Combined infection with Tomato yellow leaf curl virus and Rickettsia influences fecundity, attraction to infected plants and expression of immunity-related genes in the whitefly Bemisia tabaci.

We have recently shown that Rickettsia, a secondary facultative bacterial symbiont that infects the whitefly B. tabaci is implicated in the transmission of Tomato yellow leaf curl virus (TYLCV). Infection with Rickettsia improved the acquisition and transmission of the virus by B. tabaci adults. Here we performed a transcriptomic analysis with Rickettsia-infected and uninfected B. tabaci adults before and after TYLCV acquisition. The results show a dramatic and specific activation of the immune system in the presence of Rickettsia before TYLCV acquisition. However, when TYLCV was acquired, it induced massive activation of gene expression in the Rickettsia uninfected population, whereas in the Rickettsia-infected population the virus induced massive down-regulation of gene expression. Fitness and choice experiments revealed that while Rickettsia-infected whiteflies are always more attracted to TYLCV-infected plants, this attraction is not always beneficiary for their offspring. These studies further confirm the role of Rickettsia in many aspects of B. tabaci interactions with TYLCV, and possibly serves as an important factor in the dissemination of the virus.

Fluorescent in situ hybridization for the localization of viruses, bacteria and other microorganisms in insect and plant tissues.

Methods for the localization of cellular components such as nucleic acids, proteins, cellular vesicles and more, and the localization of microorganisms including viruses, bacteria and fungi have become an important part of any research program in biological sciences that enable the visualization of these components in fixed and live tissues without the need for complex processing steps. The rapid development of microscopy tools and technologies as well as related fluorescent markers and fluorophores for many cellular components, and the ability to design DNA and RNA sequence-based molecular probes and antibodies which can be visualized fluorescently, have rapidly advanced this field. This review will focus on some of the localizations methods which have been used in plants and insect pests in agriculture, and other microorganisms, which are rapidly advancing the research in agriculture-related fields.

The draft genome of whitefly Bemisia tabaci MEAM1, a global crop pest, provides novel insights into virus transmission, host adaptation, and insecticide resistance.

BACKGROUND: The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is among the 100 worst invasive species in the world. As one of the most important crop pests and virus vectors, B. tabaci causes substantial crop losses and poses a serious threat to global food security. RESULTS: We report the 615-Mb high-quality genome sequence of B. tabaci Middle East-Asia Minor 1 (MEAM1), the first genome sequence in the Aleyrodidae family, which contains 15,664 protein-coding genes. The B. tabaci genome is highly divergent from other sequenced hemipteran genomes, sharing no detectable synteny. A number of known detoxification gene families, including cytochrome P450s and UDP-glucuronosyltransferases, are significantly expanded in B. tabaci. Other expanded gene families, including cathepsins, large clusters of tandemly duplicated B. tabaci-specific genes, and phosphatidylethanolamine-binding proteins (PEBPs), were found to be associated with virus acquisition and transmission and/or insecticide resistance, likely contributing to the global invasiveness and efficient virus transmission capacity of B. tabaci. The presence of 142 horizontally transferred genes from bacteria or fungi in the B. tabaci genome, including genes encoding hopanoid/sterol synthesis and xenobiotic detoxification enzymes that are not present in other insects, offers novel insights into the unique biological adaptations of this insect such as polyphagy and insecticide resistance. Interestingly, two adjacent bacterial pantothenate biosynthesis genes, panB and panC, have been co-transferred into B. tabaci and fused into a single gene that has acquired introns during its evolution. CONCLUSIONS: The B. tabaci genome contains numerous genetic novelties, including expansions in gene families associated with insecticide resistance, detoxification and virus transmission, as well as numerous horizontally transferred genes from bacteria and fungi. We believe these novelties likely have shaped B. tabaci as a highly invasive polyphagous crop pest and efficient vector of plant viruses. The genome serves as a reference for resolving the B. tabaci cryptic species complex, understanding fundamental biological novelties, and providing valuable genetic information to assist the development of novel strategies for controlling whiteflies and the viruses they transmit.

Implication of the bacterial endosymbiont Rickettsia spp. in interactions of the whitefly Bemisia tabaci with tomato yellow leaf curl virus.

