The greening-causing agent alters the behavioral and electrophysiological responses of the Asian citrus psyllid to a putative sex pheromone

Abstract

The Asian Citrus Psyllid (ACP), Diaphorina citri, is a vector of the pathological bacterium Candidatus Liberibacter asiaticus (CLas), which causes the most devastating disease to the citrus industry worldwide, known as greening or huanglongbing (HLB). Earlier field tests with an acetic acid-based lure in greening-free, 'Valencia' citrus orange groves in California showed promising results. The same type of lures tested in São Paulo, Brazil, showed unsettling results. During the unsuccessful trials, we noticed a relatively large proportion of females in the field, ultimately leading us to test field-collected males and females for Wolbachia and CLas. The results showed high rates of Wolbachia and CLas infection in field populations. We then compared the olfactory responses of laboratory-raised, CLas-free, and CLas-infected males to acetic acid. As previously reported, CLas-uninfected males responded to acetic acid at 1 µg. Surprisingly, CLas-infected males required 50 × higher doses of the putative sex pheromone, thus explaining the failure to capture CLas-infected males in the field. CLas infection was also manifested in electrophysiological responses. Electroantennogram responses from CLas-infected ACP males were significantly higher than those obtained with uninfected males. To the best of our knowledge, this is the first report of a pathogen infection affecting a vector's response to a sex attractant.

Figure 1

ACP male to female ratio in an experimental organic plot in Mogi Mirim, São Paulo State, Brazil, in 2020. Twenty-five data points were collected from February to December 2020 in Mogi Mirim. Twenty adults per plant were aspirated one by one from five plants sampled within the experimental area for each data point (of 100 adults). In the laboratory, adults were sexed, and the male/female ratios were recorded. The dotted line marks an equal number of males and females. Out of 125 samples (from a 25-day collection), only 20 showed a high male ratio (male/female ratio above 1). By contrast, 93 data points are below the dotted line, thus indicating a high female proportion. The overall mean suggests that the sex ratio was biased throughout the season toward females.

Figure 2

Percent of male and female ACP naturally infected with Wolbachia and CLas. Samples were collected for eleven days from February 18 to May 23, 2020, from an experimental organic plot in Mogi Mirim in the State of São Paulo, Brazil. Each point represents the percentage of adults infected with the tested bacteria. One thousand and one hundred psyllids were collected, but only 1084 samples passed the quality test for PCR analysis.

Figure 3

Behavioral responses from non-infected and CLas-infected ACP males to acetic acid. (A) CLas-free ACP males were tested at 1 µg (source dose). (B–D) CLas+ ACP males at 1, 10, and 50 µg doses, respectively. Bars represent mean residence times in each odorant field ± SEM. N represents the number of male responders. qPCR analyses were used to select only CLas+ males for analysis groups (B,C,D). We used untransformed data for normality and significant tests. P ˂ 0.05 denotes a significant difference in the control and treatment odor fields (Wilcoxon test).

Figure 4

The violin plot represents the electroantennographic (EAG) responses from uninfected and CLas-infected ACP males. Statistical analyses were performed with the raw data after subtracting the background responses in each preparation to paraffin oil. The mean responses elicited by acetic acid in CLas+ males differed significantly (P < 0.005, t-test) from the corresponding mean responses recorded from uninfected males at the same dose. The solid and dotted lines represent the median and quartiles in each plot. Blue plot: responses recorded with uninfected ACP males; pink plot: EAG responses elicited by CLas-infected ACP males.