Mosquito Anopheles Quadrimaculatus Research Articles

Genomes and transcriptomes help unravel the complex life cycle of the blastoclad fungus, Coelomomyces lativittatus, an obligate parasite of mosquitoes and microcrustaceans

ABSTRACT Species of the phylum Blastocladiomycota, early-diverging zoosporic (flagellated) lineages of fungi, are vastly understudied. This phylum includes the genus Coelomomyces, which consists of more than 80 fungal species that are obligate parasites of arthropods. Known Coelomomyces species lack a complete asexual life cycle, instead surviving through an obligate heterecious alternation of generations life cycle. Despite their global distribution and interesting life cycle, little is known about the genomics of any Coelomomyces species. To address this, we generated three draft-level genomes and annotations for C. lativittatus representing its haploid meiospore, orange gamete, and amber gamete life stages. These draft genome assemblies ranged in size from 5002 to 5799 contigs, with a total length of 19.8–22.8 Mb and a mean of 7416 protein-coding genes. We then demonstrated the utility of these genomes by combining the draft annotations as a reference for analysis of C. lativittatus transcriptomes. We analyzed transcriptomes from across host-associated life stages, including infected larvae and excised mature sporangia from the mosquito Anopheles quadrimaculatus. We identified differentially expressed genes and enriched GO terms both across and within life stages and used these to make hypotheses about C. lativittatus biology. Generally, we found the C. lativittatus transcriptome to be a complex and dynamic expression landscape; GO terms related to metabolism and transport processes were enriched during infection and terms related to dispersal were enriched during sporulation. We further identified five high mobility group (HMG)-box genes in C. lativittatus, three belonging to clades with mating type (MAT) loci from other fungi, as well as four ortholog expansions in C. lativittatus compared with other fungi. The C. lativittatus genomes and transcriptomes reported here are a valuable resource and may be leveraged toward furthering understanding of the biology of these and other early-diverging fungal lineages.

Overcoming insecticide resistance in Anopheles mosquitoes by using faster-acting solid forms of deltamethrin

BackgroundControlling malaria-transmitting Anopheles mosquitoes with pyrethroid insecticides is becoming increasingly challenging because of widespread resistance amongst vector populations. The development of new insecticides and insecticidal formulations is time consuming and costly, however. A more active crystalline form of deltamethrin, prepared by heating the commercial crystalline form, previously was reported to be 12-times faster acting against susceptible North American Anopheles quadrimaculatus mosquitoes. Herein the potential for heat-activated deltamethrin dispersed on chalk to overcome various resistance mechanisms amongst five West African Anopheles strains is investigated, and its long-term sustained lethality evaluated.MethodsThe more active deltamethrin form was generated in a commercial dust containing deltamethrin by heating the material as purchased. Tarsal contact bioassays were conducted to investigate its efficacy, potency, and speed of action against resistant Anopheles populations compared to the commercially available form of deltamethrin dust.ResultsIn all cases, D-Fense Dust heated to generate the more active form of deltamethrin was substantially more effective than the commercially available formulation. 100% of both Banfora M and Kisumu populations were knocked down 10 min post-exposure with no recovery afterwards. Gaoua-ara and Tiefora strains exhibited 100% knockdown within 15 min, and the VK7 2014 strain exhibited 100% knockdown within 20 min. In all cases, 100% mortality was observed 24 h post-exposure. Conversely, the commercial formulation (unheated) resulted in less than 4% mortality amongst VK7 2014, Banfora, and Gaoua-ara populations by 24 h, and Tiefora and Kisumu mosquitoes experienced 14 and 47% mortality by 24 h, respectively. The heat-activated dust maintained comparable efficacy 13 months after heating.ConclusionsThe heat-activated form of commercial deltamethrin D-Fense Dust outperformed the material as purchased, dramatically increasing efficacy against all tested pyrethroid-resistant strains. This increase in lethality was retained for 13 months of storage under ambient conditions in the laboratory. Higher energy forms of commonly used insecticides may be employed to overcome various resistance mechanisms seen in African Anopheles mosquitoes through more rapid uptake of insecticide molecules from their respective solid surfaces. That is, resistant mosquitoes can be killed with an insecticide to which they are resistant without altering the molecular composition of the insecticide.

