An optical system to detect, surveil, and kill flying insect vectors of human and crop pathogens.

Sustainable and effective means to control flying insect vectors are critically needed, especially with widespread insecticide resistance and global climate change. Understanding and controlling vectors requires accurate information about their movement and activity, which is often lacking. The Photonic Fence (PF) is an optical system that uses machine vision, infrared light, and lasers to identify, track, and interdict vectors in flight. The PF examines an insect's outline, flight speed, and other flight parameters and if these match those of a targeted vector species, then a low-power, retina-safe laser kills it. We report on proof-of-concept tests of a large, field-sized PF (30 mL × 3 mH) conducted with Aedes aegypti, a mosquito that transmits dangerous arboviruses, and Diaphorina citri, a psyllid which transmits the fatal huanglongbing disease of citrus. In tests with the laser engaged, < 1% and 3% of A. aegypti and D. citri, respectfully, were recovered versus a 38% and 19% recovery when the lacer was silenced. The PF tracked, but did not intercept the orchid bee, Euglossa dilemma. The system effectively intercepted flying vectors, but not bees, at a distance of 30 m, heralding the use of photonic energy, rather than chemicals, to control flying vectors.

Source or Sink? The Role of Residential Host Plants in Asian Citrus Psyllid Infestation of Commercial Citrus Groves.

Citriculture landscapes in the U.S. are typically habitat mosaics of commercial groves interspersed with residential areas supporting a variety of unmanaged citrus. Diaphorina citri the vector of Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing feeds on citrus in both habitats. We postulated that residential citrus function as a 'source' of D. citri that infest groves, functioning as sinks. Here we report on an experimental mark-release-recapture study conducted at the interface of a residential neighborhood and groves. Adult D. citri marked with colored fluorescent powders were released in both habitats (n = 15,300) and their movement within and between milieus monitored. Although the recapture rate of marked psyllids was very low (0.23%), the results were instructive. Most of the recaptured psyllids in residential trees (84.6%) were released within that habitat. In contrast, approximately half of the marked psyllids recovered in groves were released in residential areas. Of all the recaptured psyllids, about 40% changed habitats, but the change was skewed toward movement from residential to grove habitat. These data strongly suggest that there is a constant exchange of D. citri adults between the two habitats, with residential citrus trees functioning as a source habitat of psyllids. The further the residential trees are located from groves, the less likely they will serve as sources of D. citri. Hence, to reduce the risks of citrus grove colonization by D. citri, new groves should be established away from residential habitats where possible, and psyllid management practices must also be implemented in residential habitats.

Tetranorsesquiterpenoids as Attractants of Yucca Moths to Yucca Flowers.

The obligate pollination mutualism between Yucca and yucca moths is a classical example of coevolution. Oviposition and active pollination by female yucca moths occur at night when Yucca flowers are open and strongly scented. Thus, floral volatiles have been suggested as key sensory signals attracting yucca moths to their host plants, but no bioactive compounds have yet been identified. In this study, we showed that both sexes of the pollinator moth Tegeticula yuccasella are attracted to the floral scent of the host Yucca filamentosa. Chemical analysis of the floral headspace from six Yucca species in sections Chaenocarpa and Sarcocarpa revealed a set of novel tetranorsesquiterpenoids putatively derived from (E)-4,8-dimethyl-1,3,7-nonatriene. Their structure elucidation was accomplished by NMR analysis of the crude floral scent sample of Yucca treculeana along with GC/MS analysis and confirmed by total synthesis. Since all these volatiles are included in the floral scent of Y. filamentosa, which has been an important model species for understanding the pollination mutualism, we name these compounds filamentolide, filamentol, filamental, and filamentone. Several of these compounds elicited antennal responses in pollinating (Tegeticula) and non-pollinating (Prodoxus) moth species upon stimulation in electrophysiological recordings. In addition, synthetic (Z)-filamentolide attracted significant numbers of both sexes of two associated Prodoxus species in a field trapping experiment. Highly specialized insect-plant interactions, such as obligate pollination mutualisms, are predicted to be maintained through "private channels" dictated by specific compounds. The identification of novel bioactive tetranorsesquiterpenoids is a first step in testing such a hypothesis in the Yucca-yucca moth interaction.

