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.

Asian citrus psyllid,Diaphorina citri Kuwayama (Hemiptera:Liviidae),trans-mits the causal pathogen of huanglongbing and is a global pest of citrus.D.citri pop-ulations 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 pat-terns 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 cat-alytic 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 phos-phorylation,extracellular matrix-receptor interaction,terpenoid backbone biosynthesis,and insect hormone biosynthesis in the resistant populations.Up-regulation of 60s ribo-somal 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.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is known to exhibit abdominal color polymorphisms. In the current study, susceptibility to four insecticides was compared among orange/yellow, blue/green and gray/brown color morphs of field collected D. citri. The LD50 values and 95% fiducial limits were quantified for each insecticide and color morph combination and ranged between 0.10 ng/μL (0.06-0.10) and 6.16 ng/μL (3.30-12.50). Second, we measured the detoxification enzyme activity levels of orange/yellow, blue/green and gray/brown color morphs for cytochrome P450, glutathione S-transferase, and general esterase. The mean P450 activity (equivalent units) was significantly lower in gray/brown (0.152 ± 0.006) and blue/green morphs (0.149 ± 0.005) than in the orange/yellow morphs (0.179 ± 0.008). GST activity (μmol/min/mg protein) was significantly lower in the orange/yellow morph (299.70 ±1.24) than gray/brown (350.86 ± 1.19) and blue/green (412.25 ± 1.37) morphs. The mean EST activity (μmol/min/mg protein) was significantly higher in blue/green (416.72 ± 5.12) and gray/brown morphs (362.19 ± 4.69) than in the orange/yellow morphs (282.56 ± 2.93). Additionally, we analyzed the relative expression of assortment genes involved in cuticular melanization and basal immunity. The transcripts of Dopa Decarboxylase and Tyrosine Hydroxylase were expressed higher in blue/green and gray/brown than orange/yellow morphs. The transcription results paralleled the susceptibility of D. citri to organophosphate, neonicotinoid and pyrethroid insecticides. GST and EST activity may also be correlated with low levels of insecticide susceptibility. Cuticular melanization could be a factor for the development of resistance to insecticides among different color morphs.

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), adults were collected from eight citrus groves across central Florida, and the level of insecticide resistance to ten insecticides was measured using a bottle bioassay. The gene expression of five cytochrome P450 CYP4 (CYP4C67, CYP4DA1, CYP4DB1, CYP4G70 and CYP4C68) and three glutathione S-transferase (GSTD1, GSTE2 and GSTE1) genes was characterized in seven field populations of D.citri and compared with a laboratory population. Additionally, we reared four neonicotinoid insecticide resistant field populations in the laboratory and observed susceptibility changes without exposure to insecticides over multiple generations. The eight field populations of D.citri adults showed no and very low levels of resistance (RR=1 and 2-10) to dimethoate, chlorprifos, carbaryl, fenpropathrin, bifenthrin, flupyradifurone and spinetoram. Very low to low resistance was found to imidacloprid and cyantraniliprole (RR=2-10 and RR=10-20). Moderate to high resistance was found for thiamethoxam (RR=20-50 and RR=50-100). The CYP4G70 and CYP4C68 genes were expressed at a higher level in field populations as compared with the laboratory population. Also the Davenport, Florida field population exhibited higher expression of all target genes compared to the laboratory population. Susceptibility to imidacloprid and thiamethoxam increased by 6.62- and 6.42-fold, respectively, compared to the levels initially observed in the field over six generations of breeding without exposure. These results indicate that insecticide resistance may reverse in the field if insecticide selection pressure is removed from the spray schedule or with use of a rotational scheme with insecticides of different modes of action. Also, the results support use of insecticide resistance survey program combined with effective rotation for integrated insecticide resistance management of D.citri where huanglongbing (HLB) management includes vector suppression with insecticides.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a major pest of citrus trees worldwide. A wide variety of insecticides are used to manage D. citri populations within citrus groves in Florida. However, in areas shared by citrus growers and beekeepers the use of insecticides may increase the risks of Apis mellifera  L. (Hymenoptera: Apidae) loss and contaminated honey. The objective of this research was to determine the environmental toxicity of insecticides, spanning five different modes of action used to control D. citri, to A. mellifera. The insecticides investigated were imidacloprid, fenpropathrin, dimethoate, spinetoram and diflubenzuron. In laboratory experiments, LD50 values were determined and ranged from 0.10 to 0.53 ng/μl for imidacloprid, fenpropathrin, dimethoate and spinetoram. LD50 values for diflubenzuron were >1000 ng/μl. Also, a hazard quotient was determined and ranged from 1130.43 to 10893.27 for imidacloprid, fenpropathrin, dimethoate, and spinetoram. This quotient was <0.447 for diflubenzuron. In field experiments, residual activity of fenpropathrin and dimethoate applied to citrus caused significant mortality of A. mellifera 3 and 7 days after application. Spinetoram and imidacloprid were moderately toxic to A. mellifera at the recommended rates for D. citri. Diflubenzuron was not toxic to A. mellifera in the field as compared with untreated control plots. Phenoloxidase (PO) activity of A. mellifera was higher than in untreated controls when A. mellifera were exposed to 14 days old residues. The results indicate that diflubenzuron may be safe to apply in citrus when A. mellifera are foraging, while most insecticides used for management of D. citri in citrus are likely hazardous under various exposure scenarios.

