Investigations of Phlebotomus perniciosus sand flies in rural Spain reveal strongly aggregated and gender‐specific spatial distributions and advocate use of light‐attraction traps

The spatial and temporal distribution of Phlebotomus perniciosus (Diptera: Psychodidae) (Newstead, 1911), the sand fly vector of pathogens of public and animal health importance, was investigated in a high sand fly density rural area in Spain using light‐attraction and sticky‐interception traps. Traps were placed inside animal buildings and outside at increasing distance from animals. A total of 8506 sand flies were collected, 87% with light traps. Species frequency differed between trap types. The abundance of P. perniciosus decreased exponentially with increasing distance to animals and, while females were most common in the animal enclosure, males predominated in adjoining storage places. Increasing CO₂ concentration had an additional positive effect on female abundance only. Both male and female density increased with rising temperature, and there was some indication that females were more active than males at higher relative humidity. The study confirms that P. perniciosus aggregates around animal premises, although male and female distributions differ and should be analysed separately to account for biological and behavioural differences. This provides further evidence that light traps offer an accurate estimation of the relative spatial and temporal abundance of P. perniciosus, conferring an added value for the study of this species and the risk of pathogen transmission.     

Vector Transmission of Leishmania Abrogates Vaccine-Induced Protective Immunity

Numerous experimental vaccines have been developed to protect against the cutaneous and visceral forms of leishmaniasis caused by infection with the obligate intracellular protozoan Leishmania, but a human vaccine still does not exist. Remarkably, the efficacy of anti-Leishmania vaccines has never been fully evaluated under experimental conditions following natural vector transmission by infected sand fly bite. The only immunization strategy known to protect humans against natural exposure is “leishmanization,” in which viable L. major parasites are intentionally inoculated into a selected site in the skin. We employed mice with healed L. major infections to mimic leishmanization, and found tissue-seeking, cytokine-producing CD4+ T cells specific for Leishmania at the site of challenge by infected sand fly bite within 24 hours, and these mice were highly resistant to sand fly transmitted infection. In contrast, mice vaccinated with a killed vaccine comprised of autoclaved L. major antigen (ALM)+CpG oligodeoxynucleotides that protected against needle inoculation of parasites, showed delayed expression of protective immunity and failed to protect against infected sand fly challenge. Two-photon intra-vital microscopy and flow cytometric analysis revealed that sand fly, but not needle challenge, resulted in the maintenance of a localized neutrophilic response at the inoculation site, and removal of neutrophils following vector transmission led to increased parasite-specific immune responses and promoted the efficacy of the killed vaccine. These observations identify the critical immunological factors influencing vaccine efficacy following natural transmission of Leishmania.     

Spatial distribution of sand fly species (Psychodidae: Phlebtominae), ecological niche,and climatic regionalization in zoonotic foci of cutaneous leishmaniasis,southwest of Iran

Cutaneous leishmaniasis (CL) is a complex vector‐borne disease caused by Leishmania parasites that are transmitted by the bite of several species of infected female phlebotomine sand flies. Monthly factor analysis of climatic variables indicated fundamental variables. Principal component‐based regionalization was used for recognition of climatic zones using a clustering integrated method that identified five climatic zones based on factor analysis. To investigate spatial distribution of the sand fly species, the kriging method was used as an advanced geostatistical procedure in the ArcGIS modeling system that is beneficial to design measurement plans and to predict the transmission cycle in various regions of Khuzestan province, southwest of Iran. However, more than an 80% probability of P. papatasi was observed in rainy and temperate bio‐climatic zones with a high potential of CL transmission. Finding P. sergenti revealed the probability of transmission and distribution patterns of a non‐native vector of CL in related zones. These findings could be used as models indicating climatic zones and environmental variables connected to sand fly presence and vector distribution. Furthermore, this information is appropriate for future research efforts into the ecology of Phlebotomine sand flies and for the prevention of CL vector transmission as a public health priority.     

