Pollen Patties Research Articles

Exploring the connection between food and midgut digestive enzymes to improve honey bee (Apis mellifera) nutrition

Honey bee malnutrition is the leading challenge in beekeeping. Finding a high-quality and cheap alternative to polyfloral pollen is essential. To find a quick and cheap analysis that can be used to evaluate different substitutes for pollen in bee nutrition, caged bees were fed with four types of patties based on sugar, yeast, pollen, and Tenebrio molitor flour (SG, YG, PG, and TG group, respectively). Midgut and hindgut weight, as well as amylase and protease activity in the midgut, were measured to evaluate the effect of patties. Midgut weight was lowest in SG except on days 21 and 28 when it was similar to PG, and highest in YG on day 14 and 21. In SG and TG midgut weight didn’t change over time, while in YG it decreased from day 14 to day 28 and PG from day 7 to day 21 and 28. Specific amylase activity was the highest in YG, and lowest in SG, while it decreased in the YG and PG with time. Specific protease activity decreased in all groups comparing day 7 to day 28. PG and TG bees had a higher number of enzyme isoforms in comparison to YG bees, while SG bees had the lowest number of isoforms in contrast to all other groups. Mealworm patty fed bees had the highest protein concentration in the midgut at day 14 and 28, and similar to PG bees at day 21, induced similar enzyme profiles as pollen patty indicating its superiority over YG and SG.

Nurse bees regulate the larval nutrition of developing workers (Apis mellifera) when feeding on various pollen types.

Nutrition has been identified as a key driver of colony health and productivity. Yet, in honey bees, relatively little is known about how the vast variety of natural pollen sources impact larval development. The impact of the nutritional quality of 4 naturally occurring pollen sources, of importance to the Western Australian beekeeping industry, was tested on honey bee (Apis mellifera L.) development. Bee packages consisting of 800 g of bees and a mated sister queen were assigned to 40 nucleus hives and randomly allocated to one of the 4 feed treatments (10 colonies each) of marri (Corymbia calophylla Lindl.), jarrah (Eucalyptus marginata Sm.), clover (Trifolium repens L.), and canola (Brassica napus L.) pollen. Emerging bees were collected once the first bees started hatching on the assigned feed sources. Newly emerging bees were weighed individually, and body composition was measured in batches according to the feed treatment groups. Food consumption was recorded for the duration of the experiment. Nurse bees successfully raised young adult workers from the larval stage until emergence when fed with one of 4 pollen patties with different nutritional qualities. There was no difference in the body composition or weight of emerging bees fed on the different pollen types. However, the body weight of bees increased over time, most likely related to colony size and structure. With the type of pollen patties having little impact on larval development, the availability of pollen may be more important than its composition, providing bees have access to all essential nutrients.

Chemical Composition and Nutritional Value of Royal Jelly Samples Obtained from Honey Bee (Apis mellifera) Hives Fed on Oak and Rapeseed Pollen Patties.

Young workers, i.e., nurse honey bees, synthesize and secrete royal jelly to feed the brood and queen. Since royal jelly is a protein-rich substance, the quality of royal jelly may be influenced by the consumption of feed with varying protein content. We tested whether honey bee (Apis mellifera) colonies compensates for the nutritional quality to produce royal jelly by feeding different pollen patties made of oak or rapeseed pollen. After harvesting royal jelly, we examined the chemical composition including proximate nutrients, amino acids, proteins, fatty acids, and minerals of royal jelly samples obtained from two treatments. The results revealed that pollen patties with different nutritional levels did not influence the nutritional composition except for the crude fat. The levels of 10-HDA, which serves as an indicator of the royal jelly quality, showed no significant difference between the oak and rapeseed treatments, with values of 1.9 and 2.1 g/100 g, respectively. However, we found some differences in the protein intensity, particularly the MRJP3 precursor, MRJP3-like, and glucose oxidase. This study suggests that honey bees may have mechanisms to compensate for nutritional standards to meet the brood's and queen's nutritional requirements during bee pollen collection, preserving bee bread and royal jelly secretion.

