Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

2.1 Introduction

For decades, agriculture has been the main source of food and other useful products for humans and livestock. The world population is expected to grow by one-third from the present by 2050 (www.fao.org) leading to an increase in demand for agricultural products to feed growing populations worldwide. Production of agricultural crops is at risk due to the incidence of herbivores and disease-causing pathogens. Herbivores, mainly insect pests, are undoubtedly the most important competitors for food, fiber, and other natural products. Beyond their economic importance, they have a direct impact on agricultural food production by chewing, sucking, and boring plant parts and, most importantly, by spreading devastating plant pathogens, particularly plant viruses. Many important plant viruses depend on insect vectors which mainly belong to the orders Hemiptera, Thysanoptera, and Coleoptera, which transmit a great diversity of plant viruses across the tropical and subtropical regions of the world. Among them, phloem-feeding insects (aphids, whiteflies, plant, and leafhoppers) are the most common insect vectors that transmit numerous viruses and cause significant losses to food crops of socioeconomic importance.

In the last 20 years of the twentieth century, vector biology research switched from an understanding of the general biology of virus-vector relationships to a more deep understanding of the viral and vector proteins and components involved in the transmission process. However, the insect vector component was largely ignored due to a lack of genetic and genomic resources. Developments in mass sequencing projects such as expressed sequence tags (ESTs), together with genome annotation and functional gene discovery, are greatly advancing our understanding of the transmission process of viruses by their vectors and the components required to facilitate virus transmission. The emergence of massive sequencing technologies such as 454 pyrosequencing and the Illumina sequencing platform has been widely used to understand the underlying molecular mechanisms [1] involved in virus transmission, mating behavior, and genomes of many hemipterans and the whitefly, Bemisia tabaci, its endosymbionts, and the interactions of the insect with the environment. Functional genomic projects of the whitefly will facilitate an understanding of not only its interactions with viruses but also its resistance to insecticides, its developmental patterns, and its interactions with plants and other microorganisms. This chapter reviews the recent advances in the genomics of B. tabaci and the relevance of these advancements to our understanding of its interactions with various factors.

2.2 Status of B. tabaci as a Global Agricultural Pest

The whitefly, B. tabaci (Gennadius) (Hemiptera: Aleyrodidae), is a phloem-feeding insect that inflicts a serious worldwide threat by direct feeding on plants and by the transmission of plant viruses [2–4] (Fig. 2.1). B. tabaci was first described in 1889 as a tobacco pest in Greece and named Aleyrodes tabaci [5]; later on it was described as A. inconspicua Quaintance in 1900 [6] and B. tabaci in 1957 [7]. Comprehensive recent reviews about the history of B. tabaci [8] and its cryptic species status [9–13] are available.

Fig. 2.1
figure 1

Direct and indirect plant damage caused by Bemisia tabaci. (a) B. tabaci adult. (b) Representative damage caused by B. tabaci feeding showing an infested basil leaf with B. tabaci nymphs. (c) A basil leaf showing sooty mold growth after B. tabaci infestation and massive secretion of honeydew. (d) An uninfested watermelon field. (e) Another watermelon field that was infested by two B. tabaci-transmitted begomoviruses squash leaf curl virus (SLCV) and watermelon chlorotic stunt virus (WmCSV)

Full size image

B. tabaci adults are about 1–2 mm long and have opaque-shaped wings covered with a whitish powder or wax, leading to the common name of whiteflies. B. tabaci was first reported to be a serious insect pest in the late 1920s in Northern India [14, 15] and is now globally distributed, in the USA, Africa, the Middle East, Europe, the Orient, Russia, Southeast Asia, and South America [3], except in Antarctica [16]. It is a highly polyphagous insect that feeds on over 700 plant species from 86 families, including a large number of agriculturally and industrially important crops [9, 17, 18]. Whiteflies damage plants in various ways. The delicate mouthparts of the whitefly adults and nymphal stages cause direct feeding damage by weakening plants (Fig. 2.1). They also cause indirect damage due to the secretion of honeydew that causes sooty mold growth which interferes with photosynthesis and makes the fruits become unsightly.

