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Bluetongue virus multiplied to a low level in Phlebotomus papatasi sandflies after intrathoracic inoculation but not after membrane feeding. It is considered unlikely that this sandfly species is a vector of bluetongue virus.
The spermathecal complex of Phlebotomus papatasi Scopoli (Diptera: Psychodidae) undergoes histological and physiological changes during its gonotropic cycle. The present histochemical study revealed a mucopolysaccharide secretory mass in the spermathecae of the newly emerged sandfly. Sperm competition occurs when two or more males compete to fertilize an ovum in the female reproductive tract. In this study, spermatophores of two or more competing males were deposited at the base of the spermathecal ducts, which originate from the female bursa copulatrix. This suggests that females play a role in sperm displacement, which is defined as any situation in which the last male to mate with a female fertilizes maximum number her eggs. A blood meal ingested by the female for ovary development and egg laying stimulates the release of sperm from the spermatophore. The spermatozoa then migrate to the lumen of the spermatheca. The ultrastructure of spermatozoa comprises a head with double-layered acrosomal perforatorium, an elongate nucleus, and the axoneme with a 9 + 9 + 0 flagellar pattern. This axomene differs from the flagellate axoneme of other Psychodinae. Morphological changes, such as the casting off of the acrosomal membrane, and histological changes in the spermatophore are also described. Mating plugs that have been described previously in sandflies appear to be artefacts. Females of P. papatasi may be inseminated more than once during each gonotrophic cycle, and additional inseminations may be necessary for each cycle. The relationships between the volumes of the sperm and the spermatheca were calculated to determine sperm utilization and fecundity of P. papatasi. As the females of P. papatasi mate polyandrously, the anatomical and physiological complexity of the spermathecal complex may be related to post-copulatory sexual selection.
We have carried out a T1 ribonuclease fingerprinting analysis of the RNA genomes of Toscana virus isolates from successive generations of an experimentally virus-infected laboratory colony of Phlebotomus perniciosus sandflies. This analysis detected no virus RNA genome changes during transovarial transmission of the virus over 12 sandfly generations (a period of almost 2 years). These results demonstrate that although RNA viruses can exhibit high rates of mutational change under a variety of conditions, Toscana virus RNA genomes can be maintained in a stable manner during repeated transovarial virus transmission in the natural insect host. The implications of these results for insect RNA virus evolution are discussed.
Leishmania infections in the vector sandfly are limited to the gut, where contact with tissues, secretions and the medium of sandfly food influence their cycle of development The parasites cope with and exploit their habitat by generating products that impair the function and damage the tissues of the gut In this review, Yosef Schlein concentrates on some of the recent advances in the limited knowledge of these parasite-vector interactions.
Zoonotic visceral leishmaniasis (VL) is a re-emerging disease in the Mediterranean area. A historical review on human and canine leishmaniasis in Croatia documents the presence of stable disease foci in coastal and insular territories of central and southern Dalmatia since the beginning of the 20th century. We report on a 3-year entomological investigation (2002-2004) aimed to study the current species composition and seasonality of phlebotomine sandflies in 10 localities of Dalmatia. Standard blacklight traps employed for sampling Culicoides midges in the frame of bluetongue disease surveillance were used for the first time in phlebotomine monitoring. A total of 2917 sandflies were collected and five species were identified, Phlebotomus tobbi, Phlebotomus neglectus, Phlebotomus perfiliewi, Phlebotomus mascittii and Sergentomyia minuta. Among the species, which may act as Leishmania infantum vectors, P. tobbi and P. neglectus were the most abundant.
