Vadim Khasdan

Mating behaviour, life history and adaptation to insecticides determine species exclusion between whiteflies

1. Negative interspecific interactions, such as resource competition or reproductive interference, can lead to the displacement of species (species exclusion). 2. Here, we investigated the effect of life history, mating behaviour and adaptation to insecticides on species exclusion between cryptic whitefly species that make up the Bemisia tabaci species complex. We conducted population cage experiments independently in China, Australia, the United States and Israel to observe patterns of species exclusion between an invasive species commonly referred to as the B biotype and three other species commonly known as biotypes ZHJ1, AN and Q. 3. Although experimental conditions and species varied between regions, we were able to predict the observed patterns of exclusion in each region using a stochastic model that incorporated data on development time, mating behaviour and resistance to insecticides. 4. Between-species variation in mating behaviour was a more significant factor affecting species exclusion than variation in development time. Specifically, the ability of B to copulate more effectively than other species resulted in a faster rate of population increase for B, as well as a reduced rate of population growth for other species, leading to species exclusion. The greater ability of B to evolve resistance to insecticides also contributed to exclusion of other species in some cases. 5. Results indicate that an integrative analysis of the consequences of variation in life-history traits, mating behaviours and adaption to insecticides could provide a robust framework for predicting species exclusion following whitefly invasions.

Large-Scale Survey of Cytosine Methylation of Retrotransposons and the Impact of Readout Transcription From Long Terminal Repeats on Expression of Adjacent Rice Genes

Transposable elements (TEs) represent ∼45% of the human genome and 50–90% of some grass genomes. While most elements contain inactivating mutations, others are reversibly inactivated (silenced) by epigenetic mechanisms, including cytosine methylation. Previous studies have shown that retrotransposons can influence the expression of adjacent host genes. In this study, the methylation patterns of TEs and their flanking sequences in different tissues were undertaken using a novel technique called transposon methylation display (TMD). TMD was successfully applied on a highly copied (∼1000 copies), newly amplified LTR retrotransposon family in rice called Dasheng. We determined that the methylation status of a subset of LTRs varies in leaves vs. roots. In addition, we determined that tissue-specific LTR methylation correlated with tissue-specific expression of the flanking rice gene. Genes showing tissue-specific expression were in opposite orientation relative to the LTR. Antisense transcripts were detected in the tissue where the sense transcripts from that gene were not detected. Comparative analysis of Dasheng LTR methylation in the two subspecies, japonica vs. indica revealed LTR-mediated differences in subspecies gene expression. Subspecies-specific expression was due either to polymorphic Dasheng insertion sites between the two subspecies or to subspecies-specific methylation of LTRs at the same locus accounted for observed differences in the expression of adjacent genes.

Genetic and Epigenetic Dynamics of a Retrotransposon after Allopolyploidization of Wheat

Allopolyploidy, or the combination of two or more distinct genomes in one nucleus, is usually accompanied by radical genomic changes involving transposable elements (TEs). The dynamics of TEs after an allopolyploidization event are poorly understood. In this study, we analyzed the methylation state and genetic rearrangements of a high copied, newly amplified terminal-repeat retrotransposon in miniature (TRIM) family in wheat termed Veju. We found that Veju insertion sites underwent massive methylation changes in the first four generations of a newly formed wheat allohexaploid. Hypomethylation or hypermethylation occurred in ∼43% of the tested insertion sites; while hypomethylation was significantly predominant in the first three generations of the newly formed allohexaploid, hypermethylation became predominant in the subsequent generation. In addition, we determined that the methylation state of Veju long terminal repeats (LTRs) might be correlated with the deletion and/or insertion of the TE. While most of the methylation changes and deletions of Veju occurred in the first generation of the newly formed allohexaploid, most Veju insertions were seen in the second generation. Finally, using quantitative PCR, we quantitatively assessed the genome composition of Veju in the newly formed allohexaploid and found that up to 50% of Veju LTRs were deleted in the first generation. Retrotransposition bursts in subsequent generations, however, led to increases in Veju elements. In light of these findings, the underlying mechanisms of TRIM rearrangements are discussed.

Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing toxin genes from Bacillus thuringiensis subsp. israelensis

Genes encoding the mosquito larvicidal toxins Cry4Aa, Cry11Aa, Cyt1Aa and the regulatory P20 from Bacillus thuringiensis subsp. israelensis were introduced into the nitrogen-fixing, filamentous cyanobacterium Anabaena PCC 7120 for expression under control of two strong promoters P(psbA) and P(A1). The clone pRVE4-ADRC displayed toxicity against fourth-instar larvae of Aedes aegypti, the highest ever achieved in cyanobacteria. It was about 2.5-fold more toxic than the respective clone without cyt1Aa [Wu et al., Appl. Environ. Microbiol. 63 (1997) 4971-4975]. Cyt1Aa synergized the combination of Crys by about five-fold. Consistently, the lethal times exerted by pRVE4-ADRC were also reduced (it killed exposed larvae more quickly). This clone may become a useful biological control agent which reduces the probability of resistance development in the target organisms [Wirth et al., Proc. Natl. Acad. Sci. USA 94 (1997) 10536-10540].

Variations in the mosquito larvicidal activities of toxins from Bacillus thuringiensis ssp. israelensis.

Comparing activities of purified toxins from Bacillus thuringiensis ssp. israelensis against larvae of seven mosquito species (vectors of tropical diseases) that belong to three genera, gleaned from the literature, disclosed highly significant variations in the levels of LC(50) as well as in the hierarchy of susceptibilities. Similar toxicity comparisons were performed between nine transgenic Gram-negative species, four of which are cyanobacterial, expressing various combinations of cry genes, cyt1Aa and p20, against larvae of four mosquito species as potential agents for biological control. Reasons for inconsistencies are listed and discussed. Standard conditions for toxin isolation and presentation to larvae are sought. A set of lyophilized powders prepared identically from six Escherichia coli clones expressing combinations of four genes displayed toxicities against larvae of three mosquito species, with levels that differed between them but with identical hierarchy.

