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.

Candidatus Liberibacter asiaticus (CLas) is a phloem-limited, gram-negative, fastidious bacterium that is associated with the development of citrus greening disease, also known as Huanglongbing (HLB). CLas is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri, in a circulative manner. Two major barriers to transmission within the insect are the midgut and the salivary glands. We performed a thorough microscopic analysis within the insect midgut following exposure to CLas-infected citrus trees. We observed changes in nuclear architecture, including pyknosis and karyorrhexis as well as changes to the actin cytoskeleton in CLas-exposed midgut cells. Further analyses showed that the changes are likely due to the activation of programmed cell death as assessed by Annexin V staining and DNA fragmentation assays. These results suggest that exposure to CLas-infected trees induces apoptotic responses in the psyllid midgut that should be further investigated. Understanding the adaptive significance of the apoptotic response has the potential to create new approaches for controlling HLB.

"Candidatus Liberibacter asiaticus" is the causative bacterium associated with 24 citrus greening disease. "Ca. L. asiaticus" is transmitted by Diaphorina citri more efficiently 25 when it is acquired by nymphs rather than adults. Why this occurs is not known. We compared 26 midguts of D. citri reared on healthy or "Ca. L. asiaticus"-infected citrus trees using quantitative 27 PCR, confocal microscopy, and mitochondrial superoxide staining for evidence of oxidative 28 stress. Consistent with its classification as propagative, "Ca. L. asiaticus" titers were higher in 29 adults as compared to nymphs. Our previous work showed that adult D. citri show basal levels of 30 karyorrhexis, (fragmentation of the nucleus), in midgut epithelial cells which is increased in 31 severity and frequency in response to "Ca. L. asiaticus". Here, we show that nymphs exhibit 32 lower levels of early-stage karyorrhexis as compared to adults and are refractory to the induction 33 of advanced karyorrhexis by "Ca. L. asiaticus" in the midgut epithelium. MitoSox Red staining 34showed that guts of infected adults, particularly males, experience oxidative stress in response to 35 "Ca. L. asiaticus." A positive correlation between the titers of "Ca. L. asiaticus" and the 36Wolbachia endosymbiont was observed in adult and nymph midguts, suggesting an interplay 37 between these bacteria during development. We hypothesize that the resistance of the nymph 38 midgut to late stage karyorrhexis through as yet unknown molecular mechanisms benefits "Ca. 39