Continuous cell lines derived from the Asian citrus psyllid, Diaphorina citri, harbor viruses and Wolbachia

Abstract

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a major pest of global citriculture. In the Americas and in Asia, D. citri vectors the phloem-limited bacterium, Candidatus Liberibacter asiaticus (CLas), which causes the fatal citrus disease huanglongbing, or citrus greening. Cell lines derived from D. citri can provide insight into both the basic biology of this pest and D. citri-associated pathogens including CLas. We previously identified CLG#2 as the optimal medium for long-term growth of D. citri primary cell cultures. Here we report on the establishment and characterization of three continuous D. citri cell lines, Dici1, Dici3, and Dici5, that have been passaged for > 40 times. Based on morphological and transcriptomic data, the Dici1 and Dici3 cell lines include undifferentiated and neurogenic progenitor cells. Dici1 and Dici5 are infected with Wolbachia. Both Dici1 and Dici5 are infected with D. citri reovirus, and Dici5 is also infected with D. citri-associated C virus. Dici3 is free of both Wolbachia and virus infection. These cell lines provide an ideal platform for the study of inter-microbial relationships as well as microbe interaction with host insect cells.

Keywords: Diaphorina citri; Wolbachia; Asian citrus psyllid; Citrus greening disease; Insect cell line; Transcriptome.

Fig. 1

Photomicrographs of Diaphorina citri cell lines Dici1, Dici3, and Dici5. The morphology and organization of Dici1, Dici3, and Dici5 cells remained relatively consistent between different passages. Clusters of spherical cells in Dici1 and Dici5 are indicated (arrows). The passage number (P) at which each photograph was taken is indicated.

Fig. 2

PCR of Wolbachia ftsz and wsp confirms the presence of Wolbachia in Dici1 and Dici5, but not Dici3 cells. (A) Partial fragments of Wolbachia ftsz (1043 bp) and wsp (570 bp) were amplified from Dici1 (Passage 27, GenBank: PP894982 for ftsz  and PP894984 for wsp) and Dici5 (Passage 33, GenBank: PP894983 for ftsz  and PP894985 for wsp), but not Dici3 (Passage 46), genomic DNA. NTC: no template control. Arrows indicate PCR products at the expected sizes. GeneRuler 1 kb plus DNA ladder (ThermoFisher Scientific, SM1332) was used as a DNA marker. Two weeks of doxycycline treatment reduced the levels of ftsz and wsp in Dici1 (B) and Dici5 (C) cells.

Fig. 3

Evaluation of viral infection status in Diaphorina citri cell lines. (A) DcRV RT-PCR confirms the presence of this reovirus in Dici1, but not Dici3 cells. Partial fragments (900 bp) of the DcRV p8 were amplified from the cDNA of Dici1 (P1, GenBank: PP894986), but not Dici3 (P5). (B) RT-PCR confirmed the presence of DcACV and DcRV in Dici5 cells. Partial fragments of the DcACV RNA1 (473 bp, GenBank: PP894988) and DcRV p8 (GenBank: PP894987) were amplified from the cDNA of Dici5 (Passage 33). GeneRuler 1 kb plus DNA ladder was used as a DNA marker. + control: cDNA made from purified D. citri viruses. NTC: no template control. Arrows indicate PCR products at the expected sizes.

Fig. 4

Wolbachia detection in Dici1 (Passage 43) and Dici3 (Passage 49) cells visualized using immunofluorescence confocal microscopy. (A–D) Optical cross-section of Dici1 cells visualizing Wolbachia FtsZ localization in Dici1 cells. Wolbachia FtsZ protein signal (Alexa Fluor 488) is green and DAPI counterstaining of nuclei is blue. The punctate Wolbachia FtsZ signal does not colocalize with the DAPI signal (D). (E–H) Optical cross-section of Dici3 cells showing that Wolbachia FtsZ signal is absent in Dici3 cells.