Eco-friendly control of mosquito larvae by Brachytron pratense nymph

The study reported here revealed the biocontrol efficacy of aquatic nymphs of the dragonfly Brachytron pratense against larvae of the mosquito Anopheles subpictus. It was found that during a 24-hour study period, a nymph of B. pratense would consume (mean value of three observations) 66 fourth-instar An. subpictus larvae released in a water bowl containing 3 liters of pond water. The consumption rate was significantly higher (p < .05) during the lights-on phase of the experiment than during the lights-off phase (t = 2.15). Under field conditions, a significant decrease (p < .05) in larval density in dipper samples was observed 15 days after the introduction of dragonfly nymphs (10 individuals) in concrete tanks. The biocontrol potential of the nymphs under field conditions was also indicated by a significant increase (p < .05) in the density of mosquito larvae 15 days after the removal of nymphs. In the control tanks (where no nymphs were introduced), mean larval-mosquito density did not differ significantly throughout the study period (p > .05).     

Factors affecting transmission of Wuchereria bancrofti by anopheline mosquitoes. 2. Damage to ingested microfilariae by mosquito foregut armatures and development of filarial larvae in mosquitoes

Microfilariae (mf) of Wuchereria bancrofti from the midgut of 639 Anopheles gambiae, 557 An. arabiensis, 117 An. melas and 9 An. funestus were examined immediately after the mosquitoes had fed on carriers with different densities of mf. The percentages of mf damaged during ingestion were 57.1-60.0 in An. gambiae, 33.3-50.6 in An. arabiensis and 38.7-55.7 in An. melas. In each species the percentage of mf damaged was independent of mf density in the human host. A further 3657 An. gambiae, 2875 An. arabiensis, 347 An. melas and 32 An. funestus were examined 7 d or more after feeding on mf carriers. In An. gambiae and An. arabiensis, mean numbers of larvae per mosquito were strongly correlated to mf blood density, with similar regression slopes to those obtained from the regression of mf blood density on mean uptake of mf/mosquito. The ratio of mean numbers of larvae per mosquito to mean numbers of intact mf ingested per mosquito increased as the density of mf in the human host increased in An. gambiae and An. arabiensis, but decreased in An. melas as host mf density increased.     

Predatory capacity and prey selectivity of nymphs of the dragonfly Pantala hymenaea

Predatory capacity and prey selectivity of nymphs of the dragonfly Pantala hymenaea (Odonata: Libellulidae) were evaluated on larvae of the mosquito Culex quinquefasciatus (Diptera: Culicidae) and larvae of the midge Chironomus plumosus (Diptera: Chironomidae) as prey. With functional response methodology, 7 larval densities were exposed to predator individuals in a glass jar under laboratory conditions. The study was performed in 2 experiments. The 1st was a test system with each prey species alone. The 2nd tested a mixture of both prey species in a 1:1 ratio. Prey selectivity and prey capacity were significantly greater on midge larvae than on mosquito larvae.     

A laboratory study of predation by damselfly nymphs, Enallagma civile, upon mosquito larvae, Culex tarsalis

The nymph of the damselfly, Enallagma civile, feeds on the larvae of the mosquito Culex tarsalis amongst a variety of other small aquatic invertebrates. Average daily consumption of 3rd instar Cx. tarsalis larvae by the last nymphal stage of E. civile was 6.06 larvae (range 4.64-8.67). Experiments in which density of prey (mosquito larvae) and predators (E. civile nymphs) were varied, showed that more prey was consumed as prey density increased. However, fewer prey were consumed at higher predator densities; mutual interferences among predators at higher predator densities was suspected.     

