Base-line susceptibility of Phlebotomus papatasi to insecticides

Susceptibility of the leishmaniasis vector sandfly Phlebotomus papatasi (Scopoli) to three insecticides was assessed on a colony originating in the Jordan Valley, Israel. Results are considered to show susceptibility to DDT and permethrin. The results for methoxychlor indicated the possibility of heterogeneity in the population and tolerance towards this compound.     

Bloodmeal digestion in the midgut of Phlebotomus papatasi and Phlebotomus langeroni

Abstract. Bloodmeal digestion in midguts of the sandflies Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae) was investigated in optimized assays to detect general protease, trypsin and aminopeptidase activities using synthetic substrates. Optimal activity occurred at pH 8-9 for all enzymes examined in both species. Protease activity peaked at 24-34h post human bloodmeal in midguts of P.papatasi and 34-48h in P'.langeroni; all endo- and exoprotease activities were completed by 50 h in P.papatasi compared to 72 h in P. langeroni. Hydrolysis of two chymotrypsin substrates was <2% of trypsin activity in both species. Aminopeptidase activity was associated mainly with the midgut wall, whereas trypsin activity was confined to the midgut lumen. A feature of digestion in P.langeroni was the high level of aminopeptidase recorded within 10h of the bloodmeal.     

Scanning electron microscopy of Leishmania major in Phlebotomus papatasi

The morphology of Leishmania major parasites and their interactions with various regions of the alimentary canal of Phlebotomus papatasi were studied by scanning electron microscopy. Parasites were observed to undergo development initiated with the ingestion of amastigotes and culminating in a characteristic distribution of four distinct morphological forms in various parts of the alimentary canal: namely, large numbers of elongate nectomonads in the abdominal mid-gut, haptomonad forms attached to the cuticle of the stomodeal valve, small spherical forms attached to the esophagus and masses of short promastigotes, believed to be the infective forms, lying free in the anterior thoracic mid-gut and the esophagus.     

Cytoplasmic polyhedrosis viruses in Phlebotomus papatasi inhibit development of Leishmania major

Cytoplasmic polyhedrosis viruses (CPV's) were observed in wild-caught and laboratory-reared Phlebotomus papatasi. Chronic CPV pathology of the midgut, characterized by structural aberrations in the epithelium and the peritrophic membrane, interfered with blood digestion and rendered the sand flies refractory to Leishmania major infections. Rates of natural and artificial L. major infections were inversely correlated to the incidence of CPV infections. The interaction between viruses and protozoan parasites in an insect host is of basic biological interest and in this case may be of significance in the epidemiology of cutaneous leishmaniasis.     

Altitudinal variation in morphometric and molecular characteristics of Phlebotomus papatasi populations

Four populations of the phlebotomine sandfly Phlebotomus (Phlebotomus) papatasi (Scopoli) (Diptera: Psychodidae), in different ecoregions at altitudes between 368 and 1117 m in the Sanliurfa Province of Turkey, were compared using morphometric and isoenzyme analyses. A similarity phenogram obtained from allozyme data showed that heterozygosity was extremely low, particularly for the alleles which were found to be completely fixed in populations at Hamdun (HMD) and Alitas (ALT). Populations at Akcakale (AKL) and ALT branched as a separate group from populations at Hayatiharrani (HHR) and HMD. The ALT population at the highest altitude (1117 m), and the HHR population (488 m) were clustered distinctly when linear measurements of 46 morphological characteristics were examined. A UPGMA (unweighted pair-group method using arithmetic averages) phenogram also showed that ALT and HHR clustered separately, whereas AKL and HMD formed another group.     

Relative potency of DEPA as a repellent against the sandfly Phlebotomus papatasi

Abstract. The insect repellent N,N-diethylphenylacetamide (DEPA) was compared with two commercial repellents, N,N-diethyl-m-toluamide (DEET) and dimethyl phthalate (DMP), for protection against 3-day-old unfed females of the sandfly Phlebotomus papatasi (Scopoli) under laboratory conditions, using host rabbits. Both DEPA and DEET were found to be more effective than DMP, but there was no significant difference between the efficacy of DEPA and DEET.     

