Uninfected Flies Research Articles

Laboratory Validation of the Sand Fly Fever Virus Antigen Assay.

Sandfly fever group viruses in the genus Phlebovirus (family Bunyaviridae) are widely distributed across the globe and are a cause of disease in military troops and indigenous peoples. We assessed the laboratory sensitivity and specificity of the Sand Fly Fever Virus Antigen Assay, a rapid dipstick assay designed to detect sandfly fever Naples virus (SFNV) and Toscana virus (TOSV) against a panel of phleboviruses. The assay detected SFNV and TOSV, as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian, Heartland, Rio Grande, or Rift Valley fever viruses. It did not produce false positive results in the presence of uninfected sand flies (Lutzomyia longipalpis) or Cache Valley virus, a distantly related bunyavirus. Results from this laboratory evaluation suggest that this assay may be used as a rapid field-deployable assay to detect sand flies infected with TOSV and SFNV, as well as an assortment of other phleboviruses.

Immunogenicity and Serological Cross-Reactivity of Saliva Proteins among Different Tsetse Species

Tsetse are vectors of pathogenic trypanosomes, agents of human and animal trypanosomiasis in Africa. Components of tsetse saliva (sialome) are introduced into the mammalian host bite site during the blood feeding process and are important for tsetse’s ability to feed efficiently, but can also influence disease transmission and serve as biomarkers for host exposure. We compared the sialome components from four tsetse species in two subgenera: subgenus Morsitans: Glossina morsitans morsitans (Gmm) and Glossina pallidipes (Gpd), and subgenus Palpalis: Glossina palpalis gambiensis (Gpg) and Glossina fuscipes fuscipes (Gff), and evaluated their immunogenicity and serological cross reactivity by an immunoblot approach utilizing antibodies from experimental mice challenged with uninfected flies. The protein and immune profiles of sialome components varied with fly species in the same subgenus displaying greater similarity and cross reactivity. Sera obtained from cattle from disease endemic areas of Africa displayed an immunogenicity profile reflective of tsetse species distribution. We analyzed the sialome fractions of Gmm by LC-MS/MS, and identified TAg5, Tsal1/Tsal2, and Sgp3 as major immunogenic proteins, and the 5'-nucleotidase family as well as four members of the Adenosine Deaminase Growth Factor (ADGF) family as the major non-immunogenic proteins. Within the ADGF family, we identified four closely related proteins (TSGF-1, TSGF-2, ADGF-3 and ADGF-4), all of which are expressed in tsetse salivary glands. We describe the tsetse species-specific expression profiles and genomic localization of these proteins. Using a passive-immunity approach, we evaluated the effects of rec-TSGF (TSGF-1 and TSGF-2) polyclonal antibodies on tsetse fitness parameters. Limited exposure of tsetse to mice with circulating anti-TSGF antibodies resulted in a slight detriment to their blood feeding ability as reflected by compromised digestion, lower weight gain and less total lipid reserves although these results were not statistically significant. Long-term exposure studies of tsetse flies to antibodies corresponding to the ADGF family of proteins are warranted to evaluate the role of this conserved family in fly biology.

Effect of Leishmania spp infection on the survival, life expectancy, fecundity and fertility of Lutzomyia longipalpis s.l. and Lutzomyia pseudolongipalpis

We evaluated the effects of Leishmania spp infection on several population parameters of Lutzomyia longipalpis sensu lato andLutzomyia pseudolongipalpis, vectors of visceral leishmaniasis in Venezuela, under experimental conditions during the first post-feeding period. Females of both species were allowed to feed and engorge on a suspension of fresh washed human red blood cells in foetal calf serum. These blood cells were either non-infected or infected with one of the fourLeishmania spp strains and were offered through a chicken skin membrane. The longevity, life expectancy and the fecundity of uninfected flies were similar in both species, but the fertility was significantly lower in uninfected Lu. longipalpis females. In all cases, the infection of Lu. longipalpis and Lu. pseudolongipalpis by the Leishmania strains resulted in significant detrimental effects, which exerted a fitness cost expressed by reduced survival and life expectancy, as well as decreased fertility and fecundity compared with the control groups. Nevertheless, differences in these parameters were observed between these vector species depending on whether they were infected with the autochthonous Venezuelan Leishmania infantum strain (NESA) or the Brazilian reference strain (PP75). The experimental data obtained agree with field data on the natural infection of these vector species and the significance of this scenario is discussed.

