On species delimitation, hybridization and population structure of cassava whitefly in Africa.

The Bemisia cassava whitefly complex includes species that cause severe crop damage through vectoring cassava viruses in eastern Africa. Currently, this whitefly complex is divided into species and subgroups (SG) based on very limited molecular markers that do not allow clear definition of species and population structure. Based on 14,358 genome-wide SNPs from 62 Bemisia cassava whitefly individuals belonging to sub-Saharan African species (SSA1, SSA2 and SSA4), and using a well-curated mtCOI gene database, we show clear incongruities in previous taxonomic approaches underpinned by effects from pseudogenes. We show that the SSA4 species is nested within SSA2, and that populations of the SSA1 species comprise well-defined south-eastern (Madagascar, Tanzania) and north-western (Nigeria, Democratic Republic of Congo, Burundi) putative sub-species. Signatures of allopatric incipient speciation, and the presence of a 'hybrid zone' separating the two putative sub-species were also detected. These findings provide insights into the evolution and molecular ecology of a highly cryptic hemipteran insect complex in African, and allow the systematic use of genomic data to be incorporated in the development of management strategies for this cassava pest.

The potential geographical distribution and phenology of Bemisia tabaci Middle East/Asia Minor 1, considering irrigation and glasshouse production.

The Bemisia tabaci species complex is one of the most important pests of open field and protected cropping globally. Within this complex, one species (Middle East Asia Minor 1, B. tabaci MEAM1, formerly biotype B) has been especially problematic, invading widely and spreading a large variety of plant pathogens, and developing broad spectrum pesticide resistance. Here, we fit a CLIMEX model to the distribution records of B. tabaci MEAM1, using experimental observations to calibrate its temperature responses. In fitting the model, we consider the effects of irrigation and glasshouses in extending its potential range. The validated niche model estimates its potential distribution as being considerably broader than its present known distribution, especially in the Americas, Africa and Asia. The potential distribution of the fitted model encompasses the known distribution of B. tabaci sensu lato, highlighting the magnitude of the threat posed globally by this invasive pest species complex and the viruses it vectors to open field and protected agriculture.

Testing mate recognition through reciprocal crosses of two native populations of the whitefly Bemisia tabaci (Gennadius) in Australia.

Bemisia tabaci (Gennadius) represents a relatively large cryptic species complex. Australia has at least two native populations of B. tabaci sensu lato and these were first found on different host plants in different parts of Australia. The species status of these populations has not been resolved, although their mitochondrial sequences differ by 3.82-4.20%. We addressed the question of whether these AUSI and AUSII B. tabaci populations are distinct species. We used reciprocal cross-mating tests to establish whether the insects from these different populations recognize one another as potential mating partners. The results show that the two native Australian populations of B. tabaci have a mating sequence with four phases, each of which is described. Not all pairs in the control crosses mated and the frequency of mating differed across them. Some pairs in the AUSI-M × AUSII-F did mate (15%) and did produce female progeny, but the frequency was extremely low relative to controls. Microsatellite genotyping of the female progeny produced in the crosses showed these matings were successful. None of the AUSII-M × AUSI-F crosses mated although some of the males did search for females. These results demonstrate the critical role of the mate recognition process and the need to assess this directly in cross-mating tests if the species status of different populations is to be tested realistically. In short, AUSI and AUSII B. tabaci populations are distinct species because the individual males and females do not recognize individuals of the alternative population as potential mating partners.

Novel molecular approach to define pest species status and tritrophic interactions from historical Bemisia specimens.

