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Background: Wolbachia incompatible insect technique (IIT) programs have been shown in field trials to be highly effective in suppressing populations of mosquitoes that carry diseases such as dengue, chikungunya, and Zika. However, the frequent and repeated release of Wolbachia-infected male mosquitoes makes such programs resource-intensive. While the need for optimization is recognized, potential strategies to optimize releases and reduce resource utilization have not been fully explored., Results: We developed a process-based model to study the spatio-temporal metapopulation dynamics of mosquitoes in a Wolbachia IIT program, which explicitly incorporates climatic influence in mosquito life-history traits. We then used the model to simulate various scale-down and redistribution strategies to optimize the existing program in Singapore. Specifically, the model was used to study the trade-offs between the intervention efficacy outcomes and resource requirements of various release program strategies, such as the total number of release events and the number of mosquitoes released. We found that scaling down releases in existing sites from twice a week to only once a week yielded small changes in suppression efficacy (from 87 to 80%), while requiring 44% fewer mosquitoes and release events. Additionally, redistributing mosquitoes from already suppressed areas and releasing them in new areas once a week led to a greater total suppressive efficacy (83% compared to 61%) while also yielding a 16% and 14% reduction in the number of mosquitoes and release events required, respectively., Conclusions: Both scale-down and redistribution strategies can be implemented to significantly reduce program resource requirements without compromising the suppressive efficacy of IIT. These findings will inform planners on ways to optimize existing and future IIT programs, potentially allowing for the wider adoption of this method for mosquito-borne disease control., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: We would like to declare that the primary author, Preston Lim, is an employee of Verily Life Sciences, which produces Wolbachia mosquitoes for mosquito control programs. However, he is pursuing this research in the capacity of his PhD studentship with the National University of Singapore. The opinions expressed in this article are those of the individual authors, and do not reflect those of the primary author’s employer (Verily Life Sciences LLC)., (© 2024. The Author(s).)

This paper is devoted to the study of optimal release strategies to control vector-borne diseases, such as dengue, Zika, chikungunya and malaria. Two techniques are considered: the sterile insect one (SIT), which consists in releasing sterilized males among wild vectors in order to perturb their reproduction, and the Wolbachia one (presently used mainly for mosquitoes), which consists in releasing vectors, that are infected with a bacterium limiting their vectorial capacity, in order to replace the wild population by one with reduced vectorial capacity. In each case, the time dynamics of the vector population is modeled by a system of ordinary differential equations in which the releases are represented by linear combinations of Dirac measures with positive coefficients determining their intensity. We introduce optimal control problems that we solve numerically using ad-hoc algorithms, based on writing first-order optimality conditions characterizing the best combination of Dirac measures. We then discuss the results obtained, focusing in particular on the complexity and efficiency of optimal controls and comparing the strategies obtained. Mathematical modeling can help testing a great number of scenarios that are potentially interesting in future interventions (even those that are orthogonal to the present strategies) but that would be hard, costly or even impossible to test in the field in present conditions., Competing Interests: Declarations Conflict of interest The authors have no conflict of interest to declare that are relevant to the content of this article., (© 2024. The Author(s).)

Wolbachia pipientis (= Wolbachia) has promise as a tool to suppress virus transmission by Aedes aegypti mosquitoes. However, Wolbachia can have variable effects on mosquito-borne viruses. This variation remains poorly characterized, yet the multimodal effects of Wolbachia on diverse pathogens could have important implications for public health. Here, we examine the effects of transient somatic infection with two strains of Wolbachia (wAlbB and wMel) on the alphaviruses Sindbis virus (SINV), O'nyong-nyong virus (ONNV), and Mayaro virus (MAYV) in Ae. aegypti. We found variable effects of Wolbachia including enhancement and suppression of viral infections, with some effects depending on Wolbachia strain. Both wAlbB- and wMel-infected mosquitoes showed enhancement of SINV infection rates one week post-infection, with wAlbB-infected mosquitoes also having higher viral titers than controls. Infection rates with ONNV were low across all treatments and no significant effects of Wolbachia were observed. The effects of Wolbachia on MAYV infections were strikingly strain-specific; wMel strongly blocked MAYV infections and suppressed viral titers, while wAlbB had more modest effects. The variable effects of Wolbachia on vector competence underscore the importance of further research into how this bacterium impacts the virome of wild mosquitoes including the emergent human pathogens they transmit., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dodson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Aphids harbor nine common facultative symbionts, most mediating one or more ecological interactions. Wolbachia pipientis, well-studied in other arthropods, remains poorly characterized in aphids. In Pentalonia nigronervosa and P. caladii, global pests of banana, Wolbachia was initially hypothesized to function as a co-obligate nutritional symbiont alongside the traditional obligate Buchnera. However, genomic analyses failed to support this role. Our sampling across numerous populations revealed that more than 80% of Pentalonia aphids carried an M-supergroup strain of Wolbachia (wPni). The lack of fixation further supports a facultative status for Wolbachia, while high infection frequencies in these entirely asexual aphids strongly suggest Wolbachia confers net fitness benefits. Finding no correlation between Wolbachia presence and food plant use, we challenged Wolbachia-infected aphids with common natural enemies. Bioassays revealed that Wolbachia conferred significant protection against a specialized fungal pathogen (Pandora neoaphidis) but not against generalist pathogens or parasitoids. Wolbachia also improved aphid fitness in the absence of enemy challenge. Thus, we identified the first clear benefits for aphid-associated Wolbachia and M-supergroup strains specifically. Aphid-Wolbachia systems provide unique opportunities to merge key models of symbiosis to better understand infection dynamics and mechanisms underpinning symbiont-mediated phenotypes., (© 2024 The Author(s). Environmental Microbiology published by John Wiley & Sons Ltd.)

Background: Mosquitoes serve as vectors for numerous pathogens, posing significant health risks to humans and animals. Understanding the complex interactions within mosquito microbiota is crucial for deciphering vector-pathogen dynamics and developing effective disease management strategies. Here, we investigated the nested patterns of Wolbachia endosymbionts and Escherichia-Shigella within the microbiota of laboratory-reared Culex pipiens f. molestus and Culex quinquefasciatus mosquitoes. We hypothesized that Wolbachia would exhibit a structured pattern reflective of its co-evolved relationship with both mosquito species, while Escherichia-Shigella would display a more dynamic pattern influenced by environmental factors., Results: Our analysis revealed different microbial compositions between the two mosquito species, although some microorganisms were common to both. Network analysis revealed distinct community structures and interaction patterns for these bacteria in the microbiota of each mosquito species. Escherichia-Shigella appeared prominently within major network modules in both mosquito species, particularly in module P4 of Cx. pipiens f. molestus, interacting with 93 nodes, and in module Q3 of Cx. quinquefasciatus, interacting with 161 nodes, sharing 55 nodes across both species. On the other hand, Wolbachia appeared in disparate modules: module P3 in Cx. pipiens f. molestus and a distinct module with a single additional taxon in Cx. quinquefasciatus, showing species-specific interactions and no shared taxa. Through computer simulations, we evaluated how the removal of Wolbachia or Escherichia-Shigella affects network robustness. In Cx. pipiens f. molestus, removal of Wolbachia led to a decrease in network connectivity, while Escherichia-Shigella removal had a minimal impact. Conversely, in Cx. quinquefasciatus, removal of Escherichia-Shigella resulted in decreased network stability, whereas Wolbachia removal had minimal effect., Conclusions: Contrary to our hypothesis, the findings indicate that Wolbachia displays a more dynamic pattern of associations within the microbiota of Culex pipiens f. molestus and Culex quinquefasciatus mosquitoes, than Escherichia-Shigella. The differential effects on network robustness upon Wolbachia or Escherichia-Shigella removal suggest that these bacteria play distinct roles in maintaining community stability within the microbiota of the two mosquito species., (© 2024. The Author(s).)