A sand fly salivary protein vaccine shows efficacy against vector-transmitted cutaneous leishmaniasis in nonhuman primates.

Currently, there are no commercially available human vaccines against leishmaniasis. In rodents, cellular immunity to salivary proteins of sand fly vectors is associated to protection against leishmaniasis, making them worthy targets for further exploration as vaccines. We demonstrate that nonhuman primates (NHP) exposed to Phlebotomus duboscqi uninfected sand fly bites or immunized with salivary protein PdSP15 are protected against cutaneous leishmaniasis initiated by infected bites. Uninfected sand fly-exposed and 7 of 10 PdSP15-immunized rhesus macaques displayed a significant reduction in disease and parasite burden compared to controls. Protection correlated to the early appearance of Leishmania-specific CD4(+)IFN-γ(+) lymphocytes, suggesting that immunity to saliva or PdSP15 augments the host immune response to the parasites while maintaining minimal pathology. Notably, the 30% unprotected PdSP15-immunized NHP developed neither immunity to PdSP15 nor an accelerated Leishmania-specific immunity. Sera and peripheral blood mononuclear cells from individuals naturally exposed to P. duboscqi bites recognized PdSP15, demonstrating its immunogenicity in humans. PdSP15 sequence and structure show no homology to mammalian proteins, further demonstrating its potential as a component of a vaccine for human leishmaniasis.

Physical and physiological costs of ectoparasitic mites on host flight endurance

1. Dispersal is essential for locating mates, new resources, and to escape unfavourable conditions. Parasitism can impact a host's ability to perform energetically demanding activities such as long‐distance flight, with important consequences for gene flow and meta‐population dynamics.2. Ectoparasites, in particular, can adversely affect host flight performance by diminishing flight aerodynamics and/or by inflicting physiological damage while feeding on host tissue.3. Experimental flight assays were conducted using two fruit fly‐mite systems:Drosophila nigrospiracula(Patterson andWheeler) – Macrocheles subbadius(Berlese) andD. hydei(Sturtevan) – M. muscaedomesticae(Scopoli). Flies that are burdened by mites are expected to exhibit lower flight endurance compared to uninfected flies.4. The results show that the presence of mites (attached) significantly decreased flight endurance by 57% and 78% compared to uninfectedD. nigrospiraculaandD. hydei, respectively. The physiological damage caused byM. subbadiuswas revealed through a 53% decline in flight time among previously infected flies (mites removed just prior to flight assay). Surprisingly, the presumably phoreticM. muscaedomesticaealso caused a 62% reduction in flight endurance among previously infectedD. hydei.5. These results suggest a strong deleterious effect of ectoparasitic mites on host flight performance, mediated by a reduction in flight aerodynamics and damage to host physiology. Adverse effects on host flight and/or dispersal may have broad implications for gene flow, population genetic structure, and local adaptation in both host and parasite meta‐populations.

