Enantiomeric selectivity in behavioural and electrophysiological responses of Aedes aegypti and Culex quinquefasciatus mosquitoes

1-Octen-3-ol is a kairomone for many haematophagous insects including mosquitoes. Numerous studies have examined the effects of racemic 1-octen-3-ol; however, few studies have investigated the role of individual enantiomers in relation to mosquito attraction. In the present study, we investigated the behavioural and electrophysiological responses of two mosquito species, Aedes aegypti and Culex quinquefasciatus, to individual enantiomers and mixtures of 1-octen-3-ol, employing a laboratory Y-tube olfactometer and single sensillum recordings. The olfactory receptor neurons of both Ae. aegypti and Cx. quinquefasciatus had a significantly higher response to the (R)-1-octen-3-ol enantiomer compared to the (S)-1-octen-3-ol enantiomer at 10-9 g μl-1 to 10-6 g μl-1. Behaviourally, Ae. aegypti was more responsive to the (R)-1-octen-3-ol enantiomer, showing an increase in flight activity and relative attraction compared to Cx. quinquefasciatus. The (R)-1-octen-3-ol enantiomer caused an increase in activation for Cx. quinquefasciatus. However, the most notable effect was from an (R:S)-1-octen-3-ol mixture (84:16) that caused significantly more mosquitoes to sustain their flight and reach the capture chambers (demonstrated by a reduced non-sustained flight activity), suggesting that it may have a behaviourally excitatory effect. For Cx. quinquefasciatus, a reduced relative attraction response was also observed for all treatments containing the (R)-1-octen-3-ol enantiomer, either on its own or as part of a mixture, but not with the (S)-1-octen-3-ol enantiomer. This is the first time enantiomeric selectivity has been shown for Ae. aegypti using electrophysiology in vivo. The implications of these results for exploitation in mosquito traps are discussed.     

Goniothalamin – a potent mosquito larvicide from Bryonopsis laciniosa L.

Abstract: Goniothalamin isolated from the indigenous plant, Bryonopsis laciniosa L., was highly effective against the larvae of the mosquito, Culex quinquefasciatus Say, as a mosquitocide. This is the first report of goniothalamin. The structure of the bioactive compound isolated from the EtOAc (CH₃COOC₂H₅) fraction was established through spectroscopic methods using ¹H-NMR and ¹³C-NMR in CDCl₃. The mass spectrum was also recorded using VG micromass ZAB mass spectrometer.     

昆虫JHE基因结构及与甲基化相关的CpG O/E值分析

本研究选择西方蜜蜂、黑腹果蝇、致倦库蚊和家蚕四种昆虫为例,以降解保幼激素的特异性酯酶（juvenile hormone esterase,JHE）为研究对象,首先运用NCBI的Spidey在线软件将各昆虫的JHE基因与其相应的基因组序列作比对,分别确定各昆虫JHE基因的上游2000bp的具体序列和外显子及内含子区域。发现西方蜜蜂JHE基因含有7个外显子和6个内含子,而其余三种昆虫的JHE基因均含有6个外显子和5个内含子。接着,分别统计基因各区域的CpG,G,C位点的占有量,并计算出CpG O/E值（CpG位点的实际值与期望值之间的比值）,发现各区域CpG O/E平均值的大小依次为：基因上游2000bp区>内含子区>外显子区,基因上游2000bp区的CpG O/E平均值大于1.0,而第1到第4外显子的CpG O/E平均值均只有0.8左右。表明在昆虫JHE基因中,若有CpG甲基化位点发生,应主要发生在第1到第4外显子区域。     

Genetic determinants of host ranges of Bacillus sphaericus mosquito larvicidal toxins.

