Geographic distribution of wolbachial infections in mosquitoes from Thailand

Members of the genus Wolbachia are inherited intracellular bacterial endosymbionts that infect a diverse range of arthropods. Here I report the results of a survey of these endosymbionts in different mosquito species from six geographic regions of Northern, Northeastern, Western, Central, Eastern and Southern Thailand. Using gene amplification assays with wsp and groE gene primers, wolbachiae were detected in 999 mosquitoes representing 28 species of 1622 specimens collected representing 74 species of wild-caught mosquitoes from all regions of Thailand. Results using wsp primers were similar to those using groE primers in all cases. Wolbachiae had not been reported previously from five of the species tested, namely, Aedes lineatopennis, Aedes vexans, Aedes vittatus, Culex pallidothorax and Culex whitmorei. Infections were found in all major disease vector genera except Anopheles. These results indicate that wolbachial infections are distributed throughout many mosquito species in Thailand.     

Mosquitoes of Rarotonga,Cook Islands: A survey of breeding sites

Abstract

Two surveys of Rarotonga, Cook Islands (21°20^'S, 160°16^'W) were made to determine the mosquito fauna of the island, and to identify the habitats required for breeding by searching for larvae. The first survey was made during the “dry season” in May 2001, the second during the “wet season” in February 2002. The mosquito fauna comprised four species Culex (Culex) quinquefasciatus Say, Culex (Culex) annulirostris Skuse, Aedes (Stego‐myia) aegypti (Linnaeus) and Aedes (Stegomyia) polynesiensis Marks. Larvae of the Culex species were most often found in larger natural and artificial water bodies. The Aedes species bred in both natural and artificial containers of all sizes. Ae. polynesiensis was the most widespread species, using natural holes in all regions as well as artificial containers in the urban areas. Most larvae of Ae. aegypti were located in small artificial containers. The two Aedes species are the vectors of dengue fever on the island. Mosquito control during outbreaks should specifically target the artificial containers preferred by Aedes sp. for breeding habitats.     

Successional mosquito dynamics in surrogate treehole and ground‐container habitats in the northeastern United States: Where does Aedes albopictus fit in?

This study assessed the risk of larval displacement of the eastern treehole mosquito, Aedes triseriatus, and the northern house mosquito, Culex pipiens, by Aedes albopictus, the Asian tiger mosquito, during the establishment and successional stages of novel larval mosquito treehole and ground‐container habitats in the state of New Jersey, U.S.A. Culex pipiens and Culex restuans were the first mosquito species to colonize ground‐container habitats and were the dominant larval species throughout the study period, whereas Ae. albopictus was late to colonize ground habitats and accounted for less than 15% of weekly larval collections once established. Ae. albopictus had a much stronger community presence within treehole ovitraps; however, Ae. albopictus never reached the average larval densities of the expected primary colonizer, Ae. triseriatus. Throughout the study period, the weekly abundances of Ae. triseriatus and Ae. albopictus were positively correlated and there were no significant differences between the abundances of each species. The larval dominance of Ae. triseriatus appears to be enhanced by the presence of Toxorhynchites rutilus septentrionalis, a large predatory mosquito species. When Tx. rut. septentrionalis was present, mature larvae (3^rd–4^th instar) of Ae. albopictus were also present in only 16.7% of collections, whereas mature larvae of Ae. triseriatus were collected concurrently with Tx. rut. septentrionalis in 53.8% of collections. These data suggest that Ae. triseriatus is at a greater risk of displacement by Ae. albopictus than are Cx. pipiens and Cx. restuans.     

