Patrizia Sacchetti

Relationships between the olive fly and bacteria

The relationship between the olive fly population and epiphytic bacteria of the olive tree was investigated by carrying out a 1‐year survey in the field. The olive fly population affected the number of bacteria present on the olive surface. Scanning electron microscope observations demonstrated that bacteria may be ingested by the fly’s mouth apparatus through the midline of the pseudotracheae. DNA amplification of the oesophageal bulb content using 16S bacteria universal primers and DNA sequencing evidenced that Candidatus Erwinia dacicola was the predominant species present. The role of bacteria in olive fly biology is discussed.

Characterization of olfactory sensilla of the olive fly: Behavioral and electrophysiological responses to volatile organic compounds from the host plant and bacterial filtrate

The responses of olive fly (Bactrocera oleae) antennal and palpal olfactory receptors to odors emitted by Pseudomonas putida bacterial filtrate and to volatiles from a host plant were evaluated using electrophysiological and behavioral bioassays. Morphological identification of olfactory receptors was also performed. The third antennal segment (flagellum) bears four types of multiporous sensilla: trichoid, short basiconica, clavate and grooved. Maxillary palps have mechanosensory bristles and multiporous basiconica sensilla. In wind-tunnel bioassays, olive fly responses to volatiles emitted by bacterial filtrate were higher than those to culture medium. Bacterial filtrate was more attractive than ammonium carbonate or a mixture of ethyl acetate and acetic acid in ethanol. GC-MS of bacterial filtrate identified some of the chemicals produced by bacterial activity, including methyl thiolacetate, ammonia, 2-pentanone, 2-heptanone, ethyl tiglate and methyl thiocyanate. Electrophysiological investigations proved that antennal sensilla are responsive to bacterial filtrate odor, methyl thiolacetate, olive leaves and olives, as well as to α-pinene, while acetic acid elicited an inhibitory response. Electropalpgrams recorded a specific response to bacterial filtrate by mated males and females, as well as a dose-dependent response relationship to methyl thiolacetate by mated females. The identification of new active volatile compounds in the semiochemical system of the olive fly is promising for the development of innovative control strategies in area-wide management.

Identification by suppression subtractive hybridization of genes expressed in pear (Pyrus spp.) upon infestation with Cacopsylla pyri (Homoptera: Psyllidae).

The molecular interaction between pear tree (Pyrus spp.) and the phloem-feeding psylla Cacopsylla pyri (Linnaeus) was investigated through the construction and characterization of cDNA subtracted libraries. Genes expressed upon insect infestation were identified in the susceptible pear cultivar Bartlett and in the resistant selection NY10355. In both interactions, genes involved in the plant defense response were induced, confirming the observed similarity between the response to pathogens and to insects with piercing/sucking mouthparts. However, the two expression profiles were found to be different, with more genes involved in the response to biotic and abiotic stress being activated in the resistant plant than in the susceptible one. Further characterization of the identified genes could lead to the development of molecular markers associated with tolerance/resistance to pear psylla.

Sex Pheromone Investigation of Anastrepha serpentina (Diptera: Tephritidae)

ABSTRACT Attraction of virgin female Anastrepha serpentine Wiedemann (Diptera: Tephritidae) to the odor of calling males was demonstrated. This sex pheromone-mediated attraction occurred during the latter half of a 13-h photophase but not during the first half of the day. Two major components of emissions of calling males, 2,5-dimethylpyrazine (DMP) and 3,6-dihydro-2,5-dimethylpyrazine (DHDMP), and trimethylpyrazine (TMP), a minor component, were tested for pheromonal activity. DMP and TMP elicited antennal responses using electroantennogram assays, but no response was observed for DHDMP. DMP and combinations of DMP and TMP were not attractive in laboratory bioassays. Bioassays of DHDMP were complicated by its instability making purification of the compound unfeasible. Bioassays of DHDMP in unpurified form were further complicated by the presence of reaction byproducts and unreacted reagent in crude preparations of the DHDMP. However, statistical analysis indicated that combinations of 100–500 ng of DHDMP in mixtures with DMP as the major component elicited attraction in laboratory bioassays with sexually active female flies. Taking into account all of the data, we could not conclude that any of the chemicals emitted by males are pheromones, but based on female fly responses to mixtures of DHDMP and DMP, this is the most likely scenario.

Attraction of Mexican fruit flies (Diptera: Tephritidae) to bacteria: effects of culturing medium on odour volatiles.

Effects of culturing medium on production and emission of volatiles by Pantoea agglomerans (Beijerinck 1888) Gavini et al. 1989 preparations and on attractiveness of the preparations to the Mexican fruit fly, Anastrepha ludens Loew, were investigated. Bacterial cultures in each of four biochemically different types of liquid media emitted different volatiles. Cultures in a medium containing uric acid as its primary nitrogen source emitted more ammonia and 2‐nonanone than the other media. We postulate that the high production of ammonia was because of uricase activity by this uricase (+) strain. Regardless of media type, supernatants emitted more volatiles than preparations containing cells that had been removed from whole cultures and put into distilled water. Attractiveness varied little with biochemical make‐up of the culturing medium although the uric acid and carbohydrate preparations were as a group more attractive than preparations made from the other two media. Supernatants and whole cultures generally were more attractive than cell preparations and non‐inoculated media. Bacteria grown in aqueous uric acid‐based media emitted volatiles similar but not identical to those emitted by bacteria grown on gel (agar or solid) uric acid‐based media in Petri plates.