Almendravirus: A Proposed New Genus of Rhabdoviruses Isolated from Mosquitoes in Tropical Regions of the Americas.

The Rhabdoviridae is a diverse family of negative-sense single-stranded RNA viruses, many of which infect vertebrate hosts and are transmitted by hematophagous arthropods. Others appear to be arthropod specific, circulating only within arthropod populations. Herein, we report the isolation and characterization of three novel viruses from mosquitoes collected from the Americas. Coot Bay virus was isolated from Anopheles quadrimaculatus mosquitoes collected in the Everglades National Park, Florida; Rio Chico virus was isolated from Anopheles triannulatus mosquitoes collected in Panama; and Balsa virus was isolated from two pools of Culex erraticus mosquitoes collected in Colombia. Sequence analysis indicated that the viruses share a similar genome organization to Arboretum virus and Puerto Almendras virus that had previously been isolated from mosquitoes collected in Peru. Each genome features the five canonical rhabdovirus structural protein genes as well as a gene encoding a class 1A viroporin-like protein (U1) located between the G and L genes (3'-N-P-M-G-U1-L-5'). Phylogenetic analysis of complete L protein sequences indicated that all five viruses cluster in a unique clade that is relatively deeply rooted in the ancestry of animal rhabdoviruses. The failure of all viruses in this clade to grow in newborn mice or vertebrate cells in culture suggests that they may be poorly adapted to replication in vertebrates.

Bitter-sensitive gustatory receptor neuron responds to chemically diverse insect repellents in the common malaria mosquito Anopheles quadrimaculatus.

Female mosquitoes feed on blood from animal hosts to obtain nutritional resources used for egg production. These contacts facilitate the spread of harmful human diseases. Chemical repellents are used to disrupt mosquito host-seeking and blood-feeding behaviors; however, little is known about the gustatory sensitivity of mosquitoes to known repellents. Here, we recorded electrical responses from gustatory receptor neurons (GRNs) housed within the labellar sensilla of female Anopheles quadrimaculatus to N,N-diethyl-3-methylbenzamide (DEET), picaridin, IR3535, 2-undecanone, p-menthane-3,8-diol, geraniol, trans-2-hexen-1-ol, quinine, and quinidine. A bitter-sensitive GRN responded to all tested repellents and quinine, a known feeding deterrent. Responses of the bitter-sensitive neuron to quinine and an isomer, quinidine, did not differ. Delayed bursts of electrical activity were observed in response to continuous stimulation with synthetic repellents at high concentrations. Electrophysiological recordings from bitter-sensitive GRNs associated with mosquito gustatory sensilla represent a convenient model to evaluate candidate repellents.

Phomalactone as the Active Constituent against Mosquitoes from <i>Nigrospora spherica</i>

The culture filtrate of a plant pathogenic fungus that infected Zinnia elegans and Hydrangea macrophylla was investigated for mosquitocidal constituents by bioassay guided isolation. The fungus responsible for the pathogenic effects on Zinnia elegans and Hydrangea macrophylla plants had been identified as Nigrospora spherica by molecular techniques. The mosquito adulticidal constituent in the culture filtrate was identified as phomalactone by spectroscopic techniques. Laboratory bioassays showed that phomalactone had larvicidal activity against permethrin susceptible and resistant Aedes aegypti larvae and topical adulticide activities on permethrin susceptible and resistant Aedes aegypti and Anopheles quadrimaculatus mosquitoes. Phomalactone was effective as a topical adulticide against the standard Orlando reference strain of Ae. aegypti with an LD50 of 0.64 μg/org. Activity against An. quadrimaculatus was 0.20 μg/org.