Behavioral Responses of Asian Citrus Psyllid (Hemiptera: Liviidae) to Salinity-Stressed Citrus.

Most commercial citrus varieties are intolerant of salinity stress, but some rootstocks, such as Rangpur lime, tolerate moderately saline irrigation water. Development of salinity-tolerant citrus may allow for citriculture in semiarid and arid regions where salinity stress is problematic. Because salinity stress influences shoot growth in citrus, we compared the behavioral responses of Asian citrus psyllid, Diaphorina citri Kuwayama, to salinity-stressed versus nonstressed Rangpur lime seedlings. The effects of salinity stress on key physiological processes in the seedlings were also examined. Seedlings in the control group were fertilized with a solution having a salinity of 1.7 dS m-1 while seedlings in the salinity-stressed group were fertilized with a solution having a salinity of 10 dS m-1. The seedlings were exposed to salinity stress for increasing durations (15, 20, or 60 d). Seedlings presented differential physiological responses 15 d after the imposition of salinity stress, and differences in psyllid settling rate on control versus salinity-stressed seedlings were discernable within 1 h following the imposition of salinity stress. The levels of settling, oviposition, and egg survivorship were significantly lower on salinity-stressed versus control seedlings. Olfactometer tests showed that female psyllids preferred the odor from control seedlings, suggesting that the odors of control and salinity-stressed seedlings were different. The results showed that D. citri avoids salinity-stressed seedlings; this suggests the possibility of using moderate salinity stress as a management strategy to minimize psyllid settlement and reproduction and to reduce the spread of huanglongbing, especially in citrus grown in semiarid and arid areas.

A Multimodal Attract-and-Kill Device for the Asian Citrus Psyllid Diaphorina citri (Hemiptera: Liviidae).

Phytophagous insects, including Asian citrus psyllids (Diaphorina citri Kuwayama), use multiple sensory modalities (vision, olfaction, and gustation,) to locate and accept host plants. We explored incorporation of several sensory cues into a multi-modal attract-and-kill device (AK device) using a three-dimensional shape to increase visibility, as well as elements of color, attractant, phagostimulant, UV reflectant, and toxicant. Attraction of adult D. citri to the device was mediated by a combination of a highly reflective yellow cylinder, a UV reflectant compound (magnesium oxide), and an odorant blend as a short-range attractant. The device surface was coated with a slow-release wax matrix (SPLAT™) augmented with a phagostimulant consisting of a 3-component blend (formic acid, acetic acid, and para-cymene) and an insecticide (ß-cyfluthrin). Psyllids landing on the device attempted to feed from the wax matrix, became intoxicated, died, and fell from the device. The device remained fully active over a period of 12 weeks partly because dead psyllids or nontargets did not adhere to the surface as occurs on adhesive yellow sticky cards, the industry standard. Laboratory and field assays showed that the device attracted and killed significantly more adult D. citri than ordinary yellow sticky cards. This device or a future iteration based on the design elements of this device is expected to contribute to sustainable and environmentally appropriate management of D. citri by exploiting the psyllid's innate behavioral responses to visual, olfactory, and gustatory stimuli.

Feeding Behavior of Asian Citrus Psyllid [Diaphorina citri (Hemiptera: Liviidae)] Nymphs and Adults on Common Weeds Occurring in Cultivated Citrus Described Using Electrical Penetration Graph Recordings.

