Effects of Methyl Salicylate on reproduction of Diaphorina citri and parasitization rate of D. citri by Tamarixia radiata

Nguyet-Minh T. Dao, Miwa S. Ishisaka and Dr. Anna L. SoperAbstractTamarixia radiata, a parasitoid wasp, is the primary parasitoid utilized to reduce populations of the Asian Citrus Psyllid (ACP), Diaphorina citri. Previous research has shown that D. citri and T. radiata are attracted to Methyl Salicylate (MeSA). In this study, synthetic MeSA was used on curry plants Murraya koenigii to study its effects on ACP reproduction rate and on the parasitism rate of T. radiata. Synthetic MeSA was diluted in 70% ethanol at concentrations of 1%, 0.5% and 0.25% for the increasing effect in both experiments. No significant relationship between psyllid reproduction rate and the MeSA treatment was found at any of the concentrations. However, MeSA had a promising effect in increasing parasitization rate of T. radiata. This result also suggests that ethanol can be used to dilute MeSA, instead of dichloromethane. Due to time constaints, more replications are needed to confirm our results. IntroductionDiaphorina citri Kuwayama (Hemiptera: Psyllidae), the Asian Citrus Psyllid (ACP), poses a serious threat to the citrus industry due to its ability to vector citrus greening or Huanglongbing (HLB) disease. ACP has also been detected in California but only one case of HLB infection has been reported. In order to mitigate the spread of the psyllid and pathogen, the ACP parasitic wasp, Tamarixia radiata Waterston (Hymenoptera: Eulophidae), have been reared and released in California since 2011. Here we focus on methods to increase D. citri reproduction since their nymphs are required as hosts to rear T. radiata and Tamarixia reproduction. Methyl Salicylate (MeSA) has been known as a critical volatile of plant responses to pathogen attacks, especially insects. Recently, Martini et al. (2014) have shown that T. radiata is attracted to MeSA released by HLB-infected plants. Furthermore, MeSA is not only employed by wasps for tracking psyllids, but it also highly attractive to psyllids (Mann et al. 2012). The purpose of this study was to increase wasp mass rearing efficiency by evaluating whether MeSA increases ACP and T. radiata reproduction rates.

Methodology

Figure 1. From left to right set up for ACP experiment, and for wasp experiment; nymphs on curry plants (top) and vials containing different treatments.

In both experiments, all insects were held in 17.8 x 17.8 x 35.6 cm Plexiglas cages (Figure 1) which were screened on one side with mesh (no-seeum) and a stockinette sleeve (BioQuip) on the other to allow access. The cages were held at 28◦ and 38% R.H., under 14L:10D photoperiod. Each cage contained a randomly chosen curry plant with flush and a vial containing 30 ml either of deionized (DI) water, 70% ethanol, or synthetic Methyl Salicylate (Sigma Aldrich) diluted with 70% ethanol (Sigma Aldrich) at concentrations of 1%, 0.5% and 0.25%.

These two experiment was replicated five times.Statistical analysesThe significance of both experiments were estimated by using ANOVA

ResultsExperiment 1: Reproduction of Diaphorina citri under Methyl Salicylate treatment

Figure 2. Average number of nymphs collected from each cage under two control conditions (water and ethanol) and under different concentrations of MeSA.

A one-way ANOVA test showed no significant difference between treatments (F=0.1995, DF =(4,15), P=0.9347),

Experiment 2: Methyl Salicylate and parasitization rate of D. citri by Tamarixia radiata

Figure 3. Percentage of parasitized nymphs by T. radiata under two control conditions (water and ethanol) and under different concentrations of MeSA.

A one-way ANOVA test, showed no significant difference between MeSA treatment levels (F=0.8358, DF=(4,19), P=0.5192). However, the parasitization rate at the 0.25%MeSA treatment is higher than the two controls, suggesting a potential effect of MeSA on parasitization of D. citri by T. radiata.Moreover, ethanol did not present any side effects on wasp parasitism behaviors (compare to the control with deionized water), hence MeSA could be diluted by ethanol instead of dichloromethane as in Martini et al (2014).

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DiscussionMann and his colleagues (2012) showed that ACP adults, especially females were attracted to MeSA released by HLB-infected citrus plants. We expected to see an increased reproduction rate of psyllids under MeSA conditions. However the ACP experiment showed highly variable reproduction rates of ACP adults in the control cages and cages with MeSA treatment. This lack of relationship between MeSA and psyllid reproduction in our experiment could be due to some potential factors such as female to male ACP ratio and environmental conditions (our cages were held in close proximity). Since we were working with a plant volatile, a sufficient gap between experimental cages is needed. Moreover, we also noticed that ACP adults were found dead near vials with MeSA at high concentrations (data not shown), suggesting the attraction of MeSA to D. citri. Synthetic MeSA was used on the curry plants to increase the parasitism of T. radiata on D. citri nymphs, based on Martini’s work. Even though the results were insignificant, 0.25% MeSA showed potential for increasing parasitism compared to the controls (Figure 3). The results may have been insignificant due to using only four to five replications. Future studies would focus on increasing the number of replications to increase statistical power. Other factors that may have affected the results are the age of the T. radiata and environment spacing.Further research on this plant volatile would be needed prior to applying MeSA in wasp mass rearing. Add 20 ACP adults into

each cage

Take out all ACP adults

Count number of nymphs

7 days

12 days

ACP reproduction experiment

Add 30 ACP nymphs and 1 female wasp into

each cage

Take out the wasps

Count number of parasitized nymphs

3 days

12 days

Wasp parasitization experiment

Acknowledgement Special thanks to Dr. Anna L. Soper for her advice, to Dr. Mellano, to Ashley Van Vliet and Ben Lehan for their help and to the ARI program 14-04-213 for grant support, to CDFA and USDA. Literature Cited Martini, X., Pelz-Stelinski, K. S. and Lukasz L. Stelinski, (2014), Plant pathogen-induced volatiles attract parasitoids to increase parasitism of an insect vector, Frontiers in Ecology and Evolution, 2(8).Mann R.S., Ali J.G., Hermann S.L., Tiwari S., Pelz-Stelinski K.S., et al. (2012) Induced Release of a Plant-Defense Volatile ‘Deceptively’ Attracts Insect Vectors to Plants Infected with a Bacterial Pathogen, PLoS Pathog 8(3).