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Paris polyphylla is an important traditional medicinal plant of the Himalayan region. It is extensively used for the production of natural steroidal saponins and flavonoids. Although seed dormancy of wild plants can be broken to be artificially maintained and regenerated through micropropagation in the laboratory, the success of secondary metabolite production in higher quantities and the synthesis of superior plant metabolites have been very limited. In this study, we present differential metabolic profiling of P. polyphylla plants maintained for 8 years in natural and greenhouse conditions. Untargeted profiling of the metabolites through ultra-performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS), followed by statistical analysis, identified secondary metabolites that were enriched in the naturally occurring plant roots compared with the greenhouse plant roots. A multivariate statistical analysis revealed the differential distribution of the compounds between the two groups. Overall, we identified 1,182 secondary metabolites, with 116 metabolites being differentially upregulated and 256 metabolites being downregulated. Moreover, 810 metabolites showed no significant variation under both growing conditions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the naturally forest-grown P. polyphylla plants were significantly enriched in steroidal saponins, lipids, vitamins, flavonoids, and flavonols. An analysis of the top 10 differentially upregulated secondary metabolites indicated a significantly enriched quantity of spirost-5-en-3,12-diol and kaempferol synthesis pathways, which are known to reduce the effect of free radicals scavengers inside the cell. In addition, veratramine alkaloids were also enriched under natural conditions. Our findings indicated that naturally maintained P. polyphylla plants are suitable for the extraction of medicinally important compounds. Our study established a causal relationship between the metabolic composition of the roots and their natural growth condition. This study highlighted the importance of environmental conditions in the biosynthesis of secondary metabolites of plants.

Common bean (Phaseolus vulgaris L.) is a widely cultivated crop, representing an important protein source in the human diet in developing countries. The production of this crop faces serious challenges, such as virus diseases transmitted by the whitefly Bemisia tabaci. Although there is a lot of information about some of these viruses, most of what we know has been developed using model systems, such as tomato plants and tomato yellow leaf curl virus (TYLCV). There is still very little information on the most relevant common bean viruses, such as bean golden mosaic virus (BGMV), bean golden yellow mosaic virus (BGYMV), bean dwarf mosaic virus (BDMV), cowpea mild mottle virus (CPMMV), and bean yellow disorder virus (BnYDV). In this review, we discuss the available data in the most up-to-date literature and suggest future research avenues to contribute to the development of management tools for preventing or reducing the damage caused by viruses in this important crop.

Panax notoginseng is a perennial plant well known for its versatile medicinal properties, including hepatoprotective, antioxidant, anti-inflammatory, anti-tumor, estrogen-like, and antidepressant characteristics. It has been reported that plant age affects the quality of P. notoginseng. This study aimed to explore the differential metabolome and transcriptome of 2-year (PN2) and 3-year-old (PN3) P. notoginseng plant root samples. Principal component analysis of metabolome and transcriptome data revealed major differences between the two groups (PN2 vs. PN3). A total of 1813 metabolites and 28,587 genes were detected in this study, of which 255 metabolites and 3141 genes were found to be differential (p < 0.05) between PN2 vs. PN3, respectively. Among differential metabolites and genes, 155 metabolites and 1217 genes were up-regulated, while 100 metabolites and 1924 genes were down-regulated. The KEGG pathway analysis revealed differentially enriched metabolites belonging to class lipids (“13S-hydroperoxy-9Z, 11E-octadecadionic acid”, “9S-hydroxy-10E, 12Z-octadecadionic acid”, “9S-oxo-10E, 12Z-octadecadionic acid”, and “9,10,13-trihydroxy-11-octadecadionic acid”), nucleotides and derivatives (guanine and cytidine), and phenolic acids (chlorogenic acid) were found to be enriched (p < 0.05) in PN3 compared to PN2. Further, these differentially enriched metabolites were found to be significantly (p < 0.05) regulated via linoleic acid metabolism, nucleotide metabolism, plant hormone signal transduction, and arachidonic acid metabolism pathways. Furthermore, the transcriptome analysis showed the up-regulation of key genes MAT, DMAS, SDH, gallate 1-beta-glucosyltransferase, and beta-D-glucosidase in various plants’ secondary metabolic pathways and SAUR, GID1, PP2C, ETR, CTR1, EBF1/2, and ERF1/2 genes observed in phytohormone signal transduction pathway that is involved in plant growth and development, and protection against the various stressors. This study concluded that the roots of a 3-year-old P. notoginseng plant have better metabolome and transcriptome profiles compared to a 2-year-old plant with importantly enriched metabolites and genes in pathways related to metabolism, plant hormone signal transduction, and various biological processes. These findings provide insights into the plant’s dynamic biochemical and molecular changes during its growth that have several implications regarding its therapeutic use.

The host – pathogen interaction is a multifactorial process subject to a co-evolutionary arms race consisting of rapid changes in both host and pathogen, controlled at the genetic and epigenetic levels. Previously, we showed intra-species variation in disease progression and pathogenicity in aphids for Metarhizium brunneum isolates MbK and Mb7. Herein, we compared genomic, epigenetic, and metabolomic variations between these isolates and their effects on pathogenicity. Genomic variation could not completely explain the observed differences between the isolates. However, differential N6-adenine methylation (6 mA) and its correlation to reduced expression of the essential SWC4 subunit of SWR1 chromatin-remodelling complex (SWR1-C) led us to hypothesize a role for swc4 in the varying pathogenicity. Mutagenesis of the essential swc4 gene in MbKisolate resulted in reduction of secondary-metabolite (SM) secretion and impaired virulence in Galleria mellonella. Our results suggest the role of SWC4 in the regulation of SMs and the role of both SWC4 and SWR1-C in virulence of M. brunneum isolates. A better understanding of epigenetic regulation of SM production and secretion in entomopathogenic fungi may enable theirmanipulation for better biocontrol performance, and expand possibilities for environmentally friendly pest control.