The whitefly’s major impact on agriculture is due to the fact that this insect serves as a vector of more than 110 plant viruses [9, 16–19], many of which have great impact on plants and agricultural production. Estimates of their destructive reach extend to 20 million hectares of crops and 15 million farmers [20]. Next to aphids, whiteflies are of greater importance as vectors of the most devastating plant virus groups, including Begomovirus, Crinivirus, Closterovirus, Carlavirus, and Torradovirus [17, 21]. Among whitefly-transmitted viruses, 90 % belong to the Begomovirus genera [17], which include approximately 200 species, and have emerged as the most threatening group of plant viruses globally during the past two decades as reported from dicotyledonous host-causing diseases of economic importance.

B. tabaci is considered as a cryptic species complex [11]. Individual cryptic species within the complex differ in their adaptability to hosts, in their resistance to chemicals, and importantly in their ability to transmit begomoviruses [22–26]. Exotic whitefly biotypes or cryptic species transmit Old and New World begomoviruses in many places of the world resulting from changes in environmental conditions and agricultural practices. Studies on whitefly biotypes/cryptic species and the begomoviruses they transmit worldwide reveal that grouping of begomoviruses based on CP (coat protein) sequences parallels grouping of biotypes/genotypes so that both begomoviruses and vector are grouped in similar patterns according to their geographic origin. As different members of the species complex are morphologically indistinguishable, various molecular methods have been applied over the past two decades to delimit the members of the B. tabaci species complex.

2.3 Biotypes or Cryptic Species of B. tabaci: Definitions and Distribution

B. tabaci has numerous biotypes which transmit a large number of viruses that infect many important agricultural plants, causing major economic impacts [27]. As early as the 1950s, biotypes were proposed to characterize the morphologically indistinguishable populations of B. tabaci on the basis of host range, host plant adaptability, and plant virus transmission capabilities [28–30]. During the 1980s, the A biotype became a serious problem in cotton and cucurbits in the Southwestern USA and Mexico [3]. In 1991, the introduced B biotype displaced the A biotype in the Southwestern USA [3]. In the same year, Perring et al. [31] suggested that A and B biotypes were separate species. A newly evolved B biotype, commonly referred to as the silver leaf whitefly or poinsettia strain, has a very wide host range, which has contributed to the spread of geminiviruses to new hosts [32] and has been documented as a separate species named B. argentifolii [33]. Using allozymes and the random amplified polymorphic cDNA-polymerase chain reaction (RAPD-PCR), Perring et al. [34] and Gawel and Bartlett [35] further showed consistent differences between A and B biotypes.

As the different members of the B. tabaci species complex are morphologically indistinguishable, various molecular methods have been applied over the past two decades to delimit the members of this species complex. The most popular techniques and the types of DNA markers used to study B. tabaci are sequence characterized amplified regions (SCAR) [36–41], cleaved amplified polymorphic sequences or restriction fragment length polymorphisms (CAPS/RFLP) [37, 42], amplified fragment length polymorphisms (AFLP) [43], 16S ribosomal RNA [44], mitochondrial cytochrome oxidase 1 (mtCO1) [10–12, 45–49], nuclear ribosomal internal transcribe spacer 1 (ITS1) [50–54], and microsatellites [55–65]. Among them, mtCOI has several advantages over other approaches and has been extensively used [11].

Using CO1-based Bayesian phylogenetic analysis and sequence divergence, Dinsdale et al. [10] and De Barro et al. [11] proposed a speciation system on the basis of a demarcation criterion of a 3.5 % divergence threshold. Recently, Lee et al. [49] observed that a 4.0 % genetic boundary was more realistic than 3.5 % in distinguishing B. tabaci species. Following the above criteria, 37 morphologically indistinguishable species (Africa, Asia I, Asia II 1, Asia II 2, Asia II 3, Asia II 4, Asia II 5, Asia II 6, Asia II 7, Asia II 8, Asia II 9, Asia II 10, Asia II 11, Asia II 12, Asia III, Asia IV, Australia, Australia/Indonesia, China 1, China 2, China 3, China 4, Indian Ocean, Middle East-Asia Minor (MEAM) I, MEAM II, Mediterranean (MED), New World 1, New World 2, Japan 1, Japan 2, Uganda, Italy 1, Sub-Saharan Africa 1, Sub-Saharan Africa 2, Sub-Saharan Africa 3, Sub-Saharan Africa 4, Sub-Saharan Africa 5) have been currently delimited at the global level [10–13, 46–49, 66]. Among the B. tabaci species complex, the most important biotypes worldwide are B and Q, recently termed as the MEAM1 and MED species, respectively [3, 11, 44, 67].

