Whitefly-Plant Interactions: An Integrated Molecular Perspective.

The rapid advances in available transcriptomic and genomic data and our understanding of the physiology and biochemistry of whitefly-plant interactions have allowed us to gain new and significant insights into the biology of whiteflies and their successful adaptation to host plants. In this review, we provide a comprehensive overview of the mechanisms that whiteflies have evolved to overcome the challenges of feeding on phloem sap. We also highlight the evolution and functions of gene families involved in host perception, evaluation, and manipulation; primary metabolism; and metabolite detoxification. We discuss the emerging themes in plant immunity to whiteflies, focusing on whitefly effectors and their sites of action in plant defense-signaling pathways. We conclude with a discussion of advances in the genetic manipulation of whiteflies and the potential that they hold for exploring the interactions between whiteflies and their host plants, as well as the development of novel strategies for the genetic control of whiteflies.

Comparative evolutionary analyses of eight whitefly Bemisia tabaci sensu lato genomes: cryptic species, agricultural pests and plant-virus vectors.

BACKGROUND: The group of > 40 cryptic whitefly species called Bemisia tabaci sensu lato are amongst the world's worst agricultural pests and plant-virus vectors. Outbreaks of B. tabaci s.l. and the associated plant-virus diseases continue to contribute to global food insecurity and social instability, particularly in sub-Saharan Africa and Asia. Published B. tabaci s.l. genomes have limited use for studying African cassava B. tabaci SSA1 species, due to the high genetic divergences between them. Genomic annotations presented here were performed using the 'Ensembl gene annotation system', to ensure that comparative analyses and conclusions reflect biological differences, as opposed to arising from different methodologies underpinning transcript model identification. RESULTS: We present here six new B. tabaci s.l. genomes from Africa and Asia, and two re-annotated previously published genomes, to provide evolutionary insights into these globally distributed pests. Genome sizes ranged between 616-658 Mb and exhibited some of the highest coverage of transposable elements reported within Arthropoda. Many fewer total protein coding genes (PCG) were recovered compared to the previously published B. tabaci s.l. genomes and structural annotations generated via the uniform methodology strongly supported a repertoire of between 12.8-13.2 × 103 PCG. An integrative systematics approach incorporating phylogenomic analysis of nuclear and mitochondrial markers supported a monophyletic Aleyrodidae and the basal positioning of B. tabaci Uganda-1 to the sub-Saharan group of species. Reciprocal cross-mating data and the co-cladogenesis pattern of the primary obligate endosymbiont 'Candidatus Portiera aleyrodidarum' from 11 Bemisia genomes further supported the phylogenetic reconstruction to show that African cassava B. tabaci populations consist of just three biological species. We include comparative analyses of gene families related to detoxification, sugar metabolism, vector competency and evaluate the presence and function of horizontally transferred genes, essential for understanding the evolution and unique biology of constituent B. tabaci. s.l species. CONCLUSIONS: These genomic resources have provided new and critical insights into the genetics underlying B. tabaci s.l. biology. They also provide a rich foundation for post-genomic research, including the selection of candidate gene-targets for innovative whitefly and virus-control strategies.

Glucosylation prevents plant defense activation in phloem-feeding insects.

The metabolic adaptations by which phloem-feeding insects counteract plant defense compounds are poorly known. Two-component plant defenses, such as glucosinolates, consist of a glucosylated protoxin that is activated by a glycoside hydrolase upon plant damage. Phloem-feeding herbivores are not generally believed to be negatively impacted by two-component defenses due to their slender piercing-sucking mouthparts, which minimize plant damage. However, here we document that glucosinolates are indeed activated during feeding by the whitefly Bemisia tabaci. This phloem feeder was also found to detoxify the majority of the glucosinolates it ingests by the stereoselective addition of glucose moieties, which prevents hydrolytic activation of these defense compounds. Glucosylation of glucosinolates in B. tabaci was accomplished via a transglucosidation mechanism, and two glycoside hydrolase family 13 (GH13) enzymes were shown to catalyze these reactions. This detoxification reaction was also found in a range of other phloem-feeding herbivores.