The 51.4-kDa-41.9-kDa binary toxin produced by different strains of Bacillus sphaericus shows differential activity toward Culex quinquefasciatus, Aedes atropalpus, and Aedes aegypti mosquito larvae. The patterns of larvicidal activity toward all three mosquito species and growth retardation in A. aegypti have been shown to be due to the 41.9-kDa protein. By using mutant toxins expressed in Escherichia coli, insecticidal activity and growth retardation correlated with amino acids centered around position 100 of the 41.9-kDa protein. In its response to these toxins, A. atropalpus resembled C. quinquefasciatus rather than its congener, A. aegypti.     

Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera

The economically most important honey bee species, Apis mellifera, was formerly considered to be parasitized by one microsporidian, Nosema apis. Recently, [Higes, M., Martín, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93-95] and [Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A Nosema ceranae isolate from the honeybee Apis mellifera. Apidologie 38, 30-37] used 16S (SSU) rRNA gene sequences to demonstrate the presence of Nosema ceranae in A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between N. apis and N. ceranae based on PCR-RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (N =9 isolates gave N. apis RFLPs and sequences, N =20 isolates gave N. ceranae RFLPs and sequences; 100% correct classification). We then employed the method to analyze N =115 isolates from across the world. Our data, combined with N =36 additional published sequences demonstrate that (i) N. ceranae most likely jumped host to A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii) N. ceranae is now a parasite of A. mellifera across most of the world. The rapid, long-distance dispersal of N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping.     

A polymerase chain reaction based method for the detection of Culex quinquefasciatus (Diptera: culicidae)

Culex quinquefasciatus Say is the major vector of the filarial parasite Wuchereria bancrofti (Cobbold) which causes lymphatic filariasis in humans. A repetitive DNA sequence from the genome of C. quinquefasciatus has been cloned and completely sequenced. The 693 bp cloned fragment had an A+T content of 72%. Dot matrix analysis of the fragment did not reveal any direct or inverted repeats within it. Southern blot analysis using a variety of restriction enzymes appeared to indicate that the cloned fragment was interspersed within the genome with a copy number of approximately 30,000. A search of the GenBank database did not reveal significant homologies to any previously cloned sequences. Although the probe was sensitive enough to detect picogram quantities of DNA, it was not specific for C. quinquefasciatus, as it hybridized with DNA from other mosquito species, Culex pseudovishnui Colless, Culex gelidus Theobald, Culex tritaeniorhynchus Giles, Anopheles vagus D?nitz and Mansonia uniformis (Theobald). However PCR primers derived from the cloned sequence, IpC, were found to be specific and amplified only C. quinquefasciatus DNA. The optimized PCR assay was found to be very sensitive and was capable of detecting DNA from all stages of C. quinquefasciatus thus making it an ideal diagnostic tool.     

Malathion and pyrethroid resistance in Culex quinquefasciatus from Cuba: efficacy of pirimiphos-methyl in the presence of at least three resistance mechanisms

Use of malathion for mosquito control in Cuba for 7 years up to 1986 has selected for elevated non-specific esterase and altered acetylcholinesterase (AChE) resistance mechanisms in populations of the pest mosquito Culex quinquefasciatus Say. These mechanisms are still present in relatively high frequencies in the Havana area, despite the replacement of malathion by pyrethroid insecticides for the last 3 years in the mosquito control programme. Samples of Culex quinquefasciatus populations from within a 100 km radius of Havana had high levels of resistance to malathion and lower levels of resistance to propoxur, but there was little or no cross-resistance to the organophosphorus insecticide pirimiphos-methyl. Selection with malathion for twenty-two consecutive generations in the laboratory increased the level of malathion resistance to 1208-fold and propoxur level to 1002-fold, but the maximum level of pirimiphos-methyl resistance was only 11-fold. Pirimiphos-methyl is still operationally effective, despite the resistance mechanisms segregating, so this insecticide if used for control is unlikely to select either of the known resistance factors directly in the field population. Since 1986, pyrethroids have been used extensively, and low levels of pyrethroid resistance were detected in two of five field population samples tested. Malathion selection did not increase the level of pyrethroid resistance, which indicates that one or more distinct pyrethroid resistance factors are now being selected in the field populations of Culex quinquefasciatus.     