Museum specimens represent valuable genomic resources for understanding host-endosymbiont/parasitoid evolutionary relationships, resolving species complexes and nomenclatural problems. However, museum collections suffer DNA degradation, making them challenging for molecular-based studies. Here, the mitogenomes of a single 1912 Sri Lankan Bemisia emiliae cotype puparium, and of a 1942 Japanese Bemisia puparium are characterised using a Next-Generation Sequencing approach. Whiteflies are small sap-sucking insects including B. tabaci pest species complex. Bemisia emiliae's draft mitogenome showed a high degree of homology with published B. tabaci mitogenomes, and exhibited 98-100% partial mitochondrial DNA Cytochrome Oxidase I (mtCOI) gene identity with the B. tabaci species known as Asia II-7. The partial mtCOI gene of the Japanese specimen shared 99% sequence identity with the Bemisia 'JpL' genetic group. Metagenomic analysis identified bacterial sequences in both Bemisia specimens, while hymenopteran sequences were also identified in the Japanese Bemisia puparium, including complete mtCOI and rRNA genes, and various partial mtDNA genes. At 88-90% mtCOI sequence identity to Aphelinidae wasps, we concluded that the 1942 Bemisia nymph was parasitized by an Eretmocerus parasitoid wasp. Our approach enables the characterisation of genomes and associated metagenomic communities of museum specimens using 1.5 ng gDNA, and to infer historical tritrophic relationships in Bemisia whiteflies.

Complete mitochondrial DNA genome of Bemisia tabaci cryptic pest species complex Asia I (Hemiptera: Aleyrodidae).

The complete length of the Asia I member of the Bemisia tabaci species complex mitochondrial DNA genome (mitogenome) is 15,210 bp (GenBank accession no. KJ778614) with an A-T biased nucleotide composition (A: 32.7%; T: 42.4%; G: 14.0%; C: 10.8%). The mitogenome consists of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNA (rRNAs) and a 467 bp putative control region which also includes the A+T rich repeat region. All PCGs have an ATA (n = 8) or ATG (n = 5) start codon. Gene synteny of Asia I is overall similar to B. afer and two other members of the B. tabaci species complex Mediterranean and New World 1, and contains the tRNA-Ser2 located between the Cytb and ND1 genes found in Mediterranean and New World 1, but which is absent in B. afer. The orientation of the tRNA-Arg in Asia I is on the "plus" strand and differed from Mediterranean which is found on the "minus" strand. The Asia I mitogenome size is currently ranked the second smallest after B. afer (14,968 bp) followed by New World 1 (15,322 bp) and Mediterranean (15,632 bp).

Reproductive incompatibility among genetic groups of Bemisia tabaci supports the proposition that the whitefly is a cryptic species complex.

The worldwide distribution and extensive genetic diversity of the whitefly Bemisia tabaci has long been recognized. However, whether B. tabaci is a complex species or a species complex has been a subject of debate. Recent phylogenetic analyses suggest that B. tabaci is a cryptic species complex composed of at least 24 morphologically indistinguishable species. Here, we conducted crossing experiments and demonstrated reproductive incompatibility among three of the 24 putative species. Our data and those of previously reported crossing experiments among various putative species of B. tabaci were collated to reveal the pattern of reproductive isolation. The combined results provide strong support to the proposition that B. tabaci is a cryptic species complex.

Post-release evaluation of Eretmocerus hayati Zolnerowich and Rose in Australia.

Bemisia tabaci biotype B is a significant pest of agriculture world-wide. It was first detected in Australia in 1994. Assessments of the potential of parasitoids already present in Australia to control this pest indicated that two species of Eretmocerus and 11 species of Encarsia were present, but they did not exert sufficient control with a combined average of 5.0+/-0.3% apparent parasitism of 4th instars. Further, only 25% of samples containing biotype B had parasitised individuals present. The surveys also identified that fewer B biotype were being parasitised compared with the Australian indigenous biotype. Overall, Er. mundus was the most abundant parasitoid prior to the introduction. Previous research indicated that Er. hayati offered the best prospects for Australia and, in October 2004, the first releases were made. Since then, levels of apparent parasitism have averaged 29.3+/-0.1% of 4th instars with only 24% of collections having no parasitism present. Eretmocerus hayati contributed 85% of the overall apparent parasitism. In addition, host plants of the whitefly with low or no parasitism prior to the release have had an order of magnitude increase in levels of parasitism. This study covers the establishment of the case to introduce Er. hayati and the post-release establishment period November 2004-March 2008.

