Genetic identification of members of the Bemisia tabaci cryptic species complex from South Africa reveals native and introduced haplotypes

The whitefly Bemisia tabaci cryptic species complex contains some important agricultural pest and virus vectors. Members of the complex have become serious pests in South Africa (SA) because of their feeding habit and their ability to transmit begomovirus species. Despite their economic importance, studies on the biology and distribution of B. tabaci in SA are limited. To this end, a survey was made to investigate the diversity and distribution of B. tabaci cryptic species in eight geographical locations (provinces) in SA, between 2002 and 2009, using the mitochondrial cytochrome oxidase I (mtCOI) sequences. Phylogenetic analysis revealed the presence of members from two endemic sub‐Saharan Africa (SSAF) subclades coexisting with two introduced putative species. The SSAF‐1 subclade includes cassava host‐adapted B. tabaci populations, whereas the whiteflies collected from cassava and non‐cassava hosts formed a distinct subclade, referred to as SSAF‐5, and represent a new subclade among previously recognized southern Africa clades. Two introduced cryptic species, belonging to the Mediterranean and Middle East–Asia minor 1 clades, were identified and include the B and Q types. The B type showed the widest distribution, being present in five of the eight provinces explored in SA, infesting several host plants and predominating over the indigenous haplotypes. This is the first report of the occurrence of the exotic Q type in SA alongside the more widely distributed B type. Furthermore, mtCOI PCR‐RFLP was developed for the SA context to allow rapid discrimination between the B, Q and SSAF putative species. The capacity to manage pests and disease effectively relies on knowledge of the identity of the agents causing the damage. Therefore, this study contributes to the understanding of South African B. tabaci species diversity, information needed for the development of knowledge‐based disease management practices.     

Further insight into reproductive incompatibility between putative cryptic species of the Bemisia tabaci whitefly complex

The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), with its global distribution and extensive genetic diversity, is now known to be a complex of over 35 cryptic species. However, a satisfactory resolution of the systematics of this species complex is yet to be achieved. Here, we designed experiments to examine reproductive compatibility among species with different levels of mitochondrial cytochrome oxidase I (mtCOI) divergence. The data show that putative species with mtCOI divergence of >8% between them consistently exhibited complete reproductive isolation. However, two of the putative species, Asia II 9 and Asia II 3, with mtCOI divergence of 4.47% between them, exhibited near complete reproductive compatibility in one direction of their cross, and partial reproductive compatibility in the other direction. Together with some recent reports on this topic from the literature, our data indicates that, while divergence in the mtCOI sequences provides a valid molecular marker for species delimitation in most clades, more genetic markers and more sophisticated molecular phylogeny will be required to achieve adequate delimitation of all species in this whitefly complex. While many attempts have been made to examine the reproductive compatibility among genetic groups of the B. tabaci complex, our study represents the first effort to conduct crossing experiments with putative species that were chosen with considerations of their genetic divergence. In light of the new data, we discuss the best strategy and protocols to conduct further molecular phylogenetic analysis and crossing trials, in order to reveal the overall pattern of reproductive incompatibility among species of this whitefly complex.     

Genetic diversity and geographic distribution of Bemisia tabaci species complex in Nepal

Bemisia tabaci species complex comprises at least 44 cryptic species worldwide. Here, we analyze the nucleotide sequences of mitochondrial cytochrome oxidase subunit 1 (COI) gene obtained from 76 samples of B. tabaci collected from 23 districts in Nepal. This is the first genetic and geographic study of B. tabaci species complex in Nepal. Our phylogenetic study identified the presence of three cryptic species—Asia I, Asia II 1, and Asia II 5—with high interspecific but low intraspecific variations. Among the three cryptic species, Asia II 5 was the most prevalent in Nepal, constituting 64.47% of all the sequenced samples. Based on haplotype network analysis of COI sequences, Asia II 1 was more genetically diversified than the other two cryptic species. Our results provided useful information on the genetic diversity and geographic distribution of B. tabaci in Nepal, which help monitor population changes of B. tabaci at cryptic species level and develop sustainable management strategies for its control.     

