The effect of pollen monodiets on fat body morphology parameters and energy substrate levels in the fat body and hemolymph of Apis mellifera L. workers.

Human activities associated with large-scale farms and the monocultures expose honey bees to one type of food. Moreover, there is an ongoing decline of plant species producing pollen and nectar in Europe. A poorly balanced diet affects a number of processes occurring in a bee's body. The fat body and hemolymph are the tissues that participate in all of them. Therefore, the aim of our study was to determine the effect of hazel, pine, rapeseed, buckwheat, phacelia and goldenrod pollen on the morphological parameters of fat body trophocytes, the diameters of cell nuclei in oenocytes and the concentrations of compounds involved in energy metabolism (glucose, glycogen, triglycerides and protein). In the cage tests, the bees were fed from the first day of life with sugar candy (control group) or candy with a 10% addition of one of the 6 pollen types. Hemolymph and fat body from various locations were collected from 1-, 7- and 14-day-old workers. Pollen produced by plant species such as hazel and pine increased glucose concentrations in the bee tissues, especially in the hemolymph. It can therefore be concluded that they are valuable sources of energy (in the form of simple carbohydrates) which are quickly used by bees. Pollen from plants blooming in the summer and autumn increased the concentrations of proteins, glycogen and triglycerides in the fat body, especially that from the third tergite. The accumulation of these compounds was associated with an increased the length and width of trophocytes as well as with enhanced metabolic activity, which was evidenced in the increasing diameter of oenocyte cell nuclei. It seems a balanced multi-pollen diet is more valuable for bees, but it is important to understand the effects of the particular pollen types in the context of a mono-diet. In the future, this will make it possible to produce mixtures that can ensure homeostasis in the apian body.

The Key Role of Amino Acids in Pollen Quality and Honey Bee Physiology-A Review.

When studying honey bee nutrition, it is important to pay attention not only to the quantity but also to the quality of pollen for floral visitors. The recommended way to determine the value of pollen is to determine both the protein concentration and the amino acid composition in the insect's hemolymph. In addition, the composition of pollen also includes lipids, sterols and biogenic elements such as carbon, nitrogen, etc. Very high protein concentration is observed in aloe pollen, averaging 51%. Plants with a high protein content, at the level of 27% in Europe, are rapeseed and phacelia. In turn, a plant that is poor in protein (at the level of 11%) is buckwheat. The aforementioned plants are sown over very large areas. Vast acreages in Central and Eastern Europe are occupied by pollen- and nectar-providing invasive plants, such as goldenrod. Therefore, bees are forced to use one food source-a mono diet-which results in their malnutrition. In the absence of natural pollen, beekeepers use other foods for bees; including soy protein, powdered milk, egg yolks, fish meal, etc. However, the colony is the strongest when bees are fed with pollen, as opposed to artificial protein diets. More research is needed on the relationship between bee pollen composition and nutrition, as measured by protein concentration and amino acid composition in apian hemolymph, colony strength, honey yield and good overwintering.

The Morphological Image of Fat Body and Tergal Gland Cells in Uninseminated Apis mellifera Queen Bees.

The morphological changes in fat body cells, tergal gland cells, and the surface areas of the cell nuclei were determined in queen bees of the subspecies Apis mellifera carnica. This study focused on 1-, 8-, and 20-day-old uninseminated females kept in colonies, analyzing cells from three locations in the abdomen: the sternite, and tergites III and V. The oenocytes in the sternites were large, oval/circular with a centrally located nucleus, while in tergites III and V, they were small and triangular in the 1-day-old queens. During the first week of life, these cells in tergites III and V change their shape to oval and increase their sizes. The initially light yellow and then dark yellow granularities in the oenocytes of the fat body appear along with the advancing age of the queens. The trophocytes (sternites, tergites III and V) in the 1-day-old queens were completely filled with droplets of different sizes. In the 8- and 20-day-old queens, the number and size of the droplets decreased in the trophocytes of tergites III and V. The tergal gland cells had a centrally located cell nucleus in the 1-, 8- and 20-day-old queens. The dark granularities in these cells were visible only in the 20-day-old queens. Different morphological images of the fat body at the sternite, and tergites III and V, and the difference in the size of the oenocyte cell nuclei may indicate various functions of the fat body depending on its location. Characterization of the changes in the morphology of the fat body, taking into account its segmental character, and the tergal glands requires further research in older queens, e.g., one-year-old, brooding queens.