Hemolymph was characterized from Diaphorina citri adults infected with the phytopathogen, Candidatus Liberibacter asiaticus (CLas), and compared with that from uninfected psyllids. This study identified 5531 and 3220 peptides within infected and uninfected hemolymph using nano-LC–MS/MS. A reduced number of proteins were detected for D. citri and all known endosymbionts within infected hemolymph as compared to uninfected hemolymph. A large number of immune defense proteins were absent from D. citri hemolymph; however, a single recognition protein (PGRP), two serine protease inhibitors, three prophenoloxidase (proPO) enzymes, and a single serine protease in an uninfected D. citri were detected. The hemolymph is nearly devoid of nutrient storage proteins. This is the first proteomic analysis of D. citri hemolymph that also analyses the components contributed by all the endosymbionts. By comparing the contribution of each endosymbiont (CCR, CPA, and WB) in the presence and absence of CLas infection, this study offers initial insights regarding the hemolymph response to microbial community shifts associated with D. citri infection status. Our data also present potential protein targets for analysis and disruption of CLas transmission that may facilitate management of huanglongbing (HLB) caused by CLas in citrus.

AimsFusarium graminearum is a very destructive fungal pathogen that leads to Fusarium head blight (FHB) in wheat, a disease which costs growers millions of dollars annually both in crop losses and in remediation efforts. Current countermeasures include the deployment of wheat varieties with some resistance to FHB in conjunction with timed fungicide treatments. In this article, we introduce a fungicide based on thymol, a naturally occurring plant phenolic derived from essential oils. To overcome the hydrophobicity of thymol, the thymol active was incorporated into a low-surfactant submicron emulsion with and without a carrier oil.Methods and ResultsThe minimum fungicidal concentration of F.graminearum was found to be both 002% for thymol emulsions with and without an oil component. Time-to-kill experiments showed that thymol emulsions were able to inactivate F.graminearum in as little as 10s at concentrations above 006%. Spraying the thymol emulsions (similar to 01% range) on the wheat variety Bobwhite demonstrated significant reductions in FHB infection rate (number of infected spikelets). However, with 05% thymol, the wheat heads exhibited premature senescence. Transmission and scanning electron micrographs suggest that the mechanism of antifungal action is membrane mediated, as conidia exposed to thymol showed complete organelle disorganization and evidence of lipid emulsification.ConclusionThe collective experimental data suggest that thymol emulsions may be an effective naturally derived alternative to the current thymol treatments, and chemical fungicides in ameliorating FHB.Significance and Impact of the StudyThis is the first thymol-derived nanoemulsion particles resuspended into water and not DMSO, exhibiting the same antibacterial/antifungal activity as previously described thymol and thyme oil treatments. This drastically reduces the environmental footprint thymol will leave if utilized as a fungicide treatment on field crops.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is an insect pest capable of transmitting Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening in North America. D. citri also harbors three endosymbionts, Wolbachia, Candidatus Carsonella ruddii, and Candidatus Profftella armatura, which may influence D. citri physiology and fitness. Although genomic researches on these bacteria have been conducted, much remains unclear regarding their ecology and inter-population variability in D. citri. The present work examined the densities of each endosymbiont in adult D. citri sampled from different populations using quantitative PCR. Under field conditions, the densities of all three endosymbionts positively correlated with each other, and they are associated with D. citri gender and locality. In addition, the infection density of CLas also varied across populations. Although an analysis pooling D. citri from different populations showed that CLas-infected individuals tended to have lower endosymbiont densities compared to uninfected individuals, the difference was not significant when the population was included as a factor in the analysis, suggesting that other population-specific factors may have stronger effects on endosymbiont densities. To determine whether there is a genetic basis to the density differences, endosymbiont densities between aged CLas-negative females of two D. citri populations reared under standardized laboratory conditions were compared. Results suggested that inter-population variability in Wolbachia infection density is associated with the genotypes of the endosymbiont or the host. Findings from this work could facilitate understanding of D. citri-bacterial associations that may benefit the development of approaches for managing citrus greening, such as prevention of CLas transmission.

