Evaluation of semiochemical based push-pull strategy for population suppression of ambrosia beetle vectors of laurel wilt disease in avocado.

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) bore into tree xylem to complete their life cycle, feeding on symbiotic fungi. Ambrosia beetles are a threat to avocado where they have been found to vector a symbiotic fungus, Raffaelea lauricola, the causal agent of the laurel wilt disease. We assessed the repellency of methyl salicylate and verbenone to two putative laurel wilt vectors in avocado, Xyleborus volvulus (Fabricius) and Xyleborus bispinatus (Eichhoff), under laboratory conditions. Then, we tested the same two chemicals released from SPLAT flowable matrix with and without low-dose ethanol dispensers for manipulation of ambrosia beetle populations occurring in commercial avocado. The potential active space of repellents was assessed by quantifying beetle catch on traps placed 'close' (~5-10 cm) and 'far' (~1-1.5 m) away from repellent dispensers. Ambrosia beetles collected on traps associated with all in-field treatments were identified to species to assess beetle diversity and community variation. Xyleborus volvulus was not repelled by methyl salicylate (MeSA) or verbenone in laboratory assays, while X. bispinatus was repelled by MeSA but not verbenone. Ambrosia beetle trap catches were reduced in the field more when plots were treated with verbenone dispensers (SPLAT) co-deployed with low-dose ethanol dispensers than when treated with verbenone alone. Beetle diversity was highest on traps deployed with low-dose ethanol lures. The repellent treatments and ethanol lures significantly altered the species composition of beetles captured in experiment plots. Our results indicate that verbenone co-deployed with ethanol lures holds potential for manipulating ambrosia beetle vectors via push-pull management in avocado. This tactic could discourage immigration and/or population establishment of ambrosia beetles in commercial avocado and function as an additional tool for management programs of laurel wilt

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 effect of exposure to synthetic pheromone lures on male Zygaena filipendulae mating behaviour: implications for monitoring species of conservation interest

Pheromone based monitoring of insects of conservation value has the potential to revolutionise the way in which surveys are carried out. However, due to their effective use in pest management, concerns have been raised about potential negative effects of pheromone exposure on populations of rare insects. The effect of exposure to synthetic pheromone lures on male mating behaviour was examined in laboratory and field conditions using the six spot burnet moth Zygaena filipendulae (Linnaeus, 1758). For the laboratory experiment larvae were collected and cultured separately under controlled conditions. Virgin males were exposed to a synthetic pheromone lure for 24 h; then tested for responsiveness immediately after this exposure, 1 and 24 h later. Control males were tested three times: initially, 1 h later and 24 h later. The time taken for males to detect females, shown by exposure of their anal claspers, and the time taken for males to locate females were recorded. No significant difference was found between the time taken for control and exposed males to detect or locate females, and no significant difference between the proportions of males that successfully located females in exposed and control groups was found. In the field experiment the time males spent in the presence of contained females, both with and without a pheromone lure present, was recorded. Males spent more time in the presence of the females when the pheromone lure was present. Both experiments indicate male Z. filipendulae mating behaviour is not adversely affected by exposure to synthetic pheromone lures

Synthetic blends of volatile, phytopathogen-induced odorants can be used to manipulate vector behavior

Volatile organic compounds (VOCs) are emitted from all plants and these VOCs are important means of communication between plants and insects. It has been documented that pathogen infections alter VOC profiles rendering infected plants more attractive to specific vectors transmitting these pathogens than uninfected plants, thus potentially aiding in pathogen propagation. Mimicking these chemical cues might enable insect attraction away from the plant or disruption of host finding behavior of the vector. However, the practical implications have not been fully explored. We used citrus, Diaphorina citri and huanglongbing (HLB) as a model host-vector-disease system because HLB threatens citrus production worldwide and is similar to other critical diseases of food crops, such as Zebra Chip affecting potato. We formulated a synthetic chemical blend using selected HLB-specific biomarker compounds, and tested the blend with the Attenu assay system for chemosensory proteins. The Attenu assay system is a procedure that identifies interactions between insect chemosensory proteins and their ligands. We found that an equimolar mixture of compounds mimicking the volatile profile of HLB-infected citrus bound chemosensory proteins. Further investigation of this blend in laboratory behavioral assays resulted in development of a synthetic lure that was more attractive to D. citri than natural citrus tree volatiles. This strategy could provide a new route to produce chemical lures for vector population control for a variety of plant and/or animal systems and it may result in the development of a practical lure for monitoring vectors of disease, such as D. citri