Rhesus monkey model for Leishmania major transmitted by Phlebotomus papatasi sandfly bites

Leishmaniasis research needs a near-human model for investigations of natural infection processes, immunological responses and evaluation of treatments. Therefore, we developed a reproducible system using Leishmania major Yakimoff & Schokhor (Trypanosomatidae: Kinetoplastida), the cause of Old World zoonotic cutaneous leishmaniasis (ZCL), transmitted to rhesus monkeys Macaca mulatta (Zimmerman) (Primates: Cercopithecidae) by sandfly bites of experimentally infected Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae). Eight monkeys of presumed Indian origin (Leishmania naive) were exposed to bites of female sandflies that had been infected with L. major by membrane-feeding on human blood seeded with amastigotes isolated from hamster footpad lesions. Infection rates of membrane-fed sandflies averaged > 85% seven days after the infective feed, with uniformly high numbers of promastigotes in the stomodaeal valve region of the sandfly gut. Nodules and ulcerating dermal lesions developed on 7/8 monkeys 2-4 weeks post-bite and persisted for 3-7 months. Monkeys also developed satellite lesions beyond the area of sandfly bites on the head, but not on the chest. Three re-challenged monkeys developed lesions that healed faster than lesions from their primary challenges. After infection, monkeys developed delayed type hypersensitivity (DTH) responses to a panel of Leishmania skin test antigens (LSTA) and, when tested by ELISA and IFA, showed significant post-infection antibody titres which typically rose for approximately 170 days and then gradually receded during the next 100 days following the first challenge. After the second challenge, antibody titres spiked higher within approximately 50 days and receded more rapidly. In contrast, four rhesus macaques of Chinese origin developed no lesions following infected sandfly bites, although they raised antibodies and LSTA reactions, indicating subclinical infection.     

Impact of pyrethroid-impregnated curtains on Phlebotomus papatasi sandflies indoors at Khartoum, Sudan

Laboratory and field investigations were made in an endemic focus of cutaneous leishmaniasis in Khartoum State, Sudan, to evaluate the effects of permethrin-impregnated curtains on the human-biting activity, nocturnal activity and resting behaviour of the vector sandfly Phlebotomus papatasi (Diptera: Psychodidae) indoors. Laboratory bioassays showed that curtains impregnated with 0.5, 1.0 or 1.5 g/m2 permethrin all gave 100% mortality within 24h of exposure of P. papatasi for 3 min. Under natural field conditions, the biting activity indoors and the resting density of P. papatasi were significantly reduced (P<0.001 and P=0.036, respectively) in rooms provided with permethrin-impregnated curtains as compared to control rooms left without curtains or fitted with unimpregnated curtains. No significant difference was found between the numbers of nocturnally active P. papatasi collected in rooms provided with impregnated curtains and rooms left without curtains or provided with unimpregnated curtains (P=0.377). Evidently P. papatasi was not repelled by these doses of permethrin on curtains, but the survival rate of sandflies collected from test rooms provided with permethrin-impregnated curtains was significantly reduced (P=0.036). We conclude that use of permethrin-impregnated curtains may provide a good control method for P. papatasi and other endophilic and/or endophagic sandfly vectors of leishmaniasis.     

Longitudinal monitoring of anti‐saliva antibodies as markers of repellent efficacy against Phlebotomus perniciosus and Phlebotomus papatasi in dogs

A 2‐year longitudinal study of enzyme‐linked immunosorbent assay (ELISA) antibodies against Phlebotomus perniciosus and Phlebotomus papatasi (Diptera: Psychodidae) sandfly saliva was performed in 32 Beagle dogs treated preventively with an imidacloprid–permethrin topical insecticide in an endemic area in Spain. Dogs were grouped into three sandfly exposure groups according to the time of inclusion in the study. Assays analysed immunoglobulin G (IgG) against salivary gland homogenates (SGH) of both species and recombinant P. papatasi rSP32 and P. perniciosus rSP03B proteins in serum. The dogs were participating in a Leishmania infantum (Kinetoplastida: Trypanosomatidae) vaccine trial and were experimentally infected with the parasite in the second year. No dog acquired natural L. infantum infections during the first year, but most developed anti‐saliva antibodies, and median log‐transformed optical densities (LODs) were seasonal, mimicking those of local sandflies. This indicates that the repellent efficacy of the insecticide used is below 100%. Multi‐level modelling of LODs revealed variability among dogs, autocorrelation and differences according to the salivary antigen and the dog's age. However, dog seroprevalence, estimated using pre‐exposure LODs as cut‐offs, was relatively low. This, and the fact that dogs did not become naturally infected with L. infantum, would support the efficacy and usefulness of this imidacloprid–permethrin topical insecticide in canine leishmaniasis control.     

