Effects of cryopreservation and freeze-drying on proteases enzymatic activity of entomopathogenic strains of Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metchnikoff) Sorokin.

Beauveria bassiana and Metarhizium anisopliae are fungal species known for their entomopathogenic activity; they represent an important alternative to chemical control of many pest insects and appropriate preservation methods are thus necessary to maintain their properties over time. In the present study the effects of cryopreservation and freeze­drying were investigated on virulence determinants such as proteases and specifically on subtilisin-type Pr 1 and Pr 2 that seem to play a predominant role in insect fungal penetration. Three Metharizium anisopliae strains (M 13/ I05, M 13/ I12 and M 13/ I33) and three Beauveria bassiana strains (B 13/ I03, B 13/ I57 and B 13/ I63) were tested. Data obtained from our experiments show that the decrease of Pr 1 and Pr 2 enzymatic activity in B. bassiana and M. anisopliae strains 7 months after storage does not affect their entomopathogenic ability against larvae of Tenebrio molitor compared to the fresh cultures before storage. The role of Pr 1 and Pr 2 as predominant factors in the entomopathogenicity of both fungal species is discussed. No significant differences were found in any strain tested for morphological features using both preservation methods

An effective method for Graphosoma lineatum (L.) long-term rearing.

A simple and time-saving technique for an effective and continuous rearing of Graphosoma lineatum (L.) (Heteroptera Pentatomidae), an alternative host for Trissolcus spp. and Ooencyrtus spp. production, was set for entomological research and maintained for a long period. Insects were maintained in containers as rearing units; 100x35x35cm cages hosted adults; 40x30x30cm cages hosted nymphs. Graphosoma lineatum was fed on seeds of Foeniculum vulgare Mill., Anethum graveolens L. and Pimpinella anisum L. Moreover, potted young plants of F. vulgare were also used as additional food source. Water for insects and plants was provided by small automatic irrigation systems. When each colony cage reached the density of 100 adult couples, the number of oviposited batches was followed up for 12 weeks. Batches laid per cage were approximately one hundred per week. Therefore the overall weekly production of six adult cages was about 8400 eggs. This technique was plain and cost effective, allowing a constant egg production throughout the year.

Tenebrio molitor as a Simple and Cheap Preclinical Pharmacokinetic and Toxicity Model

The progression of drugs into clinical phases requires proper toxicity assessment in animals and the correct identification of possible metabolites. Accordingly, different animal models are used to preliminarily evaluate toxicity and biotransformations. Rodents are the most common models used to preliminarily evaluate the safety of drugs; however, their use is subject to ethical consideration and elevated costs, and strictly regulated by national legislations. Herein, we developed a novel, cheap and convenient toxicity model using Tenebrio molitor coleoptera (TMC). A panel of 15 drugs-including antivirals and antibacterials-with different therapeutic applications was administered to TMC and the LD50 was determined. The values are comparable with those already determined in mice and rats. In addition, a TMC model was used to determine the presence of the main metabolites and in vivo pharmacokinetics (PK), and results were compared with those available from in vitro assays and the literature. Taken together, our results demonstrate that TMC can be used as a novel and convenient preliminary toxicity model to preliminarily evaluate the safety of experimental compounds and the formation of main metabolites, and to reduce the costs and number of rodents, according to 3R principles

Abundance and biodiversity of soil arthropods in one conventional and two organic fields of maize in stockless arable systems

Soil arthropod community was evaluated, in three different farming systems in Central Italy, in the context of a long-term experimental stockless arable system (MOLTE). The soil arthropodofauna was recorded in two organic agrosystems of different age (16-year old organic, named OldO; 6-year young organic, named YngO) and in one conventional (Co), at a fixed time on maize. Arthropods, extracted by Berlese-Tullgren funnels, were counted and identified at order or suborder taxonomic level. In the three maize fields, the farming system affected both abundance and biodiversity of arthropods. The arthropod density ranged from about 20,000 individuals/m2 in OldO to about 45,000 in YngO. The number of oribatid mites was higher in Co than in OldO, while YngO showed the highest density of collembolans. The mite/collembolan ratio was the highest in Co (6.43), the lowest in YngO (1.95). Both biodiversity indices adopted – V, synthetic index of degree of diversity change of ecological systems and QBS, index of biological soil quality – showed the highest values for YngO. On the whole, differences in the arthropod community were higher in the YngO-OldO comparison than in OldO-Co. The soil arthropod community tended to be characterized by lower density of specimens and lower number of taxa in the OldO organic system than in the YngO

Notes on Drosophila suzukii Matsumura (Diptera Drosophilidae): field survey in Tuscany and laboratory evaluation of organic products

Reported for the first time in Italy in 2009, the Spotted Wing Drosophila (SWD), Drosophila suzukii (Matsumura) (Diptera Drosophilidae), has rapidly spread throughout the country, causing serious damage on many fruit crops. The potential of infestation rate by D. suzukii is enormous: the females can complete many generations per year, and are able to attack the fresh, ripe fruit of many hosts by laying eggs under the soft skin; the larvae grow in the fruits and destroy the commercial value of them. The host range of SWD is reportedly wide, including both cultivated (soft fruits, sweet cherry, stone fruits etc…) and wild plants. In 2013, field surveys aimed at monitoring the presence of the insect on Tuscany territory, were regularly carried out: on an important district for the sweet cherry production with differently managed in control, on a biological blueberry orchard and even on other host plants neighboring the samples’ areas. D. suzukii was present in the monitored areas and its numerically large populations and heavy infestations on sweet cherries have shown that the species has settled in these areas. In laboratory tests, labeled organic products and substances of natural origin - in accordance with the requirements of EU regulations which provides for the reduction of chemicals - were evaluated as concern toxicity and residual effect on the pest. As regards effects of direct toxicity, products based on Beauveria bassiana have shown some effectiveness in the control of SWD, other organic products have shown interesting results. The evidences acquired are discussed

