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, and mammals). Honeybees play a vital role in modern agriculture by pollinating crops. In recent years, pollinators have come under threat from exposure to pesticides, an increase in pathogens and parasites, and changes in landscape management that reduce the abundance of naturally occurring pollen.

[0002] Historically, beekeepers have fed their honeybee colonies pollen-containing food sources or pollen collected by honeybees 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, commercially available honeybee food typically does not contain pollen.

[0003] However, lack of sufficient quality and quantity of pollen is a fundamental cause of observed declines in health and survival in bee colonies. Honeybees 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 significant 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 Research 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. 5,629,999 (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 and as key nutrients needed to make a "complete" diet for captive bees.

[0006] Non-Patent Document 3 discusses studies 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] Other protein sources used, such as soy protein or 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%) (Non-Patent Document 4). The concentration of sterols in these sources is below the nutritional requirements of bees.

[0008] In honeybees, isofucosterols account for 10-50% of total body sterols, which 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, which corresponds to 0.10-0.25% of the bumblebee's total 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 22 to 40 days. This indicates that current diets lack one or more essential nutrients. However, to date, no artificial (pollen-free) bee diets have been described that can support a colony's sealed larval development for more than three to four 21-day periods. The ability of a diet to support continuous larval development over an extended period is comparable to pollen and is an important indicator of its ability to function as a "complete diet." The best published data already show a significant decline in larval production beginning after two cycles (Non-Patent Document 6). Therefore, in many regions where bees are kept for pollination or honey production, the lack of pollen quality and quantity necessitates the provision of compositions and methods for feeding bees that allow for the long-term maintenance of colonies on an artificial diet that will continue to produce larvae indefinitely.

[0010] Current pollen-free artificial bee diets are incomplete, resulting in bees lacking or depleting essential nutrient stores within the colony after three to four 21-day larval production cycles (Non-Patent Document 1 (1980)). Here, the addition of isofucosterol / fucosterol and multiple sterols provides a diet that sustains colony larval production and colony development for five or more cycles, suggesting that the diet overcomes the deficiencies of one or more essential nutrients present in current artificial diets. This demonstrates that the present invention provides a new diet that provides bees with the limiting nutrients they require. Thus, such a pollen-free diet can sustain colonies for extended periods throughout the colony life cycle, even without natural pollen, representing a significant advancement that could 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 from flowers for nutrition. Such a diet would be a crucial tool for maintaining human societies' ability to sustainably pollinate 35% of the world's crops and for protecting wild pollinators and plant biodiversity.

[0012] Beehives continually require nutritionally suitable pollen from diverse sources to meet their nutritional needs. Pollen is an essential source of proteins, carbohydrates, amino acids, vitamins, lipids, sterols, and other micronutrients for the beehive. Most plant species produce pollen whose composition does not meet all of the nutritional needs of the beehive. Therefore, bees have developed physiological mechanisms 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 are still 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 specific molecules by correlating the nutrient composition of different floral pollen sources with the nutritional properties of these different pollens. However, because pollen from different flowers varies greatly in the composition of many molecules, this correlation approach cannot really provide conclusions about the relevance of single components.

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

[0016] Insects cannot synthesize sterols; instead, they obtain them from food sources, and most insects convert them to cholesterol. Unlike most insects, bees (honeybees, bumblebees, etc.) contain only trace amounts of cholesterol and are unable to dealkylate phytosterols to cholesterol. Therefore, honeybees use phytosterols instead of cholesterol as structural elements in membranes, as precursors for growth hormones, and as structural elements in protein complexes (e.g., 24-methylenecholesterol is a major 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 (listed below), which represent over 90% of the sterols present in honeybees. These phytosterols are present in varying ratios that, in part, reflect the phytosterol composition of the honeybee's floral diet. 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 published experiments have examined the importance of specific sterols in honeybee nutrition using artificial diets with defined sterol compositions. In one experiment, Herbert et al. (1980, Table 7) individually supplemented a sterol-free defined diet with the sterols cholesterol, 24-methylenecholesterol, β-sitosterol, campesterol, and stigmasterol (but not isofucosterol) and fed tented honeybee hives each of these diets, each containing a single sterol, for up to 12 weeks. These results showed that diets containing some sterols, such as 24-methylenecholesterol, produced more larvae than diets containing other sterols. The experiment did not allow for conclusions about whether certain sterols are physiologically exchangeable for other sterols, i.e., whether honeybees require a large number of sterols or only a small number of sterols, and whether the absence of certain phytosterols can be offset by the presence of other phytosterols (combinations).

