De novo sequencing, diploid assembly, and annotation of the black carpenter ant, Camponotus pennsylvanicus, and its symbionts by one person for $1000, using nanopore sequencing.

The black carpenter ant (Camponotus pennsylvanicus) is a pest species found widely throughout North America. From a single individual I used long-read nanopore sequencing to assemble a phased diploid genome of 306 Mb and 60X coverage, with quality assessed by a 97.0% BUSCO score, improving upon other ant assemblies. The mitochondrial genome reveals minor rearrangements from other ants. The reads also allowed assembly of parasitic and symbiont genomes. I include a complete Wolbachia bacterial assembly with a size of 1.2 Mb, as well as a commensal symbiont Blochmannia pennsylvanicus, at 791 kb. DNA methylation and hydroxymethylation were measured at base-pair resolution level from the same reads and confirmed extremely low levels seen in the Formicidae family. There was moderate heterozygosity, with 0.16% of bases being biallelic from the parental haplotypes. Protein prediction yielded 14 415 amino acid sequences with 95.8% BUSCO score and 86% matching to previously known proteins. All assemblies were derived from a single MinION flow cell generating 20 Gb of sequence for a cost of $1047 including consumable reagents. Adding fixed costs for equipment brings the total for an ant-sized genome to less than $5000. All analyses were performed in 1 week on a single desktop computer.

Creating reference animal genomes is typically a large, expensive process. Here I sequenced the genome of the black carpenter ant for only $1000 as a sole researcher in just one week. Along with the nuclear genome, I assembled the mitochondrial genome and two commensal bacteria species living within the ant. Nanopore technology also enabled epigenetic measurements from the same ant and replicated other studies showing very low DNA methylation. The reference genome compared favorably to other ant species in continuity and protein prediction accuracy. This method will allow other low-resource labs to create high quality genome assemblies with a low cost.

A metapopulation approach to identify targets for Wolbachia-based dengue control.

Over the last decade, the release of Wolbachia-infected Aedes aegypti into the natural habitat of this mosquito species has become the most sustainable and long-lasting technique to prevent and control vector-borne diseases, such as dengue, zika, or chikungunya. However, the limited resources to generate such mosquitoes and their effective distribution in large areas dominated by the Aedes aegypti vector represent a challenge for policymakers. Here, we introduce a mathematical framework for the spread of dengue in which competition between wild and Wolbachia-infected mosquitoes, the cross-contagion patterns between humans and vectors, the heterogeneous distribution of the human population in different areas, and the mobility flows between them are combined. Our framework allows us to identify the most effective areas for the release of Wolbachia-infected mosquitoes to achieve a large decrease in the global dengue prevalence.

Assessment of community support for Wolbachia-mediated population suppression as a control method for Aedes aegypti mosquitoes in a community cohort in Puerto Rico.

Arboviral diseases transmitted by Aedes species mosquitoes pose an increasing public health challenge in tropical regions. Wolbachia-mediated population suppression (Wolbachia suppression) is a vector control method used to reduce Aedes mosquito populations by introducing male mosquitoes infected with Wolbachia, a naturally occurring endosymbiotic bacterium. When Wolbachia-infected male mosquitoes mate with female wild mosquitoes, the resulting eggs will not hatch. Public support is vital to the successful implementation and sustainability of vector control interventions. Communities Organized to Prevent Arboviruses (COPA) is a cohort study to determine the incidence of arboviral disease in Ponce, Puerto Rico and evaluate vector control methods. Focus groups were conducted with residents of COPA communities to gather their opinion on vector control methods; during 2018-2019, adult COPA participants were interviewed regarding their views on Wolbachia suppression; and a follow-up questionnaire was conducted among a subset of participants and non-participants residing in COPA communities. We analyzed factors associated with support for this method. Among 1,528 participants in the baseline survey, median age was 37 years and 63% were female. A total of 1,032 (68%) respondents supported Wolbachia suppression. Respondents with an income of $40,000 or more were 1.34 times as likely [95% CI: 1.03, 1.37] to support Wolbachia suppression than those who earned less than $40,000 annually. Respondents who reported repellant use were 1.19 times as likely to support Wolbachia suppression [95% CI: 1.03, 1.37]. A follow-up survey in 2020 showed that most COPA participants (86%) and non-participants living in COPA communities (84%) supported Wolbachia suppression during and after an educational campaign. The most frequent questions regarding this method were related to its impact on human and animal health, and the environment. Continuous community engagement and education efforts before and during the implementation of novel vector control interventions are necessary to increase and maintain community support.