UNLABELLED: Numerous animal and plant viruses are transmitted by arthropod vectors in a persistent, circulative manner. Tomato yellow leaf curl virus (TYLCV) is transmitted by the sweet potato whitefly Bemisia tabaci. We report here that infection with Rickettsia spp., a facultative endosymbiont of whiteflies, altered TYLCV-B. tabaci interactions. A B. tabaci strain infected with Rickettsia acquired more TYLCV from infected plants, retained the virus longer, and exhibited nearly double the transmission efficiency compared to an uninfected B. tabaci strain with the same genetic background. Temporal and spatial antagonistic relationships were discovered between Rickettsia and TYLCV within the whitefly. In different time course experiments, the levels of virus and Rickettsia within the insect were inversely correlated. Fluorescence in situ hybridization analysis of Rickettsia-infected midguts provided evidence for niche exclusion between Rickettsia and TYLCV. In particular, high levels of the bacterium in the midgut resulted in higher virus concentrations in the filter chamber, a favored site for virus translocation along the transmission pathway, whereas low levels of Rickettsia in the midgut resulted in an even distribution of the virus. Taken together, these results indicate that Rickettsia, by infecting the midgut, increases TYLCV transmission efficacy, adding further insights into the complex association between persistent plant viruses, their insect vectors, and microorganism tenants that reside within these insects. IMPORTANCE: Interest in bacterial endosymbionts in arthropods and many aspects of their host biology in agricultural and human health systems has been increasing. A recent and relevant studied example is the influence of Wolbachia on dengue virus transmission by mosquitoes. In parallel with our recently studied whitefly-Rickettsia-TYLCV system, other studies have shown that dengue virus levels in the mosquito vector are inversely correlated with bacterial load. Our work here presents evidence of unifying principles between vectors of plant and animal viruses in a role for endosymbionts in manipulating vector biology and pathogen transmission. Our results demonstrate the influence of an interesting and prominent bacterial endosymbiont in Bemisia tabaci in TYLCV transmission, a worldwide disease infecting tomatoes. Besides its agricultural importance, this system provides interesting insights into Bemisia interaction with these newly discovered endosymbionts.

Sib-mating enhances fitness in a haplodiploid beetle.

Inbreeding is generally avoided in animals due to the risk of inbreeding depression following an increase in homozygous deleterious alleles and loss of heterozygosity. Species that regularly inbreed challenge our understanding of the fitness effects of these risks. We investigated the fitness consequences of extended inbreeding in the haplodiploid date stone beetle, Coccotrypes dactyliperda. We hypothesized that continuous inbreeding could result in reduced fitness, while outbreeding would either increase fitness due to heterosis or lower fitness if co-adapted gene complexes are disrupted. We established three breeding treatments with beetles from two geographically separated populations: Sib-mating (inbreeding), and outbreeding within and between populations. Between-population outbreeding groups of both populations had lower fecundity and collapsed before the experiment ended, while sib-mated and within-population breeding groups persisted for 10 generations. Sib-mated females had higher fecundity than within- and between-population outbreeding females. Inbreeding coefficients of sib-mated groups were higher than the other treatment groups, yet sib-mated beetles remained genetically polymorphic at the population level. Thus, there was no inbreeding depression, while crossing between distant populations led to outbreeding depression. Our findings are consistent with the life history of C. dactyliperda, in which sib-mating predominates within the date seed, but occasional within-population outbreeding may occur following local dispersal.

A proteomic approach reveals possible molecular mechanisms and roles for endosymbiotic bacteria in begomovirus transmission by whiteflies.

BACKGROUND: Many plant viruses are vector-borne and depend on arthropods for transmission between host plants. Begomoviruses, the largest, most damaging and emerging group of plant viruses, infect hundreds of plant species, and new virus species of the group are discovered each year. Begomoviruses are transmitted by members of the whitefly Bemisia tabaci species complex in a persistent-circulative manner. Tomato yellow leaf curl virus (TYLCV) is one of the most devastating begomoviruses worldwide and causes major losses in tomato crops, as well as in many agriculturally important plant species. Different B. tabaci populations vary in their virus transmission abilities; however, the causes for these variations are attributed among others to genetic differences among vector populations, as well as to differences in the bacterial symbionts housed within B. tabaci. RESULTS: Here, we performed discovery proteomic analyses in 9 whitefly populations from both Middle East Asia Minor I (MEAM1, formerly known as B biotype) and Mediterranean (MED, formerly known as Q biotype) species. We analysed our proteomic results on the basis of the different TYLCV transmission abilities of the various populations included in the study. The results provide the first comprehensive list of candidate insect and bacterial symbiont (mainly Rickettsia) proteins associated with virus transmission. CONCLUSIONS: Our data demonstrate that the proteomic signatures of better vector populations differ considerably when compared with less efficient vector populations in the 2 whitefly species tested in this study. While MEAM1 efficient vector populations have a more lenient immune system, the Q efficient vector populations have higher abundance of proteins possibly implicated in virus passage through cells. Both species show a strong link of the facultative symbiont Rickettsia to virus transmission.

A multi-layered mechanistic modelling approach to understand how effector genes extend beyond phytoplasma to modulate plant hosts, insect vectors and the environment.

Members of the Candidatus genus Phytoplasma are small bacterial pathogens that hijack their plant hosts via the secretion of virulence proteins (effectors) leading to a fascinating array of plant phenotypes, such as witch's brooms (stem proliferations) and phyllody (retrograde development of flowers into vegetative tissues). Phytoplasma depend on insect vectors for transmission, and interestingly, these insect vectors were found to be (in)directly attracted to plants with these phenotypes. Therefore, phytoplasma effectors appear to reprogram plant development and defence to lure insect vectors, similarly to social engineering malware, which employs tricks to lure people to infected computers and webpages. A multi-layered mechanistic modelling approach will enable a better understanding of how phytoplasma effector-mediated modulations of plant host development and insect vector behaviour contribute to phytoplasma spread, and ultimately to predict the long reach of phytoplasma effector genes.

Effects of radiation on inherited sterility in the European grapevine moth (Lobesia botrana).