2.4 Genomic and Postgenomic Resources Developed for B. tabaci and What Is Missing

As indicated, B. tabaci is a complex of biotypes that differ in their behavior, plant host, ability to induce disorders in plants, ability to transmit plant viruses, the bacterial endosymbionts they harbor, and genetic makeup [68]. In addition to the endogenous species, new invasive and better fit biotypes such as B and Q have invaded crop systems, exacerbating damage [69]. Very little is known about the virus transmission specificity, mating behavior, or genomes of either the whitefly or its bacterial endosymbionts. Collective functional genome projects of the whitefly will help to understand the whitefly genetic makeup and its interactions with environmental factors.

In 2005, the first attempt to measure nuclear DNA content of B. tabaci complex males and females was estimated using flow cytometry. This was the first step toward the exploration of the whitefly genome [70], and yielded values of 1.04 and 2.06 pg, respectively. Conversion between DNA content and genome size (1 pg DNA = 980 Mbp) indicated that the haploid genome size of B. tabaci is 1020 Mbp, which is approximately five times the size of the genome of the fruit fly, Drosophila melanogaster [70].

The first large-scale sequencing of ESTs from B. tabaci was initiated by Leshkowitz et al. in 2006 [71]. To address whitefly genomic sequence information, they constructed three cDNA libraries from non-viruliferous whiteflies (eggs, immature instars, and adults) and two libraries from adult insects with tomato yellow leaf curl virus (TYLCV) and tomato mottle virus (ToMoV). In total, 9110 sequences from the libraries were found to be involved in cellular and developmental processes. In addition, approximately 1000 bases were aligned with the genome of the B. tabaci primary endosymbiotic bacterium, Candidatus Portiera aleyrodidarum, originating primarily from the egg and immature instar libraries [71]. Apart from the mitochondrial sequences, abundant sequences in the libraries encoded vitellogenin gene sequences, indicating that much of the gene expression in B. tabaci is directed toward the production of eggs [71]. Studies thereafter were aimed to understand the mechanisms and the function of genes involved in various aspects of the whitefly interaction with its environment, and the first case was its resistance to insecticides. For the first time a DNA microarray containing 6000 unique ESTs from the whitefly was developed in 2007, and deep analysis revealed ESTs involved in insecticide resistance and xenobiotic detoxification such as those transcribed from P450 monooxygenases and oxidative stress genes, genes associated with protein, lipid and carbohydrate metabolism, and others related to juvenile hormone-associated processes in insects such as oocyte and egg development [72].

Following these pioneering studies and the development of new deep sequencing technologies and bioinformatic tools, the first de novo characterization of a whitefly transcriptome without prior genome annotation was sequenced by Wang et al. in 2010 [73], using the Illumina sequencing platform. This study obtained 168,900 unique sequences and a large number of genes associated with specific developmental stages and insecticide resistance. In 2011, using the same technology, Luan et al. [74] investigated the transcriptional response of the invasive B. tabaci MEAM1 species to tomato yellow leaf curl China virus (TYLCCNV). Results showed that 1606 genes involved in 157 biochemical pathways were differentially expressed in the viruliferous whiteflies. This indicates that TYLCCNV can perturb the cell cycle and primary metabolism, which explains the negative effect of this virus on the longevity and fecundity of B. tabaci [75]. Further results showed that TYLCCNV can activate whitefly immune responses, such as autophagy and antimicrobial peptide production, which might lead to a gradual decrease of viral particles within the body of the infected insect. Furthermore, PCR results showed that TYLCCNV can invade ovary and fat body, and LysoTracker and Western blot analyses revealed that the invasion of TYLCCNV induced autophagy in both types of tissue. Surprisingly, TYLCCNV also suppressed whitefly immune responses by downregulating the expression of genes involved in Toll-like signaling and mitogen-activated protein kinase (MAPK) pathways [74].