ABSTRACT Autophagy plays an important role against pathogen infection in many organisms; however, little has been done with regard to vector-borne plant and animal pathogens, that sometimes replicate and cause deleterious effects in their vectors. Candidatus Liberibacter solanacearum (CLso) is a fastidious gram-negative phloem-restricted plant pathogen and vectored by the carrot psyllid, Bactericera trigonica. The plant disease caused by this bacterium is called carrot yellows and has recently gained much importance due to worldwide excessive economical losses. Here, we demonstrate that calcium ATPase, cytosolic calcium, and most importantly Beclin-1 have a role in regulating autophagy and its association with Liberibacter inside the psyllid. The presence of CLso generates reactive oxygen species and induces the expression of detoxification enzymes in the psyllid midguts, a main site for bacteria transmission. CLso also induces the expression of both sarco/endoplasmic reticulum Ca2+pump (SERCA) and 1,4,5-trisphosphate receptors (ITPR) in midguts, resulting in high levels of calcium in the cellular cytosol. Silencing these genes individually disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and subsequently on Liberibacter persistence and transmission. Inhibiting Beclin1-phosphorylation through different calcium-induced kinases altered the expression of autophagy and CLso titers and persistence. Based on our results obtained from the midgut, we suggest the existence of a direct correlation between cytosolic calcium levels, autophagy, and CLso persistence and transmission by the carrot psyllid. IMPORTANCE Plant diseases caused by vector-borne Liberibacter species are responsible for the most important economic losses in many agricultural sectors. Preventing these diseases relies mostly on chemical sprays against the insect vectors. Knowledge-based interference with the bacteria-vector interaction remains a promising approach as a sustainable solution. For unravelling how Liberibacter exploits molecular pathways in its insect vector for transmission, here, we show that the bacterium manipulates calcium levels on both sides of the endoplasmic reticulum membrane, resulting in manipulating autophagy. Silencing genes associated with these pathways disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and Liberibacter transmission. These results demonstrate major pathways that could be exploited for manipulating and controlling the disease transmission.

‘Candidatus Liberibacter solanacearum’ (Lso) is a plant pathogenic bacterium transmitted by psyllids that causes significant agricultural damage. Several Lso haplotypes have been reported. Among them, LsoA and LsoB are transmitted by the potato psyllid Bactericera cockerelli and infect solanaceous crops, and LsoD is transmitted by the carrot psyllid B. trigonica and infects apiaceous crops. Several studies evaluated the transmission of these haplotypes by adult psyllids. However, fewer data are available on the transmission of different Lso haplotypes by psyllid nymphs. In this study, we investigated the transmission of these three haplotypes by psyllid nymphs to expand our basic understanding of Lso transmission. Specifically, the objective was to determine if the haplotypes differed in their transmission rates by nymphs and if LsoA and LsoB accumulated at different rates in the guts of nymphs as it occurs in adults. First, we quantified LsoA and LsoB titers in the guts of third- and fifth-instar potato psyllid nymphs. We found similar LsoA titers in the two nymphal stages, while LsoB titer was lower in the gut of the third-instar nymphs compared to fifth-instar nymphs. Second, we assessed the transmission efficiency of LsoA and LsoB by third-instar nymphs to tomato plants, revealing that LsoA was transmitted earlier and with higher efficiency than LsoB. Finally, we examined the transmission of LsoD by carrot psyllid nymphs to celery plants and demonstrated an age-related difference in the transmission rate. These findings provide valuable insights into the transmission dynamics of different Lso haplotypes by nymphal vectors, shedding light on their epidemiology and interactions with their psyllid vectors.

ABSTRACT Candidatus Liberibacter solanacearum (CLso) haplotype D, transmitted by the carrot psyllid Bactericera trigonica, is a major constraint for carrot production in Israel. Unveiling the molecular interactions between the psyllid vector and CLso can facilitate the development of nonchemical approaches for controlling the disease caused by CLso. Bacterial surface proteins are often known to be involved in adhesion and virulence; however, interactions of CLso with carrot psyllid proteins that have a role in the transmission process has remained unexplored. In this study, we used CLso outer membrane protein (OmpA) and flagellin as baits to screen for psyllid interacting proteins in a yeast two-hybrid system assay. We identified psyllid vitellogenin (Vg) to interact with both OmpA and flagellin of CLso. As Vg and autophagy are often tightly linked, we also studied the expression of autophagy-related genes to further elucidate this interaction. We used the juvenile hormone (JH-III) to induce the expression of Vg, thapsigargin for suppressing autophagy, and rapamycin for inducing autophagy. The results revealed that Vg negatively regulates autophagy. Induced Vg expression significantly suppressed autophagy-related gene expression and the levels of CLso significantly increased, resulting in a significant mortality of the insect. Although the specific role of Vg remains obscure, the findings presented here identify Vg as an important component in the insect immune responses against CLso and may help in understanding the initial molecular response in the vector against Liberibacter. IMPORTANCE Pathogen transmission by vectors involves multiple levels of interactions, and for the transmission of liberibacter species by psyllid vectors, much of these interactions are yet to be explored. Candidatus Liberibacter solanacearum (CLso) haplotype D inflicts severe economic losses to the carrot industry. Understanding the specific interactions at different stages of infection is hence fundamental and could lead to the development of better management strategies to disrupt the transmission of the bacteria to new host plants. Here, we show that two liberibacter membrane proteins interact with psyllid vitellogenin and also induce autophagy. Altering vitellogenin expression directly influences autophagy and CLso abundance in the psyllid vector. Although the exact mechanism underlying this interaction remains unclear, this study highlights the importance of immune responses in the transmission of this disease agent.

The whitefly Bemisia tabaci is one of the most important agricultural pests due to its extreme invasiveness, insecticide resistance, and ability to transmit hundreds of plant viruses. Among these, Begomoviruses and recombinant whitefly-borne Poleroviruses are transmitted persistently. Several studies have shown that upon infection, plant viruses manipulate plant-emitted volatile organic compounds (VOCs), which have important roles in communication with insects. In this study, we profiled and compared the VOCs emitted by tomato and pepper plant leaves after infection with the Tomato yellow leaf curl virus (TYLCV) (Bogomoviruses) and the newly discovered Pepper whitefly-borne vein yellows virus (PeWBVYV) (Poleroviruses), respectively. The results identified shared emitted VOCs but also uncovered unique VOC signatures for each virus and for whitefly infestation (i.e., without virus infection) independently. The results suggest that plants have general defense responses; however, they are also able to respond individually to infection with specific viruses or infestation with an insect pest. The results are important to enhance our understanding of virus- and insect vector-induced alteration in the emission of plant VOCs. These volatiles can eventually be used for the management of virus diseases/insect vectors by either monitoring or disrupting insect–plant interactions.