Gulf War Syndrome (GWS) remains a contentious diagnosis with conflicting laboratory investigation and lack of a biologically plausible aetiology. This paper discusses the potential role of maxadilan, a potent sandfly vasoactive peptide, in causing autoimmune responses in susceptible individuals through possible molecular mimicry with pituitary adenylate cyclase activating polypeptide (PACAP) and the PAC1R receptor. Gulf War Syndrome may share some causative pathology with Chronic Fatigue Syndrome (CFS), a disorder characterised by prolonged fatigue and debility mostly associated with post-infection sequelae although ongoing infection is unproven. Immunological aberration associated with an expanding group of vasoactive neuropeptides in the context of molecular mimicry and inappropriate immunological memory has been recently raised as possible cause of CFS. Vasoactive neuropeptides act as hormones, neurotransmitters, immune modulators and neurotrophes. They are readily catalysed to small peptide fragments. They and their binding sites are immunogenic and are known to be associated with a range of autoimmune conditions. Maxadilan, while not sharing substantial sequence homology with PACAP is a known agonist of the PACAP specific receptor (PAC1R) and therefore emulates these functions. Moreover a specific amino acid sequence peptide deletion within maxadilan converts it to a PACAP receptor antagonist raising the possibility of this substance provoking a CFS like response in humans exposed to it. This paper describes a biologically plausible mechanism for the development of a GWS-like chronic fatigue state based on loss of immunological tolerance to the vasoactive neuropeptide PACAP or its receptor following bites of the sandfly Phlebotomus papatasi and injection of the vasodilator peptide maxadilan. Exacerbation of this autoimmune response as a consequence of recent or simultaneous multiple vaccination exposures deserves further investigation. While the possible association between the relatively recently discovered vasoactive neuropeptides and chronic fatigue conditions has only recently been reported in the literature, this paper explores links for further research into GWS and CFS.
Midgut alpha-glucosidase (EC 3.2.1.20) activities were measured after ingestion of blood and sugar meals by the phlebotomine sandfly Phlebotomus langeroni. alpha-Glucosidase activity increased significantly within 1 hr after a blood meal and was maintained at significantly high activities until 48 hr postfeeding, when it fell to basal activity levels. Midgut alpha-glucosidase activity also increased within 1 hr of feeding on a sucrose meal, but there was no discernable peak in activity during the days postingestion. Differences in the induction of enzyme activity after a sugar meal compared to a blood meal might reflect the mode of ingestion of the two types of meal. The sugar meal is released intermittently into the midgut from the crop, in contrast to the bloodmeal, which is directed into the midgut immediately after ingestion and digested in a "batch" process. Nearly 90% of the alpha-glucosidase activity was associated with midgut cells of sugar fed sandflies compared to only 46% in blood fed insects. Isoelectrofocusing revealed the presence of seven alpha-glucosidases with isoelectric points between 4.3-5.8. No alpha-glucosidase activity was detected in the crop, indicating that glucosidases originate from the midgut epithelium rather than the salivary glands.
In this paper we report on the diversity and distribution of a set of non-LTR retrotransposon (RTP) reverse transcriptase (RT) sequences isolated from phlebotomine sandflies, and their potential for investigating the evolutionary histories of members of this subfamily of flies (Diptera:Psychodidae, Phlebotominae). The phlebotomine RT sequence families derived from one species were as different from each other as they were from RT sequences derived from other species. When each was used to probe Southern blots of sandfly genomic DNA they hybridized only to the species of source and, usually, to others of the same subgenus, but not to DNA from other subgenera-a hybridization pattern consistent with vertical evolution. There was considerable intraspecific variation in hybridization pattern, suggesting the RTs were part of non-LTR RTPs that are (or were recently) subject to flux in genomic position and copy number. Most of the RT families detected in phlebotomines are monophyletic with respect to previously described RTs, and all are monophyletic with RTs of the F/Jockey (Drosophila melanogaster) type of RTP. Orthologous sequences were isolated from the closely related species Phlebotomus perniciosus and P. tobbi (subgenus Larroussius), and different populations of P. perniciosus. The level of sequence divergence among these orthologous RTs, the subgeneric distribution of each RT family, and the intraspecific variation in hybridization pattern of many of them, indicate this class of sequence will provide genetic markers at the sub-generic level.
Saliva inoculated by sandfly females during feeding stimulated production of high levels of anti-saliva antibodies. To determine whether 3 species of the genus Phlebotomus have species-specific salivary antigens we performed dot-blots and immunoblots using sera from mice, hamsters and rabbits repeatedly bitten by sandflies. Important differences were found in the antigen components of the salivary gland lysates (SGL) of Phlebotomus papatasi, P. perniciosus and P. halepensis. In total 4-9 species-specific antigens were detected in each species by immunoblotting. Cross-reactivity was not detected between P. papatasi and the other species tested; in the SGL of P. papatasi sera from animals bitten by this species recognized 5-7 major antigens while sera from animals bitten by other species did not react. A weak cross-reaction was observed between P. perniciosus and P. halepensis; in SGL from P. perliciosus, the sera from rabbits and hamsters bitten by this species recognized about 8 intense bands while sera from animals bitten by P. halepensis reacted weakly with up to 4 saliva polypeptides.