Delayed mating in tortricid leafroller species: Simultaneously aging both sexes prior to mating is more detrimental to female reproductive potential than aging either sex alone

Abstract The effect of delayed mating on reproductive potential, longevity and oviposition period of female redbanded leafroller, Argyrotaenia velutinana (Walker) and Pandemis leafroller, Pandemis pyrusana Kearfott, was investigated in the laboratory. Virgin female or male moths of each species were held for 1, 2, 4, 6 or 10 days prior to pairing with one-day-old virgin conspecifics of the opposite sex. In addition, reproductive potential was assessed when both sexes of each species were aged for those periods prior to pairing. The expected reproduction of female A. velutinana was reduced by 34, 53, 71 and 81% for 2, 4, 6 and 10-day delays in female mating, respectively. For P. pyrusana, expected reproduction was reduced by 47, 74, 85 and 93% for 2, 4, 6 and 10-day delays in female mating, respectively. Increasing male age at mating in both species had a lesser effect on female reproductive output compared with increasing female age at mating. As male A. velutinana age at mating increased, the expected reproduction of female A. velutinana was reduced by 15, 45, 54 and 70% for 2, 4, 6 and 10-day delays, respectively. Comparing male P. pyrusana of various ages at mating, expected reproduction was reduced by 14, 42, 64 and 79% for 2, 4, 6 and 10-day delays in mating, respectively. The decrease in female reproduction when both sexes were aged prior to mating was higher than when either sex alone was aged prior to pairing with a one-day-old virgin of the opposite sex. The expected reproduction of female A. velutinana was reduced by 60, 83, 96 and 98% for 2, 4, 6 and 10-day delays in mating of both sexes, respectively. Only 7.5% of female eggs hatched when both sexes of A. velutinana were aged ten days prior to mating. When simultaneously aging both sexes of P. pyrusana prior to mating, expected reproduction was reduced by 71, 93, 96 and 99% for 2, 4, 6 and 10-day delays in mating, respectively. No P. pyrusana eggs hatched after a ten-day delay of mating for both sexes. For both species, female longevity increased and duration of oviposition period decreased with increasing female age at mating. Our results demonstrate that delayed mating in both females and males negatively affects female reproductive output in both species and that simultaneous aging of both sexes prior to mating has a greater effect than aging either sex alone. Our results suggest that laboratory studies that have paired aged females or aged males with conspecifics of optimal reproductive maturity have likely underestimated the effects of delayed mating on reproductive output

Temporal Decline in Pathogen-Mediated Release of Methyl Salicylate Associated With Decreasing Vector Preference for Infected Over Uninfected Plants

The Asian citrus psyllid, Diaphorina citri Kuwayama, is the vector of the phytopathogenic bacterium, Candidatus Liberibacter asiaticus (CLas), that causes citrus greening. Diaphorina citri is attracted to methyl salicylate (MeSA) emitted by CLas-infected citrus. MeSA is catalyzed from salicylic acid (SA) by salicylic acid methyl transferase (SAMT). In addition, salicylate hydroxylase, an enzyme that degrades SA and suppresses host plant defense, is unregulated in CLas-infected as compared with uninfected citrus. Based on these findings, we hypothesized that CLas-induced plant volatile emissions and SAMT expression may decrease over time following CLas infection. We first identified the putative Citrus sinensis SAMT (CsSAMT) that methylates SA to MeSA. Thereafter we compared changes in SAMT expression and volatile expression between uninfected citrus, citrus recently (< 6 months) infected with CLas, and citrus infected with CLas for a long (>1 year) duration. Emission of MeSA increased in citrus recently infected with CLas, whereas both MeSA emissions and SAMT expression decreased in long-infected citrus as compared with uninfected controls. Also, there was a significant decrease in release of limonene from citrus infected >1 year, and a concomitant increase in release of β-caryophyllene in both recent and long-infected citrus as compared with controls. In behavioral assays, D. citri preferred settling on asymptomatic citrus recently infected with CLas over uninfected or long-infected citrus trees. The odor of citrus recently infected with CLas was more attractive to D. citri than the odor of uninfected or long-infected citrus. There was an association between the preference for citrus odor of recently infected plants and an increase of MeSA release following CLas infection. Overall, our study demonstrated pulsed changes in citrus odor release following CLas infection. We hypothesize that allocation of resources to plant defense in citrus decreases as a function of huanglongbing disease symptom progression. These changes affected the behavior of the CLas vector. Our study confirms previous reports demonstrating that plant defenses boosted following phytopatogen infection tend to decrease over time. Implications on the CLas epidemic are discussed

