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Honeybees (Apis mellifera L.) have to face many challenges, including Varroa destructor infestation, associated with viral transmission. Oxalic acid is one of the most common treatments against Varroa. Little is known about the physiological effects of oxalic acid, especially those on honeybees’ immune systems. In this study, the short-term effects (0–96 h) of oxalic acid treatment on the immune system components (i.e., glucose oxidase, phenoloxidase, glutathione S-transferase, catalase activities, and vitellogenin contents) of house bees were preliminarily investigated. Oxalic acid contents of bee bodies and haemolymphs were also measured. The results confirm that oxalic acid is constitutively present in bee haemolymphs and its concentration is not affected by treatment. At 6 h after the treatment, a maximum peak of oxalic acid content was detected on bees’ bodies, which gradually decreased after that until physiological levels were reached at 48 h. In the immune system, the oxalic acid treatment determined a peak in glucose oxidase activity at 48 h, indicating a potential defence response and an increase in vitellogenin content at 24 h. No significant changes were recorded in phenoloxidase, glutathione S-transferase, and catalase activities. These results suggest a time-dependent response to oxalic acid, with potential immune system activation in treated bees.

Abstract Settlements are usually shared at different times by semi-fossorial mammals. Porcupine reproductive pair shows high den-site fidelity, but no data are available on the spatio-temporal inhabitation of settlements. In this investigation, the spatio-temporal inhabitation of settlements by crested porcupine families was investigated using camera-trapping as well as the ethological factors affecting the settlements selection. The crested porcupine resulted to be the main inhabitant of settlements surveyed in the present study. Each settlement was inhabited exclusively by one porcupine family. Five out of six porcupine families, each alternatively and complementarily inhabited the same two settlements. In all the five monitored families, settlements selection doesn’t follow a seasonal pattern. Settlement inhabitation of porcupines resulted positively affected by cohabitation with badger, while presence of porcupettes did not affect settlements selection. Long periods of settlement inhabitation were positively affected both by the presence of porcupettes and cohabitation with badger. The pattern of settlements inhabitation in relation to their availability and porcupine population density as well as factors promoting porcupine-badger cohabitation should be further investigated. New ethological knowledge obtained in this investigation could be involved in the evaluation of the ecological epidemiology of infectious diseases between porcupine and badger within a one health approach and may be a useful tool for a sustainable management of semi-fossorial mammals.

Searching for artificial diets positively affecting the survival, immune and antioxidant systems of honey bees is one of main challenges occurring in beekeeping. Among nutrients, lipids play a significant role in insect nutrition as structural components in cell membranes, energy sources and reserves, and are involved in many physiological processes. In this context, the aim of this work was to investigate the effect of 0.5% and 1% coconut oil-enriched diet administration on newly emerged and forager bees survival rate, feed intake, immune system, antioxidant system and both fat and vitellogenin content. In newly emerged bees, supplementation with 1% coconut oil determined a decrease in feed consumption, an increase in survival rate from the 3rd to 14th day of feeding, a short-term decrease in phenoloxidase activity, an increase in body fat and no differences in vitellogenin content. Conversely, supplementation with 0.5% coconut oil determined an increase in survival rate from the 3rd to 15th day of feeding and an increase in fat content in the long term (i.e., 20 days). Regarding the forager bee diet, enrichment with 0.5% and 1% coconut oil only determined an increase in fat content. Therefore, supplementation with coconut oil in honey bee diets at low percentages (0.5 and 1%) determines fat gain. Further investigations to evaluate the use of such supplement foods to prevent the fat loss of weak families during winter are desirable.

Abstract Previous data on crested porcupine reproduction biology were mostly collected in captivity or semi-captivity due to its elusive, burrowing and mainly nocturnal habits. Between 2017 and 2019 the reproductive behaviour (i.e., intrapair mounting and copulation, birth and parental care) of free-ranging crested porcupine was documented and described. Nightly rhythms of single and multiple mounting occurred throughout the year while only two copulation events were recorded. Three months after both copulation events, the birth of porcupettes (porcupine