Effects of pollen patties with curcumin-steviol glycoside complex on Apis mellifera

Effects of pollen patties with curcumin-steviol glycoside complex on Apis mellifera

Delivery mechanism can enhance probiotic activity against honey bee pathogens

Managed honey bee (Apis mellifera) populations play a crucial role in supporting pollination of food crops but are facing unsustainable colony losses, largely due to rampant disease spread within agricultural environments. While mounting evidence suggests that select lactobacilli strains (some being natural symbionts of honey bees) can protect against multiple infections, there has been limited validation at the field-level and few methods exist for applying viable microorganisms to the hive. Here, we compare how two different delivery systems—standard pollen patty infusion and a novel spray-based formulation—affect supplementation of a three-strain lactobacilli consortium (LX3). Hives in a pathogen-dense region of California are supplemented for 4 weeks and then monitored over a 20-week period for health outcomes. Results show both delivery methods facilitate viable uptake of LX3 in adult bees, although the strains do not colonize long-term. Despite this, LX3 treatments induce transcriptional immune responses leading to sustained decreases in many opportunistic bacterial and fungal pathogens, as well as selective enrichment of core symbionts including Bombilactobacillus, Bifidobacterium, Lactobacillus, and Bartonella spp. These changes are ultimately associated with greater brood production and colony growth relative to vehicle controls, and with no apparent trade-offs in ectoparasitic Varroa mite burdens. Furthermore, spray-LX3 exerts potent activities against Ascosphaera apis (a deadly brood pathogen) likely stemming from in-hive dispersal differences, whereas patty-LX3 promotes synergistic brood development via unique nutritional benefits. These findings provide a foundational basis for spray-based probiotic application in apiculture and collectively highlight the importance of considering delivery method in disease management strategies.

Influence of Chlorella supplemented diet on honey bee (Apis mellifera) colony health

Microalgae have received scientific attention for alternative food source for honey bees due to its remarkable nutritional composition, often comparable and even superior to bee pollen. Our previous study demonstrated that Chlorella sorokiana-supplement diet improved honey bee (Apis mellifera) longevity, development, and physiology in the cage. In the sequence of the previous study, we investigated the role Chlorella as a dietary supplement on honey bee at the colony level. We formulated feed with 0.5, 2, 5, and 10% Chlorella supplemented with commercial rapeseed pollen patty and assessed diet preference, quality of royal jelly, development of worker, honey bee colony strength for different diets. The results revealed that diets adding Chlorella represented positive effect in consumption, body weight of newly emerged worker, colony strength in terms of number of broods and worker, and 10-HDA of royal jelly compare to controls. Therefore, 2% Chlorella addition to pollen patty may be proposed as a feed additive for performance improvement of honey bee at colony level.

Discovering Aethina tumida responses to attractant and repellent molecules: A potential basis for future management strategies

Small hive beetle (Aethina tumida) management has been highly dependent upon chemical and mechanical control over the past two decades; however, many of these methods have not been consistently effective or safe for European honey bee (Apis mellifera) colonies. Here we explore the behavioral and physiological effects of the attractants isopentyl acetate and pollen patty upon A. tumida adults, and also investigate the mixture of attractants with repellent compounds, which were previously untested against A. tumida. Electroantennograms established sensitivity of A. tumida antennae to both attractants and all repellents with the exception of DEET, with antennae displaying greatest sensitivity to the repellent pyrrolidine. A walking-response olfactometer, designed specifically for A. tumida, was used for all behavioral experiments. It was found that both pollen patty and isopentyl acetate were attractive to A. tumida adults; conversely, mixes of attractants and repellent volatiles led to less attraction or avoidance of what was previously a significantly attractive source. Of all repellents tested, pyrrolidine was found to be the most repelling molecule, with significant avoidance of the attractive source at a 10 mg treatment of pyrrolidine. The results of this study indicate that, at the behavioral level, the repellent compounds pyrrolidine and 1,4-dimethylpiperazine resulted in a negative preference index indicating a repellent behavioral response. By strategically implementing a repellent source in an apiary environment, A. tumida adults could be deterred from entering and invading hives.