[0018] Chakrabarti et al. (Table 7) conducted short-term feeding experiments using 24-methylene cholesterol as the sole sterol present in an artificial diet and showed that, when present, 24-methylene cholesterol had significant physiological and metabolic effects (larval production was not measured). However, these experiments did not include control diets containing sterols different from 24-methylene cholesterol to assess whether the observed effects were specific to a particular sterol or nonspecific to a molecular class of sterol. These experiments did not distinguish whether sterols different from 24-methylene cholesterol 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 types of sterols exist in animals (among which cholesterol is by far the most abundant), a wide range of sterols has been 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 major end-products. However, sterols also occur as conjugates, for example, where the 3-hydroxyl group is esterified with a fatty acid chain or phenolic acid to give steryl esters. For purposes of this description, the term sterol refers to both free sterols and complexed sterols. However, references herein to the concentration, amount, or percentage (%) of sterol refer to the total weight of sterol groups, excluding the weight of complexed 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 herein as being fed as x grams (or other amount) over a two-week period (or other period), this is intended as a general description of the "dose given per unit time" and not a specific limitation to a particular requirement of being 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 present inventors have surprisingly demonstrated that isofucosterol is an essential nutrient for bees. Isofucosterol delivered by a pollen-free diet increases bee larval production and worker bee survival compared to a pollen-substitute bee diet that does not contain isofucosterol. The present inventors have further discovered that a combination of isofucosterol with at least one additional sterol, particularly cholesterol, 24-methylenecholesterol, and other phytosterols, particularly campesterol, β-sitosterol, or stigmasterol, results in enhanced growth and / or health performance in invertebrates, particularly bees and other pollen-feeding insects, other than isofucosterol alone.

[0025] This utility here is important in non-pollen feeding formulas for replenishing honeybee colonies during periods of environmental pollen depletion (which today is a major source of poor bee colony performance), for producing superior queens, and in the commercial rearing of pollen-feeding insects such as black soldier flies for human and animal feed, as an alternative protein source to bumblebees (for pollination), ladybugs (for aphid control), hoverflies (for pollination), and other commercially relevant insects. [Means for solving the problem]

[0026] Accordingly, 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-methylene cholesterol, campesterol, stigmasterol, and β-sitosterol, or physiologically available complexes thereof.

[0027] In another aspect, the invertebrate is a pollen-feeding insect, preferably an insect of the orders 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 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.

[0033] In another embodiment, the nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof and at least one further sterol is administered at a rate of 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 from 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 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 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 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 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 embodiment, 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-methylene cholesterol in an amount between 0% and 50% by weight, β-sitosterol, stigmasterol, and / or campesterol in an amount between 0% and 50% by weight 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 sterols 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 nutritional 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, especially honeybees (Apini), bumblebees (Bombini), or stingless bees (Meliponini).

[0042] In another aspect, the composition comprises: In solid form such as a patty 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 of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol in the total diet 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 a mixture 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-methylene cholesterol, 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, especially Thalassiosira pseudonana, Thalassiosira rotula, or Chaetoceros muelleri; and Extracts, oils or purified products of fungi, especially 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, bacterium, 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 eusocial bee colonies from a synthetic source.