Physiological Effect of Gentle Stroking in Lambs.

The aim of the study was to determine changes in physiological indicators in lambs subjected to gentle stroking. The study included 40 three-week-old lambs (20 females of the synthetic prolific-meat (BCP) line and 20 females of the Swiniarka (SW) breed). The animals were assigned to two control groups (n = 20) and two experimental groups (n = 20). Lambs from the experimental groups received treatment. Analyses consisted of determining the following parameters: heart rate (HR), saturation (S) and biochemical analysis. In the groups of sheep subjected to gentle massage, the analyses revealed a significant increase in the levels of cortisol, CAT, GST, GPx, among others, and a significant decrease in the levels of total protein, SOD, TAC, uric acid and Na+. At the same time, indicators of reduced stress levels were revealed, i.e., a significant increase in glycogen levels and a significant decrease in glucose and HR values. These results suggest that the introduction of gentle touch can induce positive states in lambs, and that the stress response may be transient and adaptive. Nevertheless, it is important to note that these interactions can be a source of stress, even if the behavioral response does not necessarily indicate stress.

The influence of selected factors on wool cortisol concentration in alpacas (Vicugna pacos).

Several internal and external factors can influence animals' hormonal activity. Cortisol level in hair and wool determines chronic stress, which is connected with the long-term HPA axis effect. Wool cortisol levels in alpacas have never been determined to this time. The study aimed to assess the influence of selected factors on wool cortisol concentration in alpacas. The study included 36 alpacas. Wool samples were collected during shearing in June 2021, cut with an electric clipper from the right shoulder and the rump. Wool samples were fragmented into proximal (winter-spring regrowth) and distal (summer-fall regrowth) segments. Alpacas' Heat Stress Index (HSI) for the summer of 2020 was 139.4, and 116 for the winter of 2021. The cortisol levels in the wool samples were determined with the General Cortisol ELISA Kit assay. The most significant differences in wool cortisol concentrations were caused by two factors: the wool segment (P < 0.001; η2 = 0.889) and the region on the body (P < 0.001; η2 = 0.876). Wool cortisol level was higher in the distal segment (referring to the summer-fall season) than in the proximal one (referring to the winter-spring season). It is suggested that alpacas can feel heat stress in summer (HSI = 139.4), which could influence higher cortisol levels in the distal segment. The wool cortisol level was higher in the rump samples than the shoulder ones. Therefore, it is essential in future studies that wool samples from all tested animals should be completed from the same body region. Differences among age and sex groups were also observed. Wool cortisol level was higher in older animals, as differences between age groups were observed in samples from the rump in the distal and proximal segments (distal, the rump younger*older: P < 0.001; η2 = 0.321; proximal, the rump older*younger: P = 0.007; η2 = 0.195). Males showed higher cortisol levels than females, as a difference between sexes was observed in samples from the rump in the proximal segment (P = 0.001, η2 = 0.271). This study emphasizes that various factors may significantly influence wool cortisol levels, which can be helpful in alpacas' welfare estimation using this hormonal indicator as a noninvasive long-term stress assessment method.

Workers of Apis mellifera Reared in Small-Cell Combs Show Higher Activity of the Proteolytic System in Hemolymph than Workers Reared in Standard-Cell Combs in Laboratory Cage Tests.

The aim of the study was to compare the activities of proteases and their inhibitors in the hemolymph of honeybee workers reared in small-cell combs (SMC) and standard-cell combs (STC) in laboratory cage tests. The analyses conducted in laboratory conditions facilitated assessment of the impact of the comb cell width (small vs. standard) along with eliminating the influence of many environmental factors on the results. The width of the comb cells in which the workers were reared had a significant effect on the protein concentrations and proteolytic system activities in the hemolymph. Irrespective of the age of the workers, higher protein concentrations were found in the hemolymph of the SMC workers. In turn, the activities of proteases and their inhibitors in the hemolymph of 1-day-old bees were higher in the STC workers. In older bees, aged 7-21 days, activity was higher in the SMC workers. The role of the considerable cell width variability in natural combs that were built without the use of an artificially produced wax foundation is worth investigating. It is highly probable that the impact of the comb cell width on the features of workers reared in these combs modifies the age polyethism in the worker caste as well. The investigation results of one-season studies of honeybees could be seriously affected by random factors. To reduce the risk of these effects, it is advisable to continue experiments over a few consecutive years.

