Accumulation and Transmission of 'Candidatus Liberibacter solanacearum' Haplotypes by the Nymphs of Two Psyllid Vectors.

'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.

Influence of juvenile hormone analog on behavior in the red imported fire ant, Solenopsis invicta.

Division of labor is a hallmark characteristic of social insect colonies. While it is understood that worker differentiation is regulated through either the queen or her brood, the understanding of the physiology behind task regulation varies within social species. Studies in eusocial insects have shown that juvenile hormone (JH) is associated with division of labor and the onset of foraging tasks. Although, outside of a few key species, this interaction has yet to be elucidated in the red imported fire ant, Solenopsis invicta. In this study, we evaluated the role of a JH analog, S-hydroprene in worker task transition in Solenopsis invicta. S-hydroprene was applied to nurses to observe behavioral changes. S-hyroprene application to nurses did not affect phototaxis, but there was a shift in behavior from internal, nest-based behaviors to external, foraging-based behaviors. These results show that JH may be implicated in worker task transition in S. invicta and may function similarly as it does in other eusocial insects.

A 'Candidatus Liberibacter solanacearum' Haplotype B-Specific Family of Candidate Bacterial Effectors.

'Candidatus Liberibacter solanacearum' (Lso) is a phloem-limited pathogen associated with devastating diseases in members of the Solanaceae and Apiaceae and vectored by several psyllid species. Different Lso haplotypes have been identified, and LsoA and LsoB are responsible for diseases in Solanaceae crops. Our efforts are aimed at identifying pathogenicity factors used by this bacterium to thrive in different hosts. Bacterial secreted proteins can play a role in host colonization or the manipulation of the host immune responses; these proteins are called effectors. In this study, we identified six LsoB-specific proteins with a conserved secretion motif as well as a conserved N-terminal domain in the mature protein. These proteins had different expression and secretion patterns but a similar subcellular localization in Nicotiana benthamiana leaves, suggesting that they play different roles regardless of their conserved secretion motif. One of these proteins, CKC_04425, was expressed at high levels in the insect vector and the host plant, indicating that it could play a role in both the plant and insect hosts, whereas the others were mainly expressed in the plant. One protein, CKC_05701, was able to efficiently suppress programmed cell death and reactive oxygen species production, suggesting that it may have a virulence role in LsoB-specific pathogenesis.

Potato psyllids mount distinct gut responses against two different 'Candidatus Liberibacter solanacearum' haplotypes.

'Candidatus Liberibacter solanacearum' (Lso) is a bacterial pathogen infecting several crops and causing damaging diseases. Several Lso haplotypes have been identified. Among the seven haplotypes present in North America, LsoA and LsoB are transmitted by the potato psyllid, Bactericera cockerelli (Sulc), in a circulative and persistent manner. The gut, which is the first organ pathogen encounters, could be a barrier for Lso transmission. However, the molecular interactions between Lso and the psyllid vector at the gut interface remain largely unknown. In this study, we investigated the global transcriptional responses of the adult psyllid gut upon infection with two Lso haplotypes (LsoA and LsoB) using Illumina sequencing. The results showed that each haplotype triggers a unique transcriptional response, with most of the distinct genes elicited by the highly virulent LsoB. The differentially expressed genes were mainly associated with digestion and metabolism, stress response, immunity, detoxification as well as cell proliferation and epithelium renewal. Importantly, distinct immune pathways were triggered by LsoA and LsoB in the gut of the potato psyllid. The information in this study will provide an understanding of the molecular basis of the interactions between the potato psyllid gut and Lso, which may lead to the discovery of novel molecular targets for the control of these pathogens.

Family before work: task reversion in workers of the red imported fire ant, Solenopsis invicta in the presence of brood.

