微小按蚊种群成员分子变异与系统发育的关系

目的：研究微小按蚊与种群之间（乌头按蚊、吉甫按蚊、微小按蚊A／C型）线粒体DNA的分子变异及系统发育关系。方法：PCR扩增和mtDNA测序。结果：在云南发现两个微小按蚊隐形种,即微小按蚊A／D型;证实了微小按蚊种团之间遗传差异,微小按蚊A／C分歧时间稍晚于乌头按蚊,吉甫按蚊分歧时间远早于前2种。结论：地理环境对mtDNA遗传差异有一定影响,但不同地理的群体之间微小按蚊的遗传没有明显变化。     

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。     

微小按蚊rDNA内转录间隔２区序列差异

目的：比较不同地株微小按蚊核糖体ＤＮＡ（rDNA）内转录间隔２区（ＩＴＳ２）序列差异。方法：通过对ＰＣＲ扩增rDNAＩＴＳ２片段测序,比较其不同地株差异。结果：对６株微小按蚊测序比较,存在有微小按蚊Ａ和Ｃ型。结论：rDNAＩＴＳ２充阢差异可用于建立Ａ、Ｃ型的分子鉴别方法。     

应用形态和分子特征对我国海南省按蚊的鉴定研究

目的利用形态特征与分子特征相结合对我国海南省按蚊进行种类鉴定。方法研究样本为海南省4个不同地理环境所采集的按蚊成虫和幼虫,依据形态特征鉴定成虫后,测定和分析部分成虫和幼虫的rDNA？ITS2和28S？D3序列以进行分子鉴定。结果对采集的336只按蚊成虫进行形态鉴定,结果显示总共分为9种,其中迷走按蚊所占比例最高,为81.55%。测序获得成虫和幼虫的ITS2序列37条,D3序列41条,Blast对比的结果显示,形态鉴定的成虫中,1只乌头按蚊错定为微小按蚊;分子鉴定的幼虫包括中华按蚊、迷走按蚊、腹簇按蚊和未定名种（与圣代克按蚊和浅色按蚊同源性高）;序列分析发现在ITS2序列中迷走按蚊和微小按蚊分别存在1处（G/A）和2处（A/T）单碱基双峰,D3序列中迷走按蚊和腹簇按蚊存在3处（G/A）和1处（G/A）单碱基双峰。结论应用形态特征结合分子特征鉴定按蚊的可靠性和实用性强;分子特征提示迷走按蚊、微小按蚊和腹簇按蚊等处于隐种的分化中。     

国境口岸常见11种蚊虫DNA条形码分子鉴定研究

目的通过研究国境口岸常见11种蚊虫的细胞色素C氧化酶亚基I(COI)基因序列差异性,并分析其系统进化关系,建立口岸常见蚊种的分子鉴定方法。方法设计l对扩增COI部分编码区的PCR引物,对广东、海南和云南等国境口岸采集的致倦库蚊、白纹伊蚊、中华按蚊等常见11种成蚊进行PCR扩增和序列测定,依据COI核苷酸序列进行系统进化分析。结果 11种蚊虫的COI基因扩增片断长度均为650 bp左右,A+T含量为66.51%~69.97%。同源性比较表明,同一蚊种间核苷酸序列同源性为94.8%~100.0%;不同蚊种间核苷酸序列同源性为77.7%~91.8%。系统进化关系显示,从种的水平上看,白纹伊蚊、致倦库蚊、中华按蚊等上述所有蚊种均聚集成簇,即同种之间呈明显的聚集,与形态学鉴定结果相一致。在属的水平上,库蚊属、阿蚊属、伊蚊属等均聚集成簇,形成各自的单独一支;各属间,阿蚊属与伊蚊属先聚类,再分别与库蚊属、曼蚊属聚类为库蚊亚科,按蚊属与上述蚊种亲缘关系较远。结论本次研究中的COI基因差异可作为国境口岸常见11种蚊虫分类鉴定的分子标记,为口岸常见蚊种的分子鉴定提供帮助,也可为口岸外来的或新发现的蚊种的鉴别提供分子水平的技术依据。     

