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Background: Huanglongbing, a devastating disease of citrus, is caused by the obligate, intracellular bacterium "Candidatus Liberibacter asiaticus" (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid. Development of transmission-blocking strategies to manage huanglongbing relies on knowledge of CLas and D. citri interactions at the molecular level. Prior transcriptome analyses of D. citri point to changes in psyllid biology due to CLas infection but have been hampered by incomplete versions of the D. citri genome, proper host plant controls, and/or a lack of a uniform data analysis approach. In this work, we present lessons learned from a quantitative transcriptome analysis of excised heads, salivary glands, midguts, and bacteriomes from CLas-positive and CLas-negative D. citri using the chromosomal length D. citri genome assembly., Results: Each organ had a unique transcriptome profile and response to CLas infection. Though most psyllids were infected with the bacterium, CLas-derived transcripts were not detected in all organs. By analyzing the midgut dataset using both the Diaci_v1.1 and v3.0 D. citri genomes, we showed that improved genome assembly led to significant and quantifiable differences in RNA-sequencing data interpretation., Conclusions: Our results support the hypothesis that future transcriptome studies on circulative, vector-borne pathogens should be conducted at the tissue-specific level using complete, chromosomal-length genome assemblies for the most accurate understanding of pathogen-induced changes in vector gene expression., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Huanglongbing (HLB) is a deadly, incurable citrus disease putatively caused by the unculturable bacterium, 'Candidatus Liberibacter asiaticus' (CLas), and transmitted by Diaphorina citri. Prior studies suggest D. citri transmits CLas in a circulative and propagative manner; however, the precise interactions necessary for CLas transmission remain unknown, and the impact of insect sex on D. citri-CLas interactions is poorly understood despite reports of sex-dependent susceptibilities to CLas. We analyzed the transcriptome, proteome, metabolome, and microbiome of male and female adult D. citri reared on healthy or CLas-infected Citrus medica to determine shared and sex-specific responses of D. citri and its endosymbionts to CLas exposure. More sex-specific than shared D. citri responses to CLas were observed, despite there being no difference between males and females in CLas density or relative abundance. CLas exposure altered the abundance of proteins involved in immunity and cellular and oxidative stress in a sex-dependent manner. CLas exposure impacted cuticular proteins and enzymes involved in chitin degradation, as well as energy metabolism and abundance of the endosymbiont 'Candidatus Profftella armatura' in both sexes similarly. Notably, diaphorin, a toxic Profftella-derived metabolite, was more abundant in both sexes with CLas exposure. The responses reported here resulted from a combination of CLas colonization of D. citri as well as the effect of CLas infection on C. medica. Elucidating these impacts on D. citri and their endosymbionts contributes to our understanding of the HLB pathosystem and identifies the responses potentially critical to limiting or promoting CLas acquisition and propagation in both sexes., Competing Interests: The authors have declared that no competing interests exist.

Presymptomatic detection of citrus trees infected with Candidatus Liberibacter asiaticus ( C Las), the bacterial pathogen associated with Huanglongbing (HLB; citrus greening disease), is critical to controlling the spread of the disease. To test whether infected citrus trees produce systemic signals that may be used for indirect disease detection, lemon ( Citrus limon ) plants were graft-inoculated with either C Las-infected or control ( C Las-) budwood, and leaf samples were longitudinally collected over 46 weeks and analyzed for plant changes associated with C Las infection. RNA, protein, and metabolite samples extracted from leaves were analyzed using RNA-Seq, mass spectrometry, and 1 H NMR spectroscopy, respectively. Significant differences in specific transcripts, proteins, and metabolites were observed between C Las-infected and control plants as early as 2 weeks post graft (wpg). The most dramatic differences between the transcriptome and proteome of C Las-infected and control plants were observed at 10 wpg, including coordinated increases in transcripts and proteins of citrus orthologs of known plant defense genes. This integrated approach to quantifying plant molecular changes in leaves of C Las-infected plants supports the development of diagnostic technology for presymptomatic or early disease detection as part of efforts to control the spread of HLB into uninfected citrus groves.

" Candidatus Liberibacter asiaticus" ( C Las) is the bacterium associated with the citrus disease Huanglongbing (HLB). Current C Las detection methods are unreliable during presymptomatic infection, and understanding C Las pathogenicity to help develop new detection techniques is challenging because C Las has yet to be isolated in pure culture. To understand how C Las affects citrus metabolism and whether infected plants produce systemic signals that can be used to develop improved detection techniques, leaves from Washington Navel orange ( Citrus sinensis (L.) Osbeck) plants were graft-inoculated with C Las and longitudinally studied using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry), and metabolomics (proton nuclear magnetic resonance). Photosynthesis gene expression and protein levels were lower in infected plants compared to controls during late infection, and lower levels of photosynthesis proteins were identified as early as 8 weeks post-grafting. These changes coordinated with higher sugar concentrations, which have been shown to accumulate during HLB. Cell wall modification and degradation gene expression and proteins were higher in infected plants during late infection. Changes in gene expression and proteins related to plant defense were observed in infected plants as early as 8 weeks post-grafting. These results reveal coordinated changes in greenhouse navel leaves during C Las infection at the transcript, protein, and metabolite levels, which can inform of biomarkers of early infection.

