The mosaic genome structure of the Wolbachia wRi strain infecting Drosophila simulans

The obligate intracellular bacterium Wolbachia pipientis infects around 20% of all insect species. It is maternally inherited and induces reproductive alterations of insect populations by male killing, feminization, parthenogenesis, or cytoplasmic incompatibility. Here, we present the 1,445,873-bp genome of W. pipientis strain wRi that induces very strong cytoplasmic incompatibility in its natural host Drosophila simulans. A comparison with the previously sequenced genome of W. pipientis strain wMel from Drosophila melanogaster identified 35 breakpoints associated with mobile elements and repeated sequences that are stable in Drosophila lines transinfected with wRi. Additionally, 450 genes with orthologs in wRi and wMel were sequenced from the W. pipientis strain wUni, responsible for the induction of parthenogenesis in the parasitoid wasp Muscidifurax uniraptor. The comparison of these A-group Wolbachia strains uncovered the most highly recombining intracellular bacterial genomes known to date. This was manifested in a 500-fold variation in sequence divergences at synonymous sites, with different genes and gene segments supporting different strain relationships. The substitution-frequency profile resembled that of Neisseria meningitidis, which is characterized by rampant intraspecies recombination, rather than that of Rickettsia, where genes mostly diverge by nucleotide substitutions. The data further revealed diversification of ankyrin repeat genes by short tandem duplications and provided examples of horizontal gene transfer across A- and B-group strains that infect D. simulans. These results suggest that the transmission dynamics of Wolbachia and the opportunity for coinfections have created a freely recombining intracellular bacterial community with mosaic genomes.     

Wolbachia strain wMel induces cytoplasmic incompatibility and blocks dengue transmission in Aedes albopictus

Wolbachia inherited bacteria are able to invade insect populations using cytoplasmic incompatibility and provide new strategies for controlling mosquito-borne tropical diseases, such as dengue. The overreplicating wMelPop strain was recently shown to strongly inhibit the replication of dengue virus when introduced into Aedes aegypti mosquitoes, as well as to stimulate chronic immune up-regulation. Here we show that stable introduction of the wMel strain of Drosophila melanogaster into Aedes albopictus, a vector of dengue and other arboviruses, abolished the transmission capacity of dengue virus-challenged mosquitoes. Immune up-regulation was observed in the transinfected line, but at a much lower level than that previously found for transinfected Ae. aegypti. Transient infection experiments suggest that this difference is related to Ae. albopictus immunotolerance of Wolbachia, rather than to the Wolbachia strain used. This study provides an example of strong pathogen inhibition in a naturally Wolbachia-infected mosquito species, demonstrating that this inhibition is not limited to naturally naïve species, and suggests that the Wolbachia strain is more important than host background for viral inhibition. Complete bidirectional cytoplasmic incompatibility was observed with WT strains infected with the naturally occurring Ae. albopictus Wolbachia, and this provides a mechanism for introducing wMel into natural populations of this species.The Asian tiger mosquito Aedes albopictus, a native of southeast Asia that in recent decades has invaded Africa, the Americas, and southern Europe, is now an important rural/semiurban vector of dengue virus across the tropics (1). It likely is involved in maintaining sylvatic cycles of transmission and acting as a bridge vector from these to urban epidemic cycles; it also transmits other Flaviviruses, such as yellow fever and West Nile, and the Alphavirus chikungunya. Like the primary urban dengue vector Aedes aegypti, Ae. albopictus is a day-biting species and thus is not amenable to control/prevention using insecticide-treated bed nets. This factor, along with the absence of a vaccine for dengue and the expanding disease range, calls for new methods of control.All known wild populations of Ae. albopictus are naturally infected with two strains of the maternally inherited bacterium Wolbachia pipientis, known as wAlbA and wAlbB (2, 3); Ae. aegypti is naturally uninfected with the bacterium. Recent work has shown that when an overreplicating strain of Wolbachia from Drosophila melanogaster, wMelPop, was transferred into Ae. aegypti (4), the dissemination of dengue virus was strongly inhibited, as was the dissemination of chikungunya virus (5). In addition, transfer of the wAlbB strain from Ae. albopictus into Ae. aegypti (6) led to reduced susceptibility to dengue (7). Both Wolbachia strains also induced cytoplasmic incompatibility (CI) in Ae. aegypti, whereby uninfected females mated with infected males produce embryos that die shortly after fertilization. This mechanism is used by Wolbachia to spread through insect populations because in contrast, infected females can mate successfully with either infected or uninfected males, giving them a frequency-dependent reproductive advantage (8–10). Thus, the combination of viral inhibition and a built-in self-spreading mechanism provides attractive prospects for the control of dengue transmission by Ae. aegypti (11).In addition to life shortening, the wMelPop strain also causes chronic immune up-regulation in Ae. aegypti (5, 12). The Toll pathway, some components of which are up-regulated in Ae. aegypti in the presence of wMelPop (5, 12), has been shown to play a role in the control of dengue dissemination in Ae. aegypti (13, 14). A general role of immune up-regulation in pathogen inhibition is also supported by the knockdown of the major immune gene TEP1, which partially rescues the inhibitory effect of the presence of wMelPop on Plasmodium berghei development in transiently infected Anopheles gambiae (15). The fact that the wAlbB transinfection caused dengue inhibition in Ae. aegypti (7) even through the original host of this Wolbachia strain Ae. albopictus is a fairly efficient dengue vector suggests a significant contribution of host background to the dengue inhibition phenotype, which possibly could be mediated by the increased immune response to Wolbachia found in a novel insect host. Thus, it is unclear whether any Wolbachia strain can produce strong dengue inhibition in a naturally Wolbachia-infected mosquito such as Ae. albopictus, which would be expected to have acquired a degree of immune tolerance to Wolbachia over time.The wMelPop strain overreplicates and can approximately halve the lifespan of both its D. melanogaster (16) and Ae. aegypti (4) hosts. However, a wMelPop transinfection into Ae. albopictus also produced a greatly reduced egg hatch from intrastrain matings, and this appeared to preclude its application to disease control in Ae. albopictus (17). The wMel strain, which is phylogenetically close to the wMelPop variant (18), does not produce the life-shortening phenotype of the latter in its native D. melanogaster host (16). However, wMel can significantly delay the accumulation of RNA viruses, such as Drosophila C virus, in D. melanogaster (19–21). Thus, we selected wMel for experimental transfer into Ae. albopictus to examine whether this strain is capable of producing dengue inhibition and CI in this new host background.     

