Life cycle of Amblyospora indicola (Microspora: Amblyosporidae), a parasite of the mosquito Culex sitiens and of Apocyclops sp. Copepods

The life cycle of Amblyospora indicola, a parasite of the mosquito Culex sitiens, was revealed by field observations and laboratory infection experiments conducted in Australia. In northern Queensland, infected C. sitiens larvae were often found breeding in association with two cyclopoid copepods: Apocyclops dengizicus and an undescribed species of the same genus. The latter species was found to be an intermediate copepod host of this microsporidium whereas A. dengizicus was not. One complete cycle of the parasite extends over two mosquito generations (by transovarial transmission from females with binucleate spores to their eggs) and by horizontal transmission between mosquitoes and copepods. The latter involves horizontal transmission from mosquitoes to copepods via meiospores produced in larval fat body infections and horizontal transmission from copepods to mosquitoes via uninucleate spores produced within infected copepods. Uninucleate clavate spores were formed in Apocyclops sp. nov. copepods 7-10 days after exposure to larval meiospores and were infectious to larvae of a microsporidian-free colony of C. sitiens. The development of A. indicola within mosquito larvae exposed to infected copepods is similar to that of A. dyxenoides infecting C. annulirostris. It proceeds from stages with a single nucleus to diplokaryotic binucleate cells in oenocytes. These stages persist through pupation to adult emergence after which time a proportion of male mosquitoes and female mosquitoes may develop binucleate spores without the need for a blood meal. A proportion of both male and female larval progeny of infected females with binucleate spores develop patent fat body infections via transovarial transmission and die in the fourth larval instar.(ABSTRACT TRUNCATED AT 250 WORDS)     

The infection of nonmosquito hosts by injection with spores of the microsporidan Nosema algerae

Nosema algerae normally infects only mosquitoes by the per os route but developed in a number of different arthropods when spores were injected into the hemocoels. Representatives of other phyla were not infected when injected with N. algerae spores. Spores produced in these injected hosts were of normal size and were infectious when fed to mosquito larvae. Many more spores were produced in some of the injected hosts than were produced in the infected mosquitoes. One corn earworm larva produced as many spores as 2,000 mosquito larvae.     

Life cycle,epizootiology, and horizontal transmission of Amblyospora (Microspora: Amblyosporidae) in a univoltine mosquito, Aedes stimulans

Amblyospora infections in Aedes stimulans are transovarially transmitted by females infected in the previous year. Pathogen development in progeny is dimorphic and host sex dependent. In males, the pathogen invades fat body tissue and undergoes an extensive developmental sequence which kills the host and results in the formation of eight haploid spores enclosed in an accessory membrane that are not infectious to other larvae. In females, the pathogen invades host oenocytes and undergoes a simple developmental sequence which has no detrimental affect on longevity, fecundity, oviposition, or egg hatch, and results in the formation of binucleated spores that infect the ovaries and ensure transmission to the next generation. Transovarial transmission is continuous and is the major way in which these microsporidia are maintained from year to year, but is incapable of maintaining infections in breeding populations because of low transmission rates and is not sufficient to account for the types and levels of infection observed in the field. Horizontal transmission is reported for the first time. It occurs sporadically during the early stages of larval subsequently disseminated to oenocytes of adult hosts and are transovarially transmitted by females to filial host generations. This pathway of transmission provides the necessary mechanism whereby these microsporidia can reenter the mosquito population and thus perpetuate themselves.     

Dry-season Mosquito Breeding Associated with Irrigation in the Northeast Kimberley Region of Western Australia: Potential Impact on Mosquito-borne Disease Transmission

Recent Ross River virus activity prior to the onset of the wet season in the Kimberley region of Western Australia points to an increased dry season mosquito-borne disease risk at Kununurra, compared to other Kimberley towns. This study describes a preliminary investigation into the role of the Ord River Irrigation Area at Kununurra in mosquito production during the dry season. Specifically, the study sought to determine whether the irrigation area has provided the opportunity for year-round breeding of arbovirus vector mosquitoes. A 10-day adult and larval mosquito sampling survey, in August 2003, revealed that mosquito breeding was occurring during the driest month of the year at Kununurra, supporting the hypothesis that mosquitoes can breed year-round. Importantly, significantly larger numbers and percentage of adult Culex annulirostris, an important disease vector in Australia, were collected within the irrigation area (44.6% of total catch) compared to nearby reference (nonirrigated) breeding sites (9.8%) (mean difference 76.2 per trap per night; 95% CI 38.6, 113.7; P <0.001). Larval Cx. annulirostris were also collected at several sites within the irrigation area, whereas none were collected at reference sites. These results indicate that mosquito breeding associated with anthropogenic environmental changes may be responsible for an increased health risk at the end of the dry season. Mosquito management needs to be given a high priority to ensure this potential health risk is not further exacerbated. Several control strategies to reduce breeding of disease vector mosquitoes are identified, primarily focusing on modification of breeding habitats and alteration of irrigation protocols.     