Modelling the Wolbachia incompatible insect technique: strategies for effective mosquito population elimination.

BACKGROUND: The Wolbachia incompatible insect technique (IIT) shows promise as a method for eliminating populations of invasive mosquitoes such as Aedes aegypti (Linnaeus) (Diptera: Culicidae) and reducing the incidence of vector-borne diseases such as dengue, chikungunya and Zika. Successful implementation of this biological control strategy relies on high-fidelity separation of male from female insects in mass production systems for inundative release into landscapes. Processes for sex-separating mosquitoes are typically error-prone and laborious, and IIT programmes run the risk of releasing Wolbachia-infected females and replacing wild mosquito populations. RESULTS: We introduce a simple Markov population process model for studying mosquito populations subjected to a Wolbachia-IIT programme which exhibit an unstable equilibrium threshold. The model is used to study, in silico, scenarios that are likely to yield a successful elimination result. Our results suggest that elimination is best achieved by releasing males at rates that adapt to the ever-decreasing wild population, thus reducing the risk of releasing Wolbachia-infected females while reducing costs. CONCLUSIONS: While very high-fidelity sex separation is required to avoid establishment, release programmes tend to be robust to the release of a small number of Wolbachia-infected females. These findings will inform and enhance the next generation of Wolbachia-IIT population control strategies that are already showing great promise in field trials.

The economic impact and cost-effectiveness of combined vector-control and dengue vaccination strategies in Thailand: results from a dynamic transmission model.

BACKGROUND AND AIMS: Dengue fever is a major public health problem in tropical/subtropical regions. Prior economic analyses have predominantly evaluated either vaccination or vector-control programmes in isolation and do not really consider the incremental benefits and cost-effectiveness of mixed strategies and combination control. We estimated the cost-effectiveness of single and combined approaches in Thailand. METHODS: The impacts of different control interventions were analysed using a previously published mathematical model of dengue epidemiology and control incorporating seasonality, age structure, consecutive infection, cross protection, immune enhancement and combined vector-host transmission. An economic model was applied to simulation results to estimate the cost-effectiveness of 4 interventions and their various combinations (6 strategies): i) routine vaccination of 1-year olds; ii) chemical vector control strategies targeting adult and larval stages separately; iii) environmental management/ public health education and awareness [EM/ PHEA]). Payer and societal perspectives were considered. The health burden of dengue fever was assessed using disability-adjusted life-years (DALYs) lost. Costs and effects were assessed for 10 years. Costs were discounted at 3% annually and updated to 2013 United States Dollars. Incremental cost-effectiveness analysis was carried out after strategies were rank-ordered by cost, with results presented in a table of incremental analysis. Sensitivity and scenario analyses were undertaken; and the impact and cost-effectiveness of Wolbachia was evaluated in exploratory scenario analyses. RESULTS: From the payer and societal perspectives, 2 combination strategies were considered optimal, as all other control strategies were dominated. Vaccination plus adulticide plus EM/ PHEA was deemed cost-effective according to multiple cost-effectiveness criteria. From the societal perspective, incremental differences vs. adulticide and EM/ PHEA resulted in costs of $157.6 million and DALYs lost of 12,599, giving an expected ICER of $12,508 per DALY averted. Exploratory scenario analyses showed Wolbachia to be highly cost-effective ($343 per DALY averted) vs. other single control measures. CONCLUSIONS: Our model shows that individual interventions can be cost-effective, but that important epidemiological reductions and economic impacts are demonstrated when interventions are combined as part of an integrated approach to combating dengue fever. Exploratory scenario analyses demonstrated the potential epidemiological and cost-effective impact of Wolbachia when deployed at scale on a nationwide basis. Our findings were robust in the face of sensitivity analyses.

A Fly on the Cave Wall: Parasite Genetics Reveal Fine-scale Dispersal Patterns of Bats.

Dispersal influences the evolution and adaptation of organisms, but it can be difficult to detect. Host-specific parasites provide information about the dispersal of their hosts and may be valuable for examining host dispersal that does not result in gene flow or that has low signals of gene flow. We examined the population connectivity of the buffy flower bat, Erophylla sezekorni (Chiroptera: Phyllostomidae), and its associated obligate ectoparasite, Trichobius frequens (Diptera: Streblidae), across a narrow oceanic channel in The Bahamas that has previously been implicated as a barrier to dispersal in bats. Due to the horizontal transmission of T. frequens, we were able to test the hypothesis that bats are dispersing across this channel, but this dispersal does not result in gene flow, occurs rarely, or started occurring recently. We developed novel microsatellite markers for the family Streblidae in combination with previously developed markers for bats to genotype individuals from 4 islands in The Bahamas. We provide evidence for a single population of the host, E. sezekorni, but 2 populations of its bat flies, potentially indicating a recent reduction of gene flow in E. sezekorni, rare dispersal, or infrequent transportation of bat flies with their hosts. Despite high population differentiation in bat flies indicated by microsatellites, mitochondrial DNA shows no polymorphism, suggesting that bacterial reproductive parasites may be contributing to mitochondrial DNA sweeps. Parasites, including bat flies, provide independent information about their hosts and can be used to test hypotheses of host dispersal that may be difficult to assess using host genetics alone.

