Zebrafish Feed Intake: A Systematic Review for Standardizing Feeding Management in Laboratory Conditions.

Zebrafish are one of the most used animal models in biological research and a cost-effective alternative to rodents. Despite this, nutritional requirements and standardized feeding protocols have not yet been established for this species. This is important to avoid nutritional effects on experimental outcomes, and especially when zebrafish models are used in preclinical studies, as many diseases have nutritional confounding factors. A key aspect of zebrafish nutrition is related to feed intake, the amount of feed ingested by each fish daily. With the goal of standardizing feeding protocols among the zebrafish community, this paper systematically reviews the available data from 73 studies on zebrafish feed intake, feeding regimes (levels), and diet composition. Great variability was observed regarding diet composition, especially regarding crude protein (mean 44.98 ± 9.87%) and lipid content (9.91 ± 5.40%). Interestingly, the gross energy levels of the zebrafish diets were similar across the reviewed studies (20.39 ± 2.10 kilojoules/g of feed). In most of the reviewed papers, fish received a predetermined quantity of feed (feed supplied). The authors fed the fish according to the voluntary intake and then calculated feed intake (FI) in only 17 papers. From a quantitative point of view, FI was higher than when a fixed quantity (pre-defined) of feed was supplied. Also, the literature showed that many biotic and abiotic factors may affect zebrafish FI. Finally, based on the FI data gathered from the literature, a new feeding protocol is proposed. In summary, a daily feeding rate of 9-10% of body weight is proposed for larvae, whereas these values are equal to 6-8% for juveniles and 5% for adults when a dry feed with a proper protein and energy content is used.

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression.

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation.

Long-Term Brain Organotypic Cultures of the Turquoise Killifish Nothobranchius furzeri.

Organotypic culture is a well-established method for culturing ex vivo tissue samples. The advantages of culturing tissue slices for prolonged time periods ex vivo are numerous and consist primarily of the maintenance of the overall in vivo architecture of the isolated sample, the lack of the ematoencephalic barrier, and the ease of pharmacological treatments and interventions that can be conducted under controlled conditions as in in vitro systems such as cell cultures. Given the extremely short life span of Nothobranchius furzeri and the emergence of aging signs only after a few months of life, it is of particular interest to establish this protocol for N. furzeri as a potential method to study brain aging ex vivo.

A Review on the Bioactivity of Cannabinoids on Zebrafish Models: Emphasis on Neurodevelopment.

For centuries, the cannabis plant has been used as a source of food, fiber, and medicine. Recently, scientific interest in cannabis has increased considerably, as its bioactive compounds have shown promising potential in the treatment of numerous musculoskeletal and neurological diseases in humans. However, the mechanisms that underlie its possible effects on neurodevelopment and nervous-system functioning remain poorly understood and need to be further investigated. Although the bulk of research on cannabis and cannabinoids is based on in vitro or rodent models, the zebrafish has now emerged as a powerful in vivo model for drug-screening studies and translational research. We here review the available literature on the use of cannabis/cannabinoids in zebrafish, and particularly in zebrafish models of neurological disorders. A critical analysis suggests that zebrafish could serve as an experimental tool for testing the bioactivity of cannabinoids, and they could thus provide important insights into the safety and efficacy of different cannabis-extract-based products. The review showed that zebrafish exhibit similar behaviors to rodents following cannabinoid exposure. The authors stress the importance of analyzing the full spectrum of naturally occurring cannabinoids, rather than just the main ones, THC and CBD, and they offer some pointers on performing behavioral analysis in zebrafish.

Quantification of noradrenergic-, dopaminergic-, and tectal-neurons during aging in the short-lived killifish Nothobranchius furzeri.