Common malaria Mosquito Anopheles quadrimaculatus Say (Insecta: Diptera: Culicidea)

EENY-491, a 6-page illustrated fact sheet by Leslie M. Rios and C. Roxanne Connelly, describes this historically most important vector of malaria in the eastern United States — synonymy, distribution, description, life cycle, medical significance, surveillance, and management. Includes selected references. Published by the UF Department of Entomology and Nematology, February 2009. EENY 491/IN791: Common Malaria Mosquito Anopheles quadrimaculatus Say (Insecta: Diptera: Culicidae) (ufl.edu)

Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus

BackgroundAminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors) for Cry toxins. We examined brush border membrane vesicle (BBMV) proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it.ResultsA 100-kDa protein with APN activity (APNAnq 100) was isolated from the brush border membrane of Anopheles quadrimaculatus. Native state binding analysis by surface plasmon resonance shows that APNAnq 100 forms tight binding to a mosquitocidal Bt toxin, Cry11Ba, but not to Cry2Aa, Cry4Ba or Cry11Aa.ConclusionAn aminopeptidase from Anopheles quadrimaculatus mosquitoes is a specific binding protein for Bacillus thuringiensis Cry11Ba.

A RETROSPECTIVE EXAMINATION OF THE EFFECT OF FEVER AND MICROGAMETOCYTE COUNT ON MOSQUITO INFECTION ON HUMANS INFECTED WITH PLASMODIUM VIVAX

A retrospective examination was made of archival data collected between 1940 and 1963 on the infection of mosquitoes with the St. Elizabeth strain of Plasmodium vivax. Patients were undergoing malariatherapy for the treatment of neurosyphilis. A total of 845 lots of Anopheles quadrimaculatus mosquitoes were fed during primary infections and 76 during secondary infections. Average percentage infection during the primary infection was 56.55% versus 49.83% during the secondary infection. There appeared to be no relationship between microgametocye density, asexual parasite count, and percentage infection. However, very high fevers appear to have a significant effect on infection rates. Persons with fever > or = 105 degrees F showed the lowest rates of infectivity regardless of parasitemia; persons with moderate (101-104.8 degrees F) fever produced somewhat higher rates, and persons with no fever had the highest levels of infection at all parasitemia levels greater than 1,500/microL

Potential of the Panama strain of Plasmodium vivax for the testing of malarial vaccines in Aotus nancymai monkeys.

Aotus monkeys were infected with a strain of Plasmodium vivax from Panama to determine its potential for the testing of malarial vaccines. After sporozoite inoculation, 3 splenectomized Aotus nancymai that had been infected previously with Plasmodium falciparum and P. vivax had prepatent periods of 13, 15, and 15 days with maximum parasite counts of 12,726/microl, 5,310/microl, and 9,180/microl. Three other A. nancymai previously infected with P. falciparum only had prepatent periods of 17, 15, and 15 days with maximum parasite counts of 44,460/microl, 31,500/microl, and 42,660/microl. One monkey with no previous history of infection had a prepatent period of 14 days after sporozoite inoculation, and a maximum parasite count of 100,000/microl; detectable parasitemia persisted for almost 500 days with 13 recognizable peaks in the parasite count. Anopheles dirus, Anopheles freeborni, Anopheles stephensi, and Anopheles quadrimaculatus mosquitoes were readily infected with the Panama strain.

Formation of basement membrane-like structure terminates the cellular encapsulation of microfilariae in the haemocoel of Anopheles quadrimaculatus.

The encapsulation of microfilariae in the haemocoels of mosquitoes combines both humoral and cellular reactions: the microfilariae are first encased in an acellular layer of melanin, followed by a cellular encapsulation by plasmatocytes. In this study, we demonstrated that cellular encapsulation of Brugia pahangi microfilariae in the haemocoel of the mosquito Anopheles quadrimaculatus was terminated by the formation of a basement membrane-like structure on the outermost surface of the cellular capsule. This structure occurred in the early stage of cellular encapsulation and was evident on the exterior surface of the plasmatocyte, when the active haemocytes were attaching to the already melanized microfilariae. The termination structure appears to be laid down by releasing the vesicle inclusions of haemocytes and has similarities in ultrastructure and cationic colloidal gold staining properties with that of mosquito basement membranes.