Background: Matings between male Aedes aegypti mosquitoes infected with wAlbB strain of Wolbachia and wildtype females yield non-viable eggs. We evaluated the efficacy of releasing wAlbB-infected Ae. aegypti male mosquitoes to suppress dengue., Methods: We specified the protocol of a two-arm cluster-randomized test-negative controlled trial (cRCT) and emulated it using a nationally representative test-negative/positive database of individuals reporting for febrile illness to any public hospital, general practitioner or polyclinic. We retrospectively built a cohort of individuals who reside in Wolbachia locations vs a comparator control group who do not reside in Wolbachia locations, using a nationally representative database of all individuals whom report for febrile illness and were tested for dengue at the Environmental Health Institute/hospital laboratories/commercial diagnostic laboratories, through general practitioner clinic, polyclinic or public/private hospital from epidemiological week (EW) 1 2019 to EW26 2022. We emulated a constrained randomization protocol used in cRCTs to balance dengue risk between intervention and control arms in the pre-intervention period. We used the inverse probability weighting approach to further balance the intervention and control groups using a battery of algorithmically selected sociodemographic, environmental and anthropogenic variables. Intention-to-treat analyses were conducted to estimate the risk reduction of dengue given Wolbachia exposure., Results: Intention-to-treat analyses revealed that, compared with controls, Wolbachia releases for 3, 6 and ≥12 months was associated to 47% (95% confidence interval: 25-69%), 44% (33-77%) and 61% (38-78%) protective efficacy against dengue, respectively. When exposed to ≥12 months of Wolbachia releases, protective efficacies ranged from 49% (13-72%) to 77% (60-94%) across years. The proportion of virologically confirmed dengue cases was lower overall in the intervention arm. Protective efficacies were found across all years, age and sex subgroups, with higher durations of Wolbachia exposure associated to greater risk reductions of dengue., Conclusion: Results demonstrated that Wolbachia-mediated sterility can strengthen dengue control in tropical cities, where dengue burden is the greatest., (© International Society of Travel Medicine 2024. Published by Oxford University Press.)

Wolbachia is an obligate endosymbiont that is maternally inherited and widely distributed in arthropods and nematodes. It remains in the mature eggs of female hosts over generations through multiple strategies and manipulates the reproduction system of the host to enhance its spreading efficiency. However, the transmission of Wolbachia within the host's ovaries and its effects on ovarian cells during oogenesis, have not been extensively studied. We used single-cell RNA sequencing to comparatively analyze cell-typing and gene expression in Drosophila ovaries infected and uninfected with Wolbachia . Our findings indicate that Wolbachia significantly affects the transcription of host genes involved in the extracellular matrix, cytoskeleton organization, and cytomembrane mobility in multiple cell types, which may make host ovarian cells more conducive for the transmission of Wolbachia from extracellular to intracellular. Moreover, the genes nos and orb , which are related to the synthesis of ribonucleoprotein complexes, are specifically upregulated in early germline cells of ovaries infected with Wolbachia , revealing that Wolbachia can increase the possibility of its localization to the host oocytes by enhancing the binding with host ribonucleoprotein-complex processing bodies (P-bodies). All these findings provide novel insights into the maternal transmission of Wolbachia between host ovarian cells.IMPORTANCE Wolbachia , an obligate endosymbiont in arthropods, can manipulate the reproduction system of the host to enhance its maternal transmission and reside in the host's eggs for generations. Herein, we performed single-cell RNA sequencing of ovaries from Drosophila melanogaster and observed the effects of Wolbachia (strain w Mel) infection on different cell types to discuss the potential mechanism associated with the transmission and retention of Wolbachia within the ovaries of female hosts. It was found that the transcriptions of multiple genes in the ovary samples infected with Wolbachia are significantly altered, which possibly favors the maternal transmission of Wolbachia . Meanwhile, we also discovered that Wolbachia may flexibly regulate the expression level of specific host genes according to their needs rather than rigidly changing the expression level in one direction to achieve a more suitable living environment in the host's ovarian cells. Our findings contribute to a further understanding of the maternal transmission and possible universal effects of Wolbachia within the host., Competing Interests: The authors declare no conflict of interest.

The mechanism of cytoplasmic incompatibility (CI) is important in the study of Wolbachia invasion in wild mosquitoes. Su et al. (Bull Math Biol 84(9):95, 2022) proposed a delay differential equation model by relating the CI effect to maturation delay. In this paper, we investigate the dynamics of this model by allowing the same density-dependent death rate and distinct density-independent death rates. Through analyzing the existence and stability of equilibria, we obtain the parameter conditions for Wolbachia successful invasion if the maternal transmission is perfect. While if the maternal transmission is imperfect, we give the ranges of parameters to ensure failure invasion, successful invasion and partially suppressing, respectively. Meanwhile, numerical simulations indicate that the system may exhibit monostable and bistable dynamics when parameters vary. Particularly, in the bistable situation an unstable separatrix, like a line, exists when choosing constant functions as initial values; and the maturation delay affects this separatrix in an interesting way., (© 2024. The Author(s), under exclusive licence to the Society for Mathematical Biology.)

Wolbachia, a prevalent intracellular symbiotic bacterium in insects, plays a significant role in insect biology. Ectropis grisescens (Warren; Lepidoptera: Geometridae) is a devastating chewing pest distributed in tea plantations throughout China. However, it is unclear how Wolbachia titers affect the fitness and reproduction of E. grisescens. In this study, the impacts of 3 different infection lines, naturally Wolbachia-infected, Wolbachia-uninfected, and Wolbachia transinfected, regarding the life history traits of E. grisescens, were evaluated using the age-stage, 2-sex life table. Wolbachia infection shortened preadult duration and preoviposition periods and increased the fecundity, net reproductive rate, and finite rate of increase. Meanwhile, population projection indicated that E. grisescens population size with Wolbachia infection can increase faster than without. These results indicate that Wolbachia plays a regulatory role in the fitness of E. grisescens. It is also noted that the life history parameters of E. grisescens may positively correlate with Wolbachia titers. These findings could aid in pest management in tea gardens., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Many arthropods carry maternally inherited endosymbionts that cause cytoplasmic incompatibility (CI), manifested as embryonic mortality in matings of infected males with uninfected females. Infected females, however, do not suffer this cost. Therefore, in populations with mixed endosymbiont infections, selection is expected to favour mechanisms that enable hosts to avoid or mitigate CI. This may include changes in mating behaviour, such as reduced female receptivity to mating and/or remating when approached by incompatible males. Here, we investigated mating behavioural traits in haplodiploid thrips naturally associated with two CI-inducing endosymbionts, Cardinium and Wolbachia . Compared with females with both endosymbionts, those with only Cardinium showed reduced receptivity to males carrying both. However, surprisingly, females without endosymbionts were not less receptive to incompatible males. Furthermore, in contrast to females without endosymbionts, females with Cardinium were far less likely to remate with incompatible than compatible males irrespective of the compatibility type of the first mating. Our results suggest that endosymbiont-specific sexual selection processes occur, whereby females carrying only Cardinium recognize Wolbachia in coinfected males to avoid CI. This may hinder a CI-driven Wolbachia spread. Endosymbiont-mediated mating behaviours may be crucial for the dynamics of CI-inducing endosymbionts and their application in pest management strategies.

