An effective entomopathogenic fungus Metarhizium anisopliae for the simultaneous control of Aedes albopictus and Culex pipiens mosquito adults

The purpose of this study was to select the most effective Metarhizium anisopliae isolates for the simultaneous control of Aedes albopictus and Culex pipiens mosquito adults. The selection was performed using 65 isolates of M. anisopliae, an entomopathogenic fungus isolated from Korean soils. The selection process was carried out through evaluations of conidial thermotolerance, UV-B tolerance, pathogenicity and virulence against adults of two mosquito species and conidial production on grain medium. The thermotolerance of 65 M. anisopliae isolates ranged from 0% to 99%, and their UV-B tolerance ranged from 7.1% to 94.8%. Among them, 30 isolates with high thermotolerance were selected for evaluation of their pathogenicity and virulence against adults of the two mosquito species. Metarhizium anisopliae isolates were more susceptible to Cx. pipiens adults than Ae. albopictus adults because 11 isolates were pathogenic to Ae. albopictus and 21 isolates were pathogenic to Cx. pipiens. Virulence and conidial production were evaluated using 6 isolates with similar pathogenicity against the two species of mosquito. As a result, the accumulated mortality was 74–100% for Ae. albopictus and 78–100% for Cx. pipiens. LT₅₀ values ranged from 2.3 to 5.2 days for Ae. albopictus and from 3.2 − 4.7 days for Cx. pipiens. Comparison of conidial production using three kinds of grain media showed various results according to isolate and medium. Based on the above results, M. anisopliae CN6S1W1 was finally selected as the most effective fungal isolate for the simultaneous control of Ae. albopictus and Cx. pipiens mosquito adults.     

Insecticidal effects of a novel polyherbal formulation (HF7) against Culex pipiens L. (Diptera: Culicidae)

Plant secondary metabolites represent the most efficient and convenient method to control and overcome environmental pollution and insecticidal resistance. This study explored the mosquitocidal activity of the combined extract of seven plants, (HF7) extracted using a Soxhlet extractor against Culex pipiens under laboratory conditions. Exposure of the 3rd instars of Cx. pipiens to HF7 hexane extract resulted in LC₅₀:114.5 μg/mL and LC₉₀:117.0 μg/mL values after 24 h. The ovicidal activities of hexane extract against Cx. pipiens eggs were 21.6%, 48.3%, and 71.6% at 187.5, 93.7, and 46.88 μg/mL, respectively. HF7-treated larvae showed the formation of irregular blebbing of epithelial cells toward the lumen and sloughing into the gut lumen. HF7 extract resulted in 100% adulticidal mortality at the concentration of 3.7 mg/test tube after 30 min of exposure. The IC₅₀ of HF7 extract was 97.03 µg/ml against larvae, at which nuclear and morphological changes were observed. The spectroscopy spectrum of HF7 hexane extract disclosed the presence of 57 different secondary metabolites, among which the dominant compound was eugenol (32.3%). HF7 hexane extract could serve as a botanical insecticide for controlling Cx. pipiens and potentially other mosquito species.     

Culex pipiens affected by joint infection of a mosquito iridescent virus and Strelkovimermis spiculatus

Dual infections with a mosquito iridescent virus (MIV) and the mermithid nematode, Strelkovimermis spiculatus were recorded in natural Culex pipiens populations around La Plata city, Argentina. S. spiculatus was detected in 82% of samples that were positive for MIV infection. Dissected larvae of Cx. pipiens with patent MIV infection presented 42% infection with S. spiculatus. Larvae of Cx. pipiens exposed to MIV and S. spiculatus under laboratory conditions produced a high joint infection rate (82.5%) while no infection was recorded on larvae exposed to virus suspension only. Field and laboratory results suggest a strong association between S. spiculatus and MIV in natural populations of Cx. pipiens, in which S. spiculatus could be a mode of entry for the virus into the mosquito hemocele.     