Fitness of wAlbB Wolbachia Infection in Aedes aegypti: Parameter Estimates in an Outcrossed Background and Potential for Population Invasion

Wolbachia endosymbionts are potentially useful tools for suppressing disease transmission by Aedes aegypti mosquitoes because Wolbachia can interfere with the transmission of dengue and other viruses as well as causing deleterious effects on their mosquito hosts. Most recent research has focused on the wMel infection, but other infections also influence viral transmission and may spread in natural populations. Here, we focus on the wAlbB infection in an Australian outbred background and show that this infection has many features that facilitate its invasion into natural populations including strong cytoplasmic incompatibility, a lack of effect on larval development, an equivalent mating success to uninfected males and perfect maternal transmission fidelity. On the other hand, the infection has deleterious effects when eggs are held in a dried state, falling between wMel and the more virulent wMelPop Wolbachia strains. The impact of this infection on lifespan also appears to be intermediate, consistent with the observation that this infection has a titer in adults between wMel and wMelPop. Population cage experiments indicate that the wAlbB infection establishes in cages when introduced at a frequency of 22%, suggesting that this strain could be successfully introduced into populations and subsequently persist and spread.     

Molecular strain typing of Wolbachia infection from Indian mosquitoes using wsp gene

ObjectiveTo determine the status of Wolbachia subgroup and phylogenetic relationships in Indian mosquitoes.MethodsRecently we reported Wolbachia infection in eight out of twenty field-caught mosquito species of India, using wsp specific primers. DNA extracted from these mosquito species were used for PCR amplification and sequencing.ResultsWolbachia A harboured in Aedes albopictus and Culex gelidus belongs to the subgroup AlbA whereas Wolbachia B harboured in Aedes albopictus and Culex quinquefasciatus belongs to the subgroup Pip and of Culex vishnui belongs to subgroup Con. However, Wolbachia harboured in Armigeres subalbatus, Armigeres kesseli, Culex sitiens and Toxorhynchites splendens could not be placed into any known subgroup and may represent other unknown strains of Wolbachia. Our phylogenetic analysis revealed eight novel Wolbachia strains, four in the A group and four in the B group. Most of the Wolbachia strains present in Indian mosquitoes belong to the Albo, Pip and Con groups.ConclusionsThe similarities and differences between Wolbachia strains infecting different mosquito species are fundamental for estimating how easily mosquitoes acquire new infections.     

