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.

No Evidence of Apoptotic Response of the Potato Psyllid Bactericera cockerelli to "Candidatus Liberibacter solanacearum" at the Gut Interface.

"Candidatus Liberibacter solanacearum" is a pathogen transmitted by the potato psyllid Bactericera cockerelli (Sulc) (Hemiptera: Triozidae) in a persistent manner. In this study, we investigated the molecular interaction between "Ca. Liberibacter solanacearum" and the potato psyllid at the gut interface. Specifically, we focused on the apoptotic response of potato psyllids to the infection by two "Ca. Liberibacter solanacearum" haplotypes, LsoA and LsoB. To this end, we first quantified and localized "Ca. Liberibacter solanacearum" in the gut of adult psyllids. We then evaluated the existence of an apoptotic response in the insect gut using microscopy analyses to visualize the nuclei and the actin cytoskeleton of the gut cells and DNA fragmentation analyses by agarose gel electrophoresis. We also performed annexin V cell death assays to detect apoptosis. Finally, we annotated apoptosis-related genes from the potato psyllid transcriptome and evaluated their expression in response to "Ca. Liberibacter solanacearum" infection. The results showed no cellular markers of apoptosis despite the large amount of "Ca. Liberibacter solanacearum" present in the psyllid gut. In addition, only three genes potentially involved in apoptosis were regulated in the psyllid gut in response to "Ca. Liberibacter solanacearum": the apoptosis-inducing factor AIF3 was downregulated in LsoA-infected psyllids, while the inhibitor of apoptosis IAPP5 was downregulated and IAP6 was upregulated in LsoB-infected psyllids. Overall, no evidence of apoptosis was observed in the gut of potato psyllid adults in response to either "Ca. Liberibacter solanacearum" haplotype. This study represents a first step toward understanding the interactions between "Ca. Liberibacter solanacearum" and the potato psyllid, which is crucial to developing approaches to disrupt their transmission.

Characterization and expression of genes encoding three small heat shock proteins in Sesamia inferens (Lepidoptera: Noctuidae).

The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens.

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.

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.

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.

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.

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.

Competitive Displacement between Bemisia tabaci MEAM1 and MED and Evidence for Multiple Invasions of MED.

Despite the severe ecological damage and economic loss caused by invasive species, the factors contributing to successful invasion or displacement remain elusive. The whitefly, Bemisia tabaci (Gennadius), is an important invasive agricultural pest worldwide, causing severe damage to numerous crops by feeding or transmitting plant viruses. In this study, we monitored the dynamics of two invasive whitefly cryptic species, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED), in Jiangsu, China, from 2005-2016. We found that B. tabaci MED quickly established and asserted dominance over MEAM1, resulting in their population displacement in Jiangsu in only three years (from 2005 to 2008). We further investigated the possible mechanisms underlying the successful invasion and competitive displacement from a genetic perspective. Based on sequencing of mitochondrial gene sequences from large numbers of whitefly samples, multiple invasion events of MED were validated by our genetic analyses. MED invaded Jiangsu starting from multiple introduction sites with secondary and/or subsequent invasive events. This may favor their invasion and displacement of MEAM1. This study advances our understanding of the mechanisms that enabled the successful invasion of MED.

Population genetics of Liriomyza trifolii (Diptera: Agromyzidae) and comparison with four Liriomyza species in China based on COI, EF-1a and microsatellites loci.

Liriomyza is a large genus that includes polyphagous and invasive species (L. trifolii, L. sativae, and L. huidobrensis), and oligophagous species such as L. Chinensis in China. Effective control of these invasive and oligophagous species is not easy due to the fast invasion rate, interspecific competition, and pesticide resistance. In this study, we investigated population genetics of five Liriomyza species L. trifolii, L. sativae, L. huidobrensis, L. bryoniae, and L. chinensis based on COI and EF-1a genes, and microsatellite DNA. These five Liriomyza species revealed highly conservative characteristics in the COI gene among populations collected from different geographical regions and host plants. By contrast, the mutation rate of the EF-1a gene was higher than COI, and phylogenetic tree based on EF-1a showed that haplotypes of L. trifolii and L. sativae were not distinguished well. Genetic differentiation in microsatellite loci was obvious among the five species. Our results also indicated that geographic isolation had a greater impact on genetic differentiation in L. trifolii than the host plant. Populations of L. trifolii in China showed a high to moderate level of genetic differentiation and they had divided into two groups representing the coastal areas of southern China and northern regions. The genetic diversity of the southern group was higher than the northern group. We speculated that the invasion of L. trifolii likely occurred in southern regions of China and then spread northward. Bottleneck analyses revealed that the L. trifolii population in China was in a steady growth period.