Field efficacy and non‐target effects of temephos granules against Culex pipiens (Diptera: Culicidae) and microorganisms in septic tanks,Republic of Korea

A granular formulation of 1.0% temephos (temephos G) was tested for its efficacy in mosquito larval control, residual effects and effects on non‐target microorganisms. Culex pipiens complex in septic tanks in Yangsan, Republic of Korea were used for this study. Microorganisms in the same septic tanks were used for study of non‐target organisms. After the application of temephos G at the rates of 2.0 and 5.0 mg/L to selected septic tanks, mortalities were recorded for 50 days. The effect of temephos G on non‐target organisms was studied by evaluating its effects on the bacterial populations in the treated septic tanks. It was observed that using 1.0% temephos G at a rate of 2.0 mg/L resulted in 100% mortality against Cx. pipiens larvae by the eighth day after treatment. The residual activity of temephos G was observed to continue for a time; for example 29 days after treatment, average larval mortality rates of >68.0% were observed for both concentrations. At the 50th day after treatment, the mortality rates of the Cx. pipiens larvae were observed to decrease to below 42.7% for both concentrations. Application of temephos G did not have significant effects on the bacteria populations in the septic tanks. Since temephos did not seem to have any major effects on the microorganisms, it was concluded that it is a safe and effective larvicide to use in the septic tanks at a rate of 2.0–5.0 mg/L of 1.0% temephos G.     

Controlling Culex pipiens: antagonists are more efficient than a neonicotinoid insecticide

Species vulnerability to pesticides depends on physiological sensitivity, the potential to recover, and the ecological context. We assessed the vulnerability of the mosquito Culex pipiens to a repeated treatment with thiacloprid in outdoor microcosms with and without antagonists (competitive and predatory invertebrates). Microcosms were treated repeatedly (three times) with thiacloprid at a concentration of 0.1, 1, or 10 µg/liter. In microcosms without antagonists, the abundance of Cx. pipiens larvae decreased moderately after the second and the third exposures to 10 µg/liter thiacloprid. In microcosms with antagonists, the abundance of Cx. pipiens larvae declined to approximately zero in the control group and the low concentration treatments during the five weeks of observation. By contrast, the abundance of Cx. pipiens larvae temporarily increased at 10 µg/liter thiacloprid after the second and third contamination. We explained this positive effect on the development of Cx. pipiens because of the decrease in competition due to the elimination of sensitive antagonists combined with the high recovery potential of Cx. pipiens. Based on these results, natural antagonists must be supported for the sustainable control of mosquitoes.     

Crustacean biodiversity as an important factor for mosquito larval control

Newly established ponds, which are highly dynamic systems with changing levels of biological interactions among species, are common larval mosquito habitats. We investigated the impact of crustacean abundance and taxa diversity on mosquito oviposition and larval development. The effects of the biological larvicide Bacillus thuringiensis israelensis (Bti) on mosquito larvae were monitored according to fluctuations in crustacean communities. Populations of the mosquito Culex pipiens colonized artificial ponds that contained crustacean communities at different time points of colonization by crustaceans: 1) ‘no colonization’ (no crustaceans), 2) ‘simultaneous colonization’ by crustaceans and mosquitoes, and 3) ‘head‐start colonization’ by crustaceans (preceding colonization by mosquitoes). All types of ponds were treated with three concentrations of Bti (10, 100, or 1,000 µg/liter). Colonization of all ponds by Cx. pipiens (in terms of oviposition, larval abundance, and larval development) decreased significantly with increasing diversity of crustacean taxa. The total abundance of crustaceans had a minor effect on colonization by Cx. pipiens. The presence of crustaceans increased the sensitivity of Cx. pipiens larvae to Bti treatment by a factor of 10 and delayed the time of recolonization. This effect of Bti was relevant in the short term. In the long term, the presence of Cx. pipiens was determined by crustacean biodiversity.     

A study of the mosquito Culex pipiens (Diptera, Culicidae) population structure in the transcaucasia using molecular identification methods

The mosquito species Culex pipiens consists of two forms, or ecotypes: the typical pipiens form and the molestus form. These forms are similar morphologically but have significant ecophysiological differences. The Cx. pipiens population structure was studied in open and underground habitats (house basements) in several localities in Abkhazia, Georgia, and Azerbaijan. The pipiens and molestus forms were identified by a molecular method, namely by restriction analysis of the amplification products with HaeIII endonuclease. Altogether, 90 individuals from 10 local populations were studied. The pipiens form was found only in surface water bodies (8 cases); in two cases both forms were found in one habitat. Homogeneous molestus populations were recorded in two underground habitats. Analysis of the Cx. pipiens population structure by the molecular method agrees with the results of biological and ecological studies of this mosquito in Georgia by Sh. Sichinava (1974, 1978, 1989). Thus, molecular diagnostics of intraspecific forms in Cx. pipiens populations is doubtlessly reliable.     