Among social insects, task allocation within its group members remains as one of the paramount pillars of social functionality. Division of labor in many eusocial insects is maintained by behavioral flexibility that can shift according to the needs of the colony they reside in. Workers typically, over time as they age, shift from intranidal nurses to extranidal foragers. If the needs of the colony change, either from the needs of the adults or the brood therein, workers shift their behavior in order to compensate for the need of a particular task to be done. This shift, either accelerating towards a behavior associated with an older worker, or regressing back into the nest, is not clearly understood in social insects outside of honeybees. In this study, evaluated how brood type affected the red imported fire ant, Solenopsis invicta, worker task reversion and acceleration. Through observation of worker behaviors performed over multiple time-points per day, we discovered that worker task reversion and acceleration does occur within this ant species. Furthermore, the type of brood influenced the rate at which this occurred, with larvae having the strongest effect of all types. Finally, there was a propensity for workers to maintain their new behavior throughout the experiment. This study shows that the needs of brood within a social insect colony can influence the behavior workers perform, reversing the age polyethism that is common among social insect species.

Treatment of Rapamycin and Evaluation of an Autophagic Response in the Gut of Bactericera cockerelli (Sulc).

Autophagy is a catabolic process that results in the autophagosomic-lysosomal degradation of bulk cytoplasmic content, abnormal protein aggregates, and excess of/or damaged organelles to promote cell survival. Autophagy is also a component of innate immunity in insects and is involved in the clearance of pathogens, including bacteria. The potato psyllid, Bactericera cockerelli, transmits the plant bacterial pathogen 'Candidatus Liberibacter solanacearum' (Lso) in the Americas and causes serious damage to solanaceous crops. Our previous studies showed that autophagy could be involved in the psyllid response to Lso and could affect pathogen acquisition. However, the tools to evaluate this response have not been validated in psyllids. To this end, the effect of rapamycin, a commonly used autophagy inducer, on potato psyllid survival and the expression of autophagy-related genes was evaluated. Further, the autophagic activity was assessed via microscopy and by measuring the autophagic flux. Artificial diet-feeding assays using rapamycin resulted in significant psyllid mortality, an increase in the autophagic flux, as well as an increase in the amount of autolysosomes. This study represents a stepping stone in determining the role of autophagy in psyllid immunity.

A New Perspective on the Co-Transmission of Plant Pathogens by Hemipterans.

Co-infection of plants by pathogens is common in nature, and the interaction of the pathogens can affect the infection outcome. There are diverse ways in which viruses and bacteria are transmitted from infected to healthy plants, but insects are common vectors. The present review aims to highlight key findings of studies evaluating the co-transmission of plant pathogens by insects and identify challenges encountered in these studies. In this review, we evaluated whether similar pathogens might compete during co-transmission; whether the changes in the pathogen titer in the host, in particular associated with the co-infection, could influence its transmission; and finally, we discussed the pros and cons of the different approaches used to study co-transmission. At the end of the review, we highlighted areas of study that need to be addressed. This review shows that despite the recent development of techniques and methods to study the interactions between pathogens and their insect vectors, there are still gaps in the knowledge of pathogen transmission. Additional laboratory and field studies using different pathosystems will help elucidate the role of host co-infection and pathogen co-transmission in the ecology and evolution of infectious diseases.

CLIBASIA_00460 Disrupts Hypersensitive Response and Interacts with Citrus Rad23 Proteins.

'Candidatus Liberibacter asiaticus' (CLas) is a bacterium that causes Huanglongbing, also known as citrus greening, in citrus plants. 'Candidatus Liberibacter solanacearum' (Lso) is a close relative of CLas and in the US it infects solanaceous crops, causing zebra chip disease in potato. Previously, we have identified the Lso hypothetical protein effector 1 (Lso-HPE1). This protein uses a signal peptide for secretion; disrupts programmed cell death; and interacts with tomato RAD23c, d, and e proteins, but not with RAD23a. In this study, we evaluated whether CLIBASIA_00460, the CLas homolog of Lso-HPE1 interacted with citrus RAD23 proteins and disrupted their programmed cell death. Based on the yeast two-hybrid assay results, CLIBASIA_00460 interacted with citrus RAD23c and RAD23d, but not with citrus RAD23b. These results were confirmed using bimolecular fluorescence complementation assays, which showed that these interactions occurred in cell puncta, but not in the nucleus or cytoplasm. Additionally, CLIBASIA_00460 was able to disrupt the PrfD1416V-induced hypersensitive response. Therefore, based on the similar interactions between Lso-HPE1 and CLIBASIA_00460 with the host RAD23 proteins and their ability to inhibit cell death in plants, we propose that these effectors may have similar functions during plant infection.