广东首次发现Q型烟粉虱种群分布

烟粉虱Bemisia tabaci(Gennadius)是全世界分布的刺吸式传毒昆虫,其中Q型烟粉虱自2003年首次在昆明一品红上发现以来,该种群已在全国10多个省份广泛传播,并有逐步取代B型烟粉虱的趋势。然而,作为较早对外开放的沿海省份,广东省一直未有Q型烟粉虱在田间分布危害的报道。本文利用常规PCR、特异B/Q型烟粉虱引物扩增及DNA测序技术,对近期在广州长湴公园扶桑寄主上采集的烟粉虱种群进行了分子鉴定,并对该种群与国内外其它Q型种群的系统发育关系进行了研究。结果表明,在广州长湴公园扶桑寄主上采集的烟粉虱种群为Q型烟粉虱,在660 bp的mtCOI序列中,该Q型烟粉虱与来自江苏、云南及浙江的Q型烟粉虱种群分别有1²个碱基差异;系统进化关系表明该Q型烟粉虱与江苏、云南的Q型烟粉虱最近,其种群的最初起源应为地中海西岸地区。相比B型烟粉虱,Q烟粉虱被认为具有更强的抗药性和传播植物病毒的能力,Q型烟粉虱在广东的首次发现,表明该烟粉虱种群已在广东野外定殖,其扩散动态、寄主范围及危害程度亟待实时监测。     

Genetic diversity of Bemisia tabaci cryptic species in Nigeria and their relationships with endosymbionts and acquired begomoviruses

Bemisia tabaci is a species complex of at least 44 cryptic species with a worldwide distribution. It is a serious pest of many crop plants as well as a successful vector of at least 100 begomoviruses. Using B. tabaci collected from cassava and tomato fields in the southwestern and north central regions of Nigeria, we determined nucleotide sequences from the mitochondrial cytochrome c oxidase subunit I (COI) of 23 B. tabaci samples, the 16S and 23S ribosomal DNA of endosymbionts, and the coat protein gene of geminiviruses ingested by the whiteflies. The COI analysis identified three different genetic groups including the indigenous Sub-Saharan Africa 1 subgroup 1 (SSA1-SG1) and 5 (SSA1-SG5, which was most prevalent), and an invasive cryptic species (Mediterranean). SSA1 was infected by five known secondary endosymbionts, Arsenophonus, Cardinium, Hamiltonella, Rickettsia, and Wolbachia, and co-infections with two or three endosymbionts were common. Five begomoviruses, okra enation leaf curl virus, squash leaf curl China virus, tobacco curly shoot virus, tomato leaf curl New Delhi virus, and tomato yellow leaf curl virus, were detected from 43.5% of the B. tabaci samples. However, cassava mosaic disease that causes devastating cassava yield losses was not detected in this study. This study improves the current understanding of the genetic diversity of B. tabaci cryptic species, and it reveals their relationships with endosymbionts and geminiviruses in the cassava and tomato fields of Nigeria.     

Genetic groups and endosymbiotic microbiota of the Bemisia tabaci species complex in Japanese agricultural sites

The whitefly Bemisia tabaci is a cryptic species complex of at least 24 genetically distinct species. Thus far, one obligate and seven facultative symbiotic bacteria have been reported from the B. tabaci species complex. Both genetic groups and infected symbionts are extremely important to estimate the pest status of B. tabaci. In this study, we collected 340 whiteflies from 39 agricultural sites, covering an entire region of the B. tabaci habitat in Japan, and examined the genotypes and symbiont community composition at subspecies level. Use of the cleaved amplified polymorphic sequence technique and mitochondrial cytochrome oxidase subunit I gene sequencing detected five genetic groups: indigenous species JpL and Asia II 6, invasive species Middle East‐Asia Minor 1 (MEAM1) and Mediterranean Subclade Q1 (MED Q1), and a genetic group previously undetected in Asia, Mediterranean Subclade Q2 (MED Q2). The genetic groups exhibited characteristic infection statuses with regard to their facultative symbionts, as observed in other countries. The endosymbiotic microbiota of the Japanese MED Q1 was different from that in neighbouring countries, but similar to that in the French or Uruguayan MED Q1. These results may indicate that Japanese MED Q1 species have not invaded from neighbouring countries, but from distant countries by international transportation. All Japanese MED Q2 species were infected with Rickettsia, some of which are regarded as conferring a female‐biased sex ratio and fitness benefit on B. tabaci. The results suggest that MED Q2 may be prevalent in Japan and neighbouring countries.     

