<- Hanokh Czosnek Home
The Whitefly Functional Genome Project
The 2006 'White Paper' Committee: Scott Atkins, Judith K. Brown (Co-Chair),
Laura Boykin, Henryk Czosnek (Chair), Paul De Barro, Don Frohlich, Murad Ghanim, Margarita
Hadjistylli, Abdel Hanafi, Moshe Lapidot, Cindy McKenzie, Shai Morin, Jane Polston, Rosemarie
Rosell, Robert Shatters (Co-Chair), Xiomara Sinisterra, and William Wintermantel.
1. Rationale
The whitefly Bemisia tabaci, a major insect pest of agricultural crops
The past two decades have witnessed a dramatic increase in the destructive impact of the
whitefly Bemisia tabaci in cotton-vegetable cropping systems worldwide, owing to its
ability to cause feeding damage and to serve as a vector for several families of plant
viruses (Brown et al., 1995; Brown and Czosnek, 2002). Whiteflies are distributed in
subtropical and tropical latitudes. The B. tabaci complex spans the dry tropics and
subtropics, and mild climates regions, including deserts, which experience seasonal rainfall.
To inhabit this diverse range of conditions, whitefly instars survive in an arrested state of
development during cold periods, and the whitefly developmental rate accelerates with
increasing temperature. The insect is able to survive long distance transportation of
agricultural products and plants (Caciagli, 2007). More than 500 crop plants are colonized by
B. tabaci (Cock 1993). A female whitefly can lay 300-400 eggs in her four-weeks
lifespan (Byrne et al. 1990). With a propensity to exploit current agricultural practices,
this once little-known insect has become a near-celebrity as a superbug both throughout
the subtropics and in temperate zones with mild climates that support multiple cropping
cycles per year.
Sibling Species Group
B. tabaci is considered by most to comprise a species complex, some of which are
recognized as distinct biological types' (Brown et al., 1995; Frohlich et
al., 1999). Phenotypic variability in the B. tabaci complex is manifest in a
variety of ways. Variability can be observed as differences in plant host-preference, host
range, fecundity, dispersal behavior, vector competency, phytotoxic feeding effects,
endosymbiont composition, invasiveness, and insecticide resistance, among others.
At a genetic level, comparisons based on the mitochondria cytochrome oxidase I gene (mtCOI)
have revealed an unexpected degree of variation within this gene, which varies within the
group by as much 26%. This knowledge has aided in the delineation of at least four major
phylogeographic clades, and within these are sister clades that generally group with a basis
in phylogeography. However, certain haplotypes do not group as such. In these cases it is
either known or suspected that human movement has been responsible for the haplotype
relocation (Berry et al. 2004; Viscarret et al. 2003).
Reproduction
B. tabaci is haplodiploid. Fertilized eggs give rise to diploid females, whereas,
males are produced from unfertilized eggs. Haplodiploidy is not uncommon among insects, and
in fact is found in all Hymenoptera including ants, wasps and bees. Homopterans employ
diverse types of reproductive systems. Many utilize parthenogenesis e.g. aphid mothers
produce live, clonal offspring without mating. In contrast, whiteflies and most other
Homoptera produce eggs that are attached to the leaf surface, giving rise to the first instar
referred to as a crawler. Whitefly crawlers seek a suitable vascular bundle as their feeding
site, and following a molt, they become sessile. After several molts they eclose as adults.
In this system diploid (2N) females are produced from fertilized eggs, while haploid (1N)
males are produced from unfertilized eggs (N=10, Blackman and Cahill 1998). When the number
of males in a population decline, fertilization is reduced, causing an increased number of
male offspring to be produced in the next generation. Hence, sex ratio bias is frequently
reported for alternate generations of this whitefly (Byrne et al. 1990; Gill 1990). Changes
of sex ratios (from predominantly males to predominantly females) have been reported to occur
during the tomato-growing season in relation to Tomato yellow leaf curl virus
infection in the Jordan Valley (Cohen et al. 1988).
Begomovirus transmission
B. tabaci transmits many begomoviruses (Bedford et al., 1994), a family of
plant viruses with ~2.8 kb circular ssDNA encapsidated in a 24x30 nm geminate particle.
Begomoviruses are transmitted in a circulative manner. The organs and cells involved in
begomovirus transmission have been described (Ghanim et al., 2001a; Brown and Czosnek,
2002) and the rate of transmission of begomoviruses has been analyzed (Rosell et al.,
1999; Ghanim et al., 2001b; Czosnek et al., 2002). Begomoviruses are
transmitted by B. tabaci in a circulative manner. Virus particles ingested through the
stylets enter the esophagus and the filter chamber, are transported through the gut into the
hemocoel, reach the salivary glands and are finally egested during feeding, about 8-12 h
after the beginning of an acquisition access period (Rosell et al. 1999; Ghanim et al.