ABSTRACT Apoptosis is generally considered the first line of defense against viral infection. However, the role of apoptosis in the interactions between plant viruses and their insect vectors has rarely been investigated. By studying plant DNA viruses of the genus Begomovirus within the family Geminiviridae, which are transmitted by whiteflies of the Bemisia tabaci species complex in a persistent manner, we revealed that virus-induced apoptosis in insect vectors can facilitate viral accumulation and transmission. We found that infection with tomato yellow leaf curl virus activated the apoptosis pathway in B. tabaci. Suppressing apoptosis by inhibitors or silencing caspase-3 significantly reduced viral accumulation, while the activation of apoptosis increased viral accumulation in vivo. Moreover, the positive effect of whitefly apoptosis on virus accumulation and transmission was not due to its cross talk with the autophagy pathway that suppresses begomovirus infection in whiteflies. We further showed that viral replication, rather than the viral coat protein, is likely the critical factor in the activation of apoptosis by the virus. These novel findings indicate that similarly to many animal and a few plant RNA viruses, plant DNA viruses may activate apoptosis in their insect vectors leading to enhanced viral accumulation and transmission. IMPORTANCE Of the approximately 1,100 known plant viruses, about one-third are DNA viruses that are vectored by insects. Plant virus infections often induce cellular and molecular responses in their insect vectors, which can, in many cases, affect the spread of viruses. However, the mechanisms underlying vector responses that affect virus accumulation and transmission are poorly understood. Here, we examined the role of virus-induced apoptosis in the transmission of begomoviruses, a group of single-stranded plant DNA viruses that are transmitted by whiteflies and cause extensive damage to many crops worldwide. We demonstrated that virus infection can induce apoptosis in the insect vector conferring protection to the virions from degradation, leading to enhanced viral accumulation and transmission to host plants. Our findings provide valuable clues for designing new strategies to block the transmission of insect-vectored plant viruses, particularly plant DNA viruses.

The egg parasitpoids Trichogramma danausicida (Nagaraja) and Trichogramma cacaeciae (Marchal) (Hymenoptera: Trichogrammatidae), are reported for the first time in Israel. Moreover, our discovery of T. danausicida is the first report of this parasitoid species outside of India. The occurrence of those trichogrammatids was first discovered and documented in May 2016 during a survey of egg parasitoids of the False codling moth Thaumatotibia leucotreta (Lepidoptera: Tortricidae). The field survey was conducted on castor bean fruits (Ricinus communis) in the Israeli central coastal plain. The identity of the parasitoids was revealed by means of sequencing a portion of the cytochrome oxidase I gene (COI) of the studied parasitoids.

Many plant viruses depend on insect vectors for their transmission and dissemination. The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is one of the most important virus vectors, transmitting more than four hundred virus species, the majority belonging to begomoviruses (Geminiviridae), with their ssDNA genomes. Begomoviruses are transmitted by B. tabaci in a persistent, circulative manner, during which the virus breaches barriers in the digestive, hemolymph, and salivary systems, and interacts with insect proteins along the transmission pathway. These interactions and the tissue tropism in the vector body determine the efficiency and specificity of the transmission. This review describes the mechanisms involved in circulative begomovirus transmission by B. tabaci, focusing on the most studied virus in this regard, namely the tomato yellow leaf curl virus (TYLCV) and its closely related isolates. Additionally, the review aims at drawing attention to the recent knowhow of unorthodox virus—B. tabaci interactions. The recent knowledge of whitefly-mediated transmission of two recombinant poleroviruses (Luteoviridae), a virus group with an ssRNA genome and known to be strictly transmitted with aphids, is discussed with its broader context in the emergence of new whitefly-driven virus diseases.

The whitefly B. tabaci is a global pest and transmits extremely important plant viruses especially begomoviruses, that cause substantial crop losses. B. tabaci is one of the top invasive species worldwide and have developed resistance to all major pesticide classes. One of the promising alternative ways for controlling this pest is studying its genetic makeup for identifying specific target proteins which are critical for its development and ability to transmit viruses. Tomato yellow leaf curl virus (TYLCV) is the most economically important and well-studied begomovirus transmitted by B. tabaci, in a persistent-circulative manner. Recently, we reported that B. tabaci Cyclophilin B (CypB) and heat shock protein 70 proteins (hsp70) interact and co-localize with TYLCV in the whitefly midgut, on the virus transmission pathway, and that both proteins have a significant role in virus transmission. Here, we extended the previous work and used the Tobacco rattle virus (TRV) plant-mediated RNA silencing system for knocking down both genes and testing the effect of their silencing on whitefly viability and virus transmission. Portions of these two genes were cloned into TRV constructs and tomato plants were infected and used for whitefly feeding and transmission experiments. Following whitefly feeding on TRV-plants, the expression levels of cypB and hsp70 in adult B. tabaci significantly decreased over 72 h feeding period. The knockdown in the expression of both genes was further shown in the first generation of silenced whiteflies, where phenotypic abnormalities in the adult, wing, nymph and bacteriosomes development and structure were observed. Additionally, high mortality rates that reached more than 80% among nymphs and adults were obtained. Finally, silenced whitefly adults with both genes showed decreased ability to transmit TYLCV under lab conditions. Our results suggest that plant-mediated silencing of both cypB and hsp70 have profound effects on whitefly development and its ability to transmit TYLCV.

