基于mtDNA COI基因的离腹寡毛实蝇属常见种DNA条形码识别和系统发育分析

【目的】离腹寡毛实蝇属Bactrocera昆虫是最具经济重要性的实蝇类害虫,本研究依据mtDNA COI基因碱基序列对离腹寡毛实蝇属常见实蝇种类进行识别鉴定与系统发育分析。【方法】以口岸经常截获的离腹寡毛实蝇属8个亚属21种实蝇为对象,采用DNA条形码技术,通过对mtDNA COI基因片段 (约650 bp)的测序和比对,以MEGA软件的K2-P双参数模型计算种内及种间遗传距离,以邻接法(NJ) 构建系统发育树。【结果】聚类分析与形态学鉴定结果一致,除11种单一序列实蝇外,其他10种实蝇均各自形成一个单系,节点支持率为99%以上。种内（10种）遗传距离为0.0003~0.0068,平均为0.0043;种间（21种）遗传距离为0.0154~0.2395,平均为0.1540;种间遗传距离为种内遗传距离的35.8倍,而且种内、种间遗传距离没有重叠区域。【结论】基于mtDNA COI基因的DNA条形码技术可以用于离腹寡毛实蝇属昆虫的快速鉴定识别,该技术体系的建立对实蝇类害虫的检测监测具有重要意义。     

Microsatellite markers for the pest fruit fly,Bactrocera papayae (Diptera: Tephritidae) and other Bactrocera species

The dorsalis complex contains some of the most economically important fruit fly pests of the Asia–Pacific regions, including Bactrocera dorsalis, Bactrocera papayae and Bactrocera carambolae. These species are morphologically indistinct and genetically very similar. We describe the development of 12 microsatellite markers isolated from a representative of the dorsalis complex, B. papayae. We show the potential utility of the B. papayae microsatellites and a set of microsatellites isolated from Bactrocera tryoni as population and species markers for the dorsalis complex.     

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) is not invasive through Asia: It's been there all along

Oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly polyphagous fruit fly which, in the last 15 years, has invaded (with or without establishment) Africa, Europe and North America. As a direct result of these invasions, there is increasing research interest in the invasion history and spread patterns of this fly. A statement being repeatedly used in the B. dorsalis invasion literature is that the species was first identified from Taiwan in 1912 and that it subsequently spread through South‐East and South Asia during the 20th century. This assumption is incorrect and stems from: (a) an incomplete knowledge of B. dorsalis taxonomic history; and (b) a confounding of first taxonomic record with first invasion record. Rather than being first detected in Taiwan in 1912, the first record of oriental fruit fly was from “East India” (India orientali) under the synonymous name of Musca ferruginea by Fabricius in 1794, and in the 1910s, it was known not only from Taiwan, but widely across tropical Asia with records from India, Burma, Bengal, Sri Lanka (as Ceylon), Singapore and Indonesia (multiple islands). The taxonomic literature is very clear that oriental fruit fly has not invaded the rest of Asia from Taiwan since 1912, and this error should not continue to be repeated in the literature.     

橘小实蝇幼虫液体人工饲料中pH值对其生长发育的影响

本文主要研究了橘小实蝇液体人工幼虫饲料中不同酸度对橘小实蝇生长发育的影响。继代饲养3代的结果表明：饲料酸度对橘小实蝇化蛹率和后代卵孵化率没有影响;对橘小实蝇蛹重、成虫羽化率和产卵量有显著影响。橘小实蝇液体人工幼虫饲料的pH值调节至3.6时仍可以获得质量较高的橘小实蝇。     

Current knowledge of the species complex Anastrepha fraterculus (Diptera,Tephritidae) in Brazil

The study of the species complex Anastrephafraterculus (Af complex) in Brazil is especially important in a taxonomical, evolutionary and pest management context, because there are evidences that some of them may occur in sympatry. In this review, we analyzed the main results supporting evidences that three cryptic species occur in Brazil. The taxonomical and phylogenetic relationships based on eggshell morphology, adult morphometrics, as well as cytotaxonomy and genetic differentiations are discussed. We also review available information on sexual behavior including acoustic communication of males during courtship and sexual incompatibility; and chemical signals involved in the communication between sexes, with a special focus on sex pheromones. We examined the role of long- and short-range pheromones (male-produced volatiles and cuticular hydrocarbons, respectively), their implications in sexual isolation, and their possible use for chemotaxonomic differentiation of the putative species of the Af complex.     