Asymmetric mating interactions drive widespread invasion and displacement in a whitefly.

The role of behavioral mechanisms in animal invasions is poorly understood. We show that asymmetric mating interactions between closely related but previously allopatric genetic groups of the whitefly Bemisia tabaci, a haplodiploid species, have been a driving force contributing to widespread invasion and displacement by alien populations. We conducted long-term field surveys, caged population experiments, and detailed behavioral observations in Zhejiang, China, and Queensland, Australia, to investigate the invasion process and its underlying behavioral mechanisms. During invasion and displacement, we found increased frequency of copulation leading to increased production of female progeny among the invader, as well as reduced copulation and female production in the indigenous genetic groups. Such asymmetric mating interactions may be critical to determining the capacity of a haplodiploid invader and the consequences for its closely related indigenous organisms.

Genetic structure of the whitefly Bemisia tabaci in the Asia-Pacific region revealed using microsatellite markers.

Bemisia tabaci (Hemiptera: Aleyrodidae) is a haplo-diploid species of sap-feeding insect belonging to the group of insects commonly known as whiteflies. From earlier analyses of mitochondrial and ribosomal markers it has been concluded that in the Asia-Pacific region there were three major indigenous races as well as a large collection of genotypes with no clear association with any race. This new study uses 15 microsatellite loci and demonstrates that the indigenous Asia-Pacific genotypes can be split into six genetic populations with little or no gene flow between them. These bare only superficial similarity to the mitochondrial and ribosomal defined races. Moreover, four of the six can be further split into two subpopulations that again show little evidence gene flow between them. While the patterns reflect a strong geographical structure, physical barriers alone cannot explain all the observed structure. Differential host-plant utilization explained some of the substructure, but could not explain the overall structure. The roles of mating interference and Wolbachia in developing the genetic structure are considered. The lack of gene flow between genetic populations and some subpopulations further suggests that the barriers were either sufficiently impermeable to immigration or that reproductive isolation and competitive interactions were sufficiently strong to prevent gene flow. If the latter is the case, it suggests that there may be as many as 10 morphologically indistinguishable species indigenous to the Asia-Pacific region.

Development, survivorship and reproduction of Eretmocerus sp. nr furuhashii (Hymenoptera: Aphelinidae) parasitizing Bemisia tabaci (Hemiptera: Aleyrodidae) on glabrous and non-glabrous host plants.

The developmental, survivorship and reproductive biology of Eretmocerus sp. nr furuhashii Rose & Zolnerowich parasitizing Bemisia tabaci (Gennadius) biotype B on tomato, cucumber, eggplant and collard at 26+/-0.5 degrees C was studied. The mean longevity ranged from 6.5 days on tomato to 8.1 days on collard. The mean lifetime fecundities on tomato, cucumber, eggplant and collard were 35.4, 37.3, 41.4 and 46.4 eggs, whereas the mean lifetime fertility was 20.2, 22.7, 28.3 and 33.6 offspring, respectively. Developmental time was longest on cucumber (17.1 days) and shortest on collard (15.9 days). Survival rates varied significantly across host plants. Sex ratio (female symbol:male symbol) varied from 1.71 to 1.93 across the four hosts. The intrinsic rate of increase (rm) was highest (0.157) on collard and lowest on tomato (0.133). Mean generation time (R0) ranged from 14.7 to 20.9 days. All the data demonstrated that the reproductive success and overall performance of E. sp. nr furuhashii increased as the density of leaf hairs declined. As leaf hairs play a key role in determining efficacy, it is unlikely that effective biological control of B. tabaci using E. sp. nr furuhashii will be achieved on non-glabrous crop varieties.

Bemisia argentifolii is a race of B. tabaci (Hemiptera: Aleyrodidae): the molecular genetic differentiation of B. tabaci populations around the world.