Malformations of the genitalia in male Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae)

Phlebotomus papatasi ( Scopoli, 1786 ) (Diptera: Psychodidae) is a major vector of Leishmania major (Kinetoplastida: Trypanosomatidae), a causative agent of zoonotic cutaneous leishmaniasis. Morphological characters of sand fly genitalia are key indicators for species identification. Various anomalies affecting male genitalia have been previously described. We take advantage of a large sand flies survey conducted in 32 stations in Central and Southern Morocco to systematically quantify the prevalence and spatial distribution of malformations affecting the genitalia of P. papatasi. Among 597 examined males, 122 were abnormal (20.4%). Malformations were widespread and largely concerned the number of spines in the lateral lobes and in the styles. Asymmetrical anomalies in lateral lobes were common. Correspondence analysis of our results highlighted the symmetrical anomalies observed in the lateral lobes, and abnormal styles of the male genitalia were found to be associated with environmental disturbances since they were prevalent in sewage dumps.     

Leishmania manipulation of sand fly feeding behavior results in enhanced transmission

In nature the prevalence of Leishmania infection in whole sand fly populations can be very low (<0.1%), even in areas of endemicity and high transmission. It has long since been assumed that the protozoan parasite Leishmania can manipulate the feeding behavior of its sand fly vector, thus enhancing transmission efficiency, but neither the way in which it does so nor the mechanisms behind such manipulation have been described. A key feature of parasite development in the sand fly gut is the secretion of a gel-like plug composed of filamentous proteophosphoglycan. Using both experimental and natural parasite-sand fly combinations we show that secretion of this gel is accompanied by differentiation of mammal-infective transmission stages. Further, Leishmania infection specifically causes an increase in vector biting persistence on mice (re-feeding after interruption) and also promotes feeding on multiple hosts. Both of these aspects of vector behavior were found to be finely tuned to the differentiation of parasite transmission stages in the sand fly gut. By experimentally accelerating the development rate of the parasites, we showed that Leishmania can optimize its transmission by inducing increased biting persistence only when infective stages are present. This crucial adaptive manipulation resulted in enhanced infection of experimental hosts. Thus, we demonstrate that behavioral manipulation of the infected vector provides a selective advantage to the parasite by significantly increasing transmission.     

The stage‐regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector,Phlebotomus papatasi

The stage‐regulated HASPB and SHERP proteins of Leishmania major are predominantly expressed in cultured metacyclic parasites that are competent for macrophage uptake and survival. The role of these proteins in parasite development in the sand fly vector has not been explored, however. Here, we confirm that expression of HASPB is detected only in vector metacyclic stages, correlating with the expression of metacyclic‐specific lipophosphoglycan and providing the first definitive protein marker for this infective sand fly stage. Similarly, SHERP is expressed in vector metacyclics but is also detected at low levels in the preceding short promastigote stage. Using genetically modified parasites lacking or complemented for the LmcDNA16 locus on chromosome 23 that contains the HASP and SHERP genes, we further show that the presence of this locus is essential for parasite differentiation to the metacyclic stage in Phlebotomus papatasi. While wild‐type and complemented parasites transform normally in late‐stage infections, generating metacyclic promastigotes and colonizing the sand fly stomodeal valve, null parasites accumulate at the earlier elongated nectomonad stage of development within the abdominal and thoracic midgut of the sand fly. Complementation with HASPB or SHERP alone suggests that HASPB is the dominant effector molecule in this process.     

The microsporidian pathogen Myrmecomorba nylanderiae: Intracolony transmission and impact upon tawny crazy ant (Nylanderia fulva) colonies

The microsporidian pathogen Myrmecomorba nylanderiae Plowes et al. infects introduced tawny crazy ants (Nylanderia fulva (Mayr)), and constitutes one of the first natural enemies known to attack this invasive ant. We assess how infection is transmitted within colonies and how infection impacts N. fulva colony fragment growth under carbohydrate‐deficient and carbohydrate‐sufficient dietary conditions. Carbohydrate scarcity is a common source of stress for ant colonies. Infected workers efficiently pass infection to developing larvae but all other potential pathways for within colony transmission are rare or non‐viable. For unknown reasons, queens within infected colony fragments are generally uninfected, limiting the role of transovarial transmission in intracolony transmission. In the laboratory, infection by M. nylanderiae primarily impacts the growth of N. fulva colonies by reducing pupal production. Colony growth showed a substantially greater impact under carbohydrate‐deficient conditions implying that the effect of the pathogen may depend on seasonally variable carbohydrate availability. In the colony growth assay, worker mortality did not differ with infection status under either nutrient regime. However, in a longer, direct test of survivorship, infected worker survivorship was significantly lower. Recently, some established N. fulva populations with high prevalence of M. nylanderiae infection have declined precipitously, though it is unknown if M. nylanderiae is a causative agent in these declines. The combination of chronic impacts, presence in North America, and potential association with population declines makes M. nylanderiae a promising prospect for the biological control of N. fulva.     