基于CLIMEX的桔小实蝇在中国适生区的预测

桔小实蝇Bactroceradorsalis(Hendel),属双翅目Diptera,果实蝇科Tetriphitidae,主要分布在热带和亚热带地区。温度和湿度是影响桔小实蝇分布的重要气候因子。根据桔小实蝇对温湿度等气候因子的反应,采用CLIMEX软件对桔小实蝇在中国大陆的适生区进行了预测。设置了CLIMEX中的相应参数17个:发育起点温度DV0、生长最适宜温度范围DV1～DV2、致死高温DV3、有效发育积温PDD。生长发育所需最低土壤湿度临界SM0、最适宜湿度范围SM1～SM2、最高土壤湿度临界SM3。冷胁迫日度临界DTCS及其积累速率DHCS,热胁迫临界温度TTHS及其积累速率THHS,干旱胁迫临界SMDS及其积累速率HDS,湿胁迫SMWS及其积累速率HWS。以印度和夏威夷为已知适生分布区,反复调试修正上述这些参数值,使之与已知广泛分布的地区达到最大程度的吻合。然后用优化后的参数和中国大陆85个气象站点的气象资料模拟桔小实蝇在中国大陆的适生分布,结果显示:广东、海南、香港、广西、四川、云南、湖南、湖北、福建、江西、浙江等11个省(区)是桔小实蝇的适生分布区。主要分布在我国的华南和西南大部分地区,以及华中和华东的部分地区。根据CLIMEX模拟结果的EI值大小,将桔小实蝇在我国大陆的适生分布情况进一步划分为最适宜、次适宜、适宜和非适宜4个气候区,即华南地区全部以及广西省全境是桔小实蝇的最适宜分布区,除桂林(EI=17)外,其余气象点的EI值均大于40;西南地区的四川、云南两省及福建沿海地区是桔小实蝇的次适宜分布区,平均EI值为29.7;适宜分布区包括湖南、湖北、江西、浙江的少数地区,除赣州(EI=17)外,其余点的EI值均小于10;长江以北的广大地区是桔小实蝇的非适宜区,这些地区不适合桔小实蝇生存。     

Improvement of protein bait produced from beer yeast waste for controlling Bactrocera dorsalis (Diptera: Tephritidae) in China

In the present study, preservatives and antibiotics were applied in a variety of doses to assess their effects on the beer waste-based protein bait, a product widely used to control populations of Bactrocera dorsalis (Diptera: Tephritidae). Screening was performed using the inhibition zone method against the dominant bacteria in deteriorated protein bait. Additionally, the attractiveness of the bait was assessed after respectively adding different doses of methyl eugenol, brown sugar solution, ammonium acetate, and guava essence, as determined by bio-cage tests. The results showed that the four selected preservatives had different inhibition effects at different dose levels, and that the level of inhibition increased with dose: at the highest dose of 1:1 (v:v), sodium benzoate can significantly inhibit Staphylococcus spp., potassium sorbate can significantly inhibit Lactobacillus spp., sodium nitrite can significantly inhibit Escherichia spp. and ethyl p-hydroxybenzoate can significantly inhibit Staphylococcus spp. and Xanthomonas spp. Among three antibiotics, kanamycin was the most effective in suppressing a mix of all four bacteria for all tested doses. In terms of lure performance, the addition of four substances in various ratios into the protein bait were found to have varying effects on its attractiveness to B. dorsalis females and males. Specifically, the addition of a high-dose of guava essence could significantly enhance the attractiveness of the bait. Our findings will help to enhance the effectiveness of protein bait applications by prolonging the quality guarantee period and improving its attractiveness toward both female and male B. dorsalis.     

Wing shape as a potential discriminator of morphologically similar pest taxa within the Bactrocera dorsalis species complex (Diptera: Tephritidae)