Evaluation of insecticide resistance and biochemical mechanisms in a population of Culex quinquefasciatus (Diptera: Culicidae) from São Paulo, Brazil

To establish an insecticidal resistance surveillance program, Culex quinquefasciatus mosquitoes from S?o Paulo, Brazil, were colonized (PIN95 strain) and analyzed for levels of resistance. The PIN95 strain showed low levels of resistance to organophosphates [malathion (3.3-fold), fenitrothion (11.2-fold)] and a carbamate [propoxur (3.0-fold)]. We also observed an increase of 7.4 and 9.9 in alpha and beta esterase activities, respectively, when compared with the reference IAL strain. An alteration in the sensitivity of acetylcholinesterase to insecticide inhibition was also found in the PIN95 mosquitoes. The resistant allele (Ace.1R), however, was found at low frequencies (0.12) and does not play an important role in the described insecticide resistance. One year later, Cx. quinquefasciatus mosquitoes were collected (PIN96 strain) at the same site and compared to the PIN95 strain. The esterase activity patterns observed for the PIN96 strain were similar to those of the PIN95 mosquitoes. However the occurrence of the Ace.1R allele was statistically higher in the PIN96 strain. The results show that esterase-based insecticide resistance was established in the PIN95 Cx. quinquefasciatus population and that an acethylcholinesterase based resistant mechanism has been selected for. A continuous monitoring of this phenomenon is fundamental for rational mosquito control and insecticide application programs.     

Biological control of Culicidae with the copepod Mesocyclops aspericornis and larvivorous fish (Poeciliidae) in a village of French Polynesia

The copepod Mesocyclops aspericornis Daday and the larvivorous fishes Gambusia affinis (B. & G.) and Poecilia reticulata R. & B., were released into mosquito breeding sites in Tuherahera village, Tikehau atoll, French Polynesia, to control larvae of Aedes aegypti (L.), Ae.polynesiensis Marks, Culex annulirostris Skuse and Cx quinquefasciatus Say. Treatments were completed within a week, in January 1990. Fish quickly eliminated mosquito larvae from the open breeding sites (ponds, wells). The impact of copepods in water tanks, drums and covered wells was inconsistent, apparently depending on the availability of microfaunal diet for growth of copepod nauplii. As the biting rate of adult Ae.aegypti seemed to be unaffected by the biological control of larvae, this village-scale experiment was judged to be unsuccessful as a means of vector control.     

Characterization of a Dopamine D1 Receptor from Apis mellifera: Cloning, Functional Expression, Pharmacology, and mRNA Localization in the Brain

Abstract: The neurotransmitter dopamine is an important regulator of physiological and behavioral functions in both vertebrates and invertebrates. We have isolated a homologue of the vertebrate dopamine D1 receptor subfamily from the honeybee Apis mellifera . [³H]Lysergic acid diethylamide specifically binds to the heterologously expressed receptor with K _D∼5 n M . Dopaminergic receptor ligands compete for this high-affinity binding, with the following order of potency: R (+)-lisuride > chlorpromazine = cis ( Z )-flupentixol > dopamine > S (+)-butaclamol > R (+)-SCH 23390 > haloperidol. Activation of the heterologously expressed receptor of Apis mellifera leads to cyclic AMP production. Receptor mRNA is expressed in perikarya of different brain neuropils, including those of mushroom body intrinsic neurons. These results suggest that this dopamine receptor is involved in signal processing of visual and olfactory information in the honeybee.     

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.     

Cyt1A of Bacillus thuringiensis delays evolution of resistance to Cry11A in the mosquito Culex quinquefasciatus

Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.     

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.     

hβ2R–Gαs complex: prediction versus crystal structure—how valuable are predictions based on molecular modeling studies?