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。     

嗜人按蚊不同地理株基因组RAPD分析

目的 为进一步确定我国嗜人按蚊是否存在不同的种型提供研究依据。方法 通过随机引物多态性DNA(RAPD)技术获取四川、云南、江苏等三个不同分布区嗜人按蚊的DNA指纹图谱，比较嗜人按蚊不同地理株基因组DNA的多态性，从分子水平上对不同分布区嗜人按蚊进行分析。结果 各地理区域嗜人按蚊之间的DNA片段在绝大部分相同的情况下存在差异，一方面三个不同地理株的嗜人按蚊基本上具有共同的扩增片段，反映了各地理区域基因组DNA的同源性；另一方面各个地理株所特有的片段及条带亮度(扩增片段强度)上又有差别。结论 不同地理株的嗜人按蚊在DNA水平上在绝大部分相同的前提下存在差异，这种差异的发现将为鉴定形态学上无法区分的嗜人按蚊地理种型提供分子水平上的分类依据。     

广西微小按蚊种群密度地理分布及种类分子鉴定研究

目的研究20世纪以来广西壮族自治区(广西)微小按蚊的种群密度和地理分布,为疟疾防治提供参考。方法收集广西20世纪50-90年代微小按蚊资料及疟疾发病率,于2004-2010年在不同经纬度原以微小按蚊为主要传播媒介的疟疾流行区收集该蚊成蚊,采用形态学和PCR方法鉴定采集的微小按蚊样品。结果1957-1998年对全自治区不同经纬度媒介按蚊调查,92个县中有56个县存在微小按蚊;2004-2007年在该蚊活动频繁的36个县的乡村收集按蚊,仅在20个县40个媒介点采集到微小按蚊244只;采用种类分子鉴定,除百色市旺甸村有微小按蚊种类A存在外,其他地区均为微小按蚊种类C。2008年后全自治区各县疟疾疫情发病率已降至0.1/万。结论目前在广西存在微小按蚊种类A和C两种,以种类C为主;全自治区不同经纬度的微小按蚊种群密度和分布范围已经明显减少,该蚊有可能不再是该区域疟疾传播的主要媒介。     

双错配碱基ARMS结合RE法快速检测FGFR3基因的突变超热点

目的建立快速简便、特异灵敏的鉴定FGFR3基因c.1138G>A突变超热点的基因诊断方法,为软骨发育不全(ACH)的产前基因诊断或植入前遗传学诊断(PGD)创造条件。方法针对突变率高达95%以上的FGFR3基因的突变热点c.1138G>A,设计双错配碱基的ARMS特异引物,对已经临床确诊的先证者家系及正常对照和非c.1138G>A突变的患者对照,分别用普通引物和特异引物进行PCR扩增和直接鉴定,然后对扩增产物进行DNA序列分析及SfcI酶切鉴定。结果用普通引物扩增,对照组和先证者均扩增阳性。SfcI酶切后,对照组仍为一条513bp的带,而患者切出205bp、308bp和513bp三条带;而用ARMS特异引物扩增,则先证者扩增阳性,而对照组扩增阴性,阳性者酶切后产生27bp和418bp两条带。这一切都与预期的结果完全吻合。结论该法快速、特异、准确性高,结合DNA序列分析和酶切鉴定(RE),可用于ACH高危胎儿的快速产前诊断。     

福建口岸三蚊种COI基因序列分析

〔目的〕分析福建口岸白纹伊蚊、中华按蚊和骚扰阿蚊细胞色素C氧化酶亚基I(COI)基因序列,为建立蚊种分子鉴定方法奠定基础。〔方法〕扩增并测定在福建口岸采集的白纹伊蚊、中华按蚊和骚扰阿蚊COI基因序列,与GenBank中相应序列进行对比分析。〔结果〕三蚊种COI基因扩增片段长度约500bp,A+T含量为65.61%~68.73%。核苷酸同源性分析结果表明,同一蚊种COI基因同源性为98.1%~100%,三蚊种间基因同源性为84.4%~87.7%;COI部分基因系统进化关系显示,三蚊种COI分子鉴定与形态学鉴定结果一致,阿蚊属与伊蚊属聚类为库蚊亚科,按蚊亚科位于系统进化树另一分支。〔结论〕COI基因核苷酸序列分析可应用于福建国境口岸三蚊种的区分,弥补形态特征信息量不足等传统分类鉴定方法的缺点,为建立外来或新发现蚊种分子鉴定方法奠定基础。     

Evidence for a new sibling species of Anopheles minimus from the Ryukyu Archipelago, Japan

