Abstract Infection of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), with ‘Candidatus' Liberibacter asiaticus (Las), the causal pathogen of citrus greening disease or huanglongbing, increases psyllid susceptibility to insecticides. Diaphorina citri populations in citrus occur year-round in tropical and sub-tropical habitats, and thus insecticide applications for managing this plant disease vector occur over a wide temperature range (10–40 °C). During the winter season, D. citri is occasionally exposed to periods of freezing temperatures, when temperatures fall below -6.5 °C. In this investigation, we compared insecticide susceptibility of uninfected and Las-infected D. citri at various temperatures (20–37 °C). Cold-acclimated (6 ± 1 °C) D. citri adults were less susceptible to neonicotinoid insecticides as compared with non-acclimated controls, but this trend was not observed for other insecticides tested. A positive correlation between temperature and percentage mortality caused by chlorpyriphos, imidacloprid, spinetoram, and thiamethoxam was found irrespective of infection status when evaluated at temperatures ranging between 20 and 37 °C. In contrast, a negative correlation between temperature and percentage mortality was observed for fenpropathrin for both infected and uninfected psyllids. Glutathione S-transferase levels were negatively correlated with temperature, whereas levels of cytochrome P450 and general esterase were not correlated with temperature fluctuations. These results indicate that altered insecticide susceptibility due to temperature may not be related to glutathione S-transferase, cytochrome P450, and general esterase levels. Diaphorina citri adults that carried the Las bacterium had reduced CYP4 transcript and protein levels, and ingested less than uninfected counterparts, as measured by the production of honeydew. Diaphorina citri adult feeding was greatest at 32 °C within the temperature range tested. Overall, annual temperature fluctuation does not appear to be a major factor impacting management of D. citri.

Candidatus Liberibacter asiaticus is a fastidious, phloem-inhabiting, gram-negative bacterium transmitted by Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The bacterium is the presumed causal agent of huanglongbing (HLB), one of the most destructive and economically important diseases of citrus. We investigated whether Las is transmitted between infected and uninfected D. citri adults during courtship. Our results indicate that Las was sexually transmitted from Las-infected male D. citri to uninfected females at a low rate (<4%) during mating. Sexual transmission was not observed following mating of infected females and uninfected males or among adult pairs of the same sex. Las was detected in genitalia of both sexes and also in eggs of infected females. A latent period of 7 days or more was required to detect the bacterium in recipient females. Rod shaped as well as spherical structures resembling Las were observed in ovaries of Las-infected females with transmission electron microscopy, but were absent in ovaries from uninfected D. citri females. The size of the rod shaped structures varied from 0.39 to 0.67 µm in length and 0.19 to 0.39 µm in width. The spherical structures measured from 0.61 to 0.80 µm in diameter. This investigation provides convincing evidence that a plant pathogenic bacterium is sexually transmitted from male to female insects during courtship and established evidence that bacteria persist in reproductive organs. Moreover, these findings provide an alternative sexually horizontal mechanism for the spread of Las within populations of D. citri, even in the absence of infected host trees.

Transmission of plant pathogens by insect vectors is a complex biological process involving interactions between the plant, insect, and pathogen. Pathogen-induced plant responses can include changes in volatile and nonvolatile secondary metabolites as well as major plant nutrients. Experiments were conducted to understand how a plant pathogenic bacterium, Candidatus Liberibacter asiaticus (Las), affects host preference behavior of its psyllid (Diaphorina citri Kuwayama) vector. D. citri were attracted to volatiles from pathogen-infected plants more than to those from non-infected counterparts. Las-infected plants were more attractive to D. citri adults than non-infected plants initially; however after feeding, psyllids subsequently dispersed to non-infected rather than infected plants as their preferred settling point. Experiments with Las-infected and non-infected plants under complete darkness yielded similar results to those recorded under light. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen. Quantification of volatile release from non-infected and infected plants supported the hypothesis that odorants mediate psyllid preference. Significantly more methyl salicylate, yet less methyl anthranilate and D-limonene, was released by infected than non-infected plants. Methyl salicylate was attractive to psyllids, while methyl anthranilate did not affect their behavior. Feeding on citrus by D. citri adults also induced release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants. Infected plants were characterized by lower levels of nitrogen, phosphorus, sulfur, zinc, and iron, as well as, higher levels of potassium and boron than non-infected plants. Collectively, our results suggest that host selection behavior of D. citri may be modified by bacterial infection of plants, which alters release of specific headspace volatiles and plant nutritional contents. Furthermore, we show in a laboratory setting that this apparent pathogen-mediated manipulation of vector behavior may facilitate pathogen spread.