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.

Projecting pest population dynamics under global warming: the combined effect of inter- and intra-annual variations.

The typical short generation length of insects makes their population dynamics highly sensitive not only to mean annual temperatures but also to their intra-annual variations. To consider the combined effect of both thermal factors under global warming, we propose a modeling framework that links general circulation models (GCMs) with a stochastic weather generator and population dynamics models to predict species population responses to inter- and intra-annual temperature changes. This framework was utilized to explore future changes in populations of Bemisia tabaci, an invasive insect pest-species that affects multiple agricultural systems in the Mediterranean region. We considered three locations representing different pest status and climatic conditions: Montpellier (France), Seville (Spain), and Beit-Jamal (Israel). We produced ensembles of local daily temperature realizations representing current and future (mid-21st century) climatic conditions under two emission scenarios for the three locations. Our simulations predicted a significant increase in the average number of annual generations and in population size, and a significant lengthening of the growing season in all three locations. A negative effect was found only in Seville for the summer season, where future temperatures lead to a reduction in population size. High variability in population size was observed between years with similar annual mean temperatures, suggesting a strong effect of intra-annual temperature variation. Critical periods were from late spring to late summer in Montpellier and from late winter to early summer in Seville and Beit-Jamal. Although our analysis suggested that earlier seasonal activity does not necessarily lead to increased populations load unless an additional generation is produced, it is highly likely that the insect will become a significant pest of open-fields at Mediterranean latitudes above 40° during the next 50 years. Our simulations also implied that current predictions based on mean temperature anomalies are relatively conservative and it is better to apply stochastic tools to resolve complex responses to climate change while taking natural variability into account. In summary, we propose a modeling framework capable of determining distinct intra-annual temperature patterns leading to large or small population sizes, for pest risk assessment and management planning of both natural and agricultural ecosystems.

Glucosinolate Desulfation by the Phloem-Feeding Insect Bemisia tabaci.

Glucosinolates are plant secondary defense metabolites confined nearly exclusively to the order Brassicales. Upon tissue rupture, glucosinolates are hydrolyzed to various bioactive breakdown products by the endogenous plant enzyme myrosinase. As the feeding of chewing insect herbivores is associated with plant tissue damage, these insects have developed several independent strategies for coping with the glucosinolate-myrosinase defense system. On the other hand, our knowledge of how phloem-feeding insects interact with the glucosinolate-myrosinase system is much more limited. In fact, phloem feeders might avoid contact with myrosinase altogether so their susceptibility to intoxication by glucosinolate hydrolysis products is unclear. Previous studies utilizing Arabidopsis thaliana plants accumulating high levels of aliphatic- or indolic-glucosinolates indicated that both glucosinolate groups have moderate negative effects on the reproductive performance of Bemisia tabaci, a generalist phloem-feeding insect. To get a deeper understanding of the interaction between B. tabaci and glucosinolate-defended plants, adults were allowed to feed on artificial diet containing intact glucosinolates or on Brussels sprout and A. thaliana plants, and their honeydew was analyzed for the presence of possible metabolites. We found that B. tabaci is capable of cleaving off the sulfate group of intact glucosinolates, producing desulfoglucosinolates that cannot be activated by myrosinases, a mechanism described to date only in several chewing insect herbivores. The presence of desulfated glucosinolates in the honeydew of a generalist phloem-feeder may indicate the necessity to detoxify glucosinolates, likely due to some level of cellular damage during feeding, which results in glucosinolate activation, or as a mechanism to circumvent the non-enzymatic breakdown of indolic glucosinolates.

Arabidopsis thaliana plants with different levels of aliphatic- and indolyl-glucosinolates affect host selection and performance of Bemisia tabaci.

