Genetic structure of the genera Psorophora (Diptera: Culicidae) in Columbian and North American populations using isoenzymes and ITS2 sequences

Two, a priori, Psorophora columbiae and one, a priori, Ps. toltecum populations from Colombia were studied by means of eleven isoenzyme loci. The levels of genetic diversity, Hardy-Weinberg equilibrium, gene flow estimates, isolation by distance and genetic relationships among these three populations were studied and the results were as follows: 1. The gene diversity levels were high as well as very similar among the three populations indicating no differences between them. 2. Within each population and from a hierarchical standpoint several loci were not in Hardy-Weinberg equilibrium simultaneously by excess and defect of heterozygous. This could reflect that several natural selection forces are acting upon these mosquito populations. 3. The gene flow estimate for these populations showed the existence of this event between them. This agrees quite well with the fact that the three populations really belong to a single species. In addition, maximum parsimony analyses with 16 isoenzyme for several individuals from four Colombian and two United States Psorophora populations showed that effectively only one species was present in the Colombian area surveyed, and that this species is Ps. columbiae, which hardly contrast with that previously suggested by other authors.     

Psorophora columbiae and Psorophora toltecum (Diptera: Culicidae) Colombian populations cannot be differentiated by isoenzymes

Two populations of the mosquito Psorophora columbiae from the central Andean area of Colombia and one population of Ps. toltecum from the Atlantic coast of Colombia were analyzed for 11 isoenzyme markers. Psorophora columbiae and Ps. toltecum are two of the main vectors of Venezuelan equine encephalitis. We found no conspicuous genetic differences between the two species. The relatively high gene flow levels among these populations indicate that these are not two different species or that there has been recent divergence between these taxa. In addition, no global differential selection among the loci was detected, although the alpha-GDH locus showed significantly less genetic heterogeneity than the remaining loci, which could mean that homogenizing natural selection acts at this locus. No isolation by distance was detected among the populations, and a spatial population analysis showed opposite spatial trends among the 31 alleles analyzed. Multiregression analyses showed that both expected heterozygosity and the average number of alleles per locus were totally determined by three variables: altitude, temperature and size of the human population at the locality. Individually, the expected heterozygosity is more related to these three variables than to the average number of alleles.     

Cibarial morphology in Psorophora Robineau-Desvoidy subgenera (Diptera: Culicidae)

We describe the cibarial morphology in eight Psorophora species Robineau-Desvoidy: Ps. (Grabhamia) cingulata (Fabricius), Ps. (Gra.) confinnis (Lynch Arribálzaga), Ps. (Janthinosoma) ferox (Humboldt), Ps. (Jan.) albipes (Theobald), Ps. (Jan.) cyanescens (Coquillett), Ps. (Psorophora) lineata (Humboldt), Ps. (Pso.) cilipes (Fabricius), y Ps. (Pso.) ciliata (Fabricius). The species belonging to subgenus Grabhamia Theobald are characterized by palatal papillae in central position and the presence of cibarial armature. The teeth in Ps. cingulata are equal meanwhile in Ps. confinnis the internal row is spatulate with apex denticulate. In Janthinosoma Lynch Arribálzaga we observed six palatal papillae (the fore pair with less size to others) and armature absent, instead we observe small spicules toward posterior plate: abundant in Ps. ferox, and few in Ps. albipes and Ps. cyanescens. The subgenus Psorophora presents four equal size palatal papillae, cibarial armature absent, also distinctive number of trichoid sensilla (12-17), in comparison to other two subgenera (5-10). We suggest to include those diagnostic characters in the future taxonomic and systematic studies in the genus Psorophora.     

Comparison of ribosomal dna nucleotide sequences of the mosquito genera Aedes and Ochlerotatus (Diptera: Culicidae: Aedini)

Nucleotide rDNA sequences of 9 species of the mosquito genus Ochlerotatus and 2 species of the genus Aedes were obtained and compared. Variation both within and between these genera was characterized. The phylogenetic relationships among the studied species of Ochlerotatus generally correspond to the grouping of species based on morphological characters.     