BACKGROUND:Color morphs of an insect species are known to vary in activities of detoxifying enzymes and associated susceptibility to insecticides. In Diaphorina citri, three color morphs are known to occur. In the present study, susceptibility to four insecticides was compared among gray/brown, blue/green and orange/yellow color morphs of field-collected D. citri.RESULTS:The orange/yellow morph was significantly more susceptible to fenpropathrin than the blue/green morph, and imidacloprid and chlorpyrifos caused higher mortality in the orange/yellow morph than in the blue/green and gray/brown morphs. To confirm the genetic basis of variable levels of susceptibility, the relative expression of five CYP4 genes was compared among the color morphs. CYP4C67 was expressed at significantly higher levels in the blue/green than in the orange/yellow and gray/brown morphs. CYP4DA1, CYP4C68, CYP4G70 and CYP4DB1 were expressed at significantly higher levels in the blue/green and gray/brown morphs than in the orange/yellow morph. Lower expression of CYP4 genes in the orange/yellow morph as compared with the others was correlated with reduced signal of 45 kD cytochrome P450 proteins, as determined by the western blot.CONCLUSIONS:The results indicate differential susceptibility of D. citri color morphs to insecticides, which will need to be accounted for in future insecticide monitoring programs and may affect management programs.

Early stage pests of sugarcane i.e. shoot borer (Chilo infuscatellus) and termites (Odontotermes spp.) are key factors in causing crop losses in terms of plant stand and number of millable canes. Post-germination application of imidacloprid 17.8. SL @ 20.03 g a.i./ha reduced the incidence of shoot borer and termites in sugarcane. The pooled data of four years (2003 to 2007) revealed that imidacloprid treatment was significantly better than control with 9.5 % incidence of shoot borer and 5.7 % incidence of termites. Imidacloprid was found to be potential by overall decrease in the incidence of shoot borer (48.8 %) and termites (77.4 %) over control with a significant increase in yield.

Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the vector of the phloem-inhabiting bacterium, Candidatus Liberibacter asiaticus, which is presumed to cause HLB in Florida citrus. Laboratory and field studies were conducted to examine the behavioral responses of male and female D. citri to their cuticular extracts. In olfactometer assays, more male D. citri were attracted to one, five, or 10 female cuticular extract equivalent units than blank controls. The results were confirmed in field studies in which clear or yellow traps baited with 10 female cuticular extract equivalent units attracted proportionately more males than clear traps baited with male cuticular extract or unbaited traps. Analyses of cuticular constituents of male and female D. citri revealed differences between the sexes in chemical composition of their cuticular extracts. Laboratory bioassays with synthetic chemicals identified from cuticular extracts indicated that dodecanoic acid attracted more males than clean air. Traps baited with dodecanoic acid did not increase total catch of D. citri as compared with blank traps at the dosages tested; however, the sex ratio of psyllid catch was male biased on traps baited with the highest lure loading dosage tested (10.0 mg).