Using Illumina sequencing, further research investigated the transcriptome of the primary salivary glands of the MED species of the B. tabaci complex [76]. This study obtained 13,615 unigenes involved in metabolism and transport. Further analysis revealed genes related to processing of secretory proteins, secretion, and virus transmission [76]. Following this sequencing, Wang et al. [77, 78] sequenced an indigenous species, Asia II 3, and compared its genetic divergence with the transcriptomes of two invasive whitefly species, MEAM1 and MED. This study revealed a conserved group of 3203 protein families among the Asia II 3, MEAM1, and MED species which might be responsible for core cellular and physiological functions of the B. tabaci complex. Further results identifying hundreds of highly diverged genes and compiling sequence annotation data into functional groups found the most divergent gene classes to be the cytochrome P450 monooxygenases, glutathione metabolism, and oxidative phosphorylation. Moreover, many of these genes are predicted to be involved in protein metabolism such as peptide deformylase, cathepsin, cysteine proteinase, and metalloendopeptidase, which might be the driving force of the MEAM1-MED divergence and MEAM1- Asia II 3 differentiation [78]. A recent pyrosequencing study of the transcriptome of an invasive B biotype from China revealed a highly diverse bacterial community and robust system for insecticide resistance [79]. As mentioned above, de novo assembly generated 178,669 unigenes including 30,980 from the insects and 17,881 from the bacteria. Further in-depth transcriptome analysis revealed additional genes potentially involved in insecticide resistance and nutrient digestion [79].

Recently, Wang et al. [80] obtained the complete mitochondrial genome of the invasive Mediterranean species and identified 37 genes, including 13 protein-coding genes (PCGs), 2 ribosomal RNAs, and 22 transfer RNAs (tRNA). Comparative analyses of the genomes from MED and New World species revealed that there are no gene arrangements. This study also revealed that atp6 and atp8, nd4 and nd4l, and nd6 and cytb were on the same cistronic transcripts, whereas the other mature mitochondrial transcripts were monocistronic.

Recent transcriptome profiling of B. tabaci revealed stage-specific gene expression signatures for thiamethoxam resistance [81, 82]. Furthermore, analysis of gut transcriptomes of two invasive whitefly species in the B. tabaci complex, MEAM1 and MED, demonstrated the important role of the gut in the metabolism of insecticides and secondary plant chemicals [83]. Interestingly, using a microarray from B. tabaci that contained about 6000 genes, it was shown that parasitization by the wasp, Eretmocerus mundus, induced genes related to immune responses and revealed a role for the bacterial symbiont, Rickettsia, in these responses [84].

The foregoing transcriptomic studies reveal that a number of highly expressed genes that belong to different and conserved metabolic pathways with other insects are involved in virus transmission, insecticide resistance, and immune responses to parasitoids. Taken together, the transcriptomic results collected so far provide not only a roadmap for further functional genomic studies and extensive whitefly research in general but also a large collection of gene and EST sequences for future genome sequencing and annotation efforts.