Parkinson's disease (PD) is characterized by phosphorylation and aggregation of the protein α-Synuclein and ensuing neuronal death progressing from the noradrenergic locus coeruleus to midbrain dopaminergic neurons. In 2019, Matsui and colleagues reported a spontaneous age-dependent degeneration of dopaminergic neurons and an even greater neurodegeneration of the noradrenergic neurons in the short-lived killifish Nothobranchius furzeri. Given the great possible relevance of a spontaneous model for PD, we assessed neurodegeneration of noradrenergic and dopaminergic neurons in two further laboratory strains of N. furzeri. We implemented, for the first time in N. furzeri, a whole-brain clarification technique and proceeded to entire 3D nuclei reconstruction to quantify total cell numbers in two different stains of N. furzeri. In both strains, we observed that age-dependent neurodegeneration is limited to the locus coeruleus and does not involve the posterior tuberculum. We also applied 3D counting to the optic tectum, an area of active adult neurogenesis, and detected an increase of neurons with age. Our results confirm age-dependent neurodegeneration of noradrenergic neurons, a condition reminiscent of the presymptomatic stage of PD indicating that N. furzeri could be used in the future to identify modifying factors for age-dependent neurodegeneration and open the intriguing possibility that natural genetic variation may influence the susceptibility of dopaminergic neurons.

Dietary Supplementation with a Blend of Hydrolyzable and Condensed Tannins Ameliorates Diet-Induced Intestinal Inflammation in Zebrafish (Danio rerio).

The current study evaluated the effects of hydrolyzable and condensed tannins from chestnut and quebracho wood, respectively (TSP, Silvafeed®), on zebrafish with intestinal inflammation induced by a plant-based diet (basal diet). Four experimental diets were prepared as follows: the basal diet + 0 TSP, the basal diet + TSP at 0.9 g/kg of feed, the basal diet + TSP at 1.7 g/kg of feed, and the basal diet + TSP at 3.4 g/kg of feed. Eighty-four zebrafish (Danio rerio) were fed for 12 days with the experimental diets. In zebrafish fed the basal diet, intestine integrity appeared to be altered, with damaged intestinal villi, high immunoexpression of tumor necrosis factor-α (TNFα) and cyclooxygenase 2 (COX2), and high expression of the cox2, interleukin 1 (il-1b), interleukin 8 (cxcl8-l1), and tnfα genes. The tannin treatment partially restored intestinal morphology and downregulated the expression of cytokines. The best activity was detected with 1.7 and 3.4 g/kg of feed. In the guts of all groups, Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes were the most represented phyla. The most represented genera were Plesiomonas and Sphingomonas, belonging to the Proteobacteria phylum; Cetobacterium, belonging to the Fusobacteria phylum; and Lactobacillus, belonging to the Firmicutes phylum. No significant differences were detected among groups, except for a slight decrease in the Fusobacteria phylum and slight increases in the Shewanella and Bacteroides genera with TSP. In conclusion, these results suggest that tannins can improve the zebrafish intestinal inflammation caused by a terrestrial-plant-based diet in a dose-dependent manner.

In Vivo Evaluation of Cannabis sativa Full Extract on Zebrafish Larvae Development, Locomotion Behavior and Gene Expression.

Historically, humans have been using Cannabis sativa for both recreational and medical purposes. Nowadays, cannabis-based products have gained scientific interest due to their beneficial effects on several syndromes and illnesses. The biological activity of cannabinoids is essentially due to the interaction with the endocannabinoid system, and zebrafish (Danio rerio) is a very well-known and powerful in vivo model for studying such specific interactions. The aim of the study was to investigate the effects of different doses of a Cannabis sativa whole extract [dissolved in dimethyl sulfoxide (DMSO)] on zebrafish eggs' hatchability, embryo post-hatching survival, larvae locomotion behavior and mRNA gene expression. The results showed the absence of toxicity, and no significant differences were observed between treatments for both embryo hatching and survival rate. In addition, larvae exposed to the cannabis extract at the highest dose [containing 1.73 nM and 22.3 nM of ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively] showed an increased locomotion compared to the control and DMSO treated groups. Moreover, qRT-PCR analysis showed that the highest dosage of cannabis induced an over-expression of cnr1 and cnr2 cannabinoid receptors. In conclusion, the exposition of zebrafish larvae to the whole extract of Cannabis sativa showed no negative effects on embryo development and survival and enhanced the larvae's locomotor performances. These findings may open up possible Cannabis sativa applications in human pharmacology as well as in other animal sectors.

Effect of Honey and Syrup Diets Enriched with 1,3-1,6 ß-Glucans on Honeybee Survival Rate and Phenoloxidase Activity (Apis mellifera L. 1758).