2001a). Path and velocity of translocation constitute intrinsic properties of the vector, not
of the virus. Once ingested by the whitefly, viral particles cross the gut barrier to the
hemolymph and engage a GroEL homologue 60S heat shock protein encoded by a primary
endosymbiont (Morin et al. 1999; 2000). The HSP60 may serve to stabilize virions
during membrane transit and/or to mask antigenic sites on the capsid to thwart host
recognition while en route in the hemolymph to the salivary glands (van den Heuvel et
al. 1994).
Several whitefly species e.g. Trialeurodes vaporariorum are capable of ingestion, but
do not transmit begomoviruses. In such species, the barrier to transmission occurs at the
gut/hemocoel interface (Rosell et al., 1999; Czosnek et al., 2002). Similarly,
several begomoviruses such as Abutilon mosaic virus AbMV have lost the ability to be
transmitted by B. tabaci; once ingested, virions accumulate in the gut and do not
cross the gut/hemocoel membrane interface (Morin et al., 2000; Czosnek et al.,
2002). Three amino acids in the coat protein of AbMV are responsible for the
non-transmissibility of this virus (Hhnle et al., 2001). The putative receptors that
mediate begomovirus gut-hemocoel translocation have not been identified.
Whitefly-begomovirus interactions
Independent but converging information suggests that the whiteflies and begomoviruses have
interacted over geological times (Czosnek, 2007). First, an ancestor of the modern whitefly
has been found in ~130 MY old amber in Lebanon (Schlee 1970). Second, geminiviral DNA
sequences are known to be present in the genome of tobacco plants, probably owing to
illegitimate recombination during Nicotiana speciation, about 25 MY ago (Bejarano et
al. 1996). Third, the endosymbiotic bacteria that produce the GroEL homologue heat shock
protein necessary for the survival of begomoviruses in their insect vector (Morin et al.
1999), have been probably been associated with whiteflies for over a 100 million years
(Baumann et al. 1993). And fourth, with the drift of continents, whitefly-begomovirus
complexes have evolved into geographically separated, co-adapted virus-vector complexes
(Bradeen et al. 1997). It is inevitable that during this long-lasting virus-vector
relationship, the virus on the one hand has evolved a shape that ensures both its survival
and probably the most efficient transmission by its whitefly vector, and on the other hand,
the vector has evolved strategies to avoid possible deleterious effects of the virus.
Despite the co-adaptation of B. tabaci with begomoviruses for which it serves as the
exclusive vector (Brown and Idris 2005), the circulation of virions in the insect body does
not appear to have a neutral outcome. Several lines of evidence inspire this rationale: 1.
Begomoviruses such as TYLCV and ToMoV influence whitefly fecundity (McKenzie et al. 2002;
Rubinstein and Czosnek 1997). 2. Virions translocate in a circulative manner requiring
receptor-mediated translocation (Czosnek et al. 2002). 3. TYLCV, but not ToMoV,
produces viral transcripts within the whitefly (Sinisterra et al. 2005). 4. TYLCV
induces the synthesis of whitefly antiviral factors (Cohen 1967; Cohen and Marco 1970). 5.
The TYLCV coat protein has a nuclear targeting signal allowing it to penetrate insect cell
nuclei (Kunik et al. 1998).
Begomoviruses studied thus far, including Tomato yellow leaf curl virus(TYLCV), may be
retained in their whitefly vector for several weeks (e.g. Tomato yellow leaf curl Sardinia
virus TYLCSV, Caciagli et al. 1995; Jiang et al. 2000) or for the entire life of the
vector (e.g. TYLCV and Tomato yellow leaf curl Chinese virusTYLCCNV, Rubinstein and
Czosnek 1997; Jiu et al. 2007). The long-term association of begomoviruses with B.
tabaci has been shown to have consequences on host longevity and the fertility. The life
span of female whiteflies (A biotype) fed for 24 h on the bipartite Squash leaf curl
virus (SLCV)-infected cucurbits was in average 25% shorter than that of whiteflies fed on
the same virus source for 4 h only (Cohen et al. 1989). The deleterious effects of the direct
association between the whitefly vector and TYLCV was established by comparing longevity and
fertility of viruliferous and non-viruliferous insects reared on cotton (a virus non-host
plant), following a short exposure to TYLCV-infected tomato plants (Rubinstein and Czosnek
1997). The life span of the viruliferous insects was shorter by 5 to 7 days compared to that
of non-viruliferous whiteflies (out of 28 to 32 days). A decrease of 25 to 50% in the number
of eggs laid (depending on the age of the adult) by viruliferous whiteflies was found.
