Isofucosterol and combinations of isofucosterol with multiple sterols as animal nutrients

Abstract

1. A method of feeding an invertebrate or aquacultured organism, comprising: providing a pollen replacement composition comprising a nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof; and administering the pollen replacement composition to the invertebrate or aquacultured organism, wherein the pollen replacement composition comprises a nutritionally effective amount of at least one further sterol, preferably at least two further sterols selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol, or physiologically available conjugates of any of these sterols.

Description

[Background technology]

[0001] Insects are increasingly being used as economically important pollinators and as cost-effective producers of animal protein for human and animal feed with less environmental impact than traditional animal protein crops (birds, fish, mammals). Honeybees play a vital role in pollinating crops in modern agriculture. In recent years, pollinators have come under threat from exposure to pesticides, increased pathogens and parasites, and changes in landscape management that reduce the abundance of naturally occurring pollen.

[0002] Historically, beekeepers have fed their honey bee colonies with pollen-containing food sources or pollen collected by honey bees in natural or agroecosystems. Pollen collection is limited in availability and scale. First, pollen collection is costly. Second, pollen is difficult to keep fresh. Finally, pollen collected in natural or agroecosystems can carry pests, diseases, and pesticides. Therefore, pollen is not usually included in commercial honey bee diets.

[0003] However, lack of sufficient quality and quantity of pollen is a root cause of observed declines in health and survival in bee colonies. Honey bees harvesting natural pollen resources puts pressure on wild bee species' resources and likely contributes to their population declines. Transporting bee hives to pollen areas is a fundamental stressor for bee colonies and a major cost to the beekeeping industry. Pollen substitutes designed for captive bee species would address these issues.

[0004] Honeybees obtain essential nutrients such as sterols from pollen. These sterols likely include cholesterol, campesterol, β-sitosterol, or stigmasterol. Original studies at the USDA Beltsville Laboratory in the 1970s described the utilization of pollen-derived sterols by honeybees fed a synthetic diet (Non-Patent Document 1). Chakrabarti et al. described the role of 24-methylenecholesterol in honeybee nutrition (Non-Patent Document 2). However, all of these experiments were performed by adding a single sterol to a different sterol-free diet. To date, it has not been described which of the many sterols, and which combinations and concentrations of sterols, are important or essential for bees.

[0005] US Patent No. 5,399,633 (Apix Biosciences) highlights the importance of plant sterols, and in particular 24-methylenecholesterol, campesterol, β-sitosterol, and cholesterol, but does not disclose isofucosterol and fucosterol, and their mixtures with sterols, as feed supplements, as important nutrients required to make a "complete" diet for farmed bees.

[0006] (2003) discusses a study on the consumption of insects and shrimp with regard to hydrolysates, their antioxidant and angiotensin converting enzyme inhibitory activity, fatty acids, cholesterol, minerals, vitamins, carotenoids, phenolic compounds and dietary fiber, but this document does not mention isofucosterol and the combination of cholesterol and isofucosterol.

[0007] Soy protein or other used protein sources such as potato contain campesterol, β-sitosterol, stigmasterol and trace amounts of desmosterol and isofucosterol (60 mg isofucosterol / kg wet weight of potato, i.e. 0.006%) (4). The concentration of sterols in these sources is below the nutritional requirements of bees.

[0008] In honeybees, isofucosterols represent 10-50% of the total body sterols. This corresponds to 0.02-0.06% of the honeybee's dry weight (Non-Patent Document 5; our data). In bumblebees, such as Bombus terrestris, isofucosterols represent 40-55% of the body sterols. This amount corresponds to 0.10-0.25% of the total bumblebee's dry weight.

[0009] Current artificial bee diets are incomplete, resulting in an initial surge in larval production followed by a rapid decline in larval production over a period of 22-40 days. This indicates that the current diets lack one or more essential nutrients. However, to date, no artificial (pollen-free) diets for bees have been described that can support the growth of sealed larvae in colonies for more than 3-4 cycles over a 21-day period. The ability of a diet to support continued larval growth over an extended period is an important indicator of its ability to function as a "complete diet," equivalent to pollen. The best published data already show a significant decline in larval production beginning after two cycles (Non-Patent Document 6). Thus, in many areas where bees are kept for pollination or honey production, the quality and quantity of pollen is insufficient, so there is a need to provide compositions and methods for feeding bees that allow for the long-term maintenance of colonies on an artificial diet that will allow the bees to continue producing larvae indefinitely.

[0010] Current pollen-free artificial bee diets are incomplete, and bees fed these diets lack or are depleted of essential nutrient reserves after 3-4 21-day larval production cycles in the colony (Non-Patent Document 1 (1980)). Here, the addition of isofucosterol / fucosterol and multiple sterols provides a diet that sustains larval production and colony development in the colony for 5 or more cycles, suggesting that the diet overcomes the deficiency of one or more essential nutrients found in current artificial diets. This shows that the present invention provides a new diet that provides bees with the necessary limiting components they require. Thus, such pollen-free diets will sustain colonies for extended periods of the colony life cycle without natural pollen, a significant advancement that can dramatically improve beekeeping practices.

[0011] Honeybees are crucial to human food security. Despite being domesticated for thousands of years, no pollen-free artificial diet has been developed that would completely eliminate the need for honeybees to forage for nectar and pollen flowers for nutrition. Such a diet would be a vital tool to maintain human societies' ability to sustainably pollinate 35% of the world's crops and to protect wild pollinators and plant biodiversity.

[0012] The honeybee hive requires a continuous supply of nutritionally suitable pollen from a variety of sources to meet its nutritional needs. Pollen is an essential source of proteins, carbohydrates, amino acids, vitamins, lipids, sterols, and other micronutrients for the honeybee hive. Most plant species produce pollen whose composition does not meet all of the nutritional needs of the honeybee hive. Therefore, honeybees have developed a physiological mechanism to balance their nutritional needs by collecting pollen from a variety of floral sources.

[0013] The nutritional relevance or requirements for honeybees of many of the complex molecules identified in pollen remain unknown. In addition to macronutrients, pollen and honeybees contain multiple members of specific chemical families (e.g., flavonoids, sterols, lipids).

[0014] Many papers have inferred the nutritional relevance of certain molecules by correlating the nutrient composition in different floral pollen sources with the nutritional properties of these different pollens. Because pollen from different flowers varies widely in the composition of many molecules, this correlation approach cannot really show any conclusions about the relevance of single components.

[0015] In another experimental approach to studying honeybee nutrition, single members of such compound families (single sterols; single antioxidants) are added to simple base samples (see Herbert, Chakrabarti, Table 7) and shown to have an effect in short-term assays, usually captive bee feeding and lifespan. This gives an indication that for each chemical, honeybees can derive a nutritional benefit from the presence of this chemical family member even when other chemical family members are not present, but does not indicate whether this is the optimal molecule of chemical required by honeybees, or whether honeybees require or benefit from more than one chemical family member to nutritionally support the hive throughout their life cycle and seasons.

[0016] Insects cannot synthesize sterols, but obtain them from food sources, which most insects convert into cholesterol. Unlike most insects, bees (honeybees, bumblebees...) contain only traces of cholesterol and are unable to dealkylate phytosterols to cholesterol. Thus, honeybees use phytosterols instead of cholesterol as structural elements in membranes, as precursors for growth hormones, and as structural elements of protein complexes (e.g., 24-methylenecholesterol is the main component of the royal jelly protein mrjp1-apimisin complex; Table 7). Pollen from different flower species has widely different phytosterol compositions and ratios. Honeybees, bumblebees, and other bee species collect pollen from a variety of species to balance their diet. The sterol composition of honeybees consists of six major sterols (described below), which represent more than 90% of the sterols present in honeybees. These phytosterols are present in various ratios that, in part, reflect the phytosterol composition of the honeybee's floral forage. Typically, honeybee larvae contain 0.1-0.3% sterols in the following ratios, depending on the food source: 40-60% 24-methylenecholesterol; 15-40% β-sitosterol; 15-50% isofucosterol; 3-10% campesterol; 1-5% stigmasterol, and 0.1-2% cholesterol.

[0017] Only two experiments have been published on artificial diets of defined sterol composition that have examined the importance of specific sterols in honeybee nutrition. In one experiment, Herbert et al. (1980, Table 7) added the sterols cholesterol, 24-methylenecholesterol, β-sitosterol, campesterol, and stigmasterol (but not isofucosterol) individually to a sterol-free defined diet and fed tented honeybee hives with each of these diets, each containing a single sterol, for up to 12 weeks. They showed that diets containing some sterols, such as 24-methylenecholesterol, produced more larvae than diets containing other sterols. From this experiment it was not possible to draw any conclusions as to whether one sterol is physiologically exchangeable for another, i.e. whether honeybees require a large number of sterols or only a small number of sterols, and whether the absence of a particular phytosterol can be counterbalanced by the presence of (combinations of) other phytosterols.

[0018] Chakrabarti et al. (Table 7) conducted short-term feeding experiments with 24-methylenecholesterol as the sole sterol present in an artificial diet and showed that when present, 24-methylenecholesterol had significant physiological and metabolic effects (larval production was not measured). However, these experiments did not include control diets containing sterols different from 24-methylenecholesterol to assess whether the observed effects were specific to a particular sterol or non-specific to a molecular class of sterol. These experiments did not distinguish whether sterols different from 24-methylenecholesterol had similar or overlapping physiological effects.

[0019] To date, it has not been shown which sterols in pollen are facultative sterols and which (if any) are nutritionally essential in the diet. To date, no nutritional experiments have been reported to evaluate the nutritional requirement of isofucosterol for honeybees. No experiments have been reported to examine whether removing 24-methylenecholesterol or isofucosterol, or both sterols, from a complete diet containing a defined number of sterols in concentrations and ratios similar to those in pollen affects the fitness of the honeybee hive and the honeybees. For further references to the state of the art, see Table 7. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] US Patent Application Publication No. 2019 / 0090507 [Non-patent literature]

[0021] [Non-Patent Document 1] Journal of Insect Physiology, Vol, 26, pp. 287-289 [Non-Patent Document 2] Chakrabarti, Evaluating effects of a critical micronutrient (24-Methylenecholesterol) on honeybee physiology, Annals of the entomological society of America, Vol 113, 176-182, 2019 [Non-Patent Document 3] Mishyna et al., Journal of Functional Foods 76 (2021) 104316, doi.org / 10.1016 / j.jff.2020.104316 [Non-Patent Document 4] Sirpa O. Kaerenlampi, Philip J. White, in Advances in Potato Chemistry and Technology, 2009 [Non-Patent Document 5] Svoboda et al.Utilization and metabolism of dietary sterols in the honeybee and the yellow fever mosquito, Lipids Volume 17 number 3, 1982 [Non-Patent Document 6] Bee Culture Education: Honeybee Nutrition - Randy Oliver - Part 1 to 4, Youtube Summary of the Invention [Problem to be solved by the invention]

[0022] While only a few sterols exist in animals (among which cholesterol is by far the most abundant), a wide range of sterols is found in plants. Their structural variations arise from various substitutions in the side chains and in the number and position of double bonds in the tetracyclic skeleton. Plant sterols can be grouped according to the presence or absence of one or more functional groups. For example, plant sterols can be divided into three groups based on the degree of ethylation at C4: 4-desmethylsterols or end-product sterols, 4alpha-monomethylsterols, and 4,4-diethylsterols. Naturally occurring 4-desmethylsterols include sitosterol, stigmasterol, brassicasterol, campesterol, avenasterol, and isofucosterol. In most higher plants, sterols with a free 3-hydroxyl group (free sterols) are the main end-products. However, sterols also occur as conjugates, for example, where the 3-hydroxyl group is esterified with a fatty acid chain or a phenolic acid to give steryl esters. In the context of this description, the term sterol refers to both free sterol and complexed sterol. However, references herein to the concentration, amount, or percentage (%) of sterol refer to the total weight of sterol groups, excluding the weight of complex groups such as fatty acids or phenolic acids. In this document, fucosterol is defined as fucosterol, its isomer isofucosterol, or a mixture of the two isomers. In this document, isofucosterol is defined as isofucosterol, its isomer fucosterol, or a mixture of the two isomers.

[0023] When a dose or amount is described in this document as being fed as x grams (or other amount) for a two week period (or other time period), this is intended as a general description of "a dose given per unit time" and not a specific limitation to a particular requirement to be fed on a two week basis. One of skill in the art can recalculate a weekly dosing regimen into a daily or biweekly dose, or any similar period.

[0024] The inventors have surprisingly demonstrated that isofucosterol is an essential nutrient for bees. The isofucosterol delivered by the pollen-free diet increases the production of bee larvae and the survival of worker bees compared to pollen-substitute bee diets that do not contain isofucosterol. The inventors have further discovered that the combination of isofucosterol with at least one additional sterol, in particular cholesterol, 24-methylenecholesterol, and other phytosterols, in particular campesterol, β-sitosterol, or stigmasterol, results in higher growth and / or health performance in invertebrates, in particular bees, and other pollen-feeding insects other than isofucosterol alone.

[0025] This utility here is important in non-pollen feeding formulations to replenish honeybee colonies during periods of environmental pollen deficiency (which today is the main source of poor bee colony performance), for the production of superior queens, and in human and animal diets, the commercial rearing of pollen-feeding insects such as the black soldier fly as an alternative protein source to bumblebees (for pollination), ladybirds (for aphid control), hoverflies (for pollination), and other commercially relevant insects. [Means for solving the problem]

[0026] Thus, a first aspect of the present invention provides a method of feeding invertebrates or aquaculture organisms, comprising the steps of: providing a pollen replacement composition comprising a nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof; administering the pollen replacement composition to an invertebrate or aquaculture organism; Including, The pollen replacement composition comprises a nutritionally effective amount of at least one additional sterol, preferably at least two additional sterols selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol, or physiologically available complexes thereof.

[0027] In another embodiment, the invertebrate is a pollen-feeding insect, preferably an insect of the order Hymenoptera and Coleoptera, more preferably a honeybee, bumblebee, soldier fly, hoverfly, or ladybug.

[0028] In another aspect, the aquaculture organisms are plankton and algae feeding aquaculture organisms, preferably fish, larvae, oysters, bivalves, mollusks, gastropods, or crustaceans.

[0029] In another embodiment, the nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof is a daily amount of 0.0006% to 0.052% by weight of the live weight of the invertebrate or aquaculture organism.

[0030] In another embodiment, the nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof is 10% to 60% by weight of the total amount of sterols from the group of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total feed of the invertebrate or aquaculture organism, or of the pollen replacement composition.

[0031] In another embodiment, isofucosterol, fucosterol, or a mixture thereof is administered at a rate of 0.14 g to 12 g of isofucosterol, fucosterol, or a mixture thereof per 30,000 bees per two week period.

[0032] In another embodiment, isofucosterol, fucosterol or a mixture thereof is administered in an amount of 10% to 60% by weight of the total amount of sterols of the group of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total feed of the invertebrate or aquaculture organism or of the pollen replacement composition.