Detection of amastigote-like forms in the valve of Phlebotomus papatasi infected with Leishmania major

A massive and homogeneous amount of amastigote-like forms was detected in the stomodeal valve (SV) and the thoracic mid-gut (TMG) of Leishmania major-infected Phlebotomus papatasi, which received a second blood meal 13 to 21 days post-infection on healthy anaesthetized hamsters. After re-feeding, the infected sand flies were dissected out to examine the morphology of the parasite in SV, TMG and the abdominal mid-gut (AMG). Different promastigote forms were seen in the infected flies. Among these included typical promastigotes (nectomonads and haptomonads), paramastigotes, metacyclic promastigotes and, in some samples, the here-reported amastigote-like forms. The Leishmania amastigote-like forms were detected in the SV of sand flies with 14, 18 and 21 days of infection as well as in the TMG at 13 and 18 days post-infection. However, the amastigote-like forms were not detected in the AMG. Factors such as the acidic pH predominating the TMG and the SV, as well as the temperature of the ingested blood, among others, are suggested as contributing to the transformation of the typical promastigotes into the amastigote-like forms. The significance of this finding is discussed and the possible biological advantage for transmission of Leishmania is considered.     

Laboratory estimation of degree-day developmental requirements of Phlebotomus papatasi (Diptera: Psychodidae).

Cutaneous leishmaniasis is one of the most important vector-borne endemic diseases in Turkey. The main objective of this study was to evaluate the influence of temperature on the developmental rates of one important vector of leishmaniasis, Phlebotomus papatasi (Scopoli, 1786) (Diptera: Psychodidae). Eggs from laboratory-reared colonies of Phlebotomus papatasi were exposed to six constant temperature regimes from 15 to 32 degrees C with a daylength of 14 h and relative humidity of 65-75%. No adult emergence was observed at 15 degrees C. Complete egg to adult development ranged from 27.89 +/- 1.88 days at 32 degrees C to 246.43 +/- 13.83 days at 18 degrees C. The developmental zero values were estimated to vary from 11.6 degrees C to 20.25 degrees C depending on life stages, and egg to adult development required 440.55 DD above 20.25 degrees C.     

Transmission and scanning EM-immunogold labeling of Leishmania major lipophosphoglycan in the sandfly Phlebotomus papatasi

Previous studies using immunostaining and light microscopy demonstrated expression of Leishmania major lipophosphoglycan (LPG) on parasites developing in the sandfly gut from 2 days post infection. By days 4 to 7 post infection, there appeared to be large amounts of parasite-free LPG deposited on/in the microvilli and epithelial cells lining the thoracic midgut, while forward migration of parasites and the morphological changes which accompany metacyclogenesis were associated with developmental modification of the LPG molecules. Studies presented here examine this process with much greater precision using electron microscopy and immunogold labeling techniques to study the different developmental forms (nectomonads, haptomonads, paramastigotes, and metacyclics) of promastigotes in the sandfly gut. Results obtained using LPG-specific monoclonal antibodies (WIC79.3, 45D3 and the metacyclic-specific 3F12) show (1) gold labeling over the cell surface, within the flagellar pocket, and extending along the entire length of the flagellum of electron-dense nectomonads observed in the abdominal and thoracic midgut regions on days 4 and 7 post infection, and of electron-lucid haptomonads in the foregut, (2) dense labeling around the flagellar tips, by which nectomonad forms bind to the midgut microvilli, but not on the microvilli themselves or within the epithelial cells lining the midgut, (3) significant metacyclic-specific (3F12) labeling on nectomonad forms in the lumen of the midgut and attached to the microvilli, and (4) dense labeling on the cell surface of electron-lucid paramastigotes in the esophagus and in the filamentous matrix surrounding paramastigote and metacyclic forms in the esophagus and pharynx. These results are discussed in the light of the proposed roles for LPG in parasite attachment to, and survival in, the sandfly gut.     