Effects of 'Candidatus Liberibacter solanacearum' haplotypes A and B on tomato gene expression and geotropism.

BACKGROUND: The tomato psyllid, Bactericera cockerelli Sulc (Hemiptera: Triozidae), is a pest of solanaceous crops such as tomato (Solanum lycopersicum L.) in the U.S. and vectors the disease-causing pathogen 'Candidatus Liberibacter solanacearum' (or Lso). Disease symptom severity is dependent on Lso haplotype: tomato plants infected with Lso haplotype B experience more severe symptoms and higher mortality compared to plants infected with Lso haplotype A. By characterizing the molecular differences in the tomato plant's responses to Lso haplotypes, the key components of LsoB virulence can be identified and, thus, targeted for disease mitigation strategies. RESULTS: To characterize the tomato plant genes putatively involved in the differential immune responses to Lso haplotypes A and B, RNA was extracted from tomato 'Moneymaker' leaves 3 weeks after psyllid infestation. Gene expression levels were compared between uninfected tomato plants (i.e., controls and plants infested with Lso-free psyllids) and infected plants (i.e., plants infested with psyllids infected with either Lso haplotype A or Lso haplotype B). Furthermore, expression levels were compared between plants infected with Lso haplotype A and plants infected with Lso haplotype B. A whole transcriptome analysis identified 578 differentially expressed genes (DEGs) between uninfected and infected plants as well as 451 DEGs between LsoA- and LsoB-infected plants. These DEGs were primarily associated with plant defense against abiotic and biotic stressors, growth/development, plant primary metabolism, transport and signaling, and transcription/translation. These gene expression changes suggested that tomato plants traded off plant growth and homeostasis for improved defense against pathogens, especially when infected with LsoB. Consistent with these results, tomato plant growth experiments determined that LsoB-infected plants were significantly stunted and had impaired negative geotropism. However, it appeared that the defense responses mounted by tomatoes were insufficient for overcoming the disease symptoms and mortality caused by LsoB infection, while these defenses could compensate for LsoA infection. CONCLUSION: The transcriptomic analysis and growth experiments demonstrated that Lso-infected tomato plants underwent gene expression changes related to abiotic and biotic stressors, impaired growth/development, impaired plant primary metabolism, impaired transport and signaling transduction, and impaired transcription/translation. Furthermore, the transcriptomic analysis also showed that LsoB-infected plants, relative to LsoA-infected, experienced more severe stunting, had improved responses to some stressors and impaired responses to others, had poorer transport and signaling transduction, and had impaired carbohydrate synthesis and photosynthesis.

Identification of Autophagy-Related Genes in the Potato Psyllid, Bactericera cockerelli and Their Expression Profile in Response to 'Candidatus Liberibacter Solanacearum' in the Gut.

Autophagy, also known as type II programmed cell death, is a cellular mechanism of "self-eating". Autophagy plays an important role against pathogen infection in numerous organisms. Recently, it has been demonstrated that autophagy can be activated and even manipulated by plant viruses to facilitate their transmission within insect vectors. However, little is known about the role of autophagy in the interactions of insect vectors with plant bacterial pathogens. 'Candidatus Liberibacter solanacearum' (Lso) is a phloem-limited Gram-negative bacterium that infects crops worldwide. Two Lso haplotypes, LsoA and LsoB, are transmitted by the potato psyllid, Bactericera cockerelli and cause damaging diseases in solanaceous plants (e.g., zebra chip in potatoes). Both LsoA and LsoB are transmitted by the potato psyllid in a persistent circulative manner: they colonize and replicate within psyllid tissues. Following acquisition, the gut is the first organ Lso encounters and could be a barrier for transmission. In this study, we annotated autophagy-related genes (ATGs) from the potato psyllid transcriptome and evaluated their expression in response to Lso infection at the gut interface. In total, 19 ATGs belonging to 17 different families were identified. The comprehensive expression profile analysis revealed that the majority of the ATGs were regulated in the psyllid gut following the exposure or infection to each Lso haplotype, LsoA and LsoB, suggesting a potential role of autophagy in response to Lso at the psyllid gut interface.