Larvicidal,ovicidal activities and histopathological alterations induced by Carum copticum (Apiaceae) extract against Culex pipiens (Diptera: Culicidae)

An experiment was carried out, firstly, to determine the possible toxicity of Carum copticum (Apiaceae) extract against Culex pipiens (Diptera: Culicidae), and, secondly, to study the histopathological alterations in the midgut of Cx. pipiens as a result of treatment with C. copticum extract. Larvicidal and ovicidal activities of C. copticum extract against the larvae of Cx. pipiens was determined according to World health organization (WHO). The inhibition effect of C. copticum was assessed by determining the mortality of the treated larvae and eggs. The histopathological effect of the C. copticum extracts on midgut epithelium of the larvae was examined under both light and transmission electron microscopy. The crude extract of C. copticum exerted 100% mortality for Cx. pipiens after 24 h at 200 μm/ml, and zero hatchability (100% mortality) at 150 μm/ml for Cx. pipiens. The histopathological study showed that larvae treated with C. copticum extract had cytopathological alterations of the midgut epithelium. The study provided information on various effects of C. copticum extract against Cx. pipiens.     

Effects of blood meal sources on the biological characteristics of Aedes aegypti and Culex pipiens (Diptera: Culicidae)

Mosquitoes transmit many diseases to humans and animals e.g., malaria, yellow fever, dengue, filariasis and encephalitis. The fundamental target of this search was to study the effect of three different blood meal sources (human; rabbit and pigeon) on some biological and behavioral properties of Aedes aegypti and Culex pipiens. The results have assured that the females of the mosquito Ae. aegypti that were fed on human blood meal has registered the highest feeding activity from feeding on the blood meal whereas the females of the other mosquito Cx. pipiens have shown the highest feeding activity after being fed on pigeons when compared with its feeding on other factors. The results have shown non-significant variation in the average time necessary to digest the blood meal on both mosquito species Ae. aegypti and Cx. pipiens that were fed on vertebrate hosts under laboratory conditions. Furthermore, results assured that the difference in blood meal sources has yielded distinct variation in the reproductive capacity and efficiency of both female mosquitoes under investigation where both species Ae. aegypti and Cx. pipiens already fed on human blood meal have yielded a pronounced distinctive increase in egg production (oviposition) when compared with females that were fed on pigeon or rabbit blood meal respectively. Moreover, feeding of the female mosquitoes under lab conditions on different blood meal sources did not affect the level of the hatching eggs that were laid by both mosquito females.     

Laboratory Evaluation of Controlled-release Repellent Treated Pulp Fabric on Human Volunteers Against Mosquito Vectors

Three controlled‐release personal‐use pulp fabric impregnated insect repellent formulations of 5% N, N‐diethyl‐3‐methylbenzamide (DEET), and 10% and 15% neem oils were evaluated in an environmental chamber on volunteers for their repellent efficacy against three mosquito species, Culex pipiens pallens, Aedes aegypti and Ochlerotatus togoi. The 5% DEET formulation showed significant repellency in pulp fabric (5 mm in width) against Culex pipiens pallens and Aedes aegypti (P < 0.05), providing an average repellency of 88.0%, 66.3%, and 46.8% of Cx. pipiens pallens, Ae. Aegypti and O. togoi bites, respectively, during the 6 hours of exposure period. Against night‐biting mosquitoes Cx. pipiens pallens, the DEET formulation provided mostly complete protection for at least 4 hours after the application. In pulp fabric of 10 mm in width, the 5% deet formulation showed significantly the highest repellency among the repellents against O. togoi (P<0.05), providing an average repellency of 52.3% during the 6 hours of exposure period. However, the pulp fabrics treated with 10% and 15% neem oil were less effective than 5% DEET against three mosquito species. This study demonstrated the potential of 5% DEET as pulp fabric repellent against both day‐ and night‐biting mosquitoes.     