Evolutionary origin of insect–Wolbachia nutritional mutualism

Obligate insect–bacterium nutritional mutualism is among the most sophisticated forms of symbiosis, wherein the host and the symbiont are integrated into a coherent biological entity and unable to survive without the partnership. Originally, however, such obligate symbiotic bacteria must have been derived from free-living bacteria. How highly specialized obligate mutualisms have arisen from less specialized associations is of interest. Here we address this evolutionary issue by focusing on an exceptional insect–Wolbachia nutritional mutualism. Although Wolbachia endosymbionts are ubiquitously found in diverse insects and generally regarded as facultative/parasitic associates for their insect hosts, a Wolbachia strain associated with the bedbug Cimex lectularius, designated as wCle, was shown to be essential for host’s growth and reproduction via provisioning of B vitamins. We determined the 1,250,060-bp genome of wCle, which was generally similar to the genomes of insect-associated facultative Wolbachia strains, except for the presence of an operon encoding the complete biotin synthetic pathway that was acquired via lateral gene transfer presumably from a coinfecting endosymbiont Cardinium or Rickettsia. Nutritional and physiological experiments, in which wCle-infected and wCle-cured bedbugs of the same genetic background were fed on B-vitamin–manipulated blood meals via an artificial feeding system, demonstrated that wCle certainly synthesizes biotin, and the wCle-provisioned biotin significantly contributes to the host fitness. These findings strongly suggest that acquisition of a single gene cluster consisting of biotin synthesis genes underlies the bedbug–Wolbachia nutritional mutualism, uncovering an evolutionary transition from facultative symbiosis to obligate mutualism facilitated by lateral gene transfer in an endosymbiont lineage.Symbiotic associations are ubiquitous in the biological world, in which obligate insect–bacterium endosymbiotic associations are among the most sophisticated forms wherein the host and the symbiont are integrated into a coherent biological entity and cannot survive without the partnership (1, 2). For example, in the aphid–Buchnera nutritional mutualism, the host depends on the symbiont for supply of essential amino acids that are needed for host’s protein synthesis but are scarce in the host’s plant sap diet (3). In the tsetse–Wigglesworthia nutritional mutualism, the symbiont provides B vitamins that are deficient in vertebrate blood the host exclusively feeds on (4). Through the intimate relationship over evolutionary time, these and other endosymbiont genomes have been reduced drastically, losing many genes needed for independent life and streamlined for specific biological roles to support their hosts (5, 6). Novel biological properties acquired through endosymbiosis have played substantial roles in adaptation, evolution, and diversification of insects and other organisms (1, 2). Although currently comprising elaborate symbiotic systems, such endosymbionts must have originally been derived from free-living ancestors. How highly specialized obligate endosymbionts have arisen from less specialized bacterial associates is of evolutionary interest.Members of the genus Wolbachia are well known as facultative bacterial endosymbionts ubiquitously associated with diverse insects, generally conferring negative fitness consequences to their hosts and often causing hosts’ reproductive aberrations to enhance their own transmission in a selfish manner (7, 8). Recently, however, a Wolbachia strain associated with the bedbug Cimex lectularius, designated as wCle, was shown to be essential for normal growth and reproduction of the blood-sucking insect host via provisioning of B vitamins (9). Hence, it is expected that a transition from facultative association to obligate mutualism may have occurred in an ancestor of wCle. What evolutionary processes and mechanisms are involved in the emergence of the insect–Wolbachia nutritional mutualism?In this study, we determined the complete genome of wCle, which was similar in size and composition to the genomes of facultative Wolbachia endosymbionts associated with other insects, except for the presence of an operon encoding biotin synthesis pathway that was presumably acquired via lateral gene transfer from an unrelated bacterium. Using wCle-infected and wCle-cured bedbug strains under the same genetic background, we experimentally demonstrated that wCle is capable of synthesizing biotin and wCle-provisioned biotin significantly contributes to the host fitness, thereby uncovering a genomic basis of the insect–Wolbachia nutritional mutualism. Through comprehensive survey of Wolbachia genomic data, we discuss evolutionary hypotheses as to how and when the biotin operon was acquired by wCle in the course of insect–Wolbachia coevolution.     

Larval Competition Extends Developmental Time and Decreases Adult Size of wMelPop Wolbachia-Infected Aedes aegypti

The intracellular endosymbiont Wolbachia has been artificially transinfected into the dengue vector Aedes aegypti, where it is being investigated as a potential dengue biological control agent. Invasion of Wolbachia in natural populations depends upon the fitness of Wolbachia-infected Ae. aegypti relative to uninfected competitors. Although Wolbachia infections impose fitness costs on the adult host, effects at the immature stages are less clear, particularly in competitive situations. We look for effects of two Wolbachia infections, wMel and wMelPop, on intra-strain and inter-strain larval competition in Ae. aegypti. Development of Wolbachia-infected larvae is delayed in mixed cohorts with uninfected larvae under crowded-rearing conditions. Slow developing wMelPop-infected larvae have reduced adult size compared with uninfected larvae, and larvae with the wMel infection are somewhat larger and have greater viability relative to uninfected larvae when in mixed cohorts. Implications for successful invasion by these Wolbachia infections under field conditions are considered.     

A single mutation weakens symbiont-induced reproductive manipulation through reductions in deubiquitylation efficiency

Animals interact with microbes that affect their performance and fitness, including endosymbionts that reside inside their cells. Maternally transmitted Wolbachia bacteria are the most common known endosymbionts, in large part because of their manipulation of host reproduction. For example, many Wolbachia cause cytoplasmic incompatibility (CI) that reduces host embryonic viability when Wolbachia-modified sperm fertilize uninfected eggs. Operons termed cifs control CI, and a single factor (cifA) rescues it, providing Wolbachia-infected females a fitness advantage. Despite CI’s prevalence in nature, theory indicates that natural selection does not act to maintain CI, which varies widely in strength. Here, we investigate the genetic and functional basis of CI-strength variation observed among sister Wolbachia that infect Drosophila melanogaster subgroup hosts. We cloned, Sanger sequenced, and expressed cif repertoires from weak CI–causing wYak in Drosophila yakuba, revealing mutations suspected to weaken CI relative to model wMel in D. melanogaster. A single valine-to-leucine mutation within the deubiquitylating (DUB) domain of the wYak cifB homolog (cidB) ablates a CI-like phenotype in yeast. The same mutation reduces both DUB efficiency in vitro and transgenic CI strength in the fly, each by about twofold. Our results map hypomorphic transgenic CI to reduced DUB activity and indicate that deubiquitylation is central to CI induction in cid systems. We also characterize effects of other genetic variation distinguishing wMel-like cifs. Importantly, CI strength determines Wolbachia prevalence in natural systems and directly influences the efficacy of Wolbachia biocontrol strategies in transinfected mosquito systems. These approaches rely on strong CI to reduce human disease.