Wolbachia are common heritable endosymbionts that influence many aspects of ecology and evolution in various insects, yet Wolbachia-mediated intracellular metabolic responses to temperature stress have been largely overlooked. Here, we introduced the Wolbachia strain wLhui from the invasive Liriomyza huidobrensis (Blanchard) into a Drosophila Schneider 2 cell line (S2) and investigated the metabolite profile of wLhui-infected (S2_wLhui) and uninfected cell lines (S2_wu) under short-term exposure to either high (37°C), moderate (27°C), or low (7 and 17°C) temperatures. We find that Wolbachia infection, temperature stress, and their interactions significantly affect cellular metabolic profiles. Most significantly, when comparing the changes in metabolites between S2_wLhui and S2_wu, glycerophospholipids, amino acids, and fatty acids associated with metabolic pathways, microbial metabolism in diverse environments, and other pathways were significantly accumulated at either low or high temperatures. Our findings suggest Wolbachia-induced cellular physiological responses to short-term temperature stress, which may in turn affect the fitness and adaptive ability of its host as an invasive species., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Background: Wolbachia are widespread intracellular bacteria in insects that often have high rates of spread due to their impact on insect reproduction. These bacteria may also affect the mating behavior of their host with impacts on the fitness of host progeny. In this study, we investigated the impact of Wolbachia on a preference for mating with young or old males in the parasitoid wasp Habrobracon hebetor., Results: Our results showed that uninfected females from a tetracycline-treated line preferred to mate with young males, whereas Wolbachia-infected females had no preference. Time to mating was relatively shorter in the infected lines. Regardless of Wolbachia infection status, progeny resulting from matings with young males showed higher fitness than those from crosses with old males, and infected females crossed with infected young males showed the highest performance., Conclusion: These results suggest an impact of Wolbachia on female mate preference and offspring fitness although it is unclear how this phenomenon increases Wolbachia transmission of infected wasps. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Background: Releasing large numbers of Aedes albopictus males, carrying the artificially introduced Wolbachia 'wPip' strain, results in a decrease in the reproductive capacity of wild females due to a phenomenon known as cytoplasmic incompatibility (CI). This vector control strategy is referred to as the incompatible insect technique (IIT). However, its widespread implementation faces various challenges, including the complexity of removing fertile females from the males intended for release. Here, we present the results of semi-field experiments comparing the impact of minimal female co-release on two IIT modes: unidirectional CI-based (UnCI IIT) and bidirectional CI-based (BiCI IIT), specifically targeting Ae. albopictus., Results: The contamination of 'wPip' infected females (2%) during male releases significantly weakened the overall effectiveness of IIT, emphasizing the need for thorough sex separation. Specifically, with UnCI IIT, despite the low rate of co-released females, there was a gradual rise in 'wPip' infection frequency, resulting in more compatible mating and subsequently higher rates of egg hatching. Conversely, this pattern was effectively mitigated in BiCI IIT owing to the reciprocal sterility between the wild-type and the 'wPip' infected populations., Conclusion: Through an experimental approach, conducted in a semi-field setting, we have contributed to advancing scientific understanding regarding the potential outcomes of implementing the IIT strategy in the absence of a complete sexing system. The results suggest that safety measures for mitigating the potential impacts of co-released females can be tailored according to the specific type of IIT being utilized. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Mosquito-borne diseases such as dengue and filariasis are a growing public health concern in endemic countries. Biological approaches, such as the trans-infection of Wolbachia pipientis in mosquitoes, are an alternative vector control strategy, especially for arthropod-borne viruses such as dengue. In the present study, the effect of Wolbachia (wMel strain) on the vectorial capacity of Aedes aegypti for Dirofilaria immitis was studied. Our results showed that Wolbachia does not affect the phenotype of mosquito survival or the prevalence, number, and molting rate of third-stage larvae in both susceptible and resistant strains of Ae. aegypti. RNA-seq analysis of Malpighian tubules at 2 days post-infection with D. immitis showed the differentially expressed genes (DEGs) with and without wMel infection. No characteristic immune-related gene expression patterns were observed among the DEGs. No significant change in the amount of Wolbachia was observed in the Ae. aegypti after D. immitis infection. Our results suggest that infection of D. immitis in Ae. aegypti populations will not interfere with Wolbachia-based vector control strategies in dengue-endemic areas where cases of D. immitis are present. This study demonstrated the veterinary medical validity of a dengue control program using Wolbachia., (© 2024. The Author(s).)

The spread and invasion of the urban malaria vector Anopheles stephensi has emerged as a significant threat to ongoing malaria control and elimination efforts, particularly in Africa. The successful use of the maternally inherited endosymbiotic bacterium Wolbachia for arbovirus control has inspired the exploration of similar strategies for managing malaria vectors, necessitating the establishment of a stable Wolbachia-Anopheles symbiosis. In this study, we successfully transferred Wolbachia wPip into An. stephensi, resulting in the establishment of a stable transinfected HP1 line with 100% maternal transmission efficiency. We demonstrate that wPip in the HP1 line induces nearly complete unidirectional cytoplasmic incompatibility (CI) and maintains high densities in both somatic and germline tissues. Despite a modest reduction in lifespan and female reproductive capacity, our results suggest the Wolbachia infection in the HP1 line has little impact on life history traits, body size, and male mating competitiveness, as well as the ability of its larvae to tolerate rearing temperatures up to 38°C, although wPip densities moderately decrease when larvae are exposed to a constant 33°C and diurnal cyclic temperatures of 27-36°C and 27-38°C. These findings highlight the potential of the HP1 line as a robust candidate for further development in malaria control., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: YKL, JLL, YLW and ZYX are affiliated with Guangzhou Wolbaki Biotech Co., Ltd. The other authors declare no competing interests., (Copyright: © 2024 Liang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dengue virus (DENV) is the most prevalent mosquito-borne Flavivirus that affects humans worldwide. Aedes albopictus, which is naturally infected with the bacteria Wolbachia, is considered to be a secondary vector of DENV. However, it was responsible for a recent DENV outbreak of unprecedented magnitude in Reunion Island, a French island in the South West Indian Ocean. Moreover, the distribution of the cases during this epidemic showed a spatially heterogeneous pattern across the island, leading to questions about the differential vector competence of mosquito populations from different geographic areas. The aim of this study was to gain a better understanding of the vector competence of the Ae. albopictus populations from Reunion Island for local DENV epidemic strains, while considering their infection by Wolbachia. Experimental infections were conducted using ten populations of Ae. albopictus sampled across Reunion Island and exposed to three DENV strains: one strain of DENV serotype 1 (DENV-1) and two strains of DENV serotype 2 (DENV-2). We analyzed three vector competence parameters including infection rate, dissemination efficiency and transmission efficiency, at different days post-exposition (dpe). We also assessed whether there was a correlation between the density of Wolbachia and viral load/vector competence parameters. Our results show that the Ae. albopictus populations tested were not able to transmit the two DENV-2 strains, while transmission efficiencies up to 40.79% were observed for the DENV-1 strain, probably due to difference in viral titres. Statistical analyses showed that the parameters mosquito population, generation, dpe and area of sampling significantly affect the transmission efficiencies of DENV-1. Although the density of Wolbachia varied according to mosquito population, no significant correlation was found between Wolbachia density and either viral load or vector competence parameters for DENV-1. Our results highlight the importance of using natural mosquito populations for a better understanding of transmission patterns of dengue., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hafsia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Vector-borne diseases leave a large footprint on global health. Notable culprits include West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV), all transmitted by Culex mosquitoes. Chemical insecticides have been widely used to reduce the spread of mosquito-borne diseases. Still, mosquitoes are becoming more and more resistant to most chemical insecticides which cause particular harm to the ecology. Wolbachia belongs to the family Ehrlichiaceae in the order Rickettsiales and is a matrilineally inherited endosymbiont present in 60% of insects in nature. Wolbachia is capable of inducing a wide range of reproductive abnormalities in its hosts, such as cytoplasmic incompatibility, and can alter mosquito resistance to pathogen infection. Wolbachia has been proposed as a biological alternative to chemical vector control, and specific research progress and effectiveness have been achieved. Despite the importance of Wolbachia, this strategy has not been tested in Culex pipiens pallens, the most prevalent mosquito species in Shandong Province, China. Little is known about how the mass release of Wolbachia-infected mosquitoes may impact the genetic structure of Culex pipiens pallens, and how the symbiotic bacterium Wolbachia interacts with mitochondria during host mosquito transmission. Based on the population genetic structure of Culex pipiens pallens in Shandong Province, this study investigated the infection rate and infection type of Wolbachia in Shandong Province and jointly analysed the evolutionary relationship between the host mosquito and the symbiotic bacterium Wolbachia. Our study showed that Wolbachia naturally infected by Culex pipiens pallens in Shandong Province was less homologous to Wolbachia infected by Aedes albopictus released from mosquito factory in Guangzhou. Our results also show that Culex pipiens pallens is undergoing demographic expansion in Shandong Province. The overall Wolbachia infection rate of Culex pipiens pallens was 92.8%, and a total of 15 WSP haplotypes were detected. We found that the genetic diversity of Wolbachia was low in Culex pipiens pallens from Shandong Province, and the mosquitoes were infected only with type B Wolbachia. Visualizing the relationship between Culex pipiens pallens and Wolbachia using a tanglegram revealed patterns of widespread associations. A specific coevolutionary relationship exists between the host mosquito and Wolbachia. Knowledge of this mosquito-Wolbachia relationship will provide essential scientific information required for Wolbachia-based vector control approaches in Shandong Province and will lead to a better understanding of the diversity and evolution of Wolbachia for its utility as a biocontrol agent., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Wolbachia pipientis (Hertig, 1936), also referred as Wolbachia, is a bacterium present across insect taxa, certain strains of which have been demonstrated to impact the fitness and capacity to transmit viruses in mosquitoes, particularly Aedes aegypti (Linnaeus, 1762). Most studies examine these impacts in limited sets of environmental regimes. Here we seek to understand the impacts of environmentally relevant conditions such as larval density, temperature, and their interaction on wAlbB-infected A. aegypti. Using a factorial design, we measured wAlbB stability (relative density, post-emergence in females, and in progeny), the ability for wAlbB to induce cytoplasmic incompatibility, and bacterial effects on mosquito fitness (fecundity, fertility, and body mass) and performance (adult survival and time to pupation) across 2 temperature regimes (fluctuating and constant) and 2 initial larval densities (low and high). Fluctuating daily regimes of temperature (27 to 40 °C) led to decreased post-emergence wAlbB density and increased wAlbB density in eggs compared to constant temperature (27 °C). An increased fecundity was found in wAlbB-carrying females reared at fluctuating temperatures compared to uninfected wild-type females. wAlbB-carrying adult females showed significantly increased survival than wild-type females. Contrarily, wAlbB-carrying adult males exhibited a significantly lower survival than wild-type males. We found differential effects of assessed treatments (Wolbachia infection status, temperature, and larval density) across mosquito sexes and life stages. Taken together, our results indicate that realistic conditions may not impact dramatically the stability of wAlbB infection in A. aegypti. Nonetheless, understanding the ecological consequence of A. aegypti-wAlbB interaction is complex due to life history tradeoffs under conditions faced by natural populations., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The mosquito microbiome significantly influences vector competence, including in Aedes albopictus, a globally invasive vector. Describing the microbiome and Wolbachia strains of Ae. albopictus from different regions can guide area-specific control strategies. Mosquito samples from Spain and São Tomé were analyzed using 16S rRNA gene sequencing and metagenomic sequencing. Wolbachia infection patterns were observed by sex and population. Female mosquitoes were blood-fed, a factor considered in analyzing their microbiota. Results revealed a dominance of dual Wolbachia infections, strains A and B, in the microbiome of both populations of Ae. albopictus, especially among females. Both populations shared a core microbiome, although 5 and 9 other genera were only present in Spain and São Tomé populations, respectively. Genera like Pelomonas and Nevskia were identified for the first time in Aedes mosquitoes. This study is the first to describe the Ae. albopictus bacteriome in Spain and São Tomé, offering insights for the development of targeted mosquito control strategies. Understanding the specific microbiome composition can help in designing more effective interventions, such as microbiome manipulation and Wolbachia-based approaches, to reduce vector competence and transmission potential of these mosquitoes., (© 2024. The Author(s).)