Oviposition diapause and other factors affecting the egg-laying of Phlebotomus papatasi in the laboratory

Stimuli which modulate oviposition of P. papatasi were investigated to improve insectary breeding efficiency. Oviposition and survival of gravid females were observed weekly during April-December 1987, in plastic cages at 28 +/- 1 degrees C with L:D 17:7. Oviposition of controls was subject to seasonal variation despite the relatively uniform insectary conditions. From April to mid-October (summer), mean weekly oviposition ranged from 11.6 to 18.6 eggs per fly, dropping to 1.4 eggs/fly in November (winter). Monthly yields of eggs were found to correlate with the seasonal cycle of ultraviolet (UV) radiation in sunlight. This was attributed to an endogenous rhythm since the flies were not normally exposed to UV under insectary conditions. Short exposure to UV sources of 254 nm and 312 nm, but not white light, raised the low rate of oviposition in October-November from 1.4 to 16.8-29.6 eggs/female/week, but female mortality also increased highly significantly. It is suggested that the seasonal oviposition cycle of P.papatasi is set by levels of UV irradiation. In the warm season oviposition was promoted, as compared to controls, by furrows in plaster of Paris lining the bottom of cages (29.6 v. 10.2 eggs/female) and by cow manure in the cages (39.7 v. 18.2 eggs/female), but the combination of both stimuli gave no greater fecundity (40.9 v. 20.9 eggs/female). Oviposition decreased when larvae were present (3.9 v. 15.0 eggs/female) and in half-volume cages (3.9 v. 12.5 eggs/female/week). Under standard insectary conditions, mean weekly mortality-rates of P.papatasi females were 18.3 +/- 4.8% in October-November and 36.5-59.1% during the warmer months. None of the experimental conditions yielded any significant improvement in survival-rates.     

Variation in life table characteristics among populations of Phlebotomus papatasi at different altitudes.

Baseline biological growth data were obtained under laboratory conditions for four local populations of the phlebotomine sand fly R papatasi (Scopoli, 1786) (Diptera: Psychodidae) in different eco-regions at altitudes between 368 and 1117 m in the Sanliurfa province of Turkey. The developmental time from egg to adult was found to be significantly different among the populations: 36 days for the AKL population (368 m), 43 days for the HHR population (488 m), 45 days for the HMD population (644 m), and 49 days for the ALT population (1117 m), respectively. Based on cohorts of adults in each population, horizontal life tables were constructed. The average longevity was determined to be in the range of 8.75 +/- 2.39 to 11.60 +/- 3.48 days for adult females, and it was found to be slightly longer for adult males. No significant difference was found in life expectancy at emergence, e(x) when x=1, between females and males in general (P>0.05) in all the populations. While significant differences could be demonstrated among populations for predictive parameters such as net reproductive rate, Ro, and generation time, Tc, no significant differences among the populations were found in terms of intrinsic rate of increase, r(m), finite rate of increase, lambda, birth (b) and death (d) rates (P>0.05). Populations that produced offspring earlier in life also produced more total female offspring, since Tc was negatively correlated with Ro among the populations (r = -0.686, 0.01相似文献   

DNA hybridizations on squash-blotted sandflies to identify both Phlebotomus papatasi and infecting Leishmania major

Epidemiological field studies on leishmaniasis have been hampered by the laborious, and often inefficient, methods used to assess the rates of infection of sandfly vectors (Diptera; Phlebotominae) by species of the causative disease organisms, protozoal parasites of the genus Leishmania (Kinetoplastida; Trypanosomatidae). We report the rapid and accurate identification of both sandfly vector (Phlebotomus (Phlebotomus) papatasi (Scopoli] and infecting Leishmania major Yakimov & Schokov by DNA hybridizations to squash-blotted sandflies. Large numbers of whole (infected) sandflies can be quickly squashed on to nylon hybridization filters and (following standard procedures) the filter-bound DNA can be hybridized sequentially to cloned, multicopy genomic sequences that are specific for species of Leishmania (kinetoplast DNA) or for the sandfly (ribosomal (r) DNA). Our sandfly probe consists of a 3.2 kb fragment of the intergenic 'non-transcribed' spacer of rDNA of P. papatasi that we have detected only in this species: it is present in all six geographically isolated populations tested (from Tunisia through to India) but cannot be detected in the morphologically similar P. (Phlebotomus) duboscqi Neveu-Lemaire, the vector of Leishmania major south of the Sahara; it also cannot be detected in Phlebotomus species of the subgenera Larroussius and Paraphlebotomus that together with P. papatasi are the dominant man-biting sandflies in north African foci of zoonotic cutaneous leishmaniasis, where (as in many arid regions of western Asia) P. papatasi is believed to be the sole vector of L. major.     

