Use of electronic nose technology to measure soil microbial activity through biogenic volatile organic compounds and gases release

Gas and volatile organic compounds (VOCs) release in soil is known to be linked to microbial activity and can differently affect the life of organisms in soil. Electronic noses (E-noses) are sensing devices composed of sensor arrays able to measure and monitor gases and VOCs in air. This is the first report on the use of such a sensing device to measure specifically microbial activity in soil. In the present study, γ-irradiated sterilised soil was inoculated with Pseudomonas fluorescens. To be sure for a rapid microbial growth and activity, two pulses of nutrient solution with organic and inorganic C, N, P and S sources were added to soil and the resulting microcosms were incubated for 23 d. During the incubation, respiration and enzyme activities of acid phosphatase, β-glucosidase, fluorescein diacetate hydrolase and protease, were measured, and microbial growth as global biomass of vital cells based on substrate-induced respiration (SIR-C_mic) and enumeration of viable and culturable cells by means of dilution plate counts (CFU) were also monitored. Concurrently, VOCs and/or gas evolution in the headspace of the soil microcosms were measured through the E-nose, upon their adsorption on quartz crystal microbalances (QCMs) comprising the sensory device. The E-nose typically generated an odorant image (olfactory fingerprint) representative of the analysed samples (soils) and resulting from the concurrent perception of all or most of the analytes in headspace, as it commonly happens when several selective but not specific sensors are used together (array). The basic hypothesis of this study was that different soil ecosystems expressing distinct microbial metabolic activities, tested through respiration and enzyme activities, might generate different olfactory fingerprints in headspace. Furthermore, the possibility to detect several substances at the same time, released from the soil ecosystems, possibly deriving from both abiotic and biotic (microbial metabolism) processes provides an “odorant image” representative of the whole ecosystem under study. The E-nose here used succeeded in discriminating between inoculated and non-inoculated ecosystems and in distinguishing different metabolic and growth phases of the inoculated bacteria during incubation. Specifically, E-nose responses were proved highly and significantly correlated with all hydrolytic activities linked to the mobilisation of nutrients from soil organic matter and their cycling, with CO₂ fluxes (respiration and presumed heterotrophic fixation) and with P. fluorescens population dynamics during exponential, stationary and starvation phases measured by SIR-C_mic and CFUs. Interestingly, the E-nose successfully detected soil microbial activity stimulated by nutrient supply, even though none of the catalytic activities tested directly produced VOCs and/or gases. The E-nose technology was then proved able to supply a real holistic image of microbial activity in the entire gnotobiotic and axenic soil ecosystems.     

THORACIC COMPUTED TOMOGRAPHY,ANGIOGRAPHIC COMPUTED TOMOGRAPHY,AND PATHOLOGY FINDINGS IN SIX CATS EXPERIMENTALLY INFECTED WITH AELUROSTRONGYLUS ABSTRUSUS

Aelurostrongylus abstrusus infection is common in endemic areas and may cause severe respiratory clinical signs. Computed tomography (CT) is an important tool to diagnose pulmonary disease, because it allows detection of small lesions and discrimination of superimposed structures. The purpose of this study was to characterize by CT and angiographic CT the pulmonary lesions in six cats before, and 48 and 81 days after inoculation with 100 or 800 A. abstrusus infective larvae. Histological examination of the accessory lung lobe was performed to determine the microscopic, pathomorphologic correlate of the CT findings. The predominant CT lesion consisted of multiple nodules of varying size distributed throughout the lungs, severity depending on infectious dose. The histological correlate of the nodular lesions was multifocal dense granulomatous to mixed inflammatory cell infiltrates, including eosinophils distributed in the parenchyma and obliterating the alveoli. Marked, multifocal, dose‐dependent thickening of the bronchi and adjacent interstitial changes blurred the margins of the outer serosal surface of the bronchi and vessels. Histologically, this was due to peribronchial mixed cell inflammation. During the course of infection some of the nodular and peribronchial changes were replaced by areas of ground‐glass opacity. In addition to providing detailed depiction of pulmonary lesions resulting from an infectious cause and clearly defining lesions with respect to time and severity of infection, CT allowed quantitative assessment of bronchial thickness and lymph node size during the course of disease. Findings indicated that CT characteristics of this disease are consistent with pathologic findings.     

