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. 2025 Jun 25;28(8):112982.
doi: 10.1016/j.isci.2025.112982. eCollection 2025 Aug 15.

Biological properties and vector competence of Diaphorina citri for Candidatus Liberibacter asiaticus modulated by an insect-specific virus

Diogo M Galdeano  1   2 , Tanvi Rawat  1 , William Ingram  1 , Curtis R Carlson  3 , Gustavo Rodrigues Alves  4 , Yen-Wen Kuo  1
Affiliations

Biological properties and vector competence of Diaphorina citri for Candidatus Liberibacter asiaticus modulated by an insect-specific virus

Diogo M Galdeano et al. iScience. .

Abstract

Insect-specific viruses (ISVs) infect only insects and have been studied primarily in mosquitoes. Here, we extend the concept of ISVs to include those found in plant-feeding insect vectors, such as Diaphorina citri, the vector of Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus Huanglongbing. Using Diaphorina citri flavi-like virus (DcFLV) as a model, we examined its effects on host biology and CLas transmission. DcFLV infection delayed development and preoviposition and reduced feeding activity, whereas overall survival, oviposition, and sex ratio were unaffected. DcFLV-infected D. citri transmitted CLas with ∼20% greater efficiency than uninfected controls, despite no significant difference in CLas titers. These findings suggest that ISVs can modulate the transmission dynamics of plant pathogens by insect vectors, adding complexity to vector-pathogen interactions.

