Rajinder S. Mann

Insecticide resistance in field populations of Asian citrus psyllid in Florida.

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.

Induced Release of a Plant-Defense Volatile 'Deceptively' Attracts Insect Vectors to Plants Infected with a Bacterial Pathogen

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.

Nicotinoid and pyrethroid insecticide resistance in houseflies (Diptera: Muscidae) collected from Florida dairies

BACKGROUND The housefly, Musca domestica L., continues to be a major pest of confined livestock operations. Houseflies have developed resistance to most chemical classes, and new chemistries for use in animal agriculture are increasingly slow to emerge. Five adult housefly strains from four Florida dairy farms were evaluated for resistance to four insecticides (beta-cyfluthrin, permethrin, imidacloprid and nithiazine). RESULTS Significant levels of tolerance were found in most field strains to all insecticides, and in some cases substantial resistance was apparent (as deduced from comparison with prior published results). At the LC(90) level, greater than 20-fold resistance was found in two of the fly strains for permethrin and one fly strain for imidacloprid. Beta-cyfluthrin LC(90) resistance ratios exceeded tenfold resistance in three fly strains. The relatively underutilized insecticide nithiazine had the lowest resistance ratios; however, fourfold LC(90) resistance was observed in one southern Florida fly strain. Farm insecticide use and its impact on resistance selection in Florida housefly populations are discussed. CONCLUSION Housefly resistance to pyrethroids is widespread in Florida. Imidacloprid resistance is emerging, and tolerance was observed to both imidacloprid and nithiazine. If these insecticides are to retain efficacy, producer use must be restrained.

Sulfur volatiles from Allium spp. affect Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), response to citrus volatiles

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.

Monitoring neonicotinoid resistance in biotype B of Bemisia tabaci in Florida

BACKGROUND Biotype B of the sweetpotato whitefly, Bemisia tabaci (Genn.), is a worldwide pest that has developed resistance to many insecticides, including the neonicotinoid class. Florida field populations were monitored for susceptibility to the neonicotinoids imidacloprid and thiamethoxam using a cut leaf petiole bioassay method. RESULTS Average RR(50) values for imidacloprid increased from 3.7 in 2000 to 12.0 in 2003; decreased to 5.0 and 2.5 in 2004 and 2005, respectively; and then increased to 26.3 and 23.9 in 2006 and 2007, respectively. Populations with RR(50) values of about 50 to 60 during generation one reverted to RR(50) values of < or =4 in six generations, when reared without further exposure to imidacloprid. Average RR(50) values for thiamethoxam increased from 2.0 in 2003 to 24.7 in 2006 and decreased to 10.4 in 2007. Populations with RR(50) values of about 22, 32 and 53 during generation one declined to 8, 5 and 6, respectively, after being reared for five generations without exposure to thiamethoxam. The correlation coefficient from the 26 populations that were bioassayed both with imidacloprid and thiamethoxam showed a significant positive correlation (R(2) = 0.58) between these populations. CONCLUSION The high level of RR(50) values to imidacloprid and thiamethoxam suggest an unstable decline in the susceptibility of B. tabaci to imidacloprid and thiamethoxam, with possible cross-resistance or predisposition for dual resistance selection.

The Scent of a Partner: Ambrosia Beetles Are Attracted to Volatiles from Their Fungal Symbionts

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.

Characterization of five CYP4 genes from Asian citrus psyllid and their expression levels in Candidatus Liberibacter asiaticus-infected and uninfected psyllids

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.

Glutathione Transferase and Cytochrome P450 (General Oxidase) Activity Levels in Candidatus Liberibacter Asiaticus-Infected and Uninfected Asian Citrus Psyllid (Hemiptera: Psyllidae)

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.

Sexual Transmission of a Plant Pathogenic Bacterium, Candidatus Liberibacter asiaticus, between Conspecific Insect Vectors during Mating

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.

Repellency and toxicity of plant-based essential oils and their constituents against Diaphorina citri Kuwayama (Hemiptera: Psyllidae)

The Asian citrus psyllid, Diaphorina citri Kuwayama, vectors Candidatus Liberibacter asiaticus (Las), the presumed causal agent of huanglongbing. D. citri generally rely on olfaction and vision for detection of host cues. Certain plant volatiles and plant‐derived essential oil products are known to repel several arthropod species and are considered minimum‐risk pesticides. We examined the effect of five essential oils and eight chemicals previously reported to have activity against various insect species on D. citri behaviour in a two‐port divided T‐olfactometer in an effort to identify repellents for further consideration and testing as crop protectants for D. citi. Volatiles from essential oils of coriander, lavender, rose, thyme, tea tree oil and 2‐undecanone, a major constituent of rue oil repelled D. citri adults compared with clean air. Also, coriander, lavender, rose and thyme oil inhibited the response of D. citri when co‐presented with citrus leaves. Volatiles from eugenol, eucalyptol, carvacrol, β‐caryophyllene, α‐pinene, α‐gurjunene and linalool did not repel D. citri adults compared with clean air. In an effort to isolate the repellents and toxicants from effective essential oils, the headspace components of coriander and lavender oil were analysed by gas chromatography‐mass spectrometry and revealed that α‐pinene and linalool were the primary volatiles present in coriander oil while linalool and linalyl acetate were the primary volatiles present in lavender oil. Coriander, lavender and garlic chive oils were also highly toxic to D. citri when evaluated as contact action insecticides using a topical application technique. The LC50 values for these three oils ranged between 0.16 and 0.25 μg/D. citri adult while LC50 values for rose and thyme oil ranged between 2.45 and 17.26 μg/insect.