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.

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 mosquitoAn. 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.

Premise: There is a general lack of consensus on the best practices for filtering of single‐nucleotide polymorphisms (SNPs) and whether it is better to use SNPs or include flanking regions (full “locus”) in phylogenomic analyses and subsequent comparative methods.Methods: Using genotyping‐by‐sequencing data from 22 Glycine species, we assessed the effects of SNP vs. locus usage and SNP retention stringency. We compared branch length, node support, and divergence time estimation across 16 datasets with varying amounts of missing data and total size.Results: Our results revealed five aspects of phylogenomic data usage that may be generally applicable: (1) tree topology is largely congruent across analyses; (2) filtering strictly for SNP retention (e.g., 90–100%) reduces support and can alter some inferred relationships; (3) absolute branch lengths vary by two orders of magnitude between SNP and locus datasets; (4) data type and branch length variation have little effect on divergence time estimation; and (5) phylograms alter the estimation of ancestral states and rates of morphological evolution.Discussion: Using SNP or locus datasets does not alter phylogenetic inference significantly, unless researchers want or need to use absolute branch lengths. We recommend against using excessive filtering thresholds for SNP retention to reduce the risk of producing inconsistent topologies and generating low support.

Among insects, Wolbachia is an exceedingly common bacterial endosymbiont with a range of consequences of infection. Despite the frequency of Wolbachia infection, very little is known about this bacteria’s diversity and role within hosts, especially within ant hosts. In this study, we analyze the occurrence and diversity of Wolbachia across the spiny ants (Polyrhachis), a large and geographically diverse genus. Polyrhachis samples from throughout the host genus’ phylogenetic and biogeographical range were first screened for single infections of Wolbachia using the wsp gene and Sanger sequencing. The multilocus sequence typing (MLST) scheme was then used on these singly infected samples to identify the Wolbachia strains. A Wolbachia phylogeny was inferred from the Polyrhachis samples analyzed in this study as well as other Formicidae MLST profiles from the MLST online database. We hypothesized that three key host factors were impacting Wolbachia diversity within the Polyrhachis genus: biogeography, phylogeny, and species level. The results suggest that the phylogeny and biogeography of Polyrhachis hosts have no impact on Wolbachia diversity; however, species level may have some limited influence. Additionally, Wolbachia strains appear to group according to being either Old World or New World strains. Among the taxa able to form complete MLST allelic profiles, all twenty are seemingly new strains.