[0033] In another embodiment, the dosage rate of the nutritionally effective amount of isofucosterol, fucosterol or a mixture thereof and at least one further sterol is 0.2 to 48 grams per 30,000 bees per two week period and the isofucosterol, fucosterol or a mixture thereof is administered in an amount of 10% to 60% by weight of the total amount of sterols of the group of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total diet of the bees or of the pollen replacement composition.

[0034] In another embodiment, isofucosterol, fucosterol or a mixture thereof and at least one further sterol are administered at a rate of 0.4 g to 36 g per 30,000 bees per two week period, and isofucosterol, fucosterol or a mixture thereof is administered in an amount of 10% to 60% by weight of the total amount of sterols of the group of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total diet of the bees or of the pollen replacement composition.

[0035] In another embodiment, isofucosterol, fucosterol or a mixture thereof and at least one further sterol are administered at a rate of 0.6 g to 20 g per 30,000 bees per two week period, and isofucosterol, fucosterol or a mixture thereof is administered in an amount of 10% to 60% by weight of the total amount of sterols of the group of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total diet of the bees or of the pollen replacement composition.

[0036] In another aspect, isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, beta-stigmasterol, stigmasterol, and / or campesterol are administered in a total amount of 0.001% to 0.087% by weight of the live weight of the invertebrate or aquaculture organism per day.

[0037] In another embodiment, the nutritionally effective amount is, relative to isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, β-sitosterol, stigmasterol, and / or campesterol: isofucosterol in an amount between 10% and 60% by weight, Cholesterol in an amount between 0% and 50% by weight, 24-methylenecholesterol in an amount between 0% and 50% by weight, β-sitosterol, stigmasterol and / or campesterol in an amount between 0% and 50% by weight It is.

[0038] In another embodiment, the total concentration of sterols is from 0.01% to 4% by weight, preferably from 0.05% to 3% by weight, and even more preferably from 0.05% to 2% by weight, and even more preferably from 0.05% to 1.5% by weight, relative to the total weight of the pollen replacement composition.

[0039] In another embodiment, the concentration of sterol is cholesterol in an amount of 0.001% to 2% by weight, preferably 0.001% to 1.5% by weight, more preferably 0.06% to 1.2% by weight, relative to the total weight of the pollen replacement composition, 24-methylene cholesterol in an amount of 0.001% to 2% by weight, preferably 0.001% to 1.5% by weight, more preferably 0.06% to 1.2% by weight, relative to the total weight of the pollen replacement composition, sitosterol, preferably β-sitosterol, in an amount of 0.001% to 2% by weight, preferably 0.001% to 1% by weight, more preferably 0.03% to 0.6% by weight, relative to the total weight of the pollen replacement composition, isofucosterol in an amount of 0.01% to 5% by weight, preferably 0.01% to 2% by weight, more preferably 0.01% to 1.0% by weight, more preferably 0.03% to 0.6% by weight, relative to the total weight of the pollen replacement composition, campesterol in an amount of 0.001% to 2% by weight, preferably 0.001% to 1% by weight, more preferably 0.02% to 0.35% by weight, relative to the total weight of the pollen replacement composition, stigmasterol in an amount of 0.001% to 2% by weight, preferably 0.001% to 1% by weight, more preferably 0.01 to 0.2% by weight, relative to the total weight of the pollen replacement composition, or - selected from the group consisting of any combination thereof.

[0040] In another embodiment, the composition is part of a whole invertebrate feed, an invertebrate feed, or a dietary supplement and the ratio of 24-methylene cholesterol to the additional sterol, or combination of additional sterols, is from 10:1 to 1:1.

[0041] In another embodiment, the composition is administered to an invertebrate of the Apidae family, in particular honey bees (Apini), bumble bees (Bombini), or stingless bees (Meliponini).

[0042] In another aspect, the composition comprises: In solid form, such as a putty or powder, or in liquid form, such as a solution, oil, or spray; It can be administered inside or outside the hive.

[0043] In one embodiment the composition is substantially pure, i.e. it comprises 50% by weight or more, preferably 70% by weight or more, even more preferably 90% by weight or more of the group isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total feed of the bees or of the pollen replacement composition.

[0044] In one embodiment, the composition is substantially pure, i.e., does not contain any additional compounds.

[0045] In another embodiment, the source of isofucosterol, fucosterol, or mixtures thereof is non-pollen tissue of one or more plant species selected from the group consisting of leaves, stems, roots, tubers, flowers, seeds, bark, and fruits, and combinations thereof.

[0046] In another embodiment, the source of the additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol is pollen substitute tissue of one or more plant species selected from the group consisting of leaves, stems, roots, tubers, flowers, seeds, bark, and fruits, and combinations thereof.

[0047] In another embodiment, the source of isofucosterol, fucosterol, or a mixture thereof, or the source of at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol, is an extract, oil, or purified product of pollen substitute tissue of one or more plant species or combinations thereof.

[0048] In another embodiment, the source of isofucosterol, fucosterol, or a mixture thereof, or the source of at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol, is pollen substitute tissue of one or more plant species selected from the group consisting of Solanaceae, Poaceae, Ranunculaceae, Fabaceae, and Bacillus subtilis.

[0049] In another embodiment, the source of isofucosterol, fucosterol, or a mixture thereof, or a source of at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol, is Extracts, oils or purified products of marine or freshwater algae, in particular Ulva lutea; Extracts, oils or purified products of marine diatoms, in particular Thalassiosira pseudonana, Thalassiosira rotula or Chaetoceros muelleri; and Extracts, oils or purified products of fungi, in particular yeasts such as Saccharomyces cerevisiae or Yarrowia lipolytica The alternative pollen source is selected from the group consisting of:

[0050] In another embodiment, isofucosterol, fucosterol, or a mixture thereof, and / or at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol, are chemically or enzymatically synthesized or obtained by a genetically modified host organism, such as a fungus, bacteria, or algae.

[0051] In another embodiment, the source of isofucosterol, fucosterol, or a mixture thereof, or at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol, is selected from the group consisting of algae, plants, fungi, algae, diatoms, and combinations thereof, and the source of isofucosterol, fucosterol, or a mixture thereof is pollen substitute tissue.

[0052] In another embodiment, isofucosterol, fucosterol, or a mixture thereof, and at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol are provided to a eusocial bee colony from a synthetic source.

[0053] In another embodiment, isofucosterol, fucosterol, or a mixture thereof, and one or more further sterols selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol, and stigmasterol, are provided as part of a pollen replacement composition, the pollen replacement composition comprising: protein in an amount between 10% and 50% by weight, preferably between 15% and 40% by weight, fatty acids in an amount between 1% and 20% by weight, preferably between 2% and 12% by weight, carbohydrates in an amount between 20% and 90% by weight, preferably between 30% and 70% by weight, Optionally, vitamins, and Optionally, minerals Including, The sum of the total amounts of the components equals 100% by weight, where weight % relates to the total dry weight of the composition.

[0054] In another embodiment, the pollen replacement composition is pollen free.

[0055] In another embodiment, the pollen replacement composition comprises: invertebrates, preferably pollen-feeding insects, more preferably insects of the orders Hymenoptera and Coleoptera, even more preferably honeybees, bumblebees, soldier flies, hoverflies or ladybirds, Aquaculture organisms, preferably plankton and algae-feeding aquaculture organisms, more preferably fish, larvae, oysters, bivalves, mollusks, gastropods, or crustaceans. The present invention is used to feed an organism selected from the group consisting of:

[0056] Another aspect is the use of the pollen replacement composition of the present invention as a concentrated patty, wherein the concentrations of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol are between 10% by weight and 33% by weight compared to the total weight of the pollen replacement composition.

[0057] In another embodiment, the pollen replacement composition comprises a bee appetite / ingestion inducer component selected from the group consisting of pollen, sugar, oil or fat, honey, or protein, or mixtures thereof. For other invertebrates, different appetite / ingestion inducer components can be used.

[0058] In another embodiment, the concentration of the bee appetite / ingestion inducing component is from 1% to 20% by weight, preferably from 2.5% to 15% by weight, even more preferably from 5% to 10% by weight, compared to the total weight of the pollen replacement composition.

[0059] In another embodiment, the pollen replacement composition is used in liquid or powder form.

[0060] In another embodiment for use as a liquid or powder, the concentrations of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol are 0.01% by weight to 99% by weight compared to the total dry weight of the pollen replacement composition.

[0061] Another aspect of the invention is a pollen-free feeding composition or a pollen replacement composition, especially for bees, comprising 0.01% to 1% by weight of isofucosterol, preferably 0.01% to 0.3% by weight of isofucosterol, relative to the total weight of the pollen replacement composition, optionally further comprising natural pollen in an amount of 1% to 15% by weight, relative to the total weight of the pollen replacement composition, to increase feed consumption.

[0062] Another aspect of the invention is a pollen-free feeding composition or a pollen replacement composition, especially for bees, comprising 0.01% to 1% by weight of fucosterol, preferably 0.01% to 0.3% by weight, relative to the total weight of the pollen replacement composition, the composition optionally further comprising natural pollen in an amount of 1% to 15% by weight, relative to the total weight of the pollen replacement composition, to increase feed consumption.

[0063] Another embodiment of the invention is a pollen-free feeding composition or a pollen replacement composition, especially for bees, comprising 0.01% to 1% by weight of isofucosterol and fucosterol, preferably 0.01% to 0.3% by weight, relative to the total weight of the pollen replacement composition, wherein the weight ratio of isofucosterol to fucosterol is 0.01:100 to 100:0.01, and the composition optionally further comprises natural pollen in an amount of 1% to 15% by weight, relative to the total weight of the pollen replacement composition, to increase feed consumption.

[0064] Another aspect of the invention is a pollen-free feeding composition or a pollen replacement composition, in particular for bees, which comprises between 0.01% and 1% by weight of isofucosterol / fucosterol, preferably between 0.01% and 0.3% by weight, relative to the total weight of the pollen replacement composition, The weight ratio of isofucosterol to fucosterol is 0.01:100 to 100:0.01; The pollen replacement composition further comprises 0.01% by weight to 0.5% by weight of 24-methylene cholesterol, compared to the total weight of the pollen replacement composition; The ratio of isofucosterol and fucosterol to 24-methylenecholesterol is 2:100 to 100:2; · The pollen replacement composition optionally further comprises natural pollen in an amount of 1% to 15% by weight compared to the total weight of the pollen replacement composition to increase feed consumption.

[0065] Another aspect of the invention is a pollen-free feeding composition or a pollen replacement composition, especially for bees, which comprises 0.01% to 1% by weight of 24-methylenecholesterol, preferably 0.01% to 0.3% by weight, relative to the total weight of the pollen replacement composition, The pollen replacement composition further comprises isofucosterol and fucosterol, and the weight ratio of isofucosterol to fucosterol is 0.01:100 to 100:0.01; The weight ratio of isofucosterol and fucosterol to 24-methylenecholesterol is 2:100 to 100:2; · The pollen replacement composition optionally further comprises natural pollen in an amount of 1% to 15% by weight compared to the total weight of the pollen replacement composition to increase feed consumption.

[0066] Another aspect of the invention is a pollen-free feeding composition or a pollen replacement composition, in particular for bees, comprising 0.02% to 3% by weight of a sterol, compared to the total weight of the pollen replacement composition, the sterol being selected from the group consisting of 24-methylenecholesterol, isofucosterol, fucosterol, β-sitosterol, campesterol, stigmasterol, ergosterol and cholesterol, the weight ratio of isofucosterol and fucosterol to 24-methylenecholesterol is between 0.5:100 and 100:0.5; and / or the weight ratio of isofucosterol to fucosterol is between 0:10 and 10:0; and / or the weight ratio of isofucosterol to the sum of one or more other sterols selected from the group consisting of 24-methylenecholesterol, fucosterol, β-sitosterol, campesterol, stigmasterol, cholesterol, ergosterol is from 2:100 to 100:2; and / or the weight ratio of fucosterol to the sum of other sterols selected from the group consisting of 24-methylenecholesterol, isofucosterol, β-sitosterol, campesterol, stigmasterol, cholesterol, ergosterol is between 2:100 and 100:2; and / or the weight ratio of 24-methylenecholesterol to the sum of one or more other sterols selected from the group consisting of isofucosterol, fucosterol, β-sitosterol, campesterol, stigmasterol, cholesterol, ergosterol is 2:100 to 100:2; and / or the weight ratio of cholesterol to the sum of one or more sterols selected from the group consisting of 24-methylenecholesterol, isofucosterol, fucosterol, β-sitosterol, campesterol, stigmasterol, ergosterol is between 15:1 and 1:100; and / or · The pollen replacement composition optionally further comprises natural pollen in an amount of 1% to 15% by weight compared to the total weight of the pollen replacement composition to increase feed consumption. [Brief description of the drawings]