Stempellia milleri sp. n. (Microsporida: Nosematidae) in the Mosquito Culex pipiens quinquefasciatus Say*

Stempellia milleri sp. n. was found in blood cells and the adipose tissue of field-collected larvae of Culex pipiens quinquefasciatus Say. Its development is different from other Stempellia species described in that it is dimorphic, producing two types of spores in adipose cells. Both were studied with the electron microscope. One spore is thin-walled and uninucleate; the other is thick-walled and binucleate. The latter spores are produced from sporonts that are somewhat similar to those of Nosema. Sporoblasts and spores of S. milleri were compared to those of Stempellia magna Kudo, and Stempellia lunata Hazard and Savage in both light and electron microscope preparations. S. milleri was transmitted experimentally to Culex pipiens pipiens, C. p. quinquefasciatus, C. salinarius, C. tarsalis, and C. territans. Transmission of S. magna to its host and other mosquito species was not possible. Although, generally low numbers of test larvae became infected with the pathogen, heavy infections were seen occasionally in individual specimens of its natural host, C. p. quinquefasciatus. Species of Aedes, Anopheles, Culiseta, Psorophora, and Uranotaenia exposed to spores of S. milleri were not susceptible to the disease.     

Hemolymph pH of the larvae of three species of mosquitoes,and the effect of Romanomermis culicivorax parasitism on the blood pH of Culex pipiens

The hemolymph pHs of late fourth-instar Culex pipiens, Aedes taeniorhynchus, and Toxorhynchites amboinensis were measured with pH-sensitive glass microelectrodes and were slightly alkaline; pH 7.51, 7.62, and 7.37, respectively. The hemolymph pH remained relatively constant during the development of C. pipiens larvae through the third and early and late fourth instars. The hemolymph pH in C. pipiens larvae parasitized with the mermithid nematode Romanomermis culicivorax was unaltered. These measurements provide an approximate pH (ca 7.4) which is normal for the hemolymph of larval mosquitoes, and should be useful for further development of a culture medium for R. culicivorax.     

Oviposition substrate selection by Florida mosquitoes in response to pathogen‐infected conspecific larvae

The impact of the presence of larval mosquito pathogens with potential for biological control on oviposition choice was evaluated for three mosquito species/pathogen pairs present in Florida. These included Aedes aegypti infected with Edhazardia aedis, Aedes albopictus infected with Vavraia culicis, and Culex quinquefasciatus infected with Culex nigripalpus nucleopolyhedrovirus (CuniNPV). Two‐choice oviposition bioassays were performed on each host and pathogen species with one oviposition cup containing infected larvae and the other cup containing uninfected larvae (control). Both uninfected and E. aedis‐infected female Ae. aegypti laid significantly fewer eggs in oviposition cups containing infected larvae. Uninfected gravid female Ae. albopictus and Cx. quinquefasciatus oviposited equally in cups containing uninfected larvae or containing larvae infected with V. culicis or CuniNPV, respectively. Gravid female Ae. albopictus infected with V. culicis did not display ovarian development and did not lay eggs. The decreased oviposition by gravid Ae. aegypti in containers containing E. aedis‐infected larvae may indicate that the infected larvae produce chemicals deterring oviposition.     

The effect of parasitism by the mermithid Romanomermis culicivorax on the dry weight and hemolymph soluble protein content of three species of mosquitoes

The dry weight, hemolymph soluble protein composition, and content of three species of mosquitoes, Culex pipiens, Aedes taeniorhynchus, and Anopheles quadrimaculatus were examined to determine the effects of parasitism by the mermithid nematode Romanomermis culicivorax. The dry weights of infected fourth-stage larvae of all three species were significantly lower than controls. The differences in weight found between infected early and late C. pipiens and A. quadrimaculatus larvae were attributed to the weight of the parasite itself. This difference was not noticeable in A. taeniorhynchus larvae. Hemolymph proteins were severely depleted in all three mosquito species during parasitism by R. culicivorax. Analysis of protein composition by PAGE showed that these depletions were accompanied by a reduction in the number of proteins. Differences between protein composition concentrations were evident between early and late fourthstage control larvae of C. pipiens and A. quadrimaculatus. The concentration of some low-molecular-weight proteins (below 68,000) remained constant between infected and control samples of all three mosquito species.     

