Seasonal trends of the ABPV, KBV, and IAPV complex in Italian managed honey bee (Apis mellifera L.) colonies.

Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), and Israeli acute paralysis virus (IAPV) usually persist as covert infections in honey bee colonies. They can cause rapid bee mortality in cases of severe infection, often associated with high Varroa destructor infestation, by which they are transmitted. In various countries, these viruses have been associated with colony collapse. Despite their potential danger, these viruses are often disregarded, and little information is available on their occurrence in many countries, including Italy. In 2021, 370 apiaries representing all of the Italian regions were investigated in four different months (June, September, November, and March) for the presence of ABPV, KBV, and IAPV. IAPV was not found in any of the apiaries investigated, whereas 16.45% and 0.67% of the samples tested positive for ABPV and KBV, respectively. Most ABPV cases occurred in late summer-autumn in both northern and southern regions. We observed a scattered pattern of KBV-positive colonies that did not allow any seasonal or regional trends to be discerned. Differences observed among regions and months were potentially related to the dynamics of varroa infestation, viral genetic variations, and different climatic conditions resulting in variations in bee behaviour. This study improves our understanding of the circulation of bee viruses and will contribute to better disease prevention and preservation of bee health.

Effect of amino acid enriched diets on hemolymph amino acid composition in honey bees.

Amino acids (AAs) are an abundant class of nectar solutes, and they are involved in the nectar attractiveness to flower visitors. Among the various AAs, proline is the most abundant proteogenic AA, and γ-amino butyric acid (GABA) and ß-alanine are the two most abundant non-proteogenic AAs. These three AAs are known to affect insect physiology, being involved in flight metabolism and neurotransmission. The aim of this study was to investigate the effects of artificial diets enriched with either ß-alanine, GABA, or proline on consumption, survival, and hemolymph composition in honey bees belonging to two different ages and with different metabolism (i.e., newly emerged and foragers). Differences in feed intake among diets were not observed, while a diet enriched with ß-alanine improved the survival rate of newly emerged honey bees compared to the control group. Variations in the hemolymph AA concentrations occurred only in newly emerged honey bees, according to the diet and the time of hemolymph sampling. A greater susceptibility of young honey bees to enriched diets than older honey bees was observed. The variations in the concentrations of hemolymph AAs reflect either the accumulation of dietary AAs or the existence of metabolic pathways that may lead to the conversion of dietary AAs into different ones. This investigation could be an initial contribution to studying the complex dynamics that regulate hemolymph AA composition and its effect on honey bee physiology.

Oxalic Acid Treatment: Short-Term Effects on Enzyme Activities, Vitellogenin Content, and Residual Oxalic Acid Content in House Bees, Apis mellifera L.

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.

Spatiotemporal evolution of the distribution of Chronic bee paralysis virus (CBPV) in honey bee colonies.

Chronic bee paralysis virus (CBPV) is a Apis mellifera viral infectious disease, exhibiting dark and hairless abdomen in workers with tremors and ataxita. Clinical signs are also typically linked to adverse weather conditions and overcrowding in the hive. The disease occurs in spring but recently it has been observed cases increase and seasonality loss of the disease incidence. This study analyses the evolution of CBPV in Italy, through data collected from 2009 to 2023 within three monitoring projects comprising nationwide extended detection networks, aimed to investigate the evolution of the CBPV spatial distribution, identifying high-risk areas for the virus spread. This study highlights an increased risk over years. Prevalence increased from 4.3% during 2009-2010 to 84.7% during 2021-2023 monitoring years. CBPV outbreaks were irregular between investigated seasons, highlighting Spring and Autumn as the most susceptible seasons. Risk of CBPV infection has increased, reaching high-risk in last years of monitoring. Sequence analysis showed a high similarity to other isolated Italian CBPVs. The study offers an epidemiological insight into the aetiology of this disease. CBPV distribution is a prerequisite to predict its future spread and factors involved in its propagation not only in honey bees but also in other pollinators and environments.

Effects of Virgin Coconut Oil-Enriched Diet on Immune and Antioxidant Enzymatic Activity, Fat and Vitellogenin Contents in Newly Emerged and Forager Bees (Apis mellifera L.) Reared in Cages.

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.

Preliminary investigation on the effect of insect-based chitosan on preservation of coated fresh cherry tomatoes.

Chitosan was produced from Hermetia illucens pupal exuviae by heterogeneous and homogeneous deacetylation. Tomato fruits (Solanum lycopersicum), that are one of the most grown and consumed food throughout the world, were coated with 0.5 and 1% chitosan, applied by dipping or spraying, and stored at room temperature or 4 °C, for a storage period of 30 days. Statistical analysis give different results depending on the analysed parameters: heterogeneous chitosan, indeed, had a better effect than the homogenous one in maintaining more stable physico-chemical parameters, while the homogenous chitosan improved the total phenols, flavonoids and antioxidant activity. Chitosan coatings applied by spraying were more effective in all the analyses. Chitosan derived from H. illucens always performed similarly to the commercial chitosan. However, a general better performance of insect-derived chitosan on the concentration of phenolics and flavonoids, and the antioxidant activity was observed as compared to the commercial one. Chitosan coating has already been successfully used for preservation of fresh fruits, as alternative to synthetic polymers, but this is the first investigation of chitosan produced from an insect for this application. These preliminary results are encouraging regarding the validation of the insect H. illucens as a source of chitosan.