Similar observations showing a decrease in longevity and fertility were made in China with
two biotypes fed on tomato plants infected with TYLCCNV: the invasive B and local ZHJ1 (Jiu
et al. 2007). Both biotypes were similarly affected by TYLCCNV (40% reduction in longevity
and 35 % in the number of eggs). In contrast to TYLCV and TYLCCNV, the bipartite begomovirus
Tomato mottle virus (ToMoV) actually increased egg production of the B biotype vector
(McKenzie, 2002). Whiteflies that acquired ToMoV produced more eggs than their
non-viruliferous counterparts. Hence some begomoviruses have deleterious effects on the
insect host, while others may actually increase fitness.
B. tabaci biotypes as a model for ongoing speciation
The origin of B. tabaci biotypes is intriguing. The question of whether they
constitute the same or different species is a heated debate. Mating and production of viable
F1 generations that produce fertile offspring is the common denominator for several
definitions of species. Sexual selection followed by mating incompatibility is one of the
mechanisms of sympatric speciation; that is, the formation of two or more descendant species
from a single ancestral species all occupying the same geographic location (Kirkpatrick and
Ravign 2002).
Mating between whitefly biotypes has been intensively studied and results are not always
definitive. Several inter-biotype crossings that resulted in unsuccessful F1 hybrids were
documented. For example A and B biotypes from the United States were crossed without
producing an F1, suggesting a lack of gametic transfer in this haplodiploid insect (Costa et
al. 1993; Perring et al. 1993; Perring and Symmes 2006). Further studies with the B
biotype provided evidence of reproductive incompatibility among members of the B.
tabaci complex. Reciprocal crosses performed between biotype B from the United States,
biotype K from Pakistan, biotype M from Turkey, biotype D from Nicaragua and biotype ZHJ1
from China resulted in no F1 hybrids (Liu et al. 2008). Compatible crosses among B.
tabaci biotypes have been documented as well. Hybrids have been identified in crosses
between biotype B from the United States and biotype L (SUD) from Sudan (Byrne et al. 1995).
Similar results were recorded between a native Australian population and biotype B (Gunning
et al. 1997) and between biotypes B and Q in Spain (Ronda et al. 2000). In the later example,
the authors did not indicate whether the F1 hybrids were fertile. Contrary to these findings,
fertile F1 hybrids between biotype A, biotype B, and the Jatropha biotype have been reported
(Caballero et al., 2001, 2008). In Israel, although B and Q biotypes coexist in the same
regions, B/Q F1 has not been reported (Horowitz et al. 2003; 2005). Recently, begomovirus
transmission has been described to occur during mating between females and males of the same
biotype, whether B or Q. However, no transmission was found when male Q and female B were
caged together, and vice versa, indicating a mating incapability (Ghanim and Czosnek 2000;
Ghanim et al. 2007b). It is clear from these results that various levels of reproductive
incompatibility operate in the B. tabaci sibling species group. It has been suggested
that the incompatibility found between the Q and B biotypes may be caused by a different
bacterial symbiont load (Cheil et al. 2007). Prezygotic mechanisms also can drive
reproductive incompatibility, and one such mechanism that may be operational in the B.
tabaci species complex is the mate recognition system (Butlin 1995). Interestingly,
several researchers observed that although males and females of distinct B. tabaci
populations did not mate, they engaged in extensive courtship behavior (De Barro and Hart
2000; Maruthi et al. 2004, Perring and Symmes 2006). Thus some haplotypes may share essential
behaviors in common, while others share only some in common but lack the necessary signals
that result in mating between the variants. These findings do not provide any definite answer
to whether B and Q biotypes have a pre- or post-zygotic barrier.
2. Some questions to be asked
Sequencing the whitefly genome
Ultimately, we wish to isolate, sequence, and study the function of the genes that enable
B. tabaci to manifest a range of phenotypes deleterious to agricultural systems, such
as are aggressiveness, invasiveness, extreme fecundity, and polyphagy, coupled with its
ability to vector begomoviruses infecting many food and ornamental crops. Availability of the
complete genome sequence is crucial for facilitating valuable genomics, proteomics, and
functional genomics applications. As genomics, proteomics, and metabolomics research is
expanded beyond model organisms, the methodologies developed through studies of model
arthropods are becoming available for application to non-model systems, many of which are
important agriculture pests. Directing these technologies to solving applied problems offers
new opportunities for developing approaches to reduce the damage they cause as agricultural
pests.
Damage reduction
Elucidating the functional genomics of the B. tabaci complex has become essential for
devising novel, sustainable pest control strategies, and directed interference of
whitefly-mediated virus transmission. Whitefly genomics research is expected to open
important avenues into the discovery of novel strategies for whitefly management based in an
improved understanding of molecular, cellular, and biological processes. The genome sequence
will synergize projects underway to develop and sequence B. tabaci expressed sequence
tags (EST) or cDNA libraries for functional genomics and proteomics analysis. The benefits
are far reaching and include their application to identify genes that combat abiotic and
biotic stresses that often leads to invasiveness and insecticide resistance, and to
understand the basis for whitefly-virus specificity.