Wolbachia bacteria encompass noteworthy reproductive manipulators of their arthropod hosts. which influence host reproduction to favour their own transmission, also exploiting toxin-antitoxin systems. Recently, multiple other bacterial symbionts of arthropods have been shown to display comparable manipulative capabilities. Here, we wonder whether such phenomena are truly restricted to arthropod hosts. We focused on protists, primary models for evolutionary investigations on eukaryotes due to their diversity and antiquity, but still overall under-investigated. After a thorough re-examination of the literature on bacterial-protist interactions with this question in mind, we conclude that such bacterial 'addictive manipulators' of protists do exist, are probably widespread, and have been overlooked until now as a consequence of the fact that investigations are commonly host-centred, thus ineffective to detect such behaviour. Additionally, we posit that toxin-antitoxin systems are crucial in these phenomena of addictive manipulation of protists, as a result of recurrent evolutionary repurposing. This indicates intriguing functional analogy and molecular homology with plasmid-bacterial interplays. Finally, we remark that multiple addictive manipulators are affiliated with specific bacterial lineages with ancient associations with diverse eukaryotes. This suggests a possible role of addictive manipulation of protists in paving the way to the evolution of bacteria associated with multicellular organisms.

Background: Incompatible insect technique (IIT) coupled with sterile insect technique (SIT) via the release of sterile male Wolbachia-infected mosquitoes is a promising tool for Aedes-borne disease control. Yet, real-world evidence on the suppressive effectiveness of IIT-SIT on mosquito abundance remains mostly limited to small semi-rural village and suburban localities over short trial durations. However, a large proportion of Aedes-borne diseases occur in dense, urban, and high-rise locations, limiting the applicability of previous studies for these settings with high disease burden. The sustainability and use of this technology over multiple years is also unknown., Methods: In this synthetic control study, we conducted a large-scale, field trial of IIT-SIT targeting Aedes aegypti among high-rise public housing estates in Singapore, an equatorial city state. Routinely collected data from a large, nationwide surveillance system of 57 990 unique mosquito traps, combined with a high-dimensional set of anthropogenic and environmental confounders were collected to ascertain mosquito abundance and its key drivers. Four townships were selected as the intervention groups (approximate population size of 607 872 residents as of 2022), wherein interventions that combined ITT with SIT over the course of the study period were conducted. Townships were subject to releases of wAlbB-SG male A aegypti mosquitoes twice a week. Data were assessed over the course of epidemiological weeks (EWs), which provide the finest temporal resolution of recorded Wolbachia release schedule and mosquito abundance data. A novel synthetic control framework was then developed to account for the non-randomised and staggered adoption setting of the intervention across trial sectors to identify the direct suppressive effectiveness of IIT-SIT on female A aegypti populations, the spillover effects in non-release areas, and the effect of the intervention on other mosquito populations such as Aedes albopictus. Furthermore, we recalculated effectiveness in terms of calendar time, time since intervention, and over multiple sites to examine heterogeneities in IIT-SIT effectiveness., Findings: Between EW27 2018 and EW26 2022, Wolbachia releases were conducted across 117 sectors, of which 97 had sufficient trap data, which were collected between EW8 2019 and EW26 2022. We found that Wolbachia-based IIT-SIT reduced wild-type female A aegypti populations by a mean of 62·01% (95% CI 60·68 to 63·26) by 3 months, 78·40% (77·56 to 79·18) by 6 months, and 91·32% (90·95 to 91·66) by at least 18 months of releases. We also found a smaller but non-negligible spillover suppression effect that gradually increased over time (mean spillover intervention effectiveness 61·02% [95% CI 57·89 to 63·72] in adjacent, non-intervention sectors). Although no consistent change in A albopictus populations was seen across the four intervention townships after Wolbachia releases, the average intervention effectiveness on the A albopictus population across all release sectors was -25·80% (95% CI -30·93 to -21·05), which was driven by increases in two towns., Interpretation: Our results demonstrate the potential of IIT-SIT for strengthening long-term, large-scale vector control in tropical cities, where dengue burden is the greatest. The effect of these interventions in different geographical settings should be assessed in future work., Funding: Singapore's Ministry of Finance, Ministry of Sustainability and the Environment, National Environment Agency, and National Robotics Program., Competing Interests: Declaration of interests DDY is a paid employee of Orinno Technology. EGK and YY are paid employees of Verily Life Sciences. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Wolbachia, an endosymbiotic bacterium, relies on nutrients from its host to complete its life cycle. The presence of Wolbachia strain wAlbB in the mosquito Aedes aegypti during egg or larval stages affects the host's development, leading to the absence of developed and visible ovaries in adult mosquito females. In this study, we investigated the impacts of egg quiescence and Wolbachia infection on lipid profiles of adult Ae. aegypti females, and discerned the role of ovaries in lipid synthesis in the reproductive process. The lipidomes of Wolbachia infected and uninfected female individuals at various developmental stages were quantitatively analyzed by LC-MS/MS. Lipidomic change patterns were systematically further investigated in wAlbB-infected fertile females and infertile females following blood feeding. Prolonged egg quiescence induced a shortage of acyl-carnitine (CAR) and potentially impacted some molecules of diacyl-phospholipid (diacyl-PL) and sphingolipid (SL) in young adult mosquitoes. After the first gonotrophic cycle, infertile females accumulated more CAR and lyso-phospholipid (lyso-PL) than fertile females. Then in the second gonotrophic cycle, the patterns of different lipid groups remained similar between fertile and infertile females. Only a small proportion of molecules of triglyceride (TG), phospholipid (lyso-PL and diacyl-PL) and ceramide (Cer) increased exclusively in fertile females from 0 h to 16 h post blood meal, suggesting that the generation or prescence of these lipids rely on ovaries. In addition, we found cardiolipins (CL) might be impacted by Wolbachia infection at the egg stage, and infected mosquitoes also showed distinct patterns between fertile and infertile females at their second gonotrophic cycle. Our study provides new insights into the long-term influence of Wolbachia on lipid profiles throughout various life stages of mosquitoes. Additionally, it suggests a role played by ovaries in lipid synthesis during mosquito reproduction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Interactions between a host organism and its associated microbiota, including symbiotic bacteria, play a crucial role in host adaptation to changing environmental conditions. Antarctica provides a unique environment for the establishment and maintenance of symbiotic relationships. One of the most extensively studied symbiotic bacteria in invertebrates is Wolbachia pipientis, which is associated with a wide variety of invertebrates. Wolbachia