This study seeks to offer a contribution to the method of subtyping major depressed patients by exploring the possible relationships between circulating brain-derived neurotrophic factor (BDNF), different peripheral inflammatory/metabolic markers in the blood and clinical characteristics. Thirty-nine patients, thoroughly diagnosed according to the DSM-5 criteria, underwent a comprehensive set of evaluations encompassing structured interviews, rating scales and a panel of blood tests. Correlation and comparison analyses were carried out by means of non-parametric statistical tests. Concurrently, a principal component analysis was performed to explain biochemical variance. The findings of our research unveiled that leukocyte counts, their ratios and other inflammatory parameters are positively correlated with depression scores. Moreover, we found variations within the BDNF pools of depressed patients. Specifically, higher levels of platelet-poor plasma BDNF (PPP-BDNF) were correlated with augmented inflammatory markers in patients showing specific episode characteristics, whereas reduced platelet BDNF (PLT-BDNF) provided a better indication of the changes that were linked to a diagnosis of long-term depression. Our findings suggest that PPP-BDNF and PLT-BDNF might differentiate depression conditions. They also imply usefulness in appraising peripheral biomarker profiles in patients for a deeper characterization of major depressive episodes. At the same time, it is plausible that they might constitute novel avenues for developing more tailored therapeutic strategies for patients with MDs.

The crested porcupine is a frequent host species in wildlife rescue centers and no guidelines for its management, liberation and post-liberation monitoring are yet available. Here, captive-grown porcupines’ behavior and survival in the wild after liberation were investigated and described for the first time. Management strategies adopted at the centers could affect porcupine adaptation to the natural environment. The detention of porcupettes in single cages and fed only human-supplied food may not ensure ethological welfare, nor a suitable behavioral development compatible with wildlife. The liberation of captive-grown porcupines should also be carefully planned to promote and increase the possibility of post-liberation success.

Senotainia tricuspis (Meigen, 1838) is a Sarcophagid dipteran endoparasitoid of Apis mellifera L., and myiasis, caused by this fly, is reported in several European, North African and Middle Eastern countries. Nevertheless, very little knowledge concerning the aggression and parasitisation behavior of S. tricuspis toward A. mellifera is available in the scientific literature, and the temporal pattern of aggression remains unclear. The aim of this investigation was to describe the aggressive behavior of S. tricuspis and to provide data on pupation and adult emergence in order to identify further tools for the control of senotainiosis in beekeeping. Data were collected in an apiary in Pisa province (Tuscany, Italy), where observations of aggressive behavior were conducted indirectly by using a VHS camera and also directly by an observer. Four behavioral categories of the attack were described. A total of 55 aggressions, 21 beecatchers, 104 chases and 6 parasitization events were recorded with the camera. Slow-motion recording analyses of the parasitization episodes resulted in contact of at least 1/6 s between the parasitoid and the host. Through four days of direct observations, a total of 1633 aggression events were recorded. The daily temporal pattern of the number of aggressions showed two main peaks: one during the morning hours (i.e., from 10:00 to 11:00) and one in the afternoon (i.e., from 15:00 to 17:00). The morphometric data on the first-instars of S. tricuspis allowed us to hypothesize a penetration in the bee through its prothoracic spiracle as a modality of entrance in the host body. Third-instars successfully pupate when sinking in topsoil or clay soil, and adults emerge when left to a 4 °C overwintering period of six months. Furthermore, the high mortality rate of those larvae that did not sink and did not pupate successfully suggests that reaching a certain depth in the soil is a determining factor for larvae survival and that mulch and/or minimum soil tillage could prevent severe senotainiosis in apiaries.

Osmia cornuta Latr. is largely managed worldwide for the pollination of orchard crops, playing a key role in the maintenance of healthy ecosystems and ensuring economic and social benefits for human society. The management techniques of this pollinator include the possibility of delaying emergence from cocoons after diapause, allowing for the pollination of later-blooming fruit crops. In this study, the mating behaviour of bees emerging at the natural time (Right Emergence Insects) and of late-emerged bees (Aged Emergence Insects) was described in order to test if a delay in emergence could affect the mating sequence of O. cornuta. Markov analysis of the mating behaviour revealed the occurrence of antenna motion episodes that were repeated in a stereotyped manner at regular intervals during the mating sequence of both Right Emergence Insects and in Aged Emergence Insects. Pouncing, rhythmic and continuous emission of sound, motion of antennae, stretching of the abdomen, short and long copulations, scratching, inactivity, and self-grooming were identified as the stereotyped behavioural units of a behavioural sequence. The occurrence of short copulations, the frequency of which increased with the age of bees, could lead to a failure in the reproduction of the mason bee.

