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Maternally transmitted endosymbiotic bacteria that cause male killing (MK) have only been described from a few insects, but this may reflect challenges in their detection rather than a rarity of MK. Here we identify MKWolbachiain populations ofDrosophila pseudotakahashii, present at a low frequency (around 4%) in natural populations and previously undetected due to a different fixedWolbachiastrain in this species expressing a different reproductive manipulation, cytoplasmic incompatibility (CI). The MK phenotype was eliminated after tetracycline treatment that removedWolbachia. Molecular analyses indicated the MK phenotype to be expressed when a secondWolbachiastrain was present alongside the CIWolbachia. A genomic analysis highlightedWolbachiaregions diverged between the strains involving 17 genes and also identified theWolbachiaas representing an outgroup to a clade ofWolbachiainfectingmelanogaster-group species, includingwRi-like andwMel-like strains. Doubly infected males induced CI with uninfected females but not females singly infected with CI-causingWolbachia. The MK phenotype manifested at the larval stage and was transmitted maternally at a high fidelity but with occasional loss of the MKWolbachiastrain. A rapidly spreading dominant nuclear suppressor genetic element affecting MK was identified through backcrossing and subsequent analysis with ddRAD SNPs of theD. pseudotakahashiigenome. These findings highlight the complexity of nuclear and microbial components affecting MK endosymbiont detection and dynamics in populations, and the challenges of making connections between endosymbionts and the host phenotypes affected by them.

The Scaptodrosophila genus represents a large group of drosophilids with a worldwide distribution and a predominance of species in Australia, but there is little information on the presence and impacts of Wolbachia endosymbionts in this group. Here we describe the first Wolbachia infection from this group, wClay isolated from Scaptodrosophila claytoni (van Klinken), a species from the east coast of Australia. The infection is polymorphic in natural populations, occurring at a frequency of around 6%-10%. wClay causes male killing, producing female-biased lines; most lines showed 100% male killing, though in 1 line it was80%. The lines need to be maintained through the introduction of males unless the infection is removed by tetracycline treatment. wClay is transmitted at a high fidelity (98.6%) through the maternal lineage and has been stable in 2 laboratory lines across 24 generations, suggesting it is likely to persist in populations. The infection has not been previously described but is closely related to the male-killing Wolbachia recently described from Drosophila pandora based on multilocus sequence typing and the wsp gene. Male-killing Wolbachia are likely to be common in drosophilids but remain difficult to detect because the infections can often be at a low frequency.

The Drosophila montium species group is a clade of 94 named species, closely related to the model species D. melanogaster. The montium species group is distributed over a broad geographic range throughout Asia, Africa, and Australasia. Species of this group possess a wide range of morphologies, mating behaviors, and endosymbiont associations, making this clade useful for comparative analyses. We use genomic data from 42 available species to estimate the phylogeny and relative divergence times within the montium species group, and its relative divergence time from D. melanogaster. To assess the robustness of our phylogenetic inferences, we use 3 non-overlapping sets of 20 single-copy coding sequences and analyze all 60 genes with both Bayesian and maximum likelihood methods. Our analyses support monophyly of the group. Apart from the uncertain placement of a single species, D. baimaii, our analyses also support the monophyly of all seven subgroups proposed within the montium group. Our phylograms and relative chronograms provide a highly resolved species tree, with discordance restricted to estimates of relatively short branches deep in the tree. In contrast, age estimates for the montium crown group, relative to its divergence from D. melanogaster, depend critically on prior assumptions concerning variation in rates of molecular evolution across branches, and hence have not been reliably determined. We discuss methodological issues that limit phylogenetic resolution - even when complete genome sequences are available - as well as the utility of the current phylogeny for understanding the evolutionary and biogeographic history of this clade.