In 2010, we predicted two models for the hβ(2)R-Gα(s) complex by combining the technique of homology modeling with a potential energy surface scan, since a complete crystal structure of the hβ(2)R-Gα(s) complex was not available. The crystal structure of opsin co-crystallized with part of the C-terminus of Gα (3DQB) was used as a template to model the hβ(2)R, whereas the crystal structure of Gα (1AZT) was used as a template to model Gα(s). Utilizing a potential energy surface scan between hβ(2)R and Gα(s), a six-dimensional potential energy surface was obtained. Two significant minimum regions were located on this surface, and each was associated with a distinct hβ(2)R-Gα(s) complex, namely model I and model II [Stra?er A, Wittmann H-J (2010) J Mol Model 16:1307-1318]. The crystal structure of the hβ(2)R-Gα(s)βγ complex has recently been published. Thus, the aim of the current study was, on the one hand, to compare our predicted structures with the true crystal structure, and on the other to discuss the question: how valuable are predictions based on molecular modeling studies?     

中华蜜蜂mrjp1 cDNA的克隆及其序列分析

构建中华蜜蜂(Apis cerana cerana)8日龄工蜂头部cDNA文库,利用中蜂基因组的mrjp3部分基因片段作为杂交探针,采用DIG标记筛选cDNA文库,获得mrjps阳性克隆120个;对阳性克隆进行PCR扩增和测序,通过NCBI的BLAST序列比对,获得12个与印度蜂(Apis cerana india)、西方蜜蜂(Apis mellifera L.)mrjp1基因同源的中蜂mtjp1 cDNA片段,并进一步对中华蜜蜂mrjp1的cDNA全序列进行测定和分析。序列比对分析表明,东方蜜蜂(Apis cerana)与西方蜜蜂mrjp1的cDNA序列相似性为93．78％,中华蜜蜂与印度蜂的相似性高达99．36％,这一结果从分子水平证实中华蜜蜂与印度蜂有较近的共同祖先,而东方蜜蜂与西方蜜蜂的亲缘关系较远。     

安徽两种蜜蜂种群的春季繁殖及数量动态特征

1997～ 1999年在皖中和皖西、皖南山区对意大利蜜蜂 (ApismelliferaLigusticaSpi.)和中华蜜蜂(ApisceranaceranaFeb .)种群数量动态进行系统观察研究 .结果表明 ,蜜蜂种群数量明显受气候和蜜粉源植物的影响 ,周年呈现“两高”(春、秋 )和两低 (夏、冬 )的变化模式曲线 ;意大利蜜蜂春繁及秋季更新速度明显快于中华蜜蜂 ,但越夏效果不及中华蜜蜂 .意大利蜜蜂的性比值为 (314.4± 2 89.9)∶1～ (32 9.4± 30 5 .8)∶1,中华蜜蜂为 (334.2± 2 35 .5 )∶1～ (413 .1± 377.2 )∶1,雄蜂呈季节性出现 .蜜蜂种群内年龄组配在不断变化 .     

Molecular characterization of the amplified aldehyde oxidase from insecticide resistant Culex quinquefasciatus.

Primary structural information including the complete nucleotide sequence of the first insect aldehyde oxidase (AO) was obtained from the common house mosquito Culex quinquefasciatus (Say) through cloning and sequencing of both genomic DNA and cDNA. The deduced amino-acid sequence encodes a 150-kDa protein of 1266 amino-acid residues, which is consistent with the expected monomeric subunit size of AO. The Culex AO sequence contains a molybdopterin cofactor binding domain and two iron-sulfur centres. A comparison of the partial sequences of AO from insecticide resistant and susceptible strains of C. quinquefasciatus shows two distinct alleles of this enzyme, one of which is amplified in the insecticide resistant strain on a 30-kb DNA amplicon alongside two resistance-associated esterases. The amplified AO gene results in elevated AO activity in all life stages, but activity is highest in 3rd instar larvae. The elevated enzyme can be seen as a separate band on polyacrylamide gel electrophoresis. The role of AO in xenobiotic oxidation in mammals and the partial inhibition of elevated AO activity by a range of insecticides in Culex, suggest that this AO may play a role in insecticide resistance.