Tabaci Species Complex Research Articles

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of <em>Bemisia tabaci</em>

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

Species-complex diversification and host-plant associations in Bemisia tabaci: A plant-defence, detoxification perspective revealed by RNA-Seq analyses.

Insect–plant associations and their role in diversification are mostly studied in specialists. Here, we aimed to identify macroevolution patterns in the relationships between generalists and their host plants that have the potential to promote diversification. We focused on the Bemisia tabaci species complex containing more than 35 cryptic species. Mechanisms for explaining this impressive diversification have focused so far on allopatric forces that assume a common, broad, host range. We conducted a literature survey which indicated that species in the complex differ in their host range, with only few showing a truly broad one. We then selected six species, representing different phylogenetic groups and documented host ranges. We tested whether differences in the species expression profiles of detoxification genes are shaped more by their phylogenetic relationships or by their ability to successfully utilize multiple hosts, including novel ones. Performance assays divided the six species into two groups of three, one showing higher performance on various hosts than the other (the lower performance group). The same grouping pattern appeared when the species were clustered according to their expression profiles. Only species placed in the lower performance group showed a tendency to lower the expression of multiple genes. Taken together, these findings bring evidence for the existence of a common detoxification “machinery,” shared between species that can perform well on multiple hosts. We raise the possibility that this “machinery” might have played a passive role in the diversification of the complex, by allowing successful migration to new/novel environments, leading, in some cases, to fragmentation and speciation.

Genetic diversity and geographic distribution of the Bemisia tabaci species complex in Bangladesh

Bemisia tabaci (Gennadius) is a species complex consisting of at least 40 cryptic species. Although the genetic diversity of B. tabaci has been studied in various regions, little is known about distribution in Bangladesh, which is covered by the Bengal delta, the largest delta on Earth. We conducted an extensive survey throughout the country and determined the nucleotide sequence of mitochondrial cytochrome c oxidase subunit 1 (COI) from 110 individuals. We then examined phylogenetic relationships. The results identified four cryptic species that expressed distinct interspecific variation but low intraspecific variation. Asia I was the most abundant, both Asia II 1 and Asia II 5 were moderately abundant, and Asia II 10 was found only in the central region. COI sequences of each cryptic species were distinctive and differentiated into many haplotypes. Our study provides important information to better understand the genetic diversity and geographic distribution of cryptic species in Bangladesh and nearby countries.

Review and guide to a future naming system of African Bemisia tabaci species

Once a pest has been correctly identified, its genus and species name can provide a link to valuable indications of its ecology, biology and life history that are critical for developing control strategies. Importantly, this link should exist even when the pest was known under other names (synonyms), or was not considered a pest at all (National Research Council, 1968). Many examples have shown that incorrect identification or classification of a pest has led to fruitless searches for biocontrol agents in the native range, incorrect assignments as disease vectors, and costly, yet misdirected, suppression measures. As new approaches for delimiting species based on molecular information become more widely used, the process of correctly identifying a species has become even more complex. Fortunately, we have good systematic frameworks and nomenclatural systems that are able to cope with these challenges. Here we review challenges associated with classification and identification within the Bemisia tabaci (Gennadius) species complex. These pests and the viruses they transmit have emerged in the past few decades as among the most damaging to food and fibre crops globally (Varma & Malathi, 2003; Pimental et al., 2005; Seal et al., 2006), especially in sub‐Saharan Africa (SSA). The systematics of the B. tabaci species group has been a highly debated topic for years (Boykin, 2014). Putative species are indistinguishable morphologically, so other biological data have been collected to investigate the species in the complex. Based on genetic differences (Colvin et al., 2004; Sseruwagi et al., 2005; Boykin et al., 2007; Boykin et al., 2013; Hsieh et al., 2014) and mating incompatibility (Colvin et al., 2004; Liu et al., 2007; Xu et al., 2010), B. tabaci is now recognized as a species complex that consists of at least 34 putative species (Boykin et al., 2012). The rapid discovery of significant species diversity has led to many changes in the informal names used over the last 10 years (Boykin, 2014), creating confusion in the literature.

Kinetics of-Amylase activity in Asia-I and Asia II-1 Genetic groups of Whitefly, Bemisia Tabaci (Hemiptera: Aleyrodidae)

Whitefly, Bemisia tabaci (Gennadius.) has drawn global attention in recent past with its wide distribution across the world as it causes severe damage to crops directly by sucking the plant sap and indirectly as vector of several viral diseases. This pest is considered as a species complex and Asia represents the largest diversity with the distribution of about 16 out of 35 genetic groups recorded so far. Investigations were carried out on kinetics of a key digestive enzyme, a-amylase in adults of isogenic female lines of B. tabaci genetic groups Asia I and Asia II-1. Asia I has shown the higher specific activity of 0.267±0.007 μmol/ml/mg compared to that of 0.209±0.009 μmol/ml/mg in Asia II-1. However, kinetics of a-amylases revealed the high affinity of this enzyme in Asia II-1 with the least Km value of 0.03372±0.001 compared to that of 0.0353 ±0.002 in Asia I genetic group. Highest enzyme activities were recorded at 35° C in both the genetic groups of B. tabaci. Kinetics study of metabolically important digestive enzymes would improve our understanding on the metabolic capabilities of B. tabaci species complex.

