An improved assay technique for matrone, a mosquito pheromone, and its application in ultrafiltration experiments.

An improved assay technique is presented for the detection of matrone, the mosquito pheromone which prevents insemination in female mosquitoes. This new technique requires 24 hours less to complete. The number of repeat tests necessary is reduced by 50% as compared to the number required utilizing the old technique and the reliability of the test is improved through the visual surveillance of the flight ability and mating activity of the females. Incorporated into this assay technique is the construction and use of a dose (dilution) response curve (calibration line) with a confidence band, establishing a threshold level of matrone activity and making it possible to assess the dilution of matrone in any non-toxic test material. Utilizing this new technique, we demonstrated by ultrafiltration that the molecular weight of matrone is between 50,000 and 100,000.     

The effect of matrone on oviposition in the mosquito, Aedes Aegypti

Male Aedes aegypti produce a substance made or stored in their accessory glands, which when passed on to females via mating, will prevent subsequent insemination. In addition to becoming refractory with regard to further insemination, mated females also oviposit far more eggs than virgins. The male accessory gland substance, called matrone, has been highly purified and when injected into virgin females, prevents any initial insemination, and will also stimulate oviposition.     

Age effects on the insemination rate ofAnopheles gambiae s.l. in the laboratory

Age effects on the insemination rate of the Galisua strain ofAnopheles gambiae s.s. (Diptera: Culicidae) and of the Nyanza strain ofAn. arabiensis were investigated in the laboratory. Batches of 7-day old males and females were kept together for 24 h with batches of mosquitoes of the opposite sex of ages ranging from 1–7 days. Males and females were also kept together continuously from emergence. The effect of increasing male/female ratios on the insemination rate was investigated as well. The insemination rate ofAn. arabiensis in the laboratory (96%) was similar to that found in nature, whereas that of our strain ofAn. gambiae s.s. was consistently low (maximum 72%), particularly after females had been together with males for a 24 h period only. The optimum age for insemination was 7 days for males and females of both species. The insemination rate of our strain ofAn. arabiensis was significantly higher at all ages than that of our strain ofAn. gambiae s.s.. The latter strain became inseminated at the earliest when 4 days old. An increase in the male/female ratio significantly enhanced the insemination rate in both strains. It was found that motility of spermatozoa inside the spermatheca, once in contact with saline, could be used as a marker for the approximate time of insemination. It is concluded that the relatively low insemination rate of the Galisua strain ofAn. gambiae s.s. is unlikely to be caused by the rearing conditions. Other factors that may be responsible are discussed.     

A study on pathogenicity and mosquito transmission success in the rodent malaria parasite Plasmodium chabaudi adami

We investigated the parasitology, pathogenicity (virulence) and infectivity to mosquitoes of blood infections in mice, of two strains, DS and DK, of the rodent malaria parasite Plasmodium chabaudi adami. Blood infections of DS were found to be highly pathogenic; the asexual parasites in these infections were fast-growing and showed no evidence of selectivity in their infection of host erythrocytes. In contrast to DS, blood infections of DK were much less pathogenic; the asexual parasites were slower-growing and showed a moderate degree of selectivity to a subset of erythrocytes which were not reticulocytes. In both DS and DK infections, infectivity to mosquitoes was highest before the peak of asexual parasitaemia had occurred; usually this did not coincide with the time when gametocyte numbers in the blood were highest. Infections with the pathogenic DS strain in CBA mice produced fewer gametocytes than did the less pathogenic DK strain. The DS strain infections in both CBA and C57 mice were also significantly much less infective to mosquitoes than the DK strain. Investigations by others on the related rodent malaria parasite subspecies, Plasmodium chabaudi chabaudi, have indicated that the mosquito infectivity of blood infections in mice tended to be higher in the more pathogenic (virulent) and lower in the less pathogenic strains of this parasite subspecies. This is the converse of the finding of the present investigation of blood infections of P. c. adami in mice in which a more pathogenic, or virulent, strain (DS) of these parasites was significantly much less infective to mosquitoes than was a less pathogenic strain (DK).     

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.     

Characterization of a novel esterase conferring insecticide resistance in the mosquito Culex tarsalis

Resistance to the organophosphate insecticide, malathion, in a strain of Culex tarsalis mosquitoes is due to increased activity of a malathion carboxylesterase (MCE). To determine whether resistance was due to a qualitative or quantitative change in the MCE, the enzyme was purified from both malathion-resistant and -susceptible mosquitoes. Enzyme kinetic measurements revealed that the two strains have one MCE in common, but resistant mosquitoes also have a unique MCE which hydrolyses malathion 18 times faster. Interestingly, this MCE does not hydrolyse α-naphthyl acetate, a substrate commonly used to detect increased levels of esterases in other organophosphate-resistant insects. Unlike the over-produced esterase of some related mosquito species, each MCE in C. tarsalis accounts for only a small fraction (0.015%) of the total extractable protein in either strain. Therefore, resistance in these insects is due to the presence of a qualitatively different enzyme, and not to a quantitative increase of a non-specific esterase. This study therefore demonstrates that the underlying biochemical mechanisms of insecticide resistance in one insect cannot necessarily be predicted from those of another, even closely related species. © 1995 Wiley-Liss, Inc.     