Generalist insects show reduced selectivity when subjected to similar, but not identical, host plant chemical signatures. Here, we produced transgenic Arabidopsis thaliana plants that over-express genes regulating the aliphatic- and indolyl- glucosinolates biosynthetic pathways with either a constitutive (CaMV 35S) or a phloem-specific promoter (AtSUC2). This allowed us to examine how exposure to high levels of aliphatic- or indolyl-glucosinolates in homogenous habitats (leaf cage apparatus containing two wild-type or two transgenic leaves) and heterogeneous habitats (leaf cage apparatus containing one wild-type and one transgenic leaf) affects host selection and performance of Bemsia tabaci, a generalist phloem-feeding insect. Data from homogenous habitats indicated that exposure to A. thaliana plants accumulating high levels of aliphatic- or indolyl-glucosinolates negatively affected the performance of both adult females and nymphs of B. tabaci. Data from heterogeneous habitats indicated that B. tabaci adult females selected for oviposition plants on which their offspring perform better (preference-performance relationship). However, the combinations of wild-type and transgenic plants in heterogeneous habitats increased the period of time until the first choice was made and led to increased movement rate on transgenic plants, and reduced fecundity on wild-type plants. Overall, our findings are consistent with the view that both performance and selectivity of B. tabaci decrease in heterogeneous habitats that contain plants with closely-related chemical signatures.

A sucrose-specific receptor in Bemisia tabaci and its putative role in phloem feeding.

In insects, specialized feeding on the phloem sap (containing mainly the sugar sucrose) has evolved only in some hemipteran lineages. This feeding behavior requires an ability to locate feeding sites buried deeply within the plant tissue. To determine the molecular mechanism involved, we hypothesized that the phloem-feeding whitefly Bemisia tabaci relies on gustatory receptor (GR)-mediated sugar sensing. We first conducted choice assays, which indicated that B. tabaci adults consistently choose diets containing higher sucrose concentrations. Next, we identified four GR genes in the B. tabaci genome. One of them, BtabGR1, displayed significant sucrose specificity when expressed in Xenopus oocytes. Silencing of BtabGR1 significantly interfered with the ability of B. tabaci adults to discriminate between non-phloem and phloem concentrations of sucrose. These findings suggest that in phloem feeders, sugar sensing by sugar receptors might allow tracking an increasing gradient of sucrose concentrations in the leaf, leading eventually to the location of the feeding site.

Evolved Transcriptional Responses and Their Trade-Offs after Long-Term Adaptation of Bemisia tabaci to a Marginally Suitable Host.

Although generalist insect herbivores can migrate and rapidly adapt to a broad range of host plants, they can face significant difficulties when accidentally migrating to novel and marginally suitable hosts. What happens, both in performance and gene expression regulation, if these marginally suitable hosts must be used for multiple generations before migration to a suitable host can take place, largely remains unknown. In this study, we established multigenerational colonies of the whitefly Bemisia tabaci, a generalist phloem-feeding species, adapted to a marginally suitable host (habanero pepper) or an optimal host (cotton). We used reciprocal host tests to estimate the differences in performance of the populations on both hosts under optimal (30°C) and mild-stressful (24°C) temperature conditions, and documented the associated transcriptomic changes. The habanero pepper-adapted population greatly improved its performance on habanero pepper but did not reach its performance level on cotton, the original host. It also showed reduced performance on cotton, relative to the nonadapted population, and an antagonistic effect of the lower-temperature stressor. The transcriptomic data revealed that most of the expression changes, associated with long-term adaptation to habanero pepper, can be categorized as "evolved" with no initial plastic response. Three molecular functions dominated: enhanced formation of cuticle structural constituents, enhanced activity of oxidation-reduction processes involved in neutralization of phytotoxins and reduced production of proteins from the cathepsin B family. Taken together, these findings indicate that generalist insects can adapt to novel host plants by modifying the expression of a relatively small set of specific molecular functions.

The molecular mechanisms that determine different degrees of polyphagy in the Bemisia tabaci species complex.

