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.

The seasonal abundance of phlebotomine sand flies, Lutzomyia species in Florida.

The seasonality of phlebotomine sand flies was studied in Florida, utilizing colored light-emitting diode- and attractant-baited Mosquito Magnet MM-X traps from September 2006 to September 2008 at San Felasco Hammock Preserve State Park, Gainesville, FL. A total of 6,278 sand flies were collected from 314 actual nights and 1,692 total trap-nights, yielding 3.7 sand flies per trap-night. Lutzomyia shannoni was the predominant species, constituting 55% to 80% of the total sand fly populations collected during the studies. Both L. shannoni and L. vexator populations were highly seasonal and were moderately influenced by weather factors. Lutzomyia shannoni populations peaked in May and showed reduced activity during December, January, and February. This species was active throughout the year and showed positive and negative correlations with average monthly temperature and relative humidity, respectively. Lutzomyia vexator showed peak activity during August and October with an activity lull from December to March. This species showed a positive correlation with average monthly temperature. No correlations were observed with either species for average daily, weekly, or 1- to 8-wk-lagging precipitation, number of rainy days, wind speed, or lunar phases. Lutzomyia shannoni abundance was weakly correlated to L. vexator abundance. No other Lutzomyia spp. were collected during the study.

Lutzomyia spp. (Diptera: Psychodidae) response to olfactory attractant- and light emitting diode-modified Mosquito Magnet X (MM-X) traps.

Mosquito Magnet-X traps were modified for use with blue, green, red, and blue-green-red light-emitting diodes and olfactory attractants to determine the response of Lutzomyia shannoni (Dyar) and Lutzomyia vexator (Coquillett) (Diptera: Psychodidae) field populations to these attractants. Red and blue-green-red-baited traps captured the highest numbers of Lu. shannoni and Lu. vexator, respectively, although, there were no significant differences between the colors. Baiting the traps with CO, attracted significantly higher numbers of Lu. shannoni but showed no effect on Lu. vexator capture. In comparison with CO, alone, Lu. shannoni preferred 1-octen-3-ol and 1-hexen-3-ol (0.05 g per trap) in combination with CO.

Abdominal color of the Asian citrus psyllid, Diaphorina citri, is associated with susceptibility to various insecticides.

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.

Chemical and behavioral analysis of the cuticular hydrocarbons from Asian citrus psyllid, Diaphorina citri.

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).

Insecticidal potency of novel compounds on multiple insect species of medical and veterinary importance.

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.

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.

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.

Colonization of Lutzomyia shannoni (Diptera: Psychodidae) utilizing an artificial blood feeding technique.

Laboratory colonization of hematophagous insects must include an efficient method of blood feeding, preferably by artificial means. Strict rules for obtaining animal use permits, extensive animal maintenance costs, and indirect anesthesia effects on animal health warrant the development of an artificial membrane feeding technique for sand fly colonization in laboratories. An attempt was made to colonize Lutzomyia shannoni using an artificial blood feeding membrane to replace the use of live animals commonly used for sand fly blood-feeding purposes. Lutzomyia shannoni readily fed through a pig intestine membrane exposed at an angle of 45°. However, it did not feed through a chicken skin membrane. Olfactory attractants were unable to improve blood-feeding efficiency. Plaster of Paris was the most suitable oviposition substrate. Female L. shannoni adults laid no eggs on moist sand substrate. Sand fly adults held in groups of ten or more laid higher numbers of eggs than did individually maintained sand flies. Inclusion of the L. longipalpis oviposition hormone dodecanoic acid or the presence of previously laid eggs did not stimulate L. shannoni oviposition. The average L. shannoni egg, larval, and pupal duration were 9.3, 36.7, and 17.8 days, respectively. The addition of a 20% sugar solution improved adult female longevity. Females survived longer (14.8 days) than males (11.9 days). Lutzomyia shannoni was successfully colonized in the laboratory for up to four generations using this artificial membrane technique.

Evaluation of semiochemical toxicity to Aedes aegypti, Ae. albopictus and Anopheles quadrimaculatus (Diptera: Culicidae).

BACKGROUND: Mosquitoes are the most important vectors of human pathogens. Wide-scale use of pesticides has led to the development of resistance to most common insecticide groups. The need to develop novel products that have a low impact on human health and the environment is well established. The toxicity of selected semiochemicals with molecular structures indicative of insecticidal activity was determined against adult Aedes aegypti (L.) and Anopheles quadrimaculatus (Say). The two most active insecticides against Ae. aegypti were also evaluated against Ae. albopictus (Skuse). RESULTS: Fifteen semiochemicals classified as terpenoid alcohols, ketones or carboxylic esters showed toxicity to both mosquito species. Geranyl acetone (LC(50) = 38.51 microg cm(-2)) followed by citronellol (LC(50) = 48.55 microg cm(-2)) were the most toxic compounds to Ae. aegypti, while geraniol and lavonax, with LC(50) values of 31.88 and 43.40 microg cm(-2), showed the highest toxicity to An. quadrimaculatus. Both geranyl acetone and citronellol were highly toxic to Ae. albopioctus. No semiochemical showed fumigation activity against either species. All semiochemicals persisted for less than 24 h when tested on filter paper. CONCLUSION: Quantification of LC(50) values of several semiochemicals against Ae. Aegypti, An. quadrimaculatus and Ae. albopioctus showed that semiochemicals not only modify insect behaviors but also hold potential as potent insecticides for mosquito control programs.

Evaluation of semiochemical toxicity to houseflies and stable flies (Diptera: Muscidae).

BACKGROUND: The housefly, Musca domestica L., and stable fly, Stomoxys calcitrans (L.) are cosmopolitan pests of both farm and home environments. Houseflies have been shown to be resistant to a variety of insecticides, and new chemistries are slow to emerge on the market. Toxicities of selected semiochemicals with molecular structures indicative of insecticidal activity were determined against adults from an insecticide-susceptible laboratory strain of houseflies. The three most active semiochemicals were also evaluated against recently colonized housefly and stable fly strains. RESULTS: Nineteen semiochemicals classified as aliphatic alcohols, terpenoids, ketones and carboxylic esters showed toxicity to houseflies and stable flies. Rosalva (LC(50) = 25.98 microg cm(-2)) followed by geranyl acetone and citronellol (LC(50) = 49.97 and 50.02 microg cm(-2)) were identified as the most toxic compounds to houseflies. Permethrin was up to 144-fold more toxic than rosalva on the susceptible strain. However, it was only 35-fold more toxic to the insecticide-tolerant field strain. The compounds generated high toxicity to stable flies, with LC(50) values ranging from 16.30 to 40.41 microg cm(-2). CONCLUSION: Quantification of LC(50) values of rosalva, citronellol and geranyl acetone against susceptible housefly and field-collected housefly and stable fly strains showed that semiochemicals could serve as potent insecticides for fly control programs.

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.

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.