The first draft genomes of the ant Formica exsecta, and its Wolbachia endosymbiont reveal extensive gene transfer from endosymbiont to host.

BACKGROUND: Adapting to changes in the environment is the foundation of species survival, and is usually thought to be a gradual process. However, transposable elements (TEs), epigenetic modifications, and/or genetic material acquired from other organisms by means of horizontal gene transfer (HGTs), can also lead to novel adaptive traits. Social insects form dense societies, which attract and maintain extra- and intracellular accessory inhabitants, which may facilitate gene transfer between species. The wood ant Formica exsecta (Formicidae; Hymenoptera), is a common ant species throughout the Palearctic region. The species is a well-established model for studies of ecological characteristics and evolutionary conflict. RESULTS: In this study, we sequenced and assembled draft genomes for F. exsecta and its endosymbiont Wolbachia. The F. exsecta draft genome is 277.7 Mb long; we identify 13,767 protein coding genes, for which we provide gene ontology and protein domain annotations. This is also the first report of a Wolbachia genome from ants, and provides insights into the phylogenetic position of this endosymbiont. We also identified multiple horizontal gene transfer events (HGTs) from Wolbachia to F. exsecta. Some of these HGTs have also occurred in parallel in multiple other insect genomes, highlighting the extent of HGTs in eukaryotes. CONCLUSION: We present the first draft genome of ant F. exsecta, and its endosymbiont Wolbachia (wFex), and show considerable rates of gene transfer from the symbiont to the host. We expect that especially the F. exsecta genome will be valuable resource in further exploration of the molecular basis of the evolution of social organization.

[New strategies for the eradication of Aedes aegypti: what challenges do we face in Latin America?]

Not available.

wPip Wolbachia contribution to Aedes albopictus SIT performance: Advantages under intensive rearing.

As a part of a project aiming at the suppression of the mosquito vector Aedes albopictus, a specific Ae. albopictus line producing sterile males, ARwP, was tested for its suitability to intense rearing conditions compatible with mass production and field release. This line was developed by the Italian National Agency for New Technologies, Energy and Sustainable Economic Development thanks to the artificial infection with a heterologous Wolbachia strain, resulting in a bidirectional incompatibility pattern with wild-type Ae. albopictus. ARwP was reared under Standard Operating Procedures at the Centro Agricoltura Ambiente and compared with a wild-type strain in terms of time of pupation onset, production of male pupae in the following 24h and mechanical sexing efficacy. Mating competitiveness of ARwP males was also evaluated in comparison with irradiated wild-type males in large field enclosures. ARwP males demonstrated a significantly shorter time of pupation onset, a higher rate of production of male pupae in the following 24h and a lower percentage of residual contaminant females when applying mechanical sexing procedures. In addition, ARwP males were more efficient than wild-types in competing for wild-type females in large enclosures, thus inducing a level of sterility significantly higher than that expected for an equal mating competitiveness. These results encourage the use of this Ae. albopictus strain as suppression tool against Ae. albopictus based on considerations thoroughly discussed in the manuscript.

Influence of oxidative homeostasis on bacterial density and cost of infection in Drosophila-Wolbachia symbioses.

The evolution of symbioses along the continuum between parasitism and mutualism can be influenced by the oxidative homeostasis, that is the balance between reactive oxygen species (ROS) and antioxidant molecules. Indeed, ROS can contribute to the host immune defence to regulate symbiont populations, but are also toxic. This interplay between ROS and symbiosis is notably exemplified by recent results in arthropod-Wolbachia interactions. Wolbachia are symbiotic bacteria involved in a wide range of interactions with their arthropods hosts, from facultative, parasitic associations to obligatory, mutualistic ones. In this study, we used Drosophila-Wolbachia associations to determine whether the oxidative homeostasis plays a role in explaining the differences between phenotypically distinct arthropod-Wolbachia symbioses. We used Drosophila lines with different Wolbachia infections and measured the effects of pro-oxidant (paraquat) and antioxidant (glutathione) treatments on the Wolbachia density and the host survival. We show that experimental manipulations of the oxidative homeostasis can reduce the cost of the infection through its effect on Wolbachia density. We discuss the implication of this result from an evolutionary perspective and argue that the oxidative homeostasis could underlie the evolution of tolerance and dependence on Wolbachia.