is known for manipulating host reproduction and having obligate or facultative mutualistic relationships with various hosts. However, there is a lack of clear understanding of the prevalence of Wolbachia in terrestrial invertebrates in Antarctica. We present the outcomes of a literature search for information on the occurrence of Wolbachia in each of the major taxonomic groups of terrestrial invertebrates (Acari, Collembola, Diptera, Rotifera, Nematoda, Tardigrada). We also performed profiling of prokaryotes based on three marker genes and Kraken2 in available whole genome sequence data obtained from Antarctic invertebrate samples. We found no reports or molecular evidence of Wolbachia in these invertebrate groups in Antarctica. We discuss possible reasons underlying this apparent absence and suggest opportunities for more targeted future research to confirm bacteria's presence or absence., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Hippoboscid flies (Diptera: Hippoboscidae) are obligate bloodsucking ectoparasites of animals. In Europe, limited research has been conducted on this family until the recent introduction of the deer ked Lipoptena fortisetosa Maa, 1965. A new species of the genus Lipoptena, Lipoptena andaluciensis sp. nov., was found in southern Spain after extensive sampling with carbon-dioxide baited suction traps. A total of 52 females and 32 males were collected at 29 out of 476 sites examined over eight months in 2023. Lipoptena andaluciensis sp. nov. was characterized morphologically and molecularly. The new Lipoptena species can be differentiated from the closely related L. fortisetosa by size, chaetotaxy of the dorsal and ventral thorax, abdominal plates, and genitalia. Based on DNA-barcoding, our specimens showed the highest similarity with Melophagus ovinus (Linnaeus, 1758) (88.4 %) and with L. fortisetosa (86-88 %). Individual screening of Lipoptena specimens (n = 76) for seven important zoonotic pathogens such as bacteria (Anaplasmataceae family: Bartonella spp., Borrelia spp., Coxiella burnetii and Rickettsia spp.) and protozoans (Babesia spp. and Theileria spp.) by conventional PCR and RT-PCR was performed. DNA of C. burnetii was detected in one specimen, while two other specimens harboured Anaplasmataceae (Wolbachia spp., 100 % homology and another endosymbiont probably related to Arsenophonus sp., 95.3 % homology, respectively), all representing the first records of these bacteria in the Lipoptena spp. from Europe. Carbon dioxide traps probed its effectiveness as a reliable passive method for keds surveillance. Our study highlights the existence of a new Lipoptena species, presumably widely distributed in southern Spain. The role of this species in the transmission cycle of pathogens of medical-veterinary relevance needs to be considered in the area., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Jordi Figuerola reports financial support was provided by LaCaixa Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Heritable endosymbionts widely occur in arthropod and nematode hosts. Among these endosymbionts, Wolbachia has been extensively detected in many arthropods, such as insects and crustaceans. Maternal inheritance is the most basic and dominant mode of transmission of Wolbachia, and it might regulate the reproductive system of the host in four ways: feminization, parthenogenesis, male killing, and cytoplasmic incompatibility. There is a relatively high percentage (10%) of thelytokous species in Oribatida, a suborder under the subclass Acari of arthropods, but the study of the endosymbionts in oribatid mites is almost negligible. In this paper, we detected endosymbiotic bacteria in two parthenogenetic oribatid species, Nothrus anauniensis Canestrini and Fanzago, 1877, which has never been tested for endosymbionts, and Oppiella nova, in which Wolbachia and Cardinium have been reported before. The results showed that Wolbachia was first found in N. anauniensis with an infection rate of 100% across three populations. Phylogenetic analysis showed that Wolbachia in N. anauniensis belonged to the supergroup K, marking the second supergroup of Wolbachia found in oribatid mites. Unlike previous studies, our study did not detect Wolbachia in O. nova, leading to the exclusion of Wolbachia's role in mediating thelytoky in this species., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: The incompatible insect technique (IIT) has been used for Aedes mosquito population suppression to curb the transmission of dengue. However, its wide application is limited owing to the low output of male mosquitoes and the risk of population replacement from the release of fertile Wolbachia-infected females. This study aims to improve IIT efficiency for broader adoption., Results: We assessed the impact of 10% pyriproxyfen (PPF) sticky powder exposure on Wolbachia (from Culex molestus)-transinfected Aedes albopictus Guangzhou line (GUA line) (GC) mosquitoes. We found that the exposure caused chronic toxicity in adult mosquitoes without affecting the cytoplasmic incompatibility (CI)-inducing capability of males. The PPF-contaminated GC females exhibited significant sterilization and the ability to disseminate lethal doses of PPF to breeding sites. Subsequently, we conducted a field trial combining PPF with IIT aiming to suppress the Ae. albopictus population. This combined approach, termed boosted IIT (BIIT), showed a notable enhancement in population suppression efficiency. The improved efficacy of BIIT was attributed to the dispersion of PPF particles in the field via the released PPF-contaminated male mosquitoes. During the BIIT field trial, no Wolbachia wPip-positive Ae. albopictus larvae were detected, indicating the effective elimination of the risk of Wolbachia-induced population replacement. Additionally, the field trial of BIIT against Ae. albopictus resulted in the suppression of the nontarget mosquito species Culex quinquefasciatus., Conclusion: Our results highlight the remarkable efficiency and feasibility of combining IIT with PPF in suppressing mosquito populations, facilitating the widespread implementation of IIT-based management of mosquito-borne diseases. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Wolbachia symbionts introduced into Aedes mosquitoes provide a highly effective dengue virus transmission control strategy, increasingly utilised in many countries in an attempt to reduce disease burden. Whilst highly effective against dengue and other positive-sense RNA viruses, it remains unclear how effective Wolbachia is against negative-sense RNA viruses. Therefore, the effect of Wolbachia on Bunyamwera virus (BUNV) infection in Aedes aegypti was investigated using w Mel and w AlbB, two strains currently used in Wolbachia releases for dengue control, as well as w Au, a strain that typically persists at a high density and is an extremely efficient blocker of positive-sense viruses. Wolbachia was found to reduce BUNV infection in vitro but not in vivo. Instead, BUNV caused significant impacts on density of all three Wolbachia strains following infection of Ae. aegypti mosquitoes. The ability of Wolbachia to successfully persist within the mosquito and block virus transmission is partially dependent on its intracellular density. However, reduction in Wolbachia density was not observed in offspring of infected mothers. This could be due in part to a lack of transovarial transmission of BUNV observed. The results highlight the importance of understanding the complex interactions between multiple arboviruses, mosquitoes and Wolbachia in natural environments, the impact this can have on maintaining protection against diseases, and the necessity for monitoring Wolbachia prevalence at release sites.