Studies on a Primaquine-Tolerant Strain of Plasmodium vivax from Brazil in Aotus and Saimiri Monkeys

A nonimmune American acquired an infection of Plasmodium vivax Type 1 malaria in Brazil in 1994. After returning to the U.S.A., he had a primary attack followed by 3 relapses. The primary attack and first 2 relapses were treated with a standard regimen of chloroquine, followed by 14 days of primaquine (15 mg/day). Following the third relapse, the primaquine treatment was extended to 28 days. No further relapses occurred. The lack of response to primaquine by this strain may recommend it as a suitable candidate for chemotherapeutic study if it can be adapted to an animal model. Anopheles quadrimaculatus mosquitoes infected by feeding on the patient during the first relapse were used to establish the strain in Aotus and Saimiri monkeys. Monkeys supported well the development of long-lasting parasitemia. Anopheles freeborni, Anopheles stephensi, and Anopheles gambiae mosquitoes were readily infected by feeding on the monkeys and by membrane feeding on diluted blood. Monkey-to-monkey transmission was obtained via the bites of infected mosquitoes and the intravenous injection of sporozoites dissected from salivary glands. This parasite is designated as the Brazil I/CDC strain of P. vivax.

Light and Electron Microscopy Studies on the Development of Parathelohania anophelis (Microspora: Amblyosporidae) in Female Anopheles quadrimaculatus (Diptera: Culicidae)

A light and electron microscopic study of Parathelohania anophelis, a heterosporous microsporidian parasite of the mosquito Anopheles quadrimaculatus, revealed both oval and cylindrical binucleate spores in adult females. Both types of spores may be responsible for transovarial transmission of the parasite. Both spore types originate from identical diplokaryotic meronts in larvae. However, the small oval spore results from plasmodia containing 10 to 40 diplokaryotic nuclei and is produced 24 hr after emergence of the adult mosquito, whereas the large cylindrical spore results from plasmodia containing 6 to 10 nuclei and is formed only after the mosquito takes a blood meal.

Biotic and abiotic parameters associated with an epizootic of Coelomomyces punctatus in a larval population of the mosquito Anopheles quadrimaculatus

Biotic and abiotic parameters associated with an epizootic of the fungus Coelomomyces punctatus in larval populations of the mosquito Anopheles quadrimaculatus were investigated for three mosquito breeding seasons (1986–1988) in two adjacent farm ponds in North Carolina. In the first pond, the prevalence of infected larvae averaged 42% (range 0–85%) for collections made weekly from May 1 to November 20, 1986, but larvae did not occur in this pond in 1987. Infection rates in the adjacent pond, sampled during the mosquito breeding seasons of 1987 and 1988, declined from 10.9% (range 0–27.5%) in 1987 to 2.5% (range 0–14.2%) in 1988. Correlation analyses between the number of female copepods and fungal infection rates in sentinel mosquitoes were significant ( P < 0.01) for Acanthocyclops robustus but insignificant for eight other species. Infections obtained in sentinel larvae placed in the ponds for 3 hr intervals indicated that C. punctatus infected larvae around sundown. Infection rates for field-collected larvae increased with the stage of larval development. However, experiments with sentinel larvae showed that early instars were more susceptible to infection than later instars, suggesting that the higher infection rates in late instars resulted from individual larvae being infected by two or more zygotes during larval development. Standard multiple regression analyses, used to determine the relationship between seasonal infection rates and water chemistry, weather variables, and the abundance of early and late instar larvae, showed that the abundance of late instars was the only independent variable common to linear models. The models only accounted for 20 and 9% of the variation in larval infection rates for 1987 and 1988, respectively. These results indicate that of the parameters examined, the seasonal abundance of the copepod, A. robustus, was the most important factor (or variable) correlated with the prevalence of mosquito infection.

