Impact of Nutritional Stress on Honeybee Gut Microbiota, Immunity, and Nosema ceranae Infection.

Honeybees are important pollinators, having an essential role in the ecology of natural and agricultural environments. Honeybee colony losses episodes reported worldwide and have been associated with different pests and pathogens, pesticide exposure, and nutritional stress. This nutritional stress is related to the increase in monoculture areas which leads to a reduction of pollen availability and diversity. In this study, we examined whether nutritional stress affects honeybee gut microbiota, bee immunity, and infection by Nosema ceranae, under laboratory conditions. Consumption of Eucalyptus grandis pollen was used as a nutritionally poor-quality diet to study nutritional stress, in contraposition to the consumption of polyfloral pollen. Honeybees feed with Eucalyptus grandis pollen showed a lower abundance of Lactobacillus mellifer and Lactobacillus apis (Firm-4 and Firm-5, respectively) and Bifidobacterium spp. and a higher abundance of Bartonella apis, than honeybees fed with polyfloral pollen. Besides the impact of nutritional stress on honeybee microbiota, it also decreased the expression levels of vitellogenin and genes associated to immunity (glucose oxidase, hymenoptaecin and lysozyme). Finally, Eucalyptus grandis pollen favored the multiplication of Nosema ceranae. These results show that nutritional stress impacts the honeybee gut microbiota, having consequences on honeybee immunity and pathogen development. Those results may be useful to understand the influence of modern agriculture on honeybee health.

Heat shock proteins in Varroa destructor exposed to heat stress and in-hive acaricides.

Varroa destructor is one of the major pests that affect honeybees around the world. Chemical treatments are common to control varroosis, but mites possess biochemical adaptive mechanisms to resist these treatments, enabling them to survive. So far, no information is available regarding whether these pesticides can induce the expression of heat shock protein (Hsp) as a common protective mechanism against tissue damage. The aims of this study were to determine differences in heat shock tolerance between mites collected from brood combs and phoretic ones, and to examine patterns of protein expression of Hsp70 that occur in various populations of V. destructor after exposure to acaricides commonly employed in beekeeping, such as flumethrin, tau-fluvalinate and coumaphos. Curiously, mites obtained from brood cells were alive at 40 °C, unlike phoretic mites that reached 100% mortality, demonstrating differential thermo-tolerance. Heat treatment induced Hsp70 in mites 4 × more than in control mites and no differences in response were observed in phoretic versus cell-brood-obtained mites. Dose-response assays were carried out at increasing acaricide concentrations. Each population showed a different stress response to acaricides despite belonging to the same geographic region. In one of them, coumaphos acted as a hormetic stressor. Pyrethroids also induced Hsp70, but mite population seemed sensitive to this treatment. We concluded that Hsp70 could represent a robust biomarker for measuring exposure of V. destructor to thermal and chemical stress, depending on the acaricide class and interpopulation variability. This is relevant because it is the first time that stress response is analyzed in this biological model, providing new insight in host-parasite-xenobiotic interaction.

A “burning” therapy for burning mouth syndrome: preliminary results with the administration of topical capsaicin.

Burning mouth syndrome is defined as an intraoral burning sensation for which no medical or dental cause can be found. Recently, researchers have demonstrated an altered trophism of the small nerve fibres and alterations in the numbers of TRPV-1 vanilloid receptors. Capsaicin is a molecule that is contained in hot peppers and is specifically detected by TRPV-1 vanilloid receptors that are distributed in the oral mucosae. We aimed at verifying if topical capsaicin could prove to be an effective treatment of Burning Mouth Syndrome. A group of 99 BMS patients were recruited. We subdivided the BMS patients into two groups: the collaborative patients, who expressed a predominantly neuropathic pattern of symptoms, and the non-collaborative patients, who were characterised by stronger psychogenic patterns of the syndrome. Both groups underwent topical therapy with capsaicin in the form of a mouth rinse 3 times a day for a long period. After 1 year of treatment, the final overall success rate was approximately 78%, but with a significant difference in the success rates of the two groups of patients (87% and 20% among the collaborative and non-collaborative patients, respectively; p=0.000). The use of topical capsaicin can improve the oral discomfort of BMS patients, especially during the first month of therapy, but it is more effective for those patients in which the neuropathic component of the syndrome is predominant. Our hypothesis is that chronic stimulation with capsaicin leads to decreases in burning symptoms. This phenomenon is called desensitisation and is accompanied by substantial improvements in oral symptoms.