HPE1, an Effector from Zebra Chip Pathogen Interacts with Tomato Proteins and Perturbs Ubiquitinated Protein Accumulation.

The gram-negative bacterial genus Liberibacter includes economically important pathogens, such as 'Candidatus Liberibacter asiaticus' that cause citrus greening disease (or Huanglongbing, HLB) and 'Ca. Liberibacter solanacearum' (Lso) that cause zebra chip disease in potato. Liberibacter pathogens are fastidious bacteria transmitted by psyllids. Pathogen manipulation of the host' and vector's immune system for successful colonization is hypothesized to be achieved by Sec translocon-dependent effectors (SDE). In previous work, we identified hypothetical protein effector 1 (HPE1), an SDE from Lso, that acts as a suppressor of the plant's effector-triggered immunity (ETI)-like response. In this study, using a yeast two-hybrid system, we identify binding interactions between tomato RAD23 proteins and HPE1. We further show that HPE1 interacts with RAD23 in both nuclear and cytoplasmic compartments in planta. Immunoblot assays show that HPE1 is not ubiquitinated in the plant cell, but rather the expression of HPE1 induced the accumulation of other ubiquitinated proteins. A similar accumulation of ubiquitinated proteins is also observed in Lso infected tomato plants. Finally, earlier colonization and symptom development following Lso haplotype B infection are observed in HPE1 overexpressing plants compared to wild-type plants. Overall, our results suggest that HPE1 plays a role in virulence in Lso pathogenesis, possibly by perturbing the ubiquitin-proteasome system via direct interaction with the ubiquitin-like domain of RAD23 proteins.

Affinity Purification-Mass Spectrometry Identifies a Novel Interaction between a Polerovirus and a Conserved Innate Immunity Aphid Protein that Regulates Transmission Efficiency.

The vast majority of plant viruses are transmitted by insect vectors, with many crucial aspects of the transmission process being mediated by key protein-protein interactions. Still, very few vector proteins interacting with viruses have been identified and functionally characterized. Potato leafroll virus (PLRV) is transmitted most efficiently by Myzus persicae, the green peach aphid, in a circulative, non-propagative manner. Using affinity purification coupled to high-resolution mass spectrometry (AP-MS), we identified 11 proteins from M. persicaedisplaying a high probability of interaction with PLRV and an additional 23 vector proteins with medium confidence interaction scores. Three of these aphid proteins were confirmed to directly interact with the structural proteins of PLRV and other luteovirid species via yeast two-hybrid. Immunolocalization of one of these direct PLRV-interacting proteins, an orthologue of the human innate immunity protein complement component 1 Q subcomponent-binding protein (C1QBP), shows that MpC1QBP partially co-localizes with PLRV in cytoplasmic puncta and along the periphery of aphid gut epithelial cells. Artificial diet delivery to aphids of a chemical inhibitor of C1QBP leads to increased PLRV acquisition by aphids and subsequently increased titer in inoculated plants, supporting a role for C1QBP in the acquisition and transmission efficiency of PLRV by M. persicae. This study presents the first use of AP-MS for the in vivo isolation of a functionally relevant insect vector-virus protein complex. MS data are available from ProteomeXchange.org using the project identifier PXD022167.

Differential gene expression in a tripartite interaction: Drosophila, Spiroplasma and parasitic wasps.