Comparison of nucleotide sequences of ribosomal DNA of the mosquito genera Aedes and Ochlerotatus (Diptera: Culicidae: Aedini)

Morphological study allowed identifying 9 species of mosquitoes of the genus Ochlerotatus and 2 species of the genus Aedes. Sequencing of the rDNA region was performed for all these speciemens. The sequences of rDNA were the following: A. cinereus -868 bp, A. vexans--889 bp, Och. cantans--803 bp, O. excrucians--801 bp, O. euedes--794 bp, Och. cyprius--777 bp, O. diantaeus--758 bp, O. intrudens--817 bp, Och. punctor--783 bp, O. dorsalis--748 bp, O. species--767 bp. On average, the size of Aedes rDNA fragments exceeds rDNA of Ochlerotatus by 90-100 bp. The sequences are characterized by a high number of insertions and deletions, and also by point substitutions of nucleotides. It is important to notice that interspecific differences include not only different regions of the internal transcribed spacers, but also the conservative site which is represented by the 5.8S gene. Among four substitutions in this gene, one (C/A) represents the difference between Aedes and Ochlerotatus, the next (T/A) distinguishes A. cinereus from Ochlerotatus speciment and A. vexans, and two substitutions (A/C, T/G) testify the similarity between O. dorsalis and O. species and specimens of Aedes. Besides, two more deletions are typical for O. dorsalis and O. species. One deletion is com- mon, it distinguishes them from the other examined species, and another one is typical only for O. dorsalis. When analyzing morphological characteristics and comparing nucleotide rDNA sequences of O. species with the database, the similarity to O. caspius has been revealed. On the whole, phylogenetic relationships among Ochlerotatus species correspond to subdivision into groups based on morphological characters. Probably, examination of the larger number of specimens will change the morphological division into groups.     

基于COI与ITS序列的杂鳞库蚊复组 ( 双翅目: 蚊科) 分子系统发育

使用杂鳞库蚊复组COI部分序列和ITS序列构建分子发育树,并基于COI序列计算该复组种内和种间的Kimura-two-Parameter(K2P)距离,探讨环带库蚊的分类地位和杂鳞库蚊复组内各亲缘种的系统发育关系。环带库蚊和杂鳞库蚊的种间K2P距离为0.24%-0.72%,支持"环带库蚊是杂鳞库蚊的同物异名"这一观点;杂鳞库蚊(环带库蚊)和伪杂鳞库蚊、三带喙库蚊的种间K2P距离为4.41%-9.68%,同时分子系统树显示各个种分别聚类,互为姐妹群,再次证明三者互为独立的种;环带库蚊和杂鳞库蚊聚类的分支最接近树的端部,三带喙库蚊分支最接近树的基部,提示三带喙库蚊最早发生分化,而杂鳞库蚊(环带库蚊)最晚发生分化;采集自日本的三带喙库蚊种内K2P距离为0.48%-2.68%,而它们与采集自中国、印度的该种K2P距离为4.17%-6.76%,日本产三带喙库蚊聚集成一支,并与中印产地的聚类分支互为姐妹群,这些结果提示日本的三带喙库蚊有种下,甚至种级分化的趋势。     

Effects of temperature and soil moisture on survival of eggs of the mosquito Psorophora columbiae (Diptera: Culicidae)

The results of laboratory tests indicated the average survival rates for Psorophora columbiae eggs remained quite high for all of the egg populations exposed to a temperature of 27°C (range 83.0–100.0% survival) after 96 days of exposure, except for the non‐diapausing eggs on dry soil (66.3%). In regard to the exposure of egg populations to moderately cold temperatures (i.e. 8°C, 4°C and ?2°C) for periods of up to 16 days, survival rates for egg populations exposed to 8°C continued to remain relatively high (average >85%) for the remainder of the experimental exposure period (i.e. 96 days). Diapausing Ps. columbiae eggs were more tolerant (82.0% survival) to low temperatures (?2°C) than non‐diapausing eggs (2.4% survival) for 64 days, particularly at temperatures of and below 4°C. Diapausing and non‐diapausing eggs were similar in their ability to survive under high temperatures (34°C and 38°C). High soil moisture (30–40%) or substrate moisture (95% relative humidity) content appeared to enhance the ability of the mosquito eggs to survive both low and high temperature extremes.     