Wolbachia are being used to reduce dengue transmission by Aedes aegypti mosquitoes around the world. To date releases have mostly involved Wolbachia strains with limited fitness effects but strains with larger fitness costs could be used to suppress mosquito populations. However, such infections are expected to evolve towards decreased deleterious effects. Here we investigate potential evolutionary changes in the wMelPop infection transferred from Drosophila melanogaster to Aedes aegypti more than ten years (~120 generations) ago. We show that most deleterious effects of this infection have persisted despite strong selection to ameliorate them. The wMelPop-PGYP infection is difficult to maintain in laboratory colonies, likely due to the persistent deleterious effects coupled with occasional maternal transmission leakage. Furthermore, female mosquitoes can be scored incorrectly as infected due to transmission of Wolbachia through mating. Infection loss in colonies was not associated with evolutionary changes in the nuclear background. These findings suggest that Wolbachia transinfections with deleterious effects may have stable phenotypes which could ensure their long-term effectiveness if released in natural populations to reduce population size., Author summary Mosquitoes infected with Wolbachia bacteria are being deployed into the field where they can suppress mosquito populations and reduce dengue transmission. These programs rely on the use of Wolbachia strains that have desirable phenotypes, which can include deleterious fitness effects, reproductive manipulation and virus blocking. However, theory predicts that Wolbachia will evolve to become less costly to their hosts, reducing the effectiveness of these programs. We investigate the potential for evolutionary changes by performing a comprehensive phenotypic analysis of a deleterious Wolbachia strain, wMelPop-PGYP, that was introduced to Aedes aegypti mosquitoes from Drosophila over ten years ago. In contrast to theoretical expectations and research from Drosophila, our results suggest that Wolbachia strains with deleterious effects may have stable phenotypes, ensuring their long-term effectiveness if released into natural populations.

Aedes aegypti mosquitoes infected with the wMel strain of Wolbachia are being released into natural mosquito populations in the tropics as a way of reducing dengue transmission. High temperatures adversely affect wMel, reducing Wolbachia density and cytoplasmic incompatibility in some larval habitats that experience large temperature fluctuations. We monitored the impact of a 43.6°C heatwave on the wMel infection in a natural population in Cairns, Australia, where wMel was first released in 2011 and has persisted at a high frequency. Wolbachia infection frequencies in the month following the heatwave were reduced to 83% in larvae sampled directly from field habitats and 88% in eggs collected from ovitraps, but recovered to be near 100% four months later. Effects of the heatwave on wMel appeared to be stage-specific and delayed, with reduced frequencies and densities in field-collected larvae and adults reared from ovitraps but higher frequencies in field-collected adults. Laboratory experiments showed that the effects of heatwaves on cytoplasmic incompatibility and density are life stage-specific, with first instar larvae being the most vulnerable to temperature effects. Our results indicate that heatwaves in wMel-infected populations will have only temporary effects on Wolbachia frequencies and density once the infection has established in the population. Our results are relevant to ongoing releases of wMel-infected Ae. aegypti in several tropical countries., Author summary Mosquitoes infected with Wolbachia bacteria are being released in the tropics to replace natural mosquito populations and suppress dengue transmission. Aedes aegypti mosquitoes with the wMel strain of Wolbachia were first released in Cairns, Australia in 2011 and releases were then expanded to the entire city and surrounding suburbs. Today, wMel is at a high frequency within the Ae. aegypti population and local dengue transmission in Cairns has declined to nearly zero. Wolbachia infections are vulnerable to high temperatures and the ability of wMel to persist in populations and block dengue may be constrained by climate. Cairns experienced a record heatwave of 43.6°C in November 2018 and we wanted to see whether this affected the wMel-infected Ae. aegypti population. Our results show that the frequency and density of wMel declined after the heatwave, with effects depending on the mosquito life stage tested. When we monitored the population again in April 2019, wMel had returned to a high frequency. We suggest that heatwaves of the magnitude experienced in Cairns will not have long-term impacts on the wMel infection but may affect invasion during releases or interfere with dengue blockage. Heatwaves may affect interventions with wMel-infected Ae. aegypti that are being deployed in several countries. Effects may depend on the proportion of larval habitats that are protected from extreme temperature fluctuations.

