Phenoloxidase activity and haemolymph cytology in honeybees challenged with a virus suspension (deformed wings virus DWV) or phosphate buffered suspension (PBS)

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<0.05) in DWV-superinfected bees. and the haemocyte population differed within the three observed groups. Although. immune responses following DWV infection have still not been completely clarified. this investigation sheds light on the relation between cell immunity and the phenoloxidase activity of DWV naturally infected honeybees exposed to additional stress such as injury and viral superinfection

Dietary supplementation of quebracho and chestnut tannins mix in rabbit: effects on live performances, digestibility, carcase traits, antioxidant status, faecal microbial load and economic value

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

Water-soluble proteinase activity of gastric fluids in the European whip snake, Hierophis viridiflavus: an experimental preliminary survey

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

Effects of nonprotein amino acids on survival and locomotion of Osmia bicornis

To investigate the effects of two non-protein amino acids (NPAAs), β-alanine and γ-amino butyric acid (GABA), on Osmia bicornis survival and locomotion, two groups of ten caged bees have been fed with a sugar syrup enriched with β-alanine and GABA, respectively. The further control group was fed with the sugar syrup. Five behavioural categories have been chosen in accordance with the principle of parsimony and with their intrinsic unitary consistency from onset to the end. Two states (staying under the paper and staying in the test tubes) and three events (walking on the net, feeding on the flower and flying) have been recorded by scan sampling. Furthermore, we analysed the amino acids content of the haemolymph that was sampled from additional forty-five bees kept under the same diet condition (15 per diet type). Bees fed with ß-alanine had a significant shorter survival than those fed with control and GABA diets. The GABA diet induced a higher locomotion than the β-alanine. The former non-protein amino acid was detected in the haemolymph of only bees fed with GABA. Results suggest that insects consuming NPAAs-reach diets can absorb and transfer these substances into the haemolymph and those NPAAs affect both survival and locomotion. Ecological consequences are discussed in the frame of plant reproductive biology

Effect of 1,3-1,6 β-Glucan on Natural and Experimental Deformed Wing Virus Infection in Newly Emerged Honeybees (Apis mellifera ligustica)

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

A proteomic investigation of soluble olfactory proteins in Anopheles gambiae

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. © 2013 Mastrobuoni et al

EFFETTO NUTRACEUTICO DEI BETA-­‐GLUCANI SU API INFETTE DA DEFORMED WING VIRUS (DWV).

Il Deformed Wing Virus (DWV) o virus delle api deformi è responsabile di una infezione spesso in forma subclinica che è molto diffusa nell'Apis mellifera. Il virus appartiene alla famiglia Picornavirales e, come gli altri membri della famiglia, è caratterizzato da un virione nudo di piccole dimensioni racchiudente un genoma a ssRNA (+). Il virus infetta le forme larvali durante il loro sviluppo e l’infezione si manifesta con la comparsa di api neo-sfarfallate con gravi deformazioni a carico delle ali e con ridotte dimensioni del corpo. La malattia può portare al collasso della colonia ed in genere le forme più gravi sono associate ad infestazioni di Varroa destructor, un acaro che agisce sia come parassita che come amplificatore e vettore biologico del virus. Le api, così come altri invertebrati, hanno sviluppato un'ampia varietà di meccanismi di difesa innata contro i vari agenti patogeni, seppur in assenza di un sistema immunitario adattativo. In molti dei meccanismi immunodifensivi degli invertebrati sono coinvolte proteine secrete da cellule dell'emolinfa dotate di attività opsonizzante, chemiotattica, battericida e perossidasica. Tali meccanismi possono essere modulati dalla presenza di varie molecole definite immunomodulatori. Tra queste i ß-glucani, polisaccaridi ramificati non amidacei costituiti da molecole di glucosio unite insieme mediante legami glicosidici β(1,3) e β(1,6), possono rappresentare una alternativa all'utilizzo di sostanze di sintesi chimica. I ß-glucani sono costituenti della parte solubile della fibra vegetale, sono presenti in molti cereali ma anche nelle pareti cellulari di vari agenti patogeni. Al fine di comprendere se i ß-glucani possano avere una efficace azione immunomodulante sulle api, la molecola è stata integrata nell’ alimentazione di gruppi sperimentali di api in un modello di infezione naturale e sperimentale con DWV. Gli esperimenti condotti hanno permesso di quantificare il tasso di mortalità, di stimare il numero e il grado di attivazione delle cellule deputate all’attività immunitaria, di quantizzare il virus presente nei vari distretti delle api (testa/addome) e di valutarne la replicazione. I risultati ottenuti hanno dimostrato che la somministrazione di ß-glucani per via orale in api infette da DWV è associata a un significativo aumento del tasso di sopravvivenza, ad un incremento del numero di emociti, e ad una riduzione della carica virale. Tali risultati suggeriscono che questa molecola possa contribuire ad incrementare la difesa delle api verso agenti patogeni