Diversity and evolution of the endosymbionts of Bemisia tabaci in China.

The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex, including members that are pests of global importance. This study presents a screening of B. tabaci species in China for infection by the primary endosymbiont, Portiera aleyrodidarum, and two secondary endosymbionts, Arsenophonus and Cardinium. The results showed that P. aleyrodidarum was detected in all B. tabaci individuals, while Arsenophonus was abundant in indigenous species of B. tabaci Asia II 1, Asia II 3, and China 1 but absent in the invasive species, Middle East-Asia Minor 1 (MEAM1); Cardinium presented in the Mediterranean (MED), Asia II 1 and Asia II 3 species but was rarely detected in the MEAM1 and China 1 species. Moreover, phylogenetic analyses revealed that the P. aleyrodidarum and mitochondrial cytochrome oxidase 1 (mtCO1) phylograms were similar and corresponding with the five distinct cryptic species clades to some extent, probably indicating an ancient infection followed by vertical transmission and subsequent co-evolutionary diversification. In contrast, the phylogenetic trees of Arsenophonus and Cardinium were incongruent with the mtCO1 phylogram, potentially indicating horizontal transmission in B. tabaci cryptic species complex. Taken together, our study showed the distinct infection status of endosymbionts in invasive and indigenous whiteflies; we also most likely indicated the co-evolution of primary endosymbiont and its host as well as the potential horizontal transfer of secondary endosymbionts.

The population genetic structure of Corythucha ciliata (Say) (Hemiptera: Tingidae) provides insights into its distribution and invasiveness.

Corythucha ciliata (Say), an invasive pest originating from North America, causes severe damage on sycamore trees. However, little is known about the population genetics and evolutionary forces underlying the invasiveness of this important pest. In the present study, we use three mitochondrial genes (COI, ND1 and ND5) and nine microsatellite markers to investigate the population genetics of C. ciliata and retrace its spread through China. The results suggest a low level of genetic diversity in Chinese and European populations of C. ciliata. Our results indicate that populations of C. ciliata have obvious genetic structure, and genetic differentiation is not caused by geographic isolation. In median-joining networks, we observed a higher frequency of shared haplotypes in groups 1 and 3. Based on gene flow and approximate Bayesian computation analyses, we discovered that C. ciliata first invaded the east coast of China and subsequently moved inland. Demographic analysis suggested that populations of C. ciliata in China may have undergone a recent bottleneck effect. Finally, our results suggest that population structure, high gene flow and environmental conditions have favored the broad invasiveness of this important pest.

New ideas about genetic differentiation of Chilo suppressalis (Lepidoptera: Pyralidae) populations in China based on the mtDNA cytochrome b gene.

The striped stem borer, Chilo suppressalis (Walker), is an important pest of rice in China and other parts of the world. To further explore the population genetic structure and genetic differentiation of C. suppressalis populations found on rice in China, we amplified 432 bp fragments of the cytochrome b (cyt b) gene for 44 C. suppressalis populations. Nineteen variable sites in the mtDNA gene were observed, and 16 haplotypes were identified. Nucleotide diversity (π) and haplotype diversity (h) ranged from 0.00274 to 0.00786 and 0.72297 to 0.87604, respectively, while genetic structure analysis found significant genetic differentiation to be present among the five regions in China - northern China (NC), northeastern China (NEC), central China (CC), southern China (SC) and southwestern China (SWC) - where C. suppresalis was collected. In addition, molecular variance (AMOVA) showed that a relatively high proportion (57.6%) of the total genetic variance was attributable to variation within the populations. N(m) and F(ST) analyses suggested that the differentiation was not significantly different between NEC and NC, CC and SC, and SC and SWC regions, but was significant between NEC and CC, SC and SWC regions, corresponding well with the geographical distribution of the sampled populations. Phylogenetic analysis divided the populations into two indistinct clades: a NEC-NC-CC clade and a CC-SC-SWC clade, while CC region acted as a transition zone between north and south China, a finding different from previous work.