Abiotic factors affecting the infectivity of Steinernema carpocapsae (Rhabditida: Steinernematidae) on larvae of Anastrepha obliqua (Diptera: Tephritidae)

The effects of soil depth, soil type and temperature on the activity of the nematode Steinernema carpocapsae (Filipjev) were examined using larvae of the West Indian fruit fly, Anastrepha obliqua (Macquart). Bioassays involved applying infective juveniles (IJs) to the surface of sterilized sand in PVC tubes previously inoculated with fly larvae of two ages. The 50% lethal concentration (LC₅₀) values estimated for 6-day-old larvae were 9, 20 and 102 IJs/cm² in tubes containing 2, 5 and 8 cm depth of sand, respectively, whereas for 8-day-old larvae, LC₅₀ values were 16, 40 and 157 IJs/cm², respectively. The effect of soil texture on the activity of S. carpocapsae was tested by applying the corresponding LC₅₀ concentrations of nematodes to sand, sand–clay and loamy–sand soils. For 6-day-old larvae, soil type had a highly significant effect on infection with the highest percentages of infection observed in the sand–clay mixture (60–82% depending on depth) compared to 45–64% infection in sand and 23–30% infection in loamy–sand soil. A very similar pattern was observed in 8-day-old larvae except that infection rates were significantly lower than in younger larvae. There was a significant interaction between soil type and soil depth. The effect of three temperatures (19, 25 and 30°C) on infection was examined in sand–clay soil. The infectivity of S. carpocapsae was affected by temperature and soil depth and by the interaction of these two factors. Response surface analysis applied to second order multiple linear regression models indicated that the optimal temperature for infection of larvae of both ages was ~26°C, at a depth of 7.9 cm for 6-day-old larvae and <2 cm for 8-day-old larvae, resulting in a predicted 91.4% infection of 6-day-old larvae and 61.2% infection of 8-day-old larvae. These results suggest that S. carpocapsae may have the potential to control fruit fly pests in tropical ecosystems with warm temperatures and high soil moisture levels, although this assertion requires field testing.     

The protective effect against Leishmania infection conferred by sand fly bites is limited to short-term exposure

Under laboratory conditions, hosts exposed twice to sand fly saliva are protected against severe leishmaniasis. However, people in endemic areas are exposed to the vector over a long term and may experience sand fly-free periods. Therefore, we exposed mice long- or short-term to Phlebotomus duboscqi bites, followed by Leishmania major infection either immediately or after a sand fly-free period. We showed that protection against leishmaniasis is limited to short-term exposure to sand flies immediately before infection. Our results may explain the persistence of leishmaniasis in endemic areas and should be taken into account when designing anti-Leishmania vaccines based on sand fly saliva.     

Predation by ants controls swallow bug (Hemiptera: Cimicidae: Oeciacus vicarius) infestations

The swallow bug (Oeciacus vicarius) is the only known vector for Buggy Creek virus (BCRV), an alphavirus that circulates in cliff swallows (Petrochelidon pyrrhonota) and house sparrows (Passer domesticus) in North America. We discovered ants (Crematogaster lineolata and Formica spp.) preying on swallow bugs at cliff swallow colonies in western Nebraska, U.S.A. Ants reduced the numbers of visible bugs on active swallow nests by 74‐90%, relative to nests in the same colony without ants. Ant predation on bugs had no effect on the reproductive success of cliff swallows inhabiting the nests where ants foraged. Ants represent an effective and presumably benign way of controlling swallow bugs at nests in some colonies. They may constitute an alternative to insecticide use at sites where ecologists wish to remove the effects of swallow bugs on cliff swallows or house sparrows. By reducing bug numbers, ant presence may also lessen BCRV transmission at the spatial foci (bird colony sites) where epizootics occur. The effect of ants on swallow bugs should be accounted for in studying variation among sites in vector abundance.     