The mosquito and predatory insect fauna inhabiting fresh-water ponds,with particular reference to Culex annulirostris Skuse (Diptera: Culicidae)

Colonization and abundance of mosquitoes and other insects were studied during four trials in man-made ponds at Mildura, Victoria. In December and February, respectively, Culex australicus and Cx annulirostris colonized the ponds within 1 day of formation. Maximum larval densities were attained within 8 days and, thereafter, densities gradually declined. The ponds were also colonized initially by predatory Coleoptera and Hemiptera, then Odonata. In three of the four trials, a significant inverse relationship existed between mosquito and predator densities. From life tables compiled during one of these trials, a survival rate of Cx annulirostris, from egg hatch to eclosion, was estimated to be 11%. Predation was considered to be largely responsible for the low survival. The results are discussed in relation to control of Cx annulirostris in temporary and permanent grassy ponds.     

A curious coincidence: mosquito biodiversity and the limits of the Japanese encephalitis virus in Australasia

Background  

The mosquito Culex annulirostris Skuse (Diptera: Culicidae) is the major vector of endemic arboviruses in Australia and is also responsible for the establishment of the Japanese encephalitis virus (JEV) in southern Papua New Guinea (PNG) as well as its incursions into northern Australia. Papua New Guinea and mainland Australia are separated by a small stretch of water, the Torres Strait, and its islands. While there has been regular JEV activity on these islands, JEV has not established on mainland Australia despite an abundance of Cx. annulirostris and porcine amplifying hosts. Despite the public health significance of this mosquito and the fact that its adults show overlapping morphology with close relative Cx. palpalis Taylor, its evolution and genetic structure remain undetermined. We address a hypothesis that there is significant genetic diversity in Cx. annulirostris and that the identification of this diversity will shed light on the paradox that JEV can cycle on an island 70 km from mainland Australia while not establishing in Australia itself.     

Mosquito seasonality and arboviral disease incidence in Murray Valley, southeast Australia

Abstract. Adult female mosquito populations were monitored at weekly intervals during spring-autumn (November-March) for 4 years (1991-95) using dry-ice-baited light traps at forty sites in the Murray Valley of Victoria, Australia. Among twenty species of mosquitoes collected, Culex annulirostriswas the most abundant (66.6 ± 9.3%) followed by Cx australicus(15.3 ± 7.7%). From a total of 476, 682 mosquitoes collected, nearly all were females and only 1295 (0.27%) were males. Mosquito population densities were generally higher in 1992-93 and 1993-94 seasons than in 1991-92 and 1994-95 seasons. Greatest densities of Cx annulirostrisand Cx australicusoccurred in 1992-93, coinciding with outbreaks of Ross River (RR) and Barman Forest (BF) arboviruses causing human polyarthritis. In the majority of shires, Cx australicuswas the predominant species from spring to early summer (November and December), then was replaced by Cx annulirostrisfrom mid-summer to autumn (January-April). In three shires, Aedes bancroftianusand Ae.sagaxpredominated during the early part of the season. Densities of both Cx annulirostrisand Cx australicuswere related to temperature. Cx australicusadults were found to be trapped when the mean ambient temperature exceeded 6d?C, with peak population recorded at 20d?C. Cx annulirostrisadult density increased when the mean temperature rose above 12d?C, reaching a peak during February and March when temperature exceeded 25d?C. Cx annulirostrisdeclined rapidly from April onwards, with no adult activity evident from May to November. Population densities of Aedesspp. were generally less than reported from earlier studies, possibly due to lower rainfall in spring and summer as well as reduced flood irrigation practices. In each year, a significant correlation was detected between Cx annulirostrisdensity and RR virus incidence in humans. As Cx annulirostrisis the predominant local mosquito species and feeds on a wide spectrum of hosts including man, it seems likely that Cx annulirostrisis the major vector of RR in the inland Murray Valley region.     

