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. 2016 Sep 15:6:33418.
doi: 10.1038/srep33418.

Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus

Murad Ghanim  1 , Somayeh Fattah-Hosseini  2 , Amit Levy  2 , Michelle Cilia  2   3   4
Affiliations

Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus

Murad Ghanim et al. Sci Rep. .

Abstract

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.

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Figures

Figure 1
Figure 1. Fluorescence in situ hybridization (FISH) using a CLas probe (red) and DAPI counterstaining (blue) of epithelial cell nuclei in midguts dissected from Diaphorina citri adults.
The left column shows DAPI staining only, the middle column show the CLas probe only, and the right column shows a merged image. Panels a–c show guts from insects reared on healthy trees (non-exposed) show no detectible CLas-FISH signal. Panels d–f show a non-exposed gut (NE) and a CLas-exposed gut (I) in the same image, showing the specificity of the CLas-FISH signal in CLas-exposed guts. The CLas probe reveals CLas within the midgut epithelial cells and the filter chamber. Midguts from CLas-exposed insects at higher magnification show FISH signal on the cell membranes (h,i) and in a punctate pattern with 0.5 μm diameter in the gut (j–l).
Figure 2
Figure 2. Immunostaining of CLas using anti-CLas OmpA antibody in dissected midguts from insects reared on healthy trees (non-exposed, (a–c)) and CLas-exposed Diaphorina citri adults (d–i).
Left panels show DAPI signal, middle panels show OmpA immunostaining signal, and right panel contained merged images. Immunostaining shows primarily intracellular CLas localization.
Figure 3
Figure 3. Microscopic analysis of cellular and morphological abnormalities are observed in midguts dissected CLas-exposed Diaphorina citri adults.
Light micrograph of a gut dissected from an insect reared on a healthy, CLas-free citrus tree (non-exposed, (a)) shows normal gut morphology. Light micrograph of a representative gut dissected from a CLas-exposed insect shows dark spots, an irregular diameter around the midgut and extensive melanization (b). Low magnification images of representative insects reared on healthy trees or (c) and CLas-infected trees (d). Midguts show nuclear abnormalities in the CLas-exposed insects. High magnification images of midguts dissected from insects reared on healthy trees (non-exposed, (e)) show uniform and round epithelial cell nuclei. In contrast, CLas-exposed midgut epithelial cell nuclei are irregular in shape, size and fragmented throughout the cytoplasm (f).
Figure 4
Figure 4. Quantification of normal and abnormal nuclei in midgut epithelial cells of psyllids exposed to healthy and CLas-infected trees.
Insects reared on healthy trees show a very low number of abnormal nuclei in contrast to insects reared on CLas-infected trees, which show a high number of abnormal nuclei.
Figure 5
Figure 5. Assessment of DNA integrity following extraction from midguts using agarose gel electrophoresis.
The DNA extracted from CLas-exposed insects (CLas+) shows an enrichment of lower molecular weight DNA fragments as compared to insects reared on healthy trees (non-exposed).
Figure 6
Figure 6. A comparison of the structure of the actin cytoskeleton in adult Diaphorina citri midguts dissected from insects reared on healthy or CLas-infected citrus trees using phalloidin staining.
CLas-exposed insects show disrupted actin filaments (b) as compared to a more organized structure of filaments in the midguts from insects reared on healthy trees (a). A qualitative analysis showed filaments in the CLas-exposed midgut samples to connect to one another at irregular angles, appear shorter in places, and thicker in diameter.
Figure 7
Figure 7. Apoptosis assay using Annexin V visualization in adult Diaphorina citri midguts reared on healthy or CLas-infected citrus trees.
Nuclei are counterstained using propidium iodide (red). Midgut samples from insects reared on healthy trees showed either no Annexin V staining or a diffuse signal distributed throughout the cytoplasm (a). In contrast, CLas-exposed samples (b) showed Annexin V binding to the cell membranes, indicating the epithelial cells are undergoing apoptosis in response to exposure to CLas-infected trees.
Figure 8
Figure 8. Differential effect of CLas on nuclear morphology and cytoskeleton organization in adult Diaphorina citri guts and oocytes.
(a) In CLas-exposed insects, changes in nuclear morphology were clearly visualized in midgut cells (MG), but not hindgut (HG). Scale bar is 100 μm. (b) Differential effect of CLas on nuclear morphology and cytoskeleton organization in adult Diaphorina citri oocytes. CLas is also visualized by immunolocalization (red) using an anti-OmpA antibody. No changes in nuclear morphology as visualized by DAPI staining (blue) or actin filaments (green) in CLas-exposed insects were observed in oocytes dissected from CLas-exposed insects. Scale bar is 50 μm.
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