Asian citrus psyllid, Diaphorina citri, transmits Candidatus Liberibacter asiaticus (CLas), the putative causal agent of Huanglongbing disease. Although they primarily feed on the phloem of Citrus and related plants, when grove or host conditions are unfavorable, D. citri may be able to use weed species as alternate food sources for survival. To explore this possibility, electrical penetration graph (EPG) recordings (18 h) were performed to investigate the feeding behavior of psyllid adults and nymphs on three common south Florida weeds (Bidens alba, Eupatorium capillifolium, and Ludwigia octovalvis). EPG recordings revealed that the proportion of time spent by D. citri feeding on xylem was similar on all tested weed species (19%-22%) and on the positive control (20%), the preferred host, Citrus macrophylla. Very little to no phloem feeding was observed on weed species by either nymphs or adults. Histological studies using epifluorescence microscopy showed that salivary sheaths were branched and extended into xylem of weed species, whereas they ended in phloem on citrus plants. No choice behavioral assays showed that adults can obtain some nutrition by feeding on weed species (xylem feeding) and they may be able to survive on them for short intervals, when host conditions are unfavorable.

Response surface methodology reveals proportionality effects of plant species in conservation plantings on occurrence of generalist predatory arthropods.

Multivariate geometric designs for mixture experiments and response surface methodology (RSM) were tested as a means of optimizing plant mixtures to support generalist predatory arthropods. The mixture design included 14 treatment groups, each comprised of six planters and having a proportion of 0.00, 0.17, 0.33, 0.66, or 1.00 of each plant species. The response variable was the frequency of predators trapped on sticky card traps placed in each group and replaced 2 times per week. The following plant species were used: Spring 2017: Euphorbia milii, E. heterophylla, and Phaseolus lunatus; Summer 2017: E. milii, Fagopyrum esculentum, and Chamaecrista fasciculata; and, Summer 2018: E. milii, F. esculentum, and Portulaca umbraticola. Predator occurrence was influenced by: 1) Linear mixture effects, which indicated that predator occurrence was driven by the amount of a single plant species in the mixture; or, 2) Nonlinear blending effects, which indicated that the plant mixture itself had emergent properties that contributed to predator occurrence. Predator abundance was highest in the Spring 2017 experiment and both linear mixture effects and nonlinear blending effects were observed. Predator occurrence decreased in subsequent experiments, which were conducted in the warmer summer months. In both Summer experiments, only linear mixture effects were observed, indicating that predator occurrence was driven by the amount of a single plant species in the test mixtures: Euphorbia milii in 2017 and Portulaca umbraticola in 2018. The results showed that not only did the species composition of a plant mixture drive predator occurrence but that proportionality of species contributed to the outcome as well. This suggests that, when formulating a plant mixture to aid in conservation biological control consideration should be given to the proportion of each plant species included in the mixture. RSM can be an important tool for achieving the goal of optimizing mixtures of plants for conservation biological control.

Development and Field Validation of a Beta-cyfluthrin-Based 'Attract-and-Kill' Device for Suppression of Asian Citrus Psyllid (Hemiptera: Liviidae) on Residential Citrus.

An 'attract-and-kill' (AK) device was evaluated for suppression of adult Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), on residential citrus. The AK device, made from weather-resistant plasticized PVC, lured D. citri adults by simulating the color of citrus flush and killed them with beta-cyfluthrin. This study evaluated: 1) lethality of AK devices weathered up to 8 wk on residential citrus; 2) survival of psyllids caged with potted plants and AK devices; 3) psyllid suppression achieved by AK devices on individual dooryard trees. AK devices weathered for up to 8 wk remained lethal to psyllids. Greenhouse trials evaluated survival of adult psyllids caged for 4 d with orange jasmine plants that were: 1) treated with an (beta-cyfluthrin-infused) AK device; 2) treated with a blank (no insecticide) AK device; or 3) 'untreated' with no AK device. After 4 d, psyllid survival was on average 95% lower among adults exposed to plants with AK devices than adults exposed to untreated plants or plants with blank AK devices. Less than half of the adults exposed to plants with AK devices were alive after 1 d and nearly all were dead after 4 d. Deployment of 20 AK devices per tree provided significant psyllid suppression on infested lemon trees from winter to summer and reduced mean reproduction (cumulative eggs) by 91% and mean attack intensity (cumulative psyllid-days) of adults by 59% and nymphs by 53%. AK devices could be an effective control option for D. citri in urban areas.