Life Cycle of Culicosporella lunata (Hazard & Savage, 1970) Weiser, 1977 (Microspora) as Revealed in the Light Microscope with a Redescription of the Genus and Species1

Light microscopy studies of Culicosporella lunata (Hazard & Savage), a parasite of the mosquito Culex pilosus (Dyar & Knab), revealed two sporogonial sequences. One sequence begins with diplokaryotic meronts that undergo repeated nuclear divisions to produce sporogonial plasmodia with nuclei in diplokaryotic arrangement. These plasmodia form rosette-like clusters of sporoblasts during incomplete cytokinesis and, eventually, binucleate spores. These spores initiate infections in healthy larvae when they ingest spores. The second sequence begins with diplokaryotic meronts that undergo karyogamy and meiosis to form Thelohania-like sporonts and haploid spores. Anomalies are often observed in these sporonts which result in aberrant spores, usually fewer than eight, in an accessory (pansporoblastic) membrane. Normal haploid spores are morphologically similar to those of species of Amblyospora. The genus and the type species are redefined based on new information presented here and it and the type species are placed in the family Amblyosporidae.     

Development of Amblyospora campbelli (Microsporida: Amblyosporidae) in the Mosquito Culiseta incidens (Thomson)

The complete life cycle of Amblyospora campbelli (Kellen and Wills, 1962) (Microsporida: Amblyosporidae) requires a two-host system involving the mosquito host, Culiseta incidens (Thomson), and an obligatory intermediate copepod host. The parasite has dimorphic spore development producing meiospores (haploid condition) and binucleated spores (diploid condition), either as an exclusive infection or simultaneously (within females only). This is the 1st known report of concurrent spore development within an adult mosquito host, and, therefore, shows the Amblyospora campbelli system to be uniquely different from other Amblyospora spp. cycles previously described. The significance of dimorphic spore development is discussed. In females, diplokaryotic meronts may invade oenocytes, causing a benign-type of infection. A blood-meal is required to initiate sporulation of the binucleate spore. The binucleate spore contains the sporoplasm involved in transovarial transmission. A 2nd sporulation sequence, primarily in adipose tissue, may involve both males and females. In this sequence, repeated merogonic division greatly increased the density of diplokaryotic meronts and generally involved most of the body of the host. Production of meiospores, unlike that for the binucleate spore, appeared to be spontaneous (i.e. no obligatory blood meal). Survivorship of male and female larval mosquitoes was nearly equal. Adult females spread the parasite in three ways: transovarial, transovum, and by meiospore deposition.     

Impacts of fungal entomopathogens on survival and immune responses of Aedes albopictus and Culex pipiens mosquitoes in the context of native Wolbachia infections

Microbial control of mosquitoes via the use of symbiotic or pathogenic microbes, such as Wolbachia and entomopathogenic fungi, are promising alternatives to synthetic insecticides to tackle the rapid increase in insecticide resistance and vector-borne disease outbreaks. This study evaluated the susceptibility and host responses of two important mosquito vectors, Ae. albopictus and Cx. pipiens, that naturally carry Wolbachia, to infections by entomopathogenic fungi. Our study indicated that while Wolbachia presence did not provide a protective advantage against entomopathogenic fungal infection, it nevertheless influenced the bacterial / fungal load and the expression of select anti-microbial effectors and phenoloxidase cascade genes in mosquitoes. Furthermore, although host responses from Ae. albopictus and Cx. pipiens were mostly similar, we observed contrasting phenotypes with regards to susceptibility and immune responses to fungal entomopathogenic infection in these two mosquitoes. This study provides new insights into the intricate multipartite interaction between the mosquito host, its native symbiont and pathogenic microbes that might be employed to control mosquito populations.     

The Plasmodium bottleneck: malaria parasite losses in the mosquito vector

Nearly one million people are killed every year by the malaria parasite Plasmodium. Although the disease-causing forms of the parasite exist only in the human blood, mosquitoes of the genus Anopheles are the obligate vector for transmission. Here, we review the parasite life cycle in the vector and highlight the human and mosquito contributions that limit malaria parasite development in the mosquito host. We address parasite killing in its mosquito host and bottlenecks in parasite numbers that might guide intervention strategies to prevent transmission.     

Life Cycle and Epizootiology of Amblyospora sp. (Microspora: Amblyosporidae) in the Mosquito,Aedes cantator1