BACKGROUND: Several facultative bacterial symbionts of insects protect their hosts against natural enemies. Spiroplasma poulsonii strain sMel (hereafter Spiroplasma), a male-killing heritable symbiont of Drosophila melanogaster, confers protection against some species of parasitic wasps. Several lines of evidence suggest that Spiroplasma-encoded ribosome inactivating proteins (RIPs) are involved in the protection mechanism, but the potential contribution of the fly-encoded functions (e.g., immune response), has not been deeply explored. METHODS: Here we used RNA-seq to evaluate the response of D. melanogaster to infection by Spiroplasma and parasitism by the Spiroplasma-susceptible wasp Leptopilina heterotoma, and the Spiroplasma-resistant wasp Ganaspis sp. In addition, we used quantitative (q)PCR to evaluate the transcript levels of the Spiroplasma-encoded Ribosomal inactivation protein (RIP) genes. RESULTS: In the absence of Spiroplasma infection, we found evidence of Drosophila immune activation by Ganaspis sp., but not by L. heterotoma, which in turn negatively influenced functions associated with male gonad development. As expected for a symbiont that kills males, we detected extensive downregulation in the Spiroplasma-infected treatments of genes known to have male-biased expression. We detected very few genes whose expression patterns appeared to be influenced by the Spiroplasma-L. heterotoma interaction, and these genes are not known to be associated with immune response. For most of these genes, parasitism by L. heterotoma (in the absence of Spiroplasma) caused an expression change that was at least partly reversed when both L. heterotoma and Spiroplasma were present. It is unclear whether such genes are involved in the Spiroplasma-mediated mechanism that leads to wasp death and/or fly rescue. Nonetheless, the expression pattern of some of these genes, which reportedly undergo expression shifts during the larva-to-pupa transition, is suggestive of an influence of Spiroplasma on the development time of L. heterotoma-parasitized flies. One of the five RIP genes (RIP2) was consistently highly expressed independently of wasp parasitism, in two substrains of sMel. Finally, the RNAseq data revealed evidence consistent with RIP-induced damage in the ribosomal (r)RNA of the Spiroplasma-susceptible, but not the Spiroplasma-resistant, wasp. Acknowledging the caveat that we lacked adequate power to detect the majority of DE genes with fold-changes lower than 3, we conclude that immune priming is unlikely to contribute to the Spiroplasma-mediated protection against wasps, and that the mechanism by which Ganaspis sp. resists/tolerates Spiroplasma does not involve inhibition of RIP transcription.

Lasting consequences of psyllid (Bactericera cockerelli L.) infestation on tomato defense, gene expression, and growth.

BACKGROUND: The tomato psyllid, Bactericera cockerelli Sulc (Hemiptera: Triozidae), is a pest of solanaceous crops such as tomato (Solanum lycopersicum L.) in the U.S. and vectors the disease-causing pathogen 'Candidatus Liberibacter solanacearum'. Currently, the only effective strategies for controlling the diseases associated with this pathogen involve regular pesticide applications to manage psyllid population density. However, such practices are unsustainable and will eventually lead to widespread pesticide resistance in psyllids. Therefore, new control strategies must be developed to increase host-plant resistance to insect vectors. For example, expression of constitutive and inducible plant defenses can be improved through selection. Currently, it is still unknown whether psyllid infestation has any lasting consequences on tomato plant defense or tomato plant gene expression in general. RESULTS: In order to characterize the genes putatively involved in tomato defense against psyllid infestation, RNA was extracted from psyllid-infested and uninfested tomato leaves (Moneymaker) 3 weeks post-infestation. Transcriptome analysis identified 362 differentially expressed genes. These differentially expressed genes were primarily associated with defense responses to abiotic/biotic stress, transcription/translation, cellular signaling/transport, and photosynthesis. These gene expression changes suggested that tomato plants underwent a reduction in plant growth/health in exchange for improved defense against stress that was observable 3 weeks after psyllid infestation. Consistent with these observations, tomato plant growth experiments determined that the plants were shorter 3 weeks after psyllid infestation. Furthermore, psyllid nymphs had lower survival rates on tomato plants that had been previously psyllid infested. CONCLUSION: These results suggested that psyllid infestation has lasting consequences for tomato gene expression, defense, and growth.

Acquisition and transmission of two 'Candidatus Liberibacter solanacearum' haplotypes by the tomato psyllid Bactericera cockerelli.