A simple and reliable technique was developed to distinguish Phlebotomine sandflies by restriction fragment length polymorphism of PCR-amplified (PCR-RFLP) 18S rDNAs. Seven morphologically identified sandflies species from several localities of Greece and Cyprus were studied, and specific patterns were developed by double digesting amplified 18S rDNAs with HpaII and RsaI. Three additional species of the subgenus Larroussius were distinguished by a second double digestion with AccI and BanI. We have successfully applied the method on samples in which morphological characters were badly distinguished due to poor storage conditions and in larval stages.
Sandfly feeding behaviour and destination of coloured sugar meals in the gut of Lutzomyia longipalpis were investigated with particular attention to the role of the crop. Sandflies were able to ingest sugar from liquid drops, microcapillaries, a slice of pear and even sugar powder. In most cases the flies adopted a 'sugar feeding mode' with raised palps. As the fruit dried, flies of both sexes fed by piercing the tissue with the proboscis. All sugar-fed flies had a full crop plus a small amount of sugary fluid in the thoracic mid-gut, i.e. past the stomodaeal valve. Dissections of flies interrupted during feeding showed that the very first trace of sugar passed through the stomodaeal valve, but that the rest of the meal was diverted into the crop. This suggests that closure of the stomodaeal valve is initiated only after a small volume of sugar solution has passed through it.
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.
Female sandflies of three Phlebotomus species were fed on membrane. The feeding apparatus is described and preliminary data obtained are also reported. A total 2,835 sandflies were fed: 2,596 P. perniciosus, 104 P. perfiliewi and 135 P. papatasi.
A total of 1,394 sandflies were collected in the domestic resting sites of Ouagadougou areas and in two villages of the East region of Burkina Faso using pyrethrum spray catch, oiled papers and miniature CDC light traps during the rainy season in 1984 and 1985. Thirteen species were identified, one belonging to the genus Phlebotomus and the others to the genus Sergentomyia. S. antennata (34.94%), S. bedfordi (16.64%), S. clydei (14.42%), S. schwetzi (13.20%), P. duboscqi (8.46%), S. adleri (7.03%) and S. africana (2.87%) were in order of percentage the more abundant species. The abdominal stages of the sandfly species caught in the human habitations are described. P. duboscqi, the suspected vector of cutaneous leishmaniasis in the area, seems to have a preferential domestic biotope.
Four species of Adlerius phlebotomine sandflies have been recorded in China, namely: P. chinensis Newstead, 1916 (Pc), P. fengi Leng & Zhang, 1994; P. longiductus Parrot, 1928 and P. sichuanensis Leng & Yin, 1983 (Ps). Adlerius phlebotomies are the main vectors of visceral leishmaniasis (VL) in China; three of them are acknowledged as VL vectors and P. fengi is considered a potential VL vector for southwestern mountainous region. Different opinion has been raised to the validity of identity of Ps by some investigators from Shanghai and Shanxi who consider Ps to be a large type of Pc instead of an isolate species. The center of controversy is whether Ps is an isolate taxon or a large type of Pc. The present authors have carried out a series of comparative studies for these two flies on: 1 quantitative and qualitative morphological characters of four Chinese Adlerius phlebotomies; and 2. differences in geographical distribution. All specimens of Pc and Ps used in the present study are collected where their holotypes-paratypes were produced--West Mountain, West Suburb, Beijing and Lixian County, Sichuan Province. The results have forcefully proved that Ps is an isolate species instead of a so-called large type Pc according to the concept of species. The clarification of their taxonomical identities is meaningful because both of them are VL vectors in different epidemic areas in China; especially Ps is an important VL vector in high mountainous regions of southwestern China and some extend to the Loess Plateau of northwestern China, where VL still exists and it is also the first Phlebotomine sandfly discovered in Tibet, the locality being near Assam in India (Leng et al. 1990).