Impact of nutritional and sanitary management on Apis mellifera colony dynamics and pathogen loads

Aim of study: The aim of this study was to assess the impact of the mite control strategies combined with nutritional management on honey bee colony dynamics and survival during winter, the following spring, and summer. Area of study: Santa Fe province in central Argentina. Material and methods: We set two apiaries with 40 colonies each and fed one apiary with high fructose corn syrup (HFCS) and the other with sucrose syrup (SS). Within each apiary, we treated half the colonies against Varroa mites and half of these treated colonies also received a pollen patty. The other half of the colonies remained untreated and did not received pollen patties. All colonies were sampled for Varroa infestation level, Nosema ceranae abundance and colony strength seven times during a year (from summer 2016 to autumn 2017). We computed autumn mite invasion and colony losses at each sampling time. Main results: Colonies fed with HFCS had more brood cells during the study that those fed with SS and treated colonies had fewer adult bees and Varroa infestation than untreated colonies. No significant effect of the protein supplementation was observed on any of the response variables. , SS colonies from all groups had significantly more mites drop counts than HFCS colonies. Research highlights: Considering that a reduced frequency of application is desirable, our results suggested that nutrition management could enhance chemical treatment effectiveness since honey bees might profit from improved nutrition. However, a better understanding of the nutritional requirements of the colonies under field conditions is needed.

A novel method in small hive beetle rearing

Research to investigate the small hive beetle, Aethina tumida, and its effects on the honey bee requires a consistent supply of healthy beetles. This protocol describes an effective rearing method for the small hive beetle using commercially available pollen patties and equipment that can easily be constructed from local sources. In this protocol, a method is described that allows for many thousands of healthy beetles to be reared weekly in a confined space. This knowledge is a crucial step for the advancement of research studies and the development of management protocols for the small hive beetle.

Differential Bacterial Community of Bee Bread and Bee Pollen Revealed by 16s rRNA High-Throughput Sequencing.

Simple SummaryGut symbionts play a crucial role in the nutrition provisioning of honeybees. However, the data on the bacterial communities of pollen collected by bees and bee bread are comparatively scarce. Therefore, the present study was designed to explore and identify the bacterial communities from them. The study reports that the bacterial diversity was significantly higher in the bee bread than in bee pollen. The higher bacterial diversity in the bee bread could presumably be due to factors such as storage period, processing of food, fermentation, and high sugar environment. The bacterial communities of bee bread possibly exhibit beneficial roles for honeybees such as polysaccharide degradation and nitrogen fixing.We investigated the bacterial community of bee bread and bee pollen samples using an approach through 16 s rRNA high-throughput sequencing. The results revealed a higher bacterial diversity in bee bread than in bee pollen as depicted in taxonomic profiling, as well as diversity indices such as the Shannon diversity index (3.7 to 4.8 for bee bread and 1.1 to 1.7 for bee pollen samples) and Simpson’s index (>0.9 for bee bread and 0.4–0.5 for bee pollen). Principal component analysis showed a distinct difference in bacterial communities. The higher bacterial diversity in the bee bread than bee pollen could presumably be due to factors such as storage period, processing of food, fermentation, and high sugar environment. However, no effect of the feed (rapeseed or oak pollen patties or even natural inflow) was indicated on the bacterial composition of bee bread, presumably because of the lack of restriction of foraged pollen inflow in the hive. The diverse bacterial profile of the bee bread could contribute to the nutritional provisioning as well as enhance the detoxification process; however, a thorough investigation of the functional role of individual bacteria genera remains a task for future studies.

Seasonal variability in physiology and behavior affect the impact of fungicide exposure on honey bee (Apis mellifera) health

Honey bee pollination services are of tremendous agricultural and economic importance. Despite this, honey bees and other pollinators face ongoing perils, including population declines due to a variety of environmental stressors. Fungicides may be particularly insidious stressors for pollinators due to their environmental ubiquity and widespread approval for application during crop bloom. The mechanisms by which fungicides affect honey bees are poorly understood and any seasonal variations in their impact are unknown. Here we assess the effects on honey bee colonies of four-week exposure (the approximate duration of the almond pollination season) of a fungicide, Pristine® (25.2% boscalid, 12.8% pyraclostrobin), that has been commonly used for almonds. We exposed colonies to Pristine® in pollen patties placed into the hive, in either summer or fall, and assessed colony brood and worker populations, colony pollen collection and consumption, and worker age of first foraging and longevity. During the summer, Pristine® exposure induced precocious foraging, and reduced worker longevity resulting in smaller colonies. During the fall, Pristine® exposure induced precocious foraging but otherwise had no significant measured effects. During the fall, adult and brood population levels, and pollen consumption and collection, were all much lower, likely due to preparations for winter. Fungicides and other pesticides may often have reduced effects on honey bees during seasons of suppressed colony growth due to bees consuming less pollen and pesticide.