The onion thrip, Thrips tabaci (Thysanoptera: Thripidae) is a major polyphagous pest that attacks a wide range of economically important crops, especially Allium species. The thrip’s damage can result in yield loss of up to 60% in onions (Allium cepa). In the past few decades, thrip resistance to insecticides with various modes of actions have been documented. These include resistance to spinosad, a major active compound used against thrips, which was reported from Israel. Little is known about the molecular mechanisms underlying spinosad resistance in T. tabaci. We attempted to characterize the mechanisms involved in resistance to spinosad using quantitative transcriptomics. Susceptible (LC50 = 0.6 ppm) and resistant (LC50 = 23,258 ppm) thrip populations were collected from Israel. An additional resistant population (LC50 = 117 ppm) was selected in the laboratory from the susceptible population. De novo transcriptome analysis on the resistant and susceptible population was conducted to identify differently expressed genes (DGEs) that might be involved in the resistance against spinosad. In this analysis, 25,552 unigenes were sequenced, assembled, and functionally annotated, and more than 1500 DGEs were identified. The expression levels of candidate genes, which included cytochrome P450 and vittelogenin, were validated using quantitative RT-PCR. The cytochrome P450 expression gradually increased with the increase of the resistance. Higher expression levels of vitellogenin in the resistant populations were correlated with higher fecundity, suggesting a positive effect of the resistance on resistant populations. This research provides a novel genetic resource for onion thrips and a comprehensive molecular examination of resistant populations to spinosad. Those resources are important for future studies concerning thrips and resistance in insect pests regarding agriculture.

By having an extensive territory and suitable climate conditions, South America is one of the most important agricultural regions in the world, providing different kinds of vegetable products to different regions of the world. However, such favorable conditions for plant production also allow the development of several pests, increasing production costs. Among them, whiteflies (Hemiptera: Aleyrodidae) stand out for their potential for infesting several crops and for being resistant to insecticides, having high rates of reproduction and dispersal, besides their efficient activity as virus vectors. Currently, the most important species occurring in South America are Bemisia afer, Trialeurodes vaporariorum, and the cryptic species Middle East-Asia Minor 1, Mediterranean, and New World, from Bemisia tabaci complex. In this review, a series of studies performed in South America were compiled in an attempt to unify the advances that have been developed in whitefly management in this continent. At first, a background of the current whitefly distribution in South American countries as well as factors affecting them are shown, followed by a background of the whitefly transmitted viruses in South America, addressing their location and association with whiteflies in each country. Afterwards, a series of management strategies are proposed to be implemented in South American fields, including cultural practices and biological and chemical control, finalizing with a section containing future perspectives and directions for further research.

Control of the crop pest African cotton leafworm, Spodoptera littoralis (Boisduval), by chemical insecticides has led to serious resistance problems. Ajuga plants contain phytoecdysteroids (arthropod steroid hormone analogs regulating metamorphosis) and clerodanes (diterpenoids exhibiting antifeedant activity). We analyzed these compounds in leaf extracts of the Israeli Ajuga iva L. by liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) and thin-layer chromatography (TLC), and their efficiency at reducing S.littoralis fitness. First and third instars of S. littoralis were fed castor bean leaves (Ricinus communis) smeared with an aqueous suspension of dried methanolic crude extract of A. iva phytoecdysteroids and clerodanes. Mortality, larval weight gain, relative growth rate and survival were compared to feeding on control leaves. We used ‘4’,6-diamidino-2-phenylindole (DAPI, a fluorescent stain) and phalloidin staining to localize A. iva crude leaf extract activity in the insect gut. Ajuga iva crude leaf extract (50, 100 and 250 µg/µL) significantly increased mortality of first-instar S. littoralis (36%, 70%, and 87%, respectively) compared to controls (6%). Third-instar larval weight gain decreased significantly (by 52%, 44% and 30%, respectively), as did relative growth rate (−0.05 g/g per day compared to the relevant controls), ultimately resulting in few survivors. Crude leaf extract (250 µg/µL) reduced gut size, with relocation of nuclei and abnormal actin-filament organization. Ajug iva extract has potential for alternative, environmentally safe insect-pest control.

Insect-borne bacterial pathogens pose a global economic threat to many agricultural crops. Candidatus liberibacter species, vectored by psyllids (Hemiptera: psylloidea), are an example of devastating pathogens related to important known diseases such as Huanglongbing or the citrus greening disease, Zebra chip disease, and carrot yellowing, along with vegetative disorders in umbellifers. Studies on liberibacter–plant interactions have gained more focus in disease control over the last few decades. However, successful and sustainable disease management depends on the early disruption of insect–pathogen interactions, thereby blocking transmission. Recent knowledge on the liberibacter genomes and various omics approaches have helped us understand this host–pathogen relationship, despite the complexity associated with the inability to culture these bacteria. Here, we discuss the cellular and molecular processes involved in the response of insect-host immunity, and the liberibacter-associated pathogenesis mechanisms that involve virulence traits and effectors released to manipulate the insect–host defense mechanism for successful transmission. Understanding such mechanisms is an important milestone for developing sustainable means for preventing liberibacter transmission by psyllids.

Endosymbionts harbored inside insects play critical roles in the biology of their insect host and can influence the transmission of pathogens by insect vectors. Bactericera trigonica infests umbelliferous plants and transmits the bacterial plant pathogen Candidatus Liberibacter solanacearum (CLso), causing carrot yellows disease. To characterize the bacterial diversity of B. trigonica, as a first step, we used PCR-restriction fragment length polymorphism (PCR-RFLP) and denaturing gradient gel electrophoresis (DGGE) analyses of 16S rDNA to identify Sodalis and Spiroplasma endosymbionts. The prevalence of both symbionts in field-collected psyllid populations was determined: Sodalis was detected in 100% of field populations, while Spiroplasma was present in 82.5% of individuals. Phylogenetic analysis using 16S rDNA revealed that Sodalis infecting B. trigonica was more closely related to symbionts infecting weevils, stink bugs and tsetse flies than to those from psyllid species. Using fluorescent in situ hybridization and immunostaining, Sodalis was found to be localized inside the nuclei of the midgut cells and bacteriocytes. Spiroplasma was restricted to the cytoplasm of the midgut cells. We further show that a recently reported Bactericera trigonica densovirus (BtDNV), a densovirus infecting B. trigonica was detected in 100% of psyllids and has reduced titers inside CLso-infected psyllids by more than two-fold compared to CLso uninfected psyllids. The findings of this study will help to increase our understanding of psyllid–endosymbiont interactions.