Plasmodium spp.: dispersion of malarial oocyst populations in anopheline and culicine mosquitoes

The numbers of malarial oocysts developing in individual, like mosquitoes fed concurrently on a single vertebrate malarial host were found to be distributed according to the negative binomial distribution in 169 experiments utilizing 6 species of Plasmodium, 6 species of mosquitoes and 3 species of vertebrate hosts. Dispersion constants ranged upward to 8.0, and mean clump sizes ranged upward to 298.4. The dispersion constant was demonstrated to be contingent on the species, strain and identity of the mosquito, the parasite and the vertebrate host; on the genetic state of the mosquito; and on the state of the infection in the vertebrate host. It was concluded that the concentration of oocyst production in particular mosquitoes was produced by varying levels and combinations of numerous factors associated with the parasite, the mosquito and the vertebrate host and that the pattern of oocyst distribution favors parasite survival and the maintenance of malaria in the field.     

Specialized Hydrocarbonoclastic Bacteria Prevailing in Seawater around a Port in the Strait of Malacca

Major degraders of petroleum hydrocarbons in tropical seas have been indicated only by laboratory culturing and never through observing the bacterial community structure in actual environments. To demonstrate the major degraders of petroleum hydrocarbons spilt in actual tropical seas, indigenous bacterial community in seawater at Sentosa (close to a port) and East Coast Park (far from a port) in Singapore was analyzed. Bacterial species was more diverse at Sentosa than at the Park, and the composition was different: γ-Proteobacteria (57.3%) dominated at Sentosa, while they did not at the Park. Specialized hydrocarbonoclastic bacteria (SHCB), which use limited carbon sources with a preference for petroleum hydrocarbons, were found as abundant species at Sentosa, indicating petroleum contamination. On the other hand, SHCB were not the abundant species at the Park. The abundant species of SHCB at Sentosa were Oleibacter marinus and Alcanivorax species (strain 2A75 type), which have previously been indicated by laboratory culturing as important petroleum-aliphatic-hydrocarbon degraders in tropical seas. Together with the fact that SHCB have been identified as major degraders of petroleum hydrocarbons in marine environments, these results demonstrate that the O. marinus and Alcanivorax species (strain 2A75 type) would be major degraders of petroleum aliphatic hydrocarbons spilt in actual tropical seas.     

Role of mermithid nematodes in biological control of mosquitoes

Mermithid nematodes have been reported from at least 63 species of mosquitoes from all over the world, but until recently they have received little attention. Such nematodes are prime candidates as biological control agents because they have adapted to the life cycle of the host; are host specific; produce high levels of parasitism; kill the hosts; are easily handled; have a high reproductive potential; are free swimming and can be disseminated easily in the infective stage; and can be used in an inundative or inoculative manner to control mosquitoes. Nevertheless, with the exception of one mermithid species, little is known about factors that influence parasitism in mosquitoes. Also, only one species of mermithid has been successfully mass cultured to date. The exception is the mermithid Reesimermis nielseni. With this mermithid, high levels of parasitism can be obtained in natural populations of mosquitoes at reasonable dosages; preparasitic nematodes can be applied easily by using many of the standard techniques used to apply insecticides; and R. nielseni can establish itself in many of the sites in which it is released. The same potentials exist for mermithids of other aquatic insects.     

Wolbachia Enhances West Nile Virus (WNV) Infection in the Mosquito Culex tarsalis

Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.     

Impact of Wolbachia on Infection with Chikungunya and Yellow Fever Viruses in the Mosquito Vector Aedes aegypti

Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever (YFV) viruses is increasing in many parts of the world. The viruses are primarily transmitted by Aedes aegypti, a highly domesticated mosquito species that is notoriously difficult to control. When transinfected into Ae. aegypti, the intracellular bacterium Wolbachia has recently been shown to inhibit replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing a potentially powerful biocontrol strategy for human pathogens. Because the extent of pathogen reduction can be influenced by the strain of bacterium, we examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and dissemination rates were significantly reduced in mosquitoes with the wMel strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar rates of infection and dissemination were observed in wMel infected and non-infected Ae. aegypti when intrathoracic inoculation was used to deliver virus. YFV infection, dissemination and replication were similar in wMel-infected and control mosquitoes following intrathoracic inoculations. In contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a 10⁴ times reduction in YFV RNA copies compared to controls. The extent of reduction in virus infection depended on Wolbachia strain, titer and strain of the virus, and mode of exposure. Although originally proposed for dengue biocontrol, our results indicate a Wolbachia-based strategy also holds considerable promise for YFV and CHIKV suppression.     