Infection parameters in the sand fly vector that predict transmission of Leishmania major

To identify parameters of Leishmania infection within a population of infected sand flies that reliably predict subsequent transmission to the mammalian host, we sampled groups of infected flies and compared infection intensity and degree of metacyclogenesis with the frequency of transmission. The percentage of parasites within the midgut that were metacyclic promastigotes had the highest correlation with the frequency of transmission. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic "cutoff" value that predicted transmission competence. Sand fly infections initiated with variable doses of parasites resulted in correspondingly altered percentages of metacyclic promastigotes, resulting in altered transmission frequency and disease severity. Lastly, alteration of sand fly oviposition status and environmental conditions at the time of transmission also influenced transmission frequency. These observations have implications for transmission of Leishmania by the sand fly vector in both the laboratory and in nature, including how the number of organisms acquired by the sand fly from an infection reservoir may influence the clinical outcome of infection following transmission by bite.     

The effects of the floral infection by a bacterial pathogen in a dioecious plant,Mallotus japonicus (Euphorbiaceae)

Among all the different organs of a plant, flowers might have one of the most dynamic microbial communities, since many microbes are transmitted during flowering by insects and pollen. However, little is known about how these microbes affect floral characteristics and plant reproduction. Among the microbes transmitted to flowers, pathogens may have highly negative effects on plants' fitness. In this study, we investigated whether a bacterial pathogen, Erwinia mallotivora, occurs on flowers of the host plant Mallotus japonicus, and whether the transmission of the pathogen to flowers can result in systemic infection and/or reduction of fruit production. The pathogen has been reported to infect through leaves, while its ecology on flowers is unknown. We first confirmed the presence of the pathogen on flowers, indicating possible transmission by visitors or pollen. Then, we showed that the bacteria can infect the plant through flowers by inoculating the pathogen to both male and female flowers. Interestingly, the symptoms on leaves appeared earlier on the female plants than on the males. Besides, the inoculation significantly decreased fruit set of the female plants. Our results suggest a higher cost of infection in a female than in a male once the pathogen infected flowers. Although the effects of pathogen infection to flowers have rarely investigated in wild plants, it would be an interesting topic for future study if such sexual differences in the infection cost can cause sexual conflict and intraspecific adaptation load.     

The development of <Emphasis Type="Italic">Trypanosoma brucei</Emphasis>within the tsetse fly midgut observed using green fluorescent trypanosomes

Background  

The protozoan pathogen, Trypanosoma brucei, undergoes complex cycles of differentiation and multiplication in its vector, the tsetse fly, genus Glossina. Flies are refractory to infection and resistance mechanisms operate at a number of levels and timepoints. Here we have used highly conspicuous green fluorescent trypanosomes to study the early events in establishment of infection in the fly midgut.     

Sand fly fauna (Diptera: Psychodidae) from the Goytacazes National Forest and surrounding areas of southeastern Brazil

Most studies of the sand fly fauna in southeastern Brazil are conducted in the peridomiciliary environment of leishmaniasis endemic regions. Therefore, to increase the knowledge about diversity and richness of sand fly conservation areas, we describe here the sand fly fauna from the National Forest of Goytacazes (NFG), state of Espírito Santo, Brazil, and its surroundings areas. We also used sand fly fauna records from eight conservations units within the state of Espírito Santo to understand the similarity and relationships among them. The sand flies were simultaneously collected from June, 2008 to May, 2009 in two different environments: a preserved environment represented by the NFG and a modified environment represented by a peridomicile. To establish the similarity among the conservation units, we used a method very similar to parsimony analysis of endemism. We collected 2,466 sand fly specimens belonging to 13 species. Pressatia choti and Nyssomyia intermedia were the most abundant sand fly species. Ny. intermedia is a known vector of Leishmania braziliensis and epidemiological surveillance must be conducted in the area. We discuss aspects regarding the diversity of sand flies as well as the risk of transmission of Leishmania parasites in the area. We also provide for the first time a hypothesis of similarity relationships among conservation units within the state of Espírito Santo.     