Evaluation in test huts of the protective effects of untreated mosquito nets against Anopheles gambiae s.s. bites

The protection against insect bites afforded by untreated mosquito bed nets was studied at the field stations of Yaokoffikro and M'bé in Ivory Coast. We tested damaged mosquito nets (tear holes covering 0.8% of the total area) in Yaokoffikro, undamaged mosquito nets and huts without mosquito nets in M'bé. The blood feeding rate of Anopheles gambiae was 70% lower in huts with undamaged mosquito nets than in huts with no mosquito net or a damaged mosquito net. The natural exophilic behavior of the mosquito was increased and mosquito mortality reached 7.4%. The blood feeding rate of An. gambiae was 83% in huts with no mosquito net and 68% in huts with damaged mosquito nets. Exophilic behavior was normal (25%) and overall mortality was 5%. Immediate mortality of An. gambiae was 54% with undamaged mosquito nets and only 17% in huts with no mosquito net. This difference may reflect the state of nutrition of the mosquitoes: 86% of the dead mosquitoes found in huts with undamaged mosquito nets had not fed, versus only 24% in huts with no mosquito net. The unfed mosquitoes may be considered to have died from starvation. Intact mosquito nets conferred some protection against An. gambiae. This protection was not total but was "better than nothing", with 25% of mosquitoes blood feeding effectively even in the presence of an intact mosquito net. This protection reduces the probability of survival of the females. In contrast, torn mosquito nets trap the mosquitoes that enter, then allowing more than two thirds of the mosquitoes to take a blood meal on the sleeper.     

广东国境口岸不同蚊种COI序列分析和分子鉴定方法

[目的]建立国境口岸不同蚊种的细胞色素C氧化酶亚基I（COI）分子和氨基酸鉴定方法并分析其系统进化关系。[方法]设计1对扩增COI部分编码区的PCR引物，对广州机场、江门和湛江等国境口岸采集的致倦库蚊、三带喙库蚊、白纹伊蚊、中华按蚊、骚扰阿蚊等成蚊和实验室喂养的蚊幼虫进行PCR扩增和序列测定，分析COI核苷酸和氨基酸的系统进化关系。[结果]5种蚊种的COI基因扩增片断长度均为415bp，A＋T含量为68．77％-70．6％。同源性比较表明，不同蚊种间COI片断碱基变异颠换数都明显高于转化数，核苷酸序列及其编码的氨基酸序列同源性分别为85．1％-93．7％和92．0％-99.3％。COI核苷酸系统进化关系显示，所有蚊虫的COI分子鉴定与其形态学结果吻合，但骚扰阿蚊位于一单独的分支上，其亲缘关系与其它蚊种最远。COI基因编码的氨基酸系统进化与蚊虫形态学亲缘关系一致，库蚊属、伊蚊属、阿蚊属聚类为库蚊亚科，中华按蚊与其它蚊种的亲缘关系最远。[结论]建立的COI核苷酸和氨基酸鉴别技术可成功地应用于国境口岸范围内成蚊和幼蚊的属和种的区分，后者更能区分高级分类阶元亚科和正确反映蚊虫的系统发育关系。这可以弥补蚊虫形态特征信息量的不足等传统分类系统的缺点，为广东口岸和其它国境口岸范围内外来的或新发现的蚊种的鉴别提供了分子水平的技术依据。     

pcDNA3．1（＋）／NspA真核表达载体的构建及鉴定

目的构建pcDNA3.1( )淋病奈瑟菌外膜蛋白—奈瑟菌表面蛋白A(Neisseria surface Protein A,NspA)基因真核表达载体,为研制淋病奈瑟菌核酸疫苗奠定基础。方法根据淋病奈瑟菌NspA基因序列,设计合成一对引物,用PCR方法从淋病奈瑟菌基因组DNA中扩增出NspA基因片段,将扩增的产物连接于测序载体pUCm-T上,并构建重组体pcDNA3.1( )/NspA,进行酶切、PCR及测序鉴定。结果NspA基因体外扩增产物大小约为525 bp。所构建的pcDNA3.1( )/NspA重组体经双酶切及PCR鉴定,与预期片断的大小相符;测序结果与GenBank中收录的NspA全长序列一致,表明pcDNA3.1( )/NspA真核表达载体构建正确。结论成功地构建了淋病奈瑟菌真核重组表达载体pcD-NA3.1( )/NspA,为NspA蛋白的功能研究和淋病奈瑟菌核酸疫苗的研制提供了物质基础。     