Many endosymbionts spread through host populations by manipulating their reproduction. For example, Rickettsiella (1), Mesenet (2), Cardinium (3), and Wolbachia (4) all cause cytoplasmic incompatibility (CI) that reduces the viability of uninfected host embryos fertilized by symbiont-modified sperm (5–8). CI is common among Wolbachia bacterial strains, being observed in at least 10 arthropod orders (6). CI strength influences Wolbachia prevalence, with stronger CI producing higher Wolbachia infection frequencies in host populations (8, 9). Indeed, CI contributes significantly to Wolbachia’s status as the most-common known endosymbionts in nature (10).CI strength directly influences the efficacy of Wolbachia biocontrol programs, with vector-control groups relying on strong CI to either suppress mosquito populations (11, 12) or to transform them with pathogen-blocking Wolbachia like wMel that naturally infects Drosophila melanogaster (13–15). The World Health Organization recommends further developing these programs (16), which are currently protecting seven million people from disease with a goal of protecting half a billion by 2030 (14, 17).Operons generally termed cifs control CI (cifA/B) (5, 18–22), and CI induction can be rescued by one factor (cifA) (5, 19, 23). Theory indicates that natural selection does not act to increase or maintain CI (24), which varies considerably among even very closely related Wolbachia (25–27), potentially due to mutational erosion of cifs (28). For example, CI strength differs significantly among model wMel from Drosophila melanogaster and closely related wMel-like Wolbachia in the Drosophila yakuba clade (wYak, wSan, and wTei) that wMel diverged from in only the last 30,000 y (25, 27, 29, 30). We sought to determine how much and why naturally observed mutations in wMel-like cifs influence CI strength.     

The role of Wolbachia bacteria in reproductive incompatibilities and hybrid zones of Diabrotica beetles and Gryllus crickets

A rickettsial bacterium in the genus Wolbachia is the cause of a unidirectional reproductive incompatibility observed between two major beetle pests of maize, the western corn rootworm, Diabrotica virgifera virgifera, and the Mexican corn rootworm, D. v. zeae. These subspecies are allopatric except for two known regions of sympatry in Texas and Mexico. We demonstrate that populations of D. v. virgifera, with the exception of two populations in southern Arizona, are infected with a strain of Wolbachia. Populations of D. v. zeae are not infected. Treatment of D. v. virgifera with tetracycline eliminated the Wolbachia and removed the reproductive incompatibility. Similar patterns of reproductive incompatibility exist among taxa of the cricket genus Gryllus. Gryllus assimilis, G. integer, G. ovisopis, G. pennsylvanicus, and G. rubens are infected with Wolbachia whereas G. firmus is usually not. Populations of G. rubens and G. ovisopis carry the same Wolbachia strain, which is distinct from that of G. integer. G. pennsylvanicus is infected with two Wolbachia strains, that found in G. rubens and one unique to G. pennsylvanicus. Moreover, a proportion of G. pennsylvanicus individuals harbors both strains. Wolbachia may have influenced speciation in some members of the genus Gryllus by affecting the degree of hybridization between species. Given that Wolbachia infections are relatively common in insects, it is likely that other insect hybrid zones may be influenced by infections with Wolbachia.     

Evolutionarily conserved Wolbachia-encoded factors control pattern of stem-cell niche tropism in Drosophila ovaries and favor infection

Wolbachia are intracellular bacteria that infect invertebrates at pandemic levels, including insect vectors of devastating infectious diseases. Although Wolbachia are providing novel strategies for the control of several human pathogens, the processes underlying Wolbachia’s successful propagation within and across species remain elusive. Wolbachia are mainly vertically transmitted; however, there is also evidence of extensive horizontal transmission. Here, we provide several lines of evidence supporting Wolbachia’s targeting of ovarian stem cell niches—referred to as “niche tropism”—as a previously overlooked strategy for Wolbachia thriving in nature. Niche tropism is pervasive in Wolbachia infecting the Drosophila genus, and different patterns of niche tropism are evolutionarily conserved. Phylogenetic analysis, confirmed by hybrid introgression and transinfection experiments, demonstrates that bacterial factors are the major determinants of differential patterns of niche tropism. Furthermore, bacterial load is increased in germ-line cells passing through infected niches, supporting previous suggestions of a contribution of Wolbachia from stem-cell niches toward vertical transmission. These results support the role of stem-cell niches as a key component for the spreading of Wolbachia in the Drosophila genus and provide mechanistic insights into this unique tissue tropism.     