Canine antibody response to Phlebotomus perniciosus bites negatively correlates with the risk of Leishmania infantum transmission

Background

Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species.

Methodology/Principal Findings

Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins.

Conclusions

Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.     

The effect of the sugar metabolism on Leishmania infantum promastigotes inside the gut of Lutzomyia longipalpis: A sweet relationship?

It is well-known that Leishmania parasites can alter the behavior of the sand fly vector in order to increase their transmission potential. However, little is known about the contribution of the infecting host’s blood composition on subsequent sand fly infection and survival. This study focused on the host’s glucose metabolism and the insulin/insulin-like growth factor 1 (IGF-1) pathway as both metabolic processes are known to impact vector-parasite interactions of other protozoa and insect species. The focus of this study was inspired by the observation that the glycemic levels in the blood of infected Syrian golden hamsters inversely correlated to splenic and hepatic parasite burdens. To evaluate the biological impact of these findings on further transmission, Lutzomyia longipalpis sand flies were infected with blood that was artificially supplemented with different physiological concentrations of several monosaccharides, insulin or IGF-1. Normoglycemic levels resulted in transiently higher parasite loads and faster appearance of metacyclics, whereas higher carbohydrate and insulin/IGF-1 levels favored sand fly survival. Although the recorded effects were modest or transient of nature, these observations support the concept that the host blood biochemistry may affect Leishmania transmission and sand fly longevity.     

Short Report: Adult Aedes abundance and risk of dengue transmission

Dengue is transmitted mainly by the adult female Aedes aegypti mosquito. However, little is known about the impact of adult Aedes abundance on the risk of dengue transmission. Here we analysed nationally representative dengue case and vector surveillance data collected from Singapore, to determine the effect of adult Aedes abundance on the risk of dengue transmission. A case was an area with active dengue transmission as indicated by the presence of dengue cluster. A control was an area where no dengue cluster was reported. Using multivariate logistic regression, we analysed 88 cases and 602 controls and estimated the odds of dengue cluster formation at various adult Aedes abundance levels, estimated by the mean number of adult female Aedes per Gravitrap per week and categorised into Low, Moderate, High and Very High abundance level. We found that the risk of dengue cluster formation was positively associated with adult Ae. aegypti abundance. We observed a three to four-fold increase in the odds of dengue clusters forming in areas with High (AOR: 3.40, 95% CI: 2.09, 5.52) and Very High (AOR: 3.99, 95% CI: 2.46, 6.46) adult Aedes aegypti abundance level compared to those with low Ae. aegypti abundance level. Our study strengthens the evidence for the use of adult Aedes indices for dengue risk assessment and early warning for dengue outbreaks. Entomological indicators of adult Ae. aegypti could be used to anticipate and prioritize areas for dengue control.     

The vector ecology of introduced Culex quinquefasciatus populations,and implications for future risk of West Nile virus emergence in the Galápagos archipelago

Culex quinquefasciatus Say (Diptera: Culicidae), an important vector of West Nile virus (WNV) in the U.S.A., was first detected on the Galápagos Islands (Ecuador) in the 1980s. However, little is known of its ecology, distribution or capacity for arbovirus transmission in the Galápagos. We characterize details of lifecycle (including gonotrophic period), temporal abundance, spatial distribution, vector competence and host‐feeding behaviour. Culex quinquefasciatus was detected on five islands of the Galápagos during 2006–2011. A period of 7–14 days was required for egg–adult emergence; water salinity above 5 ppt was demonstrated to hinder larval development. Blood‐meal analysis indicated feeding on reptiles, birds and mammals. Assessment of WNV vector competency of Galápagos C. quinquefasciatus showed a median infectious dose of 7.41 log₁₀ plaque‐forming units per millilitre and evidence of vertical transmission (minimal filial infection rate of 3.7 per 1000 progeny). The distribution of C. quinquefasciatus across the archipelago could be limited by salt intolerance, and its abundance constrained by high temperatures. Feeding behaviour indicates potential to act as a bridge vector for transmission of pathogens across multiple taxa. Vertical transmission is a potential persistence mechanism for WNV on Galápagos. Together, our results can be used for epidemiological assessments of WNV and target vector control, should this pathogen reach the Galápagos Islands.