Population Structure and Distribution Patterns of the Sibling Mosquito Species Culex pipiens and Culex torrentium (Diptera: Culicidae) Reveal Different Evolutionary Paths

Nowadays a number of endemic mosquito species are known to possess vector abilities for various diseases, as e.g. the sibling species Culex pipiens and Culex torrentium. Due to their morphological similarity, ecology, distribution and vector abilities, knowledge about these species'' population structure is essential. Culicidae from 25 different sampling sites were collected from March till October 2012. All analyses were performed with aligned cox1 sequences with a total length of 658 bp. Population structure as well as distribution patterns of both species were analysed using molecular methods and different statistical tests like distance based redundancy analysis (dbDRA), analysis of molecular variances (AMOVA) or McDonald & Kreitman test and Tajima''s D. Within both species, we could show a genetic variability among the cox1 fragment. The construction of haplotype networks revealed one dominating haplotype for Cx. pipiens, widely distributed within Germany and a more homogeneous pattern for Cx. torrentium. The low genetic differences within Cx. pipiens could be a result of an infection with Wolbachia which can induce a sweep through populations by passively taking the also maternally inherited mtDNA through the population, thereby reducing the mitochondrial diversity as an outcome of reproductive incompatibility. Pairwise population genetic differentiation (F_ST) ranged significantly from moderate to very great between populations of Cx. pipiens and Cx. torrentium. Analyses of molecular variances revealed for both species that the main genetic variability exists within the populations (Cx. pipiens [88.38%]; Cx. torrentium [66.54%]). Based on a distance based redundancy analysis geographical origin explained a small but significant part of the species'' genetic variation. Overall, the results confirm that Cx. pipiens and Cx. torrentium underlie different factors regarding their mitochondrial differentiation, which could be a result of endosymbiosis, dispersal between nearly located populations or human introduction.     

Repellent efficacy of wood vinegar against Culex pipiens pallens and Aedes togoi (Diptera: Culicidae) under laboratory and semi-field conditions

The repellent efficacy of wood vinegar was assessed against mosquitoes under laboratory conditions at 1, 5, 10, 20, 40, 60 and 80% concentrations. The study evaluated whether wood vinegar is able to repel Culex pipiens pallens Coquillet and Aedes togoi (Theobald) from the human body and if so at what concentrations. The tests were conducted using the arm-in-cage method in 80 × 40 × 40 cm screened mosquito cages. The data were analyzed and compared with those of N,N-Diethyl-3-methylbenzamide (deet) at 10.3% concentration. The results showed that wood vinegar provided mosquito repellence of varying degree depending on the concentration used. The observed repellence averaged from as low as 39.6% at 5.0% concentration to as high as 100% at 80% concentration against Ae. togoi. Repellence against Cx. pipiens pallens was high being 90.3% at 20% concentration, 92.2% at 40% concentration, 93.9% at 60% concentration and 100% at 80% concentration. The duration of protection time tests showed that the 40% and 60% concentrations of the wood vinegar give protection from landing of Ae. togoi for a period of up to 7 h, though the lower concentration gave lower protection after the first five hours. The results indicated that wood vinegar has mosquito repellent characteristics that tend to vary with the concentration used and the species of mosquitoes. Wood vinegar in this case was very effective in repelling Cx. pipiens pallens, even at lower concentrations while higher concentrations were required to repel Ae. togoi.     

Susceptibility of Anopheles messeae Fall. and Culex pipiens pipiens L. Larvae to entomopathogenic Fungi Metarhizium

The mortality rate of Anopheles messeae Fll. and Culex pipiens pipiens L. mosquito larvae infected with entopathogenic fungi Metarhizium brunneum and M. robertsii has been studied in laboratory conditions. It was shown that C. pipiens larvae are more susceptible to a water suspension of fungal conidia treatment while A. messeae larvae are more susceptible to treatment with dry conodia. The mortality of mosquito larvae infected with the most virulent strain and dose of 2 × 10⁶ conidia per cm² was 87.5–92.5%. Prospects for the use of fungi in the biocontrol of bloodsucking mosquitoes are discussed.     