2.5 B. tabaci Bacterial Symbionts

Bacterial symbionts in insects have drawn the attention of many research projects around the world because of their potential for developing friendly pest control methods by intervention with the insect-symbiont interactions. Recent surveys of facultative endosymbionts in whiteflies and aphids have used hundreds of insect populations collected around the world [85–90]. B. tabaci exhibits the highest diversity of secondary endosymbionts that infect one insect, and populations were reported to harbor seven different facultative (secondary) symbionts including the Alphaproteobacteria Rickettsia (Rickettsiales), Orentia-like organism (Rickettsiales), Wolbachia (Rickettsiales), the Gammaproteobacteria Arsenophonus (Enterobacteriales), Hamiltonella (Enterobacteriales), Cardinium (Bacteroidetes), and Fritschea (Chlamydiales) [91–97] (Fig. 2.2). All whitefly species including B. tabaci biotypes harbor the primary symbiont Portiera; however, they differ in their secondary symbiont composition [91, 98]. A study from Israel showed that B and Q biotypes differ in their infection with secondary symbionts, and while the B biotype was infected with Hamiltonella, the Q biotype was infected with Arsenophonus and Wolbachia. Both biotypes were infected with Rickettsia and none was infected with Fritschea and Cardinium [85]. A different study surveyed the distribution of secondary symbionts in whitefly species collected in the Balkan region and detected only the Q biotype of B. tabaci, which harbored Hamiltonella, Rickettsia, Wolbachia, and Cardinium, while Arsenophonus and Fritschea were not detected in any B. tabaci populations [86]. Recent studies focused on the role that these symbionts might play in the biology of their whitefly hosts. Two studies showed that Wolbachia [99] and Hamiltonella [100, 101] increase the fitness and enhance B. tabaci MED biotype performance. Other studies have shown that Rickettsia increases B. tabaci tolerance to heat [102] and influences the whitefly response to insecticides [103, 104]. A study conducted in the USA showed that Rickettsia increases the fitness of the B biotype of B. tabaci and enhances the proportion of females in the populations [105].

Fig. 2.2
figure 2

Fluorescence in situ hybridization (FISH) analysis on representative B. tabaci developmental stages and dissected organs for the localization of representative symbiotic bacteria. (a) An adult whitefly showing the primary symbiont Portiera in red and the secondary symbiont Hamiltonella in green. (b) A second-stage nymph showing the primary symbiont Portiera in red and the secondary symbiont Rickettsia in blue. (c) A fourth-stage nymph showing the primary symbiont Portiera in red and the secondary symbiont Hamiltonella in green. (d) A pupa showing the primary symbiont Portiera in red and the secondary symbiont Arsenophonus in yellow. (e) A dissected ovary showing oocytes in different developmental stages, the primary symbiont Portiera inside bacteriocyte cells in red and the secondary symbiont Rickettsia in green. (f) A portion of dissected midgut showing DAPI staining of the nuclei in blue and the secondary symbiont Rickettsia in red. Green represents FISH for localizing heat shock protein 70

Full size image

Bacterial symbionts in B. tabaci have been shown to play an important role in begomovirus transmission [106, 107]. Gottlieb et al. [108] showed that only a GroEL protein from Hamiltonella interacted with TYLCV CP and protected the virus while circulating in the whitefly hemolymph for transmission, whereas other GroEL proteins from other symbionts did not interact with TYLCV CP. It was further shown that release of virions protected by GroEL occurs adjacent to the primary salivary gland. However, CP of abutilon mosaic virus (AbMV), a begomovirus non-transmissible by B. tabaci, was also found to bind to GroEL. The midgut was shown to serve as the barrier for AbMV transmission and not interactions with GroEL [107]. A study from India showed that GroEL protein of Arsenophonus interacted with the cotton leaf curl virus (CLCuV) CP, suggesting the involvement of Arsenophonus in the transmission of CLCuV in the Asia II genetic group of B. tabaci [109]. Additionally, a recent study has demonstrated that Rickettsia plays an important role in TYLCV transmission. Rickettsia-infected females transmitted TYLCV at twice the rate of Rickettsia-uninfected females and retained the virus longer [110]. Interestingly, an antagonistic relationship was discovered between Rickettsia and TYLCV in this study, in which high levels of the bacterium in the midgut resulted in higher virus concentrations in the filter chamber, a favored site for virus translocation along the transmission pathway, whereas low levels of Rickettsia in the midgut resulted in an even distribution of the virus.

Several recent projects have sequenced the genomes of some B. tabaci symbionts, including the primary symbiont, Portiera [111–113], and secondary symbionts, Rickettsia and Hamiltonella [100, 114], and Cardinium [115]. These projects have provided unique insights into the genome reduction of these symbionts, which has made them completely dependent on their hosts and, therefore, not culturable. Additionally, the genome sequences of these symbionts have shown the dependence of the insect host on amino acid biosynthetic pathways lacking in the insect genome, providing further insights on the mutual symbiosis between the insect and its symbiotic bacteria. This is more evident when examining the genome of Portiera, the primary symbiont, which has an extremely reduced genome, yet maintains biosynthetic pathways essential for the survival of the symbiont as well as the insect.