An annotated genome sequence, together with microarray capability and other functional
genomics tools, will facilitate the analysis of whitefly genetics and of metabolic pathways.
It will help elucidate the functional characterization of genes, their differential
expression, the localization of their transcripts in situ, and the determination of
the proteome.
Whitefly-begomovirus interactions
The circulation of virions in the whitefly vector body has been shown to influence the
expression of specific whitefly genes. However, only hints of these interactions have been
uncovered to date, suggesting that many more will be discovered using a functional genomics
approach. These genes may represent targets against which novel strategies can be devised for
blocking the virus-host interaction. Functional genomics will be instrumental in 1) studying
the interactions underlying the circulative transmission of begomoviruses within vector and
non-vector whitefly species, 2) identifying the cellular determinants involved in
transmission, and 3) deciphering the evolutionary history of begomovirus-whitefly complexes.
Speciation
A sequence comparison of the DNA sequences of the nuclear and organelle genomes, and of the
endosymbiotic bacteria of the major B. tabaci biotypes, associated with comparative
functional genomics studies, e.g. by heterologous hybridizations on a B biotype microarray,
may shed some light on the question of B. tabaci speciation, and of the mechanism of
speciation in general.
Development
Whiteflies develop from eggs through four nymphal stages. Immature stages begin with a
pointed oblong yellow egg (0.2 mm) which darkens at the apex just before hatching. First
instar nymphs or crawlers (0.2-0.3 mm), which hatch from eggs walk a short distance, settle,
inserting their mouthparts into leaf tissue and feed on the phloem. The crawler goes through
three more molts as a sessile, flattened oval nymph. Late third and fourth instars develop
red-eye spots. The last nymph, or pupal stage (0.7-0.8 mm) has distinct eye spots and visible
symbionts. The adult is 0.9 - 1.2 mm in length. The patterns of gene expression that occur
during whitefly development from the egg to adult are virtually unknown. The genes underlying
the passage from one stage to another and those inducing adult emergence are not known. The
B. tabaci developmental pattern can be compared with that of other insects such as
Drosophila (Furlong et al. 2001).
3. Knowledge Base and Available Tools for the Project
Expressed sequence tags
To address the general shortage of genomic sequence information, three cDNA libraries have
been constructed: one from non-viruliferous whiteflies (eggs, immature instars, and adults)
and two from adult insects that fed on tomato plants infected by two geminiviruses: TYLCV and
ToMoV (Leshkowitz et al. 2006). In total, a sequence of 18,976 clones was determined. After
quality control and removal of clones of mitochondrial origin, 9,110 sequences remained which
included 3,843 singletons and 1017 contigs. The number of sequences from the libraries that
were assembled into contigs and singletons were: eggs (201), instars (1816), non-viruliferous
adults (2093), TYLCV-viruliferous adults (2704), and ToMoV viruliferous adults (2296).
In addition, approximately 1,000 bases aligned with the genome of the B. tabaci
endosymbiotic bacterium Candidatus Portiera aleyrodidarum, originating primarily from
the egg and instar libraries. Genes were identified representing important biological
processes, including membrane transport, sub-cellular trafficking, protein translation, and
innate immunity, development and growth, and abiotic and biotic stresses.
The sequences have been posted in GenBank. They can be found in the site of the "Whitefly
Genome Project": URL:
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genomeprj&cmd=ShowDetailView&TermToSearch=18077 (Figure 1).
The accession numbers are as follows: from egg library EE602518 to 602718, from instar
library EE602719 to EE604534, from non-viruliferous adult EE595518 to EE597607, from ToMoV
viruliferous adult EE597608 to EE599906, and from TYLCV viruliferous adults EE599905 to
EE602517. More information on these sequences can be found in three files:
1) Sequences and the contigs they assembled:
www.biomedcentral.com/content/supplementary/1471-2164-7-79-S1.xl
2) Contigs and singletons information (names, library count, length, etc.):
www.biomedcentral.com/content/supplementary/1471-2164-7-79-S2.xl
3) Top BLAST hit for each contig and singleton against the databases searched:
www.biomedcentral.com/content/supplementary/1471-2164-7-79-S3.xl
Recently normalized adult and gut EST libraries have been constructed for the B biotype and
454 sequencing (454 Life Sciences, Branford CN) of them is presently underway. These new data
are expected to substantially enrich the EST resources by providing the first near-complete
transcriptome for B. tabaci (Brown et al. unpublished). This partial transcriptome
will serve as a useful tool for whole genome annotation. Thus, we anticipate that these past
and present projects will lead to the full genome sequencing of the first tropical
homopteran.