Effect of Rearing in Small-Cell Combs on Activities of Catalase and Superoxide Dismutase and Total Antioxidant Capacity in the Hemolymph of Apis mellifera Workers.

Honeybee nests constructed without man-made wax foundation have significantly more variability of cell widths/sizes than those in commercially-kept colonies. The effects of this natural variability in comb cell widths on individual and colony traits have not been explained to date. The investigation of this problem can lead to new findings about the biology, physiology, and possibly, the evolution of the honeybee. The aim of the study was to compare the catalase and superoxide dismutase activities and the total antioxidant capacity levels in the hemolymph of honeybee workers reared in small-cell combs and standard-cell combs in colonies kept simultaneously on standard- and small-cell combs. The ratio of the small-cell combs to the standard-cell combs in the nest was 1:1. The workers reared in small-cell combs were characterized by higher antioxidant activities in the hemolymph than those reared in standard-cell combs. Consequently, their hemolymph had a greater antioxidant capacity, which indicates that they may be better predisposed to be foragers than workers reared in standard-cell combs. To describe the physiological differences between worker bees reared in small- and standard-cell combs in the same colony, the role of the considerable variation in the cell width in natural combs built without the use of artificially produced wax foundation is worth elucidating. The comparison of the apiary and cage experiments indicated that changes in antioxidant activities predominantly result from worker activities, especially those requiring the intensification of metabolism, rather than the age of the worker bees. To reduce the impact on the results of random environmental factors potentially present in one-season studies of honeybee research, investigations should preferably be carried out over a few consecutive years.

Cannabidiol (CBD) Supports the Honeybee Worker Organism by Activating the Antioxidant System.

In the experiment, we tested the effect of 30% CBD oil on the activity of the antioxidant system (superoxide dismutase, catalase, glutathione peroxidase, glutathione), the level of total antioxidant capacity, and the concentrations of ions (calcium, magnesium, and phosphorus) in honeybee workers in the hive test. For this purpose, we prepared hives containing all stages of the development of honey bees and started the experiment by adding 200 marked, one-day old bees to each colony (intended for hemolymph collection). In the test, we created three groups (two colonies per group): (1) Experimental with CBD oil mixed with sugar syrup (CSy); (2) experimental with CBD oil on textile strips (CSt); and (3) control with pure sugar syrup only (C). Every week, we collected hemolymph from the marked bees. In the experiment, all antioxidant enzyme activities were higher for the experimental groups CSy and CSt compared to group C. The highest concentrations/levels were obtained for the CSy group. Concentrations of calcium, magnesium, and phosphorus ions were also higher for the experimental groups compared to the C group (the highest concentration for the CSy group). We conclude that CBD oil positively contributes to stimulating the antioxidant system of honeybees.

Imidacloprid Pesticide Causes Unexpectedly Severe Bioelement Deficiencies and Imbalance in Honey Bees Even at Sublethal Doses.

Pesticides impair honeybee health in many ways. Imidacloprid (IMD) is a pesticide used worldwide. No information exists on how IMD impact the bees' body bioelement balance, which is essential for bee health. We hypothesized that IMD disturbs this balance and fed the bees (in field conditions) with diets containing 0 ppb (control), 5 ppb (sublethal considered field-relevant), and 200 ppb (adverse) doses of IMD. IMD severely reduced the levels of K, Na, Ca, and Mg (electrolytic) and of Fe, Mo, Mn, Co, Cu, Ni, Se, and Zn, while those of Sn, V, and Cr (enzymatic) were increased. Levels of P, S, Ti, Al, Li, and Sr were also decreased, while only the B content (physiologically essential) was increased. The increase in Tl, Pb, and As levels (toxic) was alarming. Generally, IMD, even in sublethal doses, unexpectedly led to severe bioelement malnutrition in 69% of bioelements and to a stoichiometric mismatch in the remaining ones. This points to the IMD-dependent bioelement disturbance as another, yet unaccounted for, essential metabolic element which can interfere with apian health. Consequently, there is a need for developing methods of bioelement supplementation of the honey bee diet for better preventing bee colony decline and protecting apian health status when faced with pesticides.