The whitefly Bemisia tabaci is a closely related group of >35 cryptic species that feed on the phloem sap of a broad range of host plants. Species in the complex differ in their host-range breadth, but the mechanisms involved remain poorly understood. We investigated, therefore, how six different B. tabaci species cope with the environmental unpredictability presented by a set of four common and novel host plants. Behavioral studies indicated large differences in performances on the four hosts and putative specialization of one of the species to cassava plants. Transcriptomic analyses revealed two main insights. First, a large set of genes involved in metabolism (>85%) showed differences in expression between the six species, and each species could be characterized by its own unique expression pattern of metabolic genes. However, within species, these genes were constitutively expressed, with a low level of environmental responsiveness (i.e., to host change). Second, within each species, sets of genes mainly associated with the super-pathways "environmental information processing" and "organismal systems" responded to the host switching events. These included genes encoding for proteins involved in sugar homeostasis, signal transduction, membrane transport, and immune, endocrine, sensory and digestive responses. Our findings suggested that the six B. tabaci species can be divided into four performance/transcriptomic "Types" and that polyphagy can be achieved in multiple ways. However, polyphagy level is determined by the specific identity of the metabolic genes/pathways that are enriched and overexpressed in each species (the species' individual metabolic "tool kit").

Activation and detoxification of cassava cyanogenic glucosides by the whitefly Bemisia tabaci.

Two-component plant defenses such as cyanogenic glucosides are produced by many plant species, but phloem-feeding herbivores have long been thought not to activate these defenses due to their mode of feeding, which causes only minimal tissue damage. Here, however, we report that cyanogenic glycoside defenses from cassava (Manihot esculenta), a major staple crop in Africa, are activated during feeding by a pest insect, the whitefly Bemisia tabaci, and the resulting hydrogen cyanide is detoxified by conversion to beta-cyanoalanine. Additionally, B. tabaci was found to utilize two metabolic mechanisms to detoxify cyanogenic glucosides by conversion to non-activatable derivatives. First, the cyanogenic glycoside linamarin was glucosylated 1-4 times in succession in a reaction catalyzed by two B. tabaci glycoside hydrolase family 13 enzymes in vitro utilizing sucrose as a co-substrate. Second, both linamarin and the glucosylated linamarin derivatives were phosphorylated. Both phosphorylation and glucosidation of linamarin render this plant pro-toxin inert to the activating plant enzyme linamarase, and thus these metabolic transformations can be considered pre-emptive detoxification strategies to avoid cyanogenesis.

Extraordinary resistance to insecticides reveals exotic Q biotype of Bemisia tabaci in the New World.

A strain of the whitefly Bemisia tabaci (Gennadius) possessing unusually high levels of resistance to a wide range of insecticides was discovered in 2004 in the course of routine resistance monitoring in Arizona. The multiply resistant insects, collected from poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch) plants purchased at a retail store in Tucson, were subjected to biotype analysis in three laboratories. Polyacrylamide gel electrophoresis of naphthyl esterases and sequencing of the mitochondrial cytochrome oxidase I gene (780 bp) confirmed the first detection of the Q biotype of B. tabaci in the New World. This U.S. Q biotype strain, referred to as Poinsettia'04, was highly resistant to two selective insect growth regulators, pyriproxyfen and buprofezin, and to mixtures of fenpropathrin and acephate. It was also unusually low in susceptibility to the neonicotinoid insecticides imidacloprid, acetamiprid, and thiamethoxam, relative to B biotype whiteflies. In 100 collections of whiteflies made in Arizona cotton (Gossypium spp.), vegetable, and melon (Cucumis melo L.) fields from 2001 to 2005, no Q biotypes were detected. Regions of the United States that were severely impacted by the introduction of the B biotype of B. tabaci in the 1980s would be well advised to promote measures that limit movement of the Q biotype from controlled environments into field systems and to formulate alternatives for managing this multiply-resistant biotype, in the event that it becomes more widely distributed.

Portiera Gets Wild: Genome Instability Provides Insights into the Evolution of Both Whiteflies and Their Endosymbionts.

Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are a superfamily of small phloem-feeding insects. They rely on their primary endosymbionts "Candidatus Portiera aleyrodidarum" to produce essential amino acids not present in their diet. Portiera has been codiverging with whiteflies since their origin and therefore reflects its host's evolutionary history. Like in most primary endosymbionts, the genome of Portiera stays stable across the Aleyrodidae superfamily after millions of years of codivergence. However, Portiera of the whitefly Bemisia tabaci has lost the ancestral genome order, reflecting a rare event in the endosymbiont evolution: the appearance of genome instability. To gain a better understanding of Portiera genome evolution, identify the time point in which genome instability appeared and contribute to the reconstruction of whitefly phylogeny, we developed a new phylogenetic framework. It targeted five Portiera genes and determined the presence of the DNA polymerase proofreading subunit (dnaQ) gene, previously associated with genome instability, and two alternative gene rearrangements. Our results indicated that Portiera gene sequences provide a robust tool for studying intergenera phylogenetic relationships in whiteflies. Using these new framework, we found that whitefly species from the Singhiella, Aleurolobus, and Bemisia genera form a monophyletic tribe, the Aleurolobini, and that their Portiera exhibit genome instability. This instability likely arose once in the common ancestor of the Aleurolobini tribe (at least 70 Ma), drawing a link between the appearance of genome instability in Portiera and the switch from multibacteriocyte to a single-bacteriocyte mode of inheritance in this tribe.

Inside out: microbiota dynamics during host-plant adaptation of whiteflies.

While most insect herbivores are selective feeders, a small proportion of them feed on a wide range of plants. This polyphagous habit requires overcoming a remarkable array of defenses, which often necessitates an adaptation period. Efforts for understanding the mechanisms involved mostly focus on the insect's phenotypic plasticity. Here, we hypothesized that the adaptation process might partially rely on transient associations with bacteria. To test this, we followed in a field-like experiment, the adaptation process of Bemisia tabaci, a generalist sap feeder, to pepper (a less-suitable host), after switching from watermelon (a suitable host). Amplicon sequencing of 16S rRNA transcripts from hundreds of dissected guts revealed the presence of active "core" and "transient" bacterial communities, dominated by the phyla Proteobacteria, Actinobacteria, and Firmicutes, and increasing differences between populations grown on watermelon and pepper. Insects grown on pepper for over two generations presented a significant increase in specific genera, mainly Mycobacterium, with a predicted enrichment in degradative pathways of xenobiotics and secondary metabolites. This result correlated with a significant increase in the insect's survival on pepper. Taken together, our findings suggest that gut-associated bacteria can provide an additional flexible metabolic "tool-box" to generalist sap feeders for facilitating a quick host switching process.

Can CRISPR gene drive work in pest and beneficial haplodiploid species?

Gene drives based on CRISPR/Cas9 have the potential to reduce the enormous harm inflicted by crop pests and insect vectors of human disease, as well as to bolster valued species. In contrast with extensive empirical and theoretical studies in diploid organisms, little is known about CRISPR gene drive in haplodiploids, despite their immense global impacts as pollinators, pests, natural enemies of pests, and invasive species in native habitats. Here, we analyze mathematical models demonstrating that, in principle, CRISPR homing gene drive can work in haplodiploids, as well as at sex-linked loci in diploids. However, relative to diploids, conditions favoring the spread of alleles deleterious to haplodiploid pests by CRISPR gene drive are narrower, the spread is slower, and resistance to the drive evolves faster. By contrast, the spread of alleles that impose little fitness cost or boost fitness was not greatly hindered in haplodiploids relative to diploids. Therefore, altering traits to minimize damage caused by harmful haplodiploids, such as interfering with transmission of plant pathogens, may be more likely to succeed than control efforts based on introducing traits that reduce pest fitness. Enhancing fitness of beneficial haplodiploids with CRISPR gene drive is also promising.

Molecular Evolution of the Glutathione S-Transferase Family in the Bemisia tabaci Species Complex.