Reproduction is a critical feature in the search for means to manage the Asian citrus psyllid, vector of a devastating bacterial pathogen of citrus. The importance of accuracy in functional, anatomical descriptions and interpretations for use by other disciplines, particularly molecular genetics, cannot be overstressed. The term 'sperm pump' was coined by classical authors on observational appearance of the endoskeleton of the male reproductive apparatus. They described a thimble-shaped cuticle with smooth, cylindrical columns, interpreted as muscles, that ran longitudinally around a central cylinder. They detected transverse lines on the cylinder giving the false impression of a coiled spring. These features fostered the teleological interpretation that the device is a contractile pump. Now obsolete, the term is replaced by 'drum/spout complex'. It is a hypodermis with a sclerotized cuticle that houses the phallus which transports seminal fluid through its lumen to the female for insemination. Between the spout and the external genitalia is a spout extension, conferring flexibility to the apparatus about the abdominal apex. Approximately 21 longitudinal columns extend circumferentially around the cylinder's hemolymph-side, from the thimble's basal plate to its apical plate. These columns are correctly muscle cells, and reinterpreted to exude a lipaceous, lubricating substance for mating., (Published by Elsevier Ltd.)

Huanglongbing, or citrus greening disease, is an economically devastating bacterial disease of citrus. It is associated with infection by the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid (ACP). For insect transmission to occur, CLas must be ingested during feeding on infected phloem sap and cross the gut barrier to gain entry into the insect vector. To investigate the effects of CLas exposure at the gut-pathogen interface, we performed RNAseq and mass spectrometry-based proteomics to analyze the transcriptome and proteome, respectively, of ACP gut tissue. CLas exposure resulted in changes in pathways involving the TCA cycle, iron metabolism, insecticide resistance and the insect's immune system. We identified 83 long non-coding RNAs that are responsive to CLas, two of which appear to be specific to the ACP. Proteomics analysis also enabled us to determine that Wolbachia, a symbiont of the ACP, undergoes proteome regulation when CLas is present. Fluorescent in situ hybridization (FISH) confirmed that Wolbachia and CLas inhabit the same ACP gut cells, but do not co-localize within those cells. Wolbachia cells are prevalent throughout the gut epithelial cell cytoplasm, and Wolbachia titer is more variable in the guts of CLas exposed insects. CLas is detected on the luminal membrane, in puncta within the gut epithelial cell cytoplasm, along actin filaments in the gut visceral muscles, and rarely, in association with gut cell nuclei. Our study provides a snapshot of how the psyllid gut copes with CLas exposure and provides information on pathways and proteins for targeted disruption of CLas-vector interactions at the gut interface.

Database Url: https://citrusgreening.org/., (© The Author(s) 2017. Published by Oxford University Press.)

Diaphorina citri (Hemiptera: Psyllidae), the Asian citrus psyllid, is the insect vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening disease. Sequencing of the D. citri metagenome has been initiated to gain better understanding of the biology of this organism and the potential roles of its bacterial endosymbionts. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the D. citri metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided the most support for Wolbachia and an enteric bacterium most similar to Salmonella. Wolbachia-derived reads were extracted using the complete genome sequences for four Wolbachia strains. Reads were assembled into a draft genome sequence, and the annotation assessed for the presence of features potentially involved in host interaction. Genome alignment with the complete sequences reveals membership of Wolbachia wDi in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida D. citri isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese D. citri isolates, supporting the hypothesis that the D. citri introduced into Florida did not originate from China.

Objective: The tarnished plant bug (TPB), Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), is a pest damaging many cultivated crops in North America. Although partial transcriptome data are available for this pest, a genome assembly was not available for this species. This assembly of a high-quality chromosome-length genome of TPB is aimed to develop the genetic resources that can provide the foundation required for advancing research on this species. Results: The initial genome of TPB assembled with paired-end nucleotide sequences generated with Illumina technology was scaffolded with Illumina HiseqX reads generated from a proximity ligated (HiC) library to obtain a high-quality genome assembly. The final assembly contained 3963 scaffolds longer than 1 kbp to yield a genome of 599.96 Mbp. The N50 of the TPB genome assembly was 35.64 Mbp and 98.68% of the genome was assembled into 17 scaffolds larger than 1 Mbp. This megabase scaffold number is the same as the number of chromosomes observed in karyotyping of this insect. The TPB genome is known to have high repetitive DNA content, and the reduced assembled genome size compared to flowcytometric estimates of approximately 860 Mbp may be due to the collapsed assembly of highly similar regions. [ABSTRACT FROM AUTHOR]

Objective: The redbanded stink bug (RBSB), Piezodorus guildinii (Hemiptera: Pentatomidae), is native to the Caribbean Basin and is currently considered an invasive pest in Florida, Louisiana, Mississippi, and Texas in the southern United States. Although RBSB is an economically important invasive pest in the USA, relatively few studies have been conducted to understand molecular mechanisms, population genetic structure, and the genetic basis of resistance to insecticides. The objective of this work was to obtain a high-quality genome assembly to develop genomic resources to conduct population genetic, genomic, and physiological studies of the RBSB. Results: The genome of RBSB was sequenced with Pacific Biosciences technology followed by two rounds of scaffolding using Chicago libraries and HiC proximity ligation to obtain a high-quality assembly. The genome assembly contained 800 scaffolds larger than 1 kbp and the N50 was 170.84 Mbp. The largest scaffold was 222.22 Mbp and 90% of the genome was included in the 7 scaffolds larger than 118 Mbp. The number of megabase scaffolds also matched the number of chromosomes in this insect. The genome sequence will facilitate the development of resources to conduct studies on genetics, transcriptomics, and physiology of RBSB. [ABSTRACT FROM AUTHOR]