Cytoplasmic Incompatibility Research Articles

Characterizing the Wolbachia infection in field-collected Culicidae mosquitoes from Hainan Province, China

BackgroundMosquitoes are vectors of many pathogens, such as malaria, dengue virus, yellow fever virus, filaria and Japanese encephalitis virus. Wolbachia are capable of inducing a wide range of reproductive abnormalities in their hosts, such as cytoplasmic incompatibility. Wolbachia has been proposed as a tool to modify mosquitoes that are resistant to pathogen infection as an alternative vector control strategy. This study aimed to determine natural Wolbachia infections in different mosquito species across Hainan Province, China.MethodsAdult mosquitoes were collected using light traps, human landing catches and aspirators in five areas in Hainan Province from May 2020 to November 2021. Species were identified based on morphological characteristics, species-specific PCR and DNA barcoding of cox1 assays. Molecular classification of species and phylogenetic analyses of Wolbachia infections were conducted based on the sequences from PCR products of cox1, wsp, 16S rRNA and FtsZ gene segments.ResultsA total of 413 female adult mosquitoes representing 15 species were identified molecularly and analyzed. Four mosquito species (Aedes albopictus, Culex quinquefasciatus, Armigeres subalbatus and Culex gelidus) were positive for Wolbachia infection. The overall Wolbachia infection rate for all mosquitoes tested in this study was 36.1% but varied among species. Wolbachia types A, B and mixed infections of A × B were detected in Ae. albopictus mosquitoes. A total of five wsp haplotypes, six FtsZ haplotypes and six 16S rRNA haplotypes were detected from Wolbachia infections. Phylogenetic tree analysis of wsp sequences classified them into three groups (type A, B and C) of Wolbachia strains compared to two groups each for FtsZ and 16S rRNA sequences. A novel type C Wolbachia strain was detected in Cx. gelidus by both single locus wsp gene and the combination of three genes.ConclusionOur study revealed the prevalence and distribution of Wolbachia in mosquitoes from Hainan Province, China. Knowledge of the prevalence and diversity of Wolbachia strains in local mosquito populations will provide part of the baseline information required for current and future Wolbachia-based vector control approaches to be conducted in Hainan Province.Graphical

Convergent Aedes and Drosophila CidB interactomes suggest cytoplasmic incompatibility targets are conserved.

Wolbachia-mediated cytoplasmic incompatibility (CI) is a conditional embryonic lethality induced when Wolbachia-modified sperm fertilizes an uninfected egg. The Wolbachia proteins, CidA and CidB control CI. CidA is a rescue factor that reverses lethality. CidA binds to CidB. CidB contains a deubiquitinating enzyme and induces CI. Precisely how CidB induces CI and what it targets are unknown. Likewise, how CidA prevents sterilization by CidB is not clear. To identify CidB substrates in mosquitos we conducted pull-down assays using recombinant CidA and CidB mixed with Aedes aegypti lysates to identify the protein interactomes of CidB and the CidB/CidA protein complex. Our data allow us to cross compare CidB interactomes across taxa for Aedes and Drosophila. Our data replicate several convergent interactions, suggesting that CI targets conserved substrates across insects. Our data support a hypothesis that CidA rescues CI by tethering CidB away from its substrates. Specifically, we identify ten convergent candidate substrates including P32 (protamine-histone exchange factor), karyopherin alpha, ubiquitin-conjugating enzyme, and bicoid stabilizing factor. Future analysis on how these candidates contribute to CI will clarify mechanisms.

Variation in sex determination mechanisms may constrain parthenogenesis-induction by endosymbionts in haplodiploid systems.

Endosymbionts are maternally transmitted, and therefore benefit from maximizing female offspring numbers. Parthenogenesis-induction (PI) is the most effective type of manipulation for transmission, but has solely been detected in haplodiploid species, whereas cytoplasmic incompatibility (CI) is detected frequently across the arthropod phylum, including haplodiploids. This puzzling observation led us to hypothesize that the molecular sex-determination mechanism of the haplodiploid host may be a constraining factor in the ability of endosymbionts to induce parthenogenesis. Recent insights indicate that PI-endosymbionts may be able to directly manipulate sex-determination genes to induce the necessary steps required for PI in haplodiploids. However, sex-determination cascades vary extensively, so PI-induction would require a specialized and host-dependent tool set. Contrastingly, CI-related genes target conserved cell-cycle mechanisms, are located on mobile elements, and spread easily. Finally, endosymbiont-manipulations may have a strong impact on the effectiveness of haplodiploid biocontrol agents, but can also be used to enhance their efficacy.