An entomological survey was carried out in two districts of central (Kruje) and northern (Lezhe) Albania. Six collecting sites, showing a variety of diurnal resting sites, were monitored for adult sandflies from June through October 2002. Flies were collected with CDC miniature light traps, sticky traps and mechanical or hand aspirators in peridomestic sites, in bedrooms and inside cow barns, chicken coops and pigpens. All collecting sites monitored were found positive for sandflies. A total of 849 specimens were caught (29.2% males) belonging to five Phlebotomus species. Phlebotomus neglectus (75.6%) was the most abundant species followed by P. perfiliewi (14.4%), P. papatasi (4.6%), P. tobbi (3.6%) and P. similis (1.8%). The first adult of P. neglectus appeared on June 11 and the last one was collected on October 16. The highest density for this species was observed at the end of July. A total of 111 blood-fed females were caught from the two areas studied. P. neglectus was the only species found blood fed in Lezhe and the same species was prevalent (56.1%) in Kruje followed by P. perfiliewi (30.3%), P. tobbi (10.6%); P. papatasi was represented by only two specimens. Blood meal origin was determined in 45/66 (68.2%) of the females tested from Kruie district. P. neglectus was found fed on four hosts, showing the following feeding patterns: cow (71.4%), dog (117.1%), chicken (5.7%) and human (5.7%); P. perfiliewi was found fed on cow (80.0%) and chicken (20.0%), P. tobbi on cow (50.0%), chicken (25.0%) and dog (25.0%). One specimen of P. papatasi was found fed on cow. When such prevalences were analysed by the available biomass for each host present at the collecting site, P. neglectus resulted to be an opportunistic feeder rather than exhibiting preferences for any specific animal. PCR analysis of 39 P. neglectus from the Lezhe district gave negative results for the presence of Leishmania DNA.
UNLABELLED: CASE 1: A few days after returning from a holiday in Italy a 50-year-old man developed acute gastrointestinal symptoms, followed by headache, fever and joint pains. After transitory remission he had a relapse with fever, headache and meningitis 2 weeks later. Cerebrospinal fluid contained gram-negative diplococci, but no bacteria grew on culture. Under the suspected diagnosis of meningitis he was treated with penicillin. Ten days later he suddenly developed deafness in his right ear. The various signs and symptoms gradually disappeared and the patient was discharged after 24 days in hospital. Retrospectively serological tests indicated sandfly fever (SF) virus infection with serotype Toscana. This is thought to be the first reported case of deafness associated with this disease. CASE 2: One week after returning from a holiday in Tunisia a 34-year-old man fell ill with fever, headache, rigor, nausea, joint pains and a maculopapular rash for which he was treated as an outpatient. The symptoms improved after 7 days, except for the headache which persisted another 5 days. Serology demonstrated an acute infection by SF virus, serotype Sicilian. CONCLUSION: Sandfly fever should be included in the differential diagnosis of headache, fever and signs of meningitis in persons who have recently been to mediterranean countries.
Leishmania parasites are transmitted by the bites of infected female sandflies by a mechanism that has not been clarified. Leishmania infections in the vector develop only in the gut, and the parasites' exit is through the food channel in the proboscis. The problem is how during the bite, when blood flows in, parasites are emitted through the same channel in the opposite direction. It is well documented that infected sandflies maintained on sugar diets are potent vectors, whereas transmission fails after constant feeding on blood. Hence to study the mechanism of transmission, we fed these diets to Phlebotomus papatasi infected with L. major. Histological examination demonstrated that only in the sugar-fed flies did the cuticle lining of the cardiac valve detach and other valve tissues degenerate gradually. The injury of the main valve of the food pumps hindered gorging of most flies when force-fed from capillaries, and they regurgitated the gut contents with fluids from the capillaries. We suggest that infections are caused by parasites regurgitated from the stomach that are deposited in the host tissue. We found that secretion of chitinolytic enzymes by cultured L. major parasites is inhibited by blood or hemoglobin, and hence these enzymes are apparently absent from the blood-fed infected flies, where the cardiac valve appears undamaged. We therefore presume that lysis of the chitin in the cuticle lining of the valve leads to exposure and degeneration of the underlying tissues.