'Candidatus Liberibacter solanacearum' (Lso) is a pathogen of solanaceous crops. Two haplotypes of Lso (LsoA and LsoB) are present in North America; both are transmitted by the tomato psyllid, Bactericera cockerelli (Sulc), in a circulative and propagative manner and cause damaging plant diseases (e.g. Zebra chip in potatoes). In this study, we investigated the acquisition and transmission of LsoA or LsoB by the tomato psyllid. We quantified the titer of Lso haplotype A and B in adult psyllid guts after several acquisition access periods (AAPs). We also performed sequential inoculation of tomato plants by adult psyllids following a 7-day AAP and compared the transmission of each Lso haplotype. The results indicated that LsoB population increased faster in the psyllid gut than LsoA. Further, LsoB population plateaued after 12 days, while LsoA population increased slowly during the 16 day-period evaluated. Additionally, LsoB had a shorter latent period and higher transmission rate than LsoA following a 7 day-AAP: LsoB was first transmitted by the adult psyllids between 17 and 21 days following the beginning of the AAP, while LsoA was first transmitted between 21 and 25 days after the beginning of the AAP. Overall, our data suggest that the two Lso haplotypes have distinct acquisition and transmission rates. The information provided in this study will improve our understanding of the biology of Lso acquisition and transmission as well as its relationship with the tomato psyllid at the gut interface.

Concanavalin A Toxicity Towards Potato Psyllid and Apoptosis Induction in Midgut Cells.

Concanavalin A (ConA), a legume lectin, has been drawing increasing attention in recent years concerning its toxicity against insects and its potential application in pest management. In an attempt to evaluate the effect of ConA on potato psyllid (Bactericera cockerelli), an economically important pest of solanaceous crops, the effect of ConA on potato psyllid survival, psyllid gut nuclear morphology, and expression of psyllid caspase genes were evaluated. Our results determined that artificial diet-feeding assays using ConA had deleterious effects on potato psyllids, resulting in significant psyllid mortality following ingestion. We also found that an apoptotic response was induced by ConA in psyllid midgut cells, which was demonstrated by the DNA fragmentation and abnormal nuclear architecture in the midgut cells. Following ConA ingestion, there was also upregulation of caspase genes in the psyllid midguts. Therefore, a key mechanism behind ConA toxicity towards potato psyllid probably involves the induction of apoptosis in midgut cells. This study could provide a better understanding of the mechanisms underlying ConA toxicity in insects and be a stepping stone towards the development of new psyllid control strategies based on plant lectins.

Non-invasive diagnostics of Liberibacter disease on tomatoes using a hand-held Raman spectrometer.

MAIN CONCLUSION: Hand-held Raman spectroscopy can be used for confirmatory, non-invasive and non-destructive detection and identification of two haplotypes of Liberibacter disease on tomatoes. Using this spectroscopic approach, structural changes in carotenoids, xylan, cellulose and pectin that are associ-ated with this bacterial disease can be determined. 'Candidatus Liberibacter solanacearum' (Lso) is a phloem-limited Gram-negative bacterium that infects crops worldwide. In North America, two haplotypes of Lso (LsoA and LsoB) are transmitted by the potato psyllid, Bactericera cockerelli (Sulc), and infect many solanaceous crops such as potato and tomato. Infected plants exhibit chlorosis, severe stunting, leaf cupping, and scorching. Polymerase chain reaction (PCR) and potato tuber frying are commonly used methods for diagnostics of the plant disease caused by Lso. However, they are time-consuming, costly, destructive to the sample, and often not sensitive enough to detect the pathogen in the early infection stage. Raman spectroscopy (RS) is a noninvasive, nondestructive, analytical technique, which probes chemical composition of analyzed samples. In this study, we demonstrate that Lso infection can be diagnosed by non-invasive spectroscopic analysis of tomato leaves three weeks following infection, before the development of aerial symptoms. In combination with chemometric analyses, Raman spectroscopy allows for 80% accurate diagnostics of Liberibacter disease caused by each of the two different haplotypes. This diagnostics approach is portable and sample agnostic, suggesting that it could be utilized for other crops and could be conducted autonomously.

Liberibacter, A Preemptive Bacterium: Apoptotic Response Repression in the Host Gut at the Early Infection to Facilitate Its Acquisition and Transmission.