Leishmaniasis in the Jordan Valley. V. Dispersal characteristics of the sandfly Phlebotomus papatasi

Several characteristics of dispersing and non-dispersing Phlebotomus papatasi (Scopoli) were quantified and compared. The majority of dispersing sandflies, trapped crossing fallow fields, were females (68.5% v. 51.1%); of the dispersing females, 55.4% were parous, 48.1% were inseminated and 11.6% were gravid. In the population of sandflies sampled exiting from burrows of the sand rat Psammomys obesus Cretschmar, these categories, respectively, represented 39%, 90% and 26% of the females examined. Leishmania promastigotes were found in 9% of females exiting from P. obesus burrows, and in 2.7% of the dispersing females. The anthrone test established that the reason for activity of gravid females is sugar feeding. These females do not disperse and are suitable targets for future control measures.     

An improvised method of laboratory colonization of Phlebotomus papatasi, the vector of cutaneous leishmaniasis.

A cyclic colony of P. papatasi was successfully established, using wild caught females. The major obstacle in the colonization was infestation of fungus, which was solved when bentonite, a dehydrant was mixed in the larval diet i.e., powdered and sterilized faecal pellets of rabbit. The average duration of development from egg to adult was 46.41 +/- 3.26 days. Females readily engorged on mouse, which was kept immobilized inside restrainer cages Majority of the fed females laid viable eggs, when confined in improvised styro-foam humidity chambers and survived after oviposition. In this process a stable, cyclic colony was established and it is now in F39 generation.     

Genetic structure of Mediterranean populations of the sandfly Phlebotomus papatasi by mitochondrial cytochrome b haplotype analysis

Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) is the main vector of Leishmania major Yakimoff & Schokhor; which is the cause of self-limiting cutaneous leishmaniasis in the Old World. This sandfly is found in houses, animal shelters, caves and rodent burrows. It has a large geographical range, which includes the Middle East and the Mediterranean regions. A population analysis of colony and field specimens of P. papatasi was conducted on 25 populations originating from 10 countries. The distribution of haplotypes of the maternally inherited mitochondrial cytochrome b gene were analysed to assess the population differentiation of P. papatasi. Alignment of a 442-basepair region at the 3' end of the gene identified 21 haplotypes and 33 segregating sites from 131 sandflies. The pattern of sequence variations did not support the existence of a species complex. The median-joining network method was used to describe both the origin of the haplotypes and the population structure; haplotypes tended to cluster by geographical location, suggesting some level of genetic differentiation between populations. Our findings indicate the presence of significant population differentiation for populations derived from Syria, Turkey, Palestine, Israel, Jordan and Egypt. Knowledge of population differentiation among P. papatasi populations is important for understanding patterns of dispersal in this species and for planning appropriate control measures.     

Nucleosides from Phlebotomus papatasi salivary gland ameliorate murine collagen-induced arthritis by impairing dendritic cell functions

Among several pharmacological compounds, Phlebotomine saliva contains substances with anti-inflammatory properties. In this article, we demonstrated the therapeutic activity of salivary gland extract (SGE) of Phlebotomus papatasi in an experimental model of arthritis (collagen-induced arthritis [CIA]) and identified the constituents responsible for such activity. Daily administration of SGE, initiated at disease onset, attenuated the severity of CIA, reducing the joint lesion and proinflammatory cytokine release. In vitro incubation of dendritic cells (DCs) with SGE limited specific CD4(+) Th17 cell response. We identified adenosine (ADO) and 5'AMP as the major salivary molecules responsible for anti-inflammatory activities. Pharmacologic inhibition of ADO A2(A) receptor or enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect. Importantly, CD73 (ecto-5'-nucleotidase enzyme) is expressed on DC surface during stage of activation, suggesting that ADO is also generated by 5'AMP metabolism. Moreover, both nucleosides mimicked SGE-induced anti-inflammatory activity upon DC function in vitro and attenuated establishment of CIA in vivo. We reveal that ADO and 5'AMP are present in pharmacological amounts in P. papatasi saliva and act preferentially on DC function, consequently reducing Th17 subset activation and suppressing the autoimmune response. Thus, it is plausible that these constituents might be promising therapeutic molecules to target immune inflammatory diseases.