Induction of regulatory T cells by macrophages is dependent on production of reactive oxygen species

The phagocyte NAPDH–oxidase complex consists of several phagocyte oxidase (phox) proteins, generating reactive oxygen species (ROS) upon activation. ROS are involved in the defense against microorganisms and also in immune regulation. Defective ROS formation leads to chronic granulomatous disease (CGD) with increased incidence of autoimmunity and disturbed resolution of inflammation. Because regulatory T cells (Tregs) suppress autoimmune T-cell responses and are crucial in down-regulating immune responses, we hypothesized that ROS deficiency may lead to decreased Treg induction. Previously, we showed that in p47^phox-mutated mice, reconstitution of macrophages (Mph) with ROS-producing capacity was sufficient to protect the mice from arthritis. Now, we present evidence that Mph-derived ROS induce Tregs. In vitro, we showed that Mph ROS-dependently induce Treg, using an NADPH-oxidase inhibitor. This finding was confirmed genetically: rat or human CGD Mph with mutated p47^phox or gp91^phox displayed hampered Treg induction and T-cell suppression. However, basal Treg numbers in these subjects were comparable to those in controls, indicating a role for ROS in induction of peripheral Tregs. Induction of allogeneic delayed-type hypersensitivity with p47^phox-mutated Mph confirmed the importance of Mph-derived ROS in Treg induction in vivo. We conclude that NAPDH oxidase activity in Mph is important for the induction of Tregs to regulate T cell-mediated inflammation.     

Wolbachia, normally a symbiont of Drosophila, can be virulent, causing degeneration and early death

Wolbachia, a maternally transmitted microorganism of the Rickettsial family, is known to cause cytoplasmic incompatibility, parthenogenesis, or feminization in various insect species. The bacterium–host relationship is usually symbiotic: incompatibility between infected males and uninfected females can enhance reproductive isolation and evolution, whereas the other mechanisms enhance progeny production. We have discovered a variant Wolbachia carried by Drosophila melanogaster in which this cozy relationship is abrogated. Although quiescent during the fly’s development, it begins massive proliferation in the adult, causing widespread degeneration of tissues, including brain, retina, and muscle, culminating in early death. Tetracycline treatment of carrier flies eliminates both the bacteria and the degeneration, restoring normal life-span. The 16s rDNA sequence is over 98% identical to Wolbachia known from other insects. Examination of laboratory strains of D. melanogaster commonly used in genetic experiments reveals that a large proportion actually carry Wolbachia in a nonvirulent form, which might affect their longevity and behavior.     

Wolbachia as a bacteriocyte-associated nutritional mutualist

Many insects are dependent on bacterial symbionts that provide essential nutrients (ex. aphid–Buchnera and tsetse–Wiglesworthia associations), wherein the symbionts are harbored in specific cells called bacteriocytes that constitute a symbiotic organ bacteriome. Facultative and parasitic bacterial symbionts like Wolbachia have been regarded as evolutionarily distinct from such obligate nutritional mutualists. However, we discovered that, in the bedbug Cimex lectularius, Wolbachia resides in a bacteriome and appears to be an obligate nutritional mutualist. Two bacterial symbionts, a Wolbachia strain and an unnamed γ-proteobacterium, were identified from different strains of the bedbug. The Wolbachia symbiont was detected from all of the insects examined whereas the γ-proteobacterium was found in a part of them. The Wolbachia symbiont was specifically localized in the bacteriomes and vertically transmitted via the somatic stem cell niche of germalia to oocytes, infecting the incipient symbiotic organ at an early stage of the embryogenesis. Elimination of the Wolbachia symbiont resulted in retarded growth and sterility of the host insect. These deficiencies were rescued by oral supplementation of B vitamins, confirming the essential nutritional role of the symbiont for the host. The estimated genome size of the Wolbachia symbiont was around 1.3 Mb, which was almost equivalent to the genome sizes of parasitic Wolbachia strains of other insects. These results indicate that bacteriocyte-associated nutritional mutualism can evolve from facultative and prevalent microbial associates like Wolbachia, highlighting a previously unknown aspect of the parasitism-mutualism evolutionary continuum.     

Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.Bacteria in the genus Wolbachia are maternally transmitted Rickettsia-like endosymbionts that infect an estimated 40–69% of arthropod species (1, 2). In many cases, Wolbachia manipulate host reproduction to spread throughout arthropod populations (3). Incongruence between Wolbachia and host phylogenies indicate that horizontal transfer of the symbiont has been commonplace over evolutionary time (4, 5), enabling Wolbachia to invade new species. However, there is a poor understanding of barriers to horizontal transmission and why some species remain uninfected. An understanding of these factors is important from an evolutionary perspective given that Wolbachia influences speciation (6, 7), and from an applied perspective as Wolbachia is being transinfected into vector species for the control of arthropod-borne disease (8–10).The ability to invade the host germ line is an important feature of Wolbachia biology that facilitates horizontal transmission, leading to the pervasive nature of this bacterium across invertebrate taxa. In order for Wolbachia to become established in a naïve host species, it must be acquired horizontally and successfully transmitted vertically (i.e., to offspring) to maintain the infection in the population. Multiple mechanisms of Wolbachia horizontal transmission have been proposed, including cohabitation, hemolymph transfer, predation, and parasitoid infection (11–15). After microinjection into Drosophila, Wolbachia infects the stem cell niches in the germ line (16, 17), and both Wolbachia-derived and host factors appear to influence tropism and bacterial density during oogenesis (17–20). Alternatively, somatic tissue may act as a reservoir for Wolbachia infection of the developing oocyte (20–23). Although pathways of horizontal transmission have been characterized in some species, identification of barriers to vertical transmission of the acquired Wolbachia infection remains elusive.Microbial conflict or incompatibility within arthropods is a potential barrier to transmission of heritable symbionts. Studies in the tick Dermacentor variabilis demonstrate competitive exclusion between maternally inherited bacteria. Transovarial transmission of Rickettsia montanensis (formerly Rickettsia montana) and Rickettsia rhipicephali is inhibited by infection with the reciprocal species (24). Similarly, infection exclusion has been observed in D. variabilis between conspecific strains of Anaplasma marginale where one strain inhibits the infection of the other (25). Competitive inter- and intraspecific microbial interactions have also been observed with Wolbachia (26, 27).Anopheles mosquitoes provide a unique system to examine microbial barriers to Wolbachia transmission. With few exceptions, Anophelines (which transmit the Plasmodium parasites that cause human malaria) are naturally uninfected with Wolbachia (28–31), suggesting the potential presence of innate barriers to infection in this genus. However, in vitro and in vivo studies indicate that Wolbachia are capable of infecting cultured Anopheles cells (32, 33), ex vivo cultured tissues (34), in vivo somatic tissue (35–37), and can stably infect the mosquito germ line (38). We investigated the ability of the native microbial community to influence vertical transmission of Wolbachia in Anopheles mosquitoes. We found that bacteria in the genus Asaia were responsible for inhibiting Wolbachia maternal transmission in this important mosquito genus.     

Pharmacological inhibition of NOX reduces atherosclerotic lesions,vascular ROS and immune–inflammatory responses in diabetic Apoe −/− mice

Aims/hypothesis

Enhanced vascular inflammation, immune cell infiltration and elevated production of reactive oxygen species (ROS) contribute significantly to pro-atherogenic responses in diabetes. We assessed the immunomodulatory role of NADPH oxidase (NOX)-derived ROS in diabetes-accelerated atherosclerosis.

Methods

Diabetes was induced in male Apoe ^?/? mice with five daily doses of streptozotocin (55 mg kg^?1 day^?1). Atherosclerotic plaque size, markers of ROS and immune cell accumulation were assessed in addition to flow cytometric analyses of cells isolated from the adjacent mediastinal lymph nodes (meLNs). The role of NOX-derived ROS was investigated using the NOX inhibitor, GKT137831 (60 mg/kg per day; gavage) administered to diabetic and non-diabetic Apoe ^?/? mice for 10 weeks.

Results

Diabetes increased atherosclerotic plaque development in the aortic sinus and this correlated with increased lesional accumulation of T cells and CD11c⁺ cells and altered T cell activation in the adjacent meLNs. Diabetic Apoe ^?/? mice demonstrated an elevation in vascular ROS production and expression of the proinflammatory markers monocyte chemoattractant protein 1, vascular adhesion molecule 1 and IFNγ. Blockade of NOX-derived ROS using GKT137831 prevented the diabetes-mediated increase in atherosclerotic plaque area and associated vascular T cell infiltration and also significantly reduced vascular ROS as well as markers of inflammation and plaque necrotic core area.

Conclusions/interpretation

Diabetes promotes pro-inflammatory immune responses in the aortic sinus and its associated lymphoid tissue. These changes are associated with increased ROS production by NOX. Blockade of NOX-derived ROS using the NOX inhibitor GKT137831 is associated with attenuation of these changes in the immune response and reduces the diabetes-accelerated development of atherosclerotic plaques in Apoe ^?/? mice.     

Body Size and Wing Shape Measurements as Quality Indicators of Aedes aegypti Mosquitoes Destined for Field Release

There is increasing interest in rearing modified mosquitoes for mass release to control vector-borne diseases, particularly Wolbachia-infected Aedes aegypti for suppression of dengue. Successful introductions require release of high quality mosquitoes into natural populations. Potential indicators of quality are body size and shape. We tested to determine if size, wing/thorax ratio, and wing shape are associated with field fitness of Wolbachia-infected Ae. aegypti. Compared with field-collected mosquitoes, released mosquitoes were larger in size, with lower size variance and different wing shape but similar in wing-thorax ratio and its associated variance. These differences were largely attributed to nutrition and to a minor extent to wMel Wolbachia infection. Survival potential of released female mosquitoes was similar to those from the field. Females at oviposition sites tended to be larger than those randomly collected from BG-Sentinel traps. Rearing conditions should thus aim for large size without affecting wing/thorax ratios.     