Updated mtCOI reference dataset for the Bemisia tabaci species complex.

Members of the whitefly Bemisia tabaci species complex cause millions of dollars of damage globally and are considered one of the world's most invasive species. They are capable of causing extensive damage to major vegetable, grain legume and fiber crops. All member of the species complex are morphologically identical therefore, data from the partial mitochondrial cytochrome oxidase subunit I (mtCOI) gene sequence has been used to identify the various species. The current reference dataset that is widely used is found on the CSIRO data portal. However, the reference set stored on the CSIRO data does not include newly added sequences (2013-2017), therefore an updated reference dataset is needed. All mtCOI data for the Bemisia tabaci species complex were downloaded on 22 May 2017 from GenBank and after quality checking, a dataset of 1,071 unique sequences and 696 base pairs was generated (https://doi.org/10.6084/m9.figshare.5437420.v1).

Phylogenetic Relationships among Whiteflies in the Bemisia tabaci (Gennadius) Species Complex from Major Cassava Growing Areas in Kenya.

Whiteflies, Bemisia tabaci (Gennadius) are major insect pests that affect many crops such as cassava, tomato, beans, cotton, cucurbits, potato, sweet potato, and ornamental crops. Bemisia tabaci transmits viral diseases, namely cassava mosaic and cassava brown streak diseases, which are the main constraints to cassava production, causing huge losses to many small-scale farmers. The aim of this work was to determine the phylogenetic relationships among Bemisia tabaci species in major cassava growing areas of Kenya. Surveys were carried out between 2013 and 2015 in major cassava growing areas (Western, Nyanza, Eastern, and Coast regions), for cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Mitochondrial cytochrome oxidase I (mtCOI-DNA) was used to determine the genetic diversity of B. tabaci. Phylogenetic trees were constructed using Bayesian methods to understand the genetic diversity across the study regions. Phylogenetic analysis revealed two B. tabaci species present in Kenya, sub-Saharan Africa 1 and 2 comprising five distinct clades (A–E) with percent sequence similarity ranging from 97.7 % to 99.5%. Clades B, C, D, and E are predominantly distributed in the Western and Nyanza regions of Kenya whereas clade B is dominantly found along the coast, the eastern region, and parts of Nyanza. Our B. tabaci clade A groups with sub-Saharan Africa 2-(SSA2) recorded a percent sequence similarity of 99.5%. In this study, we also report the identification of SSA2 after a 15 year absence in Kenya. The SSA2 species associated with CMD has been found in the Western region of Kenya bordering Uganda. More information is needed to determine if these species are differentially involved in the epidemiology of the cassava viruses.

Infectivity of Tomato yellow leaf curl virus isolated from imported tomato fruit in Estonia

There is a risk that Tomato yellow leaf curl virus (TYLCV) and its vector, whiteflies of the Bemisia tabaci species complex, will become established in greenhouses in temperate regions of the world ...

Identification of the B, Q, and native Brazilian biotypes of the Bemisia tabaci species complex using Scar markers

Abstract: The objective of this work was to develop sequence-characterized amplified region (Scar) markers to identify the B, Q, and native Brazilian biotypes of the sweet potato whitefly [Bemisia tabaci (Hemiptera: Aleyrodidae)]. Random amplified polymorphic DNA (RAPD) amplification products, exclusive to the B and Brazilian biotypes, were selected after the analysis of 12,000 samples, in order to design a specific Scar primer set. The BT-B1 and BT-B3 Scar markers, used to detect the B biotype, produced PCR fragments of 850 and 582 bp, respectively. The BT-BR1 Scar marker, used to identify the Brazilian biotype, produced a PCR fragment of 700 bp. The Scar markers were tested against the Q biotype, and a flowchart was proposed to indicate the decision steps to use these primers, in order to correctly discriminate the biotypes. This procedure allowed to identify the biotypes that occur in field samples, such as the B biotype. The used set of primers allowed to discriminate the B, Q, and native Brazilian biotypes of B. tabaci. These primers can be successfully used to identify the B biotype of B. tabaci from field samples, showing only one specific biotype present in all cultures.

Differential tolerance capacity to unfavourable low and high temperatures between two invasive whiteflies.