The temporal effect of a wild blueberry (Vaccinium angustifolium)-enriched diet on vasomotor tone in the Sprague-Dawley rat.

BACKGROUND AND AIMS: We have previously reported that wild blueberry (Vaccinium angustifolium)-enriched diets (WB) attenuate aortic adrenergic response through endothelial-mediated pathways. The duration of dietary intervention necessary to induce the positive changes on vasomotor tone has not been studied to date. Thus, our objective was to investigate the temporal effect of WB consumption on vascular function and reactivity in Sprague-Dawley (SD) rat aorta after 4 and 7 weeks of dietary treatment. METHODS AND RESULTS: Forty male SD rats were randomly assigned to a control (AIN-93) (C) or a WB diet for 4 or 7 weeks. Vascular ring studies were conducted in 3-mm isolated rat aortic rings to investigate vasoconstriction induced by six doses of the α(1)-adrenergic agonist, L-phenylephrine (Phe, 10(-8)-3×10(-6) M) alone or in the presence of the NOS inhibitor, L-N(G)-monomethyl-arginine (L-NMMA, 10(-4)M). The maximum force of contraction (F(max)) and vessel sensitivity (pD(2)) were determined. Analysis of variance revealed no significant differences on F(max) after 4 weeks of the WB diet but only a significant increase in pD(2) in the absence of L-NMMA. Seven week WB consumption significantly attenuated contraction in response to L-Phe and resulted in lower pD(2). Inhibition of NOS induced a significant increase in the constrictor response in both diet groups at both time periods, with the WB group fed for 7 weeks having the greater response. CONCLUSION: Thus wild blueberries incorporated into the diet at 8% w/w positively affect vascular smooth muscle contractility and sensitivity but these effects are evident only after 7 weeks of WB consumption.

Synergistic effect of surfactin from Bacillus subtilis C4 and Achyrocline satureioides extracts on the viability of Paenibacillus larvae.

The aim of this work was to determine the in vitro effect of the mixture between the lipopeptide surfactin, synthesized by Bacillus subtilis C4 (strain isolated from honey) and the most active vegetal extract from Achyrocline satureioides, a traditional medicinal plant, on local strains of Paenibacillus larvae, the agent of American Foulbrood in honeybees. Five P. larvae strains isolated in Córdoba, Argentina, were phenotypically characterized. These and 12 other P. larvae strains from different regions of Argentina were analysed. The antimicrobial activities of the essential oil, hexane (HE) and benzene extracts from A. satureioides were assessed against P. larvae and the HE showed the highest anti-P. larvae activity. A combination of the biosurfactant surfactin, produced by B. subtilis C4, and the HE of A. satureioides revealed a synergistic action on P. larvae. The effective surfactin concentration in the mixture decreased from 32 to 1 µg ml(-1) and the HE concentration from 32 to 4 µg ml(-1), values similar or equal to minimal inhibitory concentrations observed for oxytetracycline. The fractional inhibitory concentration index confirmed synergism in 4 strains and partial synergism in one strain. The combination of surfactin synthesized by B. subtilis C4 and the HE from A. satureioides could be a natural alternative to help beekeepers to combat the American foulbrood agent P. larvae.

Pathogens Detection in the Small Hive Beetle (Aethina tumida (Coleoptera: Nitidulidae)).

Aethina tumida Murray is currently a worldwide emergent pest of Apis mellifera L. hives. Although the damaging effect on the colony stores and brood is well known, the possible role of these beetles as a disease carrier is not clear. This is the first report of DNA presence of the trypanosome honeybee parasite Lotmaria passim and Crithidia bombi, and the Apis mellifera filamentous virus (AmFV) in A. tumida. Further studies will be needed to determine if A. tumida is indeed a mechanical or biological vector of these pathogens.

Blood orange juice inhibits fat accumulation in mice.