[0067] [Figure 1] 1 shows cohort selection of bees for diets with or without isofucosterol. [Diagram 2] FIG. 1 shows survival of cohorts of bees restricted to feeding diets containing specific concentrations of isofucosterol. [Diagram 3] 1 shows the threshold effect of isofucosterol in bee diets. [Figure 4]Results from Example 4A: A long-term feeding experiment is shown in an Apidea colony with bees fed multiple sterols, including isofucosterol and 24-methylenecholesterol. [Diagram 5] Results from Example 4B: Results of a long-term feeding experiment in an Apidea colony containing bees fed multiple sterols, including 24-methylenecholesterol but not isofucosterol, are shown. [Figure 6] Results from Example 4C: Shows the results of a long-term feeding experiment in Apidea colonies fed multiple sterols, where the total concentration of sterols in the diet was kept constant and the presence (concentration) of specific sterols was varied between diets. [Figure 7] Results from Example 5A: Feeding adult black soldier flies a diet containing multiple sterols: Effects on the hatchability of laid eggs and on the weight of laid egg masses. Adult survival and number of laid egg masses (as specified in US2019 / 0090507, Apix Bioscience) from populations fed the pollen replacement diet were measured daily using the rearing method described in Thinn and Kainoh 2022 JARQ 56(2), 211-217 (2022). [Figure 8] Results from Example 5B: Feeding adult soldier fly diets containing multiple sterols: Effects on adult life span and larval survival to the pre-pupal stage. A diet containing 22% protein, 4% lipid was fed to soldier fly (BSF) larvae until hatching as described in Hogsette 1992. J. Econ. Entomol. 85(6):2291-2294. Sterols were provided as pure compounds and added directly to the diet at a total amount of 0.1%. Larval and adult survival over a 50-day period was measured as described in Georgescu et al., Eur. J. Entomol. 118:297-306, 2021. [Figure 9]Results from Example 5C: Feeding adult black soldier flies various diets containing multiple sterols: Figure 1 shows the sterols found in tissues of black soldier flies fed multiple sterols. Sterols were added to the diet. Measurement of sterols in tissues was performed after saponification of BSF tissues using GC-MS as described by Boukid et al. 2022. Insects 12, 672. [Figure 10] Results from Example 6: Feeding of honeybee colonies in the Apidea experiment with a diet containing an extract of black soldier fly fed multiple sterols. [Figure 11A] Figures 11A-11D show the experimental setup for Example 7. Figure 11A is a photograph of the tent enclosure. [Figure 11B] FIG. 11B is a photograph of the nest boxes and feeders in the tent shown in FIG. 11A. [Figure 11C] FIG. 11C shows a patty on top of the bee frame of a Mini Plus hive. [Figure 11D] FIG. 11D shows the setup and device for taking photographs of the individual frames. [Figure 12] 11A-11D show the cumulative number of operculated larvae in the tent enclosures fed diet A and diet B according to Example 7 (raw data from Tables 2B and 4). The vertical axis shows the cumulative number of operculated larvae laid from days 12 to 96. The horizontal axis shows days 12 to 96 after the initiation of placement of the hives in the tent enclosure and the initiation of feeding diet A or diet B. The top solid line shows the average of all hives fed diet A. The bottom solid line shows the average of all hives fed diet B. Individual hives fed diet A are indicated by a short dashed line followed by the hive number. Individual hives fed diet B are indicated by a long dashed line followed by the hive number. [Figure 13]Figure 1 shows the cumulative weight of feed consumed in hives in tent enclosures fed diet A and diet B according to Example 7. The vertical axis shows the cumulative feed (grams) consumed from days 12 to 96. The horizontal axis shows days 12 to 96 from the start of placement of the hives in the tent enclosure and the start of feeding diet A or diet B. The top solid line shows the average for all hives fed diet A. The bottom solid line shows the average for all hives fed diet B. Individual hives fed diet A are indicated by a short dashed line followed by the hive number. Individual hives fed diet B are indicated by a long dashed line followed by the hive number. [Fig. 14ABC] The numbers of operculated larvae in hives fed diet A or diet B from 24 to 96 days after placement in the closed tent and initiation of feeding according to Example 7 are shown (raw data from Tables 2A and 3). The vertical axis shows the number of operculated larvae laid from day 24 to day 96. The horizontal axis shows the number of operculated larvae laid from day 24 to day 96 after initiation of placement of the hives in the tent enclosure and initiation of feeding diet A or diet B. [Figure 15] Photographs of all frames of larvae in hive 29 fed with diet A from days 36 to 84 according to Example 7 are shown. [Figure 16] Photographs of all frames of larvae in hive 29 fed with diet B from days 36 to 84 according to Example 7. [Figure 17] Shown are photographs of frames of larvae in hive 29 fed diet A (top two rows) and in hive 26 fed diet B (bottom two rows) at 60, 72, and 84 days after the start of the experiment according to Example 7. Both hive 29 and 26 plot close to the average of the hives fed diet A and diet B. [Figure 18]Figure 1 shows the cumulative number of occluded larvae in tent enclosures fed diet A and diet C according to Example 7 (raw data from Tables 2B and 4). The vertical axis shows the cumulative number of occluded larvae laid from days 12 to 96. The horizontal axis shows days 12 to 96 after the start of placement of the hives in the tent enclosure and the start of feeding diet A or diet B. The top solid line shows the average of all hives fed diet A. The bottom solid line is the average of all hives fed diet C. Individual hives fed diet A are indicated by a short dashed line followed by the hive number. Individual hives fed diet C are indicated by a long dashed line followed by the hive number. [Figure 19] Shown are photographs of frames of larvae in hive 29 fed diet A (top two rows) and hive 14 fed diet C (bottom two rows) at 48, 60, 72, 84, and 96 days after the start of the experiment of Example 7. Both hive 29 and 14 plot close to the average of the hives fed diet A and diet C. [Figure 20] The number of operculated larvae in hives fed diets A, B, C, and E according to Example 8 from 24 to 96 days after placement in the closed tent and initiation of diet feeding (raw data from Table 6). The vertical axis shows the number of operculated larvae produced from days 24 to 96. The horizontal axis shows the number of operculated larvae produced from days 24 to 96 after initiation of placement of the hives in the tent enclosure and initiation of feeding with diets A, B, C, and E (commercial diets). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Embodiments of the present invention are described below.

[0069] Honeybees live in colonies. Standard industry practice is for colonies of various sizes: 8 frame colonies (which is the industry standard) containing about 20,000 bees; 10 frame colonies with about 24,000; large colonies with 2 stories 12.5 frames = 30,000; and colonies with 50,000 bees (2 are extra large). Overall, bee colonies vary in size (+ / - 50% increase or decrease in size over the course of a year, depending on the season and nutrient availability).

[0070] Rortais et al. 2005 (Apidologie, 36(1), 71-83) reviewed the literature on pollen consumption in bees comprehensively and stated that between 10 and 55 kg are collected per colony per year. Crailsheim et al. 1993 (J Insect Phys 38(6):409-419) calculated the annual pollen consumption in two colonies to be 13.4 and 17.8 kg, respectively, for a colony of approximately 25,000 bees in July-August. Wille et al. 1982 (Revue Suisse de Zoologie 4:897-914) recorded between 10 and 26 kg of pollen per year. This clearly depends on the size of the colony, colony growth, environment, and pollen type.

[0071] Keller et al. 2005 (Bee world, 86(1)3-10) estimate that each worker bee requires 140-180 mg of pollen over its entire life span. For a colony of 100,000-200,000 bees per year, this requires 17-34 kg of pollen per colony. Crailsheim et al. 1993 (J Insect Phys 38(6):409-419) compiled the amount of pollen consumed per colony from five published papers. They also measured pollen consumption from two colonies they measured. They estimate that the average amount of pollen required for a colony producing about 150,000 larvae per year is 19 kg.

[0072] When bee colonies receive supplemental feeding during pollen deprivation periods, the colony typically receives supplemental food in the form of protein or pollen patties (the majority of feeding regimes are "ad libitum"), resulting in a consumption of approximately 1.5 to 2-3 pounds (0.680-1.35 kg) per 12.5 frames per two weeks. In the industry, a bee frame is defined as approximately 2400 bees (30,000 bees in a colony), and hives used in the industry can contain 5, 8, 10, or 12 frames, or even more. As the spring season begins, a hive is only partially populated (e.g., 5 frames of bees in a 10-frame hive), and as the season progresses, the bee population in that hive grows, filling a 10-frame hive with 10 frames of bees. During the winter, the colony continues to reduce its population to 5-6 frames of bees.

[0073] In calculating the dose of "mg pollen equivalent feeding" required by bees, it must be taken into account that pollen from different plant species contains a wide range of about 50% sugars, proteins, and other components, and therefore the nutritional value of pollen varies (within a range of ±50%) depending on the plant species. By supplementary feeding with pollen, pollen supplement, or protein supplement, colonies receive a dose of about 1.5 to about 3 pounds, or (680g to 1360g) of pollen supplement per 2 weeks. On an annual basis, this amounts to a pollen supplement meal in the range of 17.7kg to 35.4kg, which is within the published range of figures for a colony of 10 frames consuming pollen, taking into account the variability in the nutritional quality of pollen. For colonies of various sizes, the dose calculations are adjusted proportionally. (One bee frame = about 2400 bees).

[0074] Using calculations based on tables from Roessink et al 2021 (J Apic Res 60:659-654), a colony of 30,000 bees would consume 62 grams of pollen per day (during active flowering and bee breeding season) or 868 grams per two weeks.

[0075] A more comprehensive description is given in the text and tables in the following reference: https: / / www.insignia-bee.eu / how-much-pollen-does-a-colony-need / "Rortais et al. (2005) comprehensively reviewed the literature on pollen consumption in bees and stated that 10-55 kg is collected per colony per year. Crailsheim et al. calculated the annual pollen consumption for two colonies to be 13.4 and 17.8 kg, respectively, for a colony of approximately 25,000 bees in July-August. Wille et al. recorded pollen consumption ranging from 10 to 26 kg per year. This clearly depends on the size of the colony, the development of the colony, the environment, and the type of pollen. For example, maize pollen is consumed in approximately twice the amount compared to mixed pollen, due to its relatively low percentage of nutrients in the whole pollen grain. The percentage of protein in dry weight is comparable to that of mixed pollen (Hoecherl et al., 2012). Clearly, there is no fixed amount of pollen that can or should be collected for a colony to grow, maintain, and function. Nevertheless, there are good quality studies available to investigate the annual pollen required by a colony. As mentioned above, Rortais et al. Based on the paper by Keller et al. (2005), nurse bees consume an average of 65 mg of pollen and worker bee larvae consume an average of 5.40 mg of pollen. Consequently, bees consume at least 70.4 mg of pollen during their lifetime. This is an average estimate; twice the amount has been reported as well. In the above mentioned study by Crailsheim et al., the daily pollen consumption was 3.4-4.3 mg. For a summer honeybee's life span of 30 days, this is approximately 115 mg over the life span. Another approach to investigating pollen consumption is reported by Keller et al. al. (2005a) review paper. This is a survey of pollen consumption based on nitrogen (N) for larvae and bees. Pollen is the only source of N in the bee diet. To convert N to pollen, it is estimated that pollen protein contains 16% nitrogen, pollen contains 20% protein, and the digestive efficiency is 80%. That is, for 10 mg of N, 62.5 mg of protein is needed, and for 62.5 mg of protein, 312.5 mg of pollen is needed, and for the efficiency of the feeding process, 390.6 mg of pollen is needed to be consumed by the bee. Taking all this into account, 125 mg of pollen is needed to rear one bee.After emergence, the N in a bee increases by 0.86 mg. 0.86 mg of N corresponds to 39 mg of pollen. Thus, overall, each bee required a minimum of 125+39=164 mg of pollen, not taking into account N losses due to defecation. Assuming a 200 day active and reproductive period and an average bee life span of 35 days (Steen et al, 2012), it takes 200 / 35=6 generations. To maintain an average colony of 15,000 bees, 15,000×6=90,000 bees are kept. The number of kept bees increases year by year, and the calculations of the required pollen are summarized in Table 1 (of this paper).

[0076] The starting amount of 35 kg mentioned above is the best estimate of the amount needed for a large colony. Amounts varying between 15-25 kg seem more realistic. Again, this all depends on the pollen type, environment, colony growth, and the presence or absence of pollen traps. By depriving the colony of pollen, pollen collection has been stimulated. It is the beekeeper's responsibility to maintain an adequate pollen flow. A rough estimate of the surface / number of bee-pan cells in the colony is simply needed to ascertain whether the pollen reserve is sufficient. A healthy normal-sized colony produces approximately 0.5-2 dm2 of pollen per day. 2 (200–800 half-filled bee-pan cells). Another simple estimation is the weighting of pollen trap returns. It must be taken into account that the effectiveness of pollen traps varies from 10–50% (Keller et al., 2005b).

[0077] Table 1 in Roessink et al. 2021, results of a 6-week field study (Journal of Apicultural Research, 60:659-664), shows an exemplary calculation of pollen requirements by a hive, based on pollen consumption per day, for a colony of a given size. Larvae and nurse bees are the consumers of pollen. Older worker bees consume honey / nectar.

[0078] Pollen represents a natural food source for bees and pollen-feeding insects. It is not optimized by the plant to feed insects (it is optimized for plant use = fertilization). Pollen from different species contains a wide range of different proteins, lipids, sterols, antioxidants, molecules in widely different concentrations. Therefore, it is not yet known which of these components are beneficial, which are plant defense molecules that prevent insects from eating the pollen, and which are not important components for insect nutrition and therefore which are important to include or exclude from artificial diets.

[0079] The inventors have shown that the addition of 0.14 to 12 grams of isofucosterol over a two week period, at a dose calculated for a colony of 30,000 bees, is beneficial to colony and bee performance (as measured by bee larval production and other fitness parameters such as longevity), which represents a concentration of 0.01% to 1.76% isofucosterol in a 680 gram patty fed over a two week period for a colony of 30,000 bees.

[0080] Calculated per bee consuming non-pollen supplement in the hive (=brood bees = 50% of bees in the hive), this represents 140mg / 15000 bees = 0.009mg to 12000mg / 15000 bees = 0.8mg isofucosterol or fucosterol per nurse bee per 14 days in the hive (brood bees weigh 110mg). This represents a dose of isofucosterol or fucosterol per day (% of body weight) = 0.009 / 110*100 / 14 = 0.0006% to 0.8 / 110*100 / 14 = 0.052%.

[0081] Furthermore, the inventors show that a daily dose of isofucosterol or fucosterol of 0.0006% to 0.052% of body weight is beneficial for the production of other pollen-feeding insect larvae and adults, such as bumblebees, soldier flies adults and larvae; adult ladybirds, hoverflies, and other pollen-feeding insects. Among other things, this is important for adult reproductive and fertility, as well as high hatching percentages and survival of young larvae.

[0082] The mode of administration affects the efficiency of uptake and bioavailability.

[0083] Method of administration Administration to bee colonies or organisms is only for the period when the desired effect is needed: at the very least, a single dose to induce or support queen reproduction when bees are out of winter to improve, induce or support normal larval development, or when needed for the insects to produce gametes or eggs. It can be for longer periods when bees are fed nutritionally poor pollen producing crops (blueberries, carrots, etc.) or spanning pollen deprivation periods in spring, summer, or winter. For colonies of different sizes, dosages can be adjusted in proportion to the amount of bees or nurse bees present.

[0084] This twice weekly dose of isofucosterol can be administered to the bee hive or to feeding insects in a variety of ways. This dose of isofucosterol can be administered to the colony in a variety of formulations (incorporated in a suspended, dissolved, or emulsified form in a manner that is easily digestible by the bees or target animals). -30,000 hives per two weeks are incorporated with a standard 1.5 to 3 lb protein or pollen supplement patty, and in one embodiment the composition comprises isofucosterol or fucosterol, or a mixture thereof, in an amount of 0.01 to 5%, preferably 0.01 to 2%, even more preferably 0.01 to 1%, and even more preferably 0.03 to 0.9% of the diet for honey bees and 0.03 to 0.8% for bumble bees, as a percentage of the total weight of the pollen replacement composition. - then incorporated into or mixed with ingredients that induce the bees to eat the compound, such as pollen, sugars, oils / fats, honey, or proteins, or mixtures thereof, and further concentrated in mini-patties, with much higher concentrations, e.g., up to 33% sterols; or -Incorporated into or mixed with ingredients that induce bees to eat the formulation, such as pollen, sugars, oils / fats, honey, or proteins, or mixtures thereof, containing isofucosterol, spread within the colony, or fed outside the colony in a feeding station, as a powder or paste. -Isofucosterol is incorporated into or mixed with ingredients that induce bees to eat formulations such as pollen, sugars, oils / fats, honey, or proteins, or mixtures thereof, diffused within the colony as a syrup or liquid, or fed outside the colony in a feeding station. In another embodiment, the dose sterol molecule can be delivered by a device that injects the sterol composition into the colony, for example, in a preprogrammed manner. -or any method known to one skilled in the art that can be used to deliver the indicated doses of chemicals or nutrients, etc., to the bees so that they ingest them.

[0085] In another embodiment, the sterol mixture is not administered by patty or similar feed, but the above multiplicity and ratio of sterols is dissolved in an aqueous or oily medium, or in a nutrient slush or other edible form, and administered to the colony as a drinking liquid, liquid paste, hive spray, or powder. In this embodiment, the dry weight concentration of the sterols can be from 0.01 to 100% dry weight (as the water carrier evaporates from the administered medium).