Reduced productivity in adult yellowfever mosquito (Diptera: Culicidae) populations

Male and female Aedes aegypti (L.) mosquitoes of the laboratory strain ROCK were irradiated with 130 mw of argon 514.5 nm laser microbeams for 0.04, 0.25, 0.4, and 0.5 s, respectively. Egg production, percentage hatch, and productivity (average number of adults surviving after 3 wk) were used to assess mutagenic effects. Mortality was high for males in all laser radiation groups and increased with time of exposure. Except for the group treated for 0.25 s, significant reductions in total F1 progeny also were demonstrated for all other experimentals when male parents were exposed to laser radiation. Females showed a high mortality when subjected to 0.4- and 0.5-s laser radiation. No F1 progeny were produced when parental females were exposed for 0.25, 0.4, and 0.5 s. Numbers of F1 progeny from females exposed to 0.04 s of laser radiation were significantly reduced. A comparison of weekly mean number of progeny showed that the important differences in productivity occurred during the first and second week, respectively, when either male or female adult parents were subjected to laser radiation.     

云南中华按蚊的遗传变异和种群结构

为了掌握云南省各地中华按蚊种群间的遗传变异和种群结构特征,测序并分析了采自云南9个样本点5个种群组的89头中华按蚊的线粒体COII基因。结果表明这些中华按蚊种群的COII基因序列平均单倍型多样性指数和核苷酸多样性指数分别为h=0.933,π=0.00406,共有51个变异位点,占分析的739个碱基总数的6.9%;定义了39个单倍型,有2个频率最高的单倍型H1和H9,分别占个体序列数的20.2%和12.4%;系统发育分析表明单倍型与地理位置没有明显的对应关系,单倍型网络图显示大部分单倍型分布没有明显的亲缘地理格局,主要以单倍型H1、H9、H4、H33和H2为中心呈星状分布,但元江和元阳构成的种群组(YU)单倍型存在明显地域分布特征;AMOVA结果表明种群组间遗传变异为12.58%,达到显著水平(P=0.04888),地理种群组间具有明显种群遗传结构。不同地区两两种群组间的Fst值和Nm值显示大部分种群组间存在基因交流,没有形成明显的遗传分化,但YU种群组和其他种群组间缺乏明显的基因交流,这主要是因为哀牢山的阻隔,使云南东西部形成两种不同的气候,产生了明显的遗传分化;歧点分布图显示为明显单峰分布,中性检测结果均为显著负值,说明云南省的中华按蚊种群在近期经历过复杂的种群扩张事件。掌握中华按蚊遗传多样性及分化特征,对中华按蚊及疟疾控制具有重要的作用。     

Sequence and secondary structure comparisons of ITS rDNA in mosquitoes (Diptera: Culicidae).

Sequences of the internal transcribed spacers (ITS1 and ITS2) of the mosquito Aedes aegypti, and the ITS2 of six related species, A. simpsoni, A. albopictus, A. vexans, A. triseriatus, Haemagogus mesodentatus, and Psorophora ferox are reported. Intraspecific variation in A. aegypti ITS1 is 1.07% among four clones from three individuals, and in the ITS2 is 1.17% among 15 clones from four individuals. In A. simpsoni, intraspecific ITS2 variation is 0.46% among 10 clones from a single individual. Alignment of the ITS2 sequence of the seven species reveals several homologous domains. Secondary structure predictions for the ITS2 region indicate that these domains base pair to form a core region central to several stem features. The sequence outside the ITS2 homologous domains tends to be GC-rich and characteristically slippage generated; these areas preserve or add to the stem length of the predicted secondary structures. These ITS2 intraspacer variable regions resemble previously described expansion segments of the 28S gene region. Evolutionary analysis of the ITS2 of these species, using both sequence and secondary structure information, leads to the prediction of divergence in the mosquito tribe Aedini that is not clearly reflected in current taxonomic designations.     

Genetic differentiation of Culex pipiens (Diptera: Culicidae) in China

The population genetic structures of Culex pipiens Linnaeus were evaluated in China over a 2000 km transect that encompasses the two subspecies, C. p. pallens and C. p. quinquefasciatus. Four polymorphic allozyme loci were investigated in 1376 mosquitoes sampled from 20 populations across four provinces. These loci were not statistically dependent with no apparent heterozygote deficit or excess. On a regional scale (intra-province), a low (Fst=0.007-0.016) and significant genetic differentiation was found, with no clear geographical pattern. On a wider scale (inter-province), the genetic differentiation was higher (Fst=0.059), and an isolation by distance emerged. The results are compared with previous population genetic surveys of this mosquito species in different geographic areas over the world. The overall pattern suggests that Culex pipiens requires considerable distance (500-1000 km) to show isolation by distance, irrespective of the subspecies (C. p. pipiens, C. p. quinquefasciatus and C. p. pallens) or the geographic location.     