Field Efficacy of Autodissemination and Foliar Sprays of an Entomopathogenic Fungus, Isaria fumosorosea (Hypocreales: Cordycipitaceae), for Control of Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae), on Residential Citrus.

Autodissemination and foliar sprays of PFR-97 (Certis Inc., Columbia, MD) microbial insecticide, a blastospore formulation of Isaria fumosorosea Wize (Hypocreales: Cordycipitaceae), were evaluated for control of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), on residential citrus. Seasonal trials on dooryard trees in South Texas evaluated: 1) pathogenicity of I. fumosorosea (Ifr) spores on autodisseminators (dispensers) deployed up to 3 wk on grapefruit trees; 2) psyllid control on several citrus species by dispensers and sprays; 3) infection range of the dispenser. Decline in spore pathogenicity over time was similar among dispensers during fall, winter, or spring and decreased by 30% after 1 d, 59% after 7 d, 81% after 14 d, and 100% after 21 d. Dispensers or sprays were equally effective for psyllid control on heavily infested lime trees from fall to spring and reduced mean reproduction (cumulative eggs) by 90% and mean attack intensity (cumulative psyllid-days) of adults by 76% and nymphs by 82%. Dispensers or sprays were also equally effective for psyllid control on lightly infested lime trees from spring to mid-summer and on orange or grapefruit trees from fall to winter. Very light infestations on grapefruit trees from spring to mid-summer were not significantly reduced by dispensers or sprays. Psyllid control was not improved by combining dispensers and sprays. Adult psyllids infected by Ifr were recovered in trees located 3-4 m away from trees with dispensers but not at greater distances. PFR-97 dispensers could be a treatment option for D. citri in settings where chemical control is problematic.

Exogenous application of the plant signalers methyl jasmonate and salicylic acid induces changes in volatile emissions from citrus foliage and influences the aggregation behavior of Asian citrus psyllid (Diaphorina citri), vector of Huanglongbing.

Huanglongbing, also known as citrus greening, is a destructive disease that threatens citrus production worldwide. It is putatively caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (Las). Currently, the disease is untreatable and efforts focus on intensive insecticide use to control the vector, Asian citrus psyllid (Diaphorina citri). Emerging psyllid resistance to multiple insecticides has generated investigations into the use of exogenously applied signaling compounds to enhance citrus resistance to D. citri and Las. In the present study, we examined whether foliar applications of methyl jasmonate (MJ), a volatile signaling compound associated with the induced systemic resistance pathway, and salicylic acid, a constituent of the systemic acquired resistance pathway, would elicit the emission of defense-related volatiles in citrus foliage, and what effect this might have on the host-plant searching behavior of D. citri. Comparisons were made of volatiles emitted from growing shoots of uninfected and Las-infected 'Valencia' sweet orange (Citrus sinensis) trees over two consecutive sampling days. A settling behavioral assay was used to compare psyllid attraction to MJ-treated vs. Tween-treated citrus sprigs. All three main effects, Las infection status, plant signaler application, and sampling day, influenced the proportions of individual volatile compounds emitted in different treatment groups. MJ- and SA-treated trees had higher emission rates than Tween-treated trees. Methyl salicylate (MeSA) and ß-caryophyllene were present in higher proportions in the volatiles collected from Las-infected + trees. On the other hand, Las-infected + MJ-treated trees emitted lower proportions of MeSA than did Las-infected + Tween-treated trees. Because MeSA is a key D. citri attractant, this result suggests that MJ application could suppress MeSA emission from Las-infected trees, an approach that could be used to discourage psyllid colonization during shoot growth. MJ application enhanced emission of E-ß-ocimene, indole, volatiles attractive to many of the psyllid's natural enemies, indicating that MJ application could be used in an 'attract and reward' conservation biological control strategy. Volatile emissions in SA-treated trees were dominated by MeSA. MJ application elicited aggregation behavior in D. citri. Similar numbers of psyllids settled on MJ-treated versus Tween-treated sprigs, but a significantly greater percentage of the MJ-treated sprigs had aggregations of nine or more psyllids on them. Taken together, the results of this study indicate that exogenous applications of MJ or SA could be used to influence Asian citrus psyllid settling behavior and attract its natural enemies.