A Novel Attractant for Anastrepha ludens (Diptera: Tephritidae) from a Concord Grape Product

ABSTRACT An attractant for Mexican fruit fly, Anastrepha ludens (Loew) (Diptera: Tephritidae), was developed from a commercial product called Sabor Uva containing processed Concord grape juice. The principal volatile components of Sabor Uva aroma were identified and an aqueous mixture of 15 components that was gas chromatographically similar to Sabor Uva was prepared. This mixture was equivalent to Sabor Uva in attractiveness by using wind-tunnel bioassays. After deleting chemicals that did not contribute to attractiveness, and increasing the concentrations of the remaining chemicals, the final attractant contained propylene glycol (90,000 ppm, vol/vol), acetic acid (4,500), methyl anthranilate (1,800), ethyl 2-methylpropionate (670), and one or both of the esters ethyl 3-methylbutyrate (44) and 2-methylbutyl propionate (44), in aqueous solution. This mixture was ≈1.8× as attractive as Sabor Uva by indirect comparison. Deletion of propylene glycol, acetic acid, methyl anthranilate, or ethyl 2-methylpropionate from the mixture significantly decreased attractiveness. Deletion of either of the other two esters seemed to diminish attractiveness although effects were not statistically significant. Deletion of water from the mixture significantly decreased attractiveness. We conclude that propylene glycol, acetic acid, methyl anthranilate, water, and at least one or as many as all three of the methyl-branched esters are essential for complete attractiveness.

Volatile organic compounds emitted by bottlebrush species affect the behaviour of the sweet potato whitefly

The behavioural response of the sweet potato whitefly, Bemisia tabaci biotype B, to the volatiles emitted by three different species of Callistemon (C. laevis, C. pallidus and C. phoeniceus) was assayed in still-air, and Y-tube olfactometers. B. tabaci adults showed a preference for C. pallidus, while C. laevis and C. phoeniceus were less attractive to the insects when compared to a blank control or to C. pallidus. Volatile organic compounds emitted by the leaves of the three Callistemon species were analysed by solid phase microextraction coupled with GC–MS. 1,8-Cineole was detected in high percentages in C. phoeniceus and C. laevis headspaces, while linalool was abundant in the headspace of C. pallidus. Because of this difference, behavioural tests in the Y-tube olfactometer were carried out also with 1,8-cineole and linalool. Adults of B. tabaci were attracted by linalool, while they were repelled by 1,8-cineole compared with pure air or linalool. These results support the role of these volatiles in the host plant selection process by a herbivorous insect and lay the foundations for the development of sustainable control strategies based on the manipulation of insect pest behaviour.

Drosophila Mutant Model of Parkinson's Disease Revealed an Unexpected Olfactory Performance: Morphofunctional Evidences.

Parkinsons disease (PD) is one of the most common neurodegenerative diseases characterized by the clinical triad: tremor, akinesia, and rigidity. Several studies have suggested that PD patients show disturbances in olfaction as one of the earliest, nonspecific nonmotor symptoms of disease onset. We sought to use the fruit fly Drosophila melanogaster as a model organism to explore olfactory function in LRRK loss-of-function mutants, which was previously demonstrated to be a useful model for PD. Surprisingly, our results showed that the LRRK mutant, compared to the wild flies, presents a dramatic increase in the amplitude of the electroantennogram responses and this is coupled with a higher number of olfactory sensilla. In spite of the above reported results, the behavioural response to olfactory stimuli in mutant flies is impaired compared to that obtained in wild type flies. Thus, behaviour modifications and morphofunctional changes in the olfaction of LRRK loss-of-function mutants might be used as an index to explore the progression of parkinsonism in this specific model, also with the aim of studying and developing new treatments.

Radio frequency treatment for postharvest disinfestation of dates

Radio Frequency Treatment for Postharvest Disinfestation of Dates Francesco Garbati Pegna*, Patrizia Sacchetti, Valentina Canuti, Serena Trapani, Carlo Bergesio, Antonio Belcari, Bruno Zanoni, Ferdinando Meggiolaro a GESAAF Department of Agricultural, Food and Forestry Systems Università degli Studi di Firenze, Italy b DISPAA Department of Agri-food Production and Environmental Sciences Università degli Studi di Firenze, Italy c Stalam S.p.A. Nove, Italy francesco.garbati@unifi.it

Olive fruit fly rearing procedures affect the vertical transmission of the bacterial symbiont Candidatus Erwinia dacicola

Background: The symbiosis between the olive fruit fly, Bactrocera oleae, and Candidatus Erwinia dacicola has been demonstrated as essential for the fly’s larval development and adult physiology. The mass rearing of the olive fruit fly has been hindered by several issues, including problems which could be related to the lack of the symbiont, presumably due to preservatives and antibiotics currently used in the laboratory. To better understand the mechanisms underlying symbiont removal or loss during the rearing of lab colonies of the olive fruit fly, we performed experiments that focused on bacterial transfer from wild female flies to their eggs. In this research, eggs laid by wild females were treated with propionic acid solution, which is often used as an antifungal agent, a mixture of sodium hypochlorite and Triton X, or water (as a control). The presence of the bacterial symbiont on eggs was evaluated by real-time PCR and scanning electron microscopy. Results: DGGE analysis showed a clear band with the same migration behavior present in all DGGE profiles but with a decreasing intensity. Molecular analyses performed by real-time PCR showed a significant reduction in Ca. E. dacicola abundance in eggs treated with propionic acid solution or a mixture of sodium hypochlorite and Triton X compared to those treated with water. In addition, the removal of bacteria from the surfaces of treated eggs was highlighted by scanning electron microscopy. Conclusions: The results clearly indicate how the first phases of the colony-establishment process are important in maintaining the symbiont load in laboratory populations and suggest that the use of products with antimicrobial activity should be avoided. The results also suggest that alternative rearing procedures for the olive fruit fly should be investigated.