Chlorella-supplemented diet improves the health of honey bee (Apis mellifera)

Nutritional stress is one of the major factors affecting the health of honey bees. Supplementing the pollen patty with microalgae enhances the protein content of the patty and therefore is supposed to improve bee’s health. The objective of the present study was to investigate the effect of Chlorella as a dietary supplement on the health and physiology of the honey bee (Apis mellifera). We formulated the honey bee feed by supplementing Chlorella sorokiniana, obtained commercially, with commercially available rapeseed pollen patty in different amounts—0.5, 2, 5, and 10%, and the treatment groups were named P0.5, P2, P5, and P10, respectively. Pollen patty was set as a positive control and only 50% sucrose solution (no protein) was set as a negative control. Diets were supplied ad libitum to newly emerged workers in cages; food consumption patterns; longevity; and physiology including the development of the brain in terms of protein (i.e., total amino acids), thorax muscle, fat body mass, and glands (hypopharyngeal and venom); and gene expression of nutrition-related gene vitellogenin (Vg) of honey bee at different points of time of their age were observed. The addition of Chlorella significantly increased the food consumption pattern, longevity, gland development, muscle formation, and Vg gene expression significantly in comparison to only a pollen or sugar diet. However, the response varied depending on the level of Chlorella supplementation. As depicted in most of the cases, P2, that is, the pollen diet with 2% of Chlorella supplement exhibited the best outcome in terms of all the tested parameters. Therefore, based on the results obtained in the present study, we concluded that 2% Chlorella supplementation to pollen patty could enhance the health of honey bees, which in turn improves their performance.

In pursuit of the ultimate pollen substitute (insect larvae) for honey bee (Apis mellifera) feed

Finding a pollen substitute for honey bees that is nutritionally adequate and affordable is a scientific and practical challenge. We attempted a new rational approach and tried to exploit honey bees’ natural cannibalistic behavior. We tested processed insect larvae as a food source that is nutritionally similar to bee brood, and which can easily be produced on a large scale. In cage experiments, monitoring bee mortality, food consumption and changes in bee body parts’ weights showed that flour obtained by grinding dried yellow mealworm larvae has the potential to become an excellent component for pollen substitution. Bees from the cage group fed Tenebrio molitor patties (TG) demonstrated overall best results in comparison to sugar patties fed bee group (CG), yeast patties fed bee group (YG) and pollen patties fed bee group (PG). They did not lose weight as rapidly as the CG, did not defecate inside cages as the YG, nor show increased mortality as the PG. At the same time, TG consumed less food (mean 13.7 g/cage) than CG (16.8 g/cage), YG (20.4 g/cage) and PG (23.9 g/cage) within the period of 28 days. Bees’ gut increase in weight was lowest in the CG, followed by TG and PG and was the highest in the YG which resulted in diarrhea after 14 days. Bees from TG did not lag behind other bees in head, thorax and abdomen weight after 28 days. We demonstrated that processed yellow mealworm larvae (T. molitor) can be used as an ingredient for honey bee feed.

Temporal changes of nutrient composition from pollen patty to bee bread with special emphasis on amino and fatty acids composition

• Bee bread is made from bee pollen or pollen patty. • Amino acid contents increase from pollen patty to bee bread. • Increase of amino acid, proline is prominent. • Fatty acid contents decrease with the storing time. • Biological and chemical processes involved in the transition. Bee bread is prepared from pollen sources and salivary secretions by honey bee workers to serves the nutritional purpose of colony members. However, changes in nutrient composition occurring in bee bread from the pollen sources including pollen patty are poorly understood. We, therefore, examined the nutrient contents of pollen patty, used as source pollen feed of honey bee colony, and bee bread prepared by honey bee from this source at different time interval. Results revealed that amino acid content was increased from pollen patty to bee bread but fatty acid content was decreased. Essential amino acids like valine, lysine, and nonessential amino acids such as proline and alanine increased significantly over time. Among fatty acids, oleic acid, linoleic acid and linolenic acid decreased significantly from pollen patty to bee bread. Minerals contents in bee bread did not show any significant change during the study period. These changes could be presumably justified due to addition of nectar, honey, gland secretions and also microbial symbionts which remains as a task of further study.