Spotted cDNA microarrays
Based on the sequence of the ESTs (see above), a first microarray has been designed that
contains 6,000 entries and includes all contigs and singletons (see Figure 2). This
microarray has proven to be an excellent tool to study gene expression during whitefly
development, circulative transmission of TYLCV, comparison between B. tabaci biotypes
and parasitization by natural enemies (Mahadav et al. 2008, 2009).
Gene silencing
The function of B. tabacigenes can be determined by a reverse genetics approach.
Introduction of double-stranded RNA (dsRNA) into living cells or organisms can cause
silencing of specific genes and disruption of protein expression. In insects, significant
reduction in gene and protein expression was demonstrated in the fruitfly Drosophila
melanogaster, the mosquito Anopheles gambiae, the caterpillar Manduca
sexta, the milkweed bug Oncopeltus fasciatus, the cockroach Periplaneta
Americana, the triatomine bug Rhodnius prolixus, the light apple brown moth
Epiphyas postvittana, the ladybird beetle Harmonia axyridis and the pea aphid
Acyrthosiphon pisum (see references in Ghanim et al. 2007a). Gene silencing was
achieved in B. tabaci following injection of dsRNA between the mesothorax and the
metathoraxof newly emerged adults. The dsRNA molecules were based on exonic sequences derived
from genes specifically expressed in B. tabaci organs. The treatment resulted in
specific and significant decrease in gene expression. Furthermore, injection of dsRNA
targeting the Drosophila chickadee gene (which encodes a homolog of profilin, a small
actin binding protein) caused severe disruption of the normal B. tabaci oocyte
development, suggesting a similar function in the two insects. These experiments indicated
that it is possible to silence B. tabaci genes by microinjection of dsRNA. It might be
possible to achieve gene silencing by expressing the siRNA in host plants on which whiteflies
are feeding, as shown for several other insects.
4. The whitefly transcriptome
Genome Size of the Whitefly, B. tabaci
The nuclear DNA content of B. tabaci B biotype was estimated using flow cytometry. The
DNA content males and females was 1.04 and 2.06 pg, respectively (Brown et al. 2005).
The conversion between DNA content and genome size (1 pg DNA = 980 Mb) indicated that the
haploid genome of B. tabaci is 1,020 Mb, or about five times that of D.
melanogaster [Diptera].
It is likely that B. tabaci has a number of genes close to those of
Drosophilaand of other sequenced insect genomes, ca. 15,000 (Holt et al. 2002).
Therefore much of the whitefly genome may be found to comprise non-coding, sometimes
repetitive, DNA. Sequencing the whitefly genome will allow the user community to determine
the order of genes in whitefly euchromatin regions. This will allow direct comparisons with
that of fully sequenced genomes of other insects, facilitate the first estimations of degrees
of synteny between the whitefly and other model insect genomes, and assessment of the
likelihood that these models can serve as backbones for assembling the whitefly genome. It
also will facilitate calculations of gene density in the whitefly genome, differentiate the
various repetitive DNA families and note their dispersion in the genome. For example, in
human and mouse (3 Mbp, 30,000 genes) a gene is found every 100,000 bp, while a gene is found
every 9,000 bp in Drosophila (0.14 Mbp, 14,100 genes) and 4,000 bp in
Arabidopsis (0.11 Mbp, 25,500 genes). Hence it can be estimated that in B.
tabaci the gene density is 1/60,000 bp; this estimation is an average since in these
organisms the genes are usually clustered and the repetitive elements are mostly found in the
heterochromatin near the centromeres and telomeres. In Drosophila, about 15% of the
genome is made up of transposons, and more than 30% is satellite DNA mostly on one chromosome
(Dimitri et al. 2005). On the other hand it will point to gene clustering on chromosomes.
Finally the genome sequence will allow us to determine the size and the mean number of
introns/exons in whitefly genes, for example, in Drosophila, there are 2,470
intronless genes. On average there are about 2.5 introns per gene, compared to 4 in humans,
and the average size of exons and introns are ~230 bp and ~630 bp, respectively (Strachan and
Read 2004).
The availability of the sequence of the B. tabaci nuclear genome, combined with
bioinformatics comparisons with sequences from other whitefly biotypes and species, and other
insects will provide estimates of 1) the degree of synteny between these insects, 2) gene
density in the whitefly genome, 3) the mean number of introns/exons in whitefly genes, 4)
characterize the various repetitive DNA families and their mode of dispersion in the genome
(Sakharkarand Kangueane, 2004).