"Candidatus Liberibacter solanacearum" (Lso) is a phloem-limited Gram-negative bacterium that infects crops worldwide. In North America, two haplotypes of Lso (LsoA and LsoB) are transmitted by the potato psyllid, Bactericera cockerelli (Sulc), in a circulative and persistent manner. Both haplotypes cause damaging plant diseases (e.g., zebra chip in potatoes). The psyllid gut is the first organ Lso encounters and could be a barrier for its transmission. However, little is known about the psyllid gut immune responses triggered upon Lso infection. In this study, we focused on the apoptotic response in the gut of adult potato psyllids at the early stage of Lso infection. We found that there was no evidence of apoptosis induced in the gut of the adult potato psyllids upon infection with either Lso haplotype based on microscopic observations. However, the expression of the inhibitor of apoptosis IAPP5.2 gene (survivin-like) was significantly upregulated during the period that Lso translocated into the gut cells. Interestingly, silencing of IAPP5.2 gene significantly upregulated the expression of two effector caspases and induced apoptosis in the psyllid gut cells. Moreover, RNA interference (RNAi) of IAPP5.2 significantly decreased the Lso titer in the gut of adult psyllids and reduced their transmission efficiency. Taken together, these observations suggest that Lso might repress the apoptotic response in the psyllid guts by inducing the anti-apoptotic gene IAPP5.2 at an early stage of the infection, which may favor Lso acquisition in the gut cells and facilitate its transmission by potato psyllid.

Quenching autofluorescence in the alimentary canal tissues of Bactericera cockerelli (Hemiptera: Triozidae) for immunofluorescence labeling.

Immunofluorescence has been widely used to localize microbes or specific molecules in insect tissues or cells. However, significant autofluorescence is frequently observed in tissues which can interfere with the fluorescent identification of target antigens, leading to inaccurate or even false positive fluorescent labeling. The alimentary canal of the potato psyllid, Bactericera cockerelli Sulc, exhibits intense autofluorescence, hindering the application of immunolocalization for the detection and localization of the economically important pathogen transmitted by this insect, "Candidatus Liberibacter solanacearum" (Lso). In the present study, we tested the use of irradiation, hydrogen peroxide (H2 O2 ) and Sudan black B (SBB) treatments to reduce the autofluorescence in the B. cockerelli alimentary canal tissues. Furthermore, we assessed the compatibility of the above-mentioned treatments with Lso immunolocalization and actin staining using phalloidin. Our results showed that the autofluorescence in the alimentary canal was reduced by irradiation, H2 O2 , or SBB treatments. The compatibility assays indicated that irradiation and H2 O2 treatment both greatly reduced the fluorescent signal associated with Lso and actin. However, the SBB incubation preserved those target signals, while efficiently eliminating autofluorescence in the psyllid alimentary canal. Therefore, herein we propose a robust method for reducing the autofluorescence in the B. cockerelli alimentary canal with SBB treatment, which may improve the use of immunofluorescence labeling in this organism. This method may also have a wide range of uses by reducing the autofluorescence in other arthropod species.

Effects of "Candidatus Liberibacter solanacearum" (haplotype B) on Bactericera cockerelli fitness and vitellogenesis.

"Candidatus Liberibacter solanacearum" (Lso) are phloem-restricted and unculturable Gram-negative bacteria. Presently five haplotypes have been identified worldwide; but only haplotypes A and B are associated with the vector Bactericera cockerelli (Sulc.) in the Americas. Previous studies showed that Lso-infection reduces B. cockerelli reproductive output and that Lso haplotype B is more pathogenic than Lso haplotype A. To understand the interaction of Lso haplotype B and B. cockerelli, the fitness of Lso-free and Lso B-infected insects, and the expression of vitellogenin (BcVg1-like), a gene involved directly in the insect reproduction were analyzed. Statistical differences in the number of eggs oviposited, and the total number of progeny nymphs and adults were found among crosses of insects with or without Lso. Significant differences in sex proportions were found between Lso B-infected and Lso-free crosses: a higher proportion of F1 adult females were obtained from Lso B-infected mothers. A significant reduction of BcVg1-like was observed in crosses performed with Lso B-infected females compared to the Lso-free insects. In female cohorts of different age, a significant reduction of BcVg1-like expression was measured in 7-d-old Lso B-infected females (virgin and mated) compared with 7-d-old Lso-free females (virgin and mated), respectively. The reduction of BcVg1-like transcript was associated with a lower number of developing oocytes observed in female's reproductive systems. Overall, this study represents the first step to understand the interaction of Lso B with B. cockerelli, highlighting the effect of Lso B infection on egg production, BcVg1-like expression, and oocyte development.