[0086] Effect of feeding isofucosterol in combination with other sterols: A combination of isofucosterol and / or cholesterol, and / or 24-methylenecholesterol, and / or β-sitosterol, and / or stigmasterol, and / or campesterol, (1) Improved, better than feeding isofucosterol alone (2) More improvement than feeding the above sterol combinations without isofucosterol, (3) improved GI motility compared to feeding a combination of the above sterols lacking cholesterol, or 24-methylenecholesterol, or campesterol, or stigmasterol, or β-sitosterol. We further show that

[0087] Surprisingly, bee colonies fed diets containing isofucosterol and multiple sterols including 24-methylenecholesterol, cholesterol, isofucosterol, and campesterol, β-sitosterol, or stigmasterol, are able to support larval development for more than five cycles, whereas similar diets without isofucosterol do not. What is important in our experiments is that the larval production rate settles to a stable level. This trajectory shows that our diets provide sufficient levels of essential nutrients to allow unlimited larval production. This means that such diets can act as a pollen substitute for bee colonies over this period. Thus, isofucosterol in the diet allows beekeeping to become independent of pollen sources in natural or agricultural settings. In bumblebees, such diets with multiple sterols allow the queen to create a base for rearing larvae without the need for pollen.

[0088] The creation of such a secret is not clear from the literature. For example, Rasmont et al. (2005 J Econ Ent 98(3):656-663) interpreted data from Herbert et al. (1980) to show that sitosterol and stigmasterol are antifeedant and not essential for honeybees when they are fed an artificial diet containing only one sterol.

[0089] The inventors have extended the utility of the invented honeybee-derived feed to other pollen-feeding insects with commercial relevance in biological defense (e.g. ladybirds), insect biomass production (e.g. soldier flies) and pollination (hoverflies).

[0090] Changes to livestock production and feeding that utilize waste materials generated by food production are urgently needed. In addition, methods of protecting crops from agricultural pests that are resistant to agrochemicals, such as the protection and release of natural insect predators, such as ladybugs and hoverflies, are important tools for creating integrated pest management strategies that reduce environmental impacts and protect biodiversity.

[0091] An important emerging means of utilizing industrial agricultural waste is the cultivation of fly larvae in waste materials such as the black soldier fly (Hermetia illucens). Black soldier fly larvae are omnivorous generalists and can feed on plant or animal waste, including feces, making them an advantageous means of converting waste biomass into livestock feed. In agricultural production, these larvae and pupae are fed to poultry and fish. These insects obtain nutrients such as fatty acids and sterols from the feed they consume as larvae, which makes them a valuable addition to the food chain (Boukid et al. 2021 Insects 12,672. https: / / doi.org / 10.3390 / insects12080672). However, feed such as industrial human waste is not optimized for the black soldier fly and often lacks key essential nutrients. For example, the development time, size, and mortality of BSF-fed larvae are greatly influenced by the nature of the food on which they are cultivated (Lalander et al. 2019. J Cleaner Production, https: / / doi.org / 10.1016 / j.jclepro.2018.10.017). Delayed development time due to suboptimal diet reduces the efficiency of the rearing system.

[0092] Another problem with existing rearing methods is suboptimal diets for the life cycle stages. Holometabolous insects often consume different foods as larvae and adults. Each life cycle stage has substantially different nutritional requirements. Foods fed to larvae in laboratory or industrial production settings may not be suitable for adult reproductive potential and survival.

[0093] In the wild, BSF larvae feed on decaying organic material and adult BSF feed on flower pollen. Like larvae, adult BSF also require protein; adult BSF lay more eggs when fed protein (Bertinetti et al. 2019. Journal of Insect Science 19(1):19;1-7). Although protein improves performance, egg mass size and egg hatching in protein-fed laboratory-reared flies were less than half that of wild-fed flies (Bertinetti et al. 2019). It is likely that certain nutrients, as yet unidentified, are not present in the laboratory diet (Bertinetti et al. 2019). Adult performance is important because adult beetles lay eggs, which is the rate-limiting step in industrial production (Cickova et al. 2015 Waste Management Volume 35,January 2015,68-80).

[0094] Bertinetti et al. (2019) speculate that pollen possesses the key nutrients required by BSF. Recently, it has been shown that when black soldier flies are fed a diet containing flower pollen, their egg-laying performance is much improved (Thin and Kanoi. 2022. JARQ 56(2):211-217). For example, when female black soldier flies are fed pollen, egg-laying and egg hatching doubles and their life span increases by 30% (Thin and Kanoi 2022). However, the pollen components that cause this change are unknown.

[0095] In other beneficial insects used in agriculture, pollen feeding also affects the fertility of adult insects. This is particularly important in insects cultivated for biological defense, such as ladybirds (Coccinelidae) and hoverflies (Syrphidae). For example, ladybird larvae feed on pollen when their preferred prey, aphids, are unavailable (Berkvens et al. 2010 Eur. J. Entomol. 107:189-195, 2010, Shuldiner-Harpez and Coll. 2017 J Insect Behav 30:432-438). Importantly, pollen feeding by adult insects has been shown to be necessary for fertility; male insects that do not feed on pollen are unable to produce viable sperm (Ugine et al. 2019 Ecology Letters 22:275-283). Pollen feeding is also an essential component of reproductive success in hoverflies (Syrphidae), which are important pollinators and predators of aphids (Hickman et al. 1995 New Zealand Journal of Zoology 22:387-392). Female hoverflies are highly sensitive to the type of pollen they feed on; pollen type affects reproductive success and offspring survival (Amorous-Jimenez et al. 2014 BioControl 59:427-435).

[0096] Pollen contains many different phytosterols that are not found in other terrestrial plants or animal sources (Zu et al. 2020. New Phyt 230:1169-1184). These sterols have a wide range of functions in and outside the plant. Many phytosterols are produced by plants and inhibit insect feeding on the plant.

[0097] The inventors have surprisingly established that isofucosterol, a common sterol found in pollen, is an essential nutrient for pollen-feeding insects. Isofucosterol delivered by pollen-free diets increases the performance of adult and larval BSF. The inventors have also identified that the major sterol component of pollen selected by adult female hoverflies is isofucosterol. The inventors have further discovered that the combination of isofucosterol with at least one additional sterol, in particular cholesterol, 24-methylenecholesterol, and other phytosterols, in particular campesterol, β-sitosterol, or stigmasterol, results in higher growth and / or health performance in many invertebrates, in particular holometabolous insects, including bees and pollen-feeding flies, moths / butterflies, and insects.

[0098] Feeding a combination of isofucosterol, cholesterol, 24-methylenecholesterol, β-sitosterol, stigmasterol, and campesterol (1) More improvement than isofucosterol alone, (2) More improved than the combination of the above sterols that does not contain isofucosterol, (3) improved more than cholesterol or the combination of the above sterols lacking 24-methylenecholesterol, campesterol, stigmasterol, or β-sitosterol The present inventors have further established that

[0099] Surprisingly, feeding cultured adult black soldier flies or insects with a feed containing a mixture of isofucosterol and sterols improves the egg-laying and egg-hatching performance as well as the life span of adults. The same effect can be obtained by feeding larvae that produce larval stocks with a feed containing a mixture of isofucosterol and sterols (adults inherit substantial nutritional reserves from the larval / pupal stage). This means that such a feed can act as a pollen substitute and a form of supplemental nutrients for laboratory-reared insect colonies. This feed therefore makes it possible to optimize the larval development time and adult reproductive potential in industrial cultures of beneficial insects.

[0100] This is not clear from the literature. Svoboda, the world's leading researcher in insect sterols, teaches that in all but two species, cholesterol, when present in the diet, supplies the dietary need for sterols (Svoboda. 1999. Critical Reviews in Biochemistry and Molecular Biology, 34(1):49-57). His summary did not recognize the need for sterols from pollen as an essential nutrient for many pollen-feeding insects. He did not recognize isofucosterol as a necessary sterol for holometabolous larval growth or adult insect reproductive ability.

[0101] Thus, a first aspect of the present invention provides a method for producing a method for treating a pulmonary circulation comprising the steps of: - providing a pollen replacement formulation comprising a nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof; administering the pollen replacement formulation to animals such as honeybees and bumblebees, as well as pollen or algae consuming animals such as ladybugs, soldier flies, hoverflies, oysters, shrimp, and fish larvae; The present invention relates to a method for feeding pollen or algae consuming animals, such as bees, such as honeybees and bumblebees, ladybugs, soldier flies, hoverflies, oysters, shrimp, and larval fish.

[0102] Nutritionally Effective Dose In a preferred embodiment, the administration of a dose of a pollen replacement formulation containing isofucosterol or fucosterol or a mixture thereof and a further sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol results in a total twice weekly dose of 0.2 to 48 grams, preferably 0.4 to 36 grams, and even more preferably 0.6 to 20 grams of sterols from the above group to a colony of 30,000 animals, where 100% of sterol in the above indicated doses is divided as follows: isofucosterol-fucosterol is 10 to 60% of the sterol dose and the remainder of the sterol or sterols represents the remainder of the total sterol dose.

[0103] In a preferred embodiment, a 100% dose of the group of sterols consisting of isofucosterol / fucosterol and / or cholesterol and / or 24-methylenecholesterol and / or phytosterols consisting of campesterol and / or stigmasterol and / or β-sitosterol in a diet for a colony of 30,000 bees over a period of 14 days is 0.3 grams to 20 grams of the composition, of which 10-60% isofucosterol and / or 0-50% cholesterol and / or 0-50% 24-methylenecholesterol and / or 0-50% mixture of β-sitosterol and / or stigmasterol and / or campesterol.

[0104] This represents a daily dosage range of 0.001% (formula = 0.3 / 14 / 1650 x 100) of the body weight of the insect consuming the meal to 0.087% (formula = 20 / 14 / 1650 x 100) of the body weight of the insect consuming the meal, of a "sterol composition" that is 10-60% isofucosterol and / or 0-50% cholesterol and / or 0-50% 24-methylenecholesterol and / or 0-50% a mixture of β-sitosterol and / or stigmasterol and / or campesterol.

[0105] This calculation is based on 50% of the bees being brood bees weighing 110 mg each.

[0106] The total dose can be proportionally adapted to large or small insects, invertebrates, insect colonies, and short or long feeding intervals, with the unit doses disclosed herein being for a colony of 30,000 bees over a 14 day period (see, e.g., the table in Keller et al., 2005b, supra).

[0107] The same compositions and dose ranges apply to other pollen-feeding insects.

[0108] In another embodiment, some or all of these sterol moieties can be delivered as lipid-soluble forms, such as prodrugs such as water-soluble sterol glycosides or sterol sulfates or similar salts, or sterol-esters or other similar prodrug forms, dosed to represent a fraction of the molecular weight of the corresponding sterol nucleus in the overall molecular weight of the complexed sterol molecule.

[0109] The various methods of administration described above for isofucosterol can be applied to isofucosterol, as well as the other sterols described herein.

[0110] To calculate the dose of isofucosterol and a group of sterols that can be delivered to other pollen-feeding insects (see above) as a percentage of body weight per day, the following formula is used: -[30000 / 2]*0.110 grams = "weight of non-pollen supplement consuming insects (brood bees and / or larvae) in a 30000 hive" (@0.11 grams for the brood bees and / or larvae; @30000 bees in a hive, 50%: half of which are brood bees and / or larvae=1650 grams). -["Dose of a given sterol or group of sterols delivered over a 14 day period to a colony of 30,000 bees" / 14] = "Daily amount of sterol administered" - Daily dose administered (grams) / Weight (grams) of non-pollen supplement consuming insects (brood bees and larvae) in 30,000 hives x 100 = % body weight of sterol administered per gram of insect body weight per day -Dosage of sterols to bumblebee colonies: "Percentage of body weight of sterol administered per gram of insect body weight per day" x "Body weight of insects (brood wasps and larvae) being fed" On average - see table above; 2 / 3 of the pollen consumed in a colony is consumed by the nurse bees and 1 / 3 by the resident larvae (see table above).

[0111] In a preferred embodiment, the sterols consisting of isofucosterol / fucosterol, and / or cholesterol, and / or 24-methylenecholesterol, and / or the phytosterols consisting of campesterol and / or stigmasterol and / or β-sitosterol are administered at 1.5 to 3 pounds of patties per 14 days for 30,000 bees, the patties being: - isofucosterol (0.01-2%, preferably 0.01-1%, more preferably 0.03-0.6% of the feed dry weight) or fucosterol (0.01-2%, preferably 0.01-1%, more preferably 0.03-0.6% of the feed dry weight) or a mixture thereof (total of 0.01-2%, preferably 0.001-1%, more preferably 0.03-0.6% of the feed dry weight) and at least one further sterol (concentration as a percentage (%) of the feed dry weight) selected from the following group; resulting in a multiplicity of isofucosterol or fucosterol or a mixture thereof, comprising at least one further sterol: Cholesterol 0.001 to 2%, preferably 0.001% to 1.5%, more preferably 0.06 to 1.2%, and / or 24-methylenecholesterol 0.001 to 2%, preferably 0.001% to 1.5%, more preferably 0.06 to 1.2%, and / or β-sitosterol 0.001 to 2%, preferably 0.001 to 1%, more preferably 0.03 to 0.6%, and / or campesterol 0.001 to 2%, preferably 0.001 to 1%, more preferably 0.02 to 0.35%, and / or 0.001 to 2%, preferably 0.001 to 1%, more preferably 0.01 to 0.2% of stigmasterol, and / or or any combination thereof, and the total concentration of sterols is in the range of 0.01 to 4% (dry weight).

[0112] In another embodiment, the composition is administered to an invertebrate of the genus honeybee or bumblebee, especially a honeybee or a bumblebee.

[0113] In another embodiment, the source of isofucosterol, fucosterol, or a mixture thereof, or the source of at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, stigmasterol, campesterol, and β-sitosterol, is a pollen-substitute tissue of one or more plant species selected from the group consisting of leaves, stems, roots, tubers, flowers, seeds, bark, and fruits, and combinations thereof.

[0114] In another embodiment, the source of isofucosterol, fucosterol, or a mixture thereof, or the source of at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol, is from an animal source, chemically synthesized or in vitro enzymatically synthesized, or from natural or metabolically engineered microorganisms such as yeast, algae, or diatoms, or a combination of these sources.

[0115] In another embodiment, the source of isofucosterol, fucosterol, or a mixture thereof, or the source of at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, stigmasterol, campesterol, and β-sitosterol, is an extract, oil, or purified product of pollen substitute tissue of one or more plant species or combinations thereof.

[0116] In another embodiment, the source of isofucosterol, fucosterol, or a mixture thereof, or the source of at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, stigmasterol, campesterol, and β-sitosterol, is pollen substitute tissue of one or more plant species selected from the group consisting of Solanaceae, Poaceae, Ranunculaceae, Fabaceae, and Bacillus subtilis.

[0117] In another embodiment, the source of isofucosterol, fucosterol, or a mixture thereof is Extracts, oils or purified products of marine or freshwater algae, in particular Ulva lutea; Extracts, oils or purified products of marine diatoms, in particular Thalassiosira pseudonana, Thalassiosira rotula or Chaetoceros muelleri; and Extracts, oils or purified products of fungi, in particular Saccharomyces cerevesiae or Yarrowia lipolytica The alternative pollen source is selected from the group consisting of:

[0118] In another embodiment, isofucosterol or fucosterol is chemically or enzymatically synthesized or obtained by a genetically modified host organism, such as a fungus, bacterium, or algae.