The phylogenetic relationships between genotypes of Bemisia tabaci were compared using ITS1 and CO1 nucleotide sequences. Phylogenetic and minimum spanning network analyses identified six major races, Asia, Bali, Australia, sub-Saharan Africa, Mediterranean/Asia Minor/Africa and New World as well as a large collection of genotypes from the Asia region with no strong association with any of the races. The term race is based on its usage in Mallet (2001). Mating incompatibility occurs between some races. There is insufficient data to raise races to species status, but the data supports the recognition of the six races and an unresolved core of ungrouped genotypes under the single Bemisia tabaci (Gennadius) species name. To clarify the identity of the race to which the B. tabaci under investigation is known, the following nomenclature is suggested, B. tabaci (Asia), B. tabaci (Bali), B. tabaci (Australia) B. tabaci (sub-Saharan Africa), B. tabaci (Mediterranean/Asia Minor/Africa) and B. tabaci (New World). Further, there is insufficient molecular or biological data to support the separation of B. tabaci and B. argentifolii Bellows & Perring and its use should be discontinued.

Encarsia species (Hymenoptera: Aphelinidae) of Australia and the Pacific Islands attacking Bemisia tabaci and Trialeurodes vaporariorum (Hemiptera: Aleyrodidae)--a pictorial key and descriptions of four new species.

After the recent introduction of the pest whitefly Bemisia tabaci (Gennadius) biotype B into Australia, research was undertaken to study the parasitoids of the long established native B. tabaci and Trialeurodes vaporariorum (Westwood). The genus Encarsia contains species which are important biological control agents of whiteflies and hard scales. The taxonomy of the Encarsia species attacking B. tabaci and T. vaporariorum in Australia and the Pacific Islands is revised. DNA sequencing of the 28S D2 ribosomal DNA was used to characterize species. Sixteen species are recognized, with 12 occurring in Australia, eight in the Pacific region, and four in both regions. All except one species (E. formosa Gahan) are new records for Australia. Four species are described as new from Australia: E. accenta Schmidt & Naumann sp. n., E. adusta Schmidt & Naumann sp. n., E. oakeyensis Schmidt & Naumann sp. n., and E. ustulata Schmidt & Naumann sp. n. Diagnostic descriptions are given for all species and each species is illustrated. A pictorial key is provided to allow the identification of species by non-specialists.

Preliminary phylogeny of Encarsia Förster (Hymenoptera: Aphelinidae) based on morphology and 28S rDNA.

Species of Encarsia Förster (Hymenoptera: Aphelinidae, Coccophaginae) are economically important for the biological control of whitefly and armored scale pests (Hemiptera: Aleyrodidae, Diaspididae). Whereas some regional keys for identification of Encarsia species are now available, few studies have addressed relationships within this diverse and cosmopolitan genus because of unreliable morphological data. Nuclear sequences of the D2 expansion region of 28S rDNA were determined from 67 strains of 24 species representing 10 species groups of Encarsia, 2 strains of Encarsiella noyesi Hayat, and 1 strain of Coccophagoides fuscipennis Girault. Analysis of molecular data alone and combined with morphological data resolves many nodes not resolved by morphology alone and offer insights into which morphological characters are useful for supporting group relationships. All analyses that include molecular data reveal Encarsia to be paraphyletic with respect to Encarsiella. If monophyly of Encarsia is constrained, the relationships are the same but with a different root within Encarsia, and these trees are presented as an alternate hypothesis. The luteola and strenua species groups are shown by both morphological and molecular data to be monophyletic, whereas the inaron group, the E. nigricephala + luteola group, and the E. quericola + strenua group are supported only by molecular data. The aurantii and parvella species groups are not supported in any of the analyses. The utility of morphological characters for defining species group relationships is discussed.

Mating interactions between two biotypes of the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) in Australia.