Evaluation of synthetic repellents on mosquito nets in experimental huts against insecticide-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes

Owing to the development of pyrethroid resistance in Anopheles gambiae, there is a need to develop chemical alternatives for use on mosquito nets. Synthetic insect repellents are widely used for personal protection as skin or clothing applications. The efficacy of repellent-treated nets (RTN) was evaluated in experimental huts in C?te d'Ivoire against pyrethroid-resistant populations of An. gambiae and Culex quinquefasciatus. The repellents tested were DEET (N,N-diethyl-3-methylbenzamide) at 7.9 g/m2 and two formulations of ethyl butylacetylaminopropionate (IR3535) at 7.6 g/m2 and 7.3 g/m2. Over 45 nights there was a 74-82% reduction in the number of An. gambiae entering the huts containing RTNs but no significant reduction in entry of C. quinquefasciatus. There was a 63-64% reduction in the proportion of An. gambiae blood feeding but no reduction in the proportion of C. quinquefasciatus blood feeding in huts with RTNs. An unexpected result was the 69-76% mortality of An. gambiae and 51-61% mortality of C. quinquefasciatus in huts containing RTNs. Treated filter paper bioassays in WHO test kits confirmed that confined contact with DEET induces mortality. The DEET-based product provided better and longer protection; tunnel test bioassays confirmed that residual activity lasted for up to 6 weeks. Application of repellents to nets warrants further investigation and development.     

广东国境口岸五种蚊种核糖体基因第2内转录间隔区分子鉴定方法研究

[目的]建立国境口岸不同蚊种的核糖体基因第2内转录间隔区（rDNA-ITS2）分子鉴定方法及其系统进化关系。[方法]针对蚊虫的rDNA核酸序列保守性，设计扩增rDNA-ITS2编码区的PCR引物，对广州机场、江门和湛江等国境口岸采集的致倦库蚊、三带喙库蚊、白纹伊蚊、中华按蚊、骚扰阿蚊等成蚊和实验室喂养的蚊幼虫进行PCR扩增和序列测定，并与GenBank中已知蚊虫的rDNA-ITS2进行同源性比较和系统进化分析。[结果]不同蚊虫的rDNA-ITS2扩增片断长度不同，M2引物对致倦库蚊、三带喙库蚊、白纹伊蚊、骚扰阿蚊和中华按蚊的扩增片断分别为447bp-520bp、432bp-438bp、527bp-586bp、439bp-448bp和644bp。序列分析和系统进化关系显示，尖音库蚊组和三带喙库蚊聚类为库蚊属，再与白纹伊蚊和骚扰阿蚊聚类为库蚊亚科，库蚊亚科再与中华按蚊进行聚类，分子进化与蚊虫形态学鉴定的亲缘关系保持一致。[结论]建立的rDNA-ITS2分子鉴别技术可成功地应用于国境口岸范围内成蚊和幼蚊的亚科、属和种的区分和确定系统发育关系。这可以弥补蚊虫形态特征信息量的不足等传统分类系统的缺点，对国境口岸范围外来的或新发现的蚊种的鉴别提供了分子水平的技术依据。     

核糖体28S-D3等位基因特异扩增鉴别微小按蚊亲缘种A和C

目的 建立微小按蚊复合体不同亲缘种A和C的分子鉴别方法。方法 用单蚊蚊腿消化提取基因组DNA，PCR特异扩增28S第3结构域(D3)基因，对PCR产物进行纯化、克隆、测序和序列分析；基于序列差异设计特异引物，进行等位基因特异扩增(PCR—ASA)，根据扩增片段大小区分微小按蚊不同亲缘种。结果 发现微小按蚊不同亲缘种A和C(GeneBank登录号：AF416782，AF425594)，根据二者序列差异设计的特异引物能在普通琼脂糖凝胶上直观地区分两亲缘种，两者除在376bp处有一共同条带外，分别在294bp和112bp处出现各自的特异扩增带。结论 我们建立的ASA分子鉴别方法能有效地将我国微小按蚊不同亲缘种A和C区分开来。     