Grassland versus forest dwelling rodents as indicators of environmental contamination with the zoonotic nematode Toxocara spp.

Small mammals are suspected of contributing to the dissemination of Toxocara canis and helping with the parasite survival during periods when there is a temporary absence of suitable definitive hosts. While the primary aim of the current study was the assessment of seroprevalence of Toxocara spp. infections in wild rodents in Poland, we also explored the role of intrinsic (sex, age) and extrinsic factors (study site) influencing dynamics of this infection to ascertain whether grassland versus forest rodents play a greater role as indicators of environmental contamination with T. canis. We trapped 577 rodents belonging to four species (Myodes glareolus, Microtus arvalis, Microtus agrestis, Alexandromys oeconomus) in north-eastern Poland. Blood was collected during the parasitological examination, and serum was frozen at - 80 °C until further analyses. A bespoke enzyme-linked immunosorbent assay was used to detect antibodies against Toxocara spp. We found Toxocara spp. antibodies in the sera of all four rodent species with an overall seroprevalence of 2.8% [1.9-4.1%]. There was a significant difference in seroprevalence between vole species, with the grassland species (M. arvalis, M. agrestis and A. oeconomus) showing a 16-fold higher seroprevalence (15.7% [8.7-25.9%]) than the forest-dwelling M. glareolus (0.98% [0.5-1.8%]). We hypothesise that the seroprevalence of Toxocara spp. differs between forest and grassland rodents because of the higher contamination of grasslands by domestic dogs and wild canids. Our results underline the need for wide biomonitoring of both types of ecosystems to assess the role of rodents as indicators of environmental contamination with zoonotic pathogens.

Natural Substances, Probiotics, and Synthetic Agents in the Treatment and Prevention of Honeybee Nosemosis.

Honeybees are important pollinators, but they are continuously exposed to a variety of fungal and bacterial diseases. One of the various diseases affecting honeybees is nosemosis caused by microsporidia from the Nosema genus. Honeybees are mainly infected through consumption of infected food or faeces containing Nosema spp. spores. Nosemosis causes damage to the middle intestine epithelium, which leads to food absorption disorders and honeybee malnutrition. Fumagillin, i.e., the antibiotic used to treat nosemosis, was withdrawn in 2016 from EU countries. Therefore, researchers have been looking for compounds of both natural and synthetic origin to fight nosemosis. Such compounds should not have a negative impact on bees but is expected to inhibit the disease. Natural compounds tested against nosemosis include, e.g., essential oils (EOs), plant extracts, propolis, and bacterial metabolites, while synthetic substances tested as anti-nosemosis agents are represented by porphyrins, vitamins, antibiotics, phenolic, ascorbic acids, and others. This publication presents an 18-year overview of various studies of a number of natural and synthetic compounds used in the treatment and prevention of nosemosis cited in PubMed, GoogleScholar, and CrossRef.

CBD Supplementation Has a Positive Effect on the Activity of the Proteolytic System and Biochemical Markers of Honey Bees (Apis mellifera) in the Apiary.

We examined how CBD extract influences the activity of the immune system in the hemolymph of honey bees in the hive test. The bees were divided into 3 groups: (CSy) bees fed with CBD in sugar syrup with glycerin; (CSt) cotton strip with CBD placed in hive bees fed pure sugar syrup, (C) control bees fed sugar syrup with glycerin. CBD extract increased the total protein concentrations, proteases and their inhibitor activities in each age (the except for acidic protease activities in the 21st and 28th day and alkaline protease inhibitor activities in the 28th day in CSt group) in comparison with group C. In the groups with the extract there was also an increase in the enzymatic marker activities: ALP, AST (decrease on day 28 for CSt), ALT; and non-enzymatic marker concentrations: glucose; triglycerides; cholesterol and creatinine. The urea acid and albumin concentrations were lower in CSy and CSt groups compared to the C group (higher concentration of albumin was displayed by control bees). Higher activities/concentrations of most of biochemical parameters were obtained in the CSy compared to the CSt and C. CBD supplementation can positively influence workers' immune system.