ß-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 ß-glucans administered orally for 96 h on Apis mellifera workers (newly emerged and nurse bees). ß-glucans were included in honey and syrup. Survival rate and phenoloxidase activity were measured. In both newly emerged and nurse bees, ß-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 ß-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 ß-glucans and the control group (p > 0.05). The absence of significant variation in survival suggests that the potential negative effect of ß-glucans in healthy bees could be mitigated by their metabolism. Conversely, the inclusion of ß-glucans in a honey-based diet determined an increase of phenoloxidase activity, suggesting that the effect of ß-glucan inclusion in the diet of healthy bees on phenoloxidase activity could be linked to the type of base-diet. Further investigations on ß-glucans metabolism in bees, on molecular mechanism of phenoloxidase activation by 1,3-1,6 ß-glucans, and relative thresholds are desirable. Moreover, investigation on the combined action of honey and ß-glucans on phenoloxidase activity are needed.

ß-Glucans as Dietary Supplement to Improve Locomotion and Mitochondrial Respiration in a Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular childhood disorder that causes progressive muscle weakness and degeneration. A lack of dystrophin in DMD leads to inflammatory response, autophagic dysregulation, and oxidative stress in skeletal muscle fibers that play a key role in the progression of the pathology. ß-glucans can modulate immune function by modifying the phagocytic activity of immunocompetent cells, notably macrophages. Mitochondrial function is also involved in an important mechanism of the innate and adaptive immune responses, owing to high need for energy of immune cells. In the present study, the effects of 1,3-1,6 ß-glucans on five-day-old non-dystrophic and dystrophic (sapje) zebrafish larvae were investigated. The effects of the sonication of ß-glucans and the dechorionation of embryos were also evaluated. The results showed that the incidence of dystrophic phenotypes was reduced when dystrophic embryos were exposed to 2 and 4 mg L-1 of 1,3-1,6 ß-glucans. Moreover, when the dystrophic larvae underwent 8 mg L-1 treatment, an improvement of the locomotor performances and mitochondrial respiration were observed. In conclusion, the observed results demonstrated that 1,3-1,6 ß-glucans improve locomotor performances and mitochondrial function in dystrophic zebrafish. Therefore, for ameliorating their life quality, 1,3-1,6 ß-glucans look like a promising diet supplement for DMD patients, even though further investigations are required.

Nutraceutical Screening in a Zebrafish Model of Muscular Dystrophy: Gingerol as a Possible Food Aid.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an inherited neuromuscular disorder that causes loss of muscle mass and motor skills. In the era of genomic medicine, there is still no known cure for DMD. In clinical practice, there is a growing awareness of the possible importance of nutrition in neuromuscular diseases. This is mostly the result of patients' or caregivers' empirical reports of how active substances derived from food have led to improved muscle strength and, thus, better quality of life. In this report, we investigate several nutraceutical principles in the sapje strain of zebrafish, a validated model of DMD, in order to identify possible natural products that, if supplemented in the diet, might improve the quality of life of DMD patients. Gingerol, a constituent of fresh ginger, statistically increased the locomotion of mutant larvae and upregulated the expression of heme oxygenase 1, a target gene for therapy aimed at improving dystrophic symptoms. Although three other compounds showed a partial positive effect on locomotor and muscle structure phenotypes, our nutraceutical screening study lent preliminary support to the efficacy and safety only of gingerol. Gingerol could easily be proposed as a dietary supplement in DMD.

Effects of inulin diet supplementation on production performance, gut traits, and incidence of ascites in Haidong chicks under hypoxic conditions.

OBJECTIVE: Effects of inulin supplementation in diet of Haidong chicks under hypoxic conditions on production performance, intestinal morphologic change, microflora contents and the incidence of ascites were studied. METHODS: Commercial male chicks (360) were randomly divided into 6 groups and were fed diets supplemented with 0, 0.05, 0.075, 0.1, 0.125, and 0.15 g/kg of inulin, respectively. RESULTS: The body weight gain and feed intake were improved in chicks fed the diets supplemented with 0.1 and 0.125 g/kg of inulin, from d 1 to d 42 (p<0.05); moreover, blood parameters were positively affected when inulin was included in the diets and the thickness of the intestinal wall and muscle tissue in duodenum, jejunum, and ileum tended to increase (p<0.05), and the villi height and crypt depth in duodenum, jejunum, and ileum (p<0.05). Regarding the number of goblet cells in duodenum, jejunum and ileum tended to increase when chicks were fed the diets supplemented with 0.075, 0.1, 0.125, and 0.15 g/kg (p<0.05) of inulin. When chicks were fed diets supplemented with 0.75 or 0.1 g/kg of inulin, a significant reduction of Escherichia coli counts in the cecum was observed; for a contrary, a significant increment of Bifidobacterium and Lactobacillus was observed in cecum and ileum. Finally, supplementing the feed with inulin determined an overall reduction of ascites incidences in comparison to the control group. CONCLUSION: Thus, the results observed in the present study clearly suggest that the diet supplementation with a quantity of inulin ranging between 0.1 and 0.125 g/kg, can improve growth performances, intestinal morphology, internal microbial balance and ascites incidence, in broiler chicks raised at high altitude area. Even though these findings may be of interest for the poultry industry, they may particularly be relevant in those areas characterized by high altitude such as Northwest China regions.