Keywords: Entomology; Plant Biology; Virology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
The infectivity of Diaphorina citri flavi-like virus (DcFLV) in citrus plants and the biological effects of the infection in Diaphorina citri (A) DcFLV does not infect citrus plants but is only able to infect Diaphorina citri (Asian citrus psyllid) and gives delayed maturation development of D. citri. The D. citri used to feed on the Citrus macrophylla plants to test the DcFLV infectivity were all tested positive for DcFLV. None of the plants at any time points was tested positive for DcFLV. DcFLV-uninfected CA D. citri was used as negative controls. T0: 0 h after psyllid removal time; D3: 3 days after psyllid removal time; D7: 7 days after psyllid removal time; D14: 14 days after psyllid removal time; D14S: systemic leaf tissue collected at 14 days after psyllid removal time. (B) Egg hatching time of CA D. citri was significantly delayed by DcFLV infection. (C) The development time for nymph to adult stage of CA D. citri was delayed by DcFLV infection. (D) The overall development time egg to adult stage of CA D. citri was significantly delayed by DcFLV infection. CA/CA-D. citri: D. citri originally collected from a California population. CA-DcFLV: California D. citri infected with DcFLV. ∗p = 0.0118; ∗∗p = 0.0079; ∗∗∗p = 0.0001. The data were analyzed using two-tailed unpaired t-tests.
Figure 2
Figure 2
Effects of Diaphorina citri flavi-like virus (DcFLV) infection on the viability, sex ratio, preoviposition, oviposition time of, and total eggs laid by Diaphorina citri (A–C) (A) The viability of D. citri eggs (the number of eggs hatch into nymphs) was not significantly affected, whereas (B) the viability of nymphs (the number of nymphs emerge into adults) was affected by DcFLV infection. However, (C) the overall viability (viability total, the number of eggs developed into adults) was not statistically significantly affected by DcFLV infection. (D–G) (D) The sex ratio was not affected by DcFLV infection. Although the time for female D. citri to lay the first egg, the pre-oviposition time, (E) was significantly delayed, the oviposition time (F) and total eggs laid (G) by D. citri female were not affected. CA: D. citri originally collected from a California population. CA-DcFLV: California D. citri infected with DcFLV. ∗p = 0.0394; ∗∗∗p = 0.0003. The data were analyzed using two-tailed unpaired t tests.
Figure 3
Figure 3
The survival rate and longevity of Diaphorina citri flavi-like virus (DcFLV)-uninfected and -infected Diaphorina citri The survival rate and longevity of male D. citri were significantly higher than those of female psyllids for both DcFLV-uninfected and -infected Diaphorina citri (A–D). However, there were no significant differences of the survival rate and longevity between the DcFLV-uninfected and -infected California D. citri (E–H). (A) Survival rate of DcFLV-uninfected California D. citri (female and male). (B) Longevity of DcFLV-uninfected California D. citri (female and male). (C) Survival rate of DcFLV-infected California D. citri (female and male). (D) Longevity of DcFLV-infected California D. citri (female and male). (E) Survival rate of DcFLV-uninfected and -infected California D. citri (female). (F) Longevity of DcFLV-uninfected and -infected California D. citri (female). (G) Survival rate of DcFLV-uninfected and -infected California D. citri (male). (H) Longevity of DcFLV-uninfected and -infected California D. citri (male). CA: D. citri originally collected from a California population. CA-DcFLV: California D. citri infected with DcFLV. ∗∗∗∗p < 0.0001; ns: not significant. The data were analyzed using two-tailed unpaired t tests.
Figure 4
Figure 4
Higher titer of Diaphorina citri flavi-like virus (DcFLV) in adults and in guts and heads of Diaphorina citri while the Candidatus Liberibacter asiaticus (CLas) titers in both DcFLV-uninfected and -infected D. citri were not affected by DcFLV infection (A) Feeding consumption was performed through the reactions of honeydew and ninhydrin on filter paper. The results showed that the feeding consumption of DcFLV-infected was significantly less than that of DcFLV-uninfected D. citri. CA: D. citri originally collected from a California population. CA-DcFLV: California D. citri infected with DcFLV. The data were analyzed using two-tailed unpaired t tests. (B) DcFLV accumulation level Log per nanogram of total DNA extracted from single psyllid in different stages: egg, first instar (1st), second instar (2nd), third instar (3rd), fourth instar (4th), fifth instar (5th) of nymph, male young adults (my, one-day post emergence), female young adult (fy, one-day post emergence), male mature adult (mm, 10-day post emergence), and female mature adult (fm, 10-day post-emergence), in the D. citri life cycle. (C) DcFLV accumulation level was found to be higher in California D. citri gut (including the Malpighian tubules) and head tissues, compared to the titers in ovary, testis, and hemolymph. (D) CLas copy numbers in Log per nanogram of extracted total DNA in 7-day post-emergence adult California D. citri that were uninfected (CA 7-day-old) or infected with DcFLV (CA-FLV 7-day-old) and in 14-day post-emergence adult California D. citri that were uninfected (CA 14-day-old) or infected with DcFLV (CA-FLV 14-day-old). CLas titer in DcFLV-infected D. citri was not significantly different compared to that in the DcFLV-uninfected California D. citri. However, the CLas titer in 14-day post-emergence D. citri was significantly higher compared to that in 7-day post-emergence D. citri. (E) The Citrus macrophylla plants used in the CLas transmission assays vectored by DcFLV-infected and -uninfected D. citri were detected at 21, 60 (2 months), and 180 (6 months) days after inoculation access period (IAP) (Table 1). The results at 180 d.a.i showed statistically significant difference (p = 0.0155; paired t test) between the transmission results of DcFLV-infected and -uninfected D. citri vectored transmission. Bars headed by different letters in (B and C) indicate statistically significant differences (p < 0.05) between different insect stages (B) and between different dissected organs (C). ∗p = 0.0273 (A); ∗p = 0.0155 (E); ∗∗∗∗p < 0.0001.
Figure 5
Figure 5
Candidatus Liberibacter asiaticus (CLas) was detected in more leaves in a single plant when plants were inoculated by Diaphorina citri flavi-like virus (DcFLV)-infected Diaphorina citri at 2-month post-inoculation access period (IAP) in transmission assays (A–D) Percentage of leaves at different position infected by CLas. Individual DcFLV-infected and -uninfected D. citri that were infected by CLas was placed on the top leaf (T) of each plant, total of 21–25 plants for each assay, for 48 h (IAP). The first leaf down from leaf T (L1), the second leaf down from leaf T (L2), and the third leaf down from leaf T (L3) were tested for CLas infection. (E–H) The percentages of plants infected with 0, 1, 2, or 3 leaves were analyzed. Only the plants inoculated by 7-day post-emergence and 14-day post-emergence DcFLV-infected D. citri showed plants with all three leaves (L1, L2, and L3) infected by CLas at 2-month post-IAP but not the plants inoculated by DcFLV-uninfected D. citri. (A and E): plants inoculated by California 7-day post-emergence DcFLV-uninfected D. citri; (B and F) plants inoculated by California 7-day post-emergence DcFLV-infected D. citri; (C and G) plants inoculated by California 14-day post-emergence DcFLV-uninfected D. citri; (D and H) plants inoculated by California 14-day post-emergence DcFLV-infected D. citri. Statistical comparison showed significantly greater CLas spread in DcFLV-infected D. citri (p = 0.0041, chi-squared test); ns, not significant. The cartoon images of plants in figures (A–D) were adopted from BioRender stock images and created in Adobe Photoshop.
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