The vector-borne disease leishmaniasis, caused by Leishmania species protozoa, is transmitted to humans by phlebotomine sand flies. Development of Leishmania to infective metacyclic promastigotes in the insect gut, a process termed metacyclogenesis, is an essential prerequisite for transmission. Based on the hypothesis that vector gut microbiota influence the development of virulent parasites, we sequenced midgut microbiomes in the sand fly Lutzomyia longipalpis with or without Leishmania infantum infection. Sucrose-fed sand flies contained a highly diverse, stable midgut microbiome. Blood feeding caused a decrease in microbial richness that eventually recovered. However, bacterial richness progressively decreased in L. infantum-infected sand flies. Acetobacteraceae spp. became dominant and numbers of Pseudomonadaceae spp. diminished coordinately as the parasite underwent metacyclogenesis and parasite numbers increased. Importantly, antibiotic-mediated perturbation of the midgut microbiome rendered sand flies unable to support parasite growth and metacyclogenesis. Together, these data suggest that the sand fly midgut microbiome is a critical factor for Leishmania growth and differentiation to its infective state prior to disease transmission.

As part of the response to autochthonous Zika transmission in the United States, the City of South Miami implemented a 6-mo period in which Wolbachia-infected WB1 Aedes aegypti (L.) males were released into an ~170-acre area. Intracellular Wolbachia bacteria infections in Ae. aegypti cause early embryonic arrest (known as cytoplasmic incompatibility [CI]) and egg hatch failure, and inundative introductions have been suggested as a potential control tool. Throughout the release period, the Ae. aegypti population was monitored within both the release area and an equivalent area that did not receive WB1 male releases. The results show a significant reduction in egg hatch at the area receiving WB1 males, which is consistent with expectations for CI. Similarly, the number of Ae. aegypti was significantly reduced at the area receiving WB1 males, relative to the untreated area. The observed population reduction and results encourage additional work and replication of the Wolbachia biopesticide approach against Ae. aegypti, as an additional tool to be integrated with existing control tools for the control of this medically important vector and nuisance pest.

Human lungs are constantly exposed to bacteria in the environment, yet the prevailing dogma is that healthy lungs are sterile. DNA sequencing-based studies of pulmonary bacterial diversity challenge this notion. However, DNA-based microbial analysis currently fails to distinguish between DNA from live bacteria and that from bacteria that have been killed by lung immune mechanisms, potentially causing overestimation of bacterial abundance and diversity. We investigated whether bacterial DNA recovered from lungs represents live or dead bacteria in bronchoalveolar lavage (BAL) fluid and lung samples in young healthy pigs. Live bacterial DNA was DNase I resistant and became DNase I sensitive upon human antimicrobial-mediated killing in vitro. We determined live and total bacterial DNA loads in porcine BAL fluid and lung tissue by comparing DNase I-treated versus untreated samples. In contrast to the case for BAL fluid, we were unable to culture bacteria from most lung homogenates. Surprisingly, total bacterial DNA was abundant in both BAL fluid and lung homogenates. In BAL fluid, 63% was DNase I sensitive. In 6 out of 11 lung homogenates, all bacterial DNA was DNase I sensitive, suggesting a predominance of dead bacteria; in the remaining homogenates, 94% was DNase I sensitive, and bacterial diversity determined by 16S rRNA gene sequencing was similar in DNase I-treated and untreated samples. Healthy pig lungs are mostly sterile yet contain abundant DNase I-sensitive DNA from inhaled and aspirated bacteria killed by pulmonary host defense mechanisms. This approach and conceptual framework will improve analysis of the lung microbiome in disease.

BackgroundThe Leishmania spp. protozoa are introduced into humans through a sand fly blood meal, depositing the infectious metacyclic promastigote form of the parasite into human skin. Parasites enter a variety of host cells, although a majority are found in macrophages where they replicate intracellularly during chronic leishmaniasis. Symptomatic leishmaniasis causes considerable human morbidity in endemic regions. The Leishmania spp. evade host microbicidal mechanisms partially through virulence-associated proteins such as the major surface protease (MSP or GP63), to inactivate immune factors in the host environment. MSP is a metalloprotease encoded by a tandem array of genes belonging to three msp gene classes, whose mRNAs are differentially expressed in different life stages of the parasite. Like other cells, Leishmania spp. release small membrane-bound vesicles called exosomes into their environment. The purpose of this study was to detect MSP proteins in exosomal vesicles of Leishmania spp. protozoa.MethodsUsing mass spectrometry data we determined the profile of MSP class proteins released in L. infantum exosomes derived from promastigotes in their avirulent procyclic (logarithmic) stage and virulent stationary and metacyclic stages. MSP protein isoforms belonging to each of the three msp gene classes could be identified by unique peptides.ResultsMetacyclic promastigote exosomes contained the highest, and logarithmic exosomes had the lowest abundance of total MSP. Among the MSP classes, MSPC class had the greatest variety of isoforms, but was least abundant in all exosomes. Nonetheless, all MSP classes were present at higher levels in exosomes released from stationary or metacyclic promastigotes than logarithmic promastigotes.ConclusionsThe data suggest the efficiency of exosome release may be more important than the identity of MSP isoform in determining the MSP content of Leishmania spp. exosomes.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2937-y) contains supplementary material, which is available to authorized users.

The ecological interactions parasitic insects have with their hosts may contribute to their prodigious diversity, which is unrivaled among animals. Many insects assumed to be polyphagous generalists have been shown to consist of several differentiated races, each occupying a different host-niche. The sunflower maggot fly, Strauzia longipennis, has long been thought to consist of two or more races due to its substantial intraspecific morphological variation. Here, we use nuclear and mitochondrial markers to test the hypothesis that S. longipennis is a complex of two or more partially reproductively isolated races. We collected S. longipennis flies as pupae from roots of Jerusalem artichoke (Helianthus tuberosus) and as adults swept from leaves of mature H. tuberosus across the breadth of a field season. Flies were scored for morphological variety (typica or vittigera), mitochondrial haplotype (A or B) and a panel of 176 AFLP loci. Bayesian clustering and neighbor-joining phylogenetic analyses of AFLP data supported the existence of at least three, possibly four, genetic races of Strauzia (clusters I, II, III, and V), as well as a small number of putative interracial hybrids (cluster IV). Clusters I and III each consisted of flies of both morphological varieties and both haplotype groups, while flies in cluster II were all of variety typica and all but one was of mitochondrial haplotype B. Flies in cluster II were also collected only as adults on H. tuberosus and not among flies reared from pupae collected from H. tuberosus roots, suggesting that they use a different plant as their larval host. Mean capture date was significantly different between flies of each genetic race, indicating that partial allochronic isolation may be one contemporary barrier to gene flow between races. Evidence that mitochondrial genomes and morphological traits have moved between lineages implies a model of speciation-with-gene-flow for S. longipennis races.