Xue Dong Chen

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the vector of the phloem-limited plant pathogen ‘Candidatus’ Liberibacter asiaticus (CLas). CLas is the most destructive insect-transmitted plant pathogen affecting citrus production in the United States and worldwide. During the past 15 yr, insecticides spanning several modes of action have been widely implemented globally to suppress populations of D. citri. High selection pressure eventually resulted in insecticide resistance throughout Florida and in other countries, including China, Mexico, and Brazil. Some resistance ratios have been reported to reach up to 4,000-fold. To date, insecticide resistance in D. citri has been primarily associated with enhanced metabolic activity involving esterases, carboxylesterase, and P450 monooxygenase, but cases of target site insensitivity can also occur. Cuticular reorganization could also play a role in insecticide resistance in D. citri and ATP-binding cassette transporters are involved in efflux pump inhibitor-related defense/resistance against insecticides in D. citri. Rotating and mixing different classes of insecticides as well as providing temporal and spatial refuges from exposure to toxins have been employed with varying degrees of success for managing insecticide resistance for this pest. Although resistance will likely remain a major challenge, management strategies based on the coordinated use of insecticides coupled with the use of cultural and biological control tactics currently provide the best model for combating insecticide resistance in D. citri.

The stability of resistance to fenpropathrin was assessed using five populations of Diaphorina citri with varying initial resistances ranging from fully susceptible (SS) to fully resistant (RR). Furthermore, we quantified the relative expression of voltage-gated sodium channel (VGSC) genes in crosses of field-selected and laboratory-susceptible D. citri lines after eight months without insecticide selection. We found that resistance to fenpropathrin remained elevated up to eight months after exposure to fenpropathrin. A real-time quantitative PCR analysis using the susceptible baseline population revealed that levels of VGSC gene expression were significantly higher in the RS75 cross and the RR100 fully resistant line eight months after their last fenpropathrin exposure. Our results suggest that while fenpropathrin resistance is likely unstable under field conditions when interbreeding with susceptible individuals is possible, resistance can remain stable for at least 8 months if those populations are isolated. Further, insecticide rotation and the maintenance of susceptible reservoirs of individuals should mitigate fenpropathrin resistance in D. citri over time. The development of a VGSC gene biomarker may be a useful tool for monitoring pyrethroid resistance in D. citri going forward.

Imidacloprid is a neonicotinoid insecticide used for managing the Asian citrus psyllid, Diaphorina citri Kuwayama, which serves as vector of phytopathogens causing citrus greening. However, development of resistance to neonicotinoids among populations of D. citri has coincided with occasional control failures in the field. The objectives of this research were to (1) survey current levels of imidacloprid resistance in Florida citrus; (2) compare feeding behavior between imidacloprid‐resistant and susceptible D. citri using electrical penetration graph recordings, and (3) investigate the possible amplification of insecticide hormoligosis associated with resistance. Field surveys confirmed that the susceptibility of D. citri populations to imidacloprid has decreased in commercial Florida citrus groves compared with a laboratory‐susceptible population. Following 12 generations of selection, resistance to imidacloprid increased by 438 fold compared with the susceptible strain. Imidacloprid‐susceptible D. citri feeding on citrus exhibited significantly more bouts associated with intercellular pathway (C), phloem penetration (D), phloem salivation (E1), and nonprobing (Np) activities than imidacloprid‐resistant counterparts. However, there were no differences observed in the frequency or duration of phloem ingestion or xylem feeding between susceptible and resistant D. citri. There was no statistical difference in fecundity between resistant and susceptible strains. However, the fecundity of imidacloprid‐susceptible female D. citri treated with a sublethal concentration of imidacloprid (LC₂₅) increased significantly compared with controls, while such hormoligosis was less pronounced among imidacloprid‐resistant psyllids. Our results suggest that imidacloprid‐resistant psyllids may cease feeding sooner than susceptible counterparts following sublethal exposure to this insecticide, indicative of a behavioral resistance mechanism.

The Asian citrus psyllid, Diaphorina citri, is an important pest that transmits the causal pathogens of huanglongbing (HLB) in citrus. In Florida, HLB threatens citrus production and insecticide overuse for the vector has resulted in evolution of resistance among populations of D. citri to commonly used insecticides. Cyantraniliprole is effective against nymphal and adult D. citri. However, since the introduction of cyantraniliprole use in Florida, observations have suggested that duration of population suppression following foliar sprays against D. citri has decreased from 60 to 28 days during the period between 2012 and 2020. Here, we sampled populations of D. citri from four locations in FL and measured their susceptibility to four insecticides compared with a susceptible laboratory culture. A vial bioassay method was used to measure direct toxicity of insecticides to D. citri. For bifenthrin, cyantraniliprole, dimethoate and thiamethoxam, the resistance ratios of field populations were 6.67–11.33, 3.20–36.37, 12.50–82.50 and 4.60–10.08, respectively. Data suggested that susceptibility levels of D. citri populations to cyantraniliprole have decreased over 10‐fold at three separate citrus growing locations in Florida. Although some shifts in the resistance ratio of cyantraniliprole appear to have occurred since its use began in FL citrus, the resistance ratios are congruent with field observations indicating that D. citri should remain susceptible to label rate applications of this mode of action without control failure. However, our results indicate that sufficient field variation in susceptibility to cyantraniliprole exists among populations of D. citri to justify careful management of this insecticide for prevention of insecticide resistance development.

Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is an important economic pest of citrus crops because it vectors the causal pathogen of huanglongbing (HLB; aka citrus greening). Population suppression of D. citri with insecticides has been disproportionally relied on for HLB management and a greater diversity of more sustainable tools is needed. Periglandula spp. is a fungal endosymbiont (family Clavicipitaceae) that forms a mutualistic relationship with members of plants in family Convolvulaceae. This association results in the production of ergot alkaloids that were previously documented as having psyllicidal properties. We investigated the mortality and behavior of D. citri exposed to crude extracts from morning glories in the plant family Convolvulaceae, as well as synthetic ergot alkaloids. Nymphs and adults were exposed to the crude plant extracts from Periglandula positive species of Convolvulaceae, as well as five synthetic ergot alkaloids. Treatments were prepared by exposing clippings of citrus to 100 ng/µL of crude extract from Periglandula-positive species of Ipomoea (I. imperati, I. leptophylla, I. pandurata and I. tricolor), and Turbina corymbosa, and from one Periglandula-negative species (I. alba) (100 ng/µL). Mortality of adult and nymphal D. citri was significantly higher than the control after exposure to extracts from I. tricolor and I. imperati. The synthetic ergot alkaloids, lysergol (10–100 ng/µL), ergonovine maleate (100 ng/µL), agroclavine (10–100 ng/µL), and ergosine (10–100 ng/µL) increased mortality of D. citri nymphs, while ergosine (100 ng/µL) and agroclavine (100 ng/µL) increased mortality of adults compared to water controls. Fewer D. citri adults settled on plants treated with crude extracts or synthetic ergot alkaloids than on water controls at 48 h after release. D. citri that fed on citrus leaves treated with 10 ng/μL solution of crude extract from the Periglandula-positive species Ipomoea (I. imperati, I. leptophylla, I. pandurata, I. tricolor), and Turbina corymbosa excreted significantly less honeydew compared with a negative water control and extract from Periglandula-negative species (I. alba). Our results indicate that crude extracts and ergot alkaloids exhibit toxic and sub-lethal effects on D. citri that could be useful for management of this pest.

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits the causal pathogen of huanglongbing and is a global pest of citrus. D. citri populations exhibit resistance to multiple insecticide modes of action in areas where these chemicals have been overused. We performed genome‐wide transcriptional analysis for two field populations of D. citri (Wauchula and Lake Alfred, Florida, USA) that exhibit 1300‐fold resistance to the neonicotinoid insecticide, thiamethoxam, and compared it to that of susceptible psyllids collected from the same area and without imposed selection. The Lake Alfred population responded to insecticide resistance by up‐regulation of 240 genes and down‐regulation of 148 others. The Wauchula population exhibited similar patterns to the Lake Alfred population with up‐regulation of 253 genes and down‐regulation of 115 others. Gene Ontology annotation associated with cellular processes, cell, and catalytic activity were assigned to differentially expressed genes (DEGs). The DEGs from Lake Alfred and Wauchula populations were mapped to Kyoto Encyclopedia of Gene and Genomes pathways and implicated enrichment of metabolic pathways, oxidative phosphorylation, extracellular matrix‐receptor interaction, terpenoid backbone biosynthesis, and insect hormone biosynthesis in the resistant populations. Up‐regulation of 60s ribosomal proteins, UDP‐gluscoyltransferases, cytochrome c oxidases, and CYP and ABC transporters among thiamethoxam‐resistant D. citri implicates a broad array of novel and conventionally understood resistance mechanisms.