Bemisia tabaci is one of the most threatening pests in agriculture, causing significant losses to many important crops on a global scale. The dramatic increase and availability of sequence data for B. tabaci species complex and its bacterial endosymbionts is critical for developing emerging sustainable pest management strategies which are based on pinpointing the global diversity of this important pest and its bacterial endosymbionts. To unravel the global genetic diversity of B. tabaci species complex focusing on its associated endosymbionts, along with Israeli whitefly populations collected in this study, we combined available sequences in databases, resulting in a total of 4,253 mitochondrial cytochrome oxidase I (mtCOI) sequences from 82 countries and 1,226 16S/23S rRNA endosymbiont sequences from 32 countries that were analyzed. Using Bayesian phylogenetic analysis, we identified two new B. tabaci groups within the species complex and described the global distribution of endosymbionts within this complex. Our analyses revealed complex divergence of the different endosymbiont sequences within the species complex, with overall one Hamiltonella, two Porteria (P1 and P2), two Arsenophonus (A1 and A2), two Wolbachia (super-groups O and B), four Cardinium (C1-C4) and three Rickettsia (R1-R3) groups were identified. Our comprehensive analysis provides an updated important resource for this globally important pest and its secondary symbionts, which have been a major subject for research in last three decades.

The variegated caper bug (CB) Stenozygum coloratum (Klug, 1845) is common in the Eastern Mediterranean region and a minor agricultural pest. CB eggs were recently shown to serve as alternative hosts for Ooencyrtus pityocampae Mercet, 1921, an important natural enemy of the pine processionary moth (PPM) Thaumetopoea wilkinsoni Tams, 1924 (Lepidoptera: Notodontidae). In this study various aspects of the life history of CB were studied, including its distribution, host range, development, reproduction and ability to develop on various cultivated species of plants. CB occurs in almost all areas and habitats in Israel, except in the coldest regions, attacking several caper species (Capparis spp.). When reared on Capparis zoharyi at 25°C, one generation lasted 6 weeks and females laid a single 12-egg cluster every 3 days. Decreases in temperature, but not changes in day length, terminated reproductive activity. CB nymphs survived for up to three months, or even completed their development, on some agricultural plants. However, reproductive activity was initiated only if adults were fed capers. These findings emphasize the importance of capers in the CB life cycle and account for why other plants are rarely attacked. These plants may serve as a temporary refuge for CB when capers are scarce or unsuitable, mainly late in the season. Morphology of developmental stages, egg deposition, cannibalistic behaviour, sex ratio and effects of temperature on egg production were also studied. The possibility of using CB for enhancing the biological control of PPM is discussed.

Ooencyrtus spp. (Hymenoptera, Chalcidoidea, Encyrtidae) are important natural enemies of agricultural and forest insect pests, and are distributed worldwide. Their reduced dimensions, highly variable morphological characters and possible effect of wide host range and abiotic factors, make correct identification at the species level particularly difficult. This paper combined molecular, morphological, and biological data to characterize a group of Ooencyrtus spp. emerging from the eggs of the variegated caper bug, Stenozygum coloratum in the east Mediterranean area. COI and ITS2 sequencing revealed the presence of six and five divergent clades, respectively. Three clades were identified as Ooencyrtus telenomicida, Ooencyrtus pityocampae and O. pistaciae. Two clades represent new species which are here described and named Ooencyrtus zoeae and Ooencyrtus mevalbelus. These features were combined with reliable morphological characters to facilitate the separation of these species. A dichotomous key and a new synonymy are proposed. Ooencyrtus pistaciae had two distinct COI clades but only one ITS2 clade. Crossbreeding trials that included Ooencyrtus telenomicida, Ooencyrtus melvabelus sp. nov. and Ooencyrtus zoeae sp. nov. confirmed their reproductive isolation. COI sequences showed 0-0.8% and 4-9% within and between-species genetic differences, respectively. ITS2 showed 0.4-5.9% genetic differences between species, with no genetic differences within species. Haplotype diversity of Israeli and Turkish populations of the various species was 0-0.98 and was particularly low in Ooencyrtus pityocampae, whose Israeli population showed no diversity. The discovery of the Ooencyrtus spp. on the eggs of the caper bug, and their abundance support the idea that the bug can be used as an alternative host for augmentation of populations of these parasitoids in agricultural and forestry systems.

Candidatus Liberibacter solanacerum (CLso), transmitted by Bactericera trigonica in a persistent and propagative mode causes carrot yellows disease, inflicting hefty economic losses. Understanding the process of transmission of CLso by psyllids is fundamental to devise sustainable management strategies. Persistent transmission involves critical steps of adhesion, cell invasion, and replication before passage through the midgut barrier. This study uses a transcriptomic approach for the identification of differentially expressed genes with CLso infection in the midguts, adults, and nymphs of B. trigonica and their putative involvement in CLso transmission. Several genes related to focal adhesion and cellular invasion were upregulated after CLso infection. Interestingly, genes involved with proper functionality of the endoplasmic reticulum (ER) were upregulated in CLso infected samples. Notably, genes from the endoplasmic reticulum associated degradation (ERAD) and the unfolded protein response (UPR) pathway were overexpressed after CLso infection. Marker genes of the ERAD and UPR pathways were also upregulated in Diaphorina citri when infected with Candidatus Liberibacter asiaticus (CLas). Upregulation of the ERAD and UPR pathways indicate induction of ER stress by CLso/CLas in their psyllid vector. The role of ER in bacteria−host interactions is well-documented; however, the ER role following pathogenesis of CLso/CLas is unknown and requires further functional validation.

Persistent circulative transmission of plant viruses involves complex interactions between the transmitted virus and its insect vector. Several studies have shown that insect vector proteins are involved in the passage and the transmission of the virus. Interestingly, proteins expressed by bacterial endosymbionts that reside in the insect vector, were also shown to influence the transmission of these viruses. Thus far, the transmission of two plant viruses that belong to different virus genera was shown to be facilitated by a bacterial chaperone protein called GroEL. This protein was shown to be implicated in the transmission of Potato leafroll virus (PLRV) by the green peach aphid Myzus persicae, and the transmission of Tomato yellow leaf curl virus (TYLCV) by the sweetpotato whitefly Bemisia tabaci. These tri-trophic levels of interactions and their possible evolutionary implications are reviewed.