OBJECTIVE: To analyze the effect of the juice obtained from two varieties of sweet orange (Citrus sinensis L. Osbeck), Moro (a blood orange) and Navelina (a blond orange), on fat accumulation in mice fed a standard or a high-fat diet (HFD). METHODS: Obesity was induced in male C57/Bl6 mice by feeding a HFD. Moro and Navelina juices were provided instead of water. The effect of an anthocyanin-enriched extract from Moro oranges or purified cyanidin-3-glucoside (C3G) was also analyzed. Body weight and food intake were measured regularly over a 12-week period. The adipose pads were weighted and analyzed histologically; total RNA was also isolated for microarray analysis. RESULTS: Dietary supplementation of Moro juice, but not Navelina juice significantly reduced body weight gain and fat accumulation regardless of the increased energy intake because of sugar content. Furthermore, mice drinking Moro juice were resistant to HFD-induced obesity with no alterations in food intake. Only the anthocyanin extract, but not the purified C3G, slightly affected fat accumulation. High-throughput gene expression analysis of fat tissues confirmed that Moro juice could entirely rescue the high fat-induced transcriptional reprogramming. CONCLUSION: Moro juice anti-obesity effect on fat accumulation cannot be explained only by its anthocyanin content. Our findings suggest that multiple components present in the Moro orange juice might act synergistically to inhibit fat accumulation.

Lactobacillus kunkeei strains decreased the infection by honey bee pathogens Paenibacillus larvae and Nosema ceranae.

Due to their social behaviour, honey bees can be infected by a wide range of pathogens including the microsporidia Nosema ceranae and the bacteria Paenibacillus larvae. The use of probiotics as food additives for the control or prevention of infectious diseases is a widely used approach to improve human and animal health. In this work, we generated a mixture of four Lactobacillus kunkeei strains isolated from the gut microbial community of bees, and evaluated its potential beneficial effect on larvae and adult bees. Its administration in controlled laboratory models was safe for larvae and bees; it did not affect the expression of immune-related genes and it was able to decrease the mortality associated to P. larvae infection in larvae and the counts of N. ceranae spores from adult honey bees. These promising results suggest that this beneficial microorganism's mixture may be an attractive strategy to improve bee health. Field studies are being carried out to evaluate its effect in naturally infected colonies.

Nosema ceranae Winter Control: Study of the Effectiveness of Different Fumagillin Treatments and Consequences on the Strength of Honey Bee (Hymenoptera: Apidae) Colonies.

Overview: In Uruguay, colonies of honey bees moving to Eucalyptus grandis plantation in autumn habitually become infected with the microsporidian Nosema ceranae , a parasite that attacks the digestive system of bees. Beekeepers attributed to N. ceranae depopulation of the colonies that often occurs at the end of the blooming period, and many use the antibiotic fumagillin to reduce the level of infection. The aim of this study was to compare the effectiveness of four different fumagillin treatments and determine how this antibiotic affects the strength of the colonies during the winter season. The colonies treated with fumagillin in July showed less spore load at the end of applications, being the most effective the following treatments: the four applications sprayed over bees of 30 mg of fumagillin in 100 ml of sugar syrup 1:1, and four applications of 90 mg of fumagillin in 250 ml of sugar syrup 1:1 using a feeder. However, 2 month after the treatment applications, the colonies treated with fumagillin were the same size as the untreated colonies. In September, the colonies treated and not treated with fumagillin did not differ in colony strength (adult bee population and brood area) or spores abundance. Our study demonstrates that fumagillin treatment temporarily decreased the spore load of N. ceranae , but this was not reflected in either the size of the colonies or the probability of surviving the winter regardless of the dose or the administration strategy applied. Given the results obtained, we suggest to not perform the pharmacological treatment under the conditions described in the experiment. Resumen: En Uruguay las colonias de abejas melíferas que se trasladan a las forestaciones de Eucalyptus grandis en otoño indefectiblemente se infectan con el microsporido Nosema ceranae , parásito que ataca el sistema digestivo de las abejas. Los apicultores atribuyen a N. ceranae el despoblamiento de las colonias que ocurre con frecuencia al terminar el periodo de floración y muchos emplean el antibiótico fumagilina para reducir el nivel de infección. El objetivo de este estudio fue comparar la eficacia de cuatro tratamientos diferentes con fumagilina y determinar cómo incide en la fortaleza de las colonias durante la invernada. Las colonias tratadas con fumagilina en julio presentaron una menor carga de esporas al terminar las aplicaciones, siendo los tratamientos más eficaces el de 4 aplicaciones mediante asperjado sobre las abejas de 30 mg de fumagilina en 100 ml de jarabe de azúcar 1:1, y el de 4 aplicaciones de 90 mg de fumagilina en 250 ml de jarabe de azúcar 1:1 utilizando un alimentador. Sin embargo, durante el período de experimentación, las colonias tratadas con antibiótico presentaron igual tamaño que las colonias no tratadas. En setiembre, las colonias tratadas y no tratadas con fumagilina no se diferenciaron en la intensidad de infección ni en su tamaño. En las condiciones en que se realizó el estudio, la aplicación de fumagilina disminuyó temporalmente la carga de esporas de N. ceranae pero esto no se reflejó en el tamaño de las colonias ni en la probabilidad de sobrevivir el invierno.

