Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.Centro Regional de Estudios Genómico

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

Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies

Zika virus infection modulates the bacterial diversity associated with <i>Aedes aegypti</i> as revealed by metagenomic analysis

<div><p>Zika is a re-emerging infection that has been considered a major threat to global public health. Currently at least 100 countries are at risk of Zika virus (ZIKV) transmission. <i>Aedes aegypti</i> is the main mosquito vector in the Americas. This vector is exposed to, and interacts symbiotically with a variety of microorganisms in its environment, which may result in the formation of a lifetime association. Here, the unknown effect that ZIKV exerts on the dynamic bacterial community harbored by this mosquito vector was investigated using a metagenomic analysis of its microbiota. Groups of <i>Ae</i>. <i>aegypti</i> were experimentally fed on sugar, blood and blood mixed with ZIKV, and held for 3 to 7 days after blood meal and eggs development respectively. The infected groups were processed by qPCR to confirm the presence of ZIKV. All groups were analyzed by metagenomics (Illumina Hiseq Sequencing) and 16S rRNA amplicon sequences were obtained to create bacterial taxonomic profiles. A core microbiota and exclusive bacterial taxa were identified that incorporate 50.5% of the predicted reads from the dataset, with 40 Gram-negative and 9 Gram-positive families. To address how ZIKV invasion may disturb the ecological balance of the <i>Ae</i>. <i>aegypti</i> microbiota, a CCA analysis coupled with an explanatory matrix was performed to support the biological interpretation of shifts in bacterial signatures. Two f-OTUs appeared as potential biomarkers of ZIKV infection: <i>Rhodobacteraceae</i> and <i>Desulfuromonadaceae</i>. Coincidentally, both f-OTUs were exclusively present in the ZIKV- infected blood-fed and ZIKV- infected gravid groups. In conclusion, this study shows that bacterial symbionts act as biomarkers of the insect physiological states and how they respond as a community when ZIKV invades <i>Ae</i>. <i>aegypti</i>. Basic knowledge of local haematophagous vectors and their associated microbiota is relevant when addressing transmission of vector-borne infectious diseases in their regional surroundings.</p></div

Exclusive bacterial components associated with the <i>Ae</i>. <i>aegypti</i> group.

<p>The circular plot depicts the relative abundance of the exclusive bacterial taxa that were identified per experimental <i>Ae</i>. <i>aegypti</i> group: sugar-fed (blue), blood-fed (red), ZIKV-infected blood-fed (maroon), Gravid (purple), and ZIKV-infected gravid (lilac). The names of the bacterial f-OTUs and experimental groups are indicated next to their ribbons on the perimeter of the circle. The width of the ribbon and the position of the f-OTUs from the bottom to the top (on each segment) reflect the abundance of each bacterial taxon and their presence in each group.</p

Relative abundance of the core bacterial taxa associated with laboratory-reared adult <i>Ae</i>. <i>aegypti</i> mosquitoes.

<p>The circular plot depicts the abundance of the core bacterial taxa identified by crossing the indices of the f-OTUs per group for the experimental <i>Ae</i>. <i>aegypti</i> groups: sugar-fed (blue); blood-fed (red); ZIKV-infected blood-fed (maroon); Gravid (purple); and ZIKV-infected gravid (lilac). The bacterial taxa and experimental groups are indicated next to their ribbons on the perimeter of the circle. The width of the ribbon and the position of the f-OTUs from the bottom to the top (on each segment) reflect the abundance of the bacterial taxa and their presence within each group.</p

Raw abundance of the exclusive bacterial taxa present in the studied groups of adult mosquitoes.

<p>The raw abundance of the arbitrarily filtered f-OTUs that appeared to be present exclusively in the studied groups is shown as stacked bars revealing how community diversity is altered in the experimental groups. Each condition is represented by a color as indicated.</p

CCA triplot with explanatory biological effectors.

<p>The distribution of the experimental groups along the axes, and their orientation regarding the arrow vectors suggest that exposure to blood (red and maroon) and ZIKV infection (maroon and lilac) had an impact on the signature profiles of the bacterial community. The CCA plot, along its two axes (CCA1 = 31.15% and CCA2 = 24.25%), could explain 55.40% of the matrix variance. Experimental groups: sugar-fed (blue), blood-fed (red), ZIKV-infected blood-fed (maroon), Gravid (violet), and ZIKV-infected gravid (lilac).</p

Raw abundance of the core bacterial communities per <i>Ae</i>. <i>aegypti</i> group.

<p>The presence of arbitrarily selected core f-OTUs per group is shown as stacked bars to explore the dynamic shifts exhibited within each experimental group, which is represented by a color as indicated in the figure.</p