Cross-Mating Compatibility and Competitiveness among Aedes albopictus Strains from Distinct Geographic Origins - Implications for Future Application of SIT Programs in the South West Indian Ocean Islands

The production of large numbers of males needed for a sustainable sterile insect technique (SIT) control program requires significant developmental and operational costs. This may constitute a significant economic barrier to the installation of large scale rearing facilities in countries that are undergoing a transition from being largely dependent on insecticide use to be in a position to integrate the SIT against Aedes albopictus. Alternative options available for those countries could be to rely on outsourcing of sterile males from a foreign supplier, or for one centralised facility to produce mosquitoes for several countries, thus increasing the efficiency of the mass-rearing effort. However, demonstration of strain compatibility is a prerequisite for the export of mosquitoes for transborder SIT applications. Here, we compared mating compatibility among Ae. albopictus populations originating from three islands of the South Western Indian Ocean, and assessed both insemination rates and egg fertility in all possible cross-mating combinations. Furthermore, competitiveness between irradiated and non-irradiated males from the three studied strains, and the subsequent effect on female fertility were also examined. Although morphometric analysis of wing shapes suggested phenoptypic differences between Ae. albopictus strains, perfect reproductive compatibility between them was observed. Furthermore, irradiated males from the different islands demonstrated similar levels of competitiveness and induced sterility when confronted with fertile males from any of the other island populations tested. In conclusion, despite the evidence of inter-strain differences based on male wing morphology, collectively, our results provide a new set of expectations for the use of a single candidate strain of mass-reared sterile males for area-wide scale application of SIT against Ae. albopictus populations in different islands across the South Western Indian Ocean. Cross-mating competitiveness tests such as those applied here are necessary to assess the quality of mass reared strains for the trans-border application of sterile male release programs.     

Penetration and Development of the Mermithid Nematode Reesimermis nielseni in Eighteen Species of Mosquitoes

The susceptibility of 18 species of mosquitoes to the infective stage of the mermithid nematode Reesimermis nielseni was compared to that of Culex pipiens quinquefasciatus. Thirteen species were more susceptible, with three Anopheles species and Culiseta inornata the most susceptible. Aedes triseriatus, Culex territans, and Psorophora ferox were highly resistant. Resistance to R. nielseni appeared to be behavioral, physical, or physiological, and some host species exhibited one or more types of defense mechanisms. No noticeable differences were apparent in the degree of susceptibility of a native-and a laboratory strain of C. p. quinquqfasciatus to R. nielseni.     

Effect of Wolbachia on Replication of West Nile Virus in a Mosquito Cell Line and Adult Mosquitoes

Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that wMelpop and wMel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia-infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia. Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with wMel strain of Wolbachia was not inhibited, whereas wMelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when wMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia-free mosquitoes and were completely inhibited in the presence of Wolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in wMelPop strain of Wolbachia.     

Susceptibility and fitness cost of Aedes albopictus on their survivability after the exposure to the insecticide

The present study was undertaken to investigate the lethal and sub-lethal effects of insecticides on the life history of Aedes albopictus mosquitoes from urban and sub-urban areas in Penang, Malaysia. The female mosquitoes aged two to five days old were exposed to; (1) diagnostic dose (lethal dose) at 5% malathion and 0.75% permethrin; and (2) sub-lethal concentration of 1.5% malathion, and 0.2% permethrin, respectively and adult female survivors from sub-lethal concentrations were evaluated for fitness parameters. Fecundity, fertility, adult longevity, development time and survival of mosquitoes were the crucial point in their life history had studied. Aedes albopictus species from urban Sg. Dua strain has developed high resistance towards 0.75% permethrin and was confirmed on resistance after 24 h of reading. While sub-urban Batu Maung strain is still susceptible to both 5% malathion and 0.75% permethrin. At the sub-lethal dose, we discovered 0.2% permethrin insecticide have significantly more effects on the fitness cost of Ae. albopictus as compared to 1.5% malathion; with decreasing on fecundity, lesser time was needed to reach each development stages, and more male adult was emerged compared to female mosquitoes for both urban and sub-urban strains. Whereas, malathion insecticide only affected the number of eggs laid by the parent mosquitoes and the development time to reach adult stages. Even though, Ae. albopictus mosquitoes had developed resistance towards permethrin resulted in decreasing mortality, but subsequent effects on their fitness cost still continued on the first generation. Thus, it will benefit in reducing the transmission of mosquito-borne diseases.     