The cys‐motif gene family associated with Campoletis sonorensis ichnovirus contains 10 members, WHv1.6, WHv1.0, VHv1.1, VHv1.4, AHv1.0, A'Hv0.8, FHv1.4, LHv2.8, UHv0.8, and UHv0.8a. The results of this study indicated that, within the encapsidated virion, WHv1.6 is the most abundant cys‐motif gene, while the combined AHv genes are the least abundant. During parasitization of Heliothis virescens by Campoletis sonorenis, WHv1.6 transcripts were the mostly highly expressed, while the combined UHv genes had the lowest expression. Further proteomic analysis of WHv1.6 showed that it accumulates at high levels in parasitized plasma by 6 h, and is detectable in the haemocytes, fat body, malpighian tubules, nerve cord and epidermis by 2 days after parasitization. Localization experiments led us to conclude that WHv1.6 interacts with the cell membrane along with other organelles within a virus‐infected cell and prevents immunocytes from spreading or adhering to a foreign surface. Similarly to VHv1.4 and VHv1.1, WHv1.6 is able to inhibit the translation of haemocyte and Malpighian tubule RNAs. Our results showed that the expression of cys‐motif genes during parasitization is related to the gene copy number of each gene within the encapsidated virion and may also be dependent upon cis‐regulatory element activity in different target tissues. In addition, WHv1.6 plays a major role in inhibiting the cellular encapsulation response by H. virescens.

Understanding the molecular mechanisms underlying insect compensatory responses to plant defenses could lead to improved plant resistance to herbivores. The Mp708 inbred line of maize produces the maize insect resistant 1-cysteine protease (Mir1-CP) toxin. Reduced feeding and growth of fall armyworm larvae fed on Mp708 was previously linked to impairment of nutrient utilization and degradation of the midgut (MG) peritrophic matrix (PM) by Mir1-CP. Here we examine the biochemical and transcriptional responses of fall armyworm larvae to Mir1-CP. Insect Intestinal Mucin (IIM) was severely depleted from pure PMs treated in vitro with recombinant Mir1-CP. Larvae fed on Mp708 midwhorls excrete frass largely depleted of IIM. Cracks, fissures and increased porosity previously observed in the PM of larvae fed on Mp708 midwhorls could ensue when Mir1-CP degrades the IIM that cross-links chitin fibrils in the PM. Both targeted and global transcriptome analyses were performed to determine how complete dissolution of the structure and function of the PM is prevented, enabling larvae to continue growing in the presence of Mir1-CP. The MGs from fall armyworm fed on Mp708 upregulate expression of genes encoding proteins involved in PM production as an apparent compensation to replace the disrupted PM structure and restore appropriate counter-current MG gradients. Also, several families of digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on diets lacking Mir1-CP (artificial diet, midwhorls from Tx601 or B73 maize). Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function.

This datasheet on Campoletis sonorensis covers Identity, Distribution, Natural Enemies.

Several maize, Zea mays L., inbred lines developed from an Antiguan maize population have been shown to exhibit resistance to numerous aboveground lepidopteran pests. This study shows that these genotypes are able to significantly reduce the survival of two root feeding pests, western corn rootworm, Diabrotica virgifera virgifera LeConte, and southern corn rootworm, Diabrotica undecimpunctata howardi Barber. The results also demonstrated that feeding by the aboveground herbivore fall armyworm, Spodoptera frugiperda (J.E. Smith), before infestation by western corn rootworm reduced survivorship of western corn rootworm in the root tissues of some, but not all, genotypes. Likewise, the presence of western corn rootworm in the soil seemed to increase resistance to fall armyworm in the whorl in several genotypes. However, genotypes derived from the Antiguan germplasm with genetic resistance to lepidopterans were still more resistant to the fall armyworm and both rootworm species than the susceptible genotypes even after defense induction. These results suggest that there may be intraplant communication that alters plant responses to aboveground and belowground herbivores.