Synergistic and Antagonistic Interactions Between Varroa destructor Mites and Neonicotinoid Insecticides in Male Apis mellifera Honey Bees

Pressures from multiple, sometimes interacting, stressors can have negative consequences to important ecosystem-service providing species like the western honey bee (Apis mellifera). The introduced parasite Varroa destructor and the neonicotinoid class of insecticides each represent important, nearly ubiquitous biotic and abiotic stressors to honey bees, respectively. Previous research demonstrated that they can synergistically interact to negatively affect non-reproductive honey bee female workers, but no data exist on how concurrent exposure may affect reproductive honey bee males (drones). This is important, given that the health of reproductive females (queens), possibly because of poor mating, is frequently cited as a major driver of honey bee colony loss. To address this, known age cohorts of drones were obtained from 12 honey bee colonies—seven were exposed to field-relevant concentrations of two neonicotinoids (4.5 ppb thiamethoxam and 1.5 ppb clothianidin) during development via supplementary pollen patties; five colonies received patties not spiked with neonicotinoids. Artificially emerged drones were assessed for natural V. destructor infestation, weighed, and then allocated to the following treatment groups: 1. Control, 2. V. destructor only, 3. Neonicotinoid only, and 4. Combined (both mites and neonicotinoid). Adult drones were maintained in laboratory cages alongside attendant workers (1 drone: 2 worker ratio) until they have reached sexual maturity after 14 days so sperm concentration and viability could be assessed. The data suggest that V. destructor and neonicotinoids interacted synergistically to negatively affect adult drone survival, but that they interacted antagonistically on emergence mass. Although sample sizes were too low to assess the effects of V. destructor and combined exposure on sperm quality, we observed no influence of neonicotinoids on sperm concentration or viability. Our findings highlight the diverse effects of concurrent exposure to stressors on honey bees, and suggest that V. destructor and neonicotinoids can severely affect the number of sexually mature adult drones available for mating.

Pollen-inspired enzymatic microparticles to reduce organophosphate toxicity in managed pollinators.

Pollinators support the production of the leading food crops worldwide. Organophosphates are a heavily used group of insecticides that pollinators can be exposed to, especially during crop pollination. Exposure to lethal or sublethal doses can impair fitness of wild and managed bees, risking pollination quality and food security. Here we report a low-cost, scalable in vivo detoxification strategy for organophosphate insecticides involving encapsulation of phosphotriesterase (OPT) in pollen-inspired microparticles (PIMs). We developed uniform and consumable PIMs capable of loading OPT at 90% efficiency and protecting OPT from degradation in the pH of a bee gut. Microcolonies of Bombus impatiens fed malathion-contaminated pollen patties demonstrated 100% survival when fed OPT-PIMs but 0% survival with OPT alone, or with plain sucrose within five and four days, respectively. Thus, the detrimental effects of malathion were eliminated when bees consumed OPT-PIMs. This design presents a versatile treatment that can be integrated into supplemental feeds such as pollen patties or dietary syrup for managed pollinators to reduce risk of organophosphate insecticides.

Field cross-fostering and in vitro rearing demonstrate negative effects of both larval and adult exposure to a widely used fungicide in honey bees (Apis mellifera)

Pollinators and other insects are experiencing an ongoing worldwide decline. While various environmental stressors have been implicated, including pesticide exposure, the causes of these declines are complex and highly debated. Fungicides may constitute a particularly prevalent threat to pollinator health due to their application on many crops during bloom, and because pollinators such as bees may consume fungicide-tainted pollen or nectar. In a previous study, consumption of pollen containing the fungicide Pristine® at field-relevant concentrations by honey bee colonies increased pollen foraging, caused earlier foraging, lowered worker survival, and reduced colony population size. Because most pollen is consumed by young adults, we hypothesized that Pristine® (25.2% boscalid, 12.8% pyraclostrobin) in pollen exerts its negative effects on honey bee colonies primarily on the adult stage. To rigorously test this hypothesis, we used a cross-fostering experimental design, with bees reared in colonies provided Pristine® incorporated into pollen patties at a supra-field concentration (230 mg/kg), only in the larvae, only in the adult, or both stages. In contrast to our predictions, exposure to Pristine® in either the larval or adult stage reduced survival relative to control bees not exposed to Pristine®, and exposure to the fungicide at both larval and adult stages further reduced survival. Adult exposure caused precocious foraging, while larval exposure increased the tendency to forage for pollen. These results demonstrate that pollen containing Pristine® can induce significant negative effects on both larvae and adults in a hive, though the magnitude of such effects may be smaller at field-realistic doses. To further test the potential negative effects of direct consumption of Pristine® on larvae, we reared them in vitro on food containing Pristine® at a range of concentrations. Consumption of Pristine® reduced survival rates of larvae at all concentrations tested. Larval and adult weights were only reduced at a supra-field concentration. We conclude that consumption of pollen containing Pristine® by field honey bee colonies likely exerts impacts on colony population size and foraging behavior by affecting both larvae and adults.