Zinc protoporphyrin IX enhances chemotherapeutic response of hepatoma cells to cisplatin

AIM: To investigate the effect of zinc protoporphyrin IX on the response of hepatoma cells to cisplatin and the possible mechanism involved.METHODS: Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by a flow cytometric assay. Western blotting was used to measure protein expression. Heme oxygenase (HO)-1 activity was measured by determining the level of bilirubin generated in isolated microsomes. Reactive oxygen species (ROS) production was monitored by flow cytometry. Caspase-3 activity was measured with a colorimetric assay kit. Mice were inoculated with 1 × 10⁷ tumor cells subcutaneously into the right flanks. All mice were sacrificed 6 wk after the first treatment and tumors were weighed and measured.RESULTS: Overexpression of HO-1 in HepG2 cell line was associated with increased chemoresistance to cis-diaminedichloroplatinum (cisplatin; CDDP) compared to other cell lines in vitro. Inhibition of HO-1 expression or activity by zinc protoporphyrin IX (ZnPP IX) markedly augmented CDDP-mediated cytotoxicity towards all liver cancer cell lines in vitro and in vivo. In contrast, induction of HO-1 with hemin increased resistance of tumor cells to CDDP-mediated cytotoxicity in vitro and in vivo. Furthermore, cells treated with ZnPP IX plus CDDP exhibited marked production of intracellular ROS and caspase-3 activity, which paralleled the incidence of cell apoptosis, whereas hemin decreased cellular ROS and caspase-3 activity induced by CDDP.CONCLUSION: ZnPP IX increases cellular sensitivity and susceptibility of liver cancer cell lines to CDDP and this may represent a mechanism of increasing ROS.     

Releasing incompatible males drives strong suppression across populations of wild and Wolbachia-carrying Aedes aegypti in Australia

Releasing sterile or incompatible male insects is a proven method of population management in agricultural systems with the potential to revolutionize mosquito control. Through a collaborative venture with the “Debug” Verily Life Sciences team, we assessed the incompatible insect technique (IIT) with the mosquito vector Aedes aegypti in northern Australia in a replicated treatment control field trial. Backcrossing a US strain of Ae. aegypti carrying Wolbachia wAlbB from Aedes albopictus with a local strain, we generated a wAlbB2-F4 strain incompatible with both the wild-type (no Wolbachia) and wMel-Wolbachia Ae. aegypti now extant in North Queensland. The wAlbB2-F4 strain was manually mass reared with males separated from females using Verily sex-sorting technologies to obtain no detectable female contamination in the field. With community consent, we delivered a total of three million IIT males into three isolated landscapes of over 200 houses each, releasing ∼50 males per house three times a week over 20 wk. Detecting initial overflooding ratios of between 5:1 and 10:1, strong population declines well beyond 80% were detected across all treatment landscapes when compared to controls. Monitoring through the following season to observe the ongoing effect saw one treatment landscape devoid of adult Ae. aegypti early in the season. A second landscape showed reduced adults, and the third recovered fully. These encouraging results in suppressing both wild-type and wMel-Ae. aegypti confirms the utility of bidirectional incompatibility in the field setting, show the IIT to be robust, and indicate that the removal of this arbovirus vector from human-occupied landscapes may be achievable.

