Oviposition bioassay responses of Culex tarsalis and Culex quinquefasciatus to neem products containing azadirachtin

Neem (Azadirachta indica A. Juss) products have been shown to exert pesticidal properties against a variety of insect species. In mosquito control programs, such products may have the potential to be used successfully as larvicides. In exploring other advantages of neem products, we studied the oviposition responses of Culex tarsalis Coquillett and Cx. quinquefasciatus Say to two experimental azadirachtin (AZ) formulations, wettable powder Azad ^– WP10 (WP) and emulsifiable concentrate Azad ^– EC4.5 (EC). Gravid Cx. tarsalis exhibited a distinct preference for the neem suspension of the WP, where significantly more egg rafts were collected from the treatment than from the control. The minimum effective AZ concentration for this activity was 0.5 ppm. The aged suspensions from 1–7 days at 0.5 and 1 ppm AZ were more active in eliciting oviposition responses in Cx. tarsalis than the fresh preparations. This activity of the aged suspensions lasted up to 14 and 21 days at 0.5 and 1 ppm AZ, respectively. Negative ovipositional responses were indicated in the tests of the EC vs. Cx. tarsalis, as well as both neem formulations vs. Cx. quinquefasciatus. In the tests of the EC formulation, significantly less gravid females were trapped by oviposition cups in the treatment than in the control, and in the tests of the WP significantly less egg rafts were collected from the treatment than from the control. The minimum effective concentrations for oviposition avoidance activity were 5 ppm AZ for Cx. tarsalis and 10 ppm AZ for Cx. quinquefasciatus, which lasted up to 1 and 4 days for these two species respectively. Neem products potentially used as mosquito larvicides may have many additional benefits in mosquito control programs, the oviposition modification noted in the current studies is one such example.     

The use of morphometric wing characters to discriminate female Culex pipiens and Culex torrentium

The reliability of the length of wing radial vein r_2/3 as a character for the morphological discrimination of the two potential arbovirus vectors Culex pipiens s.s. and Cx. torrentium from Germany was reassessed, after this character had been neglected for more than 40 years. Additionally, multivariate morphometric analyses were applied to evaluate wing shape variation between both species. Although high‐throughput molecular tools are now available to differentiate the two species, a simple, low‐cost routine alternative may be useful in the absence of a molecular laboratory, such as under semi‐field conditions. A thin‐plate splines transformation confirmed that primarily the shrinkage of vein r_2/3 is responsible for the wing differences between the two species. In the bivariate analysis, the r_2/3/r₃ indices of Cx. pipiens s.s. and Cx. torrentium were 0.185 and 0.289, respectively, resulting in a correct classification of more than 91% of all tested specimens. Using the absolute length of vein r_2/3 alone still allowed for more than 90% accurate discrimination. Furthermore, classification accuracy of linear discriminant analysis exceeded 97%.     

The arbovirus vector Culex torrentium is more prevalent than Culex pipiens in northern and central Europe

Two species of arbovirus vector, Culex torrentium and Culex pipiens (Diptera: Culicidae), occur in several European countries, but difficulties in their accurate identification and discrimination have hampered both detailed and large‐scale distribution and abundance studies. Using a molecular identification method, we identified to species 2559 larvae of Cx. pipiens/torrentium collected from 138 sites in 13 European countries ranging from Scandinavia to the Mediterranean coast. In addition, samples of 1712 males of Cx. pipiens/torrentium collected at several sites in the Czech Republic were identified to species based on the morphology of their hypopygia. We found that the two species occur together in large areas of Europe, and that Cx. torrentium dominates in northern Europe and Cx. pipiens dominates south of the Alps. The transition in dominance occurs in central Europe, where both species are roughly equally common. There was a strong correlation between the length of the growing season at different sites and occurrences of the two species. As the growing season increases, the proportion and detection of Cx. torrentium decrease, whereas those of Cx. pipiens increase. The present findings have important consequences for the interpretation of the results of studies on major enzootic and link‐vectors of mosquito‐borne bird‐associated viruses (i.e. Sindbis, West Nile and Usutu viruses), especially in central Europe and Scandinavia.     