Several additional recent B. tabaci EST sequencing projects identified a total of 17,766 bacterial unigenes which were classified into 322 genera [(Proteobacteria (92 %), Betaproteobacteria (59 %), Burkholderiales (58 %), Comamonadaceae (50 %), and Delftia (43 %)] and revealed a highly diverse bacterial community which represents a high and rapid coevolution of insects and their symbionts [79]. Additional population surveys from Brazil using rRNA gene sequencing showed that Hamiltonella and Rickettsia are highly prevalent in all MEAM1 populations tested, while Cardinium was close to fixation in only three populations. Surprisingly, some MEAM1 individuals and one New World 2 population were infected with Fritschea [90]. Guo et al. [116] observed more females than males harboring Hamiltonella, and the results suggested that both the female-biased symbiont infections and female-biased TYLCV infections promote the rapid spread of TYLCV in China. Recently, Zchori-Fein et al. [97] modeled the variations in the symbiotic communities of about 2000 sweetpotato whitefly individuals and demonstrated facultative endosymbiotic combinations which were positively correlated with both distance from the equator and specificity of the genetic code of the insect host.

The reports cited here show that begomovirus translocation in the vector involves not only hypothesized insect proteins and receptors but also bacterial proteins, which could be acting directly or indirectly to influence the virus transmission. Future whitefly genomics research is expected to gain better understanding of the novel genes and mechanisms involved in virus-vector-symbiont relationships.

2.6 Studying the Interaction Between B. tabaci and Begomoviruses

B. tabaci is known to transmit a great diversity of plant viruses; among them, begomoviruses are the most devastating and cause serious viral diseases in agricultural cropping systems worldwide. The whitefly-begomovirus interactions depend on the virus, the biotype of the whitefly vector, and the endosymbionts harbored in the specific vector [68]. More than 80 % of the mono- and bipartite begomoviruses (both New and Old worlds) are transmitted by whiteflies in and around tropical and subtropical regions. Whitefly-transmitted begomoviruses are known to cause leaf curling, yellow mosaic, and yellow vein mosaic in several important crops and weeds. Selection pressure exerted by vectors of a particular geographical location is a crucial factor in the evolution of begomoviruses. Excellent reviews are available on the whitefly-transmitted viruses in New World and Old World and their interaction with insect vectors [21, 68, 117–122].

Begomoviruses are vectored by B. tabaci in a persistent and circulative manner (Fig. 2.3). The stylet of B. tabaci penetrates the plant epidermis and moves intracellularly through the parenchyma to reach the phloem, which is required for both virus acquisition and transmission. Whiteflies acquire virions during feeding from the phloem of an infected plant. The virions move through the alimentary canal into the whitefly midgut (Fig. 2.4), where they enter the hemolymph and transit to the salivary glands for transmission during the next feeding cycle [123] (Fig. 2.3).

Fig. 2.3
figure 3

Schematic representation of the circulative route of tomato yellow leaf curl virus (TYLCV) inside B. tabaci. The virus (red particles) is acquired from the plant phloem (p) through the stylet (s). The virus moves along the esophagus (e) and reaches the filter chamber (fc) in the midgut (mg) where the majority of the virus particles are translocated into the hemolymph. This translocation is aided, in part, by the heat shock protein 70 (black particles). In the hemolymph, Hamiltonella, a bacterial symbiont harbored within bacteriocyte cells (yellow organs), produces a GroEL protein (blue particles) that interacts with TYLCV and aids in its safe arrival to the primary salivary glands (psg). Once inside the salivary glands, the virus moves along the salivary duct to be injected into a new plant while the insect is feeding. hg hindgut