A Fractional-Order Density-Dependent Mathematical Model to Find the Better Strain of Wolbachia

The primary objective of the current study was to create a mathematical model utilizing fractional-order calculus for the purpose of analyzing the symmetrical characteristics of Wolbachia dissemination among Aedesaegypti mosquitoes. We investigated various strains of Wolbachia to determine the most sustainable one through predicting their dynamics. Wolbachia is an effective tool for controlling mosquito-borne diseases, and several strains have been tested in laboratories and released into outbreak locations. This study aimed to determine the symmetrical features of the most efficient strain from a mathematical perspective. This was accomplished by integrating a density-dependent death rate and the rate of cytoplasmic incompatibility (CI) into the model to examine the spread of Wolbachia and non-Wolbachia mosquitoes. The fractional-order mathematical model developed here is physically meaningful and was assessed for equilibrium points in the presence and absence of disease. Eight equilibrium points were determined, and their local and global stability were determined using the Routh–Hurwitz criterion and linear matrix inequality theory. The basic reproduction number was calculated using the next-generation matrix method. The research also involved conducting numerical simulations to evaluate the behavior of the basic reproduction number for different equilibrium points and identify the optimal CI value for reducing disease spread.

Global Dynamics for Competition between Two Wolbachia Strains with Bidirectional Cytoplasmic Incompatibility

Releasing Wolbachia-infected mosquitoes to suppress or replace wild vector mosquitoes has been carried out in 24 countries worldwide, showing great promise in controlling mosquitoes and mosquito-borne diseases. To face the instability of Wolbachia infection in different environments during the area-wide application, we should consider the overlapping of two Wolbachia strains. In this case, bidirectional cytoplasmic incompatibility occurs, which results in mating partners infected with exclusive Wolbachia strains producing inviable offspring. To determine the better Wolbachia candidate for release, we develop an ordinary differential equation model to study the global dynamics for competition between two Wolbachia strains. Our theoretical results on the sharp estimate of stable curves completely determine the fate of the two Wolbachia strains, which help choose appropriate strains for release.

A male-killing Wolbachia endosymbiont is concealed by another endosymbiont and a nuclear suppressor.

Bacteria that live inside the cells of insect hosts (endosymbionts) can alter the reproduction of their hosts, including the killing of male offspring (male killing, MK). MK has only been described in a few insects, but this may reflect challenges in detecting MK rather than its rarity. Here, we identify MK Wolbachia at a low frequency (around 4%) in natural populations of Drosophila pseudotakahashii. MK Wolbachia had a stable density and maternal transmission during laboratory culture, but the MK phenotype which manifested mainly at the larval stage was lost rapidly. MK Wolbachia occurred alongside a second Wolbachia strain expressing a different reproductive manipulation, cytoplasmic incompatibility (CI). A genomic analysis highlighted Wolbachia regions diverged between the 2 strains involving 17 genes, and homologs of the wmk and cif genes implicated in MK and CI were identified in the Wolbachia assembly. Doubly infected males induced CI with uninfected females but not females singly infected with CI-causing Wolbachia. A rapidly spreading dominant nuclear suppressor genetic element affecting MK was identified through backcrossing and subsequent analysis with ddRAD SNPs of the D. pseudotakahashii genome. These findings highlight the complexity of nuclear and microbial components affecting MK endosymbiont detection and dynamics in populations and the challenges of making connections between endosymbionts and the host phenotypes affected by them.

Functional analysis of Wolbachia Cid effectors unravels cooperative interactions to target host chromatin during replication.