Wolbachia is a widespread maternally transmitted endosymbiotic bacteria with diverse phenotypic effects on its insect hosts, ranging from parasitic to mutualistic. Wolbachia commonly infects social insects, where it faces unique challenges associated with its host's caste-based reproductive division of labor and colony living. Here, we dissect the benefits and costs of Wolbachia infection on life-history traits of the invasive pharaoh ant, Monomorium pharaonis, which are relatively short lived and show natural variation in Wolbachia infection status between colonies. We quantified the effects of Wolbachia infection on the lifespan of queen and worker castes, the egg-laying rate of queens across queen lifespan, and the metabolic rates of whole colonies and colony members. Infected queens laid more eggs than uninfected queens but had similar metabolic rates and lifespans. Interestingly, infected workers outlived uninfected workers. At the colony level, infected colonies were more productive as a consequence of increased queen egg-laying rates and worker longevity, and infected colonies had higher metabolic rates during peak colony productivity. While some effects of infection, such as elevated colony-level metabolic rates, may be detrimental in more stressful natural conditions, we did not find any costs of infection under relatively benign laboratory conditions. Overall, our study emphasizes that Wolbachia infection can have beneficial effects on ant colony growth and worker survival in at least some environments., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The Dengue virus (DENV), primarily spread by Aedes aegypti and also by Aedes albopictus in some regions, poses significant global health risks. Alternative techniques are urgently needed because the current control mechanisms are insufficient to reduce the transmission of DENV. Introducing Wolbachia pipientis into Ae. aegypti inhibits DENV transmission, however, the underlying mechanisms are still poorly understood. Innate immune effector upregulation, the regulation of autophagy, and intracellular competition between Wolbachia and DENV for lipids are among the theories for the mechanism of inhibition. Furthermore, mainly three immune pathways Toll, IMD, and JAK/STAT are involved in the host for the suppression of the virus. These pathways are activated by Wolbachia and DENV in the host and are responsible for the upregulation and downregulation of many genes in mosquitoes, which ultimately reduces the titer of the DENV in the host. The functioning of these immune pathways depends upon the Wolbachia , host, and virus interaction. Here, we summarize the current understanding of DENV recognition by the Ae. aegypti 's immune system, aiming to create a comprehensive picture of our knowledge. Additionally, we investigated how Wolbachia regulates the activation of multiple genes associated with immune priming for the reduction of DENV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mushtaq, Sarwar and Munzoor.)

Background: Wolbachia symbiosis in Aedes aegypti is an emerging biocontrol measure against dengue. However, assessing its real-world efficacy is challenging due to the non-randomised, field-based nature of most intervention studies. This research re-evaluates the spatial-temporal impact of Wolbachia interventions on dengue incidence using a large battery of quasi-experimental methods and assesses each method's validity., Methods: A systematic search for Wolbachia intervention data was conducted via PUBMED. Efficacy was reassessed using commonly-used quasi-experimental approaches with extensive robustness checks, including geospatial placebo tests and a simulation study. Intervention efficacies across multiple study sites were computed using high-resolution aggregations to examine heterogeneities across sites and study periods. We further designed a stochastic simulation framework to assess the methods' ability to estimate intervention efficacies (IE)., Results: Wolbachia interventions in Singapore, Malaysia, and Brazil significantly decreased dengue incidence, with reductions ranging from 48.17% to 69.19%. IEs varied with location and duration. Malaysia showed increasing efficacy over time, while Brazil exhibited initial success with subsequent decline, hinting at operational challenges. Singapore's strategy was highly effective despite partial saturation. Simulations identified Synthetic Control Methods (SCM) and its variant, count Synthetic Control Method (cSCM), as superior in precision, with the smallest percentage errors in efficacy estimation. These methods also demonstrated robustness in placebo tests., Conclusions: Wolbachia interventions exhibit consistent protective effects against dengue. SCM and cSCM provided the most precise and robust estimates of IEs, validated across simulated and real-world settings., (© 2024. The Author(s).)

Avian malaria is expanding upslope with warmer temperatures and driving multiple species of Hawaiian birds towards extinction. Methods to reduce malaria transmission are urgently needed to prevent further declines. Releasing Wolbachia-infected incompatible male mosquitoes could suppress mosquito populations and releasing Wolbachia-infected female mosquitoes (or both sexes) could reduce pathogen transmission if the Wolbachia strain reduced vector competence. We cleared Culex quinquefasciatus of their natural Wolbachia pipientis wPip infection and transinfected them with Wolbachia wAlbB isolated from Aedes albopictus. We show that wAlbB infection was transmitted transovarially, and demonstrate cytoplasmic incompatibility with wild-type mosquitoes infected with wPip from Oahu and Maui, Hawaii. We measured vector competence for avian malaria, Plasmodium relictum, lineage GRW4, of seven mosquito lines (two with wAlbB; three with natural wPip infection, and two cleared of Wolbachia infection) by allowing them to feed on canaries infected with recently collected field isolates of Hawaiian P. relictum. We tested 73 groups (Ntotal = 1176) of mosquitoes for P. relictum infection in abdomens and thoraxes 6-14 days after feeding on a range of parasitemias from 0.028% to 2.49%, as well as a smaller subset of salivary glands. We found no measurable effect of Wolbachia on any endpoint, but strong effects of parasitemia, days post feeding, and mosquito strain on both abdomen and thorax infection prevalence. These results suggest that releasing male wAlbB-infected C. quinquefasciatus mosquitoes could suppress wPip-infected mosquito populations, but would have little positive or negative impact on mosquito vector competence for P. relictum if wAlbB became established in local mosquito populations. More broadly, the lack of Wolbachia effects on vector competence we observed highlights the variable impacts of both native and transinfected Wolbachia infections in mosquitoes., Competing Interests: GF, PIH, AGM, BJW, and SNM report employment and equity ownership at Verily Life Sciences, a for-profit company developing new technologies for mosquito control., (Copyright: © 2024 Kilpatrick et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The symbiosis between microorganisms and host arthropods can cause biological, physiological, and reproductive changes in the host population. The present study aimed to survey facultative symbionts of the genera Wolbachia , Arsenophonus , Cardinium , Rickettsia , and Nosema in Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) and Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) in the laboratory and evaluate the influence of infection on the fitness of these hosts. For this purpose, 16S rDNA primers were used to detect these facultative symbionts in the host species, and the hosts' biological and morphological features were evaluated for changes resulting from the infection caused by these microorganisms. The bacterial symbionts studied herein were not detected in the D. saccharalis samples analysed, but the endosymbiont Wolbachia was detected in C. flavipes and altered the biological and morphological aspects of this parasitoid insect. The results of this study may help to elucidate the role of Wolbachia in maintaining the quality of populations/lineages of C. flavipes .

Symbiotic and pathogenic microorganisms such as bacteria and fungi represent promising alternatives to chemical insecticides to respond to the rapid increase of insecticide resistance and vector-borne disease outbreaks. This study investigated the interaction of two strains of Wolbachia, wAlbB and wAu, with the natural entomopathogenic fungi from Burkina Faso Metarhizium pingshaense, known to be lethal against Anopheles mosquitoes. In addition to showing the potential of Metarhizium against African Aedes aegypti wild-type populations, our study shows that the wAlbB and wAu provide a protective advantage against entomopathogenic fungal infections. Compared to controls, fungal-infected wAu and wAlbB-carrying mosquitoes showed higher longevity, without any significant impact on fecundity and fertility phenotypes. This study provides new insights into the complex multipartite interaction among the mosquito host, the Wolbachia endosymbiont and the entomopathogenic fungus that might be employed to control mosquito populations. Future research should investigate the fitness costs of Wolbachia, as well as its spread and prevalence within mosquito populations. Additionally, evaluating the impact of Wolbachia on interventions involving Metarhizium pingshaense through laboratory and semi-field population studies will provide valuable insights into the effectiveness of this combined approach., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen blocking to polymorphisms in the mosquito alpha-mannosidase-2 (αMan2) gene. Here we use CRISPR-Cas9 mutagenesis to functionally test this association. We developed αMan2 knockouts and examined effects on both Wolbachia and virus levels, using dengue virus (DENV; Flaviviridae) and Mayaro virus (MAYV; Togaviridae). Wolbachia titres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia-uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in a Wolbachia-infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication in Wolbachia-uninfected mosquitoes and did not affect Wolbachia-mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection in A. aegypti mosquitoes in a pathogen- and Wolbachia-specific manner, and that Wolbachia-mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have a significant impact for the design and use of Wolbachia-based strategies to control vector-borne pathogens., (© 2024 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Oak gall wasps typically exhibit a life cycle with one sexual and one asexual generation each year. These wasps can carry various endosymbionts, one of which is the maternally inherited bacterium Wolbachia that can induce several reproductive manipulations on its host. Cytoplasmic incompatibility (CI) has been described as the most prominent of these manipulations. CI leads to embryonic mortality in the hosts' offspring when infected males mate with either uninfected females or with females that harbour different Wolbachia strains. It has been hypothesized that Wolbachia can induce CI in oak gall wasps. To address this hypothesis, we derived a mathematical model to investigate the spread of a bacterial infection in naive populations and to determine the plausibility of CI occurrence. To validate our model, we used published data from Wolbachia-infected Belonocnema kinseyi populations in two approaches. Our first approach uses measurements of infection frequencies and maternal transmission in the sexual generation. For the second approach, we extended the model to compare predictions to estimates of mtDNA-haplotypes, which, like Wolbachia, are maternally inherited, and can therefore be associated with the infection. Both approaches indicate that CI is present in these populations. Our model can be generalized to investigate the occurrence of CI not only for oak gall wasps but also for other species., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Evolutionary Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Dengue virus, primarily transmitted by the Aedes aegypti mosquito, is a major public health concern affecting ≈3.83 billion people worldwide. Recent releases of Wolbachia-transinfected Ae. aegypti in several cities worldwide have shown that it can reduce dengue transmission. However, these releases are costly, and, to date, no framework has been proposed for determining economically optimal release strategies that account for both costs associated with disease risk and releases., Results: We present a flexible stochastic dynamic programming framework for determining optimal release schedules for Wolbachia-transinfected mosquitoes that balances the cost of dengue infection with the costs of rearing and releasing transinfected mosquitoes. Using an ordinary differential equation model of Wolbachia and dengue in a hypothetical city loosely describing areas at risk of new dengue epidemics, we determined that an all-or-nothing release strategy that quickly brings Wolbachia to fixation is often the optimal solution. Based on this, we examined the optimal facility size, finding that it was inelastic with respect to the mosquito population size, with a 100% increase in population size resulting in a 50-67% increase in optimal facility size. Furthermore, we found that these results are robust to mosquito life-history parameters and are mostly determined by the mosquito population size and the fitness costs associated with Wolbachia., Conclusions: These results reinforce that Wolbachia-transinfected mosquitoes can reduce the cost of dengue epidemics. Furthermore, they emphasize the importance of determining the size of the target population and fitness costs associated with Wolbachia before releases occur. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Drone-based mosquito releases facilitate the introduction of dengue-blocking bacteria in wild mosquito populations.