Evidence for the copepods Acanthocyclops robustus and Mesocyclops edax as competent intermediate hosts for Coelomomyces punctatus during an epizootic in a larval population of the mosquito Anopheles quadrimaculatus

Field and laboratory experiments were conducted during an epizootic of Coelomomyces punctatus (Chytridiomycetes: Blastocladiales) in a population of the mosquito Anopheles quadrimaculatus in a North Carolina farm pond to examine the interactions of several potential copepod hosts with the mosquito and fungus. The diel vertical migratory behavior of the copepod species Acanthocyclops robustus, Eucyclops serrulatus, Macrocyclops albidus, and Mesocyclops edax were monitored in relation to infection rates in sentinel mosquito larvae. Mosquito infection occurred primarily around dusk, the same period during which A. robustus and E. serrulatus were most abundant near the surface of the pond. However, exposure of A. robustus, E. serrulatus, M. albidus, M. edax, Microcyclops varicans, and Paracyclops poppei to fungal meiospores in the laboratory showed that only A. robustus and M. edax were competent intermediate hosts for C. punctatus. Laboratory studies of the diel periodicity of gametangial dehiscence in A. robustus and M. edax infected with C. punctatus revealed that gamete release and zygote formation also occurred around dusk. The combined results of the laboratory and field studies on copepod abundance, susceptibility to infection, and periodicity of gametangial dehiscence suggest that A. robustus was the principal intermediate host for C. punctatus during the epizootic, though it is probable that M. edax also contributed importantly to the overall rate of larval infection.

Speciation in an Anopheline (Diptera: Culicidae) Mosquito: Enzyme Polymorphism and the Genetic Structure of Populations

A study of enzyme variability was undertaken with the objective of measuring the level of genetic polymorphism in the mosquito Anopheles quadrimaculatus Say, with emphasis on the extent of genetic differentiation between two forms previously identified on the basis of mating compatibility. Allele and genotype frequencies were measured at 20 enzyme loci. Genotype frequencies at eight loci showed a significant deficiency of heterozygotes in four of the 10 populations studied. The IDH loci were identified as being diagnostic for the two forms and so were used to assemble gene frequency data in discrete populations. Values for genetic distance between the forms were consistent with values previously published for sibling species in the genus Anopheles , suggesting the existence of at least two sympatric sibling species. Measurements of genetic variability and the allelic composition of the two forms suggest that one may represent the ancestral form, the other recently derived from it through a process involving a significant loss of genetic variability.

In vitro study on humoral encapsulation of microfilariae: Establishment of technique and description of reaction

Recently we showed that the encapsulation of the microfilariae of Brugia pahangi in the haemocoel of the mosquito Anopheles quadrimaculatus combined both humoral and cellular encapsulations. In the present study, the formation of the humoral capsule was reproduced and studied in vitro. A simple technique for this was devised. The in vitro humoral reaction began by the deposition of scattered black granules, which gradually enlarged and coalesced to form a layer of pigmented material around the parasite. The humoral capsules produced in vitro were morphologically and utrastructurally similar to those formed in vivo. Various factors (pH value, dilution of haemolymph, temperature) were shown to affect the degree of encapsulation of the microfilariae in vitro.

Observations on the early death experienced by Dirofilaria immitis-infected mosquitoes (Diptera: Culicidae).

The phenomenon of early mortality of mosquitoes infected with Dirofilaria immitis was investigated. Anopheles quadrimaculatus mosquitoes were fed differentially filaremic blood meals, living and dead microfilariae, and special adjuncts such as homogenized mosquito body fractions. At least 25% mortality was produced in mosquitoes in the first 3 days following ingestion of living microfilariae. Dead, intact, or homogenized larvae, whole mosquito bodies, or body fractions did not produce a significant level of mortality, suggesting that early vector death is a function of active, living microfilariae.

