Getting rid of gut microbes: surface sterilization cleans symbionts from the insects'egg masses.

Diverse heteropteran insects that feed on economic important crops, commonly known as stink bugs, are associated with specific gut symbiotic bacteria within their midgut cryptic spaces or the gastric caeca. Recent studies have revealed that the stink bugs Nezara viridula, Acrosternum hilare, Murgantia histrionica, Euschistus heros, Chlorochroa ligata, Chlorochroa sayi, Chlorochroa uhleri, Plautia stali, Thyanta pallidovirens, Dichelops melacanthus, Edessa meditabunda, Loxa deducta, Pellaea stictica, Piezodorus guildinii, Thyanta perditor, (all within Pentatomidae family) carried one major bacterium in their midgut. Phylogenetic tree generated using the 16S rRNA gene sequences obtained from the midgut of these previous species placed all symbionts in a clade with the plant-associated bacteria Erwinia and Pantoea species. In this system, females of stink bug vertically transmit the symbionts by smearing them on the surface of the egg masses. When first instars of nymphs hatch, they probe the egg surface and orally acquire the symbionts. Once ingested, these microbial symbionts will reach the 4th section of the ventriculum (V4), also known as gastric caecum, where they establish. It was hypothesized that nymphs born from surface sterilized eggs do not carry the caeca-associated symbionts. Once ingested these microbial symbionts will reach the 4th section of the ventriculum (V4), also known as gastric caecum, where they establish. It was hypothesized that nymphs born from surface sterilized eggs do not carry the caeca-associated symbionts. Herne, using scanning eletron microscopy (SEM), we are showing that surface sterilized eggs do not carry the surface microbes while eggs from the field of E. heros, D. melacanthus, N. viridula, P. stitica, and Pi. Guildinii carry them. In this unique model of transmission where the symbionts located temporally on the surface of eggs, environment factors may have a great impact on bacterial survival. Additionally, climate change may impact the insect host ecology. This information may lead to design new strategies to control the stink bugs that could used in the integrated pest management

The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity

Ralstonia syzygii, the Blood Disease Bacterium and Some Asian R. solanacearum Strains Form a Single Genomic Species Despite Divergent Lifestyles

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer

Host structural carbohydrate induces vector transmission of a bacterial plant pathogen

Many insect-borne pathogens have complex life histories because they must colonize both hosts and vectors for successful dissemination. In addition, the transition from host to vector environments may require changes in gene expression before the pathogen's departure from the host. Xylella fastidiosa is a xylem-limited plant-pathogenic bacterium transmitted by leafhopper vectors that causes diseases in a number of economically important plants. We hypothesized that factors of host origin, such as plant structural polysaccharides, are important in regulating X. fastidiosa gene expression and mediating vector transmission of this pathogen. The addition of pectin and glucan to a simple defined medium resulted in dramatic changes in X. fastidiosa's phenotype and gene-expression profile. Cells grown in the presence of pectin became more adhesive than in other media tested. In addition, the presence of pectin and glucan in media resulted in significant changes in the expression of several genes previously identified as important for X. fastidiosa's pathogenicity in plants. Furthermore, vector transmission of X. fastidiosa was induced in the presence of both polysaccharides. Our data show that host structural polysaccharides mediate gene regulation in X. fastidiosa, which results in phenotypic changes required for vector transmission. A better understanding of how vector-borne pathogens transition from host to vector, and vice versa, may lead to previously undiscovered disease-control strategies

Disrupt the bacterial growth in the insect vector to block the transmission of Candidatus Liberibacter asiaticus to citrus, the causal agent of citrus greening disease

The genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR, encodingLuxR protein, one of a two component cell-to-cell communication system. However, the genome lacks the second component, luxl, that produces acyl-homoserine lactones (AHLs), suggesting that CLas has a solo LuxR system. Interestingly, we detected compounds that may act as AHLs in the insect vector (psyllids) that are healthy or infected with CLas, but not in the citrus plants. This finding suggests that the insect is the AHL source. The fact that CLas forms a biofilm on the surface of the insect gut indicates the presence of a cell-cell communication system. Here the system is solo LuxR. Moreover, we have confirmed the activity of CLas-LuxR by its expression in E. coli and detection of LuxR-AHL complex. In order to block the vector transmission of CLas, we produced plants that express LuxR. Insects will acquire CLas and luxR. LuxR will compete with the bacteria for binding to AHL and consequently, CLas will not be able to colonize the insect or form biofilm and fails in the transmission. We aim to provide an environmental friendly solution for the most destructive disease in citrus (Huanglongbing) by producing specific LuxR in citrus to interfere with the vector transmission. As an alternative, we also aim to use synthetic molecules that mimic the specific AHL as an application to disrupt CLas transmission from plant to plant by its vector. More AHL in the insect may confuse the bacteria and induce a strong sticky biofilm that hardly releases cells to plants during insect feeding. Accordingly, the transmission from plant to plant will be diminished or blocked

Generating Asian citrus Psyllid Diaphorina citri Kuwayama (Homoptera: Psyllidae) with twisting wings to prevent the spread of citrus greening disease

Huanglongbing (HLB) is seriously threatening and causing considerable economic losses to the citrus groves. Its Management depends critically on the control of the Asian citrus Psyllid (ACP), the vector of the cause of HLB, Candidatus Liberibacter asiaticus bacteria (CLas). Silencing genes by RNA interference (RNAi) is a promising technique to control pests. In this study, the abnormal disk wing (awd) has been selected from the available psyllid annotated genome. It has been known that awd gene encodes a nucleoside diphosphate kinase and is associated with wing development. This research focused on the effect of RNAi of awd gene on ACP nymph instars that acquired dsRNA. The Results provide evidence that using the dsRNA of awd gene has diminished the development and survival of ACP nymphs. Moreover, knockdown of awd gene expression was observed through malformation of adult wings. Also, the expression of awd was messured by quantitative PCR (qPCR). Furthermore, we are conducting experiments to investigate awd's possible contribution in temperature tolerance. We attempt to establish effective practical application to prevent the spread of HLB in friendly environmentally strategy

Disrupt the bacterial growth in the insect vector to block the transmission of Candidatus Liberibacter asiaticus to citrus, the causal agent of citrus greening disease

The genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR, encodingLuxR protein, one of a two component cell-to-cell communication system. However, the genome lacks the second component, luxl, that produces acyl-homoserine lactones (AHLs), suggesting that CLas has a solo LuxR system. Interestingly, we detected compounds that may act as AHLs in the insect vector (psyllids) that are healthy or infected with CLas, but not in the citrus plants. This finding suggests that the insect is the AHL source. The fact that CLas forms a biofilm on the surface of the insect gut indicates the presence of a cell-cell communication system. Here the system is solo LuxR. Moreover, we have confirmed the activity of CLas-LuxR by its expression in E. coli and detection of LuxR-AHL complex. In order to block the vector transmission of CLas, we produced plants that express LuxR. Insects will acquire CLas and luxR. LuxR will compete with the bacteria for binding to AHL and consequently, CLas will not be able to colonize the insect or form biofilm and fails in the transmission. We aim to provide an environmental friendly solution for the most destructive disease in citrus (Huanglongbing) by producing specific LuxR in citrus to interfere with the vector transmission. As an alternative, we also aim to use synthetic molecules that mimic the specific AHL as an application to disrupt CLas transmission from plant to plant by its vector. More AHL in the insect may confuse the bacteria and induce a strong sticky biofilm that hardly releases cells to plants during insect feeding. Accordingly, the transmission from plant to plant will be diminished or blocked

Sequence variation in two genes determines the efficacy of transmission of citrus tristeza virus by the brown citrus aphid

Vector transmission is an important part of the viral infection cycle, yet for many viruses little is known about this process, or how viral sequence variation affects transmission efficacy. Here we examined the effect of substituting genes from the highly transmissible FS577 isolate of citrus tristeza virus (CTV) in to the poorly transmissible T36-based infectious clone. We found that introducing p65 or p61 sequences from FS577 significantly increased transmission efficacy. Interestingly, replacement of both genes produced a greater increase than either gene alone, suggesting that CTV transmission requires the concerted action of co-evolved p65 and p61 proteins