Metagenomic sequencing reveals viral abundance and diversity in mosquitoes from the Shaanxi-Gansu-Ningxia region,China

BackgroundMosquitoes host and transmit numerous arthropod-borne viruses (arboviruses) that cause disease in both humans and animals. Effective surveillance of virome profiles in mosquitoes is vital to the prevention and control of mosquito-borne diseases in northwestern China, where epidemics occur frequently.MethodsMosquitoes were collected in the Shaanxi-Gansu-Ningxia region (Shaanxi Province, Gansu Province, and Ningxia Hui Autonomous Region) of China from June to August 2019. Morphological methods were used for taxonomic identification of mosquito species. High-throughput sequencing and metagenomic analysis were used to characterize mosquito viromes.ResultsA total of 22,959 mosquitoes were collected, including Culex pipiens (45.7%), Culex tritaeniorhynchus (40.6%), Anopheles sinensis (8.4%), Aedes (5.2%), and Armigeres subalbatus (0.1%). In total, 3,014,183 (0.95% of clean reads) viral sequences were identified and assigned to 116 viral species (including pathogens such as Japanese encephalitis virus and Getah virus) in 31 viral families, including Flaviviridae, Togaviridae, Phasmaviridae, Phenuiviridae, and some unclassified viruses. Mosquitoes collected in July (86 species in 26 families) showed greater viral diversity than those from June and August. Culex pipiens (69 species in 25 families) and Culex tritaeniorhynchus (73 species in 24 families) carried more viral species than Anopheles sinensis (50 species in 19 families) or Aedes (38 species in 20 families) mosquitoes.ConclusionViral diversity and abundance were affected by mosquito species and collection time. The present study elucidates the virome compositions of various mosquito species in northwestern China, improving the understanding of virus transmission dynamics for comparison with those of disease outbreaks.     

Modeling Dynamics of Culex pipiens Complex Populations and Assessing Abatement Strategies for West Nile Virus

The primary mosquito species associated with underground stormwater systems in the United States are the Culex pipiens complex species. This group represents important vectors of West Nile virus (WNV) throughout regions of the continental U.S. In this study, we designed a mathematical model and compared it with surveillance data for the Cx. pipiens complex collected in Beaufort County, South Carolina. Based on the best fit of the model to the data, we estimated parameters associated with the effectiveness of public health insecticide (adulticide) treatments (primarily pyrethrin products) as well as the birth, maturation, and death rates of immature and adult Cx. pipiens complex mosquitoes. We used these estimates for modeling the spread of WNV to obtain more reliable disease outbreak predictions and performed numerical simulations to test various mosquito abatement strategies. We demonstrated that insecticide treatments produced significant reductions in the Cx. pipiens complex populations. However, abatement efforts were effective for approximately one day and the vector mosquitoes rebounded until the next treatment. These results suggest that frequent insecticide applications are necessary to control these mosquitoes. We derived the basic reproductive number (ℜ₀) to predict the conditions under which disease outbreaks are likely to occur and to evaluate mosquito abatement strategies. We concluded that enhancing the mosquito death rate results in lower values of ℜ₀, and if ℜ₀<1, then an epidemic will not occur. Our modeling results provide insights about control strategies of the vector populations and, consequently, a potential decrease in the risk of a WNV outbreak.     

Charaterization of mosquito larval habitats in Qatar

Mosquito borne diseases have remained a grave threat to human health and are posing a significant burden on health authorities around the globe. The understanding and insight of mosquito breeding habitats features is crucial for their effective management. Comprehensive larval surveys were carried out at 14 sites in Qatar. A total of 443 aquatic habitats were examined, among these 130 were found positive with Culex pipiens, Cx. quinquefasciatus, Cx. mattinglyi, Ochlerotatus dorsalis, Oc. caspius and Anopheles stephensi. The majority of positive breeding habitats were recorded in urban areas (67.6%), followed by livestock (13.8%), and least were in agriculture (10.7%). An. stephensi larvae were positively correlated with Cx. pipien, Cx. quinquefasciatus, and negatively with water salinity. Large and shaded habitats were the most preferred by An. stephensi. In addition, Cx. pipiens was positively associated with the turbidity and pH, and was negatively associated with vegetation and habitat size. A negative association of Cx. quinquefasciatus with dissolved oxygen, water temperature, and salinity, while positive with habitat surface area was observed. Oc. dorsalis was negatively correlated with pH, water temperature, depth, and habitat surface area, whereas salinity water was more preferable site for females to lay their eggs. These results demonstrate that environmental factors play a significant role in preference of both anopheline and culicine for oviposition, while their effective management must be developed as the most viable tool to minimize mosquito borne diseases.     