Four morphologically cryptic species of the Bactrocera dorsalis fruit fly complex (B. dorsalis s.s., B. papayae, B. carambolae and B. philippinensis) are serious agricultural pests. As they are difficult to diagnose using traditional taxonomic techniques, we examined the potential for geometric morphometric analysis of wing size and shape to discriminate between them. Fifteen wing landmarks generated size and shape data for 245 specimens for subsequent comparisons among three geographically distinct samples of each species. Intraspecific wing size was significantly different within samples of B. carambolae and B. dorsalis s.s. but not within samples of B. papayae or B. philippinensis. Although B. papayae had the smallest wings (average centroid size=6.002 mm±0.061 SE) and B. dorsalis s.s. the largest (6.349 mm±0.066 SE), interspecific wing size comparisons were generally non-informative and incapable of discriminating species. Contrary to the wing size data, canonical variate analysis based on wing shape data discriminated all species with a relatively high degree of accuracy; individuals were correctly reassigned to their respective species on average 93.27% of the time. A single sample group of B. carambolae from locality 'TN Malaysia' was the only sample to be considerably different from its conspecific groups with regards to both wing size and wing shape. This sample was subsequently deemed to have been originally misidentified and likely represents an undescribed species. We demonstrate that geometric morphometric techniques analysing wing shape represent a promising approach for discriminating between morphologically cryptic taxa of the B. dorsalis species complex.     

Population structure and colonization of Bactrocera dorsalis (Diptera: Tephritidae) in China,inferred from mtDNA COI sequences

The oriental fruit fly, Bactrocera dorsalis, is a serious pest of fruits and vegetables in South‐east Asia, and, because of quarantine restrictions, impedes international trade and economic development in the region. Revealing genetic variation in oriental fruit fly populations will provide a better understanding of the colonization process and facilitate the quarantine and management of this species. The genetic structure in 15 populations of oriental fruit fly from southern China, Laos and Myanmar in South‐east Asia was examined with a 640‐bp sequence of the mitochondrial cytochrome oxidase subunit I (COI) gene. The highest levels of genetic diversity were found in Laos and Myanmar. Low to medium levels of genetic differentiation (F_ST ≤ 0.134) were observed among populations. Pooled populations from mainland China differed from those in Laos and Myanmar (F_ST = 0.024). Genetic structure across the region did not follow the isolation‐by‐distance model. The high genetic diversity observed in Laos and Myanmar supports the South‐east Asian origin of B. dorsalis. High genetic diversity and significant differentiation between some populations within mainland China indicate B. dorsalis populations have been established in the region for an extended period of time. High levels of genetic diversity observed among the five populations from Hainan Island and similarity between the Island and Chinese mainland populations indicate that B. dorsalis was introduced to Hainan from the mainland and has been on the island for many years. High genetic diversity in the recently established population in Shanghai (Pudong) suggests multiple introductions or a larger number of founders.     

Gene flow and genetic structure of Bactrocera carambolae (Diptera,Tephritidae) among geographical differences and sister species,B. dorsalis,inferred from microsatellite DNA data

The Carambola fruit fly, Bactroceracarambolae, is an invasive pest in Southeast Asia. It has been introduced into areas in South America such as Suriname and Brazil. Bactroceracarambolae belongs to the Bactroceradorsalis species complex, and seems to be separated from Bactroceradorsalis based on morphological and multilocus phylogenetic studies. Even though the Carambola fruit fly is an important quarantine species and has an impact on international trade, knowledge of the molecular ecology of Bactroceracarambolae, concerning species status and pest management aspects, is lacking. Seven populations sampled from the known geographical areas of Bactroceracarambolae including Southeast Asia (i.e., Indonesia, Malaysia, Thailand) and South America (i.e., Suriname), were genotyped using eight microsatellite DNA markers. Genetic variation, genetic structure, and genetic network among populations illustrated that the Suriname samples were genetically differentiated from Southeast Asian populations. The genetic network revealed that samples from West Sumatra (Pekanbaru, PK) and Java (Jakarta, JK) were presumably the source populations of Bactroceracarambolae in Suriname, which was congruent with human migration records between the two continents. Additionally, three populations of Bactroceradorsalis were included to better understand the species boundary. The genetic structure between the two species was significantly separated and approximately 11% of total individuals were detected as admixed (0.100 ≤ Q ≤ 0.900). The genetic network showed connections between Bactroceracarambolae and Bactroceradorsalis groups throughout Depok (DP), JK, and Nakhon Sri Thammarat (NT) populations. These data supported the hypothesis that the reproductive isolation between the two species may be leaky. Although the morphology and monophyly of nuclear and mitochondrial DNA sequences in previous studies showed discrete entities, the hypothesis of semipermeable boundaries may not be rejected. Alleles at microsatellite loci could be introgressed rather than other nuclear and mitochondrial DNA. Bactroceracarambolae may be an incipient rather than a distinct species of Bactroceradorsalis. Regarding the pest management aspect, the genetic sexing Salaya5 strain (SY5) was included for comparison with wild populations. The SY5 strain was genetically assigned to the Bactroceracarambolae cluster. Likewise, the genetic network showed that the strain shared greatest genetic similarity to JK, suggesting that SY5 did not divert away from its original genetic makeup. Under laboratory conditions, at least 12 generations apart, selection did not strongly affect genetic compatibility between the strain and wild populations. This knowledge further confirms the potential utilization of the Salaya5 strain in regional programs of area-wide integrated pest management using SIT.     

