Identifying the Role of Elevation, Geography, and Species Identity in Structuring Turtle Ant (Cephalotes Latreille, 1802) Bacterial Communities.

Bacterial communities in animals are often necessary for hosts to survive, particularly for hosts with nutrient-limited diets. The composition, abundance, and richness of these bacterial communities may be shaped by host identity and external ecological factors. The turtle ants (genus Cephalotes) are predominantly herbivorous and known to rely on bacterial communities to enrich their diet. Cephalotes have a broad Neotropical distribution, with high diversity in the South American Cerrado, a geologically and biologically diverse savanna. Using 16S rRNA amplicon sequencing, we examined the bacterial communities of forty-one Cephalotes samples of sixteen different species collected from multiple locations across two sites in the Cerrado (MG, Brazil) and compared the bacterial communities according to elevation, locality, species, and species group, defined by host phylogeny. Beta diversity of bacterial communities differed with respect to all categories but particularly strongly when compared by geographic location, species, and species group. Differences seen in species and species groups can be partially explained by the high abundance of Mesorhizobium in Cephalotes pusillus and Cephalotes depressus species groups, when compared to other clades via the Analysis of Composition of Microbiome (ANCOM). Though the Cephalotes bacterial community is highly conserved, results from this study indicate that multiple external factors can affect and change bacterial community composition and abundance.

The Diversity of Wolbachia across the Turtle Ants (Formicidae: Cephalotes spp.).

Wolbachia is a widespread and well-known bacterium that can induce a wide range of changes within its host. Ants specifically harbor a great deal of Wolbachia diversity and are useful systems to study endosymbiosis. The turtle ants (Cephalotes) are a widespread group of tropical ants that rely on gut microbes to support their herbivorous diet for their survival, yet little is known of the extent of this diversity. Therefore, studying their endosymbionts and categorizing the diversity of bacteria within Cephalotes hosts could help to delimit species and identify new strains and can help lead to a further understanding of how the microbiome leads to survival and speciation in the wild. In our study, 116 individual samples were initially tested for positive infection with the wsp gene. Of the initial 116 samples, 9 samples were infected with only one strain of Wolbachia, and 7 were able to be used successfully for multilocus sequence typing (MLST). We used the new MLST data to infer a phylogeny with other Formicidae samples from the MLST online database to identify new Wolbachia strains and related genes, of which only one came back as an exact match. The 18 Wolbachia-positive samples ranged across 15 different species and 7 different countries, which we further test for species identity and geographic correlation. This study is the first comprehensive look into the diversity of Wolbachia in the turtle ants, providing insight into how endosymbionts are oriented in widespread species and providing a strong foundation for further research in host-microbe interactions.

Isotopic evidence that aestivation allows malaria mosquitoes to persist through the dry season in the Sahel.

Data suggest that the malaria vector mosquito Anopheles coluzzii persists during the dry season in the Sahel through a dormancy mechanism known as aestivation; however, the contribution of aestivation compared with alternative strategies such as migration is unknown. Here we marked larval Anopheles mosquitoes in two Sahelian villages in Mali using deuterium (2H) to assess the contribution of aestivation to persistence of mosquitoes through the seven-month dry season. After an initial enrichment period, 33% of An. coluzzii mosquitoes were strongly marked. Seven months following enrichment, multiple analysis methods supported the ongoing presence of marked mosquitoes, compatible with the prediction that the fraction of marked mosquitoes should remain stable throughout the dry season if local aestivation is occurring. The results suggest that aestivation is a major persistence mechanism of An. coluzzii in the Sahel, contributing at least 20% of the adults at the onset of rains. This persistence strategy could influence mosquito control and malaria elimination campaigns.

A novel fluorescence and DNA combination for versatile, long-term marking of mosquitoes.

Current mark-release-recapture methodologies are limited in their ability to address complex problems in vector biology, such as studying multiple groups overlapping in space and time. Additionally, limited mark retention, reduced post-marking survival and the large effort in marking, collection and recapture all complicate effective insect tracking.We have developed and evaluated a marking method using a fluorescent dye (SmartWater®) combined with synthetic DNA tags to informatively and efficiently mark adult mosquitoes using an airbrush pump and nebulizer. Using a handheld UV flashlight, the fluorescent marking enabled quick and simple initial detection of recaptures in a field-ready and non-destructive approach that when combined with an extraction-free PCR on individual mosquito legs provides potentially unlimited marking information.This marking, first tested in the laboratory with Anopheles gambiae s.l. mosquitoes, did not affect survival (median ages 24-28 days, p-adj > 0.25), oviposition (median eggs/female of 28.8, 32.5, 33.3 for water, green, red dyes, respectively, p-adj > 0.44) or Plasmodium competence (mean oocysts 5.56-10.6, p-adj > 0.95). DNA and fluorescence had 100% retention up to 3 weeks (longest time point tested) with high intensity, indicating marks would persist longer.We describe a novel, simple, no/low-impact and long-lasting marking method that allows separation of multiple insect subpopulations by combining unlimited length and sequence variation in the synthetic DNA tags. This method can be readily deployed in the field for marking multiple groups of mosquitoes or other insects.

Induction of long-lived potential aestivation states in laboratory An. gambiae mosquitoes.

BACKGROUND: How anopheline mosquitoes persist through the long dry season in Africa remains a gap in our understanding of these malaria vectors. To span this period in locations such as the Sahelian zone of Mali, mosquitoes must either migrate to areas of permanent water, recolonize areas as they again become favorable, or survive in harsh conditions including high temperatures, low humidity, and an absence of surface water (required for breeding). Adult mosquitoes surviving through this season must dramatically extend their typical lifespan (averaging 2-3 weeks) to 7 months. Previous work has found evidence that the malaria mosquito An. coluzzii, survives over 200 days in the wild between rainy seasons in a presumed state of aestivation (hibernation), but this state has so far not been replicated in laboratory conditions. The inability to recapitulate aestivation in the lab hinders addressing key questions such as how this state is induced, how it affects malaria vector competence, and its impact on disease transmission. METHODS: In effort to induce aestivation, we held laboratory mosquitoes in climate-controlled incubators with a range of conditions that adjusted humidity (40-85% RH), temperature (18-27 °C), and light conditions (8-12 h of light) and evaluated their survivorship. These conditions were chosen to mimic the late rainy and dry seasons as well as relevant extremes these mosquitoes may experience during aestivation. RESULTS: We found that by priming mosquitoes in conditions simulating the late wet season in Mali, and maintaining mosquitoes in reduced light/temperature, mean mosquito survival increased from 18.34 ± 0.65 to 48.02 ± 2.87 days, median survival increased from 19 (95% CI 17-21) to 50 days (95% CI 40-58), and the maximum longevity increased from 38 to 109 days (P-adj < 0.001). While this increase falls short of the 200 + day survival seen in field mosquitoes, this extension is substantially higher than previously found through environmental or dietary modulation and is hard to reconcile with states other than aestivation. This finding will provide a platform for future characterization of this state, and allow for comparison to field collected samples.