Activation and repression of B. tabaci stress-response genes during insecticide
application, following parasitization by natural enemies and at high temperatures
Whiteflies are continuously exposed to biotic (e.g. insecticides, natural enemies, host plant
chemicals) and abiotic (climate) stresses. The genes involved in responses to stress are
hypothesized to be shared across diverse members of the Animal Kingdom (Salvucci et al.
2000). Their pattern of expression can be investigated using the spotted whitefly cDNA
microarray and confirmed by real time PCR. These genes could be targeted once their sequence
is known, and their activity could be modulated by gene silencing using siRNA-producing host
plants (see below).
Insecticide application
The heavy application of insecticides is responsible for the development of resistance
(Denholm et al. 1998). Populations of B. tabaci Q biotype resistant to
pyriproxyfen recently emerged in Israel, displacing the endogenous, less resistant, B
biotype (Horowitz et al., 2005) Pyriproxyfen is an insecticide that acts as a juvenile
hormone analogue and disrupts insect development. Gene expression in pyriproxyfen resistant
B. tabaci Q biotype was studied (Ghanim and Kontsedalov, 2007). Upon insecticide
treatment of resistant whiteflies, many genes involved in detoxification and oxidative stress
were up-regulated.
Parasitization by natural enemies
It is likely that stress induced gene activation and repression during the B. tabaci
parasitization by natural enemies is common to other biotic and abiotic stresses. Although
the whitefly is usually controlled using chemical pesticides, biological control agents
constitute an important component in integrated pest management programs. One of these agents
is the aphelinid wasp Eretmocerus mundus. E. mundus lays its egg on the leaf
underneath the B. tabaci nymph. The egg hatches and the first instar wasp larva
penetrates the host. Initiation of parasitization induces the host to form a cellular capsule
around the parasitoid. Around this capsule, epidermal cells multiply and thick layers of
cuticle are deposited (Gelman et al. 2005). The physiological and molecular processes
underlying B. tabaci-E. mundus interactions have been investigated using the spotted
whitefly cDNA microarray at two time points of the parasitization process: when the
parasitoid larva is at the pre-penetration stage and when it fully penetrates the host
(Mahadav et al. 2008). The results clearly indicated that genes known to be part of the
defense pathways described in other insects and animals (Barton and Medzhitov, 2003) are also
involved in the response of B. tabaci to parasitization by E. mundus. Some of
the responses observed included the repression of a serine protease inhibitor (serpin) and
the induction of a melanization cascade. A second set of genes that strongly responded to
parasitization included bacterial genes encoded by whitefly symbionts. Proliferation of
Rickettsia, a facultative secondary symbiont, was strongly induced following the
initiation of the parasitization process, suggesting that endosymbionts may be involved in
the insect host resistance to various environmental stresses.
High temperatures
Gene expression of B. tabaci B and Q biotypes was compared at normal (25
oC) and high (40 oC) temperatures (Mahadav et al., 2009). Gene
expression under normal temperature showed clear differences between the two biotypes: At
high temperature B exhibited higher expression of mitochondrial genes, and lower
cytoskeleton, heat-shock and stress-related genes, compared to Q. Exposing B biotype
whiteflies to heat stress was accompanied by rapid alteration of gene expression.
5. Sequencing and De Novo Assembly of Whitefly Endosymbiont Genomes-The Microbiome
Unraveling genetic/genomic relationships between the endosymbiont genes/genomes will allow
elucidatation of putative whitefly-endosymbiont genome interactions that are hypothesized to
drive whitefly biotype formation, influence phenotypic plasticity, and select for
invasivenes, monophagy, and begomovirus specificity, among others.
The smallest bacterial genome reported thus comes from Mycoplasma
genitalium, an intracellular parasite of human epithelial cells, and comprises a circular
chromosome of 580 kb with only 470 coding genes.Ca. Buchnera aphidicola is the primary
endosymbiont of the aphids Acyrthosiphon pisum. The genome size is extremely reduced (one
circular chromosome of 641 kb, with only 564 coding genes and two plasmids), compared to its
free-living counterparts, and the genome of the primary whitefly B. tabaciendosymbiont
Ca. Portiera aleyrodidarum is similar in size (Thao and Baumann 2004;Prez-Brocal et
al. 2006).
The genome sizes of the cytoplasmic incompatibility-inducing bacteria
Wolbachiaand Cardinium,vary depending on the host and bacterium.
Wolbachiais closely related to other Rickettsiaand so is expected to be small
and within a manageable range. Only several Ca. Cardinium spp. are known and promise
to be unique, in that they are divergent from Wolbachia(16S rRNA gene) but possibly
have evolved a convergent cytoplasmic incompatibility mechanism (or have acquired genes
similar to Wolbachiaspp. by lateral transfer), both intriguing possibilities.