A rural–urban latitudinal study of the distributions of Culex quinquefasciatus and Culex pipiens bioforms in their southernmost sympatric fringe

Mosquitoes grouped in the complex Culex pipiens L. (Diptera: Culicidae) are important vectors of medical and veterinary diseases. In the South American sympatric region, Cx. pipiens and Culex quinquefasciatus Say coexist and potentially hybridize. To identify key drivers of their geographical distribution, mosquito immatures were collected from flower vases of eight urban/rural cemetery pairs within a 5° latitudinal transect along Buenos Aires Province, Argentina. The specimens were identified by molecular methods and their relative proportion modelled as a function of environmental variables. At the beginning of the warm season, northern and southern cemeteries presented exclusively Cx. quinquefasciatus and Cx. pipiens, respectively, with different proportions of both at mid latitudes. By the end of the summer, Cx. quinquefasciatus was present throughout the study area, exclusively in 11 of the 16 cemeteries both rural and urban, whereas Cx. pipiens was predominant only in the southernmost pair. Mean annual temperature, photoperiod variability and time of the season were key drivers of their distributions. All specimens of Cx. pipiens were identified as form molestus and no hybrids were recognized. The reported distribution patterns and the potential absence of Cx. pipiens f. pipiens and hybrids are discussed, along with their implications in disease transmission.     

Seasonal Prevalence of Mosquitoes Collected from Light Traps in Gyeongsangnam Province,Republic of Korea (2013–2014)

Adult mosquito surveillance was conducted from 2013 through 2014 at four cattle sheds, a wild bird refuge, and two residential areas located in Gyeongnam Province in the Republic of Korea. Adult mosquitoes were collected in black light traps from April 1, through November 30. Mosquito surveillance was conducted to figure out population densities of vector mosquitoes, possibly invaded mosquitoes and identify various virus infections at the selected sites. A total of 107,466 females comprising 14 species and 7 genera were collected from 2013 to 2014. The most common species collected were Culex tritaeniorhynchus Giles (63.8%), Anopheles sinensis s.l. (18.9%), Aedes vexans nipponii (Theobald) (7.7%), and Culex pipiens Coquillett (5.1%). Trap indices (TIs) varied widely for species over their range, due to geographical distribution and degree of association with rural and urban communities . The most collected An. sinensis s.l. and Cx. tritaeniorhynchus appeared at a cow shed in Hapcheon (TI 347.5) and a pigsty in Daejeo‐1‐dong, Busan (TI 1,040.8), respectively, due in part to their situation near breeding sites such as rice paddies. The bi‐weekly population densities for mosquito species were variable for each of the years, apparently as a result of variable annual weather conditions. None of the mosquito species collected tested for the flavivirus including Japanese Encephalitis Virus, West Nile Virus, Dengue Virus, and Zika Virus infections by polymerase chain reaction (PCR) assay were positive.     

Formulation of the entomopathogenic fungus Beauveria bassiana JN5R1W1 for the control of mosquito adults and evaluation of its novel applicability

In this study, an effective control agent and control technology for adult mosquitoes using entomopathogenic fungi is proposed as a solution to the adverse effects of chemical insecticides used for mosquito control. A formulation study was conducted using Beauveria bassiana JN5R1W1 with high virulence against both Aedes albopictus and Culex pipiens adult mosquitoes. For the inverted emulsion formulation, three kinds of vegetable oils were used to evaluate the germination promotion, UV blocking effects, and adhesion rate of conidia. As a result, soybean oil was selected as the most effective vegetable oil, and the corresponding formulation was made. Direct contact treatment of the inverted emulsion by spraying resulted in a mortality rate of approximately 100 % for both species of adult mosquitoes. However, in reality, since it is difficult to directly apply fungal agent treatment against adult mosquitoes, we evaluated a control method by indirect contact with mosquitoes after applying the inverted emulsion on a net. As an indirect treatment method, the prepared inverted emulsion showed a mortality rate of approximately 93 % or 89 % against Ae. albopictus and Cx. pipiens adult mosquitoes, respectively. Therefore, these results suggested that the inverted emulsion using B. bassiana JN5R1W1 is an effective control agent for controlling adult mosquitoes.