Differential effects of Bacillus sphaericus strain 2362 on Culex quinquefasciatus and its competitor Culex cinereus in West Africa

The larval susceptibility to Bacillus sphaericus strain 2362 of the non-man-biting mosquito Culex cinereus and the urban filariasis vector Culex quinquefasciatus, two competitor mosquitoes in polluted habitats, was compared. In the laboratory, both species ingested a similar amount of B. sphaericus spores when fed c. 2 x 10(5) spores per ml for 30 min. However, in the same experiment, third-instar larvae of Cx quinquefasciatus were reduced by 98% at 24 h exposure while Cx cinereus larvae were only reduced by 6% at 72 h. In the field, preimaginal populations of Cx cinereus ingested, within a week, more than 99% of the applied spores, but showed no significant decrease through 14 days in cesspools treated at 10 g/m2 of a flowable concentrate of B. sphaericus 2362, containing 2 x 10(10) spores/g. It is proposed that specific biological control of Cx quinquefasciatus could result from appropriate treatment of breeding-sites with larvicidal B. sphaericus and competitive displacement by Cx cinereus or other mosquitoes with larvae that are more tolerant of B. sphaericus.     

A sensitive and reliable restriction enzyme assay to distinguish between the mosquitoes Culex torrentium and Culex pipiens

Culex pipiens pipiens Linnaeus and Culex torrentium Martini (Diptera: Culicidae) are closely related vector species that exist sympatrically in Europe. The two species are morphologically almost identical and can only be distinguished with certainty by characters of the male genitalia. Hence, correct species identification and conclusions on distribution and vector status are very difficult and often neglected. Therefore, we developed a reliable and simple mitochondrial cytochrome c oxidase subunit I (COI) gene restriction enzyme assay to discriminate between Cx. pipiens and Cx. torrentium, based on the analysis of morphologically identified male specimens. We sequenced approximately 830 bp in the 3′ region of the mitochondrial COI gene of 18 morphologically identified males of Cx. pipiens and Cx. torrentium. Two restriction enzymes (FspBI and SspI) that could distinguish between the two species according to species‐specific differences in these sequences were chosen. The restriction enzymes were tested on 227 samples from Sweden and verified by sequencing 44 of them. The enzyme FspBI correctly identified all investigated samples; the enzyme SspI identified all samples except one Cx. torrentium. We hope the method and the findings presented here will help to shed light on the true distribution and relative proportions of the two species in Europe.     

辽宁发现库蚊黄病毒

2011年从辽宁省丹东地区蚊虫样品中分离到6株病毒,采用逆转录-聚合酶链式扩增(RT-PCR)检测方法,结果黄病毒属通用引物和库蚊黄病毒特异性引物均为阳性。6株病毒核苷酸序列经基因库(GenBank)比对后,证实6株病毒为库蚊黄病毒,为我国首次报道。将病毒NS5基因和E基因核苷酸序列与GenBank中10株库蚊黄病毒参考毒株核苷酸序列进行比较,并构建遗传进化树,结果显示本次分离的6株病毒与美国和日本的毒株亲缘关系较近。     

光活化多炔类化合物对蚊幼虫的毒力

采用自制的光活化实验装置,测定了11个合成的多炔类化合物对致倦库蚊Culex quinquefasciatus 4龄幼虫的光活化毒力,发现部分化合物在近紫外光照射条件下,能明显地提高光活化毒杀效应,测得化合物5(1-苯基-4-(3,4-亚甲基二氧)苯基-丁二炔)光照与未光照处理LC₅₀分别是0.35 μg/mL和8.89 μg/mL。实验中发现蚊虫先接触药后,再进行光照处理,才能较好地发挥毒效,而且毒杀效应与光照时间呈正相关。模拟田间试验表明,太阳光能显著提高化合物5毒杀蚊幼虫的药效。利用抗氧化剂进行猝灭作用试验,间接地证明化合物5的光活毒杀机理是与过氧化作用有关。分析结构与活性关系,发现二苯基-丁二炔衍生物比二烷基取代丁二炔活性高,苯基上不同取代基也影响光活毒杀效果,它们的活性顺序是：亚甲基二氧基>甲氧基>邻硝基>间硝基>甲基酯。     