Italy is the only European country where the crested porcupine (Hystrix cristata) lives. A parasitological investigation was performed on faecal samples, aimed to evaluate Giardia and other parasites in a free-ranging crested porcupine population in Central Italy. Samples were collected from captured and road-killed individuals as well as from feeding areas and pathways. Collected faecal samples were examined by the Mini-FLOTAC technique and a rapid immunoassay for the search of Giardia and Cryptosporidium spp. faecal antigens. For the identification of Giardia species and genotypes, molecular analysis was performed on Giardia-positive samples, by using PCR protocols able to amplify glutamate dehydrogenase, triosephosphate isomerase and a fragment of the small subunit ribosomal RNA genes.A total of 52 crested porcupine faecal samples were collected and analysed. At microscopical examination, 39 out of 52 samples were found positive for at least a single parasite species and six different parasite taxa were identified. Forty-eight percent (25/52) of faecal samples were positive for Giardia spp. and 1.9% (1/52) for Cryptosporidium spp. at the immunoassay. Among 12 faecal samples belonging to different individuals, 33.3% (4/12) were positive for Giardia spp. By using the Mini-FLOTAC technique, positivity for Trichuris spp. (32.7%, 17/52), gastrointestinal strongyles (32.7%, 17/52), capillariid eggs (3.8%, 2/52) and coccidian oocysts (1.9%; 1/52) was also evidenced. Molecular analysis was performed on 17 out of 25 Giardia-positive isolates. At the SSU rDNA locus, expected bands were achieved for 12 out of 17 isolates and all samples were assigned to Giardia duodenalis assemblage B. Sequencing at tpi locus revealed potentially zoonotic G. duodenalis assemblage AII (two isolates) and assemblage BIV (one isolate). The present study provides the first report of G. duodenalis infection in H. cristata. More in depth studies are needed on the impact and epidemiology of G. duodenalis and other identified parasites in crested porcupines. Keywords: Hystrix cristata, Nematodes, Protozoa, Giardia duodenalis, Potential zoonotic assemblages

Gastrointestinal nematodes and protozoa and other parasite occurrences were evaluated in free-ranging wolf (Canis lupus italicus) and red fox (Vulpes vulpes) populations from natural and anthropized areas of Central Italy. Analyzed fecal samples were collected from 60 foxes and 40 wolves in the anthropized areas, and 41 foxes and 39 wolves in the natural areas. In foxes, hookworm infections (p < 0.0001) were more frequently recorded in the anthropized environment, while coccidia (p < 0.05) and Cryptosporidium spp. (p < 0.0001) were more frequent in the natural area. In wolves, a higher frequency of hookworms (p < 0.0001) was observed in natural areas, while coccidia were more common in the anthropized area (p < 0.05). Moreover, in the natural environment, trichuroid nematodes (p < 0.0001) were significantly more frequent in wolves than in foxes, while Cryptosporidium (p < 0.001) and Giardia duodenalis (p < 0.001) were more common in foxes. In the anthropic area, the occurrence of hookworms was found to be significantly higher in foxes (p < 0.0001), while trichuroid nematodes were more common in wolves (p < 0.0001). The obtained data are indicative of a different diffusion of specific parasite taxa in wolves and foxes living in the natural and/or anthropized environments examined herein.

The increase in invasive alien species is a concern for the environment. The establishment of some of these species may be changing the balance between pathogenicity and host factors, which could alter the defense strategies of native host species. Vespid species are among the most successful invasive animals, such as the genera Vespa, Vespula and Polistes. Bee viruses have been extensively studied as an important cause of honey bee population losses. However, knowledge about the transmission of honey bee viruses in Vespids is a relevant and under-researched aspect. The role of some mites such as Varroa in the transmission of honey bee viruses is clearer than in the case of Vespidae. This type of transmission by vectors has not yet been clarified in Vespidae, with interspecific relationships being the main hypotheses accepted for the transmission of bee viruses. A majority of studies describe the presence of viruses or their replicability, but aspects such as the symptomatology in Vespids or the ability to infect other hosts from Vespids are scarcely discussed. Highlighting the case of Vespa velutina as an invader, which is causing huge losses in European beekeeping, is of special interest. The pressure caused by V. velutina leads to weakened hives that become susceptible to pathogens. Gathering this information is necessary to promote further research on the spread of bee viruses in ecosystems invaded by invasive species of Vespids, as well as to prevent the decline of bee populations due to bee viruses.

Varroa destructor is a mite causing serious damage to western honey bees. Managed colonies require artificial varroa control, which may be best obtained by combining mechanical and chemical methods. This study explored the possible effects of the combination of queen caging and oxalic acid treatment on the immune system (glucose oxidase, phenoloxidase, and vitellogenin) and antioxidant enzymes (superoxide dismutase, catalase, and glutathione S transferase) of first post-treatment generation drones and workers (newly emerged, nurses, and foragers). The combination of queen caging and oxalic acid treatment caused a decrease in glucose oxidase activity only in drones. This could cause issues of cuticular sclerotization, making a drone prone to bite injuries, dehydration, and pathogens. No differences in phenoloxidase activity were recorded in both post-treatment drones and workers generation. Among worker bees, the treatment determined a lower vitellogenin content in newly emerged bees while the result was higher in nurse bees. However, the treatment did not significantly affect the antioxidant enzymes activity in either drones or workers. The results obtained in this investigation suggest that the combined anti-varroa treatments had no negative effects on oxidative stress in the first post-treatment generation bees, while effects did occur on the immune system. Further investigations on the potential effects of glucose oxidase decrease in drones and vitellogenin content variation in workers are desirable.