Improved high-throughput method for molecular identification of Culex mosquitoes

Culex pipiens, Cx. restuans, and Cx. salinarius play important and most likely different roles in transmission of West Nile virus (WNV) in the northeastern United States. While Cx. pipiens and Cx. restuans are considered the main enzootic vectors of WNV, Cx. salinarius may be involved in epizootic cycles due to its broader host preferences. Accurate morphological identification of field-collected Culex specimens may be difficult, and therefore the New York State Department of Health arbovirus surveillance program allows combined Cx. pipiens and Cx. restuans pools to be tested for WNV. We have developed a modified and improved DNA isolation protocol using proteinase K digestion without traditional mosquito trituration and nucleic acid extraction to permit high-throughput screening of a large number of Culex specimens for species identification using polymerase chain reaction (PCR). This method utilizes a 96-well-plate format and a novel 1-step crude extraction procedure using proteinase K to obtain genomic DNA template from 1 mosquito leg in sufficient quantity for at least 2 standard 50-microl PCR reactions. Proteinase K digestion of legs from individual Culex mosquitoes was performed and used for PCR amplification with previously described species-specific ribosomal DNA primers. Using these rDNA primers, our modified proteinase K method successfully identified 91% to 100% of the Culex samples.     

Factors affecting transmission of Wuchereria bancrofti by anopheline mosquitoes. 3. Uptake and damage to ingested microfilariae by Anopheles gambiae, An. arabiensis, An. merus and An. funestus in east Africa

Laboratory observations were made on the uptake of microfilariae (mf) of Wuchereria bancrofti by Anopheles gambiae, An. arabiensis, An. merus and An. funestus. Over host mf densities ranging from 450/ml to 1735 mf/ml neither the percentage of mosquitoes ingesting mf nor the mean number of mf per mosquito was correlated to host mf density. All mosquito species damaged mf during ingestion but the proportion harmed was independent of host mf density. The mean proportion damaged was 0.67 in An. gambiae, 0.51 in An. merus, 0.47 in An. Arabiensis and 0.26 in An. funestus. A comparison of the mean number of undamaged mf ingested and the number of larvae in the thoracic muscles revealed that not all undamaged mf were able to reach the thoracic muscles.     

DNA条形码技术用于原木携带蚊种的鉴定

目的对临沂口岸进境原木上截获的蚊种进行DNA条形码研究确定蚊种。方法使用双翅目COI基因国际通用引物对原木携带蚊种COI基因进行PCR扩增和序列测定,序列在NCBI网站上进行Blast,确定蚊种。结果待测蚊种COI序列与GenBank中编号为HQ398903.1的骚扰阿蚊(Armigeres subalbatus)序列相似性为99%。结论基于COI基因的DNA条形码技术可成功地对媒介蚊类进行分类鉴定,该技术可弥补蚊虫形态缺失和形态学鉴定知识不足对蚊类鉴定的影响。     

用PCR技术测定按蚊人血指数的研究

目的建立一种能替代传统免疫学方法用于按蚊人血指数测定的分子生物学检测技术。方法根据人核糖体DNA序列设计1对特异性引物,建立聚合酶链反应（PCR）鉴定按蚊胃内人血的方法。同时,对猪血、牛血、羊血、小鼠血以及未吸血按蚊中提取的DNA进行检测,验证该检测方法的特异性,并对吸饲人血后不同时间（1、6、12、18、24、27、30、33、36、40、4J4、48h）的按蚊进行检测,测试该方法的检测敏感性。结果该方法可从人血提取的DNA中扩增得到519bp大小的特异性条带,对其他动物血样及未吸血按蚊中所提取的DNA均未能扩增出特异性条带;所有吸人血24h内的中华按蚊均能扩增出特异性条带,在吸人血后27、30、33、36h的各5只中华按蚊中,分别有4、4、2、1只能扩出特异性条带,吸血40h后的中华按蚊均不能扩增出特异性条带。Logistic回归分析表明,吸血后24-40h,PCR检测阳性按蚊数与吸血后的时间呈负相关关系（P〈0．01）。结论本研究所建立的PCR方法可准确鉴定吸血24h内的中华按蚊胃内的人血液来源,可替代传统免疫学方法用于按蚊人血指数的测定。     