Evaluation of an open portable chamber system for measuring cover crop water use in a vineyard and comparison with a mini-lysimeter approach

Cover crops are largely used in viticultural areas because of the many positive agronomic and environmental benefits they provide. However, there is insufficient data describing the amount of water they use. A portable chamber used as an open measurement system and its suitability to measure the cover crop evapotranspiration (E) in a vineyard are described in this study. The performance of the chamber was tested by a calibration experiment (R² = 0.97). The lowest air flow rate used (9.2 l s⁻¹) was found to be suitable to limit the chamber from overheating beyond 3.1 K above the outside temperature. Furthermore, an experiment was designed to compare the daily cover crop (Festuca arundinacea var. barfelix) water use measured by the chamber system with measurements using a mini-lysimeter (ML) method and with estimates using the FAO-56 PM equation (Eo). The experiment was carried out in one inter row of a vineyard over the course of 4 days following an irrigation event. Although the field experiment was limited to 4 days, the results obtained together with the calibration trials support the possibility of the chamber being a useful tool for measuring the cover crop E. The ability of the MLs to represent the water use of the cover crop in the rest of the vineyard was limited to the first two days of the experiment, after which time the soil water content inside the containers was significantly (p = 0.007, p = 0.03) lower than in the surrounding field.     

Asymmetry of mating behaviour affects copulation success in two stored-product beetles

Lateralization (i.e. left–right asymmetries in the brain and behaviour) has been documented in all vertebrate classes, while evidences for invertebrates are limited. To the best of our knowledge, there is no information about behavioural asymmetries in the order Coleoptera. In this research, we investigated the lateralization of mating traits in two major pests of stored products: the rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae), and the confused flour beetle, Tribolium confusum (Coleoptera: Tenebrionidae). Both T. confusum and S. oryzae males showed population-level left-biased copulation approaches of potential mates. Interestingly, T. confusum and S. oryzae males performing left-biased copulation attempts achieved higher mating success over right-biased males. Furthermore, S. oryzae males periodically exhibited a typical head wagging behaviour, which consists of waving the rostrum laterally across the female thorax and then resting the rostrum on the mid thorax. This behaviour was right-biased, even in the majority of males that previously showed left-biased copulation attempts. The quantification of mating displays could allow comparisons with other strains, in order to evaluate the impact of a given host or of the rearing methods on the mating traits of S. oryzae and T. confusum. Overall, this is the first report of lateralization of mating traits in Coleoptera. Furthermore, this research adds basic knowledge to the reproductive behaviour of S. oryzae and T. confusum.     

Identification of Microsatellite Markers for Plasmopara viticola and Establishment of High throughput Method for SSR Analysis

The Oomycete Plasmopara viticola is the causal organism of downy mildew on grapevine (Vitis spp.). In order to set up the techniques for investigating downy mildew disease dynamics and genetic structure, co-dominant, neutral, highly reproducible and polymorphic microsatellite markers for P. viticola were developed. Five markers, two with a (TC)_n repeat (loci BER and ISA), two with a (TC)_n(AC)_n repeat (loci CES and REX) and one with a (CT)_n(CTAT)_n repeat (locus GOB), were selected. Simple sequence repeat (SSR) markers revealed different degrees of polymorphism within 190 oil spots (disease symptoms) collected from an infected Italian vineyard. The most polymorphic SSR marker GOB showed 43 alleles (Nei's expected gene diversity H_e = 0.89) while CES, ISA, BER and REX showed 14 (H_e = 0.71), 4 (H_e = 0.57), 3 (H_e = 0.24) and 1 allele (H_e = 0), respectively. A high throughput DNA extraction method, that allowed molecular analysis of this obligate pathogen directly in the host without any isolation procedure, was developed. The quality and quantity of oil spots did not influence the SSR analysis. Amplified SSR loci were separated by electrophoresis on a Beckman–Coulter 2000XL sequencer and automatically analysed. The objective of this study was to develop molecular biological tools and methods that allow high throughput analysis of the downy mildew populations.     