Whitefly (Bemisia tabaci) is an important insect pest, causing severe damage to agricultural crops. The pest was recorded in a commercial rosemary (Rosmarinus officinalis, Lamiaceae) field, colonizing rosemary variety (var.) '2', but not '11'. A series of field and controlled laboratory choice bioassays confirmed the observed phenomenon. Mature potted plants of the two varieties were randomly organized in a lemon verbena (Lippia citrodora) and lemon grass (Cymbopogon spp.) fields. Seven days later var. '2' was significantly more colonized by whiteflies than var. '11'. Under lab conditions, whiteflies were significantly more attracted to var. '2' plantlets than to var. '11' following choice bioassays. Furthermore, cotton plants dipped in an essential oil emulsion of var. '2' had significantly greater colonization than cotton plants dipped in the essential oil emulsion of var. '11'. Similar results were obtained in 'plant-plant', 'plant-no plant' as well as, 'essential oil-essential oil' choice bioassay designs. Analyses of the essential oils of the two varieties identified a set of common and unique volatiles in each variety. Among these volatiles were β-caryophyllene and limonene, two compounds known to be associated with plant-insect interactions. The attraction of B. tabaci to pure (>95%) β-caryophyllene and limonene using a range of concentrations was examined in vitro by choice bioassays. The compounds were attractive to the insect at moderate concentration, but not at the lowest or highest concentrations used, where the insect was not attracted or repelled, respectively. Limonene attracted the insects at rates that were 10-fold lower than β-caryophyllene. The results emphasized the role of host plant volatiles in shaping the structure of B. tabaci populations in nature and in agricultural systems, and provided insights into the factors that contribute to the development of insect populations with unique characteristics. The results could also serve for future development of bio-pesticides and in breeding programs.

Tomato yellow leaf curl virus (TYLCV) is a single-stranded (ssDNA) begomoviruses that causes severe damage to tomato and several other crops worldwide. TYLCV is exclusively transmitted by the sweetpotato whitefly, Bemisia tabaci in a persistent circulative and propagative manner. Previous studies have shown that the transmission, retention and circulation of TYLCV in its vector involves interaction with insect and endosymbiont proteins, which aid in the transmission of the virus, or have a protective role in response to the presence of the virus in the insect body. However, only a low number of such proteins have been identified. Here, the role of B. tabaci Cyclophilin B (CypB) in the transmission of TYLCV protein was investigated. Cyclophilins (Cyps) are a large family of cellular prolyl isomerases that have many molecular roles including facilitating protein-protein interactions in the cell. One cyclophilin protein has been implicated in aphid-luteovirus interactions. We demonstrate that the expression of CypB from B. tabaci is altered upon TYLCV acquisition and retention. Further experiments used immunocapture-PCR and co-immunolocalization and demonstrated a specific interaction and colocalization between CypB and TYLCV in the the midgut, eggs and salivary glands. Membrane feeding of anti-CypB antibodies and TYLCV infected plants showed a decrease in TYLCV transmission, suggesting a critical role that CypB plays in TYLCV transmission. Further experiments, which used membrane feeding with the CypB inhibitor Cyclosporin A (CsA) showed decrease in CypB-TYLCV colocalization in the midgut and virus transmission. Altogether, our results indicate that CypB plays an important role in TYLCV transmission by B. tabaci.

Descriptions and illustrations of the adult female and first-instar nymph of the kermesid Nidularia balachowskii Bodenheimer, based on the adult female lectotype and paralectotype (here designated), and new material collected from Israel are presented. A key for the identification of first-instar nymphs of Nidularia spp. is offered. Molecular identification of N. balachowskii, using nucleotide sequences from the D2–D3 region of the 28S ribosomal gene, and the mitochondrial Cytochrome Oxidase I (COI) gene, is presented. Morphological and molecular analyses confirm that N. balachowskii is closely related to other species within the Kermesidae. In Israel, this species develops only on Quercus ithaburensis and is univoltine. This is the first detailed report of N. balachowskii from Israel.

Begomoviruses and whiteflies have interacted for geological times. An assumed long-lasting virus-vector intimate relationship of this magnitude implies that the partners have developed co-evolutionary mechanisms that insure on one hand the survival and the efficient transmission of the virus, and on the other hand the safeguard of the insect host from possible deleterious effects of the virus. Several studies have indicated that viruses belonging to the Tomato yellow leaf curl virus (TYLCVs) family from China, Israel and Italy are reminiscent of insect pathogens. TYLCVs like all begomoviruses are transmitted in a circulative manner by the whitefly Bemisia tabaci. The survival of the virus in the haemolymph of B. tabaci is ensured by a GroEL homologue produced by a whitefly secondary endosymbiont. Following acquisition and transfer to non-host plants, the virus may remain associated with the insect for its entire 4-5 wk-long adult life. During this period, the ability of the insects to inoculate plants steadily decreased, but did not disappear. The long-term presence of TYLCVs in B. tabaci was associated with a decrease in the insect longevity and fertility. Viral DNA was transmitted to progeny, but seldom infectivity. TYLCV transcripts were found associated with the insects, raising the possibility of replication and expression in the vector. TYLCVs may spread amidst whiteflies during copulation. Functional genomics tools such as microarrays, deep sequencing, quantitative PCR and gene silencing allow revisiting the proposition that TYLCVs have retained, or acquired, some characteristics of an insect pathogen.

The sweetpotato whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is an extremely polyphagous insect pest that causes significant crop losses in Israel and worldwide. B. tabaci is a species complex of which the B and Q biotypes are the most widespread and damaging worldwide. The change in biotype composition and resistance to insecticide in Israel was monitored during the years 2008–2010 to identify patterns in population dynamics that can be correlated with resistance outbreaks. The results show that B biotype populations dominate crops grown in open fields, while Q biotype populations gradually dominate crops grown in protected conditions such as greenhouses and nethouses, where resistance outbreaks usually develop after several insecticide applications. While in previous years, Q biotype populations were widely detected in many regions in Israel, significant domination of the B biotype across populations collected was observed during the year 2010, indicating the instability of the B. tabaci population from one year to another. Reasons for the changing dynamics and the shift in the relative abundance of B. tabaci biotype, and their resistance status, are discussed.