[0119] In another aspect, the source of isofucosterol, fucosterol, or mixtures thereof, and / or at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, stigmasterol, campesterol, and β-sitosterol is selected from the group consisting of algae, plants, fungi, algae, diatoms, and combinations thereof, and the source of isofucosterol, fucosterol, or mixtures thereof, and the source of at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, stigmasterol, campesterol, and β-sitosterol is pollen substitute tissue.

[0120] In another embodiment, isofucosterol, fucosterol, or a mixture thereof, and / or at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol is provided to a eusocial or proto-eusocial bee colony from a synthetic source.

[0121] In another embodiment, isofucosterol, fucosterol, or a mixture thereof, and / or at least one further sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol, is provided as part of a pollen replacement composition, the pollen replacement composition comprising: protein in an amount between 10% and 50% by weight, preferably between 15% and 40% by weight, fatty acids in an amount between 1% and 20% by weight, preferably between 2% and 12% by weight, carbohydrates in an amount between 20% and 90% by weight, preferably between 30% and 70% by weight, Optionally, vitamins, and Optionally, minerals wherein the sum of the total amounts of the components and optional further components is 100% by weight, where the weight % is relative to the total dry weight of the composition.

[0122] Liquid or semi-liquid experimental administration: In another embodiment, isofucosterol, fucosterol, or a mixture thereof, and / or at least one further sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol, are provided as part of a liquid or semi-liquid feed supplement, optionally to which an appetite stimulant such as sugar or any other appetite stimulant is added, the liquid or semi-liquid feed supplement composition comprising: optionally, protein in an amount between 10% and 50% by weight, preferably between 15% and 40% by weight, optionally fatty acids in an amount between 1% and 20% by weight, preferably between 2% and 12% by weight, optionally carbohydrates in an amount of 20% to 90% by weight, preferably 30% to 70% by weight, Optionally, vitamins, and Optionally minerals, optionally, water or another solvent, wherein the sum of the total amounts of the components and optional further components is 100% by weight, where the weight % is relative to the total dry weight of the composition.

[0123] In another embodiment, the composition is substantially free of pollen.

[0124] Isofucosterol and fucosterol Isofucosterol is a delta-5 sterol with the structural formula (Figure I):

[0125] [ka] Fucosterol is a delta-5 sterol with the structural formula (Figure 2):

[0126] [ka] The structure of isofucosterol / fucosterol is given at https: / / en.wikipedia.org / wiki / Isofucosterol and at https: / / pubchem.ncbi.nlm.nih.gov / compound / 5281326.

[0127] 24-Methylenecholesterol The structure of 24-methylenecholesterol can be found here: https: / / pubchem.ncbi.nlm.nih.gov / compound / 24-Methylenecholesterol Isofucosterol and 24-methylenecholesterol were synthesized by chemists skilled in organic synthesis, and the final products were purified to 99% purity and characterized by LCMS, NMR, and liquid chromatography (Figures 3, 4, 5). Isofucosterol and 24-methylenecholesterol were purified from natural sources such as pollen collected by bees. Cholesterol was purchased from commercial sources. β-sitosterol and other sterols were purified from commercially available soy phytosterols using standard techniques.

[0128] Physiologically Active Sterol Complexes By physiologically active sterol complexes is meant complexes of sterols that are metabolizable by invertebrate or aquatic or plant life forms.

[0129] The sterol molecules 24-methylenecholesterol, β-sitosterol, cholesterol, campesterol, isofucosterol / fucosterol, stigmasterol can be provided in the diet in various complexes and as mixtures of complexes.

[0130] The concentration of sterol administered in the complex mixture is calculated as follows:

[0131]

number

[0132] Examples of various sterol complexes are as follows: As free alcohol Esterified with fatty acids (including but not limited to palmitic, stearic, linoleic, oleic, stearic, linoleic acids, etc.); - linked to fatty alcohols by alcohol groups (sterol alkyl ethers), - Esterification with acids such as sulfuric acid (sterol sulfates), - attached to carbohydrates (resulting in steryl glycosylation). The sterol group at the 3-OH position can be any sterol from the sterol group: 24-methylenecholesterol, β-sitosterol, cholesterol, campesterol, isofucosterol / fucosterol, stigmasterol. The sugar glucose can be glucose, galactose, mannose, xylose, arabinose. A preferred embodiment is a glucose ester from the group of sterols. · g-trans-hydroxycinnamate, ferulate (4-hydroxy-3-methoxycinnamate) and p-coumarate (see diagram below for campesteryl ferulate as an example of a steryl phenolate complex). A preferred embodiment is the coumarate ester of the sterol group.

[0133] Preferred examples of sterol complexes are as follows:

[0134] [ka]

[0135] [ka]

[0136] [ka]

[0137] Examples of β-sitosterol complexes include:

[0138] [ka]

[0139] [ka]

[0140] [ka]

[0141] [ka]

[0142] [ka]

[0143] [ka]

[0144] [ka]

[0145] [ka] As with the example of β-sitosterol above, complexes with 24-methylenecholesterol, isofucosterol, campesterol, or cholesterol, or stigmasterol can be used.

[0146] Further preferred sterol conjugates occur naturally in plants, such as (A) steryl esters (SE), (B) steryl glycosides (SG), (C) acylsteryl glycosides (ASG), having the following structures:

[0147] [ka]

[0148] [ka]

[0149] [ka] The R side chain, in this patent, can be the group cholesterol, isofucosterol, 24-methylenecholesterol, campesterol, β-sitosterol, stigmasterol.

[0150] Further examples include steryl glycosides, steryl esters, and acetylated steryl glycosides.

[0151] [ka]

[0152] [ka]

[0153] [ka]

[0154] [ka] R is the side group of the sterol. The sterol can be a delta-5 or delta-7 sterol.

[0155] Further examples of sterol complexes include:

[0156] [ka]

[0157] [ka]

[0158] [ka]

[0159] [ka]

[0160] [ka]

[0161] [ka]

[0162] [ka]

[0163] [ka]

[0164] [ka]

[0165] [ka]

[0166] [ka]

[0167] Isofucosterol as an insect nutrient Surprisingly, the inventors have discovered that isofucosterol is an essential bee and other pollen-feeding insect nutrient. Honeybees and bumblebees, for example, exhibit the following levels of isofucosterol in their body composition:

[0168] [Table 1]

[0169] Pollen substitute composition The feature "pollen substitute" means that it is essentially free of pollen. However, trace amounts of pollen may be present in the composition of the present invention. In one embodiment, the amount of pollen is 15% by weight or less, preferably 10% by weight or less, even more preferably 5% by weight or less, and even more preferably 1% by weight or less, and even more preferably 0.1% by weight or less, compared to the dry weight of the composition. The terms pollen substitute and non-pollen are used interchangeably.

[0170] Alternative pollen source of isofucosterol Isofucosterol, for example, Synthetic chemical synthesis, or Any pollen substitute plant source, can be obtained from alternative pollen sources.

[0171] Examples of plant sources of pollen substitutes for isofucosterol include algae, fungi, bacteria, or animal parts that contain isofucosterol.

[0172] In one embodiment, the pollen replacement source naturally contains isofucosterol.

[0173] In another embodiment, the alternative pollen source of isofucosterol is an organism that has been metabolically engineered to produce isofucosterol.

[0174] invertebrates As for invertebrates, Arthropods, such as insects, arachnids, crustaceans, and myriapods; Molluscs, such as chitons, snails, clams, squids and octopuses; Annelids, for example earthworms and leeches; and Cnidarians, such as hydra, jellyfish, sea anemones, and corals Examples include:

[0175] Preferred invertebrates are those cultivated or raised for human or animal nutrition purposes, such as honeybees, bumblebees, earthworms, mealworms, shrimp, prawns or crayfish, crickets, fly larvae, ladybugs, soldier flies, hoverflies, and oysters or other bivalve mollusks. Particularly preferred invertebrates are: bees of the genus Apis, and in particular Apis mellifera, Apis cerana mellifera, Apis moniliformes, or Bumblebees of the genus Bombus, in particular B. terrestris, B. impatiens, B. ignites, Stingless bees of the Tribe Apis and other animals of the Apididae or Aardvark families that are used as pollinators for agricultural or horticultural plants.

[0176] Dosage and concentration The doses and concentrations in the examples below are for the feeding of a 30,000 colony, including 1.5 to 3 pounds of bee patties, over a period of 14 days. The dose of patties can be adapted to the size of the colony, and the dose per 14 days can be repeated by the beekeeper, if desired. The same doses can be delivered in embodiments where administration is not by patties, but by other forms as described above. Formulas for calculating the dose for the different formulations, methods of administration or size of the bee colony, or as dose / day as a percentage of insect biomass, are described above.

[0177] In one embodiment, isofucosterol or fucosterol is administered in a nutritionally effective amount to an invertebrate, particularly a honeybee or a bumblebee.

[0178] In one embodiment, the nutritional effect involves feeding a colony of 30,000 bees 1 to 1.5 to 3 pounds of bee patty formulation having a concentration of isofucosterol or fucosterol or mixtures thereof in an amount of 0.01 to 5%, preferably 0.01 to 2%, and even more preferably 0.01 to 1% as a percentage of the total weight of the pollen replacement composition, over a period of 14 days. In another embodiment, the concentration of isofucosterol or fucosterol or mixtures thereof is 0.03 to 0.5% for honey bees or 0.03 to 1% for bumble bees as a percentage of the total weight of the pollen replacement composition. The dose of patty can be adapted to the size of the colony and the dose per 14 days can be repeated as desired by the beekeeper.

[0179] In one embodiment, cholesterol is administered in an amount nutritionally effective for invertebrates, particularly honeybees or bumblebees, preferably at a concentration of 0.001-2%, preferably 0.001-1.5%, more preferably 0.06-1.2% of the total pollen replacement composition dry weight.

[0180] In one embodiment, 24-methylene cholesterol is administered in an amount nutritionally effective for invertebrates, particularly honeybees or bumblebees, preferably at a concentration of 0.001-2%, preferably 0.001-1.5%, more preferably 0.06-1.2% of the dry weight of the total pollen replacement composition.

[0181] In one embodiment, sitosterol is administered in an amount nutritionally effective for invertebrates, in particular honeybees or bumblebees, preferably at a concentration of 0.001-2%, preferably 0.001-1%, more preferably 0.03-0.6% of the dry weight of the total pollen replacement composition.

[0182] In one embodiment, campesterol is administered in an amount nutritionally effective for invertebrates, in particular honeybees or bumblebees, preferably at a concentration of 0.001-2%, preferably 0.001-1%, more preferably 0.02-0.35% of the dry weight of the total pollen replacement composition.

[0183] In one embodiment, stigmasterol is administered in a nutritionally effective amount for invertebrates, in particular honeybees or bumblebees, preferably at a concentration of 0.001-2%, preferably 0.001-1%, more preferably 0.01-0.2% of the dry weight of the pollen replacement composition.

[0184] In one embodiment, the total concentration of sterols in the pollen replacement composition ranges from 0.01% to 4% by dry weight of the pollen replacement composition.

[0185] Pollen substitute composition A further aspect of the present invention is a. protein in an amount of 10% to 50% by weight, preferably 15% to 40% by weight; b. fatty acids in an amount of 1% to 20% by weight, preferably 2% to 12% by weight; c. carbohydrates in an amount of 30% to 90% by weight, preferably 30% to 70% by weight; d. optionally vitamins, and e. Optionally, minerals The sum of the total amount of the components and optional further components a) to e) is 100% by weight, the weight percentage being relative to the total dry weight of the composition, The pollen replacement composition further comprises a nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof; The pollen replacement composition further comprises at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol; In a preferred embodiment, the total concentration of sterols is 0.01% to 4% by weight, preferably 0.05% to 3% by weight, and even more preferably 0.05% to 2% by weight, and even more preferably 0.05% to 1.5% by weight, relative to the total weight of the pollen replacement composition.

[0186] Use for feeding A further aspect of the present invention is the use of a pollen replacement composition of the present invention comprising a nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof, for feeding an invertebrate or aquaculture organism, the pollen replacement composition comprising a nutritionally effective amount of at least one further sterol, preferably at least two further sterols selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol, or physiologically available complexes thereof.

[0187] A further aspect of the invention is the use of a pollen replacement composition of the invention for feeding invertebrate or aquaculture organisms, the pollen replacement composition comprising: a. protein in an amount of 10% to 50% by weight, preferably 15% to 40% by weight; b. fatty acids in an amount of 1% to 20% by weight, preferably 2% to 12% by weight; c. carbohydrates in an amount of 30% to 90% by weight, preferably 30% to 70% by weight; d. optionally vitamins, and e. Optionally, minerals the sum of the total amount of the components and optional further components a) to e) is 100% by weight, the weight % being based on the total dry weight of the composition; The pollen replacement composition further comprises a nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof; The pollen replacement composition further comprises at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol; In a preferred embodiment, the total concentration of sterols is 0.01% to 4% by weight, preferably 0.05% to 3% by weight, and even more preferably 0.05% to 2% by weight, and even more preferably 0.05% to 1.5% by weight, relative to the total weight of the pollen replacement composition.

[0188] Further composition components A further aspect is a method of feeding an invertebrate, particularly a honeybee, in which a pollen replacement composition is administered, the pollen replacement composition comprising: a. protein in an amount of 10% to 50% by weight, preferably 15% to 40% by weight; b. fatty acids in an amount of 1% to 20% by weight, preferably 2% to 12% by weight; c. carbohydrates in an amount of 30% to 90% by weight, preferably 30% to 70% by weight; d. optionally vitamins, and e. Optionally, minerals Including, The sum of the total amounts of the components and optional further components a) to e) is 100% by weight, the weight percentage being based on the total dry weight of the composition, The pollen replacement composition further comprises a nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof; The pollen replacement composition further comprises at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol; In a preferred embodiment, the total concentration of sterols is 0.01% to 4% by weight, preferably 0.05% to 3% by weight, and even more preferably 0.05% to 2% by weight, and even more preferably 0.05% to 1.5% by weight, relative to the total weight of the pollen replacement composition.

[0189] The same composition and range applies to bumblebees, soldier flies, ladybugs, and other pollen-feeding insects.

[0190] Working Example Comparative Example 1: Isofucosterol concentration in commercial honeybee feed Commercially available pollen substitute bee feeding compositions do not contain isofucosterol, as shown in Table 1 below.

[0191] [Table 2]

[0192] There is an excellent literature describing how to make artificial diets for honeybee feeding assays: see US2019 / 0090507, the aforementioned Svoboda et al., and Chakrabarti et al. for examples of various background artificial diets.

[0193] Example 1 - Preference assay: Bees prefer a specific concentration of isofucosterol in their diet.

[0194] Newly emerged adult worker honeybees (Apis mellifera) or adult worker bumblebees (Bombus terrestris) were tested in a two-choice preference assay in which bees had free access to two types of food and water.