Royal Jelly is a nutrient secretion of nurse bees and a high interest functional food in human nutrition. Very little information is available on its chemical composition integrity and enzymatic activity during shelf life and assessment of new freshness markers are desirable for its conservation. In this study, the activity of glucose oxidase, five proteases and two antioxidant enzymes in refrigerated and frozen Royal Jelly for different storage times was preliminary investigated. Refrigeration determined a significantly reduction in glucose oxidase and carboxypeptidase A-like activity in Royal Jelly after one year of storage while no differences were recorded in the activity of these enzymes in frozen samples. After one year of storage glucose oxidase and carboxypeptidase A-like activity resulted higher in frozen samples frozen than in refrigerate ones. Results obtained suggest that the activities of these enzymes may be good markers of Royal Jelly freshness within 1 year at refrigeration condition. Freezing could be a valid alternative storage method to ensure a higher preservation of glucose oxidase and carboxypeptidase A-like activities for at least 1 year. Further investigation to determine the timing of glucose oxidase inactivation/degradation under refrigerated conditions and the enzymatic activity trend under prolonged frozen conditions are desirable.

Abstract The crested porcupine is a mainly nocturnal mammal that shows both moonlight avoidance and some diurnal motor activity, the latter as an extension of its night-time foraging behaviour. Furthermore, a peculiar daytime behaviour, described as “sunbathing”, was reported as episodic in H. africaeaustralis. Between 2016 and 2019 a camera-trapping monitoring was performed within 10 porcupine settlements in order to detect the diurnal motor activity and to verify and describe the sunbathing behaviour in crested porcupine. Out of 1,003 trap days, a total of 148 events of diurnal motor activity were recorded. The diurnal motor activity occurred throughout the year mainly between December and June from 15:00 to 16:00, with no statistical difference between cubs, youngsters and adults. The sunbathing behaviour was detected for a total of 36 episodes recorded. Sunbathing was performed mainly by cubs. The sunbathing behaviour occurred only between April and June during the hottest hours of the day (11:00 to 12:00). Diurnal motor activity and sunbathing behaviour of porcupine are discussed in relation to food availability and porcupine physiology.

The effects of dietary supplementation of tannins mix (quebracho and chestnut) in rabbit diet were evaluated for productive performances, health parameters and digestibility in order to quantify their practical utilisation in the rearing system. One-hundred and twenty Martini group hybrid rabbits of 30 days old were fed four different diets for 60 days. The diets were formulated as: basal diet (negative control, C), basal diet supplemented by 0.3% of tannins mix (T0.3), basal diet supplemented by 0.6% of tannins mix (T0.6) and basal diet supplemented with coccidiostat (positive control, CC). Live performances did not show any significant differences, moreover, no significant differences were observed for all carcase traits except for gastrointestinal tract (p = .015, lowest values for T0.3 diet). Also, digestibility of the feed and faecal microbial load was not influenced by tannins addition. Slight differences between the diets were detected in catalase and glutathione peroxidase concentrations in plasma, as a common trend was revealed with higher values of C than the other diets. From an economical point of view, T0.3 diet showed to be more profitable than CC and T0.6. Tannins addition might be taken into account as potential feed additive in rabbit feeds, as it does not affect negatively the productive performances, digestibility and induce a slight increase of antioxidant status.Highlights On an economical point of view tannin diet showed to be more profitable than a diet added with coccidiostat. Addition of tannins in rabbits’ feed did not affect negatively productive performances and digestibility. Tannins represent a valuable feed additive in rabbit farming as a slightly increase of antioxidant status was induced.