Use of Glacial Acetic Acid to Enhance Bisexual Monitoring of Tortricid Pests With Kairomone Lures in Pome Fruits

Studies were conducted to assess glacial acetic acid (GAA) with various host plant volatiles (HPVs) and the sex pheromone, (E,E)-8, 10-dodecadien-1-ol, of codling moth, Cydia pomonella (L), as lures in traps for tortricid pests that often co-occur in tree fruits in the western United States. In addition to codling moth, field trapping studies were conducted with oriental fruit moth, Grapholita molesta (Busck), obliquebanded leafroller Choristoneura rosaceana (Harris), the leafroller Pandemis pyrusana Kearfott, and the eyespotted budmoth, Spilonota ocellana (Denis and Schiffermüller). HPVs included ethyl (E,Z)-2,4-decadienoate (pear ester), (E)-4,8-dimethyl-1,3,7-nonatriene, butyl hexanoate, (E)-β-ocimene, (E)-β-farnesene, and farnesol. Three types of GAA co-lures differing in a 10-fold range in weekly evaporation rates were tested. The evaporation rate of GAA co-lures was an important factor affecting moth catches. The highest rate tested captured fewer codling moth but more leafrollers and eyespotted budmoth. GAA co-lures caught both sexes of each species. The field life of butyl hexanoate and (E)-β-ocimene lures were much shorter than pear ester or sex pheromone lures. Adding GAA to pear ester or to (E)-β-ocimene significantly increased the catches of only codling moth or oriental fruit moth, respectively. Combining pear ester or (E)-β-ocimene with GAA did not affect the catch of either species compared with the single more attractive HPV. Adding HPVs to GAA did not increase the catches of either leafroller species or eyespotted budmoth. Traps baited with pear ester, sex pheromone, and GAA for monitoring codling moth were also effective in classifying pest pressure of both leafroller species within orchards.Keywords: Pandemis leafroller, Oriental fruit moth, Eyespotted budmoth, Codling moth, Obliquebanded leafrolle

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

Host Selection, Oviposition and Feeding by a Phytopathogen Vector, Diaphorina citri (Hemiptera: Liviidae), Modulated by Plant Exposure to Formic Acid

Citrus volatiles and their breakdown products, such as formic and acetic acids, guide host finding by Asian citrus psyllids (Diaphorina citri). Formic acid elicits psyllid antennal responses and stimulates probing. This study investigated how exposure to vapor phase formic acid induced changes in plant volatile emissions, expression of plant defense genes, and modified psyllid host finding and feeding behaviors on susceptible and resistant citrus cultivars. Chemical analyses of headspace volatiles and behavioral assays were performed before and after exposure of D. citri susceptible and resistant plants to vapor phase formic acid. RT-qPCR elucidated changes in plant defense-associated gene expression following exposure. Electrical Penetration Graph (EPG) measured D. citri feeding behavior on citrus with and without exposure to formic acid. Volatilized formic acid caused a 200–500% increase in release of citrus volatiles 1–3 h after exposure, increased attraction of D. citri to Poncirus trifoliata (resistant) and increased oviposition compared with controls. RT-qPCR showed significant increases in expression of WRKY22, GST1, RAR1, and EDS1 genes following exposure that also reduced phloem, but not xylem, ingestion by adult D. citri. Formic acid exposure caused increased citrus volatile release that modified host finding and feeding behaviors of an important phytopathogen vector