Synthetic Ligands of Olfactory Binding Proteins Modulate Aggregation Response of Asian Citrus Psyllid in the Presence of Host-Plant Volatiles.

There is interest in using ligands of chemosensory binding proteins (CBP) to augment an insect's responsiveness to chemosensory cues. We showed previously that combining a synthetic ligand of a CBP with limonene, a common citrus volatile, enhanced the probing response of Asian citrus psyllid (Diaphorina citri). Here, we determined whether synthetic compounds, which were ligands of D. citri olfactory binding protein (OBP) DCSAP4, influenced the settling and aggregation levels of psyllids on young citrus shoots. The test ligands and Cmac scent were dispensed from a droplet of an emulsified wax product (SPLAT) placed on the bottom of each vial. The shoots were presented: (1) alone (shoot + blank SPLAT), (2) with a mixture of citrus volatiles ("Cmac scent") (shoot + SPLAT with Cmac scent), or (3) with different concentrations of test ligands (shoot + SPLAT with test ligand at concentration 1, shoot + SPLAT with test ligand at concentration 2, etc.). Depending on the availability of test ligands, sprigs, and psyllids, each test included from two to four replicates of each treatment (i.e., shoot only, shoot + Cmac scent, shoot + test ligand at concentration 1, shoot + test ligand at concentration 2, etc.); only a single test ligand was presented in each test. For each test, 200 D. citri were released in the test area and the numbers of psyllids on each sprig were counted 24 h later. Sprigs with ≥7 psyllids were considered to be an aggregation. A total of seven ligands were tested individually. Four of the ligands (654, 717, 784, and 861) modulated psyllid settling and aggregation response, causing greater settling and aggregation to sprigs presented with the Cmac scent than to those sprigs with blank SPLAT. Presentation of one of the ligands (019) resulted in an opposite effect in which psyllid settling and aggregation levels were lower on sprigs with Cmac scent than on those with blank SPLAT. There were no differences in settling levels in the different treatment vials in the Ligand 905 experiment. In the Ligand 937 experiment, settling levels did not vary significantly between treatment vials although settling levels were relatively high in all treatment vials and there was a significant treatment effect. Increased settling and aggregation levels were largely not observed with in the vials with only the test ligands, and there was little effect of ligand concentration on psyllid response levels. This suggests that the test ligands themselves did not attract the psyllids but rather modulated the psyllid's response to the Cmac scent. The results suggest that synthetic ligands of D. citri CBPs can be used to increase the effectiveness of citrus scent lures used to attract psyllids to monitoring traps and attract and kill devices.

Laser system for identification, tracking, and control of flying insects.

Flying insects are common vectors for transmission of pathogens and inflict significant harm to humans and agricultural production in many parts of the world. We present proof of principle for an optical system capable of highly specific vector control. This system utilizes a combination of optical sources, detectors, and sophisticated software to search, detect, and identify flying insects in real-time, with the capability of eradication using a lethal laser pulse. We present data on two insect species to show species distinction; Diaphorina citri, a vector of the causal agent of citrus greening disease, and Anopheles stephensi, a malaria vector.

Oviposition Behavior and Survival of Tamarixia radiata (Hymenoptera: Eulophidae), an Ectoparasitoid of the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae), on Hosts Exposed to an Entomopathogenic Fungus, Isaria fumosorosea (Hypocreales: Cordycipitaceae), Under Laboratory Conditions.