The whitefly Bemisia tabaci is a cosmopolitan insect species complex that harbors the obligate primary symbiont Portiera aleyrodidarum and several facultative secondary symbionts including Wolbachia, which have diverse influences on the host biology. Here, for the first time, we revealed two different localization patterns of Wolbachia present in the immature and adult stages of B. tabaci AsiaII7 cryptic species. In the confined pattern, Wolbachia was restricted to the bacteriocytes, while in the scattered pattern Wolbachia localized in the bacteriocytes, haemolymph and other organs simultaneously. Our results further indicated that, the proportion of B. tabaci AsiaII7 individuals with scattered Wolbachia were significantly lower than that of confined Wolbachia, and the distribution patterns of Wolbachia were not associated with the developmental stage or sex of whitefly host. This study will provide a new insight into the various transmission routes of Wolbachia in different whitefly species.

The increased use of molecular tools for species identification in recent decades revealed that each of many apparently generalist parasitoids are actually a complex of morphologically similar congeners, most of which have a rather narrow host range. Ooencyrtus pityocampae (OP), an important egg parasitoid of the pine processionary moth (PPM), is considered a generalist parasitoid. OP emerges from PPM eggs after winter hibernation, mainly in spring and early summer, long before the eggs of the next PPM generation occurs. The occurrence of OP in eggs of the variegated caper bug (CB) Stenozygum coloratum in spring and summer suggests that OP populations alternate seasonally between PPM and CB. However, the identity of OP population on CB eggs seemed uncertain; unlike OP-PPM populations, the former displayed apparently high male/female ratios and lack of attraction to the PPM sex pheromone. We studied the molecular identities of the two populations since the morphological identification of the genus Ooencyrtus, and OP in particular, is difficult. Sequencing of COI and ITS2 DNA fragments and AFLP analysis of individuals from both hosts revealed no apparent differences between the OP-PPM and the OP-CB populations for both the Israeli and the Turkish OPs, which therefore supported the possibility of host alternation. Sequencing data extended our knowledge of the genetic structure of OP populations in the Mediterranean area, and revealed clear separation between East and West Mediterranean populations. The overall level of genetic diversity was rather small, with the Israeli population much less diverse than all others; possible explanations for this finding are discussed. The findings support the possibility of utilizing the CB and other hosts for enhancing biological control of the PPM.

Bemisia tabaci (Gennadius), Trialeurodes vaporariorum (Westwood), and Siphoninus phillyreae (Haliday) are whitefly species that harm agricultural crops in many regions of the world. These insects live in close association with bacterial symbionts that affect host fitness and adaptation to the environment. In the current study, we surveyed the infection of whitefly populations in Southeast Europe by various bacterial symbionts and performed phylogenetic analyses on the different symbionts detected. Arsenophonus and Hamiltonella were the most prevalent symbionts in all three whitefly species. Rickettsia was found to infect mainly B. tabaci, while Wolbachia mainly infected both B. tabaci and S. phillyreae. Furthermore, Cardinium was rarely found in the investigated whitefly populations, while Fritschea was never found in any of the whitefly species tested. Phylogenetic analyses revealed a diversity of several symbionts (e.g., Hamiltonella, Arsenophonus, Rickettsia), which appeared in several clades. Reproductively isolated B. tabaci and T. vaporariorum shared the same (or highly similar) Hamiltonella and Arsenophonus, while these symbionts were distinctive in S. phillyreae. Interestingly, Arsenophonus from S. phillyreae did not cluster with any of the reported sequences, which could indicate the presence of Arsenophonus, not previously associated with whiteflies. In this study, symbionts (Wolbachia, Rickettsia, and Cardinium) known to infect a wide range of insects each clustered in the same clades independently of the whitefly species. These results indicate horizontal transmission of bacterial symbionts between reproductively isolated whitefly species, a mechanism that can establish new infections that did not previously exist in whiteflies.

Begomoviruses are vectored in a circulative persistent manner by the whitefly Bemisia tabaci. The insect ingests viral particles with its stylets. Virions pass along the food canal and reach the esophagus and the midgut. They cross the filter chamber and the midgut into the haemolymph, translocate into the primary salivary glands and are egested with the saliva into the plant phloem. Begomoviruses have to cross several barriers and checkpoints successfully, while interacting with would-be receptors and other whitefly proteins. The bulk of the virus remains associated with the midgut and the filter chamber. In these tissues, viral genomes, mainly from the tomato yellow leaf curl virus (TYLCV) family, may be transcribed and may replicate. However, at the same time, virus amounts peak, and the insect autophagic response is activated, which in turn inhibits replication and induces the destruction of the virus. Some begomoviruses invade tissues outside the circulative pathway, such as ovaries and fat cells. Autophagy limits the amounts of virus associated with these organs. In this review, we discuss the different sites begomoviruses need to cross to complete a successful circular infection, the role of the coat protein in this process and the sites that balance between virus accumulation and virus destruction.

Whiteflies (Hemiptera: Aleyrodidae) are sap-sucking insect pests, and some cause serious damage in agricultural crops by direct feeding and by transmitting plant viruses. Whiteflies maintain close associations with bacterial endosymbionts that can significantly influence their biology. All whitefly species harbor a primary endosymbiont, and a diverse array of secondary endosymbionts. In this study, we surveyed 34 whitefly populations collected from the states of Sao Paulo, Bahia, Minas Gerais and Parana in Brazil, for species identification and for infection with secondary endosymbionts. Sequencing the mitochondrial Cytochrome Oxidase I gene revealed the existence of five whitefly species: The sweetpotato whitefly Bemisia tabaci B biotype (recently termed Middle East-Asia Minor 1 or MEAM1), the greenhouse whitefly Trialeurodes vaporariorum, B. tabaci A biotype (recently termed New World 2 or NW2) collected only from Euphorbia, the Acacia whitefly Tetraleurodes acaciae and Bemisia tuberculata both were detected only on cassava. Sequencing rRNA genes showed that Hamiltonella and Rickettsia were highly prevalent in all MEAM1 populations, while Cardinium was close to fixation in only three populations. Surprisingly, some MEAM1 individuals and one NW2 population were infected with Fritschea. Arsenopnohus was the only endosymbiont detected in T. vaporariorum. In T. acaciae and B. tuberculata populations collected from cassava, Wolbachia was fixed in B. tuberculata and was highly prevalent in T. acaciae. Interestingly, while B. tuberculata was additionally infected with Arsenophonus, T. acaciae was infected with Cardinium and Fritschea. Fluorescence in situ hybridization analysis on representative individuals showed that Hamiltonella, Arsenopnohus and Fritschea were localized inside the bacteriome, Cardinium and Wolbachia exhibited dual localization patterns inside and outside the bacteriome, and Rickettsia showed strict localization outside the bacteriome. This study is the first survey of whitely populations collected in Brazil, and provides further insights into the complexity of infection with secondary endosymionts in whiteflies.