Wolbachiaare being used to reduce dengue transmission byAedes aegyptimosquitoes around the world. To date releases have mostly involvedWolbachiastrains with limited fitness effects but strains with larger fitness costs could be used to suppress mosquito populations. However, such infections are expected to evolve towards decreased deleterious effects. Here we investigate potential evolutionary changes in thewMelPop infection transferred fromDrosophila melanogastertoAedes aegyptimore than ten years (~120 generations) ago. We show that most deleterious effects of this infection have persisted despite strong selection to ameliorate them. ThewMelPop infection is difficult to maintain in laboratory colonies, likely due to the persistent deleterious effects coupled with occasional maternal transmission leakage. Furthermore, female mosquitoes can be scored incorrectly as infected due to transmission ofWolbachiathrough mating. Infection loss in colonies was not associated with evolutionary changes in the nuclear background. These findings suggest thatWolbachiatransinfections with deleterious effects may have stable phenotypes which could ensure their long-term effectiveness if released in natural populations to reduce population size.Author summaryMosquitoes infected withWolbachiabacteria are being deployed into the field where they can suppress mosquito populations and reduce dengue transmission. These programs rely on the use ofWolbachiastrains that have desirable phenotypes, which can include deleterious fitness effects, reproductive manipulation and virus blocking. However, theory predicts thatWolbachiawill evolve to become less costly to their hosts, reducing the effectiveness of these programs. We investigate the potential for evolutionary changes by performing a comprehensive phenotypic analysis of a deleteriousWolbachiastrain,wMelPop, that was introduced toAedes aegyptimosquitoes fromDrosophilaover ten years ago. In contrast to theoretical expectations and research fromDrosophila, our results suggest thatWolbachiastrains with deleterious effects may have stable phenotypes, ensuring their long-term effectiveness if released into natural populations.

Wolbachia infections have been described in several Drosophila species, but relatively few have been assessed for phenotypic effects. Cytoplasmic incompatibility (CI) is the most common phenotypic effect that has been detected, while some infections cause male killing or feminization, and many Wolbachia infections have few host effects. Here, we describe two new infections in a recently described species, Drosophila pandora, one of which causes near-complete CI and near-perfect maternal transmission (the "CI" strain). The other infection is a male killer (the "MK" strain), which we confirm by observing reinitiation of male production following tetracycline treatment. No incompatibility was detected in crosses between CI strain males and MK strain females, and rare MK males do not cause CI. Molecular analyses indicate that the CI and MK infections are distantly related and the CI infection is closely related to the wRi infection of Drosophila simulans. Two population surveys indicate that all individuals are infected with Wolbachia, but the MK infection is uncommon. Given patterns of incompatibility among the strains, the infection dynamics is expected to be governed by the relative fitness of the females, suggesting that the CI infection should have a higher fitness. This was evidenced by changes in infection frequencies and sex ratios in population cages initiated at different starting frequencies of the infections.

Wolbachia bacteria are common insect endosymbionts transmitted maternally and capable of spreading through insect populations by cytoplasmic incompatibility (CI) when infected males cause embryo death after mating with uninfected females. Selection in the Wolbachia endosymbiont occurs on female hosts and is expected to favour strong maternal transmission to female offspring, even at the cost of reduced CI. With maternal leakage, nuclear genes are expected to be selected to suppress cytoplasmic incompatibility caused by males while also reducing any deleterious effects associated with the infection. Here we describe a new type of Wolbachia strain from Drosophila pseudotakahashii likely to have arisen from evolutionary processes on host and/or Wolbachia genomes. This strain is often absent from adult male offspring, but always transmitted to females. It leads to males with low or non-detectable Wolbachia that nevertheless show CI. When detected in adult males, the infection has a low density relative to that in females, a phenomenon not previously seen in Wolbachia infections of Drosophila. This Wolbachia strain is common in natural populations, and shows reduced CI when older (infected) males are crossed. These patterns highlight that endosymbionts can have strong sex-specific effects and that high frequency Wolbachia strains persist through effects on female reproduction. Female-limited Wolbachia infections may be of applied interest if the low level of Wolbachia in males reduces deleterious fitness effects on the host.