Evidence for horizontal transfer of Wolbachia by a Drosophila mite

Mites are common ectoparasites of Drosophila and have been implicated in bacterial and mobile element invasion of Drosophila stocks. The obligate endobacterium, Wolbachia, has widespread effects on gene expression in their arthropod hosts and alters host reproduction to enhance its survival and propagation, often with deleterious effects in Drosophila hosts. To determine whether Wolbachia could be transferred between Drosophila melanogaster laboratory stocks by the mite Tyrophagus putrescentiae, mites were introduced to Wolbachia-infected Drosophila vials. These vials were kept adjacent to mite-free and Wolbachia-uninfected Drosophila stock vials. The Wolbachia infection statuses of the infected and uninfected flies were checked from generation 1 to 5. Results indicate that Wolbachia DNA could be amplified from mites infesting Wolbachia-infected fly stocks and infection in the previously uninfected stocks arose within generation 1 or 2, concomitant with invasion of mites from the Wolbachia-infected stock. A possible mechanism for the transfer of Wolbachia from flies to mites and vice versa, can be inferred from time-lapse photography of fly and mite interactions. We demonstrated that mites ingest Drosophila corpses, including Wolbachia-infected corpses, and Drosophila larva ingest mites, providing possible sources of Wolbachia infection and transfer. This research demonstrated that T. putrescentiae white mites can facilitate Wolbachia transfer between Drosophila stocks and that this may occur by ingestion of infected corpses. Mite-vectored Wolbachia transfer allows for rapid establishment of Wolbachia infection within a new population. This mode of Wolbachia introduction may be relevant in nature as well as in the laboratory, and could have a variety of biological consequences.

The complex contributions of genetics and nutrition to immunity in Drosophila melanogaster.

Both malnutrition and undernutrition can lead to compromised immune defense in a diversity of animals, and “nutritional immunology” has been suggested as a means of understanding immunity and determining strategies for fighting infection. The genetic basis for the effects of diet on immunity, however, has been largely unknown. In the present study, we have conducted genome-wide association mapping in Drosophila melanogaster to identify the genetic basis for individual variation in resistance, and for variation in immunological sensitivity to diet (genotype-by-environment interaction, or GxE). D. melanogaster were reared for several generations on either high-glucose or low-glucose diets and then infected with Providencia rettgeri, a natural bacterial pathogen of D. melanogaster. Systemic pathogen load was measured at the peak of infection intensity, and several indicators of nutritional status were taken from uninfected flies reared on each diet. We find that dietary glucose level significantly alters the quality of immune defense, with elevated dietary glucose resulting in higher pathogen loads. The quality of immune defense is genetically variable within the sampled population, and we find genetic variation for immunological sensitivity to dietary glucose (genotype-by-diet interaction). Immune defense was genetically correlated with indicators of metabolic status in flies reared on the high-glucose diet, and we identified multiple genes that explain variation in immune defense, including several that have not been previously implicated in immune response but which are confirmed to alter pathogen load after RNAi knockdown. Our findings emphasize the importance of dietary composition to immune defense and reveal genes outside the conventional “immune system” that can be important in determining susceptibility to infection. Functional variation in these genes is segregating in a natural population, providing the substrate for evolutionary response to pathogen pressure in the context of nutritional environment.

Phlebotomus papatasi exposure cross-protects mice against Leishmania major co-inoculated with Phlebotomus duboscqi salivary gland homogenate

Leishmania parasites are inoculated into host skin together with sand fly saliva and multiple exposures to uninfected sand fly bites protect mice against Leishmania infection. However, sand fly vectors differ in composition of the saliva and therefore the protection elicited by their salivary proteins was shown to be species-specific. On the other hand, the optimal vaccine based on sand fly salivary proteins should be based on conserved salivary proteins conferring cross-reactivity. In the present study we therefore focused on cross-protective properties of saliva from Phlebotomus papatasi and Phlebotomus duboscqi, the two natural vectors of Leishmania major. Two groups of mice exposed to bites of P. papatasi and two control, non-immunized groups were infected with L. major promastigotes along with either P. papatasi or P. duboscqi salivary gland homogenate. All mice were followed for the development of Leishmania lesions, parasite burdens, specific antibodies, and for production of NO, urea, or cytokines by peritoneal macrophages. Protection against Leishmania infection was observed not only in exposed mice challenged with homologous saliva but also in the group challenged with P. duboscqi saliva. Comparing both exposed groups, no significant differences were observed in parasite load, macrophage activity, or in the levels of anti-L. major and anti-P. papatasi/P. duboscqi antibodies. This is the first study showing cross-protection caused by salivary antigens of two Phlebotomus species. The cross-protective effect suggests that the anti-Leishmania vaccine based on P. papatasi salivary proteins might be applicable also in areas where L. major is transmitted by P. duboscqi.