山东地区不同季节优势恙螨自然感染恙虫病东方体的分子流行病学调查

目的从分子水平探讨山东地区不同季节优势恙螨自然感染恙虫病东方体（Ot）情况及其作为恙虫病传播媒介的可能性.方法根据Ot-Sta56 kDa外膜蛋白基因部分序列设计Ot种和型特异性引物,对标本提取的DNA先采用种特异性引物扩增,然后再采用型特异性引物扩增分型,并对部分标本进行序列测定.结果从不同季节优势恙螨幼虫共得到11株Ot分离株和16份恙螨匀浆,从这27份标本提取的DNA经首轮PCR扩增后,18份标本有预期的目的带.对首轮PCR产物采用nested PCR扩增分型,结果17份为Kawasaki型,1份（LHGM2株）为Karp型.序列同源性分析结果证实了nested PCR分型结果：XDM2株首轮PCR产物碱基序列与Kawasaki型相应DNA片段的碱基序列同源性最高,为97.00%,在系统发育树上与Kawasaki株位于同一分支,应属于Kawasaki型;LHGM2株首轮PCR产物碱基序列与Karp型相应DNA片段的碱基序列同源性最高,为96.45%,在系统发育树上与Karp株位于同一分支,应属于Karp型.结论山东地区不同季节优势恙螨中存在Ot自然感染,Ot主要基因型为Kawasaki型.     

Impact of fenoxycarb, a carbamate insect growth regulator, on some aquatic invertebrates abundant in mosquito breeding habitats

The insect growth regulator, Fenoxycarb, induced various morphogenetic aberrations in Notonecta unifasciata, Anax junius and Pantala hymenaea after treatment of late nymphal stages. Most affected nymphs died while molting from nymphs to adults. Fenoxycarb is also ovicidal to young eggs of N. unifasciata. The treatment rate of 0.034 kg AI/ha fenoxycarb induced some reduction in N. unifasciata population densities and produced a few morphogenetic abnormalities in some Odonata. However, planktonic organisms and aquatic beetles regularly found in the mosquito breeding habitats showed no deleterious effect.     

Anopheles gambiae complex egg-stage survival in dry soil from larval development sites in western Kenya

The potential for Anopheles egg survival in dry soil from larval development sites was investigated in western Kenya. A total of 230 dry soil samples collected in 1987, 1988 and 1989 yielded 126 first-instar Anopheles gambiae s.l. larvae from 2 to 5 days after flooding with water. These larvae were from dried animal hoofprints along streams (57.9%), from dried edges of permanent and temporary pools (41.3%) and from dried stream beds (0.8%). Larval density was 1.2 larvae/kg of soil from positive microhabitats in 1987 and 2.4 larvae/kg in 1988. Thirteen larvae from the 1989 soil samples, reared to adults, were identified by DNA probes as Anopheles gambiae sensu strictu (n = 6) and Anopheles arabiensis (n = 7). Experimentally, eggs from field-collected females remained viable up to 12 days for An. gambiae s.l. and 10 days for An. funestus. In western Kenya, egg viability in dry soil may represent a significant, short-term survival mechanism for 2 species of the An. gambiae complex.     