The Eurasian badger (Meles meles) is widespread in Italy and occupies different habitats. The occurrence and species of gastrointestinal parasites were evaluated in a free-ranging badger population living in a highly anthropic area in central Italy. A total of 43 fecal samples were examined using the flotation test, the Mini-FLOTAC and Baermann techniques, and a rapid immunoassay for the detection of Giardia duodenalis and Cryptosporidium spp. fecal antigens. Molecular investigations were also performed that aimed at identifying Giardia genotypes. Overall, 37/43 samples (86%) were found positive. Specifically, 48.8% (21 samples) were positive for G.duodenalis, 23.2% (10/43) for Cryptosporidium spp., and 7% (3/43) for coccidian oocysts. Strongyloides sp. nematode larvae were detected in 3/43 samples (7%). Ascarid (1/43, 2.3%), capillariid (1/43, 2.3%), and strongyle-type eggs (76.7%, 33/43) were also identified. Among the 11 readable sequences of samples that were positive for G. duodenalis by end-point PCR (18/21), the zoonotic assemblage A sub-assemblage AII and mixed assemblage A and B were identified. This is the first report of zoonotic G. duodenalis genotypes in the Eurasian badger. Moreover, most of identified parasites have zoonotic potential and/or potential impact on the population health of wild badgers and other wild and domestic animals.

Protein-supplemented artificial diets are widely used by beekeepers during winter and whenever food availability is low, yet no data are available concerning their effects on bees’ health. In this work, the effects of two commercial diets enriched with 1.7% and 7.7% protein concentration on feed intake, survival rate, glucose oxidase, phenoloxidase and glutathione S-transferase in newly emerged and forager bees were tested. Administration of a 7.7% protein-enriched diet significantly reduced the lifespan of both newly emerged and forager bees, while only in foragers a significantly higher feed intake was recorded. In newly emerged bees, administration of a high-protein-enriched diet stimulated glucose oxidase production at the 10th day of feeding, determined a reduction of phenoloxidase and did not affect glutathione S-transferase activity. In forager bees, a high level of protein inclusion did not determine any significant variation in either glucose oxidase, phenoloxidase or glutathione S-transferase activity. Therefore, the results obtained in this investigation suggest that administration of commercial protein diets negatively affect honey bee health, determining an increase in mortality. Further investigations on the effect of concentration and quality of proteins are desirable to provide beekeepers with scientific evidence on protein feeding.

Propolis antimicrobial activity has been limitedly studied in food, particularly in dairy products. We studied the antimicrobial activity of an alcoholic extract of an Italian propolis in sterile skim milk, pasteurized cow’s milk, and cow’s and goat’s whey cheese (ricotta). Following the determination of the minimal inhibitory concentration on Gram+ and Gram- bacteria, the extract was employed at 2 and 5% (P2, P5), using controls with the same ethanol concentrations (E2, E5) and without any addition. In milk trials, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and Pseudomonas fluorescens were tested. P2 and P5 samples registered significant decreases of Gram+ bacteria in skim milk. The same was true for P5 in cows’ milk, but only with S. aureus for P2. Ricotta was inoculated with L. monocytogenes, S. aureus and B. cereus and stored at 8.5°C. In cow’s milk ricotta, L. monocytogenes counts in P5 were always lower than control during the storage time, significantly so from the 14th day. In goat’s ricotta, L. monocytogenes counts in P5 were at least one logarithm lower than E5, whereas the extract didn’t show a significant effect on S. aureus and B. cereus. The antimicrobial activity of propolis, particularly on L. monocytogenes, could be employed in ready-to-eat refrigerated dairy products.

β-glucans can activate the animal innate immune system by acting as immune-modulators and inducing various stimulatory effects. The aim of this study was to investigate the effect of 1,3-1,6 β-glucans administered orally for 96 h on Apis mellifera workers (newly emerged and nurse bees). β-glucans were included in honey and syrup. Survival rate and phenoloxidase activity were measured. In both newly emerged and nurse bees, β-glucans supplementation did not affect survival rate (p > 0.05). Conversely, phenoloxidase activity was higher in both newly emerged bees (p = 0.048) and nurse bees (p = 0.014) fed with a honey diet enriched with β-glucans compared to those fed with only honey. In both the newly emerged and nurse bees, no statistical differences in phenoloxidase activity were recorded between the group fed with a syrup-based diet enriched with β-glucans and the control group (p > 0.05). The absence of significant variation in survival suggests that the potential negative effect of β-glucans in healthy bees could be mitigated by their metabolism. Conversely, the inclusion of β-glucans in a honey-based diet determined an increase of phenoloxidase activity, suggesting that the effect of β-glucan inclusion in the diet of healthy bees on phenoloxidase activity could be linked to the type of base-diet. Further investigations on β-glucans metabolism in bees, on molecular mechanism of phenoloxidase activation by 1,3-1,6 β-glucans, and relative thresholds are desirable. Moreover, investigation on the combined action of honey and β-glucans on phenoloxidase activity are needed.

