The quest for a non-vector psyllid: Natural variation in acquisition and transmission of the huanglongbing pathogen 'Candidatus Liberibacter asiaticus' by Asian citrus psyllid isofemale lines

Abstract

Genetic variability in insect vectors is valuable to study vector competence determinants and to select non-vector populations that may help reduce the spread of vector-borne pathogens. We collected and tested vector competency of 15 isofemale lines of Asian citrus psyllid, Diaphorina citri, vector of 'Candidatus Liberibacter asiaticus' (CLas). CLas is associated with huanglongbing (citrus greening), the most serious citrus disease worldwide. D. citri adults were collected from orange jasmine (Murraya paniculata) hedges in Florida, and individual pairs (females and males) were caged on healthy Murraya plants for egg laying. The progeny from each pair that tested CLas-negative by qPCR were maintained on Murraya plants and considered an isofemale line. Six acquisition tests on D. citri adults that were reared as nymphs on CLas-infected citrus, from various generations of each line, were conducted to assess their acquisition rates (percentage of qPCR-positive adults). Three lines with mean acquisition rates of 28 to 32%, were classified as 'good' acquirers and three other lines were classified as 'poor' acquirers, with only 5 to 8% acquisition rates. All lines were further tested for their ability to inoculate CLas by confining CLas-exposed psyllids for one week onto healthy citrus leaves (6-10 adults/leaf/week), and testing the leaves for CLas by qPCR. Mean inoculation rates were 19 to 28% for the three good acquirer lines and 0 to 3% for the three poor acquirer lines. Statistical analyses indicated positive correlations between CLas acquisition and inoculation rates, as well as between CLas titer in the psyllids and CLas acquisition or inoculation rates. Phenotypic and molecular characterization of one of the good and one of the poor acquirer lines revealed differences between them in color morphs and hemocyanin expression, but not the composition of bacterial endosymbionts. Understanding the genetic architecture of CLas transmission will enable the development of new tools for combating this devastating citrus disease.

Fig 1. Mean ‘Candidatus Liberibacter asiaticus’ (CLas) acquisition and transmission rates of Diaphorina citri isofemale lines collected from throughout the state of Florida.

Lines are organized according to their CLas acquisition rate. Shown are means ± standard error of the means of psyllids from 15 tested isofemale lines. Corresponding inoculation/transmission rate is shown below the acquisition rate for each line.

Fig 2. Linear regression between ‘Candidatus L. asiaticus’ (CLas) Ct values in Diaphorina citri isofemale lines and CLas acquisition and transmission rates.

Correlation analysis between mean psyllid Ct values in qPCR tests, CLas-acquisition rate (A) and CLas-transmission rates (B) by psyllids of the isofemale lines tested. Shaded areas indicate 95% confidence limits.

Fig 3. Linear regression (correlation analysis) between ‘Candidatus Liberibacter asiaticus’ (CLas) -acquisition and CLas-transmission rates by Diaphorina isofemale lines.

Shaded area indicates 95% confidence limits.

Fig 4. Isofemale lines vary in proportion of color morphs.

Percentages of four color morphs between the psyllids of two isofemale lines (L8 and L16) show significant differences in the blue and gray color morphs but not yellow and intermediate.

Fig 5. Expression of Diaphorina citri hemocyanin-1 gene quantified by q-RT-PCR in two isofemale lines.

Normalized, relative expression of D. citri hemocyanin-1 gene using q-RT-PCR in L8 and L16 using hemocyanin-1 gene specific primers. The gene is expressed at higher levels in L16 as compared to L8.

Fig 6. Quantification of the three Diaphorina citri bacterial symbionts using qPCR in two isofemale lines.

The population of endosymbionts Wolbachia pipientis, ‘Candidatus Proftella armatura’ and ‘Candidatus Carsonella rudii’ show no differences in abundance between D. citri isofemale lines L8 and L16.