Wolbachia are common bacteria among terrestrial arthropods. These endosymbionts transmitted through the female germline manipulate their host reproduction through several mechanisms whose most prevalent form called Cytoplasmic Incompatibility -CI- is a conditional sterility syndrome eventually favoring the infected progeny. Upon fertilization, the sperm derived from an infected male is only compatible with an egg harboring a compatible Wolbachia strain, this sperm leading otherwise to embryonic death. The Wolbachia Cif factors CidA and CidB responsible for CI and its neutralization function as a Toxin-Antitoxin system in the mosquito host Culex pipiens. However, the mechanism of CidB toxicity and its neutralization by the CidA antitoxin remain unexplored. Using transfected insect cell lines to perform a structure-function analysis of these effectors, we show that both CidA and CidB are chromatin interactors and CidA anchors CidB to the chromatin in a cell-cycle dependent-manner. In absence of CidA, the CidB toxin localizes to its own chromatin microenvironment and acts by preventing S-phase completion, independently of its deubiquitylase -DUB- domain. Experiments with transgenic Drosophila show that CidB DUB domain is required together with CidA during spermatogenesis to stabilize the CidA-CidB complex. Our study defines CidB functional regions and paves the way to elucidate the mechanism of its toxicity.

Modeling Sustained Transmission of Wolbachia among Anopheles Mosquitoes: Implications for Malaria Control in Haiti.

Wolbachia infection in Anopheles albimanus mosquitoes can render mosquitoes less capable of spreading malaria. We developed and analyzed a mechanistic compartmental ordinary differential equation model to evaluate the effectiveness of Wolbachia-based vector control strategies among wild Anopheles mosquitoes in Haiti. The model tracks the mosquito life stages, including egg, larva, and adult (male and female). It also accounts for critical biological effects, such as the maternal transmission of Wolbachia through infected females and cytoplasmic incompatibility, which effectively sterilizes uninfected females when they mate with infected males. We derive and interpret dimensionless numbers, including the basic reproductive number and next-generation numbers. The proposed system presents a backward bifurcation, which indicates a threshold infection that needs to be exceeded to establish a stable Wolbachia infection. The sensitivity analysis ranks the relative importance of the epidemiological parameters at baseline. We simulate different intervention scenarios, including prerelease mitigation using larviciding and thermal fogging before the release, multiple releases of infected populations, and different release times of the year. Our simulations show that the most efficient approach to establishing Wolbachia is to release all the infected mosquitoes immediately after the prerelease mitigation process. Moreover, the model predicts that it is more efficient to release during the dry season than the wet season.

NDUFA8 potentially rescues Wolbachia-induced cytoplasmic incompatibility in Laodelphax striatellus.

The endosymbiont Wolbachia manipulates host reproduction by several strategies, one of the most important of which is cytoplasmic incompatibility (CI). CI can be rescued when Wolbachia-infected males mate with females infected with the same Wolbachia strain. However, the potential rescue mechanism of CI in the small brown planthopper Laodelphax striatellus is unclear. In this study, comparative transcriptome analysis was applied to explore the effect of Wolbachia on L. striatellus eggs. A total of 1387 differentially expressed genes were identified. RNA interference of 7 Wolbachia-upregulated key planthopper genes reduced egg reproduction, suggesting that Wolbachia might improve fecundity in L. striatellus by affecting these 7 genes. Suppressing the expression of another upregulated gene, NDUFA8 (encoding NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 8-like) by RNA interference significantly increased the mortality of early embryos without affecting the number of deposited eggs. Wolbachia infection upregulated the mRNA level of NDUFA8, and dsNDUFA8 treatment of Wolbachia-infected females recreated CI-like symptoms, suggesting that NDUFA8 is associated with the rescue phenotype. Because all L. striatellus populations worldwide are infected with Wolbachia, NDUFA8 is a potential pest control target.

A mosquito population suppression model with a saturated Wolbachia release strategy in seasonal succession

Releasing Wolbachia-infected male mosquitoes to suppress wild female mosquitoes through cytoplasmic incompatibility has shown great promise in controlling and preventing mosquito-borne diseases. To make the release logistically and economically feasible, we propose a saturated release strategy, which is only implemented during the epidemic season of mosquito-borne diseases. Under this assumption, the model becomes a seasonally switching ordinary differential equation model. The seasonal switch brings rich dynamics, including the existence of a unique periodic solution or exactly two periodic solutions, which are proved by using the qualitative property of the Poincaré map. Sufficient conditions are also obtained for determining the stability of the periodic solutions.