Differential Infectivities among Different Japanese Encephalitis Virus Genotypes in Culex quinquefasciatus Mosquitoes

During the last 20 years, the epidemiology of Japanese encephalitis virus (JEV) has changed significantly in its endemic regions due to the gradual displacement of the previously dominant genotype III (GIII) with clade b of GI (GI-b). Whilst there is only limited genetic difference distinguishing the two GI clades (GI-a and GI-b), GI-b has shown a significantly wider and more rapid dispersal pattern in several regions in Asia than the GI-a clade, which remains restricted in its geographic distribution since its emergence. Although previously published molecular epidemiological evidence has shown distinct phylodynamic patterns, characterization of the two GI clades has only been limited to in vitro studies. In this study, Culex quinquefasciatus, a known competent JEV mosquito vector species, was orally challenged with three JEV strains each representing GI-a, GI-b, and GIII, respectively. Infection and dissemination were determined based on the detection of infectious viruses in homogenized mosquitoes. Detection of JEV RNA in mosquito saliva at 14 days post infection indicated that Cx. quinquefasciatus can be a competent vector species for both GI and GIII strains. Significantly higher infection rates in mosquitoes exposed to the GI-b and GIII strains than the GI-a strain suggest infectivity in arthropod vectors may lead to the selective advantage of previously and currently dominant genotypes. It could thus play a role in enzootic transmission cycles for the maintenance of JEV if this virus were ever to be introduced into North America.     

Mosquito diversity (Diptera: Culicidae) and medical importance,in a bird sanctuary inside the flooded forest of Prek Toal,Cambodia

Wild birds have been identified as reservoirs of many pathogens transmissible to humans through bird-biting mosquitoes. Understanding the diversity of vector fauna in bird-conservation areas, is a cornerstone to later develop early detection systems and reduce the risk of emerging disease incidence. Prek Toal is a bird Sanctuary in the Tonle-Sap area in Cambodia hosting the largest migratory bird colony of southeast Asia. The Culicidae fauna was investigated during both dry and rainy seasons. In total, 8,224 mosquitoes from 6 genera and 25 species were collected. The two most represented genera were Anopheles (8 species) and Culex (11 species), while the dominant species was Culex vishnui (58.8%). During the rainy season, 1.6 fold more mosquitoes were caught and the number of species doubled. One of the most notable feature is the absence of any Aedinii mosquito which could be attributed to the low human density around the area, and the large populations of migratory birds. A total of 13 species from 3 genera are reported to be of medical importance. 6 medically-important species were collected in all 3 sites, and are known to bite both wild birds and humans: An. barbirostris, An. campestris, Cx. bitaeniorhynchus, Cx. vishnui, Ma. indiana and Ma. uniformis. One of the most important feature is the over-abundance of Culex vishnui, largely dominant in Prek Toal throughout the seasons and the sites, and being present everywhere in Cambodia. This finding stresses its potentiality to act as bridge vectors in the case of new disease emergence.     

Genetic variation of male reproductive success in a laboratory population of Anopheles gambiae

Background

A previous study showed for Anopheles gambiae s.s. a gradation of feeding preference on common plant species growing in a malaria holoendemic area in western Kenya. The present follow-up study determines whether there is a relationship between the mosquito's preferences and its survival and fecundity.

Methods

Groups of mosquitoes were separately given ad libitum opportunity to feed on five of the more preferred plant species (Hamelia patens, Parthenium hysterophorus, Ricinus communis, Senna didymobotrya, and Tecoma stans) and one of the less preferred species (Lantana camara). The mosquitoes were monitored daily for survival. Sugar solution (glucose 6%) and water were used as controls. In addition, the fecundity of mosquitoes on each plant after (i) only one blood meal (number of eggs oviposited), and (ii) after three consecutive blood meals (proportion of females ovipositing, number of eggs oviposited and hatchability of eggs), was determined. The composition and concentration of sugar in the fed-on parts of each plant species were determined using gas chromatography. Using SAS statistical package, tests for significant difference of the fitness values between mosquitoes exposed to different plant species were conducted.

Results and Conclusion

Anopheles gambiae that had fed on four of the five more preferred plant species (T. stans, S. didymobotrya, R. communis and H. patens, but not P. hysterophorus) lived longer and laid more eggs after one blood meal, when compared with An. gambiae that had fed on the least preferred plant species L. camara. When given three consecutive blood-meals, the percentage of females that oviposited, but not the number of eggs laid, was significantly higher for mosquitoes that had previously fed on the four more preferred plant species. Total sugar concentration in the preferred plant parts was significantly correlated with survival and with the proportion of females that laid eggs. This effect was associated mainly with three sugar types, namely glucose, fructose, and gulose. Except for P. hysterophorus, the results suggest that feeding by mosquitoes on preferred plant species under natural conditions results in higher fitness-related benefits, and that the sugar content in preferred plant parts is largely responsible for these effects.