Over the past 50 years, there has been a marked increase in diseases like dengue fever, chikungunya, and Zika. The World Mosquito Program (WMP) has developed an approach that, instead of attempting to eliminate vector species, introduces Wolbachia into native Aedes aegypti populations through the release of Wolbachia -infected mosquitoes. Using this approach, a randomized controlled study recently demonstrated a 77% reduction in dengue across a treatment area within Yogyakarta, Indonesia. Existing release methods use the ground-based release of mosquito eggs or adults that are labor-intensive, are logistically challenging to scale up, and can be restrictive in areas where staff safety is a concern. To overcome these limitations, we developed a fully automated mosquito dosing release system that released smaller cohorts of mosquitoes over a wide area and integrated it into an uncrewed aerial vehicle. We established the effectiveness of this system using an aerial mark, release, and recapture approach. We then demonstrated that using only the aerial release method, we can establish Wolbachia infection in a naive Ae. aegypti population. In both cases, the use of aerial releases demonstrated comparable outcomes to ground-based releases without the required labor or risk. These two trials demonstrated the feasibility of using an aerial release approach for large-scale mosquito releases.

The fitness effects of overt parasites, and host resistance to them, are well documented. Most symbionts, however, are more covert and their interactions with their hosts are less well understood. Wolbachia , an intracellular symbiont of insects, is particularly interesting because it is thought to be unaffected by the host immune response and to have fitness effects mostly focussed on sex ratio manipulation. Here, we use quantitative PCR to investigate whether host genotype affects Wolbachia infection density in the leaf-cutting ant Acromyrmex echinatior , and whether Wolbachia infection density may affect host morphology or caste determination. We found significant differences between host colonies in the density of Wolbachia infections, and also smaller intracolonial differences in infection density between host patrilines. However, the density of Wolbachia infections did not appear to affect the morphology of adult queens or likelihood of ants developing as queens. The results suggest that both host genotype and environment influence the host- Wolbachia relationship, but that Wolbachia infections carry little or no physiological effect on the development of larvae in this system., Competing Interests: The authors declare there are no competing interests., (©2024 Frost et al.)

Objective: Sclerodermus wasps are important biocontrol agents of a class of wood borers. Bacterial symbionts influence the ecology and biology of their hosts in a variety of ways, including the formation of life-long beneficial or detrimental parasitic infections. However, only a few studies have explored the species and content of the symbionts in the Sclerodermus species., Methods: Here, a high-throughput sequencing study of the V3-V4 region of the 16S ribosomal RNA gene revealed a high level of microbial variety in four Sclerodermus waps, and their diversities and functions were also predicted., Results: The three most prevalent phyla of microorganisms in the sample were Firmicutes, Bacteroides, and Proteus. The KEEG pathways prediction results indicated that the three pathways with the highest relative abundances in the S . sichuanensis species were translation, membrane transport, and nucleotide metabolism. These pathways differed from those observed in S . guani , S . pupariae , and S . alternatusi , which exhibited carbohydrate metabolism, membrane transport, and amino acid metabolism, respectively. Bacteroides were found to be abundant in several species, whereas Wolbachia was the most abundant among S. sichuanensis , with a significant negative correlation between temperature and carriage rate., Conclusions: These results offer insights into the microbial communities associated with the bethylid wasps, which is crucial for understanding how to increase the reproductive capacity of wasps, enhance their parasitic effects, and lower cost in biocontrol., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kang, Wang, Gong, Tang and Wei.)

The intracellular symbiont Wolbachia pipientis evolved after the divergence of arthropods and nematodes, but it reached high prevalence in many of these taxa through its abilities to infect new hosts and their germlines. Some strains exhibit long-term patterns of co-evolution with their hosts, while other strains are capable of switching hosts. This makes strain selection an important factor in symbiont-based biological control. However, little is known about the ecological and evolutionary interactions that occur when a promiscuous strain colonizes an infected host. Here, we study what occurs when two strains come into contact in host cells following horizontal transmission and infection. We focus on the faithful wMel strain from Drosophila melanogaster and the promiscuous wRi strain from Drosophila simulans using an in vitro cell culture system with multiple host cell types and combinatorial infection states. Mixing D. melanogaster cell lines stably infected with wMel and wRi revealed that wMel outcompetes wRi quickly and reproducibly. Furthermore, wMel was able to competitively exclude wRi even from minuscule starting quantities, indicating that this is a nearly deterministic outcome, independent of the starting infection frequency. This competitive advantage was not exclusive to wMel's native D. melanogaster cell background, as wMel also outgrew wRi in D. simulans cells. Overall, wRi is less adept at in vitro growth and survival than wMel and its in vivo state, revealing differences between the two strains in cellular and humoral regulation. These attributes may underlie the observed low rate of mixed infections in nature and the relatively rare rate of host-switching in most strains. Our in vitro experimental framework for estimating cellular growth dynamics of Wolbachia strains in different host species and cell types provides the first strategy for parameterizing endosymbiont and host cell biology at high resolution. This toolset will be crucial to our application of these bacteria as biological control agents in novel hosts and ecosystems., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mirchandani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mosquitoes of the Culex genus are responsible for a large burden of zoonotic virus transmission globally. Collectively, they play a significant role in the transmission of medically significant diseases such as Japanese encephalitis virus and West Nile virus. Climate change, global trade, habitat transformation and increased urbanisation are leading to the establishment of Culex mosquitoes in new geographical regions. These novel mosquito incursions are intensifying concerns about the emergence of Culex -transmitted diseases and outbreaks in previously unaffected areas. New mosquito control methods are currently being developed and deployed globally. Understanding the complex interaction between pathogens and mosquitoes is essential for developing new control strategies for Culex species mosquitoes. This article reviews the role of Culex mosquitos as vectors of zoonotic disease, discussing the transmission of viruses across different species, and the potential use of Wolbachia technologies to control disease spread. By leveraging the insights gained from recent successful field trials of Wolbachia against Aedes -borne diseases, we comprehensively discuss the feasibility of using this technique to control Culex mosquitoes and the potential for the development of next generational Wolbachia -based control methods.