Are Hair Cortisol Levels of Humans, Cats, and Dogs from the Same Household Correlated?

Human-animal interactions and the emotional relationship of the owner with the pet are the subjects of many scientific studies and the constant interest of not only scientists but also pet owners. The aim of this study was to determine and compare the hair cortisol levels of dogs, cats, and their owners living in the same household. The owners were asked to complete a questionnaire concerning the frequency of their interactions with pets and emotional relationship with each of their cats and each of their dogs. The study involved 25 women who owned at least one dog and at least one cat. In total, 45 dogs and 55 cats from 25 households participated in the study. The average level of hair cortisol of the owners was 4.62 ng/mL, of the dogs 0.26 ng/mL, and in the hair of cats 0.45 ng/mL. There was no significant correlation between the hair cortisol level of the owner and dog or the owner and the cat and between dogs and cats living together. A significant positive correlation was observed between the hair cortisol level in the owner and the pet, for dogs in which the owner performs grooming treatments once a week and for cats which are never kissed. Although our study did not find many significant correlations, studies using other stress markers might have yielded different results.

Do the different life history strategies of ants and honeybees determine fat body morphology?

The separation of two sister groups such as ants and bees in the Cretaceous involved the development of distinctive characteristics to occupy separate ecological niches. From the point of view of biology and ecology, it is important to see how different life history strategies affect the physiology of these insects. The fat body is the most metabolically important tissue in the organism of each insect. Therefore, we conducted a comparative analysis of the morphological image of the subcuticular fat body in different localisation/segments in Formica (Seviformica) cinerea and Apis mellifera mellifera foragers, because of the similarity of their functions in colonies. We observed that the fat bodies of ants and bees were composed of the same cell types: trophocytes and oenocytes. However, in each of the segments, the fat body cells in ants were bigger and there were fewer of them in comparison with bees. The dorsal part of the fat body of ants had a bilayer structure, where the outer layer was formed by binucleated oenocytes. Binucleated oenocytes were also found in the inner layer near the heart and tracheole. In bees, the fat body was unilayered and the trophocytes and oenocytes were present side by side. The similarities and, in particular, the differences in the structure of the fat body are the adaptation of these sister groups to life in a diverse environment.

Activities of Antioxidant and Proteolytic Systems and Biomarkers in the Fat Body and Hemolymph of Young Apis mellifera Females.

The proteolytic and antioxidant systems are important components of humoral immunity, and these biomarkers indicate the immune status. These compounds are synthesized in the bees' fat body and released into the hemolymph. Their functions maintain the organism's homeostasis and protect it against adverse environmental factors (including pathogens). We determined the activities of acidic, neutral, and alkaline proteases and their inhibitors, as well as superoxide dismutase (SOD), catalase (CAT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and the level of total antioxidant potential (TAC). These compounds were investigated in the fat body and hemolymph in the females with increased reproductive potential, i.e., queens and rebels, and in normal (non-reproductive sterile non-rebel) workers. The phenoloxidase (PO) activities were determined in the hemolymph. The normal workers had higher activities of proteases and their inhibitors, SOD and CAT, in the fat body and hemolymph, compared to the queens and rebels. The protease inhibitors were not usually active in the queens. As we predicted, the rebels revealed values between those of the queens and normal workers. The highest activities of proteases and antioxidants were identified in the fat body from the third tergite in comparison with the sternite and the fifth tergite. These results are important for oxidative stress ecology and give a better understanding of the functioning of the fat body and the division of labor in social insects.

Morphology of Nasonov and Tergal Glands in Apis mellifera Rebels.

Social insect societies are characterized by a high level of organization. This is made possible through a remarkably complex array of pheromonal signals produced by all members of the colony. The queen's pheromones signal the presence of a fertile female and induce daughter workers to remain sterile. However, the lack of the queen mandibular pheromone leads to the emergence of rebels, i.e., workers with increased reproductive potential. We suggested that the rebels would have developed tergal glands and reduced Nasonov glands, much like the queen but contrary to normal workers. Our guess turned out to be correct and may suggest that the rebels are more queen-like than previously thought. The tergal gland cells found in the rebels were numerous but they did not adhere as closely to one another as they did in queens. In the rebels, the number of Nasonov gland cells was very limited (from 38 to 53) and there were fat body trophocytes between the glandular cells. The diameters of the Nasonov gland cell nuclei were smaller in the rebels than in the normal workers. These results are important for understanding the formation of the different castes of Apis mellifera females, as well as the division of labor in social insect societies.