Protein patterns and enzyme activities differences between healthy, ill and cured honey bees investigated by 2D electrophoresis and enzymatic assays

The honey bees are the most important commercial pollinators of those crops which depend on animal pollination for reproduction. Beekeeping is an eco-compatible activity that offers more than one type of production: honey, wax, royal jelly, pollen and propolis. Beekeepers in Western countries have been reporting slow declines of bee stocks for many years, that seem unprecedented in recent history. This phenomena has been named "Colony collapse disorder" (CCD). It seems to be caused by a combination of various contributing factors rather than a single pathogen or poison. The beekeeping of Apis mellifera L. in different types of beehive and the the import of honeybees from other countries due to globalization, have caused the presence of new pathogens. Beekeepers use treatments against these pathogens, in particular against the Varroa destructor, a mite that can in some cases cause the weakening of the colony. For these reasons we have decided to investigate the effects that some treatments, that the beekeepers generally use in their apiary, and some prevailing pathologies cause on the honey bees immune system. The aim of this work is to research new potential wellness indicators for honey bees. The old indicators which are family force, flight activity, pollen importation, are nowadays not sufficient anymore. In our research we used the proteomic and enzymatic assay approaches. The 2D approach has been an immediate instrument to give a qualitative analysis of treated/ill samples compared to control samples. This analysis was used to investigate the effects of formic acid treatments. A small quantity of spots was identified by mass spectrometry but one protein was particularly interesting, glutathione-S-transferase. The 2D zymography approach was used to investigate the proteases present in samples infected with American foulbrood. At least 4 proteases were revealed in healthy prepupa samples and absent (P1, P2, P3) or partially present (P4) in diseased prepupa samples. These proteases could be grouped in two families: the serine proteases or the cysteine proteases. Analysis with 2D zymography have also revealed a protease inhibitor (trypsin or α-chymotrypsin inhibitor) that was present in the healthy prepupa samples but was absent in diseased prepupa samples. Regarding to the phenoloxidase and glucose oxidase activities, some spectrophotometer and activity staining protocols were identified. Several analysis with treated/ill samples (adult or brood bees) revealed different phenoloxidase and glucose oxidase activities compared to the control samples (healthy or not treated samples). With the study of treated samples 30 days after the treatment by beekeepers, we could see a return of the enzymatic activity (phenoloxidase and glucose oxidase) to the initial values before the treatment. We can suppose that the honeybees have detoxification processes that eliminate the effects of the treatments. After a double stress represented by the infection with a parasite and a treatment, the honeybees presented a phenoloxidase or glucose oxidase activity frequently opposite or enhanced compared to a single stress scenario. We can suppose that after a stress the honey bees could be “prepared” to respond to a second stress. A small pilot project was made with the aim to understand if the phenoloxidase and the glucose oxidase activities change in function of the season. The changes in the phenoloxidase and glucose oxidase activities were attributed particularly to the apiary condition, meteorological conditions, to the tasks which the honey bees carry out in a particular month and the cares of the beekeepers. The use of the glucose oxidase and its products (gluconic acid and hydrogen peroxide) against Paenibacillus larvae represents a new approach against this terrible pathology. It opens the possibility to try to “reinforce” the immune system of the honeybees against pathogens