Chromosome-level assembly of Culex pipiens molestus and improved reference genome of Culex pipiens pallens (Culicidae,Diptera)

Culex pipiens molestus and Culex pipiens pallens are two distinct bioforms in the Culex pipiens complex that are important vectors of several pathogens and are widely distributed around the world. In the current study, we present a high-quality chromosome-level genome of Cx. pipiens f. molestus and describe the genetic characteristics of this genome. The assembly genome was 559.749 Mb with contig and scaffold N50 values of 200.952 Mb and 0.370 Mb, and more than 94.78% of the assembled bases were located on 3 chromosomes. A total of 19,399 protein-coding genes were predicted. Many gene families were expanded in the genome of Cx. pipiens f. molestus, particularly those of the chemosensory protein (CSP) and gustatory receptor (GR) gene families. In addition, utilizing Hi-C data, we improved the previously assembled draft genome of Cx. pipiens f. pallens, with scaffold N50 of 186.195 Mb and contig N50 of 0.749 Mb, and more than 97.02% of the assembled bases were located on three chromosomes. This reference genome provides a foundation for genome-based investigations of the unique ecological and evolutionary characteristics of Cx. pipiens f. molestus, and the findings in this study will help to elucidate the mechanisms involved in species divergence in the Culex pipiens complex.     

Wide recognition of Culex pipiens and lack of detection of Culex torrentium through biomolecular differentiation of mosquitoes in the Emilia‐Romagna region,Northern Italy

The Culex pipiens complex includes species with reported differences in vector competence for arthropod‐borne viruses, many of which are of significant importance to human health such as the West Nile virus and the Sindbis virus. This group of mosquitoes is difficult to distinguish morphologically; particularly as adult females. In Europe, the two species of the complex, Culex pipiens Linnaeus 1758 and Culex torrentium Martini 1925, are often found sympatrically. With the aim to characterize the presence and spread of both species in the Emilia‐Romagna region, Northern Italy, mosquitoes of the complex – collected during the West Nile virus surveillance plans – were tested by multiplex real‐time PCR for the detection of the two species Cx. pipiens and Cx. torrentium. A total of 24 165 mosquitoes, collected between 2012 and 2014 from 105 sites, and sorted in 204 pools, were tested. All tested pools were found to be composed of Cx. pipiens, whereas Cx. torrentium was not detected. These results indicate a likely absence of Cx. torrentium mosquitoes within the surveyed territory, whereas Cx. pipiens is widely distributed in the area mentioned. This is in line with previous reports, which describe a pre‐alpine distribution of Cx. torrentium in Italy.     

淡色库蚊对四种化学物质的嗅觉反应

在室内条件下通过Y型嗅觉仪测试了淡色库蚊Culexpipiens pallens雌成虫对乳酸、丙酮、氨水、辛醇、正庚酸、对甲酚、间甲酚等7种化学物质的嗅觉反应。结果表明:与对照相比,1和10mg/L氨水,1,10和100mg/L正庚酸,1mg/L辛醇,0.1和1mg/L对甲酚对淡色库蚊雌成虫具有显著的引诱作用。比较各处理的相对引诱率,最高的为10mg/L正庚酸,达50%以上;其他处理相对引诱率均低于40%,最低的为0.1mg/L对甲酚。     

Persistent natural infection of a Culex tritaeniorhynchus cell line with a novel Culex tritaeniorhynchus rhabdovirus strain

Culex tritaeniorhynchus rhabdovirus (CTRV) is a mosquito virus that establishes persistent infection without any obvious cell death. Therefore, occult infection by CTRV can be present in mosquito cell lines. In this study, it is shown that NIID‐CTR cells, which were derived from Cx. tritaeniorhynchus, are persistently infected with a novel strain of CTRV. Complete genome sequencing of the infecting strain revealed that it is genetically similar but distinct from the previously isolated CTRV strain, excluding the possibility of contamination. These findings raise the importance of further CTRV studies, such as screening of CTRV in other mosquito cell lines.     