Wolbachia and Spiroplasma endosymbionts in the Anurida maritima (Collembola) species group

Abstract A large proportion of arthropods carry maternally-inherited endosymbiotic bacteria with which they have developed close relationships. Some of these endosymbionts are selfish genetic elements and manipulate their hosts’ reproduction to their own advantage, for example via cytoplasmic incompatibility (CI) or male killing. Here we report that the sexually reproducing collembolan Anurida maritima carries two endosymbionts (Wolbachia and Spiroplasma) and provide genome sequences for both bacteria. Phylogenomic analyses indicate that the Wolbachia belong to the A supergroup and that the Spiroplasma are sister to the Citri-Chrysopicola-Mirum lineage. A. maritima is considered a species group and consists of at least two distinct genetic lineages. We show that both lineages carry both endosymbionts. No homologs of the Spiroplasma male-killing gene SpAID were observed within our datasets. Homologs of the male-killing associated wmk and the CI inducing cifA and cifB prophage genes were detected in the Wolbachia genome. Phylogenetic analyses placed the cif genes in the poorly characterised Type V clade. The cifA and cifB gene sequences of the two Anurida lineages are identical. It therefore seems unlikely that the genetic divergence within the species group stems from cifA and cifB induced CI. Laboratory controlled genetic crosses and sex ratio studies will be needed to reveal any potential effect of the two endosymbionts on A. maritima’s reproduction.

Pangenomic analysis of Wolbachia provides insight into the evolution of host adaptation and cytoplasmic incompatibility factor genes.

The genus Wolbachia provides a typical example of intracellular bacteria that infect the germline of arthropods and filarial nematodes worldwide. Their importance as biological regulators of invertebrates, so it is particularly important to study the evolution, divergence and host adaptation of these bacteria at the genome-wide level. Here, we used publicly available Wolbachia genomes to reconstruct their evolutionary history and explore their adaptation under host selection. Our findings indicate that segmental and single-gene duplications, such as DNA methylase, bZIP transcription factor, heat shock protein 90, in single monophyletic Wolbachia lineages (including supergroups A and B) may be responsible for improving the ability to adapt to a broad host range in arthropod-infecting strains. In contrast to A strains, high genetic diversity and rapidly evolving gene families occur in B strains, which may promote the ability of supergroup B strains to adapt to new hosts and their large-scale spreading. In addition, we hypothesize that there might have been two independent horizontal transfer events of cif genes in two sublineages of supergroup A strains. Interestingly, during the independent evolution of supergroup A and B strains, the rapid evolution of cif genes in supergroup B strains resulted in the loss of their functional domain, reflected in a possible decrease in the proportion of induced cytoplasmic incompatibility (CI) strains. This present study highlights for reconstructing of evolutionary history, addressing host adaptation-related evolution and exploring the origin and divergence of CI genes in each Wolbachia supergroup. Our results thus not only provide a basis for further exploring the evolutionary history of Wolbachia adaptation under host selection but also reveal a new research direction for studying the molecular regulation of Wolbachia- induced cytoplasmic incompatibility.

Developing Wolbachia-based disease interventions for an extreme environment.

Aedes aegypti mosquitoes carrying self-spreading, virus-blocking Wolbachia bacteria are being deployed to suppress dengue transmission. However, there are challenges in applying this technology in extreme environments. We introduced two Wolbachia strains into Ae. aegypti from Saudi Arabia for a release program in the hot coastal city of Jeddah. Wolbachia reduced infection and dissemination of dengue virus (DENV2) in Saudi Arabian mosquitoes and showed complete maternal transmission and cytoplasmic incompatibility. Wolbachia reduced egg hatch under a range of environmental conditions, with the Wolbachia strains showing differential thermal stability. Wolbachia effects were similar across mosquito genetic backgrounds but we found evidence of local adaptation, with Saudi Arabian mosquitoes having lower egg viability but higher adult desiccation tolerance than Australian mosquitoes. Genetic background effects will influence Wolbachia invasion dynamics, reinforcing the need to use local genotypes for mosquito release programs, particularly in extreme environments like Jeddah. Our comprehensive characterization of Wolbachia strains provides a foundation for Wolbachia-based disease interventions in harsh climates.

Harnessing Wolbachia cytoplasmic incompatibility alleles for confined gene drive: A modeling study.