[0195] One treatment diet contained isofucosterol and the other contained no sterol. Newly emerged bees were removed from the larval frames and housed in plastic rearing cages in cohorts of 30 bees per replicate. In all experiments, 10 cohorts of approximately 30 bees each were used for each treatment group. In all diets, carbohydrate was maintained at 60% using sucrose and fat was maintained at 8%. Consumption of each diet was measured every 24 hours for 5 days. Preference index was calculated as (treatment consumed - control consumed) / (total amount of food consumed).

[0196] In Example 1, a cohort of bees was given a choice of diets containing or not containing isofucosterol. The bees preferred to consume foods containing at least 0.05% isofucosterol in the diet, as shown in FIG.

[0197] Example 2 - Survival: Bees live longer on foods containing isofucosterol Newly emerged adult worker honey bees were fed the treatment diet and had free access to water. The treatment diet contained isofucosterol. Newly emerged bees were removed from the larval frames and housed in plastic rearing cages in cohorts of 30 bees per replicate. In all experiments, 10 cohorts of approximately 30 bees each were used for each treatment group. In all diets, carbohydrate was maintained at 60% using sucrose and fat was maintained at 8%. Consumption of each diet was measured daily over the course of the experiment. The number of live bees in the box was counted daily for 14 days.

[0198] Example 2 presents the survival of cohorts of bees restricted to feeding on diets containing specific concentrations of isofucosterol (0%, 0.5%, and 1% by weight of the diet), as shown in FIG.

[0199] Example 3 - Larvae production: Honeybees produce more larvae and produce larvae for a longer period with isofucosterol.

[0200] Bees: Five miniframes housing adult worker bees and Apidea nucs of fully functional insulating Styrofoam containing one mated queen were inhabited by 300–400 mL of young adult worker bees (approximately N < 1000 bees (various ages)).

[0201] Colonies were placed in closed greenhouses with ventilation to prevent bees from accessing nectar or pollen. Each treatment was tested with 3-6 colonies, each of which was fed 60-100 g of patty (solid food) in a top feeder with a mesh floor. Food was offered again on the first and sixth day, and the amount consumed was measured on the sixth and fifteenth day. If no larvae or eggs / queens were observed by the sixth day, the experiment was terminated. The number of lidded larval cells was counted on the fifteenth day. Between each inspection, the number of hives was estimated. Sugar syrup (34%) and water were provided in the tent at the feeder to prevent carbohydrate depletion and stimulate foraging activity.

[0202] In Example 3, bees were fed a diet containing 10-18% protein, 6% fat, 1% vitamins / minerals, and >75% carbohydrates.

[0203] Figure 9 shows the threshold value of Example 3 for the effect of isofucosterol in bee diet. Increasing the concentration of isofucosterol increased the amount of larvae produced per colony (N>3 colonies / treatment). The number of pupae and new adults were counted over the entire 10 week period.

[0204] Example 4A: Long-term Apidea experiments with bees fed multiple sterols including isofucosterol and 24-methylenecholesterol Bees: Three to five miniframes housing adult worker bees and Apidea nucs of fully functional insulating Styrofoam containing one mated queen bee were inhabited by 300–400 mL of young adult worker bees (approximately N < 1000 bees (various ages)).

[0205] Colonies were placed in closed screen houses with ventilation to prevent bees from accessing nectar or pollen. Each treatment was tested with 3-6 colonies, each colony was fed solid food ad libitum in a top feeder with a mesh floor and the amount consumed was checked at each feeding. The number of lidded larval cells was checked every 12 days. The number of hives was recorded between each inspection. When colonies reached the maximum size of the Apidea box, oversize items were added. Sugar syrup (34%) and water were provided in the tent at the feeder to prevent carbohydrate depletion and stimulate foraging activity.

[0206] In Example 4A, bees were fed a diet containing 15-30% protein, 4-12% fat, 1% vitamins / minerals, and greater than 40% carbohydrates (Diet 1).

[0207] The experiment included the following treatments: Feed 1: No sterols added, same as Feed 1 above Feed 2 = Feed 1, in which isofucosterol, 24-methylenecholesterol, campesterol, β-sitosterol, stigmasterol, and cholesterol are added in the ratios and concentrations according to the preferred embodiments described above. Feed 3 = Feed 2 without 24-methylenecholesterol Feed 4 = Feed 2 without isofucosterol Diet 5 = Pollen-based patty (Pollen Patty) consisting of 60.5% pollen (fresh frozen honeybee collected pollen from BioBest, Antalya, Turkey), 25.2% powdered sugar, and 14.3% honey. Feed 6 = Cholesterol-free Feed 2 Diet 7 = Diet 2 without β-sitosterol, stigmasterol, and campesterol Feed 8 = Commonly used, commercially available feed Figure 4 - Results of experiment 4A: Mean area of ​​enclosed larvae (cm2) surveyed every 12 days over a 15-week period. Each colony was fed one treatment diet: diet 1 (x, thin dotted line), diet 2 (full circle, thick solid line), diet 3 (full diamond, thin dashed line), diet 4 (open diamond, thick dotted line), diet 5 (open circle, thick dashed line), diet 6 (full square, thick solid line), diet 7 (open square, thick dashed-dotted line), and diet 8 (asterisk, thin dashed-dotted line).

[0208] Example 4B: Long-term Apidea experiments with bees fed multiple sterols, including 24-methylenecholesterol but not isofucosterol Bees: Three to five miniframes housing adult worker bees and Apidea nucs of fully functional insulating Styrofoam containing one mated queen bee were inhabited by 300–400 mL of young adult worker bees (approximately N < 1000 bees (various ages)).

[0209] Colonies were placed in closed screen houses with ventilation to prevent bees from accessing nectar or pollen. Each treatment was tested with 3-6 colonies, each colony was fed solid food ad libitum in a top feeder with a mesh floor and the amount consumed was checked at each feeding. The number of lidded larval cells was checked every 12 days. The number of hives was recorded between each inspection. When colonies reached the maximum size of the Apidea box, oversize items were added. Sugar syrup (34%) and water were provided in the tent at the feeder to prevent carbohydrate depletion and stimulate foraging activity.

[0210] In Example 4B, bees were fed a diet containing 15-30% protein, 4-12% fat, 1% vitamins / minerals, and greater than 40% carbohydrates (Diet 1).

[0211] The experiment included the following treatments: Feed 1 = Feed containing 15-30% protein, 5-10% fat, 1% vitamins / minerals, and >40% carbohydrate with no added sterols.

[0212] Diet 2 = Pollen Patties (60.5% bee-collected mixed pollen pellets, 25.2% powdered sugar, and 14.3% honey).

[0213] Feed 3 = Feed 4 without 24-methylenecholesterol Feed 4 = Feed 1, with 24-methylenecholesterol, campesterol, β-sitosterol, stigmasterol and cholesterol added in ratios and concentrations according to the preferred embodiments above.

[0214] Feed 5 = commonly used, commercial feed Figure 5 shows the results of experiment 4B: the average area (cm2) of enclosed larvae surveyed every 12 days over a 15-week period. Each colony was fed one treatment diet: diet 1 (x, thin dotted line), diet 2 (full circle, thick solid line), diet 3 (open diamond, thin line), diet 4 (open circle, thick dashed line), diet 5 (asterisk, thin dashed-dotted line).

[0215] Example 4C: A long-term Apidea experiment with bees fed multiple sterols, where the total concentration of sterols in the diet was kept constant and the presence (concentration) of specific sterols was varied between diets.

[0216] Bees: Three to five miniframes housing adult worker bees and Apidea nucs of fully functional insulating Styrofoam containing one mated queen bee were inhabited by 300–400 mL of young adult worker bees (approximately N < 1000 bees (various ages)).

[0217] Colonies were placed in closed screen houses with ventilation to prevent bees from accessing nectar or pollen. Each treatment was tested with 3-6 colonies, each colony was fed solid food ad libitum in a top feeder with a mesh floor and the amount consumed was checked at each feeding. The number of lidded larval cells was checked every 12 days. The number of hives was recorded between each inspection. When colonies reached the maximum size of the Apidea box, oversize items were added. Sugar syrup (34%) and water were provided in the tent at the feeder to prevent carbohydrate depletion and stimulate foraging activity.

[0218] The experiment included the following treatments: Feed 1 = Feed containing 15-30% protein, 5-10% fat, 1% vitamins / minerals, and >40% carbohydrate with no added sterols.

[0219] Feed 2 = Feed 1, where 24-methylenecholesterol and isofucosterol are added in lower concentrations than in the preferred embodiment above, and campesterol, β-sitosterol, stigmasterol and cholesterol are added in ratios and concentrations according to the preferred embodiment above.

[0220] Diet 3 = Diet 2 with higher concentrations of 24-methylenecholesterol and isofucosterol, cholesterol than the preferred embodiment above, and lower concentrations of campesterol, β-sitosterol, and stigmasterol, maintaining a similar total sterol percentage as in Diet 2.

[0221] Diet 4 = Similar to diet 3 but without 24-methylenecholesterol and cholesterol, maintaining the percentage of total sterols in diet 7.

[0222] Diet 5 = Similar to diet 3 but without isofucosterol.

[0223] Diet 6 = Similar to diet 3 but without 24-methylenecholesterol and isofucosterol.

[0224] Diet 7 = Similar to diet 3, but lacking 24-methylenecholesterol, isofucosterol, and cholesterol, maintaining the % total sterols in diet 4.

[0225] Diet 8 = Pollen Patties (60.5% bee-collected mixed pollen pellets, 25.2% powdered sugar, and 14.3% honey).

[0226] FIG. 6: Results from Example 4C: Results of a long-term feeding experiment in Apidea colonies fed multiple sterols, where the total concentration of sterols in the diet was kept constant and the presence (concentration) of specific sterols was varied between diets.

[0227] Example 5: Black soldier fly experiment 7 shows the results of Example 5A: Feeding adult soldier flies a diet containing multiple sterols: Effects on the hatchability of laid eggs and on the weight of laid egg masses. Adult survival and number of laid egg masses from populations fed the pollen replacement diet (as specified in US2019 / 0090507, Apix Bioscience) were measured daily using the rearing method described in Thinn and Kainoh 2022JARQ 56 (2), 211-217 (2022).

[0228] FIG. 8 shows the results of Example 5B: Feeding adult soldier fly diets containing multiple sterols: Effects on adult longevity and larval survival to the pre-pupal stage. A diet containing 22% protein and 4% lipid was fed to soldier fly (BSF) larvae until hatching as described in Hogsette 1992. J. Econ. Entomol. 85(6): 2291-2294. Sterols were provided as pure compounds and added directly to the diet at a total amount of 0.1%. Larval and adult survival over a 50-day period was measured as described in Georgescu et al., Eur. J. Entomol. 118: 297-306, 2021.

[0229] FIG. 9 shows the results of Example 5C: Feeding adult black soldier flies various diets containing multiple sterols: sterols found in tissues of black soldier flies fed multiple sterols. Sterols were added to the diet. Measurement of sterols in tissues was performed after saponification of BSF tissues using GC-MS as described in Boukid et al. 2022. Insects 12, 672.

[0230] Example 6: Experiments with black soldier flies Nutritional composition of BSF larvae for the target species (honeybee) is changed by incorporating multiple sterols into the diet of BSF larvae, a diet formulation containing larvae is made and these larvae are fed the target species. Larvae of American soldier fly were fed diets as in Figure 10. Sterols required by honeybees were added to the diet in the amounts specified in diet 2 or as single sterols (24-methylenecholesterol, 24MC, or isofucosterol). In some groups, no sterols were added directly to the diet. Extracts were made from freeze-dried BSF larvae with hexane. Hexane solvent containing the fat portion was added to the diet; after hexane was evaporated, the diet was used to feed the larvae.

[0231] This example generally shows a method of feeding multiple sterols adapted to meet the needs of the target species, which can benefit the performance of the target species. Since BSF grown on different waste streams does not contain an optimal sterol profile for cultured fish, crustaceans, and cultured marine invertebrates and the algae or plankton that feed on these larvae, fucosterol or isofucosterol, 24-methylenecholesterol (all enriched in algae but not present in current non-algae artificial feeds for the above species) are added to the BSF feed, and then the algae-fed aquaculture organisms are fed lipids or proteins or total extracts of these BSF larvae, which is a method of improving the performance of aquaculture artificial feeds for organisms that normally feed on plankton and larvae and require isofucosterol / fucosterol and / or 24-methylenecholesterol.

[0232] FIG. 10 shows the results of Example 6: Feeding of honeybee colonies in the Apidea experiment with a diet containing an extract of black soldier fly fed multiple sterols.

[0233] Example 7: Development of a complete diet for honeybees: The importance of isofucosterol and multiple sterols The following experiment tested whether removing a single phytosterol or a pair of phytosterols from a pollen-free diet containing multiple added sterols would affect long-term larval production in hives fed exclusively with this diet.

[0234] There, we set up an assay in which beehives were completely housed in large tents (Figures 11A-11D), had no access to outside food, and were fed a pollen-free diet that differed in sterol composition but had identical total sterol concentrations similar to those found in pollen and honeybees.

[0235] Three diets were prepared: Diet A: A pollen-free honey bee diet containing protein, lipids, minerals, vitamins and antioxidants, as well as trace amounts of sterols, but no added sterols. This investigational diet was formulated by APIX Biosciences NV Belgium using commercially available ingredients as a patty that can be placed on top of the bee frames inside the hive, as standard in the industry (PCT / GB2016 / 053573) for "protein supplements". This diet is similar to the diet used by Herbert et al., except that the nutrients are more complete and balanced, with a better nutritional base. This diet contained 0.375% added sterols (dissolved in lipids) [24-methylenecholesterol (0.125%), isofucosterol / fucosterol isomers (0.0625%), and 0.1875% of a mixture of sterols typically found in pollen sourced by honeybees, including β-sitosterol, campesterol, and stigmasterol (but not isofucosterol or 24-methylenecholesterol)], resulting in phytosterol concentrations and ratios that, on average, reflect the natural composition of pollen. Semi-synthetic 24-methylenecholesterol (97% pure) and semi-synthetic isofucosterol (97% purity; 75% isofucosterol and 25% EZ isomer fucosterol; hereafter interchangeably named "isofucosterol / fucosterol" or "isofucosterol") were obtained from Phytant NV Belgium. Background diet A was a pollen-free, proprietary diet (developed by APIX Biosciences NV, Belgium) (PCT / GB2016 / 053573) formulated with registered animal feed ingredients. Diet B: Pollen-free and isofucosterol / fucosterol-free diet. This is diet A with the 0.0625% added isofucosterol / fucosterol mixture removed. In order to keep the total added sterol concentration in the diet constant, the concentration of the other sterols added to the diet was increased proportionately to the total sterol concentration of diet A at 0.375% added sterol. This increased the concentration of the other individually added sterols by a factor of 1.2, which places the concentration of these sterols in the centre of the range present in the pollen on which the bees successfully feed. Diet C: diet without pollen and without 24-methylenecholesterol. This is diet A with the 0.125% added 24-methylenecholesterol removed. To keep the total added sterol concentration in the diet constant, the concentration of other sterols present in the diet was increased proportionately to the total sterol concentration of diet A at 0.375% added sterol. This places the concentration of added other sterols in the centre of the range present in the pollen on which the bees successfully feed.