[0053] In another embodiment, isofucosterol, fucosterol, or a mixture thereof, and one or more additional 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 of 20% to 90% by weight, preferably 30% to 70% by weight, optionally vitamins, and Optionally, minerals Including, The sum of the total amounts of the components is 100% by weight, with the weight percentages relating to the total dry weight of the composition.

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

[0055] In another aspect, 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 method 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 concentration of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol is 10% to 33% by weight compared to the total weight of the pollen replacement composition.

[0057] In another aspect, the pollen replacement composition comprises a bee appetite / feeding inducer component selected from the group consisting of pollen, sugar, oil or fat, honey, or protein, or mixtures thereof. For other invertebrates, different appetite / feeding 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, and even more preferably from 5% to 10% by weight, relative 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 relative to the total dry weight of the pollen replacement composition.

[0061] Another aspect of the invention is a pollen-free feeding composition or 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, and optionally the composition 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.

[0062] Another aspect of the invention is a pollen-free feeding composition or 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 aspect of the invention is a pollen-free feeding composition or 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 pollen replacement composition, in particular for bees, which comprises 0.01% to 1% by weight of isofucosterol / 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 pollen replacement composition further comprises 0.01% by weight to 0.5% by weight of 24-methylene cholesterol relative to the total weight of the pollen replacement composition; The ratio of isofucosterol and fucosterol to 24-methylene cholesterol is 2:100 to 100:2, The pollen replacement 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.

[0065] Another aspect of the invention is a pollen-free feeding composition or pollen replacement composition, especially for bees, comprising 0.01% to 1% by weight of 24-methylene cholesterol, 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 relative 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, especially for bees, comprising 0.02% to 3% by weight of a sterol, relative 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, and ergosterol is between 2:100 and 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, and 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, and 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, and 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 relative to the total weight of the pollen replacement composition to increase feed consumption. [Brief explanation of the drawings]

[0067] [Figure 1] 1 shows cohort selection of bees for diets with or without isofucosterol. [Figure 2] 1 shows survival of a cohort of bees restricted to feeding on a diet containing a specific concentration of isofucosterol. [Figure 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. [Figure 5] Results from Example 4B: Results of a long-term feeding experiment in an Apidea colony containing bees fed multiple sterols, including 24-methylene cholesterol, 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 the weight of laid egg masses. Adult survival and number of egg masses laid by 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 2022 JARQ 56(2), 211-217 (2022). [Figure 8] Results from Example 5B: Feeding adult black soldier flies a diet containing multiple sterols: Effects on adult lifespan and larval survival to the prepupal stage. Black soldier fly (BSF) larvae were fed a diet containing 22% protein and 4% lipid 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 concentration 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 the 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 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] Figure 11C shows the 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] Figures 11A-11D show the cumulative number of operculated larvae fed diet A and diet B according to Example 7 in the tent enclosures shown in Figures 11A-11D (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 12-96 from the initiation of hive placement in the tent enclosure and the initiation 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. [Figure 13]Figure 1 shows the cumulative weight of feed consumed by 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 through 96. The horizontal axis shows days 12 through 96 from the start of hive placement 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. [Figure 14ABC] The numbers of operculated larvae in hives fed diet A or diet B are shown from 24 to 96 days after placement in the closed tent and initiation of feeding according to Example 7 (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 days 24 to 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 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 diet B from days 36 to 84 according to Example 7 are shown. [Figure 17] Figure 1 shows photographs of frames of larvae in hive 29 fed diet A (top two rows) and 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 near the average of the hives fed diet A and diet B. [Figure 18]Figure 1 shows the cumulative number of occluded larvae in hives within 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 through 96. The horizontal axis shows days 12 through 96 from the start of placement of the hives within 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 is the average for 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 in Example 7. Both hive 29 and 14 plot near the average of the hives fed diet A and diet C. [Figure 20] The numbers of operculated larvae in hives fed diets A, B, C, and E according to Example 8 are shown from 24 to 96 days after placement in the enclosed tent and initiation of diet feeding (raw data from Table 6). The vertical axis shows the number of operculated larvae laid from day 24 to day 96. The horizontal axis shows 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 INVENTION