Biotype B of the sweetpotato whitefly, Bemisia tabaci (Gennadius), is a worldwide pest of many agronomic and horticultural crops, including tomato. Extensive pesticide use targeting this pest has led to the development of resistance to every major class of insecticides. The baseline toxicity of spiromesifen was established for laboratory susceptible and field collected B tabaci biotype B. populations in Florida in 2005 and 2006, respectively, using a leaf dip bioassay method for 2nd instar. LC50 values for field populations of B. tabaci ranged from 0.63 mg[AI]l(-1) to 0.86 mg[AI]l(-1) in 2005 and from 0.46 mg[AI]l(-1) to 2.08 mg[AI]l(-1) in 2006. No population had a RR50 value over 3.5 in either year and the fiducial limits of the LC50 values for the laboratory and field populations overlapped, indicating no differences among them. The laboratory and field baseline toxicity data generated in this study confirmed the susceptibility of field populations of B. tabaci to spiromesifen and will be useful in documenting any future changes in the susceptibility of the whitefly to the insecticide.

Natural chemicals have considerable potential for vector management because these chemicals are safer than conventional insecticides on account of their rapid environmental biodegradation and low toxicity to natural enemies, humans and other mammals and they suffer less from problems of registration difficulties. However, isolation and chemical characterization of the active compounds from plants with strong biological activities can be a tedious process compared to synthesizing new synthetic compounds because natural compounds are generally isolated in small amounts. In addition, the purity of natural products is highly variable and is dependent upon the extraction method, plant part, plant age, geographic origin and location, climate and the overall growth and health of the plant from which the chemical is extracted. Furthermore, the research and development of natural pesticides against insect vectors is constrained because of the perceived lack of economic return to the manufacturers on investment in insect vector control and also due to the difficulties in registration. Despite these difficulties, research in plant-derived pesticides has increased considerablly. In this paper we provide an overview of the compounds isolated from plants that have been evaluated for control of insect vectors of human and animal pathogens.

Abstract Candidatus Liberibacter asiaticus (Las) has been reported to increase the susceptibility of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), to selected insecticides. Reduced general esterase activity in Las-infected, compared with uninfected, D. citri has been proposed as a possible explanation for this difference in insecticide susceptibility. The current study was conducted to quantify glutathione transferase (GST) and cytochrome P450 (general oxidase) activities in Las-infected D. citri to further explain the possible mechanisms for altered susceptibility to insecticides due to Las infection. GST and cytochrome P450 activities (indirectly through general oxidase levels) were quantified in Las-infected and uninfected D. citri nymphs and adults. Mean (±SEM) GST activity was significantly lower in Las-infected (468.23 ± 26.87 /µmol/min/mg protein) than uninfected (757.63 ± 59.46 µmol/min/mg protein) D. citri adults. Likewise, mean cytochrome P450 activity was significantly lower in Las-infected (0.23 ± 0.02 equivalent units [EU] cytochrome P450/mg protein) than uninfected (0.49 ± 0.05 EU cytochrome P450/mg protein) D. citri adults. Immature stages (second and fifth instars) were characterized by significantly lower GST activity than adults for uninfected D. citri. However, cytochrome P450 activity was significantly higher in second instar nymphs than adults and fifth-instar nymphs for uninfected D. citri. Lower activities of GST and general oxidase in Las-infected D. citri indicate that infection with Las alters D. citri physiology in a manner that could increase insecticide susceptibility. The reduced activities of these detoxifying enzymes due to Las infection may be explained by examining expression levels of associated genes in Las-infected and uninfected D. citri.

Most Drosophila flies are associated with rotten or over-ripened fruits and are nuisance pests. However, a few species such as the spotted wing drosophila, D. suzukii (Matsumura), can infest un-ripened fruits and are of economic significance. First detected within the continental United States in August 2008, D. suzukii has become a serious threat to fruit crops. This 6-page fact sheet was written by Rajinder S. Mann and Lukasz L. Stelinski and published by the UF Department of Entomology and Nematology, May 2011. EENY492/IN887: Spotted Wing Drosophila Drosophila suzukii (Matsumura) (Insecta: Diptera: Drosophilidae) (ufl.edu)