Thermal response and tolerance to ambient temperature play important roles in determining the geographic distribution and seasonal abundance of insects. We examined the survival and performance, as well as expression of three heat shock protein related genes, of two species of invasive whiteflies, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED), of the Bemisia tabaci species complex following exposure to a range of low and high temperatures. Our data demonstrated that the MED species was more tolerant to high temperatures than the MEAM1 species, especially in the adult stage, and this difference in thermal responses may be related to the heat shock protein related genes hsp90 and hsp70. These findings may assist in understanding and predicting the distribution and abundance of the two invasive whiteflies in the field.

Culturable bacteria in Asia I and Asia II genetic groups of whiteflyBemisia tabaci(Gennadius) species complex

This study enumerats the cultivable gut bacteria in the developmental stages of whitefly (Bemisia tabaci species complex) populations belonging to the Asia II-1 and Asia I genetic groups. The gut bacterial density was assessed by growing them in growth media for all the developmental stages viz., egg, four nymphal instars and adult. The highest bacterial density was observed with adults in both Asia II-1 (2.09×106± 0.19 cfu/ml) and Asia I (6.4×105±0.146 cfu/ml genetic groups). Analysis of morphological and biochemical characteristics revealed the presence of Pseudomonas and Sphingomonas. Specific association of Proteus spp., and Acinetobacter respectively, with Asia II-1 and Asia I genetic group was also noticed. The use of diverse growth media and biochemical kits for isolation and identification of culturable gut bacteria are also discussed.

Temporal changes of symbiont density and host fitness after rifampicin treatment in a whitefly of the Bemisia tabaci species complex.

Microbial symbionts are essential or important partners to phloem-feeding insects. Antibiotics have been used to selectively eliminate symbionts from their host insects and establish host lines with or without certain symbionts for investigating functions of the symbionts. In this study, using the antibiotic rifampicin we attempted to selectively eliminate certain symbionts from a population of the Middle East-Asia Minor 1 whitefly of the Bemisia tabaci species complex, which harbors the primary symbiont "Candidatus Portiera aleyrodidarum" and two secondary symbionts "Candidatus Hamiltonella defensa" and Rickettsia. Neither the primary nor the secondary symbionts were completely depleted in the adults (F0) that fed for 48 h on a diet treated with rifampicin at concentrations of 1-100 μg/mL. However, both the primary and secondary symbionts were nearly completely depleted in the offspring (F1) of the rifampicin-treated adults. Although the F1 adults produced some eggs (F2), most of the eggs failed to hatch and none of them reached the second instar, and consequently the rifampicin-treated whitefly colony vanished at the F2 generation. Interestingly, quantitative polymerase chain reaction assays showed that in the rifampicin-treated whiteflies, the density of the primary symbiont was reduced at an obviously slower pace than the secondary symbionts. Mating experiments between rifampicin-treated and untreated adults demonstrated that the negative effects of rifampicin on host fitness were expressed when the females were treated by the antibiotic, and whether males were treated or not by the antibiotic had little contribution to the negative effects. These observations indicate that with this whitefly population it is not feasible to selectively eliminate the secondary symbionts using rifampicin without affecting the primary symbiont and establish host lines for experimental studies. However, the extinction of the whitefly colony at the second generation after rifampicin treatment indicates the potential of the antibiotic as a control agent of the whitefly pest.

An Updated List of Parasitoid Hymenoptera Reared from theBemisia tabaciSpecies Complex (Hemiptera: Aleyrodidae)

An updated list to the world fauna of parasitoid Hymenoptera reared from members in the Bemisia tabaci species complex (Hemiptera: Aleyrodidae) is provided. In total, 112 parasitoid species in 5 families and 7 genera are tabulated along with their global distributions. Pertinent references are given to aid in the accurate identification of these minute insects. We also reviewed published host-genera associations and consider some dubious, possibly due to contamination of rearing vessels with non-target insects or to misidentification of the host whitefly or parasitoid species.

Morphometric Analysis of Three Putative Species ofBemisia tabaci(Hemiptera: Aleyrodidae) Species Complex from India

ABSTRACT The Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a pest of agricultural and horticultural crops. The B. tabaci species complex consists of 36 morphologically indistinguishable putative species. This study evaluates the morphometric variations of developmental stages, puparia, and adults in three putative species of B. tabaci occurring in India. The genetic identity of these studied populations were confirmed by mtCO1 analysis and revealed that the population from Amravati, Ludhiana, and Delhi were clustered with Asia I, Asia II1, and Asia II7 putative species, respectively. The morphological comparisons showed that fourth instar and adult of Asia-II1 was comparatively larger than Asia-I and Asia-II7. The positioning of sensorial cone on antennal segment 7 is much apart, and away from the sensorium in Asia-II1 while these are comparatively adjacent in Asia-I and Asia-II7 for both the sexes. The multivariate statistical analyses reveal that 31 measurements in puparia, 23 of male and 22 of...