The biological consequences of mating interactions between indigenous and exotic biotypes of Bemisia tabaci (Gennadius) in Australia were studied using a combination of field and laboratory experiments. The key results of the interaction between the B and eastern Australian biotypes were reduced population increase, a marked increase in the proportion of male progeny, fewer eggs produced by females paired with males of different biotype and no difference in the numbers of eggs per unmated female and females paired with males of the same biotype. In addition, there was no change in the proportion of eggs hatching, mixed biotype pairs spent more time courting than single biotype pairs and a low level of hybridization in field cages and small containers was observed. These observations suggest three possibilities. The first is the 'distracting male hypothesis' in which mating pairs made up of different biotypes apportion more time to courtship and less time to egg laying than single biotype pairs. The second invokes the 'single-locus complementary sex determination model' in which the production of non-viable diploid male zygotes may explain the reduction in eggs laid. The third is cytoplasmic incompatibility between biotypes caused by Wolbachia. The results also suggest that the geographical distribution of clusters of related biotypes both overseas and in Australia may be explained by between-biotype interactions leading to the formation of parapatric populations.

Phylogenetic relationships of world populations of Bemisia tabaci (Gennadius) using ribosomal ITS1.

A phylogeny of Bemisia tabaci is presented based on the ITS1 region of ribosomal DNA. The monophyly of each biotype of Bemisia is supported, and a strong biogeographic pattern is evident in the data. Populations from the Americas (including the A biotype) form a clade sister to a clade comprising the silverleafing or B biotype and a nonsilverleafing biotype from the North Africa/Mediterranean region. Polymorphisms in rDNA suggest that silverleafing is a recent phenomenon, while the nonsilverleafing form is the ancestral or plesiomorphic state. Based on this phylogeny, if B. argentifolii is accepted as a separate species then one would have to review the taxonomic status of all biotypes of B. tabaci. In particular, a new name clearly would be needed for an Egypt/Spain/Sudan/Nigeria clade, and the monophyly of the haplotypes remaining in B. tabaci would be open to serious doubt. To make new species assignments in these circumstances seems premature. The phylogenetic relationships of the different populations of B. tabaci and the origins of effective natural enemies of the B biotype suggest that knowing the origin of the B biotype is not essential to finding effective agents and supports the notion that crop management is the key aspect to control.

Genetic variation within a parthenogenetic lineage.

A clone of the grain aphid Sitobion avenae F. was maintained parthenogenetically over thirty-two generations (n = 344) in a constant environment: a new generation being set up by a female selected at random from the preceding generation. Genomic DNA from individual aphids was screened for genetic stability using RAPD-PCR with a previously tested ten-mer primer. A putative germ-line mutation was noted in generation 14 and somatic mutations were noted in generations 12, 25, 27 and 29. There were no differences in the RAPD-PCR profiles of winged and wingless morphs and samples tested for symbiotic DNA. No endoblotic fungal organism was associated with the clone. Southern blotting and hybridization studies indicated that band additions were of aphid origin. However, the RAPD-PCR profiles of the germ-line and somatic mutation samples were unique from other aphid clones cultured during the experimental period. This paper documents discernible genetic changes occurring within an animal clonal lineage over time and Impacts on the consequences this may have for clonal systems.

Genetic structure of an aphid studied using microsatellites: cyclic parthenogenesis, differentiated lineages and host specialization.

In a previous study, samples of the grain aphid Sitobion avenae (F.) were collected from wheat and adjacent cocksfoot hosts in a population thought to be primarily parthenogenetic, and DNA from individual aphids was analysed with a multilocus technique. Here we have applied single-locus microsatellites and a mitochondrial DNA marker to a subset of the same DNA extracts, and have made several additional inferences about important genetic and population processes in S. avenae. Microsatellite analysis indicated very high levels of genic and genotypic variation. S. avenae fell into three genotypic groups inferred to be almost noninterbreeding, while analysis of linkage and Hardy-Weinberg equilibria suggested high levels of sexual recombination within each genotypic group. Host specialization was evident: one lineage was found only on wheat, and one (bearing many alleles inferred to be introgressed from the blackberry-grass aphid S. fragariae (Walker)) was found only on cocksfoot. The third group of interrelated genotypes was found commonly on both hosts. Although most genotypes were found only once, some were much more numerous in the sample than expected from the frequency of the alleles they contained. This, and rapid temporal changes in genotypic composition of samples, indicates strong selective differences between genotypes and lineages. In the major genotypic group, the commonest genotypes were significantly more homozygous than were rare ones: thus these data may help to explain the frequent observation of homozygous excess in aphid allozymes. The genotype group showing S. avenae-like as well as S. fragariae-like alleles also carried S. fragariae-like mitochondrial DNA in at least 25/31 cases, indicating gender-asymmetrical hybridization.