The untapped cell biology of neglected tropical diseases.

The World Health Organization lists a constellation of 17 tropical diseases that afflict approximately one in six individuals on the planet and, until recently, few resources have been devoted to the treatment and eradication of those diseases. They are often referred to as the diseases of the "bottom billion," because they are most prevalent among the poorest individuals in impoverished tropical nations. However, the few studies that have been performed reveal an extraordinary world of molecular and cellular adaptations that facilitate the pathogens' survival in hosts ranging from insects to humans. A compelling case can be made that even a modest investment toward understanding the basic molecular and cell biology of these neglected pathogens has a high probability of yielding exciting new cellular mechanisms and insights into novel ways of combating these diseases.

Costs of Three Wolbachia Infections on the Survival of Aedes aegypti Larvae under Starvation Conditions.

The mosquito Aedes aegypti, the principal vector of dengue virus, has recently been infected experimentally with Wolbachia: intracellular bacteria that possess potential as dengue biological control agents. Wolbachia depend on their hosts for nutrients they are unable to synthesize themselves. Consequently, competition between Wolbachia and their host for resources could reduce host fitness under the competitive conditions commonly experienced by larvae of Ae. aegypti in the field, hampering the invasion of Wolbachia into natural mosquito populations. We assess the survival and development of Ae. aegypti larvae under starvation conditions when infected with each of three experimentally-generated Wolbachia strains: wMel, wMelPop and wAlbB, and compare their fitness to wild-type uninfected larvae. We find that all three Wolbachia infections reduce the survival of larvae relative to those that are uninfected, and the severity of the effect is concordant with previously characterized fitness costs to other life stages. We also investigate the ability of larvae to recover from extended food deprivation and find no effect of Wolbachia on this trait. Aedes aegypti larvae of all infection types were able to resume their development after one month of no food, pupate rapidly, emerge at a large size, and exhibit complete cytoplasmic incompatibility and maternal transmission. A lowered ability of Wolbachia-infected larvae to survive under starvation conditions will increase the threshold infection frequency required for Wolbachia to establish in highly competitive natural Ae. aegypti populations and will also reduce the speed of invasion. This study also provides insights into survival strategies of larvae when developing in stressful environments.

The microbiome of Folsomia candida: an assessment of bacterial diversity in a Wolbachia-containing animal.

The springtail Folsomia candida is an important model organism for soil ecology, ecotoxicology and ecogenomics. The decomposer activities of soil invertebrates like Folsomia depend on their relationship with microbial communities including gut symbionts. In this paper, we apply high-throughput sequencing to provide a detailed characterization of the bacterial community associated with parthenogenetic F. candida. First, we evaluated a method to suppress the amplification of DNA from the endosymbiont Wolbachia, to prevent it from interfering with the identification of less abundant operational taxonomic units (OTUs). The suppression treatment applied was effective against Wolbachia and did not interfere with the detection of the most abundant OTUs (59 OTUs, contributing over 87% of the reads). However, this method did affect the inferred community composition. Significant differences were subsequently observed in the composition of bacterial communities associated with two different strains of F. candida. A total of 832 OTUs were found, of which 45% were only present in one strain and 17% only in the other. Among the 20 most abundant OTUs, 16 were shared between strains. Denaturing gradient gel electrophoresis and clone libraries, although unable to capture the full diversity of the bacterial community, provided results that supported the NGS data.

Bad guys turned nice? A critical assessment of Wolbachia mutualisms in arthropod hosts.

Wolbachia are the most abundant bacterial endosymbionts among arthropods. Although maternally inherited, they do not conform to the widespread view that vertical transmission inevitably selects for beneficial symbionts. Instead, Wolbachia are notorious for their reproductive parasitism which, although lowering host fitness, ensures their spread. However, even for reproductive parasites it can pay to enhance host fitness. Indeed, there is a recent upsurge of reports on Wolbachia-associated fitness benefits. Therefore, the question arises how such instances of mutualism are related to the phenotypes of reproductive parasitism. Here, we review the evidence of Wolbachia mutualisms in arthropods, including both facultative and obligate relationships, and critically assess their biological relevance. Although many studies report anti-pathogenic effects of Wolbachia, few actually prove these effects to be relevant to field conditions. We further show that Wolbachia frequently have beneficial and detrimental effects at the same time, and that reproductive manipulations and obligate mutualisms may share common mechanisms. These findings undermine the idea of a clear-cut distinction between Wolbachia mutualism and parasitism. In general, both facultative and obligate mutualisms can have a strong, and sometimes unforeseen, impact on the ecology and evolution of Wolbachia and their arthropod hosts. Acknowledging this mutualistic potential might be the key to a better understanding of some unresolved issues in the study of Wolbachia-host interactions.