Abstract The PREDICTS project—Projecting Responses of Ecological Diversity In Changing Terrestrial Systems (https://www.nhm.ac.uk/our-science/our-work/biodiversity/predicts.html)—has collated from published studies a large, reasonably representative database of comparable samples of biodiversity from multiple sites that differ in the nature or intensity of human impacts relating to land use. We have used this evidence base to develop global and regional statistical models of how local biodiversity responds to these measures. We describe and make freely available this 2016 release of the database, containing more than 3.2 million records sampled at over 26,000 locations and representing over 47,000 species. We outline how the database can help in answering a range of questions in ecology and conservation biology. To our knowledge, this is the largest and most geographically and taxonomically representative database of spatial comparisons of biodiversity that has been collated to date; it will be useful to researchers and international efforts wishing to model and understand the global status of biodiversity.

The health status of the honey bee populations has attracted a great amount of interest in recent years. We investigated honey bee health in five natural protected areas in Italy from October 2009 to December 2010. Areas were selected to represent a wide range of bio‑geographical zones including alpine, continental, and Mediterranean. Within each of these natural protected areas, one apiary of 20 colonies near potential pollution sources (e.g., agricultural areas, industrial areas, or urban settlements) and another apiary of 20 colonies far from possible sources of pollutants have been placed. To monitor honey bee health, colony mortality was related to: honey bee pathologies, environment (Naturality Index, plant protection products and heavy metal exposure), and apiary management. Anthropogenic pollutants and pathogens did not have significant effects on colony mortality while environment and the poor colony management skills of the beekeepers did.

ABSTRACT: The innate immune system of honeybees mainly consists in antimicrobial peptides, cellular immunity and melanisation. In order to investigate the immune response of honeybees to immune stressors, three stress degrees were tested. Newly emerged bees naturally DWV-infected were collected from a Varroa mite-free apiary and divided into three experimental groups: naturally DWV infected bees, PBS injected bees, and artificially DWV super infected bees. Phenoloxidase activity and haemolymph cellular subtype count were investigated. Phenoloxidase activity was highest (P

The tiletamine-zolazepam mixture is a widely used anesthetic for chemical immobilization of wild mammals due to its short induction time, good muscle relaxation, smooth recovery with low convulsions occurrence, and minimal effect on respiration. An injection dose of 7–8 mg/kg of tiletamine-zolazepam has been proven to be an effective and safe immobilizing mixture for crested porcupines under field conditions. However, the occurrence of long immobilization and recovery times, with high excitement and convulsion during awakening, were recorded. In order to reduce such side effects after recovery, the effectiveness of a lower dosage (4–6 mg/kg) of tiletamine-zolazepam (Zoletil®) was tested. The results obtained confirm that the use of tiletamine-zolazepam in crested porcupine immobilization provides a quick induction, wide safety margin, and predictable awakening under field conditions. A smaller injection dosage of 5 mg/kg has been proven to be sufficient to ensure a short induction time (average: 7.1 min), with good muscle relaxation and little excitement of the animals during awakening. The lower dosage of tiletamine-zolazepam, while providing a shorter recovery time (average: 53.6 min), proves to be adequate for standard handling procedures. Furthermore, the smaller amount of tiletamine-zolazepam also ensures safe immobilization for pregnant individuals and porcupettes.

The crested porcupine is a widespread naturalized Italian rodent of African origin; nevertheless, very little information on the population abundance and its health status is available. In this study, the hematological and serum chemistry profile of 10 free-ranging captured crested porcupines was established for the first time. The mean hematological values resulted: 5.7 SD 0.4 M/μL for red blood cells; 13.6 SD 0.8 g/dL for hemoglobin; 77.3 SD 5.7 fL for mean corpuscular volume and 30.1 SD 4.7 g/dL for mean corpuscular hemoglobin concentration; 14.4 SD 7.2 K/μL for white blood cells; and 557.0 SD 469.9 K/μL for platelets. The mean urea and creatinine values resulted with 19.8 SD 8.3 mg/dL and 1.6 SD 3.0 mg/dL, respectively. The mean value of total protein was 6.7 SD 1.0 g/dL, with values of albumin higher than globulins. The mean activity of creatine kinase, aspartate transaminase, gamma-glutamyl transpeptidase, and alkaline phosphatase was 927.3 SD 607.6 U/L, 199.2 SD 70.8 U/L, 16.9 SD 13.7 U/L, and 256 SD 75.8 U/L, respectively. Highest values of alkaline phosphatase were recorded in two porcupines presenting severe injuries with clear signs of infection. These preliminary results may be a helpful tool in order to assess porcupine health status.