Spatial and temporal genetic variation in British field populations of the grain aphid Sitobion avenae (F.) (Hemiptera: Aphididae) studied using RAPD-PCR.

The grain aphid Sitobion avenae (F.) was collected from winter wheat and adjacent cocksfoot grass at two locations in southern England and at four times in the year (April-July). Genetic variation between individual aphids was then investigated using random amplified polymorphic DNA polymerase chain reaction. Individuals caught in wheat and cocksfoot during April provided very different and highly diagnostic banding patterns that were independent of location. This host-based genetic differentiability was less evident as the season progressed, largely as a result of genetic drift and local movement between adjacent host species, which appeared to be predominantely in the direction from cocksfoot to wheat. The diversity of putative clones fell significantly, the mean number of individuals per clone rose and clones became more exclusively associated with certain sites which suggests that long-distance migration may have less of a homogenizing effect than hitherto thought for this species.

Geographic and microgeographic genetic differentiation in two aphid species over southern England using the multilocus (GATA)4 probe.

Samples of the grain aphid Sitobion avenae (F.) and the rose-grain aphid Metopolophium dirhodum (Walker) were collected in late March from wheat fields and adjacent road-side grasses at a number of locations in southern England. Unparasitized aphids were DNA fingerprinted using the multilocus (GATA)4 probe. Over all locations, the fingerprints of individual S. avenae caught in wheat had lower overall average distances of band migration (ADBM) and shared a higher proportion of bands, than fingerprints of individuals caught in adjacent road-side grasses. The ADBM of fingerprints of S. avenae collected on road-side grasses altered significantly with geographical location, while the ADBM of fingerprints of S. avenae caught on wheat did not. A comparison of the fingerprints of individual M. dirhodum caught in wheat and neighbouring road-side grasses did not reveal any genetic differentiation. Fingerprints of M. dirhodum that were caught in the same host type did however, show significant variation in ADBM between different locations. With both S. avenae and M. dirhodum, spatial autocorrelation revealed that locations that were close together were no more likely to have individuals with similar ADBM than locations that were far apart. Our results suggest that (i) particular clones of S. avenae prefer to colonize wheat, and/or that (ii) particular clones of S. avenae perform better on wheat than other clones. It is unclear why M. dirhodum did not show any genetic structuring according to host type, but this species appears to engage in sexual reproduction much more frequently than S. avenae in southern England.(ABSTRACT TRUNCATED AT 250 WORDS)

An investigation of the differential performance of clones of the aphid Sitobion avenae on two host species.

Individuals of the grain aphid Sitobion avenae were collected from wheat and cocksfoot stands around Hampshire, UK, during March and April 1994. Eight wheat and eight cocksfoot aphid clones were chosen on the basis of readily distinguishable RAPD-PCR fingerprint profiles. The performances (weight, fecundity and survival) of successive generations of each of these clones were then carefully monitored in the laboratory as new generations of aphids were transferred either to winter wheat or to cocksfoot in planned sequences. Even those clones that were originally caught on the same host showed significant variability in performance. Clones generally performed better on their host of origin than they did on the alternate host, and they performed less well on the alternate host compared to the clones that had originated there. A comparison of the performance of third generation aphids with first generation aphids showed that the experience of the mother in the second generation often influences the subsequent performance of their offspring. As the sequence of host transfers had more effect on the performance of wheat clones than cocksfoot clones, it is likely that wheat clones are more specialised, such that wheat is a satisfactory host for cocksfoot clones but not vice versa. The study provides evidence of genetic variation in performance on host and evidence for clonal adaptation to particular host species. This adaptation may well be a major cause of the observed consistent genetic differentiability of populations of S. avenae found on wheat and roadside grasses in early spring in southern England.