Diaphorina citri Nymphs Are Resistant to Morphological Changes Induced by “Candidatus Liberibacter asiaticus” in Midgut Epithelial Cells

Abstract

"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

Immuno-Ultrastructural Localization and Putative Multiplication Sites of Huanglongbing Bacterium in Asian Citrus Psyllid Diaphorina citri

“…In our study, the near systemic invasion of CLas in most of the ACP organs and tissues examined, including the epidermis of a leg ( Figure 7A), supported previous FISH and qPCR studies indicating a wide distribution of CLas in infected ACP [5,6,10,22,29], as well as ultrastructural, qPCR and FISH studies that have indicated an almost systemic invasion of the zebra chip pathogen CLso in the potato psyllid vector [11,27,30]. The near systemic distribution of both Liberibacter pathogens in their psyllid vectors was similar to that of other circulative-propagative hemipteran born bacterial phytopathogens, viz.…”

“…However, no immunolabeling or molecular labeling methods were available to confirm the identity of such bacteria in these studies. Later, using fluorescent in situ hybridization (FISH) or qPCR indicated the presence of CLas in most organs and tissues of CLas-infected D. citri adults from Florida [6,7,10,22]. More recently, Ghanim et al [16] used FISH and immunogold labeling to describe CLas accumulation in the cells of ACP midgut.…”

“…It was shown that CLas also propagates more efficiently in the nymphal stages of ACP compared to the adults, and this can also contribute to the higher transmission efficiency of ACP that acquire CLas as nymphs [4,7]. Differences in the innate immunity between D. citri nymphs and adults have been suggested, based on quantitative proteomic and microscopic studies of healthy and CLas-infected ACP [9,10]. Understanding the interactions between D. citri and CLas at the cellular and subcellular levels is important to our understanding of pathogen-vector interactions.…”

Spatiotemporal dynamics and quantitative analysis of phytoplasmas in insect vectors

“…3D-G and 5A-E), in agreement with our previous findings 27 . Electron and immunofluorescence microscopy analyses have revealed similar findings in spiroplasmas [6][7][8] and liberibacter 40,41 . Thus, the visceral muscles of the alimentary canal appear to be a common infection site for insect-borne plant pathogenic bacteria that are transmitted in a persistent propagative manner.…”

Evaluation of North American Asian Citrus Psyllid (Hemiptera: Liviidae) Population Genetics using cytochrome P450 melt curve analysis

“…Given that Las is known to affect expression of CYP4 genes, this may also be the result of co-evolution. Indicating that one population is more tolerant of Las infection than the other (Ammar et al, 2016; Arp et al, 2016; Mann et al, 2018). The exact effect of these changes and why this movement would not affect Louisiana populations is unknown.…”