Wolbachia are maternally-inherited bacteria, which can spread rapidly in populations by manipulating reproduction. cifA and cifB are genes found in Wolbachia phage that are responsible for cytoplasmic incompatibility, the most common type of Wolbachia reproductive interference. In this phenomenon, no viable offspring are produced when a male with both cifA and cifB (or just cifB in some systems) mates with a female lacking cifA. Utilizing this feature, we propose new types of toxin-antidote gene drives that can be constructed with only these two genes in an insect genome, instead of the whole Wolbachia bacteria. By using both mathematical and simulation models, we found that a drive containing cifA and cifB together creates a confined drive with a moderate to high introduction threshold. When introduced separately, they act as a self-limiting drive. We observed that the performance of these drives is substantially influenced by various ecological parameters and drive characteristics. Extending our models to continuous space, we found that the drive individual release distribution has a critical impact on drive persistence. Our results suggest that these new types of drives based on Wolbachia transgenes are safe and flexible candidates for genetic modification of populations.

Single-cell transcriptome sequencing reveals Wolbachia-mediated modification in early stages of Drosophila spermatogenesis.

Wolbachia are the most widely distributed intracellular bacteria, and their most common effect on host phenotype is cytoplasmic incompatibility (CI). A variety of models have been proposed to decipher the molecular mechanism of CI, among which the host modification (HM) model predicts that Wolbachia effectors play an important role in sperm modification. However, owing to the complexity of spermatogenesis and testicular cell-type heterogeneity, whether Wolbachia have different effects on cells at different stages of spermatogenesis or whether these effects are linked with CI remains unknown. Therefore, we used single-cell RNA sequencing to analyse gene expression profiles in adult male Drosophila testes that were infected or uninfected by Wolbachia. We found that Wolbachia significantly affected the proportion of different types of germ cells and affected multiple metabolic pathways in germ cells. Most importantly, Wolbachia had the greatest impact on germline stem cells, resulting in dysregulated expression of genes related to DNA compaction, and Wolbachia infection also influenced the histone-to-protamine transition in the late stage of sperm development. These results support the HM model and suggest that future studies on Wolbachia-induced CI should focus on cells in the early stages of spermatogenesis.

Low copy numbers for mitochondrial DNA moderates the strength of nuclear-cytoplasmic incompatibility in plants.

Plant cells contain only small amounts of mitochondrial DNA (mtDNA), with the genomic information shared among multiple mitochondria. The biological relevance and molecular mechanism underlying this hallmark of plant cells has been unclear. Here, we report that Arabidopsis thaliana plants exhibited significantly reduced growth and mitochondrial dysfunction when the mtDNA copy number was increased to the degree that each mitochondrion possessed DNA. The amounts of mitochondrion-encoded transcripts increased several fold in the presence of elevated mtDNA levels. However, the efficiency of RNA editing decreased with this excess of mitochondrion-encoded transcripts, resulting in impaired assembly of mitochondrial complexes containing mtDNA-encoded subunits, such as respiratory complexes I and IV. These observations indicate the occurrence of nuclear-mitochondrial incompatibility in the cells with increased amounts of mtDNA and provide an initial answer to the fundamental question of why plant cells have much lower mtDNA levels than animal cells. We propose that keeping mtDNA levels low moderates nuclear-mitochondrial incompatibility and that this may be a crucial factor driving plant cells to restrict the copy numbers of mtDNA.

Rich and complex dynamics of a time-switched differential equation model for wild mosquito population suppression with Ricker-type density-dependent survival probability

<abstract><p>Dengue presents over 390 million cases worldwide yearly. Releasing <italic>Wolbachia</italic>-infected male mosquitoes to suppress wild mosquitoes via cytoplasmic incompatibility has proven to be a promising method for combating the disease. As cytoplasmic incompatibility causes early developmental arrest of the embryo during the larval stage, we introduce the Ricker-type survival probability to assess the resulting effects. For periodic and impulsive release strategies, our model switches between two ordinary differential equations. Owing to a Poincaré map and rigorous dynamical analyses, we give thresholds $ T^*, c^* $ and $ c^{**} (>c^*) $ for the release period $ T $ and the release amount $ c $. Then, we assume $ c > c^* $ and prove that our model admits a globally asymptotically stable periodic solution, provided $ T > T^* $, and it admits at most two periodic solutions when $ T < T^* $. Moreover, for the latter case, we assert that the origin is globally asymptotically stable if $ c\ge c^{**} $, and there exist two positive numbers such that whenever there is a periodic solution, it must initiate in an interval composed of the aforementioned two numbers, once $ c^* < c < c^{**} $. We also offer numerical examples to support the results. Finally, a brief discussion is given to evoke deeper insights into the Ricker-type model and to present our next research directions.</p></abstract>