BACKGROUND: The sterile insect technique (SIT) is an alternative, environmentally friendly method for controlling insect pests. In the Lepidoptera, a low dose of gamma irradiation causes inherited sterility (SIT-IS), leading to full sterility in females but only partial sterility in males, which successfully compete with wild males for mates. This study examined the effect of a low radiation dose (150 Gy) on the fitness parameters of male and female Lobesia botrana, a polyphagous and major pest of vineyards found in the Middle East, Europe and the Americas. RESULTS: Irradiation of the pupae did not affect their emergence rate, flight ability out of a cylinder, male response to sex pheromone in a field cage or male or female mating success. A major effect of irradiation was observed in the significantly reduced number of irradiated females' offspring reaching pupation, and as a consequence a limited number of F2 offspring. The effect of irradiation on male partial sterility (also called inherited sterility) was reflected in the male-biased sex ratio of F1 offspring of irradiated males, the reduced number of F1 offspring and the very low number of F2 descendants. CONCLUSION: This study demonstrates the feasibility of controlling L. botrana using SIT-IS. Adding this method to the arsenal of environmentally friendly tools to control this pest may assist in further reducing the use of insecticides on edible crops.

Is there a role for symbiotic bacteria in plant virus transmission by insects?

During the process of circulative plant virus transmission by insect vectors, viruses interact with different insect vector tissues prior to transmission to a new host plant. An area of intense debate in the field is whether bacterial symbionts of insect vectors are involved in the virus transmission process. We critically review the literature in this area and present a simple model that can be used to quantitatively settle the debate. The simple model determines whether the symbiont is involved in virus transmission and determines what fraction of the pathogen transmission phenotype is contributed by the symbiont. The model is general and can be applied to any vector-pathogen-symbiont interactions.

Persistent, circulative transmission of begomoviruses by whitefly vectors.

Begomoviruses comprise an emerging and economically important group of plant viruses exclusively transmitted by the sweetpotato whitefly Bemisia tabaci in many regions of the world. The past twenty years have witnessed significant progress in studying the molecular interactions between members of this virus group and B. tabaci. Mechanisms and proteins encoded by the insect vector and its bacterial symbionts, which have been shown to be important for virus transmission, have been identified and thoroughly studied. Despite the economic importance of this group of viruses and their impact on the global agriculture, progress in investigating the virus-vector interactions is moving slowly when compared with similar virus-vector systems in plants and animals. Major advances in this field and future perspectives will be discussed in this review.

Adaptation to nicotine in the facultative tobacco-feeding hemipteran Bemisia tabaci.

BACKGROUND: Plant defensive metabolites such as nicotine can provide barriers to host-range expansion by generalist herbivores. Nicotine is one of the most abundant and toxic plant secondary metabolites in nature and is defined by high toxicity to plant-feeding insects. There is significant variation in nicotine tolerance among Bemisia tabaci (tobacco whitefly) isolates. Some nicotine-tolerant B. tabaci strains can consume 40-fold higher nicotine levels than susceptible strains, and also show cross-resistance to neonicotinoid insecticides. In this study, biological and molecular assays were used to investigate the responses of B. tabaci strains that differ in their ability to tolerate dietary nicotine. RESULTS: Egg laying and honeydew secretion bioassays as well as gene expression microarrays were used to measure B. tabaci biological parameters and gene transcripts misregulated in response to nicotine in resistant and susceptible strains. The resistant B. tabaci strain laid significantly fewer eggs and excreted more honeydew on a tobacco strain with high levels of nicotine, suggesting a fitness cost effect. The molecular response was drastic in the susceptible strain, while the resistant strain exhibited moderate response. Higher expression of the previously identified CYP6CM1 P450 monooxygenase gene related to the resistance to neonicotinoids, as well as other P450s and metabolic genes, was identified in the resistant and susceptible strains after exposure to nicotine. CONCLUSIONS: Nicotine is a very toxic plant natural compound, and its mode of action resembles that of synthetic neonicotinoids. The biological and molecular responses observed in this study suggest that nicotine may play an important role in providing barriers for host-plant expansion by generalists, and may act as a natural factor that contributes to the development of insect populations resistant to synthetic pesticides.

Diversity and localization of bacterial endosymbionts from whitefly species collected in Brazil.

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.

Fluorescence in situ hybridizations (FISH) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues.

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5' or 3' ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

The role of bacterial chaperones in the circulative transmission of plant viruses by insect vectors.

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.

Fitness costs associated with insecticide resistance.

Insects are exposed to a variety of stress factors in their environment, and, in many cases for insect pests to agriculture, those factors include toxic chemical insecticides. Coping with the toxicity of insecticides can be costly and requires energy and resource allocation for adaptation and survival. Several behavioural, physiological and genetic mechanisms are used by insects to handle toxic insecticides, sometimes leading to resistance by constitutive overexpression of detoxification enzymes or inducing mutations in the target sites. Such actions are costly and may affect reproduction, impair dispersal ability and have several other effects on the insect's fitness. Fitness costs resulting from resistance to insecticides has been reported in many insects from different orders, and several examples are given in this mini-review.