桔小实蝇超氧化物歧化酶基因SOD3的克隆及表达分析

采用反转录聚合酶链式反应(RT-PCR)和快速扩增c DNA末端(RACE)技术克隆桔小实蝇SOD3基因,并命名为Bdor SOD3。Bdor SOD3阅读框全长531 bp,编码176个氨基酸,第1-20位氨基酸为其信号肽区域;该蛋白序列与桔小实蝇的另外一种SOD蛋白AGE89778.1序列的一致性最高,达98.7%;采用Swiss-model在线软件模拟构建Bdor SOD3蛋白的三维结构;采用半定量PCR方法,研究Bdor SOD3基因大肠杆菌诱导后的表达情况,结果表明,Bdor SOD3在处理与对照的24 h、48 h都有表达,但Bdor SOD3在处理后48 h表达量明显升高,结果暗示Bdor SOD3与桔小实蝇蛹对大肠杆菌的免疫机制有关。     

Population dynamics of the oriental fruit fly, Bactrocera dorsalis （Diptera： Tephritidae） in the Kunming area,southwestern China

Population dynamics of Bactrocera dorsalis Hendel （Diptera： Tephritidae） were studied through pheromone trapping over 4 years （1997, 1999, 2000, 2003） in the Kunming region, a high plateau area in southwestern China. B. dorsalis immigrates from southern Yunnan to Kunming each year, and occurs during early May through November. Annual trap captures recorded an increase in the B. dorsalis populations from May to July, when they peaked in abundance, and a decline until November. No flies were detected from November to April. The fruit flies had two generations. There was considerable overlapping due to the continuous arrival of immigrating flies during the summer months. Annual capture rates were significantly related to numbers of flies caught in July when peak captures were recorded; whereas the peak captures, in turn, positively depended on numbers of flies recorded in May, the first month of fly appearance in the current year. It suggested that the annual population abundance was mainly dependent on the size of the initial emigrating population. A daily average temperature of 18℃ was probably the threshold temperature required for the flies to undertake long-range dispersal, which partially explained the start of the fly in May each year on this high plateau. Under field conditions, the fruit flies can withstand 13℃ as a daily average temperature. No flies were recorded in any of the study years at a daily temperature colder than 10 ℃.     

Effect of sex,age and morphological traits on tethered flight of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) at different temperatures

The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is a pest of fruit and vegetable production that has become established in 42 countries in Africa after its first detection in 2003 in Kenya. It is likely that this rapid expansion is partly due to the reported strong capacity for flight by the pest. This study investigated the tethered flight performance of B. dorsalis over a range of constant temperatures in relation to sex and age. Tethered flight of unmated B. dorsalis aged 3, 10 and 21 days was recorded for 1 h using a computerized flight mill at temperatures of 12, 16, 20, 24, 28, 32 and 36 °C. Variations in fly morphology were observed as they aged. Body mass and wing loading increased with age, whereas wing length and wing area reduced as flies aged. Females had slightly larger wings than males but were not significantly heavier. The longest total distance flown by B. dorsalis in 1 h was 1559.58 m. Frequent short, fast flights were recorded at 12 and 36 °C, but long-distance flight was optimal between 20 and 24 °C. Young flies tended to have shorter flight bouts than older flies, which was associated with them flying shorter distances. Heavier flies with greater wing loading flew further than lighter flies. Flight distances recorded on flight mills approximated those recorded in the field, and tethered flight patterns suggest a need to factor temperature into the interpretation of trap captures.     