The glutathione S-transferase (GST) family plays an important role in the adaptation of herbivorous insects to new host plants and other environmental constrains. The family codes for enzymes that neutralize reactive oxygen species and phytotoxins through the conjugation of reduced glutathione. Here, we studied the molecular evolution of the GST family in Bemisia tabaci, a complex of >35 sibling species, differing in their geographic and host ranges. We tested if some enzymes evolved different functionality, by comparing their sequences in six species, representing five of the six major genetic clades in the complex. Comparisons of the nonsynonymous to synonymous substitution ratios detected positive selection events in 11 codons of 5 cytosolic GSTs. Ten of them are located in the periphery of the GST dimer, suggesting a putative involvement in interactions with other proteins. Modeling the tertiary structure of orthologous enzymes, identified additional 19 mutations in 9 GSTs, likely affecting the enzymes' functionality. Most of the mutation events were found in the environmentally responsive classes Delta and Sigma, indicating a slightly different delta/sigma tool box in each species. At a broader genomic perspective, our analyses indicated a significant expansion of the Delta GST class in B. tabaci and a general association between the diet breadth of hemipteran species and their total number of GST genes. We raise the possibility that at least some of the identified changes improve the fitness of the B. tabaci species carrying them, leading to their better adaptation to specific environments.

Asymmetric reproductive interference between two closely related spider mites: Tetranychus urticae and T. turkestani (Acari: Tetranychidae).

Tetranychus turkestani Ugarov and Nikolskii and Tetranychus urticae Koch RF (red form) (Acari: Tetranychidae) are closely related species. Previously, the two species were found in separate agricultural habitats in Israel. Here, additional collections were undertaken and mixed populations of the two species were found. Manipulation experiments were conducted in order to test whether sexual interactions occur when T. turkestani and T. urticae RF share the same host. Interspecific crosses showed that the two species are capable of producing viable F(1) females, but that these females are sterile as their F(2) eggs failed to hatch. This indicates a post-zygotic reproductive barrier, supporting the current placement of T. turkestani as a separate taxon. Mating behavior parameters revealed that males of both species courted virgin conspecific and heterospecific females at the same rate and readily tried to copulate with them. Female mate recognition seemed to be more reliable in T. turkestani than in T. urticae RF as the number of copulations was significantly higher and their duration significantly shorter in the T. turkestani interspecific (T. turkestani female x T. urticae RF male) as compared to the intraspecific crosses, a phenomenon not observed in T. urticae RF. In mixed cultures, a significant reduction in female production was observed for T. urticae RF but not for T. turkestani, suggesting an asymmetric reproductive interference effect in favor of T. turkestani. The long term outcome of this effect is yet to be determined since additional reproductive factors such as oviposition rate and progeny survival to adulthood may reduce the probability of demographic displacement of one species by the other in overlapping niches.

Complete Assembly of the Genome of an Acidovorax citrulli Strain Reveals a Naturally Occurring Plasmid in This Species.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit crop production worldwide. Based on genetic and phenotypic properties, A. citrulli strains are divided into two major groups: group I strains have been generally isolated from melon and other non-watermelon cucurbits, while group II strains are closely associated with watermelon. In a previous study, we reported the genome of the group I model strain, M6. At that time, the M6 genome was sequenced by MiSeq Illumina technology, with reads assembled into 139 contigs. Here, we report the assembly of the M6 genome following sequencing with PacBio technology. This approach not only allowed full assembly of the M6 genome, but it also revealed the occurrence of a ∼53 kb plasmid. The M6 plasmid, named pACM6, was further confirmed by plasmid extraction, Southern-blot analysis of restricted fragments and obtention of M6-derivative cured strains. pACM6 occurs at low copy numbers (average of ∼4.1 ± 1.3 chromosome equivalents) in A. citrulli M6 and contains 63 open reading frames (ORFs), most of which (55.6%) encoding hypothetical proteins. The plasmid contains several genes encoding type IV secretion components, and typical plasmid-borne genes involved in plasmid maintenance, replication and transfer. The plasmid also carries an operon encoding homologs of a Fic-VbhA toxin-antitoxin (TA) module. Transcriptome data from A. citrulli M6 revealed that, under the tested conditions, the genes encoding the components of this TA system are among the highest expressed genes in pACM6. Whether this TA module plays a role in pACM6 maintenance is still to be determined. Leaf infiltration and seed transmission assays revealed that, under tested conditions, the loss of pACM6 did not affect the virulence of A. citrulli M6. We also show that pACM6 or similar plasmids are present in several group I strains, but absent in all tested group II strains of A. citrulli.