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

The wheat curl mite (WCM), Aceria tosichella Keifer (Trombidiformes: Eriophyidae), is a major pest in cropping regions of the world and is recognised as the primary vector of several yield-reducing pathogens, primarily affecting wheat. Management of WCM is complicated due to several aspects of the mite's biology and ecology; however, commercially viable mite resistant wheat varieties may offer practical long-term management options. Unfortunately, mite populations have adapted to previously identified sources of resistance, highlighting the need for further sources of resistance and the value of stacking different resistances to give greater degrees and longevity of control. In this study we assessed the susceptibility of 42 wheat-derived genotypes to mite population growth using a new experimental method that overcomes methodological limitations of previous studies. Experimental wheat lines included a variety of wheat genotypes, related Triticeae species, wheat-alien chromosome amphiploids, and chromosome addition or substitution lines. From these we identify new promising sources of WCM resistance associated with Thinopyrum intermedium, Th. ponticum and Hordeum marinum chromosomes. More specifically we identify group 1J and 5J chromosomes of the L3 and L5 wheat-Th. intermedium addition lines as new sources of resistance that could be exploited to transfer resistance onto homoeologous wheat chromosomes. This study offers new methods for reliable in situ estimations of mite abundance on cereal plants, and new sources of WCM resistance that may assist management of WCM and associated viruses in wheat.

In Australia, the dengue vector Aedes aegypti is presently restricted to Queensland but was historically more widespread. Future spread may be facilitated by changes in the availability of suitable ovipositing sites (artificial containers) in response to climate change. We undertook a replicated comparison of thermal and hydric conditions in a selection of water containers commonly used by Ae. aegypti under sun and shade conditions in a tropical (Cairns) and temperate (Melbourne) location. We assessed the implications of thermal and hydric regimes for development rates and thermal stress. Container type had no effect on potential development rate in Cairns but mosquitoes in tyres were predicted to have consistently slower development than those in other containers in Melbourne. Our dataset included the hottest day on record for Melbourne (46.4°C) yet few containers exhibited lethal water temperatures in this location. Similarly high water temperatures were reached in Cairns at more benign air temperatures due to high solar radiation loads. The tyres had unique thermal profiles that exhibited a plateau at shaded air temperature even when in full sun. Overall, our results suggest that chronic cold stress would prevent development in Melbourne during spring, drying of containers would be limiting in Melbourne in summer, and heat stress in unshaded small containers would be limiting in Cairns. Tyres could be an important and unappreciated buffered habitat in open areas in the tropics. These results are of value for directing water storage and waste policy to prevent the further spread of Ae. aegypti and dengue fever as well as other mosquitoes. The methodology can be applied to identify priority containers for surveillance in other parts of the world.

An understanding of physiological sensitivity to temperature and its variability is important for predicting habitat suitability for disease vectors under different climatic regimes. In this study, we characterized the thermal sensitivity of larval developmental rates and survival in several Australian mainland populations of the dengue virus vector Aedes aegypti. Males developed more rapidly than females, but there were no differences among populations for development time or survival despite previously demonstrated genetic differentiation for neutral markers. Optimal development and survival temperatures were 37 degrees C and 25 degrees C, respectively. The values for maximal development and survival were similar to standard functions used in the container inhabiting simulation (CIMSIM) model for predicting population dynamics ofAe. aegypti populations, but CIMSIM assumed a lower optimal temperature. Heat stress experiments indicated that larvae could withstand water temperatures up to 44 degrees C regardless of the rate at which temperature was increased. Results from development time measured under constant temperatures could predict development time under fluctuating conditions, whereas CIMSIM predicted faster rates of development. This difference acts to reduce the predicted potential number of generations of Ae. aegypti per year in Australia, although it does not influence its predicted distribution, which depends critically on the nature of the aquatic breeding sites.

Forty percent of the world's population is at risk of contracting dengue virus, which produces dengue fever with a potentially fatal hemorrhagic form. The wMelPop Wolbachia infection of Drosophila melanogaster reduces life span and interferes with viral transmission when introduced into the mosquito Aedes aegypti, the primary vector of dengue virus. Wolbachia has been proposed as an agent for preventing transmission of dengue virus. Population invasion by Wolbachia depends on levels of cytoplasmic incompatibility, fitness effects, and maternal transmission. Here we characterized these traits in an outbred genetic background of a potential target population of Ae. aegypti using two crossing schemes. Cytoplasmic incompatibility was strong in this background, and the maternal transmission rate of Wolbachia was high. The infection substantially reduced longevity of infected adult females, regardless of whether adults came from larvae cultured under high or low levels of nutrition or density. The infection reduced the viability of diapausing and nondiapausing eggs. Viability was particularly low when eggs were laid by older females and when diapausing eggs had been stored for a few weeks. The infection affected mosquito larval development time and adult body size under different larval nutrition levels and densities. The results were used to assess the potential for wMelPop-CLA to invade natural populations of Ae. aegypti and to develop recommendations for the maintenance of fitness in infected mosquitoes that need to compete against field insects.