The Epidemiological Situation of the Managed Honey Bee (Apis mellifera) Colonies in the Italian Region Emilia-Romagna.

The recent decades witnessed the collapse of honey bee colonies at a global level. The major drivers of this collapse include both individual and synergic pathogen actions, threatening the colonies' survival. The need to define the epidemiological pattern of the pathogens that are involved has led to the establishment of monitoring programs in many countries, Italy included. In this framework, the health status of managed honey bees in the Emilia-Romagna region (northern Italy) was assessed, throughout the year 2021, on workers from 31 apiaries to investigate the presence of major known and emerging honey bee pathogens. The prevalence and abundance of DWV, KBV, ABPV, CBPV, Nosema ceranae, and trypanosomatids (Lotmaria passim, Crithidia mellificae, Crithidia bombi) were assessed by molecular methods. The most prevalent pathogen was DWV, followed by CBPV and N. ceranae. Trypanosomatids were not found in any of the samples. Pathogens had different peaks in abundance over the months, showing seasonal trends that were related to the dynamics of both bee colonies and Varroa destructor infestation. For some of the pathogens, a weak but significant correlation was observed between abundance and geographical longitude. The information obtained in this study increases our understanding of the epidemiological situation of bee colonies in Emilia-Romagna and helps us to implement better disease prevention and improved territorial management of honey bee health.

Effects of Two Commercial Protein Diets on the Health of Two Imago Ages of Apis mellifera L. Reared in Laboratory.

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.

Queen Caging and Oxalic Acid Treatment: Combined Effect on Vitellogenin Content and Enzyme Activities in the First Post-Treatment Workers and Drones, Apis mellifera L.

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.

Impact of Different Storage Temperature on the Enzymatic Activity of Apis mellifera Royal Jelly.

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.

Antimicrobial properties of chitosan from different developmental stages of the bioconverter insect Hermetia illucens.

Growing antimicrobial resistance has prompted researchers to identify new natural molecules with antimicrobial potential. In this perspective, attention has been focused on biopolymers that could also be functional in the medical field. Chitin is the second most abundant biopolymer on Earth and with its deacetylated derivative, chitosan, has several applications in biomedical and pharmaceutical fields. Currently, the main source of chitin is the crustacean exoskeleton, but the growing demand for these polymers on the market has led to search for alternative sources. Among these, insects, and in particular the bioconverter Hermetia illucens, is one of the most bred. Chitin can be extracted from larvae, pupal exuviae and dead adults of H. illucens, by applying chemical methods, and converted into chitosan. Fourier-transformed infrared spectroscopy confirmed the identity of the chitosan produced from H. illucens and its structural similarity to commercial polymer. Recently, studies showed that chitosan has intrinsic antimicrobial activity. This is the first research that investigated the antibacterial activity of chitosan produced from the three developmental stages of H. illucens through qualitative and quantitative analysis, agar diffusion tests and microdilution assays, respectively. Our results showed the antimicrobial capacity of chitosan of H. illucens, opening new perspectives for its use in the biological area.

Characterization of chitin and chitosan derived from Hermetia illucens, a further step in a circular economy process.

Due to their properties and applications, the growing demand for chitin and chitosan has stimulated the market to find more sustainable alternatives to the current commercial source (crustaceans). Bioconverter insects, such as Hermetia illucens, are the appropriate candidates, as chitin is a side stream of insect farms for feed applications. This is the first report on production and characterization of chitin and chitosan from different biomasses derived from H. illucens, valorizing the overproduced larvae in feed applications, the pupal exuviae and the dead adults. Pupal exuviae are the best biomass, both for chitin and chitosan yields and for their abundance and easy supply from insect farms. Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscope analysis revealed the similarity of insect-derived polymers to commercial ones in terms of purity and structural morphology, and therefore their suitability for industrial and biomedical applications. Its fibrillary nature makes H. illucens chitin suitable for producing fibrous manufacts after conversion to chitin nanofibrils, particularly adults-derived chitin, because of its high crystallinity. A great versatility emerged from the evaluation of the physicochemical properties of chitosan obtained from H. illucens, which presented a lower viscosity-average molecular weight and a high deacetylation degree, fostering its putative antimicrobial properties.

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.

Microbial Profile of the Ventriculum of Honey Bee (Apis mellifera ligustica Spinola, 1806) Fed with Veterinary Drugs, Dietary Supplements and Non-Protein Amino Acids.

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.GABA; beta-alanine; oxalic acid; diet effect; microbiota.