An integrated system for the production of gnotobiotic Anopheles quadrimaculatus

This paper reports the first successful gnotobiotic rearing of the mosquito Anopheles quadrimaculatus, a major vector of Dirofilaria immitis, the etiologic agent of canine heartworm disease in the United States. Mosquito eggs were sterilized in Zephiran, and emerging larvae were transferred to flat-sided rearing flasks which contained a sterile easily prepared formulation of commercial dietary products. Maturing pupae were withdrawn in a syringe and were injected with a small amount of water into the gnotobiotic arthropod module (GAM), a sterile Lucite capsule which allows sugar feeding at one end and blood feeding through a heated artificial membrane at the other end. The GAM provided a convenient sterilizable unit to support 12–15 gnotobiotic mosquitoes for a variety of experimental purposes.

Coelomomyces dodgei: Establishment of an in vivo laboratory culture

An in vivo laboratory culture of the fungus Coelomomyces dodgei (Chytridiomycetes: Blastocladiales) was established, using the copepod Cyclops vernalis as an intermediate host and the larvae of the mosquito Anopheles quadrimaculatus as the definitive host. The culture was perpetuated by infecting copepods and mosquitoes using separate procedures. Copepods were infected by being combined with dehiscing sporangia. Patently infected copepods, which contained either light amber, bright orange, or both light amber and bright orange mycelia, were collected daily beginning 12 days later. Mosquitoes were infected by combining 100 first-instar larvae for 48 hr with a mixture of 12 infected copepods, four of each of the above types. The mean rate of infection for the first 100 trials was 41%. When groups of 100 first-, second-, third-, and fourth-instar larvae were exposed to a similar mixture of infected copepods for 48 hr, the mean rates of infection were 37.4, 27.0, 17.8, and 2.4%, respectively. Observations and experimental evidence suggest that the differentially pigmented mycelia found in infected copepods are gametophytes which develop into gametangia that subsequently release gametes of opposite mating types, the light amber gametangia producing female and the bright orange gametangia producing male gametes. Zygotes resulting from the fusion of these gametes lead to the infection of mosquito larvae. Thus, C. dodgei appears to have an Euallomyces type of life cycle with sporophyte and gametophyte generations alternating between mosquito and copepod hosts, respectively, with differentially pigmented sexual structures present in the gametophyte phase.

Ultrastructural changes in the cell surface of Coelomomyces punctatus infecting mosquito larvae

As the fungus Coelomomyces punctatus develops in the coelomic cavity of the mosquito Anopheles quadrimaculatus, the conformation of the plasma membrane and extracellular coat of the fungus changes markedly. The vegetative stage was surrounded by a granular and fibrillar extracellular coat which reacted positively in the silver methenamine procedure for the localization of polysaccharides. Numerous simple, branched or contorted cytoplasmic protuberances covered the irregularly shaped hyphal bodies. The surface of the hyphal body adjacent to the fat body of the mosquito had occasional involutions of the plasma membrane sheathed by cisternae of endoplasmic reticulum. In contrast with these hyphal bodies, cytoplasmic protuberances were spaced at wide intervals along filamentous hyphae. Aborting thalli were contorted and deeply lobed. The plasma membrane was smooth, and cytoplasmic protuberances were absent on other hyphae and hyphal bodies, particularly at advanced stages of infection. Instead unattached vesicles, morphologically similar to the protuberances found on some thalli, were embedded in granular material clustered around the smooth plasma membrane of these thalli. Mosquito hemocytes appeared to engulf these vesicles and granular material. As the vegetative stage was transformed into the reproductive stage, a newly formed, compact extracellular layer surrounded the sporangial initial. Later, a darkly staining wall appeared around the resting sporangium. Cisternae of endoplasmic reticulum consistently subtended thin areas in this pitted wall.