As Wolbachia pipientis is more widely being released into field populations of Aedes aegypti for disease control, the ability to select the appropriate strain for differing environments is increasingly important. A previous study revealed that longer-term quiescence in the egg phase reduced the fertility of mosquitoes, especially those harboring the wAlbB Wolbachia strain. This infertility was also associated with a greater biting rate. Here, we attempt to quantify the effect of this heightened biting behavior on the transmission potential of the dengue virus using a combination of assays for fitness, probing behavior, and vector competence, allowing repeat feeding, and incorporate these effects in a model of R0. We show that Wolbachia-infected infertile mosquitoes are more interested in feeding almost immediately after an initial blood meal relative to wild type and Wolbachia-infected fertile mosquitoes and that these differences continue for up to 8 days over the period we measured. As a result, the infertile Wolbachia mosquitoes have higher virus prevalence and loads than Wolbachia-fertile mosquitoes. We saw limited evidence of Wolbachia-mediated blocking in the disseminated tissue (legs) in terms of prevalence but did see reduced viral loads. Using a previously published estimate of the extrinsic incubation period, we demonstrate that the effect of repeat feeding/infertility is insufficient to overcome the effects of Wolbachia-mediated blocking on R0. These estimates are very conservative, however, and we posit that future studies should empirically measure EIP under a repeat feeding model. Our findings echo previous work where periods of extensive egg quiescence affected the reproductive success of Wolbachia-infected Ae. aegypti. Additionally, we show that increased biting behavior in association with this infertility in Wolbachia-infected mosquitoes may drive greater vector competence. These relationships require further exploration, given their ability to affect the success of field releases of Wolbachia for human disease reduction in drier climates where longer egg quiescence periods are expected., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lau et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rickettsia , a group of intracellular bacteria found in eukaryotes, exhibits diverse lifestyles, with some acting as vertebrate pathogens transmitted by arthropod vectors and others serving as maternally transmitted arthropod endosymbionts, some of which manipulate host reproduction for their own benefit. Two phenotypes, namely male-killing and parthenogenesis induction are known as Rickettsia -induced host reproductive manipulations, but it remains unknown whether Rickettsia can induce other types of host manipulation. In this study, we discovered that Rickettsia induced strong cytoplasmic incompatibility (CI), in which uninfected females produce no offspring when mated with infected males, in the predatory insect Nesidiocoris tenuis (Hemiptera: Miridae). Molecular phylogenetic analysis revealed that the Rickettsia strain was related to Rickettsia bellii , a common insect endosymbiont. Notably, this strain carried plasmid-encoded homologues of the CI-inducing factors (namely cifA -like and cifB -like genes), typically found in Wolbachia , which are well-known CI-inducing endosymbionts. Protein domain prediction revealed that the cifB -like gene encodes PD-(D/E)XK nuclease and deubiquitinase domains, which are responsible for Wolbachia -induced CI, as well as ovarian tumour-like (OTU-like) cysteine protease and ankyrin repeat domains. These findings suggest that Rickettsia and Wolbachia endosymbionts share underlying mechanisms of CI and that CI-inducing ability was acquired by microbes through horizontal plasmid transfer.

The obligate endosymbiont Wolbachia induces pathogen interference in the primary disease vector Aedes aegypti, facilitating the utilization of Wolbachia-based mosquito control for arbovirus prevention, particularly against dengue virus (DENV). However, the mechanisms underlying Wolbachia-mediated virus blockade have not been fully elucidated. Here, we report that Wolbachia activates the host cytoplasmic miRNA biogenesis pathway to suppress DENV infection. Through the suppression of the long noncoding RNA aae-lnc-2268 by Wolbachia wAlbB, aae-miR-34-3p, a miRNA upregulated by the Wolbachia strains wAlbB and wMelPop, promoted the expression of the antiviral effector defensin and cecropin genes through the Toll pathway regulator MyD88. Notably, anti-DENV resistance induced by Wolbachia can be further enhanced, with the potential to achieve complete virus blockade by increasing the expression of aae-miR-34-3p in Ae. aegypti. Furthermore, the downregulation of aae-miR-34-3p compromised Wolbachia-mediated virus blockade. These findings reveal a novel mechanism by which Wolbachia establishes crosstalk between the cytoplasmic miRNA pathway and the Toll pathway via aae-miR-34-3p to strengthen antiviral immune responses against DENV. Our results will aid in the advancement of Wolbachia for arbovirus control by enhancing its virus-blocking efficiency., Competing Interests: Z.X. is affiliated with Guangzhou Wolbaki Biotech Co., Ltd. The other authors have declared that no competing interests exist., (Copyright: © 2024 She et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Many insects and other animals carry microbial endosymbionts that influence their reproduction and fitness. These relationships only persist if endosymbionts are reliably transmitted from one host generation to the next. Wolbachia are maternally transmitted endosymbionts found in most insect species, but transmission rates can vary across environments. Maternal transmission of wMel Wolbachia depends on temperature in natural Drosophila melanogaster hosts and in transinfected Aedes aegypti, where wMel is used to block pathogens that cause human disease. In D. melanogaster, wMel transmission declines in the cold as Wolbachia become less abundant in host ovaries and at the posterior pole plasm (the site of germline formation) in mature oocytes. Here, we assess how temperature affects maternal transmission and underlying patterns of Wolbachia localization across 10 Wolbachia strains diverged up to 50 million years-including strains closely related to wMel-and their natural Drosophila hosts. Many Wolbachia maintain high transmission rates across temperatures, despite highly variable (and sometimes low) levels of Wolbachia in the ovaries and at the developing germline in late-stage oocytes. Identifying strains like closely related wMel-like Wolbachia with stable transmission across variable environmental conditions may improve the efficacy of Wolbachia-based biocontrol efforts as they expand into globally diverse environments., (© 2024. The Author(s).)

Wolbachia pipientis is a maternally inherited intracellular bacterium that infects a wide range of arthropods. Wolbachia can have a significant impact on host biology and development, often due to its effects on reproduction. We investigated Wolbachia-mediated effects in the Asian citrus psyllid, Diaphorina citri Kuwayama, which transmits Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease. Diaphorina citri are naturally infected with Wolbachia; therefore, investigating Wolbachia-mediated effects on D. citri fitness and CLas transmission required artificial reduction of this endosymbiont with the application of doxycycline. Doxycycline treatment of psyllids reduced Wolbachia infection by approximately 60% in both male and female D. citri. Psyllids treated with doxycycline exhibited higher CLas acquisition in both adults and nymphs as compared with negative controls. In addition, doxycycline-treated psyllids exhibited decreased fitness as measured by reduced egg and nymph production as well as adult emergence as compared with control lines without the doxycycline treatment. Our results indicate that Wolbachia benefits D. citri by improving fitness and potentially competes with CLas by interfering with phytopathogen acquisition. Targeted manipulation of endosymbionts in this phytopathogen vector may yield disease management tools., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The Wolbachia strain that infects the parasitoid wasp Encarsia formosa induces female-producing parthenogenesis. A new study shows that a Wolbachia-encoded gene has replaced the use of the ancestral wasp homologue that normally controls sexual reproduction, resulting in parthenogenesis., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Host reproduction can be manipulated by bacterial symbionts in various ways. Parthenogenesis induction is the most effective type of reproduction manipulation by symbionts for their transmission. Insect sex is determined by regulation of doublesex (dsx) splicing through transformer2 (tra2) and transformer (tra) interaction. Although parthenogenesis induction by symbionts has been studied since the 1970s, its underlying molecular mechanism is unknown. Here we identify a Wolbachia parthenogenesis-induction feminization factor gene (piff) that targets sex-determining genes and causes female-producing parthenogenesis in the haplodiploid parasitoid Encarsia formosa. We found that Wolbachia elimination repressed expression of female-specific dsx and enhanced expression of male-specific dsx, which led to the production of wasp haploid male offspring. Furthermore, we found that E. formosa tra is truncated and non-functional, and Wolbachia has a functional tra homolog, termed piff, with an insect origin. Wolbachia PIFF can colocalize and interact with wasp TRA2. Moreover, Wolbachia piff has coordinated expression with tra2 and dsx of E. formosa. Our results demonstrate the bacterial symbiont Wolbachia has acquired an insect gene to manipulate the host sex determination cascade and induce parthenogenesis in wasps. This study reveals insect-to-bacteria horizontal gene transfer drives the evolution of animal sex determination systems, elucidating a striking mechanism of insect-microbe symbiosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)