The effect of predatory fish exudates on the ovipostional behaviour of three mosquito species: Culex quinquefasciatus, Aedes aegypti and Culex tarsalis

Abstract.  Three mosquito species, Culex tarsalis Coquillett, Culex quinquefasciatus Say and Aedes aegypti L. (Diptera: Culicidae), were examined in laboratory binary choice experiments to investigate whether fish exudates from the mosquitofish, Gambusia affinis (Baird & Girard) (Cyprinodontiformes: Poecilliidae), deter oviposition and whether the responses of these mosquito species to fish exudates in oviposition sites are consistent with the risk of predation from fish experienced by each species in their respective natural breeding habitats. Culex tarsalis was deterred significantly from egg laying by the presence of fish exudates in oviposition cups, consistent with high levels of predation by fish in natural breeding sites. Egg laying by Cx quinquefasciatus was slightly reduced in water with fish exudates, but was not consistently deterred by water conditioned by mosquitofish, consistent with the species' relatively low risk of fish predation in natural habitats. Oviposition by container-breeding Ae. aegypti was not deterred by the presence of fish exudates in oviposition cups, consistent with a low risk of predation by fish in natural habitats.     

Culex torrentium mosquitoes from Germany are negative for Wolbachia

Wolbachia (Rickettsiales: Anaplasmataceae) infects a wide range of arthropods, including several mosquito species. The bacterium is known to induce a plethora of phenotypes in its host, examples being the reproductive phenotype cytoplasmic incompatibility or resistance against infection with arboviruses. The latter is especially relevant when assessing the vector competence of mosquito species for emerging arboviruses. Thus, knowledge of Wolbachia infection status is important for the assessment of vector competence. To facilitate Wolbachia screening in mosquito populations, a quantitative polymerase chain reaction (qPCR) assay was developed to enable high‐throughput analysis of mosquito samples. Using this assay, the Wolbachia infection status of the two most common Culex mosquito species in Germany, Culex pipiens biotype pipiens Linnaeus (Diptera: Culicidae) and Culex torrentium Martini (Diptera: Culicidae), was assessed. About 93% of all tested C. pipiens biotype pipiens individuals were positive for Wolbachia, whereas none of the C. torrentium samples was found to be infected. Furthermore, other applications of the qPCR assay were explored by assessing a potential link between the levels of Wolbachia and West Nile virus (WNV) infections in German C. pipiens biotype pipiens mosquitoes. No relationship was found between the two variables, indicating that a Wolbachia‐induced antiviral phenotype in this mosquito population is not exclusively attributable to the general level of bacterial infection.     

四个不同地理区域的库蚊复合组居群的遗传多样性研究

库蚊种群在农药强选择压下的生存及抗性进化是与其种群的遗传多样性密切相关的。本文主要从居群的遗传多样性角度出发 ,研究其抗性遗传行为。分别对采自 4个不同地理区域的库蚊复合组样群 ,采用水平切片淀粉凝胶电泳的方法 ,分析了 3个酶系统 (苹果酸酶Me、苹果酸脱氢酶Mdh、甘油醛-3-磷酸脱氢酶Gpd) ,获得了 5个位点共 2 7个等位基因的资料。并计算了 4个库蚊居群的遗传多样性指标为 (A ,P ,He,Ho) ,同时分析了 5种居群间的遗传距离。分析结果表明 :遗传多样性主要存在于各居群之内 (FIS=0 .5 4 37) ,但居群之间的遗传分化程度也相对较大 (Gst =0 .4 498)。     

Vertical transmission of hepatitis B virus in Culex quinquefasciatus

An investigation of the vertical transmission of hepatitis B virus (HBV) in Culex quinquefasciatus Say revealed the presence of low levels of the virus in adult F1 progeny from the first ovarian cycle of mosquitoes infected by feeding on HBV positive human blood. HBV was not transmitted vertically during the second, third and fourth ovarian cycles nor to the F2 generation. The salivary glands, ovaries and faeces of the F1 generation did not contain detectable levels of HBV. Progeny of female Cx quinquefasciatus mated with F1 males were negative for HBV.     