Spatial and host-plant partitioning between coexisting Bemisia tabaci cryptic species in Tunisia

The whitefly Bemisia tabaci is a species complex including at least 24 morphologically indistinguishable species among which the Mediterranean (Med) and Middle East-Asia Minor I (MEAMI) species containing the biotypes commonly known as Q and B, respectively. These B and Q biotypes (hereafter referred to as MEAMI and Med species) are the most invasive agricultural pests of the B. tabaci complex worldwide. The spread of MEAMI and more recently of Med species into regions already invaded by other B. tabaci populations has been frequently seen to lead to their displacement by Med species. In Tunisia, in contrast to usual observations in the Mediterranean basin, Med and MEAMI species have been seen to co-occur in the main crop producing regions. Based on fine population genetics and field spatial distribution analyses, we found that the co-existence of these two interacting species was based on habitat partitioning including spatial and host-plant partitioning. Although they co-occurred at larger spatial scales, they excluded one another at sample scale. We observed neither spatial overlapping nor hybridization between MEAMI and Med B. tabaci. Vegetable crops were the main hosts for MEAMI specimens while 99.1% of the B. tabaci collected on the ornamental, Lantana camara, were Med specimens. Different patterns of genetic diversity were observed between the two species, as well as among Med specimens sampled on the ornamental versus vegetables, with the highest genetic diversity found in Med B. tabaci sampled on L. camara. These findings lead us to focus our discussion on the role played by lantana, human pressure, and competition, in the spatial and genetic patterns observed in the whitefly B. tabaci.     

The presence of six cryptic species of the whitefly Bemisia tabaci complex in China as revealed by crossing experiments

Abstract Recent phylogenetic analysis using mitochondrial cytochrome oxidase I (mtCOI) sequences of Bemisia tabaci worldwide indicates that the whitefly comprises at least 24 morphologically indistinguishable but genetically distinct cryptic species. While evidence of reproductive isolation has been reported for some of the putative species, more extensive crossing experiments are required to clarify the systematics of this species complex. In this study, we established laboratory cultures for six putative species of B. tabaci collected in China. We conducted 22 inter‐species crosses among the six putative species. The data and those reported previously were collated, and the combined dataset covered all the 30 possible inter‐species crosses among the six putative species. Intra‐species controls always produced female and male progeny and the proportions of females in the first generation (F1) ranged from 56% to 70%. However, in inter‐species crosses female progeny were rarely produced, and the few F1 females produced in four of the 30 inter‐species crosses were either sterile or significantly weaker in viability. These results demonstrate a pattern of complete reproductive isolation among the six putative species and show that they are six cryptic species in the B. tabaci complex.     

First mtCO-I based molecular identification of two cryptic species of Bemisia tabaci from Afghanistan

B. tabaci exists as a complex of morphologically very similar but genetically different variants previously termed as biotypes and more recently as cryptic species. Using a region of mitochondrial cytochrome oxidase, I (mtCO-I) gene sequences this study for the first time identified at molecular level the presence of two cryptic species of B. tabaci; Asia-I and Asia-II-5 from cotton grown in the fields of Bland Ghar, Bihsud district, Nangarhar province of Afghanistan. It is the first report of its type as this insect was not reported earlier from any crop or area of this country at species level.     

Use of consensus sequences and genetic networks to identify members of the Bemisia tabaci cryptic species complex in Egypt and Syria