6. Top Candidate Biotypes for Genome-Transcriptome Sequencing
B biotype
The worldwide introduction of the B biotype was the single most significant impetus behind
the recent molecular phylogenetic and population genetics studies carried out to investigate
extent of variation for this little known cryptic species. The B biotype is the most
aggressive B. tabaci to date and represents extreme polyphagy, is widely distributed
and the best known as an important pest and virus vector. Additionally, it is the only B.
tabaci biotype to cause physiological disorders such as squash silverleaf and tomato
irregular ripening. The B biotype has all of the hallmarks of an invasive species, given its
cryptic nature, propensity for developing insecticide resistance, high fecundity, and
capacity to disperse long distance.
A Biotype
During the 1980's the polyphagous A biotype reached extreme pest status in cotton and
vegetable growing areas in the southwestern U.S. deserts, and was responsible for the first
outbreaks of begomovirus and crinivirus-incited diseases in vegetable crops in the region.
The A type shares many of the same traits with B type but exhibits lower fecundity, more
moderate polyphagy with a distinct host range, is not resistant to the same classes of
insecticides, originated in the New World, and does not disperse long-distance. The A and B
biotypes have identical primary symbionts (Zchori-Fein and Brown 2002). However, the A
biotype is infected by Ca. Cardinium, a putative cytoplasmic incompatibility-inducer,
whereas, the B biotype is not (Caballero et al., 2001). In terms of other
endosymbionts, the A and B biotypes each harbor a distinct suite of secondary symbionts
(Zchori-Fein and Brown 2002) and so these additional genomes will be made available if de
novo methods are used.
Q biotype
The Q-biotype is thought to have originated from the Mediterranean region,
and has been associated with insecticide resistance there. It exhibits resistance to
pyriproxyfen (Horowitz et al. 2003), buprofezin and reduced susceptibility to the
neonicotinoid insecticides imidicloprid, acetamiprid and thimethoxam. While the B biotype is
defined by high fecundity, aggressiveness and wide host range, the Q biotype is known to
develop greater resistance to insecticides (Horowitz et al. 2003; 2005). It also harbors
Ca. P. aleyrodidarum, the obligatory primary symbiotic bacterium of whiteflies, as
well as several secondary symbionts including Rickettsia, Hamiltonella, Wolbachia,
Arsenophonus, Cardinium and Fritschea (Baumann 2005; Gottlieb et al. 2006). The
relative abundance of secondary symbionts was recently determined in populations of B.
tabaci from Israel (Cheil et al., 2007). Hamiltonella was detected only in the B
biotype, while Wolbachia and Arsenophonus were found in the Q biotype.
Rickettsia was abundant in both biotypes, while Cardinium and Fritschea
were not found in any of the populations. The association found between whitefly biotypes and
secondary symbionts suggests a possible contribution of these bacteria to host
characteristics such as insecticide resistance, and virus transmission ability (Cheil et al.
2007). In Spain, the Q biotype transmits TYLCV more efficiently than the B biotype does
(Sanchez-Campos et al. 1999; Jiang et al. 2004).
7. Available Annotation Tools
Model insects
The haploid genome of B. tabaci is estimated to be approximately 1020 Mbp, or 5 times
the size of the Drosophila melanogaster genome. The closest relatives to B.
tabaci for which genome sequences are being worked out are the, pea aphid (furthest
advanced), the green peach aphid, and a cereal aphid (in progress), which are hompterans
(genome size range for Insecta = 0.18-0.89 pg), but represent a different family (Aphididae
vs Aleyrodidae) and region (temporate vs tropical) . These aphid genomes together with
Drosophila, Anopheles, and others will aid significantly in the bioinformatic
annotation.
B. tabaci transcriptome
Additional resources available or under construction for investigating the functional
genomics of B. tabaci and also as a scaffold for annotation of the genome include:
1. EST libraries (contigs available presently number >10,000) generated by the Hebrew
University of Jerusalem, the Volcani Center, USDA-Florida, and the University of Arizona,
(supported by funding from The United States-Israel Binational Agricultural Research and
Development and the Israel Science Foundation).
2. The transcriptome (subtracted EST library) of the B biotype using 454 sequencing, underway
at The University of Arizona (supported by funding from The United States-Israel Binational
Agricultural Research and Development, and The University of Arizona ARL Biotechnology Core
Facility http://biotech.arl.arizona.edu/index.php/core-facilities.html).
3. Microarray resources developed by the Hebrew University of Jerusalem and the Volcani
Center that are presently being employed toward management of the whitefly as an important
agricultural pest.