The comparative activity of the Australian and united states strains of Culicinomyces clavosporus bioassayed in mosquito larvae of three different genera

Anopheles hilli, Culex quinquefasciatus, and Aedes aegypti were used as test insects to compare the activity of the Australian and United States strains of Culicinomyces clavosporus. To minimize the variability incurred by using different larval batches, both strains were bioassayed at the same time using one batch of larvae. Six pairs of assays for each of the three test species were conducted in this manner. It was found that there was no difference in potency of the two strains in any one of the three species. A between species comparison, with the data pooled for both strains, showed that A. aegypti was more susceptible to the fungus than A. hilli. The susceptibility of C. Quinquefasciatus appeared to be intermediate but the fiducial limits of the weighted mean LC₅₀ overlapped with those of the other two species. From the results of these experiments it would seem that, with regard to potency, both strains of Culicinomyces may be equally promising for the biological control of mosquitoes.     

Larval Gnathostomes and Zoonotic Trematode Metacercariae in Fish from a Local Market in Yangon City,Myanmar

A survey was performed to investigate the infection status of zoonotic helminth larvae in fish from a local market of North Dagon District in Yangon City, Myanmar. A total of 486 fish in 13 species were collected 8 times from December 2015 to December 2019. All fish were transported under ice to a laboratory in Korea and examined for helminth larvae using artificial digestion method. Larval gnathostomes and metacercariae of more than 8 zoonotic trematode species, i.e., Opisthorchis viverrini, Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus spp., Stellantchasmus falcatus, Pygidiopsis cambodiensis, and Procerovum sp., were detected. Larval gnathostomes were found in 58 (16.0%) out of 362 fish of 6 species, with mean intensity of 2.8 per fish infected. Metacercariae of O. viverrini were detected in 10 (2.9%) out of 349 fish of 5 species, with mean intensity of 16.9 per fish infected. Metacercarial prevalences of 4 intestinal flukes, H. taichui, H. pumilio, H. yokogawai, and Centrocestus spp., were 16.8%, 26.0%, 12.5%, and 15.0% in the positive fish species, respectively, and mean metacercarial intensity was 63.3, 26.8, 86.2, and 8.7 per fish infected. Metacercariae of S. falcatus and P. cambodiensis were detected only from the mullet, Chelon macrolepis. Metacercariae of Procerovum sp. were found in Channa striata and Anabas testudineus. Collectively, it was confirmed that the fish were infected with gnathostome larvae and metacercariae of O. viverrini and intestinal flukes in Yangon City, Myanmar.     

Understanding the mechanism of Chikungunya virus vector competence in three species of mosquitoes

Chikungunya virus (CHIKV) is primarily transmitted by Aedes spp. mosquitoes. The present study investigated vector competence for CHIKV in Aedes aegypti and Aedes albopictus mosquitoes found in Madurai, South India. The role of receptor proteins on midguts contributing to permissiveness of CHIKV to Aedes spp. mosquitoes was also undertaken. Mosquitoes were orally infected with CHIKV DRDE‐06. Infection of midguts and dissemination to heads was confirmed by immunofluorescence assay at different time points. A plaque assay was performed from mosquito homogenates at different time points to study CHIKV replication. Presence of putative CHIKV receptor proteins on mosquito midgut epithelial cells was detected by virus overlay protein binding assay (VOPBA). The identity of these proteins was established using mass spectrometry. CHIKV infection of Ae. aegypti and Ae. albopictus midguts and dissemination to heads was observed to be similar. A plaque assay performed with infected mosquito homogenates revealed that CHIKV replication dynamics was similar in Aedes sp. mosquitoes until 28 days post infection. VOPBA performed with mosquito midgut membrane proteins revealed that prohibitin could serve as a putative CHIKV receptor on Aedes mosquito midguts, whereas an absence of CHIKV binding protein/s on Culex quinquefasciatus midguts can partially explain the non‐permissiveness of these mosquitoes to infection.     

Elaphoidella taroi: The intermediate copepod host in Fiji for the mosquito pathogenic fungus Coelomomyces

Five species of copepods were screened for their ability to transmit Coelomomyces sp. in Fiji. Only one, a common treehole copepod, Elaphoidella taroi, was found to be the intermediate host. When E. taroi was used, the mosquitoes Aedes aegypti, A. pseudoscutellaris, and A. polynesiensis were susceptible to Coelomomyces sp. infection.     