Full size image
Fig. 2.4
figure 4

Immunolocalization of TYLCV in the midgut of B. tabaci following acquisition of the virus from infected plants for several days. First antibody was used against the virus CP and secondary antibody attached to Cy3 dye (red). (a) Shows the virus in red, scattered along the midgut and concentrating in the filter chamber (fc). (b) The same midgut as in the left panel showing DAPI staining of the nuclei (blue). am ascending midgut, dm descending midgut, ca ceca, CP coat protein

Full size image

The capsid protein plays a crucial role in the transmission of begomoviruses by B. tabaci. Evidence for a CP role in virus transmission emerged from following observations: (a) exchange of an African cassava mosaic virus (ACMV) CP gene with that of beet curly top virus (BCTV) altered the insect specificity of ACMV from whiteflies to leafhoppers [124]; (b) whiteflies have been shown to be unable to acquire coat protein mutants of geminiviruses that did not form capsids [125, 126]; and (c) replacing the CP of the non-transmissible begomovirus, Abutilon mosaic virus (AbMV), by that of a transmissible isolate of Sida golden mosaic virus (SiGMV), restored transmission by B. tabaci [127].

Site-directed mutagenesis in the CP domain was used to map the functional regions in tomato yellow leaf curl Sardinia virus (TYLCSV) which are involved in the transmission of the virus by B. tabaci [128, 129]. Briefly, a region between amino acids 129 and 152, including Q129, Q134, and D152, was found to be relevant for virion assembly, systemic infection, and transmission by the vector. In addition, it was shown that crossing the salivary gland barrier may not be sufficient for transmission. These results were confirmed in other bipartite begomoviruses, namely, watermelon chlorotic stunt virus (WmCSV) [130] and AbMV [131]. Using immunogold labeling studies, the CP mutants PNHD and PNHE were detected in B. tabaci salivary glands similar to the wild-type virus, but the mutant QDQD CP was not detected in salivary gland cells. The most interesting result was that although the mutants PNHD and PNHE were present in primary salivary gland, transmission did not occur, suggesting that their presence in salivary gland does not ensure transmission. Thus, molecular interactions with saliva might be necessary to maintain infectivity, which, perhaps, are absent from these mutants [129].

Further studies to unravel the importance of TYLCV CP in virus transmission showed its interaction with a member of the small heat shock protein family (BtHSP16), which was identified using a yeast two-hybrid system screen against TYLCSV CP [132]. Another study recently demonstrated that another heat shock protein, HSP70, interacts with the TYLCV CP in vivo and in vitro, and membrane feeding with anti-HSP70 antibodies resulted in an increase in TYLCV transmission. This result suggested that under normal conditions HSP70 restricts virus activity, thereby protecting the insect from deleterious effects of the circulating virus [133]. Interactions with these proteins may be necessary for refolding of the virion particle and facilitating its crossing the barrier between the midgut and the hemolymph or hemolymph and the salivary glands.

2.7 Interaction of B. tabaci with Plants Using Genomics Tools

Many plants have developed various chemical defenses to deter or combat insect pests. To date, some genomic studies were conducted for understanding the plant responses to B. tabaci. This has limited our understanding of the plant response to hemipteran species in general. Hemipterans cause minimal cellular wounding, and although the duration of feeding can be very long, the molecular plant responses can be minimal; however, they can also be dramatic [134]. Salicylic acid (SA) and jasmonic acid (JA) are two important components in the signal transduction cascades that regulate plant defense responses against biotic stresses. JA affects plant resistance to necrotrophic pathogens and tissue-damaging insects as well as some phloem-feeding insects [135]. Other plant defense proteins such as proteinase inhibitors (PI) [136, 137], PI II [138–140], LOX genes [140], and others are inducible during insect feeding.

To understand the major plant defenses in responding to feeding by the whitefly, studies were conducted with the model plant Arabidopsis thaliana [141, 142]. Zarate et al. [141] reported that the whitefly induces SA defenses and suppresses effectual JA defenses. Supporting this study, using the Affymetrix ATH1 GeneChip to monitor the A. thaliana transcriptome, 700 transcripts were found to be upregulated and 556 downregulated by feeding of the silverleaf whitefly nymphs [142]. Closer examination of the regulation of secondary metabolite (glucosinolate) and defense pathway genes after feeding showed that responses were qualitatively and quantitatively different from chewing insects and aphids. Furthermore, the JA genes PDF1.2, PR4, and CORI3 were repressed and SA-regulated genes were induced after whitefly feeding. Cytological staining of whitefly-infected tissue showed that pathogen defenses, such as localized cell death and hydrogen accumulation, were not observed [142].