Occurrence of inter-species hybrids in natural populations might be evidence of gene flow between species. In the present study we found evidence of gene flow between two sympatric, genetically related scale insect species--the citrus mealybug Planococcus citri (Risso) and the vine mealybug Planococcus ficus (Signoret). These species can be distinguished by morphological, behavioral, and molecular traits. We employed the sex pheromones of the two respective species to study their different patterns of male attraction. We also used nuclear ITS2 (internal transcribed spacer 2) and mitochondrial COI (Cytochrome c oxidase sub unit 1) DNA sequences to characterize populations of the two species, in order to demonstrate the outcome of a possible gene flow between feral populations of the two species. Our results showed attraction to P. ficus pheromones of all tested populations of P. citri males but not vice versa. Furthermore, ITS2 sequences revealed the presence of 'hybrid females' among P. citri populations but not among those of P. ficus. 'hybrid females' from P. citri populations identified as P. citri females according to COI sequences. We offer two hypotheses for these results. 1) The occurrence of phenotypic and genotypic traits of P. ficus in P. citri populations may be attributed to both ancient and contemporary gene flow between their populations; and 2) we cannot rule out that an ancient sympatric speciation by which P. ficus emerged from P. citri might have led to the present situation of shared traits between these species. In light of these findings we also discuss the origin of the studied species and the importance of the pherotype phenomenon as a tool with which to study genetic relationships between congener scale insects.

Bacterial symbionts often enhance the physiological capabilities of their arthropod hosts and enable their hosts to expand into formerly unavailable niches, thus leading to biological diversification. Many arthropods, including the worldwide invasive whitefly Bemisia tabaci, have individuals simultaneously infected with symbionts of multiple genera that occur in different locations in the host. This study examined the population dynamics of symbionts that are located in different areas within B. tabaci. While densities of Portiera and Hamiltonella (which are located in bacteriocytes) appeared to be well-regulated during host development, densities of Rickettsia (which are not located in bacteriocytes) were highly variable among individual hosts during host development. Host mating did not significantly affect symbiont densities. Infection by Tomato yellow leaf curl virus did not affect Portiera and Hamiltonella densities in either sex, but increased Rickettsia densities in females. High and low temperatures did not affect Portiera and Hamiltonella densities, but low temperature (15 °C) significantly suppressed Rickettsia densities whereas high temperature (35 °C) had little effect on Rickettsia densities. The results are consistent with the view that the population dynamics of bacterial symbionts in B. tabaci are regulated by symbiont location within the host and that the regulation reflects adaptation between the bacteria and insect.

A functional capsid protein (CP) is essential for host plant infection and insect transmission of Tomato yellow leaf curl virus (TYLCV) and other monopartite begomoviruses. We have previously shown that TYLCV CP specifically interacts with the heat shock protein 70 (HSP70) of the virus insect vector, Bemisia tabaci. Here we demonstrate that during the development of tomato plant infection with TYLCV, a significant amount of HSP70 shifts from a soluble form into insoluble aggregates. CP and HSP70 co-localize in these aggregates, first in the cytoplasm, then in the nucleus of cells associated with the vascular system. CP-HSP70 interaction was demonstrated by co-immunopreciptation in cytoplasmic - but not in nuclear extracts from leaf and stem. Inhibition of HSP70 expression by quercetin caused a decrease in the amount of nuclear CP aggregates and a re-localization of a GFP-CP fusion protein from the nucleus to the cytoplasm. HSP70 inactivation resulted in a decrease of TYLCV DNA levels, demonstrating the role of HSP70 in TYLCV multiplication in planta. The current study reveals for the first time the involvement of plant HSP70 in TYLCV CP intracellular movement. As described earlier, nuclear aggregates contained TYLCV DNA-CP complexes and infectious virions. Showing that HSP70 localizes in these large nuclear aggregates infers that these structures operate as nuclear virus factories.

Insects and other arthropods are the most important vectors of plant pathogens. The majority of plant pathogens are disseminated by arthropod vectors such as aphids, beetles, leafhoppers, planthoppers, thrips and whiteflies. Transmission of plant pathogens and the challenges in managing insect vectors due to insecticide resistance are factors that contribute to major food losses in agriculture. RNA interference (RNAi) was recently suggested as a promising strategy for controlling insect pests, including those that serve as important vectors for plant pathogens. The last decade has witnessed a dramatic increase in the functional analysis of insect genes, especially those whose silencing results in mortality or interference with pathogen transmission. The identification of such candidates poses a major challenge for increasing the role of RNAi in pest control. Another challenge is to understand the RNAi machinery in insect cells and whether components that were identified in other organisms are also present in insect. This review will focus on summarizing success cases in which RNAi was used for silencing genes in insect vector for plant pathogens, and will be particularly helpful for vector biologists.

The whitefly Bemisia tabaci is a major pest to agricultural crops. It transmits begomoviruses, such as Tomato yellow leaf curl virus (TYLCV), in a circular, persistent fashion. Transcriptome analyses revealed that B. tabaci knottin genes were responsive to various stresses. Upon ingestion of tomato begomoviruses, two of the four knottin genes were upregulated, knot-1 (with the highest expression) and knot-3. In this study, we examined the involvement of B. tabaci knottin genes in relation to TYLCV circulative transmission. Knottins were silenced by feeding whiteflies with knottin dsRNA via detached tomato leaves. Large amounts of knot-1 transcripts were present in the abdomen of whiteflies, an obligatory transit site of begomoviruses in their circulative transmission pathway; knot-1 silencing significantly depleted the abdomen from knot-1 transcripts. Knot-1 silencing led to an increase in the amounts of TYLCV ingested by the insects and transmitted to tomato test plants by several orders of magnitude. This effect was not observed following knot-3 silencing. Hence, knot-1 plays a role in restricting the quantity of virions an insect may acquire and transmit. We suggest that knot-1 protects B. tabaci against deleterious effects caused by TYLCV by limiting the amount of virus associated with the whitefly vector.