The acetate switch of an intestinal pathogen disrupts host insulin signaling and lipid metabolism.

Vibrio cholerae is lethal to the model host Drosophila melanogaster through mechanisms not solely attributable to cholera toxin. To examine additional virulence determinants, we performed a genetic screen in V. cholerae-infected Drosophila and identified the two-component system CrbRS. CrbRS controls transcriptional activation of acetyl-CoA synthase-1 (ACS-1) and thus regulates the acetate switch, in which bacteria transition from excretion to assimilation of environmental acetate. The resultant loss of intestinal acetate leads to deactivation of host insulin signaling and lipid accumulation in enterocytes, resulting in host lethality. These metabolic effects are not observed upon infection with ΔcrbS or Δacs1 V. cholerae mutants. Additionally, uninfected flies lacking intestinal commensals, which supply short chain fatty acids (SCFAs) such as acetate, also exhibit altered insulin signaling and intestinal steatosis, which is reversed upon acetate supplementation. Thus, acetate consumption by V. cholerae alters host metabolism, and dietary acetate supplementation may ameliorate some sequelae of cholera.

Nitric oxide levels regulate the immune response of Drosophila melanogaster reference laboratory strains to bacterial infections.

Studies on the innate immune response against microbial infections in Drosophila melanogaster involve mutant strains and their reference strains that act as experimental controls. We used five standard D. melanogaster laboratory reference strains (Oregon R, w1118, Canton-S, Cinnabar Brown, and Yellow White [YW]) and investigated their response against two pathogenic bacteria (Photorhabdus luminescens and Enterococcus faecalis) and two nonpathogenic bacteria (Escherichia coli and Micrococcus luteus). We detected high sensitivity among YW flies to bacterial infections and increased bacterial growth compared to the other strains. We also found variation in the transcription of certain antimicrobial peptide genes among strains, with Oregon and YW infected flies showing the highest and lowest gene transcription levels in most cases. We show that Oregon and w1118 flies possess more circulating hemocytes and higher levels of phenoloxidase activity than the other strains upon infection with the nonpathogenic bacteria. We further observed reduced fat accumulation in YW flies infected with the pathogenic bacteria, which suggests a possible decline in physiological condition. Finally, we found that nitrite levels are significantly lower in infected and uninfected YW flies compared to w1118 flies and that nitric oxide synthase mutant flies in YW background are more susceptible to bacterial infection compared to mutants in w1118 background. Therefore, increased sensitivity of YW flies to bacterial infections can be partly attributed to lower levels of nitric oxide. Such studies will significantly contribute toward a better understanding of the genetic variation between D. melanogaster reference strains.

Characterization of the early inflammatory infiltrate at the feeding site of infected sand flies in mice protected from vector-transmitted Leishmania major by exposure to uninfected bites.