Septic tank mosquitoes: competition between species in central Nigeria

Exit traps, placed over the air vents of septic tanks, were used to examine species diversity and relative abundance of mosquitoes breeding in ammonia-rich waters of septic tanks. Of the six species found, Culex decens Theobald and Culex cinereus Theobald appeared to be competing successfully with Culex quinquefasciatus Say during the wet season but not during the long dry season. The seasonal timing of their displacement by Cx quinquefasciatus was variable and did not correlate well with climatic factors. The three other species present, generally during the wet season and early dry season, were Culex tigripes G. & C., Culex horridus Edwards and Aedes aegypt (L.). Experimental bucket ovitraps were used to assess preference towards covered (dark) septic tank water in comparison with sunlit septic tank water, covered and sunlit compost water. These were colonized by Cx quinquefasciatus, Cx decens, Ae. aegypti and Ae. vittatus Bigot. The covered septic tank water was more abundantly colonized by Cx quinquefasciatus and marginally so by the two Aedes species. Cx decens appeared to colonize the exposed compost water more readily in the dry season, but changed to the covered septic tank water in the wet season. The discussion centres around competition between these mosquitos species and concludes that it would be useful to know what environmental factors, or what aspects of competition, lead to severe natural reductions in the abundance of the major pest species Cx quinquefasciatus.     

Incrimination of the mosquito,Aedes taeniorhynchus,as the primary vector of heartworm,Dirofilaria immitis,in coastal Yucatan,Mexico

Mosquito collections were carried out on microfilaraemic dogs, positive for Dirofilaria sp., for 18 consecutive nights in the coastal town of Celestún, Yucatan, southeast Mexico, during the rainy season (August) of 2007. A total of 292 female mosquitoes representing 12 species of dipteran Culicidae were collected: Anopheles albimanus (Wiedemann); Anopheles crucians (Wiedemann); Anopheles pseudopunctipennis (Theobald); Culex coronator (Dyar & Knab); Culex interrogator (Dyar & Knab); Culex nigripalpus (Theobald); Culex quinquefasciatus (Say); Culex salinarius (Coquillett); Aedes aegypti (Linnaeus); Aedes scapularis (Rondani); Aedes sollicitans (Walker), and Aedes taeniorhynchus (Wiedemann). Aedes taeniorhynchus and Cx. quinquefasciatus were the species found most commonly feeding on the dogs. Filarial nematodes were observed by microscopy in nine of the mosquito species collected; however, third‐instar larvae were only observed in Ae. taeniorhynchus and An. crucians. Of 76 Ae. taeniorhynchus specimens found positive for Dirofilaria sp. by dissection, 14 were confirmed to be positive for Dirofilaria immitis (Leidy) by polymerase chain reaction (PCR). The resulting infection rate for D. immitis confirmed by PCR (6.2%) is higher than any infection rate for Ae. taeniorhynchus previously reported from the Americas.     

Susceptibility of aposymbiotic Culex quinquefasciatus to Wuchereria bancrofti

Larvae of the mosquito Culex quinquefasciatus originating from Kenya were reared in 0.025 mg/ml tetracycline hydrochloride. Some of the resulting progeny were shown, by electron microscopy and crossing experiments, to have been rendered free of the rickettsia like symbiont Wolbachia pipientis and from these progeny, symbiont-free lines were established. In experimental feedings on infected human volunteers and on cryopreserved microfilariae, the aposymbiotic stocks were found to be fully susceptible to the filaria Wuchereria bancrofti. This contrasts with some recently published data on Aedes polynesiensis, from which it has been suggested that rickettsia like symbionts have an important role in the development of filaria in the mosquito.     

Reactive Oxygen Species Production and Brugia pahangi Survivorship in Aedes polynesiensis with Artificial Wolbachia Infection Types