桔小实蝇幼体及成虫残体DNA条形码识别技术的建立与应用

实蝇类害虫多为国内外检疫对象, 其鉴定识别方法主要依据成虫的外部形态特征, 而传统的形态学识别法对口岸经常截获的幼体及残缺的虫体, 则无能为力。本研究以桔小实蝇Bactrocera dorsalis的幼体（卵、 幼虫、 蛹）以及成虫残体（足、 翅、 头部、 胸部、 腹部）为对象, 利用 DNA 条形码技术, 构建实蝇类害虫快速鉴定技术体系, 并以其他4种常见实蝇（包括番石榴实蝇B. correcta、 瓜实蝇B. cucurbitae、 南亚果实蝇B. tau、 柑桔大实蝇B. minax）为对象对该技术体系进行应用验证。结果显示, 桔小实蝇幼体以及成虫残体的碱基序列与数据库中靶标种COⅠ基因碱基序列的一致性为99.51%~99.84%, 其他4种实蝇相应序列与数据库中靶标种COⅠ基因序列的一致性分别为100%, 100%, 99.81%~99.83%和100%; 以邻接法（NJ法）构建系统发育树, 靶标种实蝇均与数据库中对应种实蝇聚为一支, 且置信度均为100%。以K2-P模型计算种内及种间遗传距离得出, 5种实蝇的种间遗传距离为0.0597~0.2363, 平均为0.1693; 种内遗传距离为0.0000~0.0041, 平均为0.0019。这些结果表明, 基于DNA条形码的物种识别技术完全可用于口岸截获的实蝇类害虫幼体及残体的准确鉴定。     

Intraspecific variation of cuticular hydrocarbon profiles in the Anastrepha fraterculus (Diptera: Tephritidae) species complex

The goal of this study was to define whether cuticular hydrocarbons (CHs) could be used for taxonomic determination of putative species hidden in the Anastrepha fraterculus cryptic species complex, widespread from Argentina to Mexico. Recently, increasing evidence of phenotypic and genetic variability has resulted in the characterization of eight morphotypes within this complex. The CH profiles of six A. fraterculus populations from Argentina, Brazil, Peru, Colombia and Mexico were analysed in this study by two‐dimensional gas chromatography coupled with mass spectrometry. In parallel, multiple factorial analyses were used to elucidate population structures. Vector populations segregated into four distinct groups. The analysis demonstrated that the studied populations from Peru, Argentina and southern Brazil (Vacaria) might be classified in accordance with the earlier division of the A. fraterculus complex into Peruvian, and Brazilian‐1 cryptic species, using the specific CH profiles. Population from south‐eastern Brazil (Piracicaba) formed separated group. Mexican and Andean (Colombian) putative species had similar CH signatures, when compared to each other.     

Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application

Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage to horticulture industries. Immature stages of these two fruit fly species have been intercepted in New Zealand a number of times. Identification to species was not possible using morphological characters; thus, it is important to develop an assay for their species‐level identification. Here, the real‐time PCR assays for rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity by amplifying the two target species successfully, with no cross‐reactions observed in the tested tephritid species. The in silico test of the primer and probe binding sites of the two assays also demonstrated the assays’ specificity by no mismatches present in the binding regions of the target species, but 1–4 mismatches in the binding regions of the non‐target fruit fly species. The thresholds of detection for the two assays are as low as 10 copies/µl of the target DNA, indicating that the assays have a very high sensitivity. The application of the real‐time PCR assays has greatly assisted in routine pest identifications at the New Zealand border and surveillance programme. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations worldwide.     

桔小实蝇V-ATPase G亚基基因的克隆及组织表达特异性分析

空泡型ATP酶（vacuolar-type H⁺-ATPase, V-ATPase）作为质子泵几乎在所有的真核生物细胞中发挥重要作用。本研究利用RT-PCR和RACE技术获得了桔小实蝇Bactrocera dorsalis (Hendel)V-ATPase G亚基序列全长, 命名为BdorATPG。测序结果表明, BdorATPG阅读框全长354 bp, 编码117个氨基酸。氨基酸序列比对表明, BdorATPG的N端序列与其他物种的ATPG亚基对应区域具有较高的序列一致性。BdorATPG与拟暗果蝇Drosophila pseudoobscura ATPG亚基的氨基酸序列一致性最高, 为88.9%。三维结构模建结果表明, BdorATPG N端（第1~59位氨基酸）序列为α-螺旋结构, 亲水性和疏水性氨基酸在螺旋两侧呈对称分布。BdorATPG在不同组织中的荧光定量PCR分析表明, BdorATPG在各组织中都有表达, 其中在触角中的表达量最高; 在雄虫生殖节中的表达量是雌虫中的6.04倍。结果提示BdorATPG可能在雄虫生殖生理过程中发挥重要作用。     

Spatio‐temporal analysis of the relationship between landscape structure and the olive fruit fly Bactrocera oleae (Diptera: Tephritidae)

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