BackgroundMice exposed to sand fly saliva are protected against vector-transmitted Leishmania major. Although protection has been related to IFN- γ producing T cells, the early inflammatory response orchestrating this outcome has not been defined.Methodology/Principal findingsMice exposed to uninfected P. duboscqi bites and naïve mice were challenged with L. major-infected flies to characterize their early immune response at the bite site. Mostly, chemokine and cytokine transcript expression post-infected bites was amplified in exposed compared to naïve mice. In exposed mice, induced chemokines were mostly involved in leukocyte recruitment and T cell and NK cell activation; IL-4 was expressed at 6 h followed by IFN-γ and iNOS2 as well as IL-5 and IL-10 expression. In naïve animals, the transcript expression following Leishmania-infected sand fly bites was suppressed. Expression profiles translated to an earlier and significantly larger recruitment of leukocytes including neutrophils, macrophages, Gr+ monocytes, NK cells and CD4+ T cells to the bite site of exposed compared to naïve mice post-infected bites. Additionally, up to 48 hours post-infected bites the number of IFN-γ-producing CD4+T cells and NK cells arriving at the bite site was significantly higher in exposed compared to naïve mice. Thereafter, NK cells become cytolytic and persist at the bite site up to a week post-bite.Conclusion/SignificanceThe quiet environment induced by a Leishmania-infected sand fly bite in naïve mice was significantly altered in animals previously exposed to saliva of uninfected flies. We propose that the enhanced recruitment of Gr+ monocytes, NK cells and CD4 Th1 cells observed at the bite site of exposed mice creates an inhospitable environment that counters the establishment of L. major infection.

Wolbachia-induced paternal defect in Drosophila is likely by interaction with the juvenile hormone pathway

Wolbachia are endosymbionts that infect many insect species. They can manipulate the host's reproduction to increase their own maternal transmission. Cytoplasmic incompatibility (CI) is one such manipulation, which is expressed as embryonic lethality when Wolbachia-infected males mate with uninfected females. However, matings between males and females carrying the same Wolbachia strain result in viable progeny. The molecular mechanisms of CI are currently not clear. We have previously reported that the gene Juvenile hormone-inducible protein 26 (JhI-26) exhibited the highest upregulation in the 3rd instar larval testes of Drosophila melanogaster when infected by Wolbachia. This is reminiscent of an interaction between Wolbachia and juvenile hormone (JH) pathway in flies. Considering that Jhamt gene encodes JH acid methyltransferase, a key regulatory enzyme of JH biosynthesis, and that methoprene-tolerant (Met) has been regarded as the best JH receptor candidate, we first compared the expression of Jhamt and Met between Wolbachia-infected and uninfected fly testes to investigate whether Wolbachia infection influence the JH signaling pathway. We found that the expressions of Jhamt and Met were significantly increased in the presence of Wolbachia, suggesting an interaction of Wolbachia with the JH signaling pathway. Then, we found that overexpression of JhI-26 in Wolbachia-free transgenic male flies caused paternal-effect lethality that mimics the defects associated with CI. JhI-26 overexpressing males resulted in significantly decrease in hatch rate. Surprisingly, Wolbachia-infected females could rescue the egg hatch. In addition, we showed that overexpression of JhI-26 caused upregulation of the male accessory gland protein (Acp) gene CG10433, but not vice versa. This result suggests that JhI-26 may function at the upstream of CG10433. Likewise, overexpression of CG10433 also resulted in paternal-effect lethality. Both JhI-26 and CG10433 overexpressing males resulted in nuclear division defects in the early embryos. Finally, we found that Wolbachia-infected males decreased the propensity of the mated females to remating, a phenotype also caused by both JhI-26 and CG10433 overexpressing males. Taken together, our results provide a working hypothesis whereby Wolbachia induce paternal defects in Drosophila probably by interaction with the JH pathway via JH response genes JhI-26 and CG10433.

Behavioral decline and premature lethality upon pan-neuronal ferritin overexpression in Drosophila infected with a virulent form of Wolbachia.