Few studies have shown that virus proteins can act as suppressors for plant defense systems which are directed to block or reduce defenses against insect feeding. Coinfection of the begomovirus TYLCCNV and its tomato yellow leaf curl China virus betasatellite (TYLCCNB) could repress the JA response but not the SA response [143]. Microarray analysis of the A. thaliana transcriptome in response to cabbage leaf curl virus (CabLCV) infection uncovered 5365 differentially expressed genes; data mining revealed that CabLCV triggers a pathogen response via the SA pathway and induces expression of genes involved in programmed cell death, genotoxic stress, and DNA repair [144]. Further, coinfection of plants by TYLCCNV and TYLCCNB increased expression of the whitefly vitellogenin gene [145]. Recently, Shi et al. [146] demonstrated that SA content was always higher in leaves infested with the viruliferous B biotype compared with the viruliferous Q biotype. Additional recent studies by Shi et al. [147, 148] showed that B. tabaci Q carrying TYLCV strongly suppressed host plant defenses.

2.8 Interactions of B. tabaci with Parasitoids and Pathogens Using Genomic Tools

Parasitoids that specifically attack B. tabaci have been used commercially for the biocontrol of whiteflies. Most whitefly parasitoids kill the insect by piercing the body contents and feeding on host fluids. Parasitoids from the genera Encarsia, Eretmocerus (Aphelinidae), and Amitus (Platygastridae) can attack whiteflies [149, 150]. Recent studies identified Encarsia sophia and Eretmocerus hayati (Hymenoptera: Aphelinidae) as two key parasitoids of B. tabaci (MEAM1) [151, 152], and En. formosa [153], for which it was suggested that begomoviruses transmitted by the whitefly can manipulate the host suitability of a parasitoid and hence the parasitoid-host interactions.

Mahadav et al. [84] investigated the response of B. tabaci to parasitization by the wasp, E. mundus, using a whitefly cDNA-based microarray. The results clearly indicated that genes known to be part of defense pathways described in other insects are also involved in the response of B. tabaci to parasitization by E. mundus. Some of these responses include repression of a serine protease inhibitor (serpin) and induction of a melanization cascade. Quantitative real-time RT-PCR (qRT-PCR) and fluorescence in situ hybridization (FISH) analyses showed that the proliferation of Rickettsia, a facultative secondary symbiont, was strongly induced upon initiation of the parasitization process [84].

Recently, using RNA-seq technology, Zhang et al. [154] conducted a comprehensive investigation of the whitefly defense response to infection by Pseudomonas aeruginosa. Compared to uninfected whiteflies, 6- and 24-h post-infected whiteflies showed 1348 upregulated and 1888 downregulated genes that were differentially expressed. Functional analysis of these genes revealed that the mitogen-associated protein kinase (MAPK) pathway was activated after P. aeruginosa infection. Additional research included investigating the response of B. tabaci to the entomopathogenic fungus, Beauveria bassiana, using next-generation sequencing, which showed that conserved pathways such as the DNA repair and MAPK pathways are responsive to attack by this fungus [155].

2.9 Concluding Remarks and Future Perspective

B. tabaci is undoubtedly one of the major worldwide insect pests in agriculture. Despite many years of research, conventional chemical control methods for reducing the damage caused by this pest are still dominating. The past two decades have witnessed a dramatic increase in the research aimed toward understanding the interactions of B. tabaci with its environment and other organisms, including those that are part of its damage cycle such as plant viruses. However, those interactions also include plants, parasitoids, symbionts, and abiotic factors such as heat. The use of newly developed molecular and genomic tools will facilitate our understanding of the genetic makeup of the whitefly and will enable further development of novel tools for better means of controlling this insect pest.