Co-inoculated Plasmopara viticola genotypes compete for the infection of the host independently from the aggressiveness components

During Plasmopara viticola epidemics only few genotypes produce most of the secondary lesions and dominate in the population. Selection of dominant genotypes is hypothesized to be linked to environmental conditions and can occur rapidly, particularly if there is also difference between genotypes in terms of fitness and aggressiveness. Measurements of aggressiveness components can largely determine the rate of epidemic development, although the components of aggressiveness do not take into account potential direct competition between genotypes. Differences in aggressiveness have been also reported to be greater under non-optimal conditions suggesting for genotype adaptation to different conditions. To evaluate differences in latency at non-optimal conditions, we characterized genotypes deriving from different climatic regions at three different temperatures (15, 25 and 35 °C) and we found no differences. To investigate whether other factors may impact on competition between P. viticola genotypes, we evaluated polycyclic infections of P. viticola by co-inoculating three genotypes with similar aggressiveness components in two different co-inoculation experiments and an increasing prevalence of one of the two genotypes was observed. Competition was not related to the origin of the genotype and we hypothesize that competitive selection is modulated by differences in the secretion of effector molecules which can contribute to the establishment of dominant genotypes over an epidemic season.     

New Methods for Selecting Stallion Spermatozoa for Assisted Reproduction

Improved sperm selection techniques are needed to increase the efficiency of equine-assisted reproduction. Single layer centrifugation (SLC) of spermatozoa has been shown to improve the quality of stallion sperm samples. In this study, the functionality of selected stallion spermatozoa was tested by intracytoplasmic sperm injection of equine oocytes after selection by SLC through Androcoll-E or by discontinuous density gradient centrifugation (DGC) through Redigrad and Tyrode's medium with added albumin, lactate, and pyruvate. The mean cleavage rates of the injected oocytes from SLC- and DGC-selected spermatozoa were 67% and 66%, respectively, whereas the proportion of blastocysts developing from cleaved oocytes was 28% and 22%, respectively (P > .05, not significant). An incidental finding was that there was a tendency for SLC-selected spermatozoa to have a higher percentage of spermatozoa with normal morphology than DGC (70% ± 22% vs. 58% ± 38%) and for more blastocysts to be obtained from subfertile ejaculates (21 [19.6%] vs. 15 [14.4%], respectively). In further experiments, stallion spermatozoa bound to hyaluronan, although binding may depend on the semen extender and sperm treatment as well as incubation time. In conclusion, SLC-selected stallion spermatozoa function normally when injected into oocytes. SLC may potentially be better than DGC at selecting spermatozoa from subfertile ejaculates, but this effect needs rigorous investigation with a much larger sample size. Use of the hyaluronan-binding assay for assessing the potential fertility of stallion spermatozoa may be useful but requires further evaluation.     

Efficacy of a single administration of a spot-on solution containing imidacloprid 10%/moxidectin 2.5% in eliminating Dirofilaria repens microfilariae in naturally infected dogs

In the past decade reports of canine subcutaneous dirofilariosis, caused by the mosquito-transmitted nematode Dirofilaria repens, increased in number in several countries in Europe, along with a rise of human cases. Given the merit to the new approaches for the control and treatment of this infection, the present study evaluated the efficacy of a single application of the spot-on formulation containing imidacloprid 10%/moxidectin 2.5% (Advocate(?), Bayer Animal Health) in the elimination of D. repens microfilariaemia in naturally infected dogs. In September 2009, 18 dogs with a natural infection by D. repens were enrolled in the study. In October 2009 all the dogs were treated once with Advocate(?) and the presence/absence of circulating MF and skin lesions after treatment was evaluated monthly until April 2010. From November 2009 to April 2010 15 dogs scored negative for D. repens while one dog remained negative till March 2010 when it died. Two dogs had a recurrence of microfilariaemia in December 2009 and January 2010 respectively. Nine infected dogs showed skin lesions at the beginning of the trial, which disappeared after treatment in 7 dogs, whereas the other two symptomatic dogs did not show any dermatological improvement until the end of the trial even though they scored negative for D. repens microfilariae. This study demonstrated that a single dermal administration of Advocate(?) is effective in eliminating microfilariae of D. repens and likely has a certain degree of activity in killing subcutaneous adult worms as well. This study demonstrates the efficacy of Advocate(?) in the treatment of dermatitis caused by D. repens. Also, these results are of importance towards further control programs aiming to reduce the number of bites infectious for mosquitoes and the risk of infection for both humans and dogs.