The employment of natural substances such as beehive products with a preventive and therapeutic purpose has been a widespread custom since ancient times. In this investigation, the antibacterial activity of 41 honey samples from different Ukraine regions has been evaluated. For each honey, melissopalynological and physico-chemical analysis were performed in order to determine botanical origin, pH, glucose and fructose contents and free acidity. So, antibacterial activity against Staphylococcusaureus CCM 4223, Listeria monocytogenes ATCC 7644, Salmonella enterica serovar Typhimurium CCM 3807 and Escherichia coli ATCC 25922 was assessed through the determination of MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values by the microdilutions method. The results show that the most susceptible bacterial strain was L. monocytogenes. Its growth was inhibited at a honey concentration ranging from 0.094 to 0.188 g/mL. The most resistant bacterial strain was S. aureus. As concerns MBC values, L. monocytogenes was the most susceptible bacteria, while S. aureus was the most resistant. Helianthus spp. honeys was the most effective against all tested bacterial strains, followed by Robinia spp. and multifloral honeys. Promising results for MIC tests have been found for Brassica spp.

The effects of veterinary drugs, dietary supplements and non-protein amino acids on the European honey bee (Apis mellifera ligustica Spinola, 1806) ventriculum microbial profile were investigated. Total viable aerobic bacteria, Enterobacteriaceae, staphylococci, Escherichia coli, lactic acid bacteria, Pseudomonas spp., aerobic bacterial endospores and Enterococcus spp. were determined using a culture-based method. Two veterinary drugs (Varromed® and Api-Bioxal®), two commercial dietary supplements (ApiHerb® and ApiGo®) and two non-protein amino acids (GABA and beta-alanine) were administered for one week to honey bee foragers reared in laboratory cages. After one week, E. coli and Staphylococcus spp. were significantly affected by the veterinary drugs (p < 0.001). Furthermore, dietary supplements and non-protein amino acids induced significant changes in Staphylococcus spp., E. coli and Pseudomonas spp. (p < 0.001). In conclusion, the results of this investigation showed that the administration of the veterinary drugs, dietary supplements and non-protein amino acids tested, affected the ventriculum microbiological profile of Apis mellifera ligustica.

Honeybee pathogens have an important role in honeybee colony mortality and colony losses; most of them are widely spread and necessitate worldwide solutions to contrast honeybee’s decline. Possible accepted solutions to cope with the spread of honeybee’s pathogens are focused on the study of experimental protocols to enhance the insect’s immune defenses. Honeybee’s artificial diet capable to stimulate the immune system is a promising field of investigation as ascertained by the introduction of 1,3-1,6 β-glucans as a dietary supplement. In this work, by collecting faecal samples of honeybees exposed to different dietary conditions of 1,3-1,6 β-glucans (0.5% and 2% w/w), it has been possible to investigate the Deformed wing virus (DWV) viral load kinetic without harming the insects. Virological data obtained by a one-step TaqMan RT-PCR highlighted the ability of 1,3-1,6 β-glucans to reduce the viral load at the 24th day of rearing. The results indicated that the diet supplemented with 1,3-1,6 β-glucans was associated with a dose-dependent activation of phenoloxidase. The control group showed a higher survival rate than the experimental groups. This research confirmed 1,3-1,6 β-glucans as molecules able to modulate honeybees’ defense pathways, and this is the first report in which the kinetic of DWV infection in honeybee faeces has been monitored by a RT-qPCR.

Abstract Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species’ threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project – and avert – future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local‐scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups – including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems – https://www.nhm.ac.uk/our-science/our-work/biodiversity/predicts.html). We make site‐level summary data available alongside this article. The full database will be publicly available in 2015.

The Western Honeybee is a key pollinator for natural as well as agricultural ecosystems. In the last decade massive honeybee colony losses have been observed worldwide, the result of a complex syndrome triggered by multiple stress factors, with the RNA virus Deformed Wing Virus (DWV) and the mite Varroa destructor playing crucial roles. The mite supports replication of DWV to high titers, which exert an immunosuppressive action and correlate with the onset of the disease. The aim of this study was to investigate the effect of 1,3-1,6 β-glucan, a natural innate immune system modulator, on honeybee response to low-titer natural and high-titer experimental DWV infection. As the effects exerted by ß-glucans can be remarkably different, depending on the target organism and the dose administered, two parallel experiments were performed, where 1,3-1,6 ß-glucan at a concentration of 0.5% and 2% respectively, was added to the diet of three cohorts of newly emerged honeybees, which were sampled from a Varroa-free apiary and harboured a low endogenous DWV viral titer. Each cohort was subjected to one of the following experimental treatments: no injection, injection of a high-copy number DWV suspension into the haemocel (experimental DWV infection) or injection of PBS into the haemocoel (physical injury). Control bees fed a ß-glucan-free diet were subjected to the same treatments. Viral load, survival rate, haemocyte populations and phenoloxidase activity of each experimental group were measured and compared. The results indicated that oral administration of 0.5% ß-glucan to naturally infected honeybees was associated with a significantly decrease of the number of infected bees and viral load they carried, and with a significant increase of the survival rate, suggesting that this natural immune modulator molecule might contribute to increase honeybee resistance to viral infection.