Does tomato consumption effectively increase the resistance of lymphocyte DNA to oxidative damage?

BACKGROUND: Lycopene, the main carotenoid in tomato, has been shown to be a potent antioxidant in vitro. However, there is no significant evidence of its antioxidant action in vivo. OBJECTIVE: We evaluated the effect of tomato intake on plasma carotenoid concentrations and lymphocyte resistance to oxidative stress. DESIGN: Ten healthy women (divided into 2 groups of 5 subjects each) ate a diet containing tomato puree (providing 16.5 mg lycopene) and a tomato-free diet for 21 d each in a crossover design. Before and after each diet period, plasma carotenoid concentrations and primary lymphocyte resistance to oxidative stress (evaluated by means of single-cell gel electrophoresis) were analyzed. RESULTS: After the first 21-d experimental period, total plasma lycopene concentrations increased by 0.5 micromol/L (95% CI: 0.14, 0.87) in the group that consumed the tomato diet and decreased by 0.2 micromol/L (95% CI: -0.11, -0.30) in the group that consumed the tomato-free diet (P < 0.001). Tomato consumption also had an effect on cellular antioxidant capacity: lymphocyte DNA damage after ex vivo treatment with hydrogen peroxide decreased by 33% (95% CI: 0.8%, 61%; P < 0.05) and by 42% (95% CI: 5.1%, 78%; P < 0.05) in the 2 groups of subjects after consumption of the tomato diet. CONCLUSION: The consumption of tomato products may reduce the susceptibility of lymphocyte DNA to oxidative damage.

Relation between diet composition and coronary heart disease risk factors.

STUDY OBJECTIVE: The aim was to evaluate dietary intakes and their correlation to some risk factors for coronary heart disease. DESIGN: The study was a population based survey with random sample selection stratified by age and sex. PARTICIPANTS: 352 adults living in a small town in Northern Italy took part in the study. Response rate was 46% among females and 48% among males. Refusal to take part was mainly due to the large number of tests involved. MEASUREMENTS AND MAIN RESULTS: Diets were high in protein (animal/vegetable ratio 1.7 in women and 1.4 in men) and in fat and low in carbohydrates. The hypercholesterolaemic and atherogenic potential of the diet, evaluated by the cholesterol/saturated fat index, was high in about 50% of the population. The thiamin and riboflavin intakes were lower than the Italian recommended allowances in more than 60% of the people tested, whereas the vitamin A intake was more than adequate in about 70%. A positive association was found in the younger groups (men and women 20-39 years old) between some nutrient components (energy, alcohol, total and saturated fats) and some blood lipids. In the older people blood lipids were correlated with body mass index. CONCLUSIONS: The overall data indicate that a correlation exists between dietary intake and some risk factors for coronary heart disease; dietary intervention, at least in young adults, is suggested.

Effects of physical and chemical characteristics of food on specific and general satiety.