ACLEESCF. SP. FOVEATUS(COLEOPTERA CURCULIONIDAE), AN EXOTIC PESTOF FICUS CARICAIN ITALY: A SUSTAINABLE APPROACH TO DEFENCE BASEDON ALUMINOSILICATE MINERALS AS HOST PLANT MASKING SOLIDS

The exceptionally frequent entries of alien pest are a major source of concern for the farmers who haveto protect their crops from unknown insects, often without natural enemies in the new areas. A new pestbelonging to the Molytinae family (Coleoptera: Curculionidae), tribe Hylobiini, reported as Acleessp. cf.foveatusVoss, was recently introduced in Italy. The species is responsible for severe damages in many Italianfig nurseries and orchards, particularly in the Italian Central Northern regions, i.e. Tuscany, Ligury andLatium. Currently, no active ingredients are registered against this insect on fig crops. An innovative and eco-friendly approach for controlling this exotic weevil infestation was investigated, by using montmorillonite-based clays, either in their native state or containing copper(II) species, and clinoptilolite zeolites, in order tocheck the perception of the adults’ weevil towards the different solid materials and, subsequently, to evaluatethe capability of these innovative products to act as masking agent with respect to the host plant and/or asrepellent upon contact. The formulations containing copper(II)-exchanged clay and clinoptilolite zeoliteshowed preliminary promising results in terms of efficacy and environmental sustainability

Terpene arms race in the Seiridium cardinale - Cupressus sempervirens pathosystem

The canker-causing fungus Seiridium cardinale is the major threat to Cupressus sempervirens worldwide. We investigated the production of terpenes by canker-resistant and susceptible cypresses inoculated with S. cardinale, the effect of these terpenes on fungal growth, and the defensive biotransformation of the terpenes conducted by the fungus. All infected trees produced de novo terpenes and strongly induced terpenic responses, but the responses were stronger in the cankerresistant than the susceptible trees. In vitro tests for the inhibition of fungal growth indicated that the terpene concentrations of resistant trees were more inhibitory than those of susceptible trees. The highly induced and de novo terpenes exhibited substantial inhibition (more than a fungicide reference) and had a high concentration-dependent inhibition, whereas the most abundant terpenes had a low concentration-dependent inhibition. S. cardinale biotransformed three terpenes and was capable of detoxifying them even outside the fungal mycelium, in its immediate surrounding environment. Our results thus indicated that terpenes were key defences efficiently used by C. sempervirens, but also that S. cardinale is ready for the battle

Influences of Wolbachia (Rickettsiales Rickettsiaceae) on the cellular response to cold stress in Drosophila melanogaster (Diptera Drosophilidae)

Wolbachia pipiensis (Hertig et Wolbach, 1924) is known to manipulate the expression of genes implicated in the metabolism, immunity and reproduction in Drosophila melanogaster (Meigen, 1830). Under stress, cells activate the cellular stress response (CSR). The CSR is a conserved network of pathways regulating identification, check and response to stress, preserving the cellular homeostasis. The CSR involves the unfolded protein response, autophagy, the heat shock response and other subcellular pathways. How Wolbachia affects the CSR has not yet been investigated. Here, we report the influence of Wolbachia infection and cold stress on the expression of the Heat-shock-protein-70Aa (Hsp70Aa), Autophagy-related gene-1 (Atg1) and X box binding protein-1 (Xbp1) genes and the influence of cold stress on the Wolbachia surface protein gene (wsp). The Hsp70Aa, Atg1, and Xbp1 genes were affected by Wolbachia infection since they were found to be up-regulated in the Wolbachia-free flies. After cold stress, the Wolbachia-infected flies showed high expression of the Atg1 and Hsp70Aa genes in comparison to the Wolbachia-free flies. Moreover, cold stress negatively influenced the expression of the wsp gene

Soil ecosystem functions in a high-density olive orchard managed by different soil conservation practices

The long-term effects of two different soil management practices, natural grass cover (NC) and conservation tillage (CT), on soil functions (carbon sequestration, habitat for organisms, and water movement and retention) were determined in a high-density, mature olive orchard (Olea europaea L. cv. Frantoio) growing in a sandy loam soil (Typic Haploxeralf) in a Mediterranean environment. Ten years after the beginning of the different soil management, soil samples were collected at 0–10 and 10–20 cm depth and at two distances from the trunk, underneath the olive canopy (UC) and in the inter-row (IR). There were no differences in fruit yield, oil yield, and yield efficiency between the two soil management systems during the 2011–2013 period. CT negatively affected soil organic carbon pools (total and humified), but only at the IR position. The distance from the plant did not significantly influence soil structure and hydrological properties, while NC treatment increased water movement and retention. Tillage reduced the microarthropod abundance, in particular Collembola and eu-edaphic forms, which were the most sensitive groups to soil perturbation. We conclude that natural grass cover was more effective than conservation tillage in maintaining or improving elements of soil functionality