The effect of Aspergillus niger as a dietary supplement on blood parameters, intestinal morphology, and gut microflora in Haidong chicks reared in a high altitude environment.

AIM: The effects of the inclusion of Aspergillus niger in the diet of Haidong chicks reared in the Qing-Zang high altitude area (China) under hypoxic conditions. MATERIALS AND METHODS: A total of 720 Haidong chicks were randomly divided into six groups and fed diets supplemented with 0%, 0.5%, 0.75%, 1.0%, 1.25%, and 1.5% of A. niger to determine blood parameters, intestinal morphology, and gut microflora in Haidong chicks reared in a high altitude environment. RESULTS: Packed cell volume (PCV), red blood cell (RBC) count, white blood cell count, and hemoglobin concentration increased in the groups fed diets containing A. niger. The administration of A. niger in 1.0% and 1.25% significantly decreased the concentration of Escherichia coli in the cecum, while the concentration of Bifidobacterium and Lactobacillus in the cecum and ileum was increased in the treated groups. When compared to the control groups, villi height, crypt depth, and goblet cell density in the intestine was raised, in general, in the groups treated with A. niger. CONCLUSION: These findings suggest that 1.25% A. niger as dietary supplement may improve the resistance to ascites among birds reared under hypoxic conditions.

Effect of Oral Administration of 1,3-1,6 ß-Glucans in DWV Naturally Infected Newly Emerged Bees (Apis mellifera L.).

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 ß-glucans as a dietary supplement. In this work, by collecting faecal samples of honeybees exposed to different dietary conditions of 1,3-1,6 ß-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 ß-glucans to reduce the viral load at the 24th day of rearing. The results indicated that the diet supplemented with 1,3-1,6 ß-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 ß-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.

Social Preference Tests in Zebrafish: A Systematic Review.

The use of animal models in biology research continues to be necessary for the development of new technologies and medicines, and therefore crucial for enhancing human and animal health. In this context, the need to ensure the compliance of research with the principles Replacement, Reduction and Refinement (the 3 Rs), which underpin the ethical and human approach to husbandry and experimental design, has become a central issue. The zebrafish (Danio rerio) is becoming a widely used model in the field of behavioral neuroscience. In particular, studying zebrafish social preference, by observing how an individual fish interacts with conspecifics, may offer insights into several neuropsychiatric and neurodevelopmental disorders. The main aim of this review is to summarize principal factors affecting zebrafish behavior during social preference tests. We identified three categories of social research using zebrafish: studies carried out in untreated wild-type zebrafish, in pharmacologically treated wild-type zebrafish, and in genetically engineered fish. We suggest guidelines for standardizing social preference testing in the zebrafish model. The main advances gleaned from zebrafish social behavior testing are discussed, together with the relevance of this method to scientific research, including the study of behavioral disorders in humans. The authors stress the importance of adopting an ethical approach that considers the welfare of animals involved in experimental procedures. Ensuring a high standard of animal welfare is not only good for the animals, but also enhances the quality of our science.

Fishing in the Cell Powerhouse: Zebrafish as A Tool for Exploration of Mitochondrial Defects Affecting the Nervous System.

The zebrafish (Danio rerio) is a small vertebrate ideally suited to the modeling of human diseases. Large numbers of genetic alterations have now been modeled and could be used to study organ development by means of a genetic approach. To date, limited attention has been paid to the possible use of the zebrafish toolbox in studying human mitochondrial disorders affecting the nervous system. Here, we review the pertinent scientific literature discussing the use of zebrafish in modeling gene mutations involved in mitochondria-related neurological human diseases. A critical analysis of the literature suggests that the zebrafish not only lends itself to exploration of the pathological consequences of mitochondrial energy output on the nervous system but could also serve as an attractive platform for future drugs in an as yet untreatable category of human disorders.