Since the beginning of agriculture approximately 10,000 years ago, farmers have struggled to protect their crops from insect pests. This is particularly true for maize, an important agricultural foodstuff that is vulnerable to attack by wide variety of herbivores. Plant breeding is one strategy successfully employed to develop genotypes with improved resistance to these pests. One example is the maize inbred Mp708 that has resistance to several caterpillar species that feed in the whorls and stalks.1 Mp708 was developed from exotic germplasm that originated in Antigua by selecting for plants resistant to feeding by fall armyworm (Spodoptera frugiperda) and southwestern cornborer (Diatraea grandiosella).2,3 Research to understand the mechanism of caterpillar resistance in Mp708 has revealed that it is a multigene trait regulated by several quantitative trait loci (QTL).4,5

Exploiting the ability of insect pathogens, parasites, and predators to control natural and damaging insect populations is a cornerstone of biological control. Here we focus on an unusual group of viruses, the polydnaviruses (PDV), which are obligate symbionts of some hymenopteran insect parasitoids. PDVs have a variety of important pathogenic effects on their parasitized hosts. The genes controlling some of these pathogenic effects, such as inhibition of host development, induction of precocious metamorphosis, slowed. or reduced feeding, and immune suppression, may have use for biotechnological applications. In this chapter, we consider the physiological functions of both wasp and viral genes with emphasis on the Cys-motif gene family and their potential use for insect pest control.

Polydnaviruses are obligate symbionts of some parasitic hymenopteran wasps responsible for modifying the physiology of their host lepidopteran larvae to benefit the endoparasite. Injection of Campoletis sonorensis ichnovirus (CsIV) into Heliothis virescens larvae alters larval growth, development and immunity but genes responsible for alterations of host physiology are not well described. Recent studies of polydnavirus genomes establish that these genomes encode families of related genes expressed in parasitized larvae. Here we evaluate five members of the CsIV cys-motif gene family for their ability to inhibit growth and development of lepidopteran larvae. To study the function of cys-motif proteins, recombinant proteins were produced from baculovirus expression vectors and injected or fed to H. virescens larvae in diet. rVHv1.1 was identified as the most potent protein tested causing a significant reduction in growth of H. virescens and Spodoptera exigua larvae. H. virescens larvae ingesting this protein also exhibited delayed development, reductions in pupation and increased mortality. Increased mortality was associated with chronic sub-lethal baculovirus infections. Taken together, these data indicate that the cys-motif proteins have pleiotropic effects on lepidopteran physiology affecting both development and immunity.

Protamine is a naturally occurring cationic antimicrobial peptide (CAP) that has shown some promise for control of microorganisms in food. It was hypothesized that the antibacterial effect is partially due to protamine's electrostatic affinity to the negatively charged cell envelopes of actively growing bacteria. However, nonspecific binding of the CAPs to negatively charged food particles may reduce the effect in food systems. To test the hypothesis, the antibacterial efficacies of native and reduced charge protamines (chemically modified by randomly blocking 10 to 71% of the guanido groups of the arginine residues) were compared in model and food systems. In Tryptic Soy Broth, moderate reductions of charge (<26%) resulted in either a similar or slightly improved antimicrobial efficacy, measured as the minimum inhibitory concentration (MIC) toward 21 food-related bacteria. Further reductions in positive charge led to lower antimicrobial activity. Compared to protamine, the affinity of reduced charge protamines (10 and 20%) for binding to Listeria monocytogenes cells was higher at pH 7 and 8. As perhaps would be expected, L. monocytogenes is most sensitive to modified protamines in this pH range. Protamine with reduced charge (14 and 23%) inhibited growth of L. monocytogenes in milk as well as total bacteria and coliforms in ground beef significantly (P<0.05) better than native protamine, demonstrating that the reduced charge peptides were more inhibitory in these high protein food matrices. Electrophoretic analysis of the 21 bacteria revealed a statistically significant (P<0.01) relationship with antimicrobial activity, where the most negatively charged bacteria were also the most susceptible to protamine. In conclusion, components of food matrices interfered with the antibacterial effects of the peptides, however; these undesirable interferences were reduced by altering the electrostatic properties of protamine.