Antagonistic interactions between the nymphal parasitoid, Tamarixia radiata Waterston (Hymenoptera: Eulophidae), and the ARSEF 3581 strain of the entomopathogenic fungus, Isaria fumosorosea Wize (Hypocreales: Cordycipitaceae), could disrupt biological control of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). Three interactions were evaluated under laboratory conditions at 25 °C: 1) parasitoid survival if parasitized hosts were exposed to ARSEF 3581 blastospores before or after host mummification; 2) parasitoid survival if mummies containing larva or pupa were exposed to ARSEF 3581 hyphae; 3) parasitoid oviposition on infected hosts with visible or without visible hyphae. Topical application of blastospore formulation onto the dorsal surfaces of live nymphs parasitized with second-instar wasp larva (3 d after parasitism) reduced host mummification by 50% and parasitoid emergence by 85%. However, parasitoid emergence was not affected by topical application of blastospore formulation onto mummies that contained fourth-instar wasp larva (6 d after parasitism). Parasitoid emergence was reduced by 80% if mummies containing fourth-instar wasp larva were covered with blastospore formulation colonized by fungal hyphae. In comparison, parasitoid emergence was not affected if mummies containing wasp pupa (9 d after parasitism) were covered with formulation colonized by fungal hyphae. Female parasitoids oviposited on infected hosts without visible hyphae but not on infected hosts with visible hyphae. Our findings suggest that I. fumosorosea could detrimentally affect T. radiata, if both natural enemies are simultaneously deployed for biological control of D. citri However, temporal separation of the fungus and parasitoid could reduce antagonism and enhance control of D. citri.

The Influence of Learning on Host Plant Preference in a Significant Phytopathogen Vector, Diaphorina citri.

Although specialist herbivorous insects are guided by innate responses to host plant cues, host plant preference may be influenced by experience and is not dictated by instinct alone. The effect of learning on host plant preference was examined in the Asian citrus psyllid, Diaphorina citri; vector of the causal agent of citrus greening disease or huanglongbing. We investigated: a) whether development on specific host plant species influenced host plant preference in mature D. citri; and b) the extent of associative learning in D. citri in the form of simple and compound conditioning. Learning was measured by cue selection in a 2-choice behavioral assay and compared to naïve controls. Our results showed that learned responses in D. citri are complex and diverse. The developmental host plant species influenced adult host plant preference, with female psyllids preferring the species on which they were reared. However, such preferences were subject to change with the introduction of an alternative host plant within 24-48 hrs, indicating a large degree of experience-dependent response plasticity. Additionally, learning occurred for multiple sensory modalities where novel olfactory and visual environmental cues were associated with the host plant. However, males and females displayed differing discriminatory abilities. In compound conditioning tasks, males exhibited recognition of a compound stimulus alone while females were capable of learning the individual components. These findings suggest D. citri are dynamic animals that demonstrate host plant preference based on developmental and adult experience and can learn to recognize olfactory and visual host plant stimuli in ways that may be sex specific. These experience-based associations are likely used by adults to locate and select suitable host plants for feeding and reproduction and may suggest the need for more tailored lures and traps, which reflect region-specific cultivars or predominate Rutaceae in the area being monitored.

Variability in Small Hive Beetle (Coleoptera: Nitidulidae) Reproduction in Laboratory and Field Experiments.

Experiments were conducted to examine how several key factors affect population growth of the small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae). Laboratory experiments were conducted to examine effects of food quantity and temperature on reproduction of cohorts of young A. tumida adults (1:1 sex ratio) housed in experimental arenas. Daily numbers and total mass of larvae exiting arenas were highly variable within treatment. Either one or two cohorts of larvae were observed exiting the arenas. Food quantity, either 10 g or 20 g, did not significantly affect the number of larvae exiting arenas at 32°C, but did at 28°C; arenas provided 20 g food produced significantly more larvae than arenas provided 10 g. Temperature did not affect the total mass of larvae provided 10 g food, but did affect larval mass provided 20 g; beetles kept at 28°C produced more larval mass than at 32°C. Field experiments were conducted to examine A. tumida reproductive success in full strength bee colonies. Beetles were introduced into hives as egg-infested frames and as adults, and some bee colonies were artificially weakened through removal of sealed brood. Efforts were unsuccessful; no larvae were observed exiting from, or during the inspection of, any hives. Possible reasons for these results are discussed. The variability observed in A. tumida reproduction even in controlled laboratory conditions and the difficulty in causing beetle infestations in field experiments involving full colonies suggest that accurately forecasting the A. tumida severity in such colonies will be difficult.