Existence and stability of periodic solutions for a mosquito suppression model with incomplete cytoplasmic incompatibility

Cytoplasmic incompatibility (CI) caused by endosymbiotic bacteria Wolbachia has been an effective way to suppress and even eradicate wild vector mosquitoes. By assuming that CI is driven by impulsive and periodic releases of Wolbachia-infected males, we develop a time-switching ordinary differential equation model to characterize the impact of the release amount, the release period and the incomplete CI on the suppression efficiency. Sufficient conditions to guarantee the existence and uniqueness, or the existence of exactly two periodic solutions are obtained, together with their stability analyses.

A new species of Andricus Hartig, 1840 (Hymenoptera, Cynipidae) from China, with references to DNA taxonomy and Wolbachia infection.

In the present paper, a new species of cynipid gall wasp, Andricuselodeoides Liu & Pang, is described from several provinces in southern China. The new species is closely related to the recently redescribed A.mairei (Kieffer, 1906). In addition to differences in adult and gall morphology, the new species is also readily separated by COI sequences, with a 6.2-8.9% genetic distance between populations of the new species and those of A.mairei. A contrasting difference in sex ratios was also observed between the two species, with A.elodeoides extremely female-biased (95.5-97.8% female) while A.mairei male-biased to more balanced (5.4-43.5% female). PCR screening for Wolbachia infection further revealed contrasting infection rates between populations of A.elodeoides and A.mairei: the Wolbachia infection rate was 0% in A.elodeoides and 100% in A.mairei. Cytoplasmic incompatibility induced by Wolbachia is proposed as a potential mechanism of speciation of the sympatric A.elodeoides and A.mairei.

Male sex pheromone in the parasitoid wasp Nasonia longicornis: Chemical and behavioral analyses

The use of sex pheromones for the attraction of potential mating partners is widespread in insects. Species-specificity of these chemical signals is essential, particularly in closely related species with overlapping habitats. In parasitoid wasps of the genus Nasonia, it is the males that produce sex pheromones in their rectal vesicles. The genus consists of four species: N. vitripennis (Nv), N. giraulti (Ng), N. oneida (No), and N. longicornis (Nl). The cosmopolitan species Nv is sympatric with Ng and No in eastern North America and with Nl in the west. Interspecific courtship is common in Nasonia although hybridization is prevented in most combinations by Wolbachia-mediated cytoplasmic incompatibility. The pheromone of Nv males differs from all Nasonia congeners by an additional component that is believed to ensure precopulatory isolation from the sympatric congeners. Detailed investigations on these interactions, however, exist only for the species combination Nv/Ng. Here, we report the results of chemical and behavioral investigations on the sex pheromone of Nl males. The pheromone consists of (4R,5S)-5-hydroxy-4-decanolide (RS) and 4-methylquinazoline (MQ) as a minor component which are produced only after eclosion. Pheromone titers peaked 2 days after eclosion and remained constant on day three. The pheromone is deposited by abdomen dipping which males increasingly exhibited after mating or brief contact with a female. The presence of hosts containing females about to emerge did not increase marking behavior. Site fidelity of males at their own pheromone markings is mediated by MQ. Both natural and synthetic pheromone attracted virgin but not mated females and both RS and MQ are required for female attraction. Females collected during emergence from the host responded likewise to the pheromone and 84% of them produced mixed-sex offspring showing that most of them emerge unmated. Nl females preferred pheromone extract from conspecific males to extract from Nv males, and the addition of the Nv-specific component (4R,5R)-5-hydroxy-4-decanolide (RR) to the Nl pheromone made them unresponsive. The present study demonstrates that Nl uses the male sex pheromone in a similar way as previously shown for Nv and Ng. Furthermore, it shows that Nl females use the Nv-specific pheromone component RR to avoid costly sexual interactions with Nv males in regions of sympatry.