Impressive Impact of Hemp Extract on Antioxidant System in Honey Bee (Apis mellifera) Organism.

We examined the effect of hemp extract on the activity of the antioxidant system (catalase, peroxidase, glutathione, superoxide dismutase, and total antioxidant capacity) in the hemolymph of adult honey bees (Apis mellifera). The bees were divided into three groups: (1) an experimental group fed with pure sugar syrup with cotton strips soaked with hemp extract put inside the cage; (2) an experimental group fed with a mixture of sugar syrup with hemp extract; and (3) a control group fed with a mixture of sugar and a water-glycerine solution. Hemolymph samples were collected on the 1st day of this study and then every week, until all bees in the group died. The activities of all antioxidant enzymes were higher for the experimental groups, compared to those for the control group. The highest antioxidant activities were noted in the group supplemented with cannabis with the use of syringes. Supplementation with hemp also increased the lifespan of bees in this group compared to that of the bees consuming only sugar syrup (control: 35 days), with 49 and 52 days for groups of cannabis on strips and in syrup, respectively. Hemp extract, thanks to its antioxidant properties, increased the activities of key antioxidant enzymes that protect the bee's organisms against free radicals and thus delay the aging processes.

Effect of Comb Cell Width on the Activity of the Proteolytic System in the Hemolymph of Apis mellifera Workers.

This study is a continuation of the innovative research of the impact of rearing of bee colonies simultaneously on standard- and small-cell combs on the traits of worker bees and bee colonies. Its aim was to compare the activities of proteases and their inhibitors in the hemolymph of workers reared in a small-cell comb (SMC) and a standard-cell comb (STC) in colonies kept simultaneously on standard- and small-cell combs. The width of comb cells in which workers are reared has a significant effect on the protein concentration and the proteolytic system in the hemolymph, which is reflected in the activities of proteases and their inhibitors. The protein concentrations in the 1-day-old workers were always higher (p ≤ 0.05) in the SMC than STC workers. The opposite was found in the older bee workers (aged 7, 14 and 21 d). The activities of proteases and their inhibitors in the 1-day-old workers were always higher (usually significantly at p ≤ 0.05) in STC than SMC workers, and opposite results were observed in the groups of the older workers (aged 7, 14 and 21 d). The differences between the workers from small-cell combs and those reared in standard-cell combs may be related to their different tasks. Workers reared in small-cell combs probably work as foragers outside the nest, whereas bees reared in standard-cell combs work in the nest. This hypothesis requires confirmation. To reduce the impact of accidental determinants on the results of single-season research on honeybees, it is advisable that such investigations should be conducted for several consecutive years.

Reproductive Potential Impacts Body Maintenance Parameters and Global DNA Methylation in Honeybee Workers (Apis mellifera L.).

The widely accepted hypothesis in life history evolution about the trade-off between fecundity and longevity is not confirmed by long-living and highly fecund queens in eusocial insects. The fact that the queens and facultatively sterile workers usually arise from genetically identical eggs but differ in DNA methylation makes them a good model for studies on senescence, eusocial evolution, and epigenetics. Therefore, honeybees seem to be especially useful here because of long living rebel-workers (RW) with high reproductive potential recently described. Longevity, ovariole number, nosema tolerance, and global DNA methylation have been assayed in normal workers (NW) versus RW in hives and cages. RW always lived longer than NW and unexpectedly extended longevity of NW when they were together, similarly as the presence of a queen did. RW lived longer despite the fact that they had higher Nosema spore load; surprisingly they became infected more easily but tolerated the infection better. Global DNA methylation increased with age, being lower in RW than in NW. Therefore, RW are queen-like considering global DNA methylation and the link between fecundity, longevity, and body maintenance. Presented features of RW expands possibilities of the use of honeybees as a model for studies on senescence, nosemosis, eusocial evolution, and epigenetics.