Maternally transmitted Wolbachia, Spiroplasma, and Cardinium bacteria are common in insects [1], but their interspecific spread is poorly understood. Endosymbionts can spread rapidly within host species by manipulating host reproduction, as typified by theglobal spread of wRi Wolbachia observed in Drosophila simulans [2, 3]. However, because Wolbachia cannot survive outside host cells, spread between distantly related host species requires horizontal transfers that are presumably rare [4-7]. Here, we document spread of wRi-like Wolbachia among eight highly diverged Drosophila hosts (10-50 million years) over only about 14,000 years (5,000-27,000). Comparing 110 wRi-like genomes, we find ≤0.02% divergence from the wRi variant that spread rapidly through California populations of D.simulans. Thehosts include both globally invasive species (D.simulans, D.suzukii, and D.ananassae) andnarrowly distributed Australian endemics (D.anomalata and D.pandora) [8]. Phylogenetic analyses that include mtDNA genomes indicate introgressive transfer of wRi-like Wolbachia between closely related species D.ananassae, D.anomalata, and D.pandora but no horizontal transmission within species. Our analyses suggest D.ananassae as theWolbachia source for the recent wRi invasion ofD.simulans and D.suzukii as the source of Wolbachia in its sister species D.subpulchrella. Although six of these wRi-like variants cause strong cytoplasmic incompatibility, two cause no detectable reproductive effects, indicating that pervasive mutualistic effects [9, 10] complement the reproductive manipulations for which Wolbachia are bestknown. "Super spreader" variants like wRi may beparticularly useful for controlling insect pestsand vector-borne diseases with Wolbachia transinfections [11].

Influenza infections have high frequency and morbidity in HIV-infected pregnant women, underscoring the importance of vaccine-conferred protection. To identify the factors that determine vaccine immunogenicity in this group, we characterized the relationship of B- and T-cell responses to pandemic H1N1 (pH1N1) vaccine with HIV-associated immunologic and virologic characteristics. pH1N1 and seasonal-H1N1 (sH1N1) antibodies were measured in 119 HIV-infected pregnant women after two double-strength pH1N1 vaccine doses. pH1N1-IgG and IgA B-cell FluoroSpot, pH1N1- and sH1N1-interferon γ (IFNγ) and granzyme B (GrB) T-cell FluoroSpot, and flow cytometric characterization of B- and T-cell subsets were performed in 57 subjects. pH1N1-antibodies increased after vaccination, but less than previously described in healthy adults. pH1N1-IgG memory B cells (Bmem) increased, IFNγ-effector T-cells (Teff) decreased, and IgA Bmem and GrB Teff did not change. pH1N1-antibodies and Teff were significantly correlated with each other and with sH1N1-HAI and Teff, respectively, before and after vaccination. pH1N1-antibody responses to the vaccine significantly increased with high proportions of CD4+, low CD8+ and low CD8+HLADR+CD38+ activated (Tact) cells. pH1N1-IgG Bmem responses increased with high proportions of CD19+CD27+CD21- activated B cells (Bact), high CD8+CD39+ regulatory T cells (Treg), and low CD19+CD27-CD21- exhausted B cells (Bexhaust). IFNγ-Teff responses increased with low HIV plasma RNA, CD8+HLADR+CD38+ Tact, CD4+FoxP3+ Treg and CD19+IL10+ Breg. In conclusion, pre-existing antibody and Teff responses to sH1N1 were associated with increased responses to pH1N1 vaccination in HIV-infected pregnant women suggesting an important role for heterosubtypic immunologic memory. High CD4+% T cells were associated with increased, whereas high HIV replication, Tact and Bexhaust were associated with decreased vaccine immunogenicity. High Treg increased antibody responses but decreased Teff responses to the vaccine. The proportions of immature and transitional B cells did not affect the responses to vaccine. Increased Bact were associated with high Bmem responses to the vaccine.