Our objective during this past quarter was to investigate the evolution of insecticide resistant phenotypes (physical characteristics) in Asian citrus psyllid (ACP) and their underlying genotype (genes responsible for those characteristics). Specifically, we investigated resistance to the neonicotinoid insecticide, thiamethoxam, in a field investigation and also addressed cross-resistance to other insecticide modes of action by investigating field populations of ACP where we identified and documented neonicotinoid resistance. The obtained results are contributing to development of rotation strategies for improved resistance management of ACP.
Two types of rotation models have been developed by us over time based on our understanding of how resistance develops in ACP. These two rotations of insecticide modes of action are meant to prevent development of resistance in the field. While any rotation of modes of action is better than not rotating at all, our data to date with ACP from the laboratory indicate that certain rotation schemes should be superior to others in preventing resistance. This is because there can be varying levels of multiple-resistance between modes of action. Also, previous exposure to a certain mode of action in a sequence can effect the response of an ACP population to a second modes of action. These two rotation schedules compared here are referred to as “A” and “B”. First, insecticides were applied in these two rotational schemes (treatments), each consisting of four different insecticide modes of action (rotations A and B). The third treatment was a type of control in which neonicotinoid insecticides were applied in sequence four times with no rotation of mode of action (this is referred to as no rotation or NR). Rotation A consisted of dimethoate followed by cyantraniliprole, fenpropathrin, and diflubenzuron. Rotation B consisted of fenpropathrin followed by dimethoate, cyantraniliprole, and imidacloprid. NR consisted of thiamethoxam followed by clothianidin, thiamethoxam, and imidacloprid. Although different chemicals were rotated in the NR treatment, these are all different types of neonicotinoids. The field experiment consisted of five randomized replicate blocks and was conducted in two different groves: Grove 1 and Grove 2.
Insecticide toxicity bioassay were performed on ACP collected from the replicated treatment blocks from each of the three treatments compared: 1) NR, 2) rotation treatment A, and 3) rotation treatment B. A leaf dip bioassay technique was used to determine susceptibility levels of field-collected adult ACP after each insecticide application for two full rotations of all 4 insecticides comprising each treatment for a total of 9 evaluations, which included a pre-treatment evaluation. During each of the evaluations, the susceptibility of ACP adults from each replicated treatment plot was compared with the susceptibility of a known susceptible laboratory population maintained at the Citrus Research and Education Center. In Grove 1, we found that resistance of the ACP population in the NR control treatment, where neonicotinoids were applied in sequence rose 1,394 fold after 4 consecutive applications of neonicotonoids. In Grove 2, resistance of the ACP to thiamethoxam in the NR (no rotation) treatment rose by 1,266 fold after only three consecutive applications of neonicotinoids. However, the susceptibility of ACP to thiamethoxam in plots that were treated with rotations A and B only changed by 1.71 and 4.57 fold in Grove 1, respectively, and by 3.71 and 5.28 fold in Grove 2, respectively. Our results indicate that we have developed two rotation schedules that can robustly prevent development of resistance to neonicotinoids in locations where resistance to these insecticides has been previously documented for ACP in Florida. Furthermore, the results indicate that rotation A is slightly more effective than rotation B.
In addition, we have investigated the possible underlying mechanisms that have caused resistance in the populations of ACP in the NR (no rotation) treatment plots. We have also investigated the mechanism(s) involved in possible cross-resistance to different insecticide modes of action after an ACP populations develops resistance to neonicotinoids. We investigated susceptibility of ACP to the insecticides: dimethoate, cyantraniliprole, fenpropathrin, clothianidin, and imidacloprid from each of the three treatments evaluated in both Grove 1 and Grove 2. Resistance ratio (RR) were calculated by comparing the susceptibility of ACP to insecticides in the field with that our our laboratory susceptible strain reared at CREC. The results indicated that we did not find an increase in resistance that would cause product failure to fenpropathrin (RR=8.19); cyantraniliprole (RR=1.57); and dimethoate (RR=9.72) in Grove 1, and fenpropathrin (RR=4.24); cyantraniliprole (RR=1.58); and dimethoate (RR=7.22) in Grove 2. However, we documented significantly higher resistance for imidacloprid (RR=1059.65 in Grove 1 and RR = 1595.43 in Grove 2) and clothianidin (RR = 1798.77 in Grove 1 and RR = 1270.57 in Grove 2). These results indicate that there was significant cross-resistance developed between the neonicotinoid insecticides in plots where neonicotinoids were not rotated; however, the results demonstrate that multiple resistance did not occur, where a decrease in susceptibility to neonicotinoids did not impact the susceptibility of neonicotinoid-resistant ACP to other insecticide modes of action.
Currently and into the future, we are continuing to monitor stability of insecticide resistance to thiamethoxam in ACP populations using combined laboratory and field experiments. We have plans to evaluate a new rotation schedule treated by a different sequence of modes of action insecticide. Second, we will be analyzing the expression of seven P450, four GST and one EST gene and comparing their expression between the laboratory susceptible populations and identified resistant strains from the field. Finally, we will systematically examine differential gene expression using RNA sequencing (RNA-seq) to identify genes involved in general insecticide resistance in ACP. Again, here we will compare the laboratory susceptible strain with populations that are resistant to neonicotinoids. Furthermore, we are developing a new method to compare genes involved ACP fitness and reproduction, that are affected by development of resistance to thiamethoxam. The assembly, transcriptome annotation, the sequencing provides valuable genomic resources for further understanding the molecular basis of resistance and will allow us to precisely define the mechanisms conferring insecticide resistance in ACP. Figuring out the underlying genetic mechanisms confirming resistance allows us to tailor effective rotation schedules for management of resistance.