Innate and Conditioned Responses to Chemosensory and Visual Cues in Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae), Vector of Huanglongbing Pathogens.

Asian citrus psyllid (Diaphorina citri) transmits Huanglongbing, a devastating disease that threatens citrus trees worldwide. A better understanding of the psyllid's host-plant selection process may lead to the development of more efficient means of monitoring it and predicting its movements. Since behavioral adaptations, such as associative learning, may facilitate recognition of suitable host-plants, we examined whether adult D. citri could be conditioned to visual and chemosensory stimuli from host and non-host-plant sources. Response was measured as the frequency of salivary sheaths, the residue of psyllid probing activity, in a line of emulsified wax on the surface of a test arena. The psyllids displayed both appetitive and aversive conditioning to two different chemosensory stimuli. They could also be conditioned to recognize a blue-colored probing substrate and their response to neutral visual cues was enhanced by chemosensory stimuli. Conditioned psyllids were sensitive to the proportion of chemosensory components present in binary mixtures. Naïve psyllids displayed strong to moderate innate biases to several of the test compounds. While innate responses are probably the psyllid's primary behavioral mechanism for selecting host-plants, conditioning may enhance its ability to select host-plants during seasonal transitions and dispersal.

Changes in free amino acids and polyamine levels in Satsuma leaves in response to Asian citrus psyllid infestation and water stress.

The effects of biotic and abiotic stresses on changes in amino acids and polyamine levels in Satsuma orange (Citrus unshiu; cultivar Owari) leaves were investigated. Asian citrus psyllids Diaphorina citri (Kuwayama) (ACP) infestation was used to induce biotic stress while a water deficit was imposed to induce abiotic stress. Potted trees were infested by placing 50 psyllids on 3 citrus leaves enclosed in nylon mesh bags for 5 d. A parallel set of plants were kept water stressed by maintaining the soil at 20% water holding capacity for 5 d. Levels of total free amino acids were higher in water stressed and ACP infested leaves. Polyamine putrescine increased in infested leaves but not in water stressed leaves. Proline was the most abundant amino acid and its levels significantly increased by both biotic and abiotic stresses. Proline levels in infested leaves were significantly higher than the water stressed leaves. Histidine, methionine, asparagine, arginine, serine, and leucine levels also increased significantly in infested leaves, but in water stressed leaves only leucine, methionine, and threonine increased. Levels of amino acids, such as tyrosine, isoleucine, phenylalanine, glutamic acid, and alanine, declined in infested leaves. Under water stress asparagine, phenylalanine, serine, and histidine also declined compared to controls. This indicates that while proteolysis occurred under both stresses, metabolic conversion of amino acids was different under the two stresses. In ACP infested leaves some amino acids may be used as feeding material and/or converted into secondary metabolites for defense.

Intraspecific competition effects on Aethina tumida (Coleoptera: Nitidulidae).