In Aedes cantator, Amblyospora sp. is transovarially transmitted and has two developmental sequences. The life cycle is initiated in the adult female with the release of sporoplasms from binucleated spores not bounded by membranes, lying free within host oenocytes. Sporoplasms infect the developing oocytes and are transmitted to the filial generation when the eggs are laid. In some of the female progeny that hatch from infected eggs, diplokaryotic cells infect host oenocytes and divide by binary fission during merogony. Sporulation and spore formation do not occur until a blood meal is taken by the host and they coincide with the development and maturation of the oocytes to complete the cycle. In other female and all male progeny, pathogen development occurs within fat body tissue of the host where diplokaryotic cells divide by multiple fission during merogony to spread the infection. Sporulation in this developmental sequence is characterized by the secretion of an accessory membrane and the meiotic division of diplokaryotic sporonts, which result in the formation of octonucleated plasmodia that undergo cytokinesis to form eight haploid spores which are not perorally infectious to other mosquito larvae. There is no increase in the prevalence of either type of infection in field populations during juvenile development, indicating that there is no direct horizontal transmission of the pathogen within any one generation. Data obtained from laboratory rearings of infected progeny, however, show that infections cannot persist relying solely upon maternal-mediated transmission and that some other mode of transmission must be operative for continued maintenance of this microsporidium in A. cantator.     

Malaria Vector Surveillance in Ganghwa-do, a Malaria-Endemic Area in the Republic of Korea

We investigated the seasonality of Anopheles mosquitoes, including its species composition, density, parity, and population densities of mosquitoes infected with the parasite in Ganghwa-do (Island), a vivax malaria endemic area in the Republic of Korea. Mosquitoes were collected periodically with a dry-ice-tent trap and a blacklight trap during the mosquito season (April-October) in 2008. Anopheles sinensis (94.9%) was the most abundant species collected, followed by Anopheles belenrae (3.8%), Anopheles pullus (1.2%), and Anopheles lesteri (0.1%). Hibernating Anopheles mosquitoes were also collected from December 2007 to March 2008. An. pullus (72.1%) was the most frequently collected, followed by An. sinensis (18.4%) and An. belenrae (9.5%). The composition of Anopheles species differed between the mosquito season and hibernation seasons. The parous rate fluctuated from 0% to 92.9%, and the highest rate was recorded on 10 September 2008. Sporozoite infections were detected by PCR in the head and thorax of female Anopheles mosquitoes. The annual sporozoite rate of mosquitoes was 0.11% (2 of 1,845 mosquitoes). The 2 mosquitoes that tested positive for sporozoites were An. sinensis. Malarial infections in anopheline mosquitoes from a population pool were also tried irrespective of the mosquito species. Nine of 2,331 pools of Anopheles mosquitoes were positive. From our study, it can be concluded that An. sinensis, which was the predominant vector species and confirmed as sporozoite-infected, plays an important role in malaria transmission in Ganghwa-do.     

Wolbachia increases susceptibility to Plasmodium infection in a natural system

Current views about the impact of Wolbachia on Plasmodium infections are almost entirely based on data regarding artificially transfected mosquitoes. This work has shown that Wolbachia reduces the intensity of Plasmodium infections in mosquitoes, raising the exciting possibility of using Wolbachia to control or limit the spread of malaria. Whether natural Wolbachia infections have the same parasite-inhibiting properties is not yet clear. Wolbachia–mosquito combinations with a long evolutionary history are, however, key for understanding what may happen with Wolbachia-transfected mosquitoes after several generations of coevolution. We investigate this issue using an entirely natural mosquito–Wolbachia–Plasmodium combination. In contrast to most previous studies, which have been centred on the quantification of the midgut stages of Plasmodium, we obtain a measurement of parasitaemia that relates directly to transmission by following infections to the salivary gland stages. We show that Wolbachia increases the susceptibility of Culex pipiens mosquitoes to Plasmodium relictum, significantly increasing the prevalence of salivary gland stage infections. This effect is independent of the density of Wolbachia in the mosquito. These results suggest that naturally Wolbachia-infected mosquitoes may, in fact, be better vectors of malaria than Wolbachia-free ones.     

Fine scale spatiotemporal clustering of dengue virus transmission in children and Aedes aegypti in rural Thai villages

Background

Based on spatiotemporal clustering of human dengue virus (DENV) infections, transmission is thought to occur at fine spatiotemporal scales by horizontal transfer of virus between humans and mosquito vectors. To define the dimensions of local transmission and quantify the factors that support it, we examined relationships between infected humans and Aedes aegypti in Thai villages.