Wolbachia strain wAlbB confers both fitness costs and benefit on Anopheles stephensi.

BACKGROUND: Wolbachia is a maternally transmitted intracellular bacterium that is estimated to infect up to 65% of insect species, but it is not naturally present in Anopheles malaria vectors. Wolbachia-based strategies for malaria vector control can be developed either through population replacement to reduce vectorial capacity or through population suppression to reduce the mosquito population. We have previously generated An. stephensi mosquitoes carrying a stable wAlbB Wolbachia infection and have demonstrated their ability to invade wild-type laboratory populations and confer resistance to Plasmodium on these populations. METHODS: We assessed wAlbB-associated fitness by comparing the female fecundity, immature development and survivorship, body size, male mating competiveness, and adult longevity of the infected An. stephensi to that of wild-type mosquitoes. RESULTS: We found that wAlbB reduced female fecundity and caused a minor decrease in male mating competiveness. We also observed that wAlbB increased the life span of both male and female mosquitoes when they were maintained solely on sugar meals; however, there was no impact on the life span of blood-fed females. In addition, wAlbB did not influence either immature development and survivorship or adult body sizes. CONCLUSIONS: These results provide significant support for developing Wolbachia-based strategies for malaria vector control.

Endosymbiont costs and benefits in a parasitoid infected with both Wolbachia and Cardinium.

Theory suggests that maternally inherited endosymbionts can promote their spread and persistence in host populations by enhancing the production of daughters by infected hosts, either by improving overall host fitness, or through reproductive manipulation. In the doubly infected parasitoid wasp Encarsia inaron, Wolbachia manipulates host reproduction through cytoplasmic incompatibility (CI), but Cardinium does not. We investigated the fitness costs and/or benefits of infection by each bacterium in differentially cured E. inaron as a potential explanation for persistence of Cardinium in this population. We introgressed lines infected with Wolbachia, Cardinium or both with the cured line to create a similar genetic background, and evaluated several parasitoid fitness parameters. We found that symbiont infection resulted in both fitness costs and benefits for E. inaron. The cost was lower initial egg load for all infected wasps. The benefit was increased survivorship, which in turn increased male production for wasps infected with only Cardinium. Female production was unaffected by symbiont infection; we therefore have not yet identified a causal fitness effect that can explain the persistence of Cardinium in the population. Interestingly, the Cardinium survivorship benefit was not evident when Wolbachia was also present in the host, and the reproduction of doubly infected individuals did not differ significantly from uninfected wasps. Therefore, the results of our study show that even when multiple infections seem to have no effect on a host, there may be a complex interaction of costs and benefits among symbionts.

High Wolbachia density correlates with cost of infection for insecticide resistant Culex pipiens mosquitoes.

In the mosquito Culex pipiens, insecticide resistance genes alter many life-history traits and incur a fitness cost. Resistance to organophosphate insecticides involves two loci, with each locus coding for a different mechanism of resistance (degradation vs. insensitivity to insecticides). The density of intracellular Wolbachia bacteria has been found to be higher in resistant mosquitoes, regardless of the mechanism involved. To discriminate between costs of resistance due to resistance genes from those associated with elevated Wolbachia densities, we compared strains of mosquito sharing the same genetic background but differing in their resistance alleles and Wolbachia infection status. Life-history traits measured included strength of insecticide resistance, larval mortality, adult female size, fecundity, predation avoidance, mating competition, and strength of cytoplasmic incompatibility (CI). We found that: (1) when Wolbachia are removed, insecticide resistance genes still affect some life-history traits; (2) Wolbachia are capable of modifying the cost of resistance; (3) the cost of Wolbachia infections increases with their density; (4) different interactions occurred depending on the resistance alleles involved; and (5) high densities of Wolbachia do not increase the strength of CI or maternal transmission efficiency relative to low Wolbachia densities. Insecticide resistance genes generated variation in the costs of Wolbachia infections and provided an interesting opportunity to study how these costs evolve, a process generally operating when Wolbachia colonizes a new host.