present in sand fly saliva. We propose that salivary hyaluronidase may facilitate the spread of other vector-borne microorganisms, especially those transmitted by insects with high hyaluronidase activity, namely blackflies (Simuliidae), biting midges (Ceratopogonidae) and horse flies (Tabanidae).

Isoenzymatic characterization of Phlebotomus papatasi (Diptera: Psychodidae) of the Marrakech area, Morocco.

This study reports the genetic characterization of urban and rural populations of Phlebotomus (Phlebotomus) papatasi Scopoli (Diptera: Psychodidae) in Marrakech, Morocco. Using isoenzymatic analysis, four Moroccan populations were compared with other Mediterranean basin populations from Spain, Cyprus, and Syria. Morphological anomalies were noted in the male genitalia of 5.3% of the specimens collected from Marrakech area. Qualitative analysis of zymogram profiles revealed nine polymorphic enzymes (HK, PGM, PGI, 6PGD, MDH1, MDH2, ICD2, FUM, and ACO) and three monomorphic enzymes (ME, ICD1, and alphaGPDH). Genetic distances clearly separated the populations of western Mediterranean countries (Morocco and Spain) from eastern countries (Syria and Cyprus), but they could not be used to differentiate between urban and rural populations in Marrakech area.

Morphological and isoenzymatic differentiation of Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Psychodidae) in Southern Morocco.

Morphological and enzymatic characterization of Phlebotomus perniciosus and Phlebotomus longicuspis in Morocco is reported. Twenty-nine localities in central and southern of Morocco were sampled and compared with three localities from the Rif (northern Morocco). For morphological study, sand flies were collected by sticky-paper traps. For males, the morphology of the copulatory valves (aedeagi) was examined and the number of coxite hairs was recorded. For isoenzyme analyses, specimens were collected in CDC light traps and immediately conserved at -80 degrees C. P. perniciosus samples from the south of Morocco, up to 150 km from Marrakech, showed single-pointed aedeagi curved at their apices, indistinguishable from the atypical morph of P. perniciosus, previously described in northern Morocco. Twelve enzyme systems were tested and the qualitative analysis of zymogram profiles revealed eight polymorphic loci (glucosephosphate isomerase (GPI), phosphoglucomutase (PGM), hexokinase (HK), fumarate hydratase (FUM), malate dehydrogenase 1 (MDH1), malate dehydrogenase 2 (MDH2), 6 phosphogluconate dehydrogenase (6PGD) and aconitase (ACO)). Enzyme loci showed fixed alleles diagnostic for P. perniciosus (aconitase) and P. longicuspis (aconitase and hexokinase).

Identification and characterization of a salivary adenosine deaminase from the sand fly Phlebotomus duboscqi, the vector of Leishmania major in sub-Saharan Africa.

Two transcripts coding for an adenosine deaminase (ADA) were identified by sequencing a Phlebotomus duboscqi salivary gland cDNA library. Adenosine deaminase was previously reported in the saliva of the sand fly Lutzomyia longipalpis but it was not present in the saliva of the sand flies Phlebotomus papatasi, P. argentipes, P. perniciosus and P. ariasi, suggesting that this enzyme is only present in the saliva of sand flies from the genus Lutzomyia. In the present work, we tested the hypothesis that the salivary gland transcript coding for ADA in Phlebotomus duboscqi, a sister species of Phlebotomus papatasi, produces an active salivary ADA. Salivary gland homogenates of P. duboscqi converted adenosine to inosine, suggesting the presence of ADA activity in the saliva of this species of sand fly; furthermore, this enzymatic activity was significantly reduced when using either salivary glands of recently blood-fed sand flies or punctured salivary glands, suggesting that this enzyme is secreted in the saliva of this insect. This enzymatic activity was absent from the saliva of P. papatasi. In contrast to other Phlebotomus sand flies, we did not find AMP or adenosine in P. duboscqi salivary glands as measured by HPLC-photodiode array. To confirm that the transcript coding for ADA was responsible for the activity observed in the saliva of this sand fly, we cloned this transcript into a prokaryotic expression vector and produced a soluble and active recombinant protein of approximately 60 kDa that was able to convert adenosine to inosine. Extracts of bacteria transformed with control plasmids did not show this activity. These results suggest that P. duboscqi transcripts coding for ADA are responsible for the activity detected in the salivary glands of this sand fly and that P. duboscqi acquired this activity independently from other Phlebotomus sand flies. This is another example of a gene recruitment event in salivary genes of blood-feeding arthropods that may be relevant for blood feeding and, because of the role of ADA in immunity, it may also play a role in parasite transmission.

Molecular and biochemical characterization of a sand fly population from Sri Lanka: evidence for insecticide resistance due to altered esterases and insensitive acetylcholinesterase.

With an increasing incidence of cutaneous leishmaniasis in Sri Lanka, particularly in northern provinces, insecticide-mediated vector control is under consideration. Optimizing such a strategy requires the characterization of sand fly populations in target areas with regard to species composition and extant resistance, among other parameters. Sand flies were collected by human bait and cattle-baited net traps on Delft Island, used as an illegal transit location by many refugees returning to the north of Sri Lanka from southern India where leishmaniasis is endemic. For species identification, genomic DNA was extracted and a fragment of the ribosomal 18S gene amplified. The sequence from all flies analysed matched that of Phlebotomus argentipes Annandale & Brunetti, the primary vector in India and the most likely vector in Sri Lanka. Independent morphological analysis also identified P. argentipes. To establish the current susceptibility status of vector species, data were obtained at the biochemical level, from which potential cross-resistance to alternative insecticides can be predicted. The Delft Island collection was assayed for the activities of four enzyme systems involved in insecticide resistance (acetylcholinesterase, non-specific carboxylesterases, glutathione-S-transferases and cytochrome p450 monooxygenases), establishing baselines against which subsequent collections can be evaluated. There was preliminary evidence for elevated esterases and altered acetylcholinesterase in this population, the first report of these resistance mechanisms in sand flies to our knowledge, which probably arose from the malathion-based spraying regimes of the Anti-Malarial Campaign.

Postglacial dispersal of Phlebotomus perniciosus into France.

Phlebotomus perniciosus was identified morphologically in samples from France and northeast Spain, and individuals were then characterized at three polymorphic isoenzyme loci (by isoelectrofocusing) and at the mitochondrial DNA locus (by comparative DNA sequence analysis of a fragment of the Cytochrome b gene). The four polymorphic loci gave conflicting patterns of population relationships, which can be explained by hypothesizing different amounts of gene introgression at each locus when two distinctive lineages met in southern France or northeast Spain after isolation in southern Italy and Spain during the Pleistocene Ice Ages. P. perniciosus is an important vector of leishmania infantum and so these population differentiation studies are relevant for predicting the emergence and spread of leishmaniasis in relation to environmental changes, including climate.