Iron is required for organismal growth. Therefore, limiting iron availability may be a key part of the host’s innate immune response to various pathogens, for example, in Drosophila infected with Zygomycetes. One way the host can transiently reduce iron bioavailability is by ferritin overexpression. To study the effects of neuronal-specific ferritin overexpression on survival and neurodegeneration we generated flies simultaneously over-expressing transgenes for both ferritin subunits in all neurons. We used two independent recombinant chromosomes bearing UAS-Fer1HCH, UAS-Fer2LCH transgenes and obtained qualitatively different levels of late-onset behavioral and lifespan declines. We subsequently discovered that one parental strain had been infected with a virulent form of the bacterial endosymbiont Wolbachia, causing widespread neuronal apoptosis and premature death. This phenotype was exacerbated by ferritin overexpression and was curable by antibiotic treatment. Neuronal ferritin overexpression in uninfected flies did not cause evident neurodegeneration but resulted in a late-onset behavioral decline, as previously reported for ferritin overexpression in glia. The results suggest that ferritin overexpression in the central nervous system of flies is tolerated well in young individuals with adverse manifestations appearing only late in life or under unrelated pathophysiological conditions.

Infection withHaemoproteus iwaaffects vector movement in a hippoboscid fly—frigatebird system

Haemosporidian parasites, which require both a vertebrate and invertebrate host, are most commonly studied in the life stages occurring in the vertebrate. However, aspects of the vector's behaviour and biology can have profound effects on parasite dynamics. We explored the effects of a haemosporidian parasite, Haemoproteus iwa, on a hippoboscid fly vector, Olfersia spinifera. Olfersia spinifera is an obligate ectoparasite of the great frigatebird, Fregata minor, living among bird feathers for all of its adult life. This study examined the movements of O.spinifera between great frigatebird hosts. Movement, or host switching, was inferred by identifying host (frigatebird) microsatellite genotypes from fly bloodmeals that did not match the host from which the fly was collected. Such host switches were analysed using a logistic regression model, and the best-fit model included the H.iwa infection status of the fly and the bird host sex. Uninfected flies were more likely to have a bird genotype in their bloodmeal that was different from their current host's genotype (i.e. to have switched hosts) than infected flies. Flies collected from female birds were more likely to have switched hosts than those collected on males. Reduced movement of infected flies suggests that there may be a cost of parasitism for the fly. The effect of host sex is probably driven by differences in the sex ratio of bird hosts available to moving flies.

Comparative diagnostic and analytical performance of PCR and LAMP-based trypanosome detection methods estimated using pooled whole tsetse flies and midguts

Detection of trypanosomes that cause disease in human beings and livestock within their tsetse fly hosts is an essential component of vector and disease control programmes. Several molecular-based diagnostic tests have been developed for this purpose. Many of these tests, while sensitive, require analysis of trypanosome DNA extracted from single flies, or from pooled tsetse fly heads and amplified trypanosome DNA. In this study, we evaluated the relative analytical and diagnostic sensitivities of two PCR-based tests (ITS and TBR) and a Trypanozoon specific LAMP assay using pooled whole tsetse flies and midguts spiked with serially diluted procyclics of a laboratory strain of Trypanosoma brucei brucei (KETRI 3386). Test sensitivity was also evaluated using experimentally infected tsetse flies. The aim was to determine the most appropriate pooling strategy for whole tsetse and midguts. RIME-LAMP had the highest diagnostic sensitivity (100%) followed by TBR-PCR (95%) and ITS-PCR (50%) in detecting trypanosome DNA from pooled tsetse midguts. RIME-LAMP also had the best diagnostic specificity (75%) followed by ITS-PCR (68%) and TBR-PCR (50%). The relative detection limit determined by serial dilution of procyclics was below 10−6 (equivalent to 1parasite/ml). Using TBR-PCR, ITS-PCR and RIME-LAMP, it was possible to detect trypanosome DNA in single flies or in pools of 2, 3, 4, 5, 10, or 15 flies/midguts. The proportion of positive pools declined by up to 60% when testing pools of 15 whole flies as opposed to testing pools of 5–10 flies. Additionally, it was possible to detect DNA in a single infected tsetse fly in the background of 4, 9, or 14 uninfected tsetse flies. Averaged across pool sizes and tsetse species, RIME-LAMP detected the highest proportion of positive pools in spiked whole tsetse and midguts (86.6% and 87.2%) followed by TBR-PCR (78. 6% and 79.2%) and ITS-PCR (34.3% and 40.2%). There were no significant differences between the proportions of positive pools detected in whole flies and midguts. We conclude that pooling of whole tsetse/midguts is an effective strategy to reduce hands-on-time and hence has potential application in large scale xenomonitoring to generate epidemiological data for decision making. RIME-LAMP offers the best diagnostic sensitivity and specificity on pooled tsetse midguts, thus demonstrating its superior diagnostic performance when compared with TBR-PCR and ITS-PCR. Using pools of whole tsetse or midguts as source of DNA does not have any significant effect on test results and is more representative of the field conditions where the proportion of flies with infected midguts tends to be higher than flies with infected salivary glands. Therefore to save time and minimize costs, pooling of whole tsetse flies is recommended.