Genetic Structure of Water Chestnut Beetle: Providing Evidence for Origin of Water Chestnut.

Water chestnut beetle (Galerucella birmanica Jacoby) is a pest of the water chestnut (Trapa natans L.). To analyze the phylogeny and biogeography of the beetle and provide evidence for the origin of T. natans in China, we conducted this by using three mitochondrial genes (COI, COII and Cytb) and nuclear ITS2 ribosomal DNA of G. birmanica. As for mtDNA genes, the beetle could be subdivided into three groups: northeastern China (NEC), central-northern-southern China (CC-NC-SC) and southwestern China (SWC) based on SAMOVA, phylogenetic analyses and haplotype networks. But for ITS2, no obvious lineages were obtained but individuals which were from NEC region clustered into one clade, which might be due to sequence conservation of ITS2. Significant genetic variation was observed among the three groups with infrequent gene flow between groups, which may have been restricted due to natural barriers and events in the Late Pleistocene. Based on our analyses of genetic variation in the CC-NC-SC geographical region, the star-like haplotype networks, approximate Bayesian computation, niche modelling and phylogeographic variation of the beetle, we concluded that the beetle population has been lasting in the lower, central reaches of the Yangtze River Basin with its host plant, water chestnut, which is consistent with archaeological records. Moreover, we speculate that the CC-NC-SC population of G. birmanica may have undergone a period of expansion coincident with domestication of the water chestnut approximately 113,900-126,500 years ago.

Genetic differentiation of geographical populations of Liriomyza sativae (Diptera: Agromyzidae) in China based on mitochondrial COI gene sequences.

In this study, partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene of four Liriomyza sativae (Blanchard) geographic populations in China were sequenced. As for the 784 bp mtDNA-COI gene obtained, six variable sites were found which were all transitions and no base composition was insertions or deletions. Six haplotypes were identified in all the sequences, with five showing polymorphism and one was exclusive. Nucleotide diversity (π) and haplotype diversity (h) ranged from 0.00068 to 0.00300 and 0.53571 to 0.82857, respectively. The phylogenetic trees suggested that six haplotypes constructed two clades. Molecular variance analysis (AMOVA) demonstrated that the genetic variation was not obvious and mainly occurred within geographic populations (94.8%). Most molecular variance within the species was due to the difference of haplotypes among different geographic populations. The genetic characters of the four populations were analyzed by FST value and gene flow (Nm), and the FST and Nm values were 0.174-0.464 and 0.577-2.367, respectively. All results showed that not only the gene flow presented among the four populations but also the genetic differences did. The main reason causing the genetic differences among the four populations was supposed to be related to geographic isolation and host plants aggravated the differences.

Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae).

The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and ß-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

Microsatellite-based analysis of the genetic structure and diversity of Aleurocanthus spiniferus (Hemiptera: Aleyrodidae) from tea plants in China.

Although Aleurocanthus spiniferus (Quaintance) (Hemiptera: Aleyrodidae) is a well known insect pest of tea plants, little information is available about its genetic structure and diversity. The present study used microsatellite markers to assess the genetic structure and diversity of this species on tea plants in China. For this purpose, 193 individuals from ten natural populations were analyzed using ten microsatellite markers. Our results indicated that the average number of alleles (A) across populations was 35.6, and all observed heterozygosities (HO) were greater than 0.7, indicating an excess of heterozygosity and a relatively high level of genetic diversity among populations, and the number of private alleles per population ranged from 3 to 26. Pairwise FST analysis suggested that the number of genetic differentiation events was moderate (0.05