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), populations exhibit varying levels of resistance to several insecticide classes, including pyrethroids. Artificial laboratory selection was conducted to investigate mechanisms underlying fenpropathrin resistance. After ten generations of selection, the LC₅₀ for fenpropathrin increased from 0.18 to 11.60 ng/μL, and the associated resistance ratio increased by 96.67-fold. The realized heritability of resistance (h² ) to fenpropathrin changed from 0.17 to 0.44 during the first and second round of selection, respectively. We did not find high cross-resistance to imidacloprid (RR = 3.58) or dimethoate (RR = 1.77) in the fenpropathrin-selected strain, but found cross-resistance to bifenthrin (RR = 28.21). RT-qPCR analysis showed that expression of CYP6A2-1 was significantly increased in the selected population relative to the laboratory susceptible population. Our data indicate that D. citri has the capacity to develop a high level of fenpropathrin resistance following persistent selection and that cytochrome P450 (CYP) and glutathione S-transferase (GST) metabolic detoxification may be contributing mechanisms. Rotation programs that include neonicotinoids and organophosphates should manage resistance to fenpropathrin for D. citri.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a major pest that transmits phloem-limited, gram negative bacteria including ‘Candidatus’ liberibacter asiaticus (Clas), causing huanglongbing. Management of this pest relies primarily on insecticides, such as the neonicotinoid imidacloprid, that may affect secondary pests including the brown citrus aphid, Toxoptera citricida (Kirkaldy) (Hemiptera: Aphididae). Here, we report on direct toxicity and sublethal dose effects of imidacloprid on T. citricida nymphs and adults following direct contact and ingestion. We also examined transgenerational dose-response and hormetic effects following exposure of T. citricida to a sublethal concentration of imidacloprid. Toxicity of imidacloprid was similar for nymphs (0.6 ng per µL) and adults (1 ng per µL) at 72 h. Fecundity and finite rate of increase were greater for populations exposed to systemic and foliar treatments at a sublethal concentration (0.1 ng per µL) compared with untreated controls. Development times of first instar nymphs in the F1 generation and third instar nymphs in the F2 generation were significantly greater for individuals treated with the sublethal dose than an untreated control. Survival of second instar T. citricida on plants treated with a sublethal concentration also was significantly greater than controls. There also was a significant increase in fecundity of F1 and F2 individuals after sublethal treatment compared with controls. Our results indicated that a sublethal concentration of imidacloprid increased the reproductive performance and induced possible physiologically stimulative (hormetic) effects in T. citricida. Hormesis in secondary pests should be considered when developing a management program for pathogen vectors such as D. citri.

Background: We investigated rotation using insecticides with multiple modes of action as a resistance management strategy for Asian citrus psyllid, Diaphorina citri, Kuwayama (Hemiptera: Liviidae), in Florida. The stability of thiamethoxam resistance was investigated in the laboratory by establishing populations of field‐collected, resistant D. citri and rearing them under no insecticide exposure. Furthermore, recovery of susceptibility was investigated in the field by initiating rotation to insecticides in plots that previously were treated with consecutive thiamethoxam applications.Results: The resistance ratio (RR) for thiamethoxam reached between 1266.29‐ and 1395.00‐fold after three and four consecutive applications of thiamethoxam, respectively. However, the RR for thiamethoxam remained low (1.71–5.28‐fold) under both rotations at both Lake Alfred and Wauchula. Thiamethoxam was cross‐resistant with imidacloprid (RR _{site 1} = 1059.65‐fold, RR _{site 2} = 1595.43‐fold) and clothianidin (RR _{site 1} = 1798.78‐fold, RR _{site 2} = 1270.57‐fold) in the nonrotated treatment at both sites. There was very low cross‐resistance to other insecticides with different modes of action. Both laboratory and field investigations indicated that susceptibility to thiamethoxam fully recovered after five D. citri generations. Expression of CYP4C67 was significantly increased in resistant populations.Conclusion: Our results revealed that D. citri populations develop a high level of resistance following only three or four consecutive neonicotinoid sprays; this was associated with subsequent product failure. Our data suggest that metabolic detoxification by cytochrome P450s contributes to thiamethoxam resistance in D. citri. Overall, the investigation demonstrated that resistance to thiamethoxam can be managed readily in populations of D. citri by rotating modes of action.

Insecticide resistance is an increasing problem in citrus production. The Asian citrus psyllid,Diaphornia citriKuwayama, is recognized as one of the most important citrus pests worldwide and it has developed resistance in areas where insecticides have been overused. The development of insecticide resistance is often associated with fitness costs that only become apparent in the absence of selection pressure. Here, the fitness costs associated with resistance to thiamethoxam and imidacloprid were investigated in three agricultural populations ofD. citrias compared with susceptible laboratory colonies. Results showed that all field populations had greater resistance than laboratory susceptible colonies. For both thiamethoxam and imidacloprid, aCandidatusLiberibacter asiaticus-positive (CLas⁺) colony was more susceptible than theCLas⁻colony. Resistance ratios ranged from 7.65–16.11 for imidacloprid and 26.79–49.09 for thiamethoxam in field populations as compared with a susceptible,CLas⁻laboratory strain. Among three resistant field populations, a significantly reduced net reproductive rate and finite rate of population increase were observed in a population from Lake Wales, FL as compared to both susceptible strains. The fecundity of field populations from Lake Wales, FL was statistically lower than both laboratory susceptible populations. Certain changes in morphological characteristics were observed among resistant, as compared, with susceptible strains. Our data suggest fitness disadvantages associated with insecticide resistance inD. citriare related to both development and reproduction. The lower fitness ofD. citripopulations that exhibit resistance to neonicotinoid insecticides should promote recovery of sensitivity when those populations are no longer exposed to thiamethoxam and/or imidacloprid in the field. The results are congruent with a strategy of insecticide rotation for resistance management.