Orientation of Culex mosquitoes to carbon dioxide-baited traps: flight manoeuvres and trapping efficiency

Females of Culex quinquefasciatus Say and Culex tarsalis Coquillet (Diptera: Culicidae) in the host-seeking stage were released and video recorded in three dimensions in a large field wind tunnel as they flew to four kinds of CO2-baited mosquito traps. The trapping efficiency (number of mosquitoes approaching compared to the number caught) was determined for each trap type. The Encephalitis Virus Surveillance (EVS), Mosquito Magnet Freedom (MMF) and Mosquito Magnet Liberty (MML) traps captured only 13-16% of approaching Cx. quinquefasciatus females, whereas the Mosquito Magnet-X (MMX) trap captured 58%. Similar results were obtained for Cx. tarsalis. Orientation behaviour and flight parameters of mosquitoes approaching the four traps were compared. Mosquitoes spent the most time orienting to the EVS trap. Flight speed decreased as mosquitoes entered the vicinity of each trap and a large portion of their time was spent within 30 cm downwind of the traps. Flights became highly tortuous downwind of the poorly performing traps and just upwind of the MMX trap. Differences between traps and possible explanations for the superior performance of the MMX trap are considered.     

球形芽孢杆菌对致倦库蚊的后致死作用

研究了球形芽孢杆菌Bacillus sphaericus C3-41菌株对致倦库蚊Culex quinquefasciatus幼虫的毒力及其后致死作用。生物测定表明,该菌株对目标蚊幼虫具有很高的毒力,其丙酮粉剂对3～4龄幼虫48 h的半致死浓度(LC₅₀)为(6.92±0.22) μg/L。用不同亚致死浓度处理2～3龄致倦库蚊幼虫,存活幼虫在后期发育中存在明显的延续死亡和损伤现象,经LC₃₀、LC₅₀、LC₇₀、LC₉₀和LC₉₈剂量的C3.41粉剂处理的致倦库蚊羽化前的总死亡率分别为84%、91%、95%、97%和100%,同时存活的幼虫、蛹和成蚊的发育和行为也受到一定的影响。这种后致死作用随处理浓度的升高而增强,可能同球形芽孢杆菌毒素蛋白对处理期间蚊幼虫中肠上皮细胞造成的损伤相关。     

Overwintering of Culex mosquitoes in Sweden and their potential as reservoirs of human pathogens

1. Culex pipiens L. and Cx torrentium Martini are suspected enzootic vectors in Sweden of Sindbis virus causing Ockelbo disease in man. To estimate their potential as virus reservoirs over the winter, mosquitoes were collected from August to May from a cellar in a rural area, central Sweden. The mosquitoes were dissected and examined for blood content, gonotrophic stage, parity and insemination, and tested for fructose. 2. Cx pipiens, Cx torrentium and Cx territans Walker constituted 94% of 887 females caught. A few Culiseta annulata (Schrank), Cs. alaskaensis (Ludlow) and An. maculipennis Meigen s.l. were also collected. 3. Data suggested that all blood-fed, parous Culex females disappeared during the winter and that only non-bloodfed, nulliparous females survived. 4. Fructose, detected in some of these females, and other plant sugars may be the main energy sources for winter survival. 5. Unless transovarially or venerally infected, Culex females are presumably of no or very limited importance in rural, central Sweden as winter reservoirs for blood-borne pathogens of vertebrates.