Effects of dietary yeast beta-1,3-1,6-glucan on growth performance, intestinal morphology and chosen immunity parameters changes in Haidong chicks.

Objective: This study investigated the effects of 1,3-1,6 beta-glucan added to the diet of Haidong chicks reared under hypoxic conditions, to ascertain the growth performances, immunity and intestinal morphology changes. Methods: A total of 750 chicks were divided into five groups and fed diets containing 0.5g/kg, 1.0g/kg and 2.0g/kg 1,3-1,6 beta-glucan from yeast (G1, G2, G3, respectively), 0.2g/kg Taylor rhizomorph (T) and a control feed (C). Results: The body weight and body weight gain was higher in chicks fed 1,3-1,6 beta-glucan and Taylor rhizomorph than in control group. Feed conversion ratio significantly differed for G2 and G3 groups in comparison to control group. The relative weight of bursa was higher in G1, G2 and G3 groups. The white blood cells and lymphocytes were significantly increased in groups fed 1,3-1,6 beta-glucan. The IgG of serum peak appeared in the G3 group. The villous height of the duodenum was higher in 1,3-1,6 beta-glucan feed groups. In the jejunum, the villous height was higher in G2 and G3 groups and crypt depth for all the groups fed ß-glucan;. At ileum level the villous height and crept depth was higher for groups G1, G2 and G3. Conclusion: The growth performance of Haidong chicks is improved when 10 and 20 g/kg 1,3-1,6 beta-glucan is included in the diet; hence, it is suggested to diet include 1,3-1,6 beta-glucan in poultry diet to reduce and replace the use of antibiotics.

In Vitro Activity of Essential Oils against Saprolegnia parasitica.

Saprolegnia spp. water molds severely impact fish health in aquaculture, fish farms and hobby fish tanks colonizing mature and immature stages of fishes, as well as eggs. Considering that there are no drugs licensed for treating and/or control the organism, efficient and environmental low-impact methods to control these oomycetes in aquaculture are needed. The aim of the present report was to evaluate the in vitro sensitivity of Saprolegnia parasitica to essential oils (EOs) from Citrus aurantium L., Citrus bergamia Risso et Poiteau, Citrus limon Burm. f., Citrus paradisi Macfad, Citrussinensis Osbeck, Cinnamomum zeylanicum Blume, Cymbopogon flexuosum (Nees ex Steud.) Watson, Foeniculum vulgare Mill., Illicium verum Hook.f., Litsea cubeba (Lour.) Pers., Origanum majorana L., Origanum vulgare L., Pelargonium graveolens L'Hér., Syzygium aromaticum Merr. & L.M.Perry, and Thymus vulgaris L., by microdilution test. The most effective EOs assayed were T. vulgaris and O. vulgare, followed by C. flexuosum, L. cubeba and C. bergamia. These EOs could be of interest for controlling Saprolegnia infections. Nevertheless, further safety studies are necessary to evaluate if these products could be dispersed in tank waters, or if their use should be limited to aquaculture supplies.

Feeding of nano scale oats ß-glucan enhances the host resistance against Edwardsiella tarda and protective immune modulation in zebrafish larvae.

In this study, we prepared and characterized the oats origin of nano scale ß-glucan (NBG) and investigated the immunomodulatory properties in zebrafish larvae. Newly prepared NBG (average particle size of 465 nm) was fully soluble in water. Zebrafish larvae survival rate was increased against pathogenic bacteria Edwardsiella tarda, when NBG was added to the water (500 µg/mL) compared to NBG non-exposed controls. Moreover, quantitative real time PCR (qRT-PCR) results showed up-regulation of immune functional genes including TNF-α, IL-1ß, ß-defensin, lysozyme, IL 10, IL 12 and C-Rel indicating higher survival rate could be due to stronger immunomodulatory function of NBG (500 µg/mL). Thus, non-toxic, water soluble and biodegradable NBG from oats could be considered as the potential immunostimulant for larval aquaculture.

Effect of 1,3-1,6 ß-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 ß-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.