Two high-energy-dense and two low-energy-dense Italian dishes were employed to study the effects of chemical and physical characteristics of foods on satiety. The specific satiety was firstly investigated. Then the satiating efficiency was evaluated when each dish was divided into two calorie levels (preloads) before an ad lib meal. Our results suggest that specific satiety differs, depending on the food itself rather than on energy intake. More calories were ingested with the high-energy-dense foods. The Satiating Efficiency Index (SEI) was calculated: fruit salad was the most satiating (SEI = 3.7), followed by mixed boiled vegetables (SEI = 2.4), meat balls (SEI = 1.0), and baked macaroni (SEI = 0.4). Among the variables considered, energy density, volume, protein, and firmness were the most effective in inducing satiety. In conclusion, the consumption of an adequate amount of low-energy-dense foods, high in firmness, as a first course of a meal, can help to decrease short-term intake.

Weight, protein, fat, and timing of preloads affect food intake.

Two foods, one rich in protein (HP) and one rich in fat (HF), were employed to evaluate the effect of macronutrients on food intake and to underline the differences that occurred when the foods were served as uniform meal, as first course of a varied meal, and as a snack 2 h before a varied meal. Our results showed that HP food always exerted a higher effect on both intrameal satiation and postingestive satiety than HF food. When a uniform meal was consumed, satiation for the specific food was reached before fullness; in this condition, sensory characteristics of foods played an important role in controlling food intake and made the uniform meal more satiating than the varied one. The consumption of a snack far from a meal did not contribute to satiety; consequently, gastric filling seems to be an important factor determining the amount consumed in a varied meal.

The influence of thermic effect of food on satiety.

OBJECTIVES: To evaluate energy expenditure after three isoenergetic meals of different nutrient composition and to establish the relationship between the thermic effect of food (TEF), subsequent energy intake from a test meal and satiety sensations related to consumption. DESIGN: The study employed a repeated measures design. Ten subjects received, in a randomized order, three meals of 2331+/-36 kJ (557+/-9 kcal). About 68% of energy from protein in the high protein meal (HP), 69% from carbohydrate in the high carbohydrate meal (HC) and 70% from fat in the high fat meal (HF). SETTING: The experiments were performed at the University of Milan. SUBJECTS: Ten normal body-weight healthy women. METHODS: Energy expenditure was measured by indirect calorimetric measurements, using an open-circuit ventilated-hood system; intake was assessed 7h later by weighing the food consumed from a test meal and satiety sensations were rated by means of a satiety rating questionnaire. RESULTS: TEF was 261+/-59, 92+/-67 and 97+/-71 kJ over 7 h after the HP, HC and HF meals, respectively. The HP meal was the most thermogenic (P < 0.001) and it determined the highest sensation of fullness (P=0.002). There were no differences in the sensations and thermic effect between fat and carbohydrate meals. A significant relationship linked TEF to fullness sensation (r=0.41, P=0.025). Energy intake from the test meal was comparable after HP, HC and HF meals. CONCLUSIONS: Our results suggest that TEF contributes to the satiating power of foods.

Lycopene and vitamin C concentrations increase in plasma and lymphocytes after tomato intake. Effects on cellular antioxidant protection.

OBJECTIVE: This study seeks to verify whether the regular consumption of small amounts of tomato products can protect lymphocyte DNA and lipids from oxidative damage. DESIGN: Standardized dietary intervention. SUBJECTS: Twelve healthy female subjects (mean age 25.2 y). INTERVENTION: Subjects were instructed to follow a standardized diet for 1 week, followed by 3 weeks consumption of the same diet enriched with small amounts of different tomato products providing as a mean 8 mg lycopene, 0.5 mg beta-carotene and 11 mg vitamin C per day. Plasma and lymphocyte concentrations of carotenoids, vitamin C and vitamin E were analysed. Ex vivo protection of lymphocyte DNA from oxidative injury produced by iron ions was evaluated by means of the Comet assay, and lipid peroxidation by HPLC analysis of malondialdehyde (MDA). RESULTS: Dietary intervention with tomato products increased lycopene concentration both in plasma (P < 0.001) and lymphocytes (P < 0.01). Vitamin C concentrations increased by approximately 35% in plasma (P < 0.05) and by approximately 230% in lymphocytes (P < 0.005). Vitamin E decreased significantly in plasma (P < 0.0001) but not in lymphocytes. Finally, there was an improved protection from DNA oxidative damage (P < 0.05) with no significant effect on MDA levels. CONCLUSIONS: Our results suggest that tomato products are not only good sources of lycopene but also sources of bioavailable vitamin C. A Regular intake of small amounts of tomato products can increase cell protection from DNA damage induced by oxidant species. This effect may originate from the synergism of different antioxidants present in tomatoes.