Failure of the Asian longhorned tick,Haemaphysalis longicornis,to serve as an experimental vector of the Lyme disease spirochete,Borrelia burgdorferi sensu stricto

The invasive, human-biting Asian longhorned tick, Haemaphysalis longicornis, was detected in New Jersey in the eastern United States in August of 2017 and by November of 2018 this tick had been recorded from 45 counties across 9 states, primarily along the Eastern Seaboard. The establishment of H. longicornis in the United States has raised the questions of how commonly it will bite humans and which native pathogens may naturally infect this tick. There also is a need for experimental vector competence studies with native pathogens to determine if H. longicornis can acquire a given pathogen while feeding, pass it transstadially, and then transmit the pathogen in the next life stage. In this experimental study, we evaluated the vector competence of a population of H. longicornis originating from the United States (New York) for a native isolate (B31) of the Lyme disease spirochete, Borrelia burgdorferi sensu stricto (s.s.). In agreement with a previous experimental study on the vector competence of H. longicornis for Borrelia garinii, we found that uninfected H. longicornis larvae could acquire B. burgdorferi s.s. while feeding on infected Mus musculus mice (infection prevalence >50% in freshly fed larvae) but that the infection was lost during the molt to the nymphal stage. None of 520 tested molted nymphs were found to be infected, indicating that transstadial passage of B. burgdorferi s.s. is absent or rare in H. longicornis; and based on the potential error associated with the number of nymphs testing negative in this study, we estimate that the upper 95% limit for infection prevalence was 0.73%. An Ixodes scapularis process control showed both effective acquisition of B. burgdorferi s.s. from infected mice by uninfected larvae and transstadial passage to the nymphal stage (infection prevalence of 80–82% for both freshly fed larvae and molted nymphs). We also observed that although H. longicornis larvae could be compelled to feed on mice by placing the ticks within feeding capsules, attachment and feeding success was minimal (<0.5%) when larvae were placed freely on the fur of the mice. We conclude that H. longicornis is unlikely to contribute more than minimally, if at all, to transmission of Lyme disease spirochetes in the United States.     

Short-range attractiveness of pregnant women to Anopheles gambiae mosquitoes

Malaria is a major cause of illness and an indirect cause of mortality in pregnant women. It can also cause stillbirths and low-birthweight babies. We have shown previously that pregnant women attracted twice as many Anopheles gambiae mosquitoes, the principal African malaria vector, as their non-pregnant counterparts over distances of about 15 m. In the current study (in 1998/99) we compared the short-range attractiveness of both pregnant and non-pregnant women sleeping under untreated bednets in Gambian villages. First, we measured the rate of mosquito entry under bednets and, second, we calculated the proportion of mosquitoes biting mothers under each bednet compared to their children. The feeding preference of An. gambiae collected under nets was determined by DNA fingerprinting blood samples from human subjects sleeping under each bednet and comparing these to fingerprints obtained from mosquito bloodmeals. Pregnant women were more attractive to An. gambiae mosquitoes than non-pregnant women under an untreated bednet. The number of mosquitoes entering bednets each night was 1.7-4.5 times higher in the pregnant group (P = 0.02) and pregnant women also received a higher proportion of bites under the bednets than did non-pregnant women (70% vs 52%, P = 0.001). This study clearly demonstrates that pregnant women are more exposed to malaria parasites than other women, which contributes to the greater vulnerability of pregnant women to malaria.     

3种物质不同配比对美洲大蠊若虫生长的影响

目的筛选美洲大蠊若虫饲料的优化配方。方法采用L（934）设计方法,研究小麦粉、奶粉、干酵母粉3种物质不同配比对美洲大蠊若虫生长发育的影响。结果实验结果表明,若虫饲养120d后,取食配方S5的若虫体长增量（19.21mm/只）、日取食量（25.94mg/只）、体重（532.5mg/只）及若虫对饲料的利用率（96.69%）均高于取食其他配方饲料的若虫。结论美洲大蠊若虫最佳的饲料配方为小麦粉：奶粉：干酵母粉=2：4：1。干酵母粉是美洲大蠊若虫体长增量的关键因子,有利于若虫的生长。