Developmental timing of DNA elimination following allopolyploidization in wheat

The elimination of DNA sequences following allopolyploidization is a well-known phenomenon. Yet, nothing is known about the biological significance, the mechanism, or the precise developmental timing of this event. In this study, we have observed reproducible elimination of an Aegilops tauschii allele in the genome of the second generation (S2) of a newly synthesized allohexaploid derived from a cross between Triticum turgidum and Ae. tauschii. We show that elimination of the Ae. tauschii allele did not occur in germ cells but instead occurred during S2 embryo development. This work shows that deletion of DNA sequences following allopolyploidization might occur also in a tissue-specific manner.

Synergy Between Aedes aegypti Trypsin Modulating Oostatic Factor and δ-Endotoxins

Starved first instar Aedes aegypti larvae were 35-fold more sensitive to Bacillus thuringiensis subsp. israelensis (Bti) toxins than fed larvae. Feeding larvae Pichia pastoris yeast cells expressing tmfA (synthetic gene coding for the Trypsin Modulating Oostatic Factor of Ae. aegypti) together with Escherichia coli cells expressing Bti toxin genes (cry4Aa, cry11Aa, cyt1Aa and p20) indicate that TMOF and Cry toxins are synergisitic. tmfA was cloned and expressed in the cyanobacterium Anabaena PCC 7120 and the hormone was purified by HPLC and identified by ELISA. The amount of TMOF synthesized by Anabaena was low (0.5 - 1 μg in 10 8 cells). P. pastoris, which synthesizes high amounts of heterologous proteins in the presence of methanol and is readily consumed by mosquito larvae, was genetically engineered to produce more TMOF. Codon-optimized synthetic genes, cry11Aa- tmfA and gst-cry11Aa- tmfA, that were cloned into P. pastoris and fed to Ae. aegypti larvae caused 87.5% mortality in 5 days. GST (glutathione-S-transferase) enhanced the activity of Cry11A-TMOF and protected it from heat denaturation. Cell free extracts of recombinant P. pastoris cells killed 40% of tested 4 th instar larvae within 24 h, and mass spectra analysis confirmed that the recombinants synthesize Cry11Aa. This report shows for the first time that Cry toxins and TMOF are synergists to Ae. aegypti larvae when jointly fed or expressed in recombinant P. pastoris.

Transposable elements generate population-specific insertional patterns and allelic variation in genes of wild emmer wheat (Triticum turgidum ssp. dicoccoides)

BackgroundNatural populations of the tetraploid wild emmer wheat (genome AABB) were previously shown to demonstrate eco-geographically structured genetic and epigenetic diversity. Transposable elements (TEs) might make up a significant part of the genetic and epigenetic variation between individuals and populations because they comprise over 80% of the wild emmer wheat genome. In this study, we performed detailed analyses to assess the dynamics of transposable elements in 50 accessions of wild emmer wheat collected from 5 geographically isolated sites. The analyses included: the copy number variation of TEs among accessions in the five populations, population-unique insertional patterns, and the impact of population-unique/specific TE insertions on structure and expression of genes.ResultsWe assessed the copy numbers of 12 TE families using real-time quantitative PCR, and found significant copy number variation (CNV) in the 50 wild emmer wheat accessions, in a population-specific manner. In some cases, the CNV difference reached up to 6-fold. However, the CNV was TE-specific, namely some TE families showed higher copy numbers in one or more populations, and other TE families showed lower copy numbers in the same population(s).Furthermore, we assessed the insertional patterns of 6 TE families using transposon display (TD), and observed significant population-specific insertional patterns. The polymorphism levels of TE-insertional patterns reached 92% among all wild emmer wheat accessions, in some cases. In addition, we observed population-specific/unique TE insertions, some of which were located within or close to protein-coding genes, creating allelic variations in a population-specific manner. We also showed that those genes are differentially expressed in wild emmer wheat.ConclusionsFor the first time, this study shows that TEs proliferate in wild emmer wheat in a population-specific manner, creating new alleles of genes, which contribute to the divergent evolution of homeologous genes from the A and B subgenomes.

The basis for rootstock resilient to Capnodis species : screening for genes encoding δ-endotoxins from Bacillus thuringiensis

BACKGROUND Conventional methods often fail to control the flatheaded borers Capnodis spp., major pests of stone fruit trees; the larvae are protected from insecticides and predation because they feed deep in the roots. A potential solution is transgenic trees producing in their roots toxic compounds such as Cry proteins of Bacillus thuringiensis (Bt). RESULTS Toxicities against Capnodis larvae were demonstrated by exploiting a recently designed artificial larval diet and an available collection of field isolated Bt. An isolate of Bt tenebrionis (Btt) from commercial bioinsecticide (Novodor) displayed LC50 and LC95 values of 3.2 and 164 mg g(-1) , respectively, against neonates of Capnodis tenebrionis, whereas values of the most toxic field isolate K-7 were 1.9 and 25.6 mg g(-1) respectively. Weights of surviving larvae after 1 month on diets containing low concentrations of K-7 (0.1-1.0 mg g(-1) ) were lower than on Btt or untreated larvae. K-7 was also toxic against larvae of C. cariosa and C. miliaris and found to harbour genes encoding Cry9Ea-like and Cry23Aa/Cry37Aa binary toxins. CONCLUSION Larvae of Capnodis spp. are susceptible to Bt Cry toxins. Expressing cry genes active against these pests thus seems a feasible solution towards production of transgenic rootstock trees resilient to the pest.