Intragenomic rDNA ITS2 variation in the neotropical Anopheles (Nyssorhynchus) albitarsis complex (Diptera: Culicidae)

We cloned and sequenced the rDNA internal transcribed spacer 2 (ITS2) of 4 species belonging to the neotropical Anopheles (Nyssorhynchus) albitarsis complex, that is, A. albitarsis; A. albitarsis B; Anopheles marajoara, a proven malaria vector; and Anopheles deaneorum, a suspected vector. Even though the ITS2 sequences of these species were very similar (< or =1.17% divergence), we found differences suitable for species identification and intragenomic variation of possible consequence in phylogenetic reconstruction. Variation came from 2 microsatellite regions and a number of indels and base substitutions. The existence of partially correlated subsets of clones in A. albitarsis is hypothesized either to be separate rDNA loci or to be semi-independently evolving portions of a single rDNA locus. No differences were found between males and females, suggesting that similar rDNA arrays exist on both the X and Y chromosomes. In addition, highly variant clones, possibly pseudogenes, were found in A. marajoara from Venezuela.     

基于mtDNA-COI基因序列的雷氏按蚊分子群体遗传结构研究

【目的】探讨我国雷氏按蚊Anopheles lesteri的群体差异和分化程度。【方法】采用PCR方法, 从分子水平鉴别了采自我国和韩国的雷氏按蚊共9个群体139个样本, 扩增其线粒体DNA细胞色素氧化酶亚基Ⅰ基因, 并进行序列测定和分析。【结果】本研究共获得49个单倍型, 各单倍型呈高水平的平行演化, 来自云南群体的单倍型显示是扩张的源头。分子变异等级分析(AMOVA)的计算结果显示, 群体内变异占总变异的比例（64.95%）大于群体间（35.05%）, FST值为0.3504, 各群体间出现遗传分化。Mantel检验结果显示基因流水平与地理距离呈负相关关系（R²＝0.1322）, 群体遗传结构符合距离隔离模型。【结论】雷氏按蚊韩国和辽宁群体与其他分布地群体间差异大, 已出现明显分化, 我国其他分布地群体间的遗传差异小。     

Phytogeny of genusGlossina (Diptera:Glossinidae) according to ITS2 sequences

The flies of genusGlossina (Diptera: Glossinidae) are an important vector of African trypanosomiases which cause diseases in humans and animals. The ribosomal DNA Internal Transcribed Spacer-2 (ITS-2) region sequences from differentGlossina species were PCR-amplified and analyzed in order to construct a molecular phylogeny for genusGlossina. Trees generated by parsimony confirmed the monophyletic taxonomic placement of genusGlossina wherefusca group species formed the deepest branch followed bymorsitans andpalpalis groups, respectively. The placement ofGlossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on ITS-2 locus sequence analysis, suggest thatGlossina austeni can be placed into a separate subgenerus which forms a sister-group relationship with themorsitans group species.     

Density-dependent regulation of Aedes cantans (Diptera: Culicidae) in natural and artificial populations

Abstract.

• 1 Mortality and size variation (siphon length) in Aedes cantans larvae were examined in natural populations in northern England in 1989 and 1990.
• 2 Under crowded conditions, density-dependent competition led to reduction in the size of both larvae and adults and increased larval mortality.
• 3 Larvae were also maintained in cages in the field at different densities. Results paralleled those for the natural populations in the ponds; larvae maintained at high densities showed increased mortality and reduced size.
• 4 Possible density-dependent factors leading to mortality and size reduction include cannibalism and contact inhibition leading to food shortages.
• 5 The main density-independent factor contributing to larval mortality was habitat desiccation.

     

Phylogeny of genus Glossina (Diptera: Glossinidae) according to ITS2 sequences

The flies of genus Glossina (Diptera: Glossinidae) are an important vector of African trypanosomiases which cause diseases in humans and animals. The ribosomal DNA Internal Transcribed Spacer-2 (ITS-2) region sequences from different Glossina species were PCR-amplified and analyzed in order to construct a molecular phylogeny for genus Glossina. Trees generated by parsimony confirmed the monophyletic taxonomic placement of genus Glossina where fusca group species formed the deepest branch followed by morsitans and palpalis groups, respectively. The placement of Glossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on ITS-2 locus sequence analysis, suggest that Glossina austeni can be placed into a separate subgenerus which forms a sister-group relationship with the morsitans group species.