The sweet potato whitefly, Bemisia tabaci (Gennadius), is a cryptic species complex composed of at least 24 different morphologically indistinguishable species. The considerable differences in the pest status across the complex, and the ability of some to develop resistance to, insecticides make awareness of their identity critical in terms of developing effective control measures. Previously, phylogenetic reconstructions have been used to identify different B. tabaci, but this approach is no longer necessary because of the existence of mitochondrial cytochrome oxidase one consensus sequences for each of the known species. We therefore use these consensus sequences to determine the identities of the members of the complex in Syria and Egypt and then used genetic networks to reveal the pattern of their genetic relatedness to other haplotypes in the species to which they were assigned. The results showed the presence of three species in Syria, AsiaII 1, Middle East–Asia Minor 1 (this equates to the global invader known commonly as the B biotype) and Mediterranean (this equates to the global invader known commonly as the Q biotype). Egypt was shown to have two cryptic species, Middle East–Asia Minor 1 and Mediterranean. In Syria, Middle East–Asia Minor 1 was found around Damascus only (south‐west Syria), while Mediterranean was found throughout Aleppo (northern Syria) and Hama (north central Syria). AsiaII 1 was found around Hims (south central Syria) and Damascus (south‐western Syria). In Egypt, Mediterranean was found in Cairo and Ismailia (central Upper Egypt), while Middle East–Asia Minor 1 was found in the remaining all parts of Upper Egypt, Suez, North Sinai, Port Said, Dakahila, Behera and Alexandria which cover the main agricultural zone of Egypt. Genetic relatedness of Syrian and Egyptian populations with each other and with rest of world is also discussed.     

The Bemisia tabaci species complex: Additions from different parts of the world

Bemisia tabaci is one of the most threatening pests in many crops. We sequenced part of the mitochondrial cytochrome oxidase I gene from fifty whitefly populations collected in Indonesia, Thailand, India and China. Nineteen unique sequences (haplotypes) of the cytochrome oxidase I were identified in these populations. They were combined with sequences available in databases, resulting in a total of 407 haplotypes and analyzed together with nine outgroup accessions. A phylogenetic tree was calculated using the maximum likelihood method. The tree showed that all groups that were found in previous studies were also present in our study. Additionally, seven new groups were identified based on the new haplotypes. Most B. tabaci haplotypes grouped based on their geographical origin. Two groups were found to have a worldwide distribution. Our results indicate that our knowledge on the species complex around B. tabaci is still far from complete.     

At least two indigenous species of the Bemisia tabaci complex are present in Brazil

Bemisia tabaci is one of the most important global agricultural insect pests, being a vector of emerging plant viruses such as begomoviruses and criniviruses that cause serious problems in many countries. Although knowledge of the genetic diversity of B. tabaci populations is important for controlling this pest and understanding viral epidemics, limited information is available on this pest in Brazil. A survey was conducted in different locations of São Paulo and Mato Grosso states, and the phylogenetic relationships of B. tabaci individuals from 43 populations sampled from different hosts were analysed based on partial mitochondrial cytochrome oxidase 1 gene (mtCOI) sequences. According to the recently proposed classification of the B. tabaci complex, which employs the 3.5% mtCOI sequence divergence threshold for species demarcation, most of the specimens collected were found to belong to the Middle East‐Asia Minor 1 species, which includes the invasive populations of the commonly known B biotype, within the Africa/Middle East/Asia Minor high‐level group. Three specimens collected from Solanun gilo and Ipomoea sp. were grouped together and could be classified in the New World species that includes the commonly known A biotype. However, six specimens collected from Euphorbia heterophylla, Xanthium cavanillesii and Glycine maxima could not be classified into any of the 28 previously proposed species, although according to the 11% mtCOI sequence divergence threshold, they belong to the New World high‐level group. These specimens were classified into a new recently proposed species named New World 2 that includes populations from Argentina. Middle East‐Asia Minor 1, New World and New World 2 were differentiated by RFLP analysis of the mtCOI gene using TaqI enzyme. Taq I analysis in silico also differentiates these from Mediterranean species, thus making this method a convenient tool to determine population dynamics, especially critical for monitoring the presence of this exotic pest in Brazil.     

烟粉虱成虫空间分布型的研究

对烟粉虱Bemisiatabaci成虫在单株茄子(SolanummelongenaL.)、西瓜(CitrullusvulgarisSchard)和甜瓜(CucumismeloL.)上的分布规律及其空间分布型进行了研究。在植株上中部较幼嫩且已充分展开之叶片上烟粉虱成虫数量最多,成虫呈聚集分布。成虫在黄瓜(CucumissativusL.)上亦呈聚集分布,确定了不同虫口密度情况下的最适抽样数。     

Genetic analysis reveals multiple cryptic invasive species of the hydrozoan genus Cordylophora