8. Other genome projects and user communities
The User Community is global
A particularly exciting aspect of this work is the opportunity to expand the reach of
interrelated genomics projects for arthropods, and most specifically other insects, that may
shed new light on all of the organisms, owing to a comparative approach between closely and
distantly related species. Eventually, the combination of tools and informatics developed by
other/the collective members of the Whitefly Genome Consortium can be extended to
other clade-specific arthropod genome projects. Thus, an important synergy has been already
formed with the establishment of the Whitefly Genome Consortium, which consists of a large
number of interested and active members from over 12 countries worldwide with an enthusiastic
interest in this undertaking and its future goals. In total over forty-eight supporting
communications were received from industry (Syngenta, Dupont, Bayer Crop Science, HM-Vilmorin
and Seminis-Monsanto vegetable seed, KeyGene, Cotton Incorporated, Arizona Cotton Growers. A
broad representation among researchers, systematists, virologists, entomologists, producers,
and industry partners, indicated enthusiastic support, and included individuals from
Australia, Brazil, France, Germany, Japan, China, Israel, Egypt, South Africa, Morocco,
Taiwan, Uganda, Spain, Mexico, United Kingdom, and United States (represented by members from
Arizona, California, Florida, and Texas).
Insect Genome Projects
http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Search&db=genomeprj&term="Hexapoda"%5BOrganism%5D:
Examples
of genomes and/or transcriptomes (among 51
current entries) in progress or completed include:Aedes
aegypti, A. albopictus,Anopheles
gambiae str. PEST,Apis
mellifera, Bemisia tabaci,Bombyx
mori,Drosophila
melanogaster,D.
persimilis,D.
pseudoobscura ,D.
sechellia,D.
simulans,D.
yakuba,Helicoverpa armigera, Toxoptera citricida (brown citrus aphid), Homalodisca
vitripennis (glassy-winged
sharpshooter), Acyrthosiphon pisum (pea aphid), Rhodnius
prolixus (vector of Chagas'
disease), Nasonia vitripennis , N. giraulti, N.
longicornis, Triboliumcastaneum (red
flour beetle).
Homopteran endosymbionts
[rtedi.ebc.uu.se/Projects/Buchnera/];{Microbiomes}.
Some relevant websites
IWSN/EWSN Website:http://www.whitefly.org/
Whitefly Discussion Forum: http://www.whitefly.org/whiteflyforum/
Membership & Publications: http://www.whitefly.org/DataEWSN/EWSN-Contacts.asp;http://www.whitefly.org/IWSN_2/1-IWSN-DirectorySearch.asp
About Whiteflies http://gemini.biosci.arizona.edu/whitefly/about_wf/index.htm
North American Whitefly Studies Network http://cals.arizona.edu/nawhiteflystudies/
European Whitefly Studies Network http://www.whitefly.org/
Global Invasive Species Database http://issg.appfa.auckland.ac.nz/database/species/;
Whitefly Collection & Specimen Preparation http://www.sel.barc.usda.gov:591/1WF/wf-prep-instructions.html
Whitefly Knowledgebase-USDA & University of Florida http://whiteflies.ifas.ufl.edu/;
Whitefly IPM Project http://www.tropicalwhiteflyipmproject.cgiar.org/project-results.jsp;
Crop Loss-Cotton http://msucares.com/insects/cotton/profile/whiteflies.pdf;
Tropical
whiteflies www.spipm.cgiar.org/;
www.ars.usda.gov/is/AR/archive/jul98/soft0798.htm;
www.ars.usda.gov/
research/programs/
Control Unwanted Pests-Solutions/Whiteflies www.AntiPest.com
www.ipm.ucdavis.edu/PMG/PESTNOTES/pn7401.html
Aleyrodidae; http://ctd.mdibl.org/voc.go;jsessionid
9. Conclusions
Collectively genomics, proteomics, and functional genomics efforts will initiate further
local, regional, national and international partners to expand present and future efforts
aimed at determining the B. tabaci genome and proceed to undertake functional genomics
aspects that are of high interest amongst a broad user community, but for which sufficient
tools are yet unavailable or inaccessible. This will truly be a case where homopterans, as a
diverse group. The exciting possibility that commercial, academic, and outreach efforts can
benefit from one another is an excellent example of how internationally conducted genomics
projects are able to cross-feed and create more powerful resources than any one project could
accomplish on its own.
10. Acknowledgements
Supported by The United States-Israel Binational Agricultural Research and Development
(awards IS-3479-03 and IS-4062-07, The Israel Science Foundation (award 884/07) and by The
University of Arizona ARL Biotechnology Core Facility.
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12. Figures
Figure 1. Home page of The Whitefly Genome Project at NCBI. URL:
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genomeprj&
cmd=ShowDetailView&TermToSearch=18077
Figure 2. Spotted cDNA representing 6,000 clones from the whitefly B. tabaci B
biotype. The figure shows hybridization of transcripts from the B and Q biotype, using the B
biotype array.
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