Organophosphates' resistance in the B-biotype of Bemisia tabaci (Hemiptera: Aleyrodidae) is associated with a point mutation in an ace1-type acetylcholinesterase and overexpression of carboxylesterase.

Organophosphate (OP) insecticides are inhibitors of the enzyme acetylcholinesterase (AChE), which terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine. Previous biochemical studies in Bemisia tabaci (Hemiptera: Aleyrodidae) proposed the existence of two molecular mechanisms for OPs' resistance: carboxylesterase- (COE) mediated hydrolysis or sequestration and decreased sensitivity of AChE. Here, two acetylcholinesterase genes, ace1 and ace2, have been fully cloned and sequenced from an OP-resistant strain and an OP-susceptible strain of B. tabaci. Comparison of nucleic acid and deduced amino acid sequences revealed only silent nucleotide polymorphisms in ace2, and one mutation, Phe392Trp (Phe331 in Torpedo californica), in ace1 of the resistant strain. The Phe392Trp mutation is located in the acyl pocket of the active site gorge and was recently shown to confer OP insensitivity in Culex tritaeniorhynchus. In addition, we also report on the isolation of two carboxylesterase genes (coe1 and coe2) from B. tabaci, the first carboxylesterases to be reported from this species. We show that one of the genes, coe1, is overexpressed ( approximately 4-fold) in the OP-resistant strain, and determine, by quantitative PCR, that the elevated expression is not related to gene amplification but probably to modified transcriptional control. Lastly, we bring new biochemical evidence that support the involvement of both AChE insensitivity and COE metabolism in resistance to OP insecticides in the resistant strain.

Over-expression of cytochrome P450 CYP6CM1 is associated with high resistance to imidacloprid in the B and Q biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae).

The two most damaging biotypes of Bemisia tabaci, B and Q, have both evolved strong resistance to the neonicotinoid insecticide imidacloprid. The major mechanism in all samples investigated so far appeared to be enhanced detoxification by cytochrome P450s monooxygenases (P450s). In this study, a polymerase chain reaction (PCR) technology using degenerate primers based on conserved P450 helix I and heme-binding regions was employed to identify P450 cDNA sequences in B. tabaci that might be involved in imidacloprid resistance. Eleven distinct P450 cDNA sequences were isolated and classified as members of the CYP4 or CYP6 families. The mRNA expression levels of all 11 genes were compared by real-time quantitative RT-PCR across nine B and Q field-derived strains of B. tabaci showing strong resistance, moderate resistance or susceptibility to imidacloprid. We found that constitutive over-expression (up to approximately 17-fold) of a single P450 gene, CYP6CM1, was tightly related to imidacloprid resistance in both the B and Q biotypes. Next, we identified three single-nucleotide polymorphic (SNP) markers in the intron region of CYP6CM1 that discriminate between the resistant and susceptible Q-biotype CYP6CM1 alleles (r-Q and s-Q, respectively), and used a heterogeneous strain to test for association between r-Q and resistance. While survivors of a low imidacloprid dose carried both the r-Q and s-Q alleles, approximately 95% of the survivors of a high imidacloprid dose carried only the r-Q allele. Together with previous evidence, the results reported here identify enhanced activity of P450s as the major mechanism of imidacloprid resistance in B. tabaci, and the CYP6CM1 gene as a leading target for DNA-based screening for resistance to imidacloprid and possibly other neonicotinoids in field populations.