Changes in nutritional composition from bee pollen to pollen patty used in bumblebee rearing

Abstract Pollen patties are generally supplied to augment the nourishment of honey bee or bumblebee colonies during late winter or early spring. In the present study, we examined nutrient content of bee collected pollen (BP) and pollen patties (PT) prepared from those bee collected pollens in order to understand the chemical compositional change of pollen to pollen patty and figure out the possible benefits for bee’s health. Protein and fat contents of pollen patties were found lower than that reported for the respective bee pollens. Amino and fatty acids followed the same trend for the simple logical reason. In contrast, carbohydrate contents of pollen patties were found much higher than the bee pollen. The addition of sugar solution in the process of pollen patty preparation could presumably explain the change. Another possible determinant factor of bee’s preference of feed protein to fat ratio was found the highest for PT 2 (5.5) followed by PT 1 and BP 2 (4.1), BP Ref. (2.9) and the least value obtained for BP 1 (2.2). Additionally, we discuss the possible health benefits if the bee collected pollen is used as a human food supplement.

The development of an attract-and-kill bait for controlling the small hive beetle (Coleoptera: Nitidulidae)

This research investigates the development of an attract-and-kill bait for in-hive control of the small hive beetle, Aethina tumida. The control method employs attracting the beetles to an in-hive trap with a feeding attractant/stimulant where a toxicant is delivered by consuming an edible bait. Investigations into mild insecticides led to the use of boric acid. At low doses, boric acid is non-toxic to humans but lethal to insects. This research was designed to identify key compounds that would attract small hive beetles, develop an edible bait using those compounds and if the diet would be consumed by the beetle, determine the lethal dose with the lowest amount of toxicant, and the effects the toxicant would have on the honey bee. Attractive compounds were identified from honey bee pollen patties inoculated Kodamaea ohmeri yeast and resulted in the identification of three key components: ethyl propionate, isobutyl propionate, and ethyl butyrate. A diet comprised of corn gluten meal, barley flour, soy flour, Brewer’s yeast, and glycerin containing the attractant/feeding stimulant was highly attractive and readily consumed. In laboratory trials, the treatments containing the attract-and-kill with 2% boric acid reduced the beetle population to zero within a few days. There was no significant difference between the sex of the beetle for survival on any of the treatments. Honey bee survival was reduced by ingesting the boric acid. The development of an inexpensive small hive beetle trapping system is essential for in-hive control of this devastating pest. This system has the potential to provide beekeepers a tool for control of this pest species that affects honey bee health and survival worldwide.

Impact of nutritional stress on the honeybee colony health

Honeybees Apis mellifera are important pollinators of wild plants and commercial crops. For more than a decade, high percentages of honeybee colony losses have been reported worldwide. Nutritional stress due to habitat depletion, infection by different pests and pathogens and pesticide exposure has been proposed as the major causes. In this study we analyzed how nutritional stress affects colony strength and health. Two groups of colonies were set in a Eucalyptus grandis plantation at the beginning of the flowering period (autumn), replicating a natural scenario with a nutritionally poor food source. While both groups of colonies had access to the pollen available in this plantation, one was supplemented with a polyfloral pollen patty during the entire flowering period. In the short-term, colonies under nutritional stress (which consumed mainly E. grandis pollen) showed higher infection level with Nosema spp. and lower brood and adult bee population, compared to supplemented colonies. On the other hand, these supplemented colonies showed higher infection level with RNA viruses although infection levels were low compared to countries were viral infections have negative impacts. Nutritional stress also had long-term colony effects, because bee population did not recover in spring, as in supplemented colonies did. In conclusion, nutritional stress and Nosema spp. infection had a severe impact on colony strength with consequences in both short and long-term.