In recent years, Brucella suis has been sporadically reported in Italy in domestic and wild swine. Since standard serological tests can determine false positive results, the development of alternative tests with improved sensitivity and specificity is rather essential. We analyzed 1212 sera collected at slaughterhouse from healthy pigs belonging to 62 farms of North-Central Italy. Sera were tested by Rose Bengal Test, Complement Fixation Test, and subsequently by a Dot Blot (DB) and Western Blot assays (WB). Only one serum resulted positive to all tests, indicating that swine brucellosis has a very limited spread. DB and WB could represent a support to the available serological tests; however, further studies to validate these tests are needed. In the presence of reemerging diseases, a prompt and continuous monitoring design is necessary to acquire epidemiological information for the subsequent application of specific health emergency plans.

Deformed wing virus (DWV) is a honeybee pathogen whose presence is generally associated with infestation of the colony by the mite Varroa destructor, leading to the onset of infections responsible for the collapse of the bee colony. DWV contaminates bee products such as royal jelly, bee-bread and honey stored within the infected hive. Outside the hive, DWV has been found in pollen loads collected directly from infected as well as uninfected forager bees. It has been shown that the introduction of virus-contaminated pollen into a DWV-free hive results in the production of virus-contaminated food, whose role in the development of infected bees from virus-free eggs has been experimentally demonstrated. The aim of this study was twofold: (i) to ascertain the presence of DWV on pollen collected directly from flowers visited by honeybees and then quantify the viral load and (ii) determine whether the virus associated with pollen is infective. The results of our investigation provide evidence that DWV is present on pollen sampled directly from visited flowers and that, following injection in individuals belonging to the pollinator species Apis mellifera, it is able to establish an active infection, as indicated by the presence of replicating virus in the head of the injected bees. We also provide the first indication that the pollinator species Osmia cornuta is susceptible to DWV infection.

Understanding the mechanism of metabolic digestion and of food assimilation in wild snake species is of fundamental importance to compare trophic niches, their adaptive significance and evolutionary patterns of diet selection for different snake species. From two adult Hierophis viridiflavus males in fasting condition, we sampled gastric fluids using fiberoptic endoscopy. We analyzed the proteolytic activities (optimal pH and temperature, response to inhibitors) of snake samples by zymography techniques. The two major protease activity bands were always detected at every tested pH. Activity bands were also detectable over 37 °C; in particular both bands showed the highest activity ranging from 55 °C to 60 °C. Ranging from 65 °C to 85 °C, one band remained visible while the other band disappeared over 60 °C. The pattern of proteolytic enzymes activity of sampled gastric fluids highlighted a scenario composed of few active proteases, reflecting the feeding status (e.g., fasting) of studied snakes. Detection of proteolytic activity until 85 °C, as in prokaryotic organisms, supported the hypothesis about the presence of proteases of exogenous source.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector.

MALDI profiling and imaging mass spectrometry (IMS) are novel techniques for direct analysis of peptides and small proteins in biological tissues. In this work we applied them to the study of Anopheles gambiae antennae, with the aim of analysing expression of soluble proteins involved in olfaction perireceptor events. MALDI spectra obtained by direct profiling on single antennae and by the analysis of extracts, showed similar profiles, although spectra obtained through profiling had a richer ion population and higher signal to noise ratio. Male and female antennae showed distinct protein profiles. MALDI imaging experiments were also performed and differences were observed in the localization of some proteins. Two proteins were identified through high resolution measurement and top-down MS/MS experiments. A 8 kDa protein only present in the male antennae matched with an unannotated sequence of the An. gambiae genome, while the presence of odorant binding protein 9 (OBP-9) was confirmed through experiments of 2-DE, followed by MS and MS/MS analysis of digested spots. This work shows that MALDI MS profiling is a technique suitable for the analysis of proteins of small and medium MW in insect appendices, and allows obtaining data for several specimens which can be investigated for differences between groups. Proteins of interest can be identified through other complementary MS approaches.