The Anopheles minimus complex is known to comprise at least 2 sibling species (A and C) in Thailand and Vietnam. This study investigated the specific status of An. minimus on Ishigaki Island, the Ryukyu Archipelago, Japan using morphological and genetic analyses. Morphological studies revealed that almost all (99.5%) of the adult mosquitoes are characterized by the humeral pale spot on the costa of their wings, a character that partially differentiates species A and C elsewhere. A high frequency (81.4%) have a pale fringe spot at the tip of vein 1A, a character rarely observed in other An. minimus populations. Significant seasonal variation in the size of wild An. minimus mosquitoes on the island was observed, with the largest size in the winter. Scanning micrographs of the cibarial armature of females from Ishigaki Island revealed that over 90% had cone filaments clearly differing in shape from those of species A or C. The Giemsa-stained metaphase karyotypes of larval brain cells were somewhat similar to those of species A, with a few exceptions, but were very different from those reported for species C. Crossing experiments between species A (CM strain) from Thailand and the progeny of An. minimus from Ishigaki Island (ISG strain) revealed postzygotic genetic incompatibility, although no prezygotic isolation. Hybrid progeny were only obtained from CM female x ISG male. F2 hybrid progeny were not obtained, since the hybrid males were sterile or almost sterile with atrophied testes or abnormal spermatozoa, although the polytene chromosomes of hybrid larvae showed synapsis. The hybrid females backcrossed with either CM or ISG males laid eggs with significantly lowered fertility and viability. The sequence for the D3 region of the 28S gene of ribosomal DNA of the ISG strain differed from those of species A and C. In addition, sequence data from Vietnamese mosquitoes suggest that the An. minimus complex may contain additional species. The morphological, cytogenetic, molecular, and hybridization evidence together suggest the existence of another sibling species of the An. minimus complex on Ishigaki Island, which is provisionally designated An. minimus species E.     

A diagnostic polymerase chain reaction assay for species A and D of the Anopheles Dirus (Diptera: Culicidae) species complex based on ribosomal DNA second internal transcribed spacer sequence

A polymerase chain reaction assay based on differences in the internal transcribed spacer regions of ribosomal DNA was developed for distinguishing 2 members of the Anopheles dirus sibling species complex. This assay distinguished An. dirus species A from species D by producing diagnostic bands, 374 base pairs (bp) in length for species A and 663 bp in length for species D. Both laboratory colonies and field collections from Hainan and Yunnan provinces of China were identified with 100% accuracy.     

How reliable is the humeral pale spot for identification of cryptic species of the Minimus Complex?

The Anopheles minimus Complex Theobald (Diptera: Culicidae) is composed of the 3 sibling species A, C, and E. The malaria vectors An. minimus A and C are distributed over the Southeast Asian region, whereas species E is restricted to the Ryukyu Japanese Islands. Because species A and C can be sympatric and present specific behaviors and have a role in malaria transmission, it is important to differentiate them. The literature mentioned the presence of a presector pale spot on the wing costa of An. minimus A, whereas species C may exhibit both presector and humeral pale spots. However, the reliability of their diagnostic power has not been established over large temporal and geographic surveys. From the analyses of 9 populations throughout Southeast Asia, including published data and field populations from 2 sites in Thailand, we showed that the wing patterns present spatial and temporal variations that make these two morphological characters unreliable for the precise identification of An. minimus A and C. Therefore, molecular identification remains the most efficient method to obtain an unambiguous differentiation of these 2 species. Correct species identification is essential and mandatory for any relevant study on the Minimus Complex and for the application of successful control strategies.     

First record of Anopheles minimus C and significant decrease of An. minimus A in central Vietnam

Before August 1998, in the Khanh Phu commune (central Vietnam), Anopheles minimus s.l. individuals were identified as species A and showed the typical species A wing form. After a significant decrease over the 3 years 1999-2001, an increase in 2002 of An. minimus s.l. possessing a different wing pattern was observed. To determine the specific status of the An. minimus species collected in 2002 and to follow changes in the species composition, an allele-specific polymerase chain reaction was applied to samples collected from 1993 to 2002. This study reports the first record of An. minimus C in central Vietnam and, since 1998, a significant reduction of An. minimus A that coincided with the wide use of permethrin-treated bednets. This change in anopheline composition may have important consequences on malaria transmission. This work shows that the geographic distribution of malaria vectors in southeast Asia is only partially known and highlights the importance of species identification for understanding changes in the vector composition as a result of selective vector control.     