Understanding the patterns and dynamics of biological invasions is a crucial prerequisite to predicting and mitigating their potential ecological and economic impacts. Unfortunately, in many cases such understanding is limited not only by ignorance of invasion history, but also by uncertainty surrounding the ecology, physiology, and even systematics of the invasive taxa themselves. The invasive, colonial euryhaline hydroid Cordylophora has invaded multiple regions outside of its native Ponto-Caspian range. However, extensive morphological and ecological plasticity has prevented consensus on both species-level classification within the genus and the environmental conditions conducive to establishment. The goal of this research was to explore the invasive history and species composition of the genus Cordylophora through molecular analyses. We addressed both issues using DNA sequence data from two mitochondrial loci [the small subunit 16S rRNA and cytochrome c oxidase subunit I (COI)] and one nuclear locus (28S large nuclear rRNA), generated from 27 invasive Cordylophora populations collected throughout the global range of the taxon. Phylogenetic analysis and comparisons of genetic distances between populations suggest the presence of multiple cryptic species within the genus. This conclusion is further supported by the observation of significantly different habitat preferences between invasive lineages. Geographic distribution of lineages is consistent with the introduction of multiple lineages to some non-native regions, indicating that repeated introductions may contribute to the current global distribution of Cordylophora. Applying molecular and morphological analyses to additional populations of Cordylophora is likely to assist in clarifying the taxonomy of this genus and in providing a better understanding of the invasive history of this hydroid.     

烟粉虱复合种内共生菌多样性及其生物学意义

烟粉虱复合种Bemisia tabaci（Gennadius） complex内共生菌的种类可分为5类：变形菌门（Proteobacteria）γ变形菌纲的初级内共生菌Candidatus Portiera aleyrodidarum和次级内共生菌,变形菌门α变形菌纲Wolbachia属细菌,衣原体门（Chlamydiae）衣原体纲细菌CandidatusFritschea bemisiae以及拟杆菌门（Bacteroidetes）类噬胞细菌（Cytophaga-like-organism, CLO）。系统发育分析表明,烟粉虱初级内共生菌分为2支,一些初级内共生菌和宿主系统发育并不严格一致; 次级内共生菌分为3支,一些次级内共生菌可能存在水平传播或多次侵入的现象。上述5类内共生菌在烟粉虱不同生物型/地理种群内的分布存在差异。B型烟粉虱可能具有独特的初级、次级内共生菌,并且不含有或很少含有Wolbachia属细菌、衣原体纲细菌Candidatus Fritschea bemisiae以及类噬胞细菌（CLO）。最后探讨了不同内共生菌对烟粉虱宿主的生物学意义及其研究方向。     

A multiple PCR‐LDR assay for identification of two invasive cryptic species of whiteflies Bemisia tabaci

The MEAM1 and MED species of the cryptic species complex Bemisia tabaci are important invasive pests that cause tremendous crop losses worldwide. A rapid and highly reliable molecular technique is necessary to identify these species because they are morphologically indistinguishable. Therefore, a multiple polymerase chain reaction coupled with a ligase detection reaction (PCR‐LDR) that was based on polymorphisms in the mitochondrial cytochrome oxidase I (mtCOI) gene of B. tabaci was developed to distinguish the two cryptic species. An assessment of the method indicated that PCR‐LDR provided high specificity and sensitivity in discriminating MEAM1 (SHB) and MED (SHQ) whiteflies. In field tests, PCR‐LDR genotyping was performed in one 96‐well plate to identify 93 individuals collected from 8 districts in the suburbs of Shanghai. Complete concordance was observed between PCR‐LDR and sequencing methods. The method was used to confirm that MEAM1 and MED were found in two districts, but only the MED was found in the other six districts. PCR‐LDR, which is a transplantable platform, provides an alternative method for species identification of B. tabaci at low cost.     