Two kinds of experiments were conducted with Aethina tumida Murray larvae over four temperatures: "consumption" experiments, in which larvae and diet were weighed to determine food consumption rates under conditions of unlimited food and few conspecifics; and "competition" experiments, in which varying numbers of larvae were presented with the same amount of honey and pollen diet, and larval weight at final instar was used to determine competition effects. In consumption experiments temperature, diet and their interaction all had significant effects on the ratio of larval weight to the weight of food consumed, which was higher at 24 degrees C than at any other temperature. In competition experiments, three relationships were examined and modeled: that between the number of larvae per experimental unit and the average weight of those larvae; that between average larval and adult weights; and that between average adult weight and survivorship to adult (emergence rate). An exponential decay function was fit to the relationship between number of larvae per experimental unit and their average weight. Average adult weight was linearly correlated with larval weight. Likewise, emergence rates for adults < 11.6 mg in weight were linearly correlated with adult weights, but no significant relationship was observed for heavier adults. Using these relationships, the reproductive potential for A. tumida were estimated for a frame of honey and pollen. Information on resource acquisition by A. tumida will be useful in evaluating the impact of different factors on beetle population dynamics, such as bee hygienic behavior or control strategies used by the beekeeper.

The effects of temperature, diet, and other factors on development, survivorship, and oviposition of Aethina tumida (Coleoptera: Nitidulidae).

Developmental rate and survivorship of small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae), life stages were measured across different temperatures (21, 25, 28, 32 and 35 degrees C) and diets, which included natural and artificial pollen, honey, and bee pupae. Temperature affected hatch success, time to hatching, and larval growth. Eggs hatched in 61 h at 21 degrees C but in < 22 h at 35 degrees C. Larvae achieved peak weight in < 8 d at 35 degrees C but needed 17 d at 21 degrees C. Diet had comparatively little effect on larval survivorship or maximum weight, although larvae fed only bee pupae had lower survivorship. Access to soil influenced pupation success. Duration of the life stage spent in the soil, during which pupation occurs, was also affected by temperature: adults emerged after 32.7 d at 21 degrees C but after only 14.8 d at 35 degrees C, albeit with high mortality. Minimum temperature for development was estimated at 13.5 degrees C for eggs, and 10.0 degrees C for larvae and pupae. Temperature influenced adult longevity and oviposition: on a honey and pollen diet average adult lifespan was 92.8 d at 24 degrees C but only 11.6 d at 35 degrees C. Beetles lived longer at 28 degrees C or lower but produced the most eggs per female, regardless of diet, at 32 degrees C. Beetle density influenced fecundity: beetles kept at three pairs per vial laid 6.7 times more eggs per female than those kept as single pairs. Overall, beetles fared best at 28-32 degrees C with mortality of all stages highest at 35 degrees C.

In situ cell-by-cell imaging and analysis of small cell populations by mass spectrometry.

Molecular imaging by mass spectrometry (MS) is emerging as a tool to determine the distribution of proteins, lipids, and metabolites in tissues. The existing imaging methods, however, mostly rely on predefined rectangular grids for sampling that ignore the natural cellular organization of the tissue. Here we demonstrate that laser ablation electrospray ionization (LAESI) MS can be utilized for in situ cell-by-cell imaging of plant tissues. The cell-by-cell molecular image of the metabolite cyanidin, the ion responsible for purple pigmentation in onion (Allium cepa) epidermal cells, correlated well with the color of cells in the tissue. Chemical imaging using single-cells as voxels reflects the spatial distribution of biochemical differences within a tissue without the distortion stemming from sampling multiple cells within the laser focal spot. Microsampling by laser ablation also has the benefit of enabling the analysis of very small cell populations for biochemical heterogeneity. For example, with a ∼30 µm ablation spot we were able to analyze 3-4 achlorophyllous cells within an oil gland on a sour orange (Citrus aurantium) leaf. To explore cell-to-cell variations within and between tissues, multivariate statistical analysis on LAESI-MS data from epidermal cells of an A. cepa bulb and a C. aurantium leaf and from human buccal epithelial cell populations was performed using the method of orthogonal projections to latent structures discriminant analysis (OPLS-DA). The OPLS-DA analysis of mass spectra, containing over 300 peaks each, provided guidance in identifying a small number of metabolites most responsible for the variance between the cell populations. These metabolites can be viewed as promising candidates for biomarkers that, however, require further verification.