Wolbachia Elimination Induced Cytoplasmic Incompatibility, a Latent Tool For the Management of Uzi Fly, Exorista sorbillans (Wied.) (Diptera: Tachinidae)

The endosymbiotic bacteria Wolbachia that occurs commonly in arthropods, often manipulate host reproduction through cytoplasmic incompatibility (CI). The crosses between Wolbachia infected and uninfected uzi fly Exorista sorbillans populations (cured with antibiotic oxytetracycline) indicated strong CI, signifying their role in host reproduction. Significant reduction in brood hatch and increased sterility was observed.

Identifying salivary antigens of Phlebotomus argentipes by a 2DE approach

In the Indian subcontinent visceral leishmaniasis, also known as kala-azar, is caused by the protozoa Leishmania donovani and is transmitted to humans by the bite of infected female sand flies Phlebotomus argentipes in an anthroponotic cycle. Sand fly saliva is known to play an important role in host infection outcome after an infective bite. Immunogenicity of P. argentipes saliva has already been described. However, specific antigens that can contribute to these immunogenic properties are unknown. This work focuses on the identification of antigens present in P. argentipes saliva through the combination of two-dimensional electrophoresis (2DE) and Western blot (WB). Analysis of the salivary protein profile showed a gradual increase of the protein content in relation to the age of sand flies, reaching the complete salivary protein pattern at day five, which marked the minimum age for dissections. The 2DE revealed a reproducible protein profile that matched the classic monodimensional SDS-PAGE pattern (1DE). The resulting salivary proteomic map consisted of at least 30 spots located between 10 and 60kDa. According to their isoelectric points, spots were mostly distributed around pH ranges: 5–6 and 9–10. In the proteomic maps, the presence of isoforms or posttranslational modifications was also highlighted since several spots were identified as the same protein. Analysis by in silico prediction programs located several potential glycosylation and phosphorylation sites in the aminoacidic sequences. On the other hand, pooled sera of immunized hamsters through the bite of uninfected sand flies showed elevated anti-saliva IgG levels. These sera permitted the detection of 4 protein bands and at least 20 protein spots in 1DE and 2DE respectively, followed by WB. The antigens were identified by MALDI-TOF, MALDI-TOF/TOF and de novo sequencing as D7-related proteins, PpSP15-like proteins, antigen 5-related proteins, apyrases, and several proteins without assigned protein family. Absence of cross-reactivity between P. argentipes and Phlebotomus perniciosus saliva antibodies determined by ELISA and WB was highlighted in this study, confirming that specific salivary antigens from different sand fly vectors need to be sought when designing vector-borne vaccines and markers for vector exposure assays.