Multiple Gene Markers to Understand Genetic Diversity in theBemisia tabaci(Hemiptera: Aleyrodidae) Species Complex

Abstract The analysis of sequence variation in the mitochondrial and ribosomal regions has been shown to provide an efficient method for the identification of species in a wide range of animal taxa. In order to assess its effectiveness in the discrimination of genetic groups of Bemisia tabaci (Gennadius) species complex, populations from ten cotton cultivars were analyzed. MtCOI, and ITS1 sequences were evaluated to find out the genetic diversity through phylogenetic methods, viz., maximum likelihood and network neighbor-net analysis. Analysis of mtCOI-1 or barcoding region did not reveal significant variation. On the contrary, the mtCOI-II region revealed the presence of three B. tabaci genetic groups or subgroups corresponded with the global data. However, the ITS1 region could not discriminate these groups of the species complex. Our results indicate that more than the barcoding and ITS1 regions, mtCOI-II region or 3′ end of the gene will be more appropriate for the identification of variations among t...

Regional co-occurrence between distinct Bemisia tabaci species in Tunisia with new insights into the role of host plants

Among the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex, the Middle East–Asia Minor I (MEAMI) and the Mediterranean (Med) species are the most invasive and widespread agricultural pests worldwide. Currently, only Tunisia and a few other countries are reported to still “host” these two competing species. The objective was thus to improve our knowledge on the factors, particularly the host plant, which contribute to this unusual situation. To that end, we analyzed 47 samples collected from protected and outdoor plants (ten vegetables and ornamentals) in the two main crop-producing regions of Tunisia to (i) better document the co-occurrence of several B. tabaci species and populations, and (ii) confirm the role of the host plant in the distribution pattern of each species. B. tabaci specimens were identified using at least two molecular diagnostic tests. Taken together, the tests confirmed the equivalent prevalence of Med and MEAMI species and, for the first time, the presence of some Sub-Saharan Africa 2 B. tabaci, in Tunisia. The regional co-occurrence between Med and MEAM1 was based on spatial and host-plant partitioning. Our results indicate that Med species are closely associated with ornamentals and MEAM1 with vegetables. Med displayed a higher level of genetic diversity than MEAM1, and another Med specimen characterized by a mtCOI haplotype which perfectly matched the ancestral Gennadius specimen was discovered. The role of host plant type and possible associated factors in the distribution patterns of MEAM1 and Med is discussed in the context of the co-occurrence of cryptic species.

Scientific Opinion on the pest categorisation ofTomato yellow leaf curl virusand related viruses causing tomato yellow leaf curl disease in Europe

The Panel on Plant Health performed for the European Union (EU) territory a pest categorisation of Tomato yellow leaf curl virus (TYLCV) and three related viruses, Tomato yellow leaf curl Sardinia virus (TYLCSV), Tomato yellow leaf curl Axarquia virus (TYLCAxV) and Tomato yellow leaf curl Malaga virus (TYLCMaV), which collectively cause the tomato yellow leaf curl disease (TYLCD) in Europe. The viruses are well-defined species of the genus Begomovirus, are exclusively transmitted by members of the Bemisia tabaci species complex and have tomato, as well as a few other crops or weeds, as their hosts. TYLCV is listed on tomato plants for planting, other than seeds, in Annex IIAII of Directive 2000/29/EC. While establishment and local spread rely on the Bemisia vector, the viruses can also be disseminated over long distances by movement of infected plants for planting or by consignments of non-host plants carrying viruliferous whiteflies. Establishment outdoors and spread are limited to regions with ecoclimatic conditions suitable for the establishment of vector populations in the open. Outbreaks can nevertheless occur in other regions under protected cultivation conditions. Because of the very high potential impact of TYLCD, tomato production in affected regions requires intensive crop management efforts to reduce impact. TYLCV appears to be present in almost all EU regions with suitable ecoclimatic conditions for its establishment in open fields, while the other three viruses do not appear to have reached their full establishment potential. All four viruses are absent from other regions of the EU but have the potential to cause temporary outbreaks there. © European Food Safety Authority, 2014

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

AbstractThe 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.

A new record for Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) species complex of Turkey

AbstractIn this study, species complex of Turkish Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) populations was determined by PCR‐based DNA analysis. According to phylogenetic analyses, the B. tabaci samples have been identified within three generic groups. A major part of the samples belonged to two invasive species, either Middle East–Asia Minor 1 (MEAM1) or Mediterranean (MED). In addition to these two invasive species, several samples collected from greenhouses and cotton fields have been found to be related to Middle East–Asia Minor 2 (MEAM2), which is the first record of Turkish B. tabaci species complex.