Methodology/Principal Findings

Geographic cluster investigations of 100-meter radius were conducted around DENV-positive and DENV-negative febrile “index” cases (positive and negative clusters, respectively) from a longitudinal cohort study in rural Thailand. Child contacts and Ae. aegypti from cluster houses were assessed for DENV infection. Spatiotemporal, demographic, and entomological parameters were evaluated. In positive clusters, the DENV infection rate among child contacts was 35.3% in index houses, 29.9% in houses within 20 meters, and decreased with distance from the index house to 6.2% in houses 80–100 meters away (p<0.001). Significantly more Ae. aegypti were DENV-infectious (i.e., DENV-positive in head/thorax) in positive clusters (23/1755; 1.3%) than negative clusters (1/1548; 0.1%). In positive clusters, 8.2% of mosquitoes were DENV-infectious in index houses, 4.2% in other houses with DENV-infected children, and 0.4% in houses without infected children (p<0.001). The DENV infection rate in contacts was 47.4% in houses with infectious mosquitoes, 28.7% in other houses in the same cluster, and 10.8% in positive clusters without infectious mosquitoes (p<0.001). Ae. aegypti pupae and adult females were more numerous only in houses containing infectious mosquitoes.

Conclusions/Significance

Human and mosquito infections are positively associated at the level of individual houses and neighboring residences. Certain houses with high transmission risk contribute disproportionately to DENV spread to neighboring houses. Small groups of houses with elevated transmission risk are consistent with over-dispersion of transmission (i.e., at a given point in time, people/mosquitoes from a small portion of houses are responsible for the majority of transmission).     

Significance of transovarial infections of Amblyospora sp. (Microspora:Thelohaniidae) in relation to parasite maintenance in the mosquito Culex salinarius

Adult females of Culex salinarius, transovarially infected with the microsporidium Amblyospora sp., showed no significant differences in overall fecundity, physiological longevity, and preoviposition periods when compared to healthy adults under laboratory conditions. Development times and survival rates for congenitally infected young to reproductive age were also indistinguishable from those of healthy controls. A significant reduction of 52% in egg hatch was observed for infected eggs when compared to healthy eggs. Prevalence rates of infection for progeny produced by infected females declined with each successive gonotrophic cycle and averaged 90%. Transovarial transmission is not sufficient for the maintenance of the microsporidium in a population of mosquitoes. An alternate host is suggested as a mechanism whereby the microsporidium can reenter a healthy mosquito population.     

What does not kill them makes them stronger: larval environment and infectious dose alter mosquito potential to transmit filarial worms

For organisms with complex life cycles, larval environments can modify adult phenotypes. For mosquitoes and other vectors, when physiological impacts of stressors acting on larvae carry over into the adult stage they may interact with infectious dose of a vector-borne pathogen, producing a range of phenotypes for vector potential. Investigation of impacts of a common source of stress, larval crowding and intraspecific competition, on adult vector interactions with pathogens may increase our understanding of the dynamics of pathogen transmission by mosquito vectors. Using Aedes aegypti and the nematode parasite Brugia pahangi, we demonstrate dose dependency of fitness effects of B. pahangi infection on the mosquito, as well as interactions between competitive stress among larvae and infectious dose for resulting adults that affect the physiological and functional ability of mosquitoes to act as vectors. Contrary to results from studies on mosquito–arbovirus interactions, our results suggest that adults from crowded larvae may limit infection better than do adults from uncrowded controls, and that mosquitoes from high-quality larval environments are more physiologically and functionally capable vectors of B. pahangi. Our results provide another example of how the larval environment can have profound effects on vector potential of resulting adults.     

A decade of stability for wMel Wolbachia in natural Aedes aegypti populations

Mosquitoes carrying Wolbachia endosymbionts are being released in many countries for arbovirus control. The wMel strain of Wolbachia blocks Aedes-borne virus transmission and can spread throughout mosquito populations by inducing cytoplasmic incompatibility. Aedes aegypti mosquitoes carrying wMel were first released into the field in Cairns, Australia, over a decade ago, and with wider releases have resulted in the near elimination of local dengue transmission. The long-term stability of Wolbachia effects is critical for ongoing disease suppression, requiring tracking of phenotypic and genomic changes in Wolbachia infections following releases. We used a combination of field surveys, phenotypic assessments, and Wolbachia genome sequencing to show that wMel has remained stable in its effects for up to a decade in Australian Ae. aegypti populations. Phenotypic comparisons of wMel-infected and uninfected mosquitoes from near-field and long-term laboratory populations suggest limited changes in the effects of wMel on mosquito fitness. Treating mosquitoes with antibiotics used to cure the wMel infection had limited effects on fitness in the next generation, supporting the use of tetracycline for generating uninfected mosquitoes without off-target effects. wMel has a temporally stable within-host density and continues to induce complete cytoplasmic incompatibility. A comparison of wMel genomes from pre-release (2010) and nine years post-release (2020) populations show few genomic differences and little divergence between release locations, consistent with the lack of phenotypic changes. These results indicate that releases of Wolbachia-infected mosquitoes for population replacement are likely to be effective for many years, but ongoing monitoring remains important to track potential evolutionary changes.