[0236] The ability of these three diets, and a fourth commercial diet (Diet E, see Figure 20 and Example 8) widely used in the United States, to sustain brood production in hives for 96 days (over four brood generations) when fed as the sole source of nutrition (other than sugar syrup and water provided to the tents by sugar and water feeders) was investigated. Six small hives per diet, each containing 800-850 mL (approximately 2000 bees) and one queen, were distributed into adjacent tent enclosures, each 2 m x 4.5 m x 3 m (three hives per tent; Figures 11A-11D). The tent enclosures were located in Wingene, Belgium (day 0 = May 23). On day 0, only traces of pollen and bee bread were present in the hives. Queens were newly mated sister queens of the same age and genetic background (Apis mellifera).

[0237] Food was provided to each hive every 6 days by placing patties on the top of the frame (above the larval nest) according to standard beekeeping practices (Figures 11A-11D). All three hives in the same tent received the same diet (two tents per diet). Bees had free access to water and 400 mL of 50% sucrose sugar syrup was placed on the table inside the tent every 6 days (sugar was not provided ad libitum to prevent the cells from filling up with syrup and limiting the space available to the larvae) (Figure 11C). Food consumption was measured by weighing the uneaten patties in each hive and replacing them with fresh patties every 6 days. Photographs of each frame containing larvae were taken in each hive every 12 days over a 96-day period (Figure 11D). The number of operculated larvae in each photograph was manually counted by 2-3 different individuals (Figures 15, 16, and 17), and the results were verified (quality assurance checked) by another individual. Hive boxes in which the queen had stopped laying eggs during the first 24 days (hive stability period) and boxes in which the queen had been accidentally crushed during observation were removed from the experiment. This observation regime ensured that all bees born in the hive (all larvae that had developed into operculated larvae) were photographed at least once, and only once. On day 84, a sample of 25-30 bees was taken from each hive for further study.

[0238] This design is similar to a series of experiments by Herbert et al. (reference) in which single phytosterols (but not isofucosterol) were incorporated into a simple artificial diet and small nest boxes were maintained on this diet until egg production ceased. However, our experimental design involved the addition of a defined number of sterols, a more complex and complete background diet, and the investigation of the effect of removing single sterols from the diet while keeping the total sterol concentration constant.

[0239] The experiment was carried out for 96 days (4.5 larval cycles on day 21 on artificial diet). It takes approximately 24 days, or 1.5 life cycles, for the nurse bees to deplete any pools of food and nutrients stored in the hive that were present at the start of the experiment. All operculated larvae observed on day 24 are from eggs laid in the tents and are therefore expected to be fed on artificial diet. Occlusive larvae observed from day 36 onwards are expected to be entirely fed by the nurse bees, who themselves were reared on artificial diet. The same applies to nutrients fed to the worker bees by the nurse bees.

[0240] The Importance of Isofucosterol / Fucosterol The bees and hives behaved normally in the tent as indicated by the normal diurnal cycle of flying away from and back to the hive at dusk or when it was raining, foraging for sugar syrup and water, orientation adjustments, and defecation flights. However, these conditions caused significant stress to the hives: (1) they were surrounded and flying against the mesh, and (2) more importantly, the hives were opened and all frames were removed every 12 days for photography, a procedure that exposed the larvae to low temperatures and substantially disturbed the hives. The latter procedure would have a significant negative impact on the health and reproductive potential of the hives. Another factor to consider is that we started with newly established small hives (800-850 mL of bees and a newly mated queen). Small hives are more sensitive to disturbance and cannot heat / cool themselves as well as larger colonies of, for example, 4000 bees. During a heatwave, spraying from a single nozzle reduced the temperature inside the tent, keeping temperatures at a maximum of 37.5°C.

[0241] Despite these stresses, hives could be fed comprehensively with pollen-free diet A and sugar solution in good health and produced stable larvae for over 96 days in tent enclosures. Hives fed commercial protein patties showed a steep decline in larvae after 36 days (Diet E, Figure 20).

[0242] To date, no artificial pollen-free complete diet for honey bees has been reported.

[0243] The results are shown in Figures 12 to 20 and Tables 2 to 4 listed below.

[0244] [Table 3]

[0245] [Table 4]

[0246] [Table 5]

[0247] [Table 6]

[0248] Figure 12 and Tables 2, 3, and 4 show that, on average, 2353 operculated larvae were produced between days 12 and 96 for hives fed diet B, whereas, on average, 890 operculated larvae were produced during the same period for hives fed diet B. Figure 12 also shows that after 60 days, the amount of larvae produced in all but one hive fed diet B (hive no. 12) decreased, while all hives fed diet A showed a linear increase in growth. Three of the five hives fed diet B stopped producing operculated larvae by day 84, whereas all hives fed diet A continued to produce larvae. This indicates that removing isofucosterol-fucosterol from diet A affects the ability of the hive to produce larvae, and that other sterols cannot compensate for this deficit. The production of larvae is a vital function for the survival of the hive.

[0249] Tables 2 and 3 and Figures 12, 14, 15, 16 and 17 show that for diet A, the amount of larvae at each time point is stable (Figure 14A), whereas for diet B, the amount of larvae produced at each time point is less than for diet A. It can be concluded that for diet A, the hives are stable under the test conditions for 96 days or 4.5 generations of nurse bees (despite the severe stresses mentioned above). When 0.0625% isofucosterol-fucosterol is removed from the diet and replaced by proportionally increasing concentrations of each of the other added sterols, the hives perform significantly worse in larval production and are not stable.

[0250] Figure 13 shows the consumption of diets A and B. Since the main use of pollen in the hive is to feed the nurse bees and subsequently the larvae and queen, it is logical that the consumption of diet would decrease when the number of larvae produced decreases (Figure 13).

[0251] Photographs of the frames with larvae clearly show differences in the number and pattern of operculated larvae in hive number 29 fed diet A (Figure 15) and in hive number 26 fed diet B (Figure 16).

[0252] Figure 17 shows that hives fed diet A produced more larvae than hives fed diet B.

[0253] We conclude that the addition of isofucosterol-fucosterol to an incomplete diet containing cholesterol, campesterol, β-sitosterol, stigmasterol, and 24-methylenecholesterol in a concentration similar to that of pollen, without isofucosterol-fucosterol, provides a diet that will sustain a hive for 4.5 larval cycles (generations) of nurse bees (without access to pollen or other nutrients, except sugar solution). Conversely, the embodiments show that removing isofucosterol-fucosterol from the complete diet reduces the fitness of the hive and the bees, and that this removal is not compensated for by proportionally compensating for the removal of isofucosterol-fucosterol with other sterols.

[0254] This is the first clear indication that isofucosterol-fucosterol is functionally important and essential in the honeybee hive diet. The data show that, despite the presence of all other major sterols present in pollen and honeybees, isofucosterol-fucosterol is required for honeybees to produce larvae, and its physiological role cannot be assumed by other sterols present in the diet (cholesterol, 24-methylenecholesterol, isofucosterol, stigmasterol, β-sitosterol, which usually represent more than 80% of the total sterols present in honeybees - the remainder is isofucosterol-fucosterol). Diet A is also the first reported complete artificial diet for honeybees that is pollen-free.

[0255] The Importance of 24-Methylene Cholesterol Hives fed diet C (= diet A with 24-methylenecholesterol removed) started to show increasing differences in the amount of eggs hatched from day 48 onwards (Figures 18 and 19, Tables 2, 3 and 4). Between days 36 and 96, hives fed diet C cumulatively hatched 1126 operculated larvae, whereas hives fed diet A cumulatively hatched 1588 operculated larvae. Note that the difference between operculated larvae hatched by diet A and diet B (isofucosterol-no fucosterol) was already evident between days 24 and 36 (Tables 2 and 3). The delayed phenotypic expression of 24-methylenecholesterol depletion observed in diet C compared to the effect of isofucosterol depletion in diet B can be explained by the observation by Sbovoda et al. (Table 7) that honeybees have mechanisms to maintain (some of) the pool of 24-methylenecholesterol in the hive by recycling it to larvae and nurse bees. The data reported here further emphasize the increased importance of isofucosterol-fucosterol in artificial bee diets and 24-methylenecholesterol, since the effects of its depletion become evident earlier.

[0256] These data provide the first technical demonstration that 24-methylenecholesterol is functionally required in the honeybee diet and that its physiological function cannot be fully compensated for by the presence of other sterols present in the diet (cholesterol, isofucosterol / fucosterol, β-sitosterol, campesterol, stigmasterol) and in the concentrations and ratios normally present in pollen.

[0257] Example 8: Behavioral phenotype of isofucosterol / fucosterol and 24-methylenecholesterol deficiency.

[0258] While the number of bees and nurse bees within a hive is important, the quality and physiological fitness of the bees is equally important to the fitness of the hive and its ability to pollinate a crop.

[0259] Bees have to perform complex behavioral functions inside the hive (cleaning, feeding the larvae, building the cell, defense, ventilation, heating…) and outside the hive (flying long distances – up to 2 km from the hive, finding the homing route, signaling the location of food sources to hive-mates, collecting nectar, pollen, minerals, water, beeswax…). The number of hives, and their lifespan, determine how many resources they can gather to maintain and grow the hive. From a commercial point of view, much more active and well-fed hives (fitter, longer live food search) are better able to pollinate crops and to withstand exposure to pesticides and other stressors (heat, transport, disease). In short, not only the quantity but also the quality of bees in the hive is important.

[0260] It is known that different sterols have different effects on membrane structure in cells. The role of sterols in the membranes of animal cells is to control membrane fluidity, which is important for proper functioning among other muscle cells and neurons, the organization of proteins in intramembrane rafts, membrane curvature and vesicle formation, and adaptation to different environmental temperatures.

[0261] In this embodiment, we investigate whether hives in tented enclosures fed various diets that differ only in sterol composition emerge with honeybees of equal behavioral fitness.

[0262] Following the same procedures as described above and in the same tent set-up, the behavior and activity of bees fed the following diets were characterized (see Figures 11A-11D).

[0263] 1. Outside hives: These hives initially contained the same sister queens and the same number of bees as the hives inside the tent, but were placed outside adjacent to the tent, allowing them full access to pollen flow and natural nectar. 2. Tent nests - Diet A: Diet A see above (same nests and time points as in Example 7). Diet A' = Diet A with the same composition and ratios as Diet A, but with twice the concentration of added sterols (0.75%). Diets A and A' have the same phenotype. 3. Hive in tent - Diet B: Diet A - isofucosterol / fucosterol (concentrations of other sterols added proportionally adjusted upwards as described above; same hive and time points as in embodiment 1) 4. Tent nests - Diet C: Diet A-24-methylenecholesterol (concentrations of other sterols added proportionally adjusted upwards as described above; same nests and time points as in embodiment 1) 5. Nests in tents - Diet D: Diet A lacking isofucosterol / fucosterol and 24-methylenecholesterol (concentration of other sterols added (0.75%) adjusted upwards to that of Diet A'; carried out in parallel tents at the same time points as in embodiment 1; feeding and nesting examination procedures were generally the same) 6. Nests in tents - Diet E: Commercial diet (completely different diet; carried out at the same time points as in embodiment 1, in a parallel tent; feeding and nest disturbance procedures were generally the same)

[0264] In hives fed diet E (commercial diet), essentially no larvae were produced after 36 days (Table 5, Figure 20). In hives fed diets A, B, C, and D, larvae were still produced at 96 days. Diets and experimental design are described in Example 7.

[0265] On days 98-99, the hives were opened and visually scored for several phenotypes (scores: no phenotype (-), present phenotype (+), more severe phenotype (++), even more severe phenotype (+++)).

[0266] These were scored independently by two observers for the following phenotypes: · Lethargy: Bees that remain motionless in the hive. Tremors and lack of coordination: bees dragging their hind legs, walking unsteadily, taking a long time to move their wings from open to closed position (indicators of uncoordinated neuromuscular activity) Poor response - no flight: reacts slowly to being disturbed by removing the frame from the hive or follows the tweezers with bees on the frame, crawls more slowly upwards after being pushed off the frame and does not fly when disturbed. Slow movement: bees moving more slowly on the frame and / or around the sugar feeders and / or at the hive entrance · Responsiveness: This is an integrated score of 1-5 (low to high) that describes the activity of the entire hive and the vitality of the bees inside and outside the hive.

[0267] These observations are shown in Tables 5 and 6.

[0268] [Table 7]

[0269] [Table 8]

[0270] [Table 9]

[0271] Diet A and A' bees were equally active, responsive and mobile as bees in natural food-collecting hives placed next to the tent enclosure.

[0272] Nests fed diet C (diet A-24-methylenecholesterol) exhibited a slow migration phenotype (+) and a reduced reactivity score of 3.

[0273] Nests fed diet B (diet A-isofucosterol / fucosterol) showed lethargy (++); trembling / lack of coordination (++); poor response / no flying (++) and slow movement (++) phenotypes, lowering the reactivity score to 2.

[0274] Nests fed diet D (diet A-24 methylenecholesterol and -isofucosterol) showed a much greater trembling / lack of coordination (+++) phenotype than diet B, but no lethargy phenotype. Because of the absence of lethargy phenotype in diet D, nests fed diet D received a reactivity score of 3. The poor reactivity / no flying (++) and slow movement phenotype (++) phenotypes were similar to nests fed diets B and D, but distinct from diet D.

[0275] We conclude that removing isofucosterol / fucosterol or 24-methylenecholesterol from Diet A, which contains the major sterols present in honeybees (cholesterol, 24-methylenecholesterol, β-sitosterol, campesterol, stigmasterol, isofucosterol / fucosterol), not only results in fewer operculated larvae (embodiment 1), but also in bees that exhibit a number of phenotypes that are likely to affect physiological fitness and perform complex hive tasks efficiently.

[0276] It is further shown that the phenotype due to the lack of isofucosterol-fucosterol or 24-methylenecholesterol, or both, cannot be physiologically compensated for by an increase in the other sterol, and it is further shown that removing both isofucosterol and 24-methylenecholesterol has a more severe effect on the nervous and muscular systems (lack of coordination) and thus on fitness, than removing either one of these sterols at a time from Diet A'.

[0277] The observation that the effect of removing isofucosterol / fucosterol from Diet A was more profound than the effect of removing 24-methylenecholesterol is a surprising finding to the field, since it has been widely argued in the field that 24-methylenecholesterol is a critical and essential phytosterol in honeybees, as confirmed in “Sterol and Lipid metabolism in bees” Furse et al 2023,https: / / doi.org / 10.1007 / s11306-023-02039-1).

[0278] Data Examples 7 and 8 show that honeybees require multiple sterols because both isofucosterol and 24-methylenecholesterol, either alone or in combination, are functionally non-substitutable in honeybee physiology by other sterols present in pollen.

[0279] We conclude that diet A is a complete diet not only in terms of maintaining brood in the hive, but also in terms of producing quality bees. We further conclude that isofucosterol / fucosterol is essential to this diet and cannot be replaced by proportionally increasing other key phytosterols present in the pollen. The same was shown for 24-methylenecholesterol, but to a lesser extent. Figure 20 and Table 5 show the superior performance of complete diet A compared to a commercial protein patty widely used in the United States.