BACKGROUND Vector-borne diseases continue to present significant threats to human, animal and plant health. Mosquitoes, houseflies, sand flies and stable flies are well-known vectors of several human and animal pathogens. The toxicity of selected semiochemicals with molecular structures indicative of insecticidal activity was determined against these insect species with the aim of developing novel insecticides toxic to multiple insect species. RESULTS Three semiochemicals, namely beta-damascone, cyclemone A and melafleur, showed remarkable toxicity to three mosquitoes, Aedes aegypti L., Ae. albopictus (Skuse) and Anopheles quadrimaculatus Say, the housefly, Musca domestica L., the stable fly, Stomoxys calcitrans L., and the sand fly, Lutzomyia shannoni (Dyar). The chemicals were equally toxic to several field-collected permethrin-tolerant housefly strains. When formulated as 500 mL L(-1) emulsifiable concentrates, the chemicals demonstrated stability and toxicity on filter paper and camouflage military fabrics, with persistence up to 8 days under laboratory conditions. The chemicals were equally effective under field conditions when evaluated on unpainted plywood panels, although a higher dosage was required under field conditions to achieve similar efficacy. CONCLUSION Laboratory quantification of LC(50) values and field efficacy of three semiochemicals as formulated compounds on mosquitoes, houseflies, stable flies and sand flies showed that these semiochemicals could serve as potent insecticides for multiple insect species.

BACKGROUND:Asian citrus psyllid (ACP), Diaphorina citri, is a major pest of citrus because it vectors the putative causal agent of huanglongbing disease. Insecticides are currently the basis of psyllid management programs, and the number of annual insecticide applications has increased significantly. In this paper, a series of investigations of insecticide resistance among field populations of adult and immature ACP in Florida is described.RESULTS:In 2009, the highest level of resistance for adult ACP, as compared with the laboratory susceptible (LS) population, was found with imidacloprid with an LD(50) resistance ratio (RR(50) ) of 35 in one population. This was followed by chlorpyriphos (RR(50) = 17.9, 13.3, 11.8 and 6.9), thiamethoxam (RR(50) = 15 and 13), malathion (RR(50) = 5.4 and 5.0) and fenpropathrin (RR(50) = 4.8). In 2010, mortality of adults from all five sites sampled was lower than with the LS population at three diagnostic concentrations of each insecticide tested. Among nymph populations, indications of resistance were observed with carbaryl (RR(50) = 2.9), chlorpyriphos (RR(50) = 3.2), imidacloprid (RR(50) = 2.3 and 3.9) and spinetoram (RR(50) = 4.8 and 5.9). General esterase, glutathione S-transferase and monooxygenase levels were also elevated in field-collected adult and nymph ACP as compared with the LS population.CONCLUSION:The present results suggest that varying levels of insecticide susceptibility exist in ACP populations across the citrus-growing areas of Florida. Increased levels of detoxifying enzymes in these populations may partially explain these differences. The present results indicate that insecticide resistance may become an emerging problem for ACP control if effective resistance management is not practiced.

Ambrosia beetles are wood-degrading insects that live in nutritional symbiosis with ambrosia fungi. Usually we consider ambrosia beetles beneficial because they accelerate the decay of dead trees, which is important for nutrient cycling in healthy forests. However, the redbay ambrosia beetle and its fungal symbiont transmit the causal pathogen of laurel wilt disease among plants in the Laurel family (Lauraceae). They are considered a “very high risk” invasive disease pest complex having potential equal to that of Dutch elm disease or chestnut blight. Laurel wilt is a relatively new disease, and much is still unknown about how it will impact the flora of North America. This revised 7-page fact sheet highlights what we do know about this important new pest. Written by Rajinder Mann, Jiri Hulcr, Jorge Peña, and Lukasz Stelinski and published by the UF Department of Entomology and Nematology, May 2011. EENY491/IN886: Redbay Ambrosia Beetle Xyleborus glabratus Eichhoff (Insecta: Coleoptera: Curculionidae: Scolytinae) (ufl.edu)