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

[0069] Honeybees live in colonies. Standard industry practice involves colonies of various sizes: 8-frame colonies containing approximately 20,000 bees (which is the industry standard); 10-frame colonies with approximately 24,000 bees; large, two-story colonies (12.5 frames = 30,000 bees); and colonies with 50,000 bees (2 are extra-large). Overall, bee colonies vary in size (they can fluctuate in size by ±50% over the course of a year, depending on the season and nutrient availability).

[0070] Rortais et al. 2005 (Apidologie, 36(1), 71-83) comprehensively reviewed the literature on pollen consumption in bees and stated that 10-55 kg of pollen is collected per colony per year. Crailsheim et al. 1993 (J Insect Phys 38(6):409-419) calculated the annual pollen consumption of 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 pollen consumption of 10-26 kg per year. This clearly depends on colony size, colony growth, environment, and pollen type.

[0071] Keller et al. 2005 (Bee World, 86(1)3-10) estimated that each worker bee requires 140-180 mg of pollen over its entire lifespan. For a colony with a total of 100,000-200,000 bees per year, this translates to a requirement of 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 studies. They also measured pollen consumption from two colonies they measured. They estimated that the average amount of pollen required for a colony producing approximately 150,000 larvae per year is 19 kg.

[0072] When bee colonies receive supplemental feeding during pollen deprivation periods, they typically receive supplemental food in the form of protein or pollen patties (most 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 2,400 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, with 10 frames of bees filling a 10-frame hive. Over the winter, the colony continues to reduce its population to 5-6 frames of bees.

[0073] When calculating the "mg pollen equivalent feeding" required by bees, it is important to consider that pollen from different plant species contains a wide range of approximately 50% sugars, proteins, and other components, and therefore the nutritional value of pollen varies by plant species (within a ±50% range). Depending on the supplemental feeding of pollen, pollen supplements, or protein supplements, colonies receive a dose of approximately 1.5 to 3 pounds, or (680g to 1360g) of pollen supplement per two weeks. On an annual basis, this translates to a range of 17.7kg to 35.4kg of pollen supplement diet, which, considering the variability in the nutritional quality of pollen, is within the published range for a 10-frame colony consuming pollen. For colonies of various sizes, the dose calculation should be adjusted proportionally (one bee frame = approximately 2400 bees).

[0074] Using calculations based on the table in 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 citation: https: / / www.insignia-bee.eu / how-much-pollen-does-a-colony-need / Rortais et al. (2005) comprehensively reviewed the literature on bee pollen consumption and noted that 10-55 kg of pollen 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 10-26 kg per year. This clearly depends on colony size, colony development, environment, and pollen type. 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 mixed pollen (Hoecherl et al., 2012). Clearly, there is no fixed amount of pollen that can or should be collected per colony in order to grow, maintain, and function. Nevertheless, there are good studies available to investigate the annual pollen requirements of a colony. Rortais et al., cited above, 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 in their lifetime. This is an average estimate; twice the amount has been reported as well. In the study by Crailsheim et al. mentioned above, the daily pollen consumption was 3.4-4.3 mg. For a summer honeybee's 30-day lifespan, this is approximately 115 mg over the lifespan. Another approach to investigating pollen consumption is by Keller et al. (2005a) is a review article. This is a survey of pollen consumption based on nitrogen (N) for larvae and bees. Pollen is the only source of N in bee diets. 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 required, and for 62.5 mg of protein, 312.5 mg of pollen is required, and for the efficiency of the feeding process, 390.6 mg of pollen is required to be consumed by the bee. Taking all this into consideration, 125 mg of pollen is required to raise one bee.After emergence, bees gain 0.86 mg of N. 0.86 mg of N corresponds to 39 mg of pollen. Therefore, not taking into account N losses due to defecation, overall, each bee required a minimum of 125 + 39 = 164 mg of pollen. Assuming a 200-day active and reproductive period and an average bee lifespan of 35 days (Steen et al., 2012), it takes 200 / 35 = 6 generations. To maintain an average colony of 15,000 bees, 15,000 x 6 = 90,000 bees are kept. The number of bees kept increases each year, and the calculated amount of pollen required is summarized in Table 1 (of this paper).