Differential effect of unsaturated oils and butter on blood glucose and insulin response to carbohydrate in normal volunteers.

Plasma glucose and insulin responses were monitored in normal volunteers after a standard carbohydrate meal with unsaturated oils (olive and corn oil, in identical volumes) or butter. Both unsaturated oils almost totally blunted the glycaemic response; butter, instead, only delayed the plasma glucose rise, without significantly changing the area under the curve. The insulin rise was essentially unaffected by the three experimental meals, only a slight delay in the peak being observed with no differences between unsaturated oils and butter. These findings support previous data indicating reduced glucose levels after prolonged administration of olive oil, and reduced glycaemic response, in the absence of clear-cut insulin changes, differentiates unsaturated oils from indigestible fibres; it may be linked to an altered polysaccharide handling at the intestinal level.

Tomato consumption does not affect the total antioxidant capacity of plasma.

This study was performed to evaluate the effect of tomato intake on total antioxidant activity of plasma measured by the radical trapping antioxidant parameter assay in 11 healthy female subjects. After 7 d of a diet low in carotenoids and free from lycopene, subjects ate 25 g tomato puree daily (containing 7.0 mg lycopene and 0.25 mg beta-carotene) for 14 consecutive days. At the beginning and end of tomato supplementation, the carotenoid plasma concentration and the total antioxidant activity of plasma were assessed. Before tomato puree consumption, mean +/- SE total lycopene and beta-carotene plasma concentrations were 0.13 +/- 0.02 micromol/L and 0.24 +/- 0.04 micromol/L, respectively. After tomato puree supplementation, both concentrations increased significantly (0.57 +/- 0.06 micromol/L, P < 0.0001 for total lycopene, and 0.31 +/- 0. 04 micromol/L, P = 0.0036 for beta-carotene); however, total plasma antioxidant capacity values did not change significantly. From our results, intake of a food rich in carotenoids does not seem to modify the antioxidant capacity of plasma as evaluated by the radical trapping antioxidant parameter assay.

Determination of carotenoids in vegetable foods and plasma.

In this paper a HPLC method for the determination of lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, beta-carotene and lycopene in mixed vegetables and fruit and in human plasma is described. The carotenoids were well separated and the separation was achieved within fifteen minutes using a HPLC system consisting of a 5 microns Vydac 201TP54C18 column, an UV detector, methanol-tetrahydrofuran (95:5 v/v) as mobile phase and a flow rate of 1.0 ml/min. The validity of the separation method was determined by evaluating the linearity of the calibration graphs of each carotenoid (between 0.1 and 1.0 microgram/ml for all compounds except lycopene between 0.1 and 0.8 microgram/ml, r = 0.999) and the accuracy of the chromatographic response (CV < 10%). The reproducibility of the retention times was also good. In the foods samples the extraction procedure was very effective whereas, the saponification step significantly damaged some of the carotenoids. In the plasma the extraction and separation of these compounds were also effective and the qualitative data obtained comparable with those reported in literature. The use of echinenone as internal standard helped to improve quality control.

Chemical composition of Italian cooked dishes.

50 different food items, including "first courses", main and side dishes, cheese and pork products, have been analysed for proteins, carbohydrates, fats, fiber, thiamin, riboflavin and vitamin A. The results obtained have been compared with the values determined on food samples by means of food composition tables so as to have information about the quantitative variations occurring after food processing. It can be concluded that composition tables report sufficiently reliable data with regard to the protein, fat and carbohydrate content for most of the considered food items. On the other hand, values for vitamins are lower by analysis than by calculation, suggesting that it is necessary to continually check data.

Vitamin A, E and C nutriture of elderly people in North Italy.

The vitamin A, E and C nutritional status of 313 non institutionalized elderly people living in a small town and in an agricultural village of North Italy has been evaluated. From results obtained it is possible to say that all the population tested is at low risk for vitamin A deficiency, while 10-20% of people over 70 living in the small town have low levels of vitamin E. On the contrary, the nutritional status of ascorbic acid, where the levels are inadequate in more than 50% of the population, is worrying.