Previously, we reported that Candidatus Liberibacter asiaticus (Las)-infected Diaphorina citri are characterized by lower levels of cytochrome P450 monooxygenases than uninfected counterparts. In the present study, we investigated expression levels of family 4 cytochrome P450 (CYP4) genes in Las-infected and uninfected D. citri adults. Five novel CYP4 genes (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1 and CYP4G70) were identified. Four of the five CYP4 genes were expressed at significantly higher levels in uninfected than Las-infected males, whereas only one was expressed at significantly higher levels in uninfected than Las-infected females. These results suggest that levels of cytochrome P450 monooxygenases in D. citri may be linked to expression levels of these CYP4 genes. Expression of all five CYP4 genes was induced by exposure of D. citri to imidacloprid, suggesting their possible involvement in metabolism of this toxin. Higher expression of the five CYP4 genes was found in nymphs than adults, which is congruent with previous results indicating higher levels of cytochrome P450 monooxygenases in nymphs than adults. These five CYP4 genes may be promising candidates for RNA-interference to silence overexpression of genes associated with insecticide resistance in D. citri. These newly identified genes may also serve as DNA-based screening markers for cytochrome P450-mediated insecticide resistance in field populations of D. citri.

The Asian citrus psyllid, Diaphorina citri Kuwayama, vectors Candidatus Liberibacter asiaticus (Las) and Candidatus Liberibacter americanus (Lam), the presumed causal agents of huanglongbing. D. citri generally rely on olfaction and vision for detection of host cues. Plant volatiles from Allium spp. (Alliaceae) are known to repel several arthropod species. We examined the effect of garlic chive (A. tuberosum Rottl.) and wild onion (A. canadense L.) volatiles on D. citri behaviour in a two-port divided T-olfactometer. Citrus leaf volatiles attracted significantly more D. citri adults than clean air. Volatiles from crushed garlic chive leaves, garlic chive essential oil, garlic chive plants, wild onion plants and crushed wild onion leaves all repelled D. citri adults when compared with clean air, with the first two being significantly more repellent than the others. However, when tested with citrus volatiles, only crushed garlic chive leaves and garlic chive essential oil were repellent, and crushed wild onions leaves were not. Analysis of the headspace components of crushed garlic chive leaves and garlic chive essential oil by gas chromatography-mass spectrometry revealed that monosulfides, disulfides and trisulfides were the primary sulfur volatiles present. In general, trisulfides (dimethyl trisulfide) inhibited the response of D. citri to citrus volatiles more than disulfides (dimethyl disulfide, allyl methyl disulfide, allyl disulfide). Monosulfides did not affect the behaviour of D. citri adults. A blend of dimethyl trisulfide and dimethyl disulfide in 1:1 ratio showed an additive effect on inhibition of D. citri response to citrus volatiles. The plant volatiles from Allium spp. did not affect the behaviour of the D. citri ecto-parasitoid Tamarixia radiata (Waterston). Thus, Allium spp. or the tri- and di-sulphides could be integrated into management programmes for D. citri without affecting natural enemies.

Invasive fungus-growing ambrosia beetles are an emerging threat to forest ecosystems and fruit industries, but management tools are lacking. Here we explored the potential of beetle symbionts–ambrosia fungi–as a source of attractants. Our focus was the redbay ambrosia beetle, Xyleborus glabratus, and its symbiotic fungus, Raffaelea lauricola, which are devastating lauraceous hosts in the southeastern United States. We also tested three additional co-occurring beetle species and their symbionts. Each beetle species was consistently attracted to the odors of its symbiotic fungal species, occasionally also to symbionts of other species, but never to non-symbiotic Trichoderma. We further confirmed attraction to ethanol (positive control) in some species. Thus, ambrosia fungi produce volatiles attractive to their vector beetles, which may have potential as novel lures for ambrosia beetle management.