[0076] The starting amount of 35 kg mentioned above is the amount needed for a large colony and is a best guess. 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. To ensure that pollen reserves are sufficient, a rough estimate of the surface / number of bee cells in the colony is simply required. A healthy, normal-sized colony produces approximately 0.5-2 dm per day. 2 A pollen trap requires / consumes between 200 and 800 half-filled bee-pan cells. Another simple estimation is the weighting of pollen trap collections. It should be noted that the effectiveness of pollen traps varies from 10 to 50% (Keller et al., 2005b).

[0077] Table 1 in Roessink et al. (2021) shows that bee bread consumption by honeybees is rapid, and the results of a 6-week field study (Journal of Apicultural Research, 60:659-664) provide an example calculation of the pollen requirement 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. Pollen is not optimized by plants to feed insects (pollen is optimized for plant use = fertilization). Pollen from different species contains a wide range of different proteins, lipids, sterols, antioxidants, and molecules in widely differing 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 adding 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). This dose represents a concentration of 0.01% to 1.76% isofucosterol in a 680 gram patty fed to a 30,000 bee colony over a two-week period.

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

[0081] Furthermore, the inventors have shown that daily doses of isofucosterol or fucosterol of 0.0006% to 0.052% of body weight are beneficial to the production of larvae and adults of other pollen-feeding insects, such as bumblebees and adult soldier flies; 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] Administration method Administration to bee colonies or organisms is only for the period needed for the desired effect: at the very least, it can be a single dose to induce or support queen reproduction as bees emerge from winter to improve, induce, or support normal larval development, or as 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-deficient periods in spring, summer, or winter. For colonies of different sizes, dosages can be adjusted proportionally to the amount of bees or nurse bees present.

[0084] This twice-weekly dose of isofucosterol can be administered to the bee hive or 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 way that is easily digestible by the bees or target animals). - Incorporated into 30,000 hives per two weeks with a standard 1.5 to 3 lb protein or pollen supplement patty, 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 total weight of the pollen replacement composition for honey bees and 0.03 to 0.8% of the total weight of the pollen replacement composition for bumblebees. - then incorporated into or mixed with ingredients that induce bees to feed on the compound, such as pollen, sugars, oils / fats, honey, or proteins, or mixtures thereof, and further concentrated in mini-patties at much higher concentrations, e.g., up to 33% sterols; or - isofucosterol-containing powders or pastes incorporated into or mixed with ingredients that induce bees to feed on formulations such as pollen, sugars, oils / fats, honey, or proteins, or mixtures thereof, spread within the colony, or fed to the outside of the colony in feeding stations. -Isofucosterols are incorporated into or mixed with ingredients that induce bees to feed on formulations such as pollen, sugars, oils / fats, honey, or proteins, or mixtures thereof, or are diffused within the colony as a syrup or liquid, or are fed outside the colony in feeding stations. In another embodiment, the dose sterol molecules can be delivered by a device that injects the sterol composition into the colony, for example, in a pre-programmed manner. -or any method known to those skilled in the art that can be used to deliver the indicated doses so that the bees ingest the chemicals or nutrients, etc.