House flies delay fungal infection by fevering: at a cost

1. Many ectothermic species have evolved the ability to invoke a ‘behavioural fever’ when infected with a pathogen. The relative costs and benefits of this response, however, have rarely been quantified.2. The aim of this study was investigate the nature and consequences of behavioural fever in the house fly, Musca domestica L., in response to infection with a possible biocontrol agent, the fungal entomopathogen, Beauveria bassiana (Balsamo) Vuillemin.3. It was found that infected flies preferred higher temperatures and allocated more effort to thermoregulation than uninfected flies. Flies could not overcome infection but the altered thermal behaviour allowed infected flies to extend their survival and to lay more eggs relative to infected flies maintained under constant conditions. However, flies allowed to fever had lower egg viability suggesting a possible cost.4. Under the present experimental conditions, the putative costs and benefits fever balanced one another resulting in no net change in fitness. Fever did not, therefore, limit the control potential of the fungus. We discuss whether the costs and benefits of behavioural fever might differ in other ecological contexts.

Molecular and Immunogenic Properties of Apyrase SP01B and D7-Related SP04 Recombinant Salivary Proteins ofPhlebotomus perniciosusfrom Madrid, Spain

Sand fly salivary proteins are on the spotlight to become vaccine candidates against leishmaniasis and to markers of exposure to sand fly bites due to the host immune responses they elicit. Working with the whole salivary homogenate entails serious drawbacks such as the need for maintaining sand fly colonies and the laborious task of glands dissection. In order to overcome these difficulties, producing recombinant proteins of different vectors has become a major task. In this study, a cDNA library was constructed with the salivary glands of Phlebotomus perniciosus from Madrid, Spain, the most widespread vector of Leishmania infantum in the Mediterranean basin. Analysis of the cDNA sequences showed several polymorphisms among the previously described salivary transcripts. The apyrase SP01B and the D7-related protein SP04 were successfully cloned, expressed in Escherichia coli, and purified. Besides, recombinant proteins were recognized by sera of hamsters and mice previously immunized with saliva through the exposure to uninfected sand fly bites. These results suggest that these two recombinant proteins conserved their immunogenic properties after expression in a prokaryote system. Therefore, this work contributes to expand the knowledge of P. perniciosus saliva that would be eventually used for the development of tools for vector control programs.

Wolbachia Infection Decreased the Resistance of Drosophila to Lead

BackgroundThe heavy metal lead has been shown to be associated with a genotoxic risk. Drosophila melanogaster is a model organism commonly utilized in genetic toxicology testing. The endosymbionts — Wolbachia are now very common in both wild populations and laboratory stocks of Drosophila. Wolbachia may induce resistance to pathogenic viruses, filarial nematodes and Plasmodium in fruit fly and mosquito hosts. However the effect of Wolbachia infection on the resistance of their hosts to heavy metal is unknown.Methodology/Principal FindingsManipulating the lead content in the diet of Drosophila melanogaster, we found that lead consumption had no different effects on developmental time between Wolbachia-infected (Dmel wMel) and –uninfected (Dmel T) flies. While in Pb-contaminated medium, significantly reduced amount of pupae and adults of Dmel wMel were emerged, and Dmel wMel adults had significantly shorter longevity than that of Dmel T flies. Lead infusion in diet resulted in significantly decreased superoxide dismutase (SOD) activity in Dmel T flies (P<0.05), but not in Dmel wMel flies. Correspondingly, lead cultures induced a 10.8 fold increase in malonaldehyde (MDA) contents in Dmel T larvae (P<0.05). While in Dmel wMel larvae, it resulted in only a 1.3 fold increase. By quantitative RT-PCR, we showed that lead infused medium caused significantly increased expression level of relish and CecA2 genes in Dmel T flies (P<0.01). Lead cultures did not change dramatically the expression of these genes in Dmel wMel flies.Conclusions/SignificanceThese results suggest that Wolbachia infection decreased the resistance of Drosophila to lead likely by limiting the production of peroxides resulted from lead, thus being unable to activate the immunological pathway in the host to prevent them from lead damage. This represents a novel Wolbachia–host interaction and provides information that researchers working on Drosophila toxicology should take in consideration the presence of Wolbachia in the stocks they are analyzing.