Mosquitoes transmit parasites and viruses that infect hundreds of millions of humans annually. Globally, one invasive species—Aedes aegypti (Linnaeus)—is responsible for the greatest transmission of arboviruses, causing diseases including yellow fever, dengue, chikungunya, and Zika (1). The paucity of effective vaccines for most of these diseases and observed concerns for both growing insecticide resistance and insecticide’s adverse effects on other beneficial species have prompted renewed interest in species-specific biological control methods (2).Releasing sterile or incompatible male insects en masse is one method of insect population control currently under development (3). The sterile insect technique (SIT) uses the release of sterile male insects that search and mate with wild females to prevent subsequent production of offspring. The SIT has been successfully used to control various insect pest species (4), including the New World screwworm fly (5), the tsetse fly (6), the Mediterranean fruit fly (7), and the apple codling moth (8). Previous SIT technologies have been hampered by their need for radiation or chemical sterilents, which can compromise male fitness, requiring the release of more sterile males to compensate for their fitness decline (9, 10). Furthermore, the development of genetic modification to generate sterile male Ae. aegypti mosquitoes has been hampered by perceived environmental risks as well as strong regulatory issues and community acceptance (11).The first applications of SIT to mosquitoes encountered mixed successes during the 1970s and ’80s (12), as it proved difficult to produce sufficient numbers of competitive sterile males to suppress natural populations. More recently, elegant transgenic approaches have generated sterile male mosquitoes for the malaria vector Anopheles gambiae (13), the dengue vector Ae. aegypti (3, 14), and the Asian tiger mosquito Aedes albopictus (15). Releases of transgenic sterile Ae. aegypti by Oxitec Ltd in Grand Cayman have demonstrated an effective reduction of these mosquitoes (16).Reproductively incompatible males can now be produced using a maternally inherited gram-negative endosymbiotic bacterium Wolbachia pipientis (17, 18). Many of these strains carry a cytoplasmic incompatibility (CI) phenotype, where males of one Wolbachia strain can be reproductively incompatible with females that do not carry the strain; thus, females lay eggs that fail to hatch (18). This scenario provides an opportunity to reassess the SIT using what is now termed the incompatible insect technique [IIT (19)]. The SIT is generally more effective when females are not released (20), as sterile female insects can still damage crops, transmit disease, or simply distract sterile males from searching out wild mates. In the case of mosquito SIT and IIT, releasing males means only releasing mosquitoes which do not bite, have fewer risks, and encourage community acceptance.The primary aim for this study was to demonstrate the effectiveness of an Ae. aegypti IIT in a replicated treatment control study using males infected with a Wolbachia wAlbB strain from Ae. albopictus. Australia’s northern Queensland towns have endemic populations of Ae. aegypti (21), some of which have recently been transformed to carry the wMel Wolbachia strain (transfected from Drosophila melanogaster) in order to reduce arbovirus transmission risk (22): this phenotype provides arbovirus-blocking properties (23). We hypothesized that by selecting isolated towns and suburbs in tropical northern Queensland, we could demonstrate strong Ae. aegypti suppression over one season. By monitoring over the following season, we also sought to test whether suppression during one season would have a subsequent impact on populations the following season. At the outset of this study, all Ae. aegypti populations in the study site were wild type (no Wolbachia). However, during preparations for our IIT experiment—and at the request of the Queensland state health authority—all experimental landscape populations were transformed by high levels of wMel-infected Ae. aegypti released by Eliminate Dengue (now the World Mosquito Program [WMP]) (24). Subsequently, we released wAlbB2-infected males into a mosaic population of both wild type and wMel-Ae. aegypti to induce incompatible mating. We observed strong suppression over 20 wk, with bidirectional incompatibility in a field setting—and evidence of an ongoing suppression effect lasting into the next season.     

Filamentation temperature-sensitive protein Z (FtsZ) of Wolbachia, endosymbiont of Wuchereria bancrofti: A potential target for anti-filarial chemotherapy

Lymphatic filariasis (LF) is a leading cause of morbidity in the tropical world. It is caused by the filarial parasites Wuchereria bancrofti, Brugia malayi and Brugia timori and transmitted by vector mosquitoes. Currently a programme for the elimination of LF, Global programme for Elimination of Lymphatic Filariasis (GPELF), is underway with the strategy of mass administration of single dose of diethylcarbamazine or ivermectin, in combination with an anthelmintic drug, albendazole. However, antifilarial drugs used in the programme are only microfilaricidal but not or only partially macrofilaricidal. Hence, there is a need to identify new targets for developing antifilarial drugs. Filarial parasites harbor rickettsial endosymbionts, Wolbachia sp., which play an important role in their biology and hence are considered as potential targets for antifilarial chemotherapy development. In this study, one of the cell division proteins of Wolbachia of the major lymphatic filarial parasite, W. bancrofti, viz., filamentation temperature-sensitive protein Z (FtsZ), was explored as a drug target. The gene coding for FtsZ protein was amplified from the genomic DNA of W. bancrofti, cloned and sequenced. The derived amino acid sequence of the gene revealed that FtsZ protein is 396 amino acids long and contained the tubulin motif (GGGTGTG) involved in GTP binding and the GTP hydrolyzing motif (NLDFAD). The FtsZ gene of endosymbiont showed limited sequence homology, but exhibited functional homology with β-tubulin of its host, W. bancrofti, as it had both the functional motifs and conserved amino acids that are critical for enzymatic activity. β-tubulin is the target for the anti-helminthic activity of albendazole and since FtsZ shares functional homology with, β-tubulin it may also be sensitive to albendazole. Therefore, the effect of albendazole was tested against Wolbachia occurring in mosquitoes instead of filarial parasites as the drug has lethal effect on the latter. Third instar larvae of Culex quinquefasciatus were treated with 0.25 mg/ml of albendazole (test) or tetracycline (positive control) in the rearing medium for different intervals and tested for the presence of Wolbachia by FtsZ PCR. All the treated larvae were negative for the presence of the FtsZ band, whereas all the control larvae were positive. The findings of the study, thus indicated that FtsZ is sensitive to albendazole. In view of this albendazole appears to have dual targets; FtsZ in Wolbachia and β-tubulin in W. bancrofti. Further, the functional domain of the gene was assessed for polymorphism among recombinant clones representing 120 W. bancrofti parasites, prevalent across wide geographic areas of India and found to be highly conserved among them. Since it is highly conserved and plays an important role in Wolbachia cell division it appears to be a potential target for anti-filarial chemotherapy development.