Heterologous transinfection with the endosymbiotic bacterium Wolbachia has been shown previously to induce pathogen interference phenotypes in mosquito hosts. Here we examine an artificially infected strain of Aedes polynesiensis, the primary vector of Wuchereria bancrofti, which is the causative agent of Lymphatic filariasis (LF) throughout much of the South Pacific. Embryonic microinjection was used to transfer the wAlbB infection from Aedes albopictus into an aposymbiotic strain of Ae. polynesiensis. The resulting strain (designated “MTB”) experiences a stable artificial infection with high maternal inheritance. Reciprocal crosses of MTB with naturally infected wild-type Ae. polynesiensis demonstrate strong bidirectional incompatibility. Levels of reactive oxygen species (ROS) in the MTB strain differ significantly relative to that of the wild-type, indicating an impaired ability to regulate oxidative stress. Following a challenge with Brugia pahangi, the number of filarial worms achieving the infective stage is significantly reduced in MTB as compared to the naturally infected and aposymbiotic strains. Survivorship of MTB differed significantly from that of the wild-type, with an interactive effect between survivorship and blood feeding. The results demonstrate a direct correlation between decreased ROS levels and decreased survival of adult female Aedes polynesiensis. The results are discussed in relation to the interaction of Wolbachia with ROS production and antioxidant expression, iron homeostasis and the insect immune system. We discuss the potential applied use of the MTB strain for impacting Ae. polynesiensis populations and strategies for reducing LF incidence in the South Pacific.     

Isolation and identification of entomopathogenic fungus from Eastern Ghats of South Indian forest soil and their efficacy as biopesticide for mosquito control

The repeated usage of chemical insecticides, responsible for insecticide resistance in mosquitoes and environmental toxicity. Currently effective and environmental-safe control strategies are needed for the control disease-vector mosquitoes. Entomopathogens can be an effective alternative to chemical insecticide. Herein we isolated and tested 46 soil-borne entomopathogenic fungi belonging to six genera, namely Beauveria sp., Metarhizium sp., Fusarium sp., Aspergillus sp., Trichoderma sp., and Verticillium sp., fungi conidia were tested on Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus larvae. Bioassays results show that M. anisopliae fungal isolate causes a 100%, 98.6% and 92% mortality within six days, on Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus, respectively. M. anisopliae treated three mosquito larvae have lower lifetime with LT₅₀ values in A. stephensi, 2.931 days; A. aegypti, 2.676 days and C. quinquefasciatus, 3.254 days. 18 s rDNA sequence analysis confirmed that the isolated fungus are belonging to the genus of M. anisopliae-VKKH3, B. bassiana-VKBb03, and V. lecanii-VKPH1. Our results clearly show that M. anisopliae has good potential, as a low-cost, environmentally safe tool for the control of A. aegypti, A. stephensi, and C. quinquefasciatus mosquitoes.     

Evaluation of a briquet formulation of Bacillus thuringiensis var. israelensis (H‐14) against aedes spp. and culex spp. larvae in dambos in Kenya

Bacillus thuringiensis Berliner var. israelensis serotype H‐14 (Bti) in briquet formulation (Bactimos) was tested in a field trial against ground‐pool breeding mosquitoes in a dambo located in a Rift Valley fever virus‐enzootic/epizootic area in central Kenya. Bactimos (10% Bti; 7000 AA International Toxic Units/mg) was tested for 30 days in 3 separate treatment areas at the rate of 1 briquet/9 m2, later increased to 1 briquet/1.5 m2 on day 13 postflood; 1 briquet/9 m2; and 1 briquet(4.6 m2). An estimate of the daily survival rate of larvae at different periods during the study revealed that mosquitoes in the area treated with 1 briquet/9 m2 had a significantly lower (64%) survival rate than those in the control site (92%) against Aedes spp. and was not significantly different from the site treated with 1 briquet/4.6 m². Mosquitoes in the site treated with I briquet/1.5 m² on day 13 postflood had a much‐reduced survival rate (25%;) when compared to those in the control site (67%) during the 3rd Culex spp. generation (22‐ 26 days postflood). This five‐fold increase above standard label dosage still failed to prevent Culex spp. emergence. There was no significant difference in survival rates between the control and any of the three treatment sites during the first or second Culex generation (9–20 days postflood). Emerged adults collected as pupae from control and treatment sites indicated that Aedes mcintoshi Huang and Culex antennatus (Becker) were the most predominant Aedes and Culex species.     