[0280] Table 7: List of references Chakrabarti et al. ·doi:10.3390 / molecules25030571 Herbert and Svoboda Group doi:10.1007 / BF02534310 ·doi:10.1016 / 0022-1910(80)90135-3 ·doi:10.1016 / 0022-1910(80)90136-5 ·doi:10.1016 / 0020-1790(86)90024-7 ·doi:10.1007 / BF02535107 Field runner ·doi.org / 10.1002 / arch.940030502 Furse S et al. ·doi:10.1007 / s11306-023-02039-1 Tian et al. ·doi:10.1038 / s41467-018-05619-1

Claims

1. 1. A method for feeding invertebrates or aquaculture organisms, comprising: Providing a pollen replacement composition comprising a nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof; - administering the pollen replacement composition to an invertebrate or aquaculture organism; Including, The pollen replacement composition comprises a nutritionally effective amount of at least one additional sterol, preferably at least two additional sterols selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol, or physiologically available complexes thereof.

2. the invertebrate is a pollen-feeding insect, preferably an insect of the order Hymenoptera or Coleoptera, more preferably a honeybee, a bumblebee, an American soldier fly, a hoverfly or a ladybird, The aquaculture organisms are plankton and algae-feeding aquaculture organisms, preferably fish, larvae, oysters, bivalves, mollusks, gastropods, or crustaceans; The method of claim 1.

3. 3. The method of claim 1 or 2, wherein the nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof is a daily amount of 0.0006% to 0.052% by weight of the live weight of the invertebrate or aquaculture organism.

4. 4. The method according to any one of claims 1 to 3, wherein the nutritionally effective amount of isofucosterol, fucosterol or a mixture thereof is between 10% and 60% by weight of the total amount of sterols of the group of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total feed of the invertebrate or aquaculture organism or of the pollen replacement composition.

5. 5. The method of any one of claims 1 to 4, wherein the isofucosterol, fucosterol or mixture thereof is administered at a rate of 0.14g to 12g of isofucosterol, fucosterol or mixture thereof per 30,000 bees per two week period.

6. 6. The method of claim 5, wherein the isofucosterol, fucosterol or mixtures thereof are administered in an amount of 10% to 60% by weight of the total amount of sterols of the group of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total feed of the invertebrate or aquaculture organism or of the pollen replacement composition.

7. the dosage rate of said nutritionally effective amount of isofucosterol, fucosterol or a mixture thereof and said at least one further sterol is between 0.2 and 48 grams per 30,000 bees per two week period; the isofucosterol, fucosterol or mixture thereof is administered in an amount of 10% to 60% by weight of the total amount of sterols of the group isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total feed of the bees or of the pollen replacement composition; 7. The method according to any one of claims 1 to 6.

8. the isofucosterol, fucosterol or mixture thereof and the at least one further sterol are administered at a rate of between 0.4 g and 36 g per 30,000 bees per two week period; the isofucosterol, fucosterol or mixture thereof is administered in an amount of 10% to 60% by weight of the total amount of sterols of the group isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total feed of the bees or of the pollen replacement composition; 8. The method according to any one of claims 1 to 7.

9. the isofucosterol, fucosterol or mixture thereof and the at least one further sterol are administered at a rate of 0.6 g to 20 g per 30,000 bees per two week period; the isofucosterol, fucosterol or mixture thereof is administered in an amount of 10% to 60% by weight of the total amount of sterols of the group isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total feed of the bees or of the pollen replacement composition; 9. The method according to any one of claims 1 to 8.

10. 10. The method of any one of claims 1 to 9, wherein the isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, beta-stigmasterol, stigmasterol, and / or campesterol are administered in an amount of 0.001% to 0.087% by weight of the live weight of the invertebrate or aquaculture organism per day.

11. The nutritionally effective amount is, relative to isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, β-sitosterol, stigmasterol, and / or campesterol, isofucosterol in an amount between 10% and 60% by weight, - cholesterol in an amount between 0% and 50% by weight, 24-methylenecholesterol in an amount between 0% and 50% by weight, β-sitosterol, stigmasterol and / or campesterol in an amount between 0% and 50% by weight The method according to any one of claims 1 to 10, wherein

12. 12. The method according to any one of claims 1 to 11, wherein the total concentration of sterols is from 0.01% to 4% by weight, preferably from 0.05% to 3% by weight, and even more preferably from 0.05% to 2% by weight, and even more preferably from 0.05% to 1.5% by weight compared to the total weight of the pollen replacement composition.

13. The concentration of sterols is cholesterol in an amount of from 0.001% to 2% by weight, preferably from 0.001% to 1.5% by weight, more preferably from 0.06% to 1.2% by weight, relative to the total weight of the pollen replacement composition; 24-methylene cholesterol in an amount of from 0.001% to 2% by weight, preferably from 0.001% to 1.5% by weight, more preferably from 0.06% to 1.2% by weight, relative to the total weight of the pollen replacement composition; sitosterol, preferably β-sitosterol, in an amount of 0.001% to 2% by weight, preferably 0.001% to 1% by weight, more preferably 0.03% to 0.6% by weight, compared to the total weight of the pollen replacement composition; isofucosterol in an amount of from 0.01% to 5% by weight, preferably from 0.01% to 2% by weight, more preferably from 0.01% to 1.0% by weight, more preferably from 0.03% to 0.6% by weight, relative to the total weight of the pollen replacement composition; campesterol in an amount of from 0.001% to 2% by weight, preferably from 0.001% to 1% by weight, more preferably from 0.02% to 0.35% by weight, relative to the total weight of the pollen replacement composition; stigmasterol, in an amount of from 0.001% to 2% by weight, preferably from 0.001% to 1% by weight, more preferably from 0.01 to 0.2% by weight, relative to the total weight of the pollen replacement composition, or Any combination of these The method according to any one of claims 1 to 12, wherein the compound is selected from the group consisting of:

14. 14. The method of any one of claims 1 to 13, wherein the composition is part of a whole invertebrate feed, an invertebrate feed or a nutritional supplement and the ratio of 24-methylene cholesterol to the further sterol or combination of further sterols is from 10:1 to 1:

1.

15. 15. The method according to any one of claims 1 to 14, wherein the composition is administered to an invertebrate of the family Apidae, in particular to honey bees (Apini), bumble bees (Bombini) or stingless bees (Meliponini).

16. The composition comprises: In a solid form, such as a patty or powder, or in a liquid form, such as a solution, oil, or spray; - Inside or outside the hive 16. The method of claim 1 , wherein the patient is administered

17. the source of isofucosterol, fucosterol or a mixture thereof is a pollen-substitute tissue of one or more plant species selected from the group consisting of leaves, stems, roots, tubers, flowers, seeds, bark and fruits, and combinations thereof; and / or the source of the further sterols selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol, in particular the source of cholesterol, is a pollen substitute tissue of one or more plant species selected from the group consisting of leaves, stems, roots, tubers, flowers, seeds, bark, and fruits, and combinations thereof; 17. The method according to any one of claims 1 to 16.

18. 18. The method of any one of claims 1 to 17, wherein the source of isofucosterol, fucosterol, or a mixture thereof, or the source of the at least one further sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol, is an extract, oil, or purified product of non-pollen tissues of one or more plant species or combinations thereof.

19. 19. The method according to any one of claims 1 to 18, wherein the source of isofucosterol, fucosterol or a mixture thereof, or the source of the at least one further sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, stigmasterol, is a pollen substitute tissue of one or more plant species selected from the group consisting of Solanaceae, Poaceae, Ranunculaceae, Fabaceae, and Bacillus subtilis.

20. The source of isofucosterol, fucosterol, or a mixture thereof, or the source of the at least one additional sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol, - extracts, oils or purified products of marine or freshwater algae, in particular Ulva lutea; - extracts, oils or purified products of marine diatoms, in particular Thalassiosira pseudonana, Thalassiosira rotula or Chaetoceros muelleri; and - extracts, oils or purified products of fungi, especially yeasts such as Saccharomyces cerevisiae or Yarrowia lipolytica 20. The method of claim 1, wherein the pollen replacement source is selected from the group consisting of:

21. 21. The method according to any one of claims 1 to 20, wherein the isofucosterol or fucosterol and the at least one further sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol are chemically or enzymatically synthesized or obtained by a genetically modified host organism, such as a fungus, a bacterium, or an algae.

22. 22. The method of any one of claims 1 to 21, wherein the source of isofucosterol, fucosterol, or mixtures thereof, or the at least one further sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol, is selected from the group consisting of algae, plants, fungi, algae, diatoms, and combinations thereof, and the source of isofucosterol, fucosterol, or mixtures thereof is pollen substitute tissue.

23. 23. The method of any one of claims 1 to 22, wherein the isofucosterol, fucosterol, or mixtures thereof and the at least one further sterol selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, and β-sitosterol, and stigmasterol are provided to the eusocial bee colony from a synthetic source.

24. The isofucosterol, fucosterol, or mixtures thereof, and one or more further sterols selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol, are provided as part of the pollen replacement composition, the pollen replacement composition comprising: protein in an amount of 10% to 50% by weight, preferably 15% to 40% by weight, fatty acids in an amount between 1% and 20% by weight, preferably between 2% and 12% by weight, carbohydrates in an amount of 20% to 90% by weight, preferably 30% to 70% by weight, Optionally, vitamins, and Optionally, minerals Including, 24. The method of any one of claims 1 to 23, wherein the sum of the total amounts of components is 100% by weight, and weight % relates to the total dry weight of the composition.

25. 25. The method of any one of claims 1 to 24, wherein the pollen replacement composition is substantially free of pollen.

26. invertebrates, preferably pollen-feeding insects, more preferably insects of the orders Hymenoptera and Coleoptera, even more preferably honeybees, bumblebees, soldier flies, hoverflies or ladybirds, Aquaculture organisms, preferably plankton and algae-feeding aquaculture organisms, more preferably fish, larvae, oysters, bivalves, mollusks, gastropods, or crustaceans.

26. Use of a pollen replacement composition according to any one of claims 1 to 25 for feeding an organism selected from the group consisting of:

27. 27. Use of the pollen replacement composition according to claim 26 as a concentrated patty, wherein the concentration of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol is between 10% and 33% by weight compared to the total weight of the pollen replacement composition.

28. 28. Use of a pollen replacement composition according to claim 27, wherein the composition comprises a bee-ingesting / appetite-inducing component selected from the group consisting of pollen, sugar, oil or fat, honey, or protein, or mixtures thereof.

29. 29. Use of a pollen replacement composition according to claim 28, wherein the concentration of the bee appetite / ingestion inducing component is from 1% to 20% by weight, preferably from 2.5% to 15% by weight, even more preferably from 5% to 10% by weight compared to the total weight of the pollen replacement composition.

30. 27. Use of the pollen replacement composition according to claim 26, wherein the pollen replacement composition is in liquid or powder form.

31. 31. Use according to claim 30, wherein the concentration of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol is between 0.01% and 99% by weight compared to the total dry weight of the pollen replacement composition.

32. - 0.01% to 1% by weight of isofucosterol, preferably 0.01% to 0.3% by weight of isofucosterol, compared to the total weight of the pollen replacement composition; the composition optionally further comprises natural pollen in an amount of 1% to 15% by weight, compared to the total weight of the pollen replacement composition, to increase feed consumption; or - 0.01% to 1% by weight of fucosterol, preferably 0.01% to 0.3% by weight, compared to the total weight of the pollen replacement composition; the composition optionally further comprises natural pollen in an amount of 1% to 15% by weight, compared to the total weight of the pollen replacement composition, to increase feed consumption; or - 0.01% to 1% by weight of isofucosterol and fucosterol, preferably 0.01% to 0.3% by weight, relative to the total weight of the pollen replacement composition; the weight ratio of isofucosterol to fucosterol is 0.01:100 to 100:0.01; the composition optionally further comprises natural pollen in an amount of 1% to 15% by weight, relative to the total weight of the pollen replacement composition, to increase feed consumption; or - 0.01% to 1% by weight of isofucosterol / fucosterol, preferably 0.01% to 0.3% by weight, compared to the total weight of the pollen replacement composition; wherein the weight ratio of isofucosterol to fucosterol is between 0.01:100 and 100:0.01, - the pollen replacement composition further comprises from 0.01% to 0.5% by weight of 24-methylene cholesterol compared to the total weight of the pollen replacement composition; the ratio of isofucosterol and fucosterol to 24-methylenecholesterol is between 2:100 and 100:2; - the pollen replacement composition optionally further comprises natural pollen in an amount of 1% to 15% by weight compared to the total weight of the pollen replacement composition to increase feed consumption; or - 0.01% to 1% by weight of 24-methylenecholesterol, preferably 0.01% to 0.3% by weight, compared to the total weight of the pollen replacement composition; wherein the pollen replacement composition further comprises isofucosterol and fucosterol, and the weight ratio of isofucosterol to fucosterol is from 0.01:100 to 100:0.01; the weight ratio of isofucosterol and fucosterol to 24-methylenecholesterol is between 2:100 and 100:2, - the pollen replacement composition optionally further comprises natural pollen in an amount of 1% to 15% by weight compared to the total weight of the pollen replacement composition to increase feed consumption; 2. A pollen-free feeding composition or a pollen replacement composition, particularly for bees, comprising any of the following:

33. A pollen-free feeding composition or a pollen replacement composition, in particular for bees, comprising 0.02% to 3% by weight of a sterol, compared to the total weight of said pollen replacement composition, said sterol being selected from the group consisting of 24-methylenecholesterol, isofucosterol, fucosterol, β-sitosterol, campesterol, stigmasterol, ergosterol and cholesterol, the weight ratio of isofucosterol and fucosterol to 24-methylenecholesterol is from 0.5:100 to 100:0.5; and / or the weight ratio of isofucosterol to fucosterol is between 0:10 and 10:0; and / or the weight ratio of isofucosterol to the sum of one or more other sterols selected from the group consisting of 24-methylenecholesterol, fucosterol, β-sitosterol, campesterol, stigmasterol, cholesterol, ergosterol is from 2:100 to 100:2; and / or the weight ratio of fucosterol to the sum of other sterols selected from the group 24-methylenecholesterol, isofucosterol, β-sitosterol, campesterol, stigmasterol, cholesterol, ergosterol is from 2:100 to 100:2; and / or the weight ratio of 24-methylenecholesterol to the sum of one or more other sterols selected from the group: isofucosterol, fucosterol, β-sitosterol, campesterol, stigmasterol, cholesterol, ergosterol is from 2:100 to 100:2; and / or the weight ratio of cholesterol to the sum of one or more sterols selected from the group consisting of 24-methylenecholesterol, isofucosterol, fucosterol, β-sitosterol, campesterol, stigmasterol, ergosterol is from 15:1 to 1:100; and / or - the pollen replacement composition optionally further comprises natural pollen in an amount of 1% to 15% by weight compared to the total weight of the pollen replacement composition to increase feed consumption; A pollen-free feeding composition or a pollen replacement composition, especially for bees.

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