Ｂ型烟粉虱抗噻虫嗪品系的遗传分化

采用扩增片段长度多态性(Amplified fragment len gth polymorphism, AFLP )技术, 研究了Ｂ型烟粉虱Bemisia tabaci 噻虫嗪 (thiamethoxam ) 抗性品系在DNA分子水平上的遗传分化。实验数据用STATISTICA (4.5版本) 软件进行分析, 遗传距离用UPGMA (非加权对组算术平均值聚类) 方法进行聚类。在聚类树中, 首先是抗性个体与敏感个体聚为2枝, 然后雌、雄个体聚为2枝, 说明抗性品系在DNA分子水平上已存在明显分化。另外, 利用Eco RⅠ+ACT/MseⅠ+CTG引物, 分别在全部抗性个体中扩增出了敏感个体所没有的特异性同一片段和在全部敏感个体中扩增出了抗性个体所没有的特异性同一片段, 经克隆、测序, 这2个片段分别是150 bp 和315 bp。此2个片段, 可作为烟粉虱抗噻虫嗪品系和敏感品系的分子标记而在抗性监测中得以应用。     

田间系统调查表明山东省农区烟粉虱优势种为Q隐种

【目的】为了揭示山东省农区烟粉虱 Bemisia tabaci 隐种的分布情况,对2013年7-8月份期间在该省不同农区采集的烟粉虱隐种组成进行了系统调查研究。【方法】在山东省15市22个地点作物上采集了69份烟粉虱样品,利用mtCOI基因PCR-RFLP方法进行了分子鉴定,并分析了烟粉虱Q隐种在不同寄主作物及地理区域内的分布状况。【结果】22个采集地点中,17个地点的采集样品全部为烟粉虱Q隐种;其他5个地点（潍坊、菏泽、泰安、淄博和临沂）的烟粉虱Q隐种所占比例均大于90%,B隐种比例较低（<10%）。烟粉虱Q隐种比例在5种寄主（茄子、辣椒、番茄、黄瓜和棉花）种群间以及在山东省东部丘陵、中部山地和西部平原地区间均没有显著差异。【结论】2013年田间调查发现烟粉虱Q隐种在山东各地已广泛取代B隐种成为该地区优势种群。     

利用mtCOI PCR-RFLP技术鉴定中国境内九个烟粉虱隐种

烟粉虱Bemisia tabaci (Gennadius)是一个物种复合体, 包括31个以上形态上无法区分的隐种, 其中少数隐种是世界性入侵害虫。目前, 在中国境内分布有2个入侵隐种和13个土著隐种。快速、 高效的鉴别方法对掌握烟粉虱田间发生规律及制定相关防控策略具有重要意义。然而到目前为止, 除了mtCOI基因测序比对外, 尚未有一种简便的方法可以有效地区分多个烟粉虱隐种。本研究采用mtCOI PCR-RFLP技术, 单独或组合使用TaqI, VspI, Van91I, NcoI和FokI这5种限制性内切酶, 酶切烟粉虱mtCOI的PCR扩增片段, 鉴别分布在中国境内的9个烟粉虱隐种。结果表明, 单独使用TaqI酶切, 可鉴别出MEAM1和China 1两个隐种, 使用TaqI+NcoI分步酶切可鉴定出MED和Asia 1两个隐种, 使用TaqI+Van91I分步酶切可鉴定出Asia II 3和Asia II 9两个隐种, 使用TaqI+VspI+FokI分步酶切可鉴定出Asia II 1, Asia II 6和Asia II 7 3个隐种。本研究为高效鉴别中国境内的多个烟粉虱隐种提供了方法。     

Genetic diversity of a Botrytis cinerea cryptic species complex in Hungary

Botrytis cinerea has been described as a species complex containing two cryptic species, referred to as groups I and II. The first B. cinerea group I strains outside of Western Europe were collected in Hungary in 2008 from strawberry and rape plants. Sympatric B. cinerea cryptic species were analyzed using a population genetic approach and phenotypic markers. Statistically significant, but moderate population differentiation was found between the two groups in Hungary. Group I was originally typified by the lack of the transposable elements Boty and Flipper. However, all the Hungarian group I isolates carried the Boty element and one isolate additionally contained Flipper, indicating a much wider genetic variation than previously believed. Vegetative compatibility analyses showed that twelve of the thirteen B. cinerea group I isolates studied belonged to a unique vegetative compatibility group (VCG), but VCGs overlapped between groups. Phenotypic markers such as fenhexamid resistance or asexual spore size were found unsuitable to differentiate between the cryptic species. The results did not confirm the complete separation of the two cryptic species, previously determined with genealogical concordance of the phylogenetic species recognition using multiple gene sequences, and suggest instead the possibility of information exchange between them.