Crossing experiments of Anopheles minimus species C and putative species E

In the Anopheles minimus complex, 2 sibling species (A and C) are generally accepted. Recently, a 3rd species, provisionally designated An. minimus species E, has been described from the Ryukyu Archipelago, Japan, based on crossing experiments (A and E), DNA analysis, mitotic karyotypes, and some morphological characteristics. The present study reports the results of crossing experiments between species C and putative species E. Hybridization between the progeny of An. minimus species C from Thailand and putative species E from Japan revealed postzygotic genetic incompatibility. Although F1 hybrid progeny were obtained from both directions of crosses, the hybrid males from C female x E male crosses were completely sterile, with atrophied testes and accessory glands. In addition, the external terminalia of all of these males never completely rotated and the males failed to copulate by artificial mating. In E female x C male crosses, the hybrid males showed partially sterile testes in which most spermatozoa were abnormal (enlarged head) and inactive, and they had very little success in inseminating females. The salivary gland polytene chromosomes of F1 hybrid larvae from species C female x species E male showed a fixed heterozygous inversion on the 3L arm. Those F1 hybrids from species E female x species C male showed partial asynapsis on identified arms (2R and 3L) and a fixed heterozygous inversion on the 3R arm. When the F1 hybrid females from both directions of crosses were backcrossed with either C or E males, they produced male progeny with abnormal spermatozoa. Study of mating behavior in a 30 x 30 x 30-cm cage showed that the C males failed to mate with either C or E females, indicating that species C males cannot breed in confined spaces (lack stenogamy). Putative species E males had little success in inseminating species C females. This study provides strong evidence of genetic incompatibility between An. minimus species C and putative species E, supporting previous data that species E is a distinct species in the An. minimus complex.     

Locomotor behavioral responses of Anopheles minimus and Anopheles harrisoni to alpha-cypermethrin in Thailand

Excito-repellency responses of 3 test populations, representing 2 sibling species within the Minimus Complex, Anopheles minimus and An. harrisoni, were characterized for contact irritant and noncontact repellent actions of chemicals during and after exposure to alpha-cypermethrin at half the recommended field (0.010 g/m2), the recommended field (0.020 g/m2), and double the recommended field concentration (0.040 g/m2), using an excito-repellency escape chamber system. Two field populations of An. minimus and An. harrisoni collected from the malaria-endemic areas in Tak and Kanchanuburi provinces in western Thailand, respectively, were tested along with a laboratory population of An. minimus maintained since 1993. Females of all 3 test populations rapidly escaped after direct contact with treated surfaces for each concentration. In general, increased escape responses in the An. minimus test populations were proportionate to increased insecticide dosages. The greatest escape response for An. harrisoni was observed at the operational field concentration of alpha-cypermethrin. The noncontact repellency response to alpha-cypermethrin was comparatively weak for all 3 test populations, but significantly different from each paired contact test and respective noncontact controls. We conclude that strong contact irritancy is a major action of alpha-cypermethrin, whereas noncontact repellency plays no role in the escape responses of 2 species in the Minimus Complex in Thailand.     

Cytogenetic evidence for a fifth species within the taxon Anopheles dirus in Thailand

Crossbreeding and chromosomal evidence are presented for the existence of a fifth sibling species within the taxon of Anopheles dirus in Thailand. The new species is morphologically identifiable as Anopheles balabacensis "Fraser's Hill form." Structural differences in mitotic chromosomes and extensive asynapsis in hybrid polytene chromosomes indicate that significant genetic divergence exists between this species and its closest relatives, An. dirus species A, B, C and D and An. balabacensis.     

Multiplex PCR assay and phylogenetic analysis of sequences derived from D2 domain of 28S rDNA distinguished members of the Anopheles culicifacies complex into two groups,A/D and B/C/E

A multiplex PCR assay was developed using the sequences of the D2 region of 28S ribosomal DNA (rDNA) to discriminate the five members of the Anopheles culicifacies complex provisionally designated as species A, B, C, D and E. Two minus strand primers derived from sequence differences in the D2 variable region and a universal plus strand primer derived from the conserved 28S (rDNA) has delimited five members into species A and D (group 1) and species B, C and E (group 2) in a PCR diagnostic assay. The complete 28S rDNA-D2 region sequence of A. culicifacies sibling species is reported for the first time. Inter-specific sequence divergence was greater than the intra-specific divergence. The phylogenetic relationships inferred from maximum likelihood, maximum parsimony and the neighbor joining analysis confirmed the presence of two unambiguous monophyly clades one consisting of species A and D and the other of species B, C and E and that the A. culicifacies sibling species diverged relatively recently in evolutionary terms despite their considerable differences in bionomics.