Laboratory evaluation of lactic acid on attraction of Culex spp. (Diptera: Culicidae)

The role of lactic acid was evaluated for attraction of Culex nigripalpus, Culex quinquefasciatus, Culex tarsalis, and Aedes aegypti in the laboratory using a dual‐port olfactometer. When lactic acid was combined with chicken odor, attraction was increased for Cx. quinquefasciatus compared to chicken odor alone but not for Cx. nigripalpus, Cx. tarsalis, and Ae. aegypti. Lactic acid combined with hand odor did not change attraction of Cx. tarsalis and Ae. aegypti but decreased attraction of Cx. nigripalpus and Cx. quinquefasciatus. The addition of lactic acid to CO₂ increased attraction of Ae. aegypti and Cx. quinquefasciatus but reduced attraction of Cx. nigripalpus and Cx. tarsalis. Use of commercial lactic acid baits with CO₂ resulted in a similar trend except for Cx. nigripalpus which showed no difference. A blend of lactic acid, acetone, and dimethyl disulfide was attractive to Ae. aegypti (63.4%) but elicited low responses by all Culex spp. (1.3–26.8%). Addition of the blend to CO₂ increased attraction of Ae. aegypti and Cx. quinquefasciatus but reduced attraction of Cx. nigripalpus and Cx. tarsalis. The mixture of compounds plus CO₂ was as attractive as a hand for Cx. quinquefasciatus, Cx. tarsalis, and Ae. aegypti.     

Bamboo stumps as mosquito larval habitats in Darjeeling Himalayas, India: A spatial scale analysis

Bamboo stumps can be a congenial breeding habitat of the mosquitoes. In view of this, a preliminary assessment of the dipteran immatures inhabiting the stumps of bamboo groves in the Darjeeling Himalayas was carried out at a spatial scale. Of the 104 stumps of Dendrocalamus hamiltoni surveyed, 70 were found to host immatures of three dipteran species, the mosquitoes Aedes aegypti and Culex quinquefasciatus and the midges Chironomus sp. in varying densities. Though the stumps varied in diameter, in each stump on average 12. 1 immatures were found. The abundance of the immatures was positively correlated with the diameter of the stumps (r=+0.382; P < 0.001) but negatively with the pH of the water present in the stumps (r=–0.336; P < 0.01). The coefficient of association was found to be +8.4 for the Ae. aegypti and Chironomus immatures, while in the rest of the species pair the association seemed to be independent. Thus it can be concluded that the stumps in the bamboo groves of Darjeeling Himalayas provides a favourable habitat for the mosquito and chironomid immatures.     

Cytogenetic analysis of metaphase chromosomes from pupal testes of four mosquito species using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization technique (FISH)

The DNA probes, P1887, P2405, P2056 (being specific tags for Aedes aegypti genes coding for ribosomal RNA) and a centric heterochromatin probe, K20-1A5, were chosen to hybridize the metaphase chromosomes from the testes of four mosquito species, Culex pipiens, Aedes aegypti, Aedes albopictus and Aedes triseriatus. In addition, a single plasmid, P2392, which contained the three probes, P1887, P2405 and P2056, was also used as chromosome landmark in aedine species. Only the Aedes aegypti metaphase chromosome 1-specific tag, P1887, was conserved in Aedes albopictus, Aedes triseriatus, and Culex pipiens metaphase chromosomes. Aedes triseriatus exhibited two gene loci, on chromosomes 1 and 3, coding for ribosomal RNA per haploid genome. When the specific probes for chromosomes 2 and 3, 2405 and 2056, were used in the fluorescence in situ hybridization technique against the metaphase chromosomes the fluorescent signals were not seen in Aedes albopictus, Aedes triseriatus or Culex pipiens. Also the centric heterochromatin probe, K20-1A5, exhibited strong fluorescent signals on chromosomes 1, 2 and 3 of Aedes aegypti. These fluorescent signals were not observed in metaphase chromosomes derived from the other aedine species, indicating that the centromere sequence can vary within the species.This paper was presented at the Second Arab Conference on Biotechnology and Genetic Engineering, held in the Kingdom of Bahrain, 15–17 April, 2002 and is published here with the endorsement of the Co-ordinator of the Scientific Committee, Professor Essam H. Ghanem, University of Bahrain. Its publication has been delayed because of the ill health of the senior author. Other papers from this conference were published in the July 2003 issue (vol. 19, no. 5).