[0085] In another aspect, the sterol mixture is not administered via patty or similar feed, but rather 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 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) Compared to feeding isofucosterol alone, (2) Improvement compared to feeding the above sterol combinations without isofucosterol. (3) Improvement over feeding cholesterol, or 24-methylenecholesterol, or campesterol, or stigmasterol, or a combination of the above sterols lacking 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, were able to support larval development for more than five cycles. Similar diets lacking isofucosterol were unable to do this. Importantly, our experiments demonstrate that larval production rates stabilize. This trajectory demonstrates that our diets provide sufficient levels of essential nutrients to allow unlimited larval production. This means that such diets can serve as a pollen substitute for bee colonies over this period. Therefore, the isofucosterol in these diets allows beekeeping in natural or agricultural settings to become independent of pollen sources. In bumblebees, such diets containing multiple sterols allow queen bees to establish 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) and showed that when honeybees were fed an artificial diet containing only one sterol, sitosterol and stigmasterol were antifeedant and not essential for honeybees.

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

[0090] Changes in 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 avenue for utilizing industrial agricultural waste is cultivating fly larvae, such as those of the American black soldier fly (Hermetia illucens), in discarded materials. Black soldier fly larvae are omnivorous generalists capable of feeding 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, making them valuable additions to the food chain (Boukid et al. 2021 Insects 12,672. https: / / doi.org / 10.3390 / insects12080672). However, feeds such as industrial human waste are not optimized for black soldier flies and often lack key essential nutrients. For example, the development time, size, and mortality of BSF-fed larvae are greatly affected by the nature of the food on which they are cultured (Lalander et al. 2019. J Cleaner Production, https: / / doi.org / 10.1016 / j.jclepro.2018.10.017). Delayed development due to suboptimal diet reduces the efficiency of the rearing system.

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

[0093] In the wild, BSF larvae feed on decaying organic material, while 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 laboratory-reared flies fed protein were less than half that of wild-fed flies (Bertinetti et al. 2019). It is likely that certain nutrients, as yet unidentified, are absent from 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 may provide key nutrients required for BSF. Recently, it has been shown that black soldier flies have significantly improved egg-laying performance when fed a diet containing flower pollen (Thin and Kanoi. 2022. JARQ 56(2):211-217). For example, when female black soldier flies are fed pollen, egg-laying and egg hatching rates double, and their lifespan increases by 30% (Thin and Kanoi 2022). However, the pollen components that cause these changes are unknown.

[0095] Pollen feeding in other beneficial agricultural insects also affects adult insect reproductive performance. This is particularly important in insects cultivated for biological defense, such as ladybugs (Coccinelidae) and hoverflies (Syrphidae). For example, ladybug 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 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 both inside and outside the plant. Many phytosterols are produced by plants and inhibit insect feeding on the plant.

[0097] The present inventors have surprisingly established that isofucosterol, a common sterol found in pollen, is an essential nutrient for pollen-feeding insects. Isofucosterol delivered by a pollen-free diet increases the performance of adult and larval BSF. The present inventors have also identified that isofucosterol is the major sterol component of pollen selected by adult female hoverflies. The present inventors have further discovered that a combination of isofucosterol with at least one additional sterol, particularly cholesterol, 24-methylenecholesterol, and other phytosterols, particularly campesterol, β-sitosterol, or stigmasterol, results in higher growth and / or health performance in many invertebrates, particularly 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) Improvement compared to isofucosterol alone (2) Improvement compared to the above sterol combinations that do not contain isofucosterol (3) improvement over cholesterol or the combination of the above sterols lacking 24-methylenecholesterol, campesterol, stigmasterol, or β-sitosterol. The inventors have further established that

[0099] Surprisingly, feeding adult cultured black soldier flies or insects with a diet containing a mixture of isofucosterol and sterols improves egg-laying and egg hatching performance, as well as the lifespan of the adults. The same effect can be achieved by feeding larvae producing larval stocks with a diet containing a mixture of isofucosterol and sterols (adults inherit substantial nutritional reserves from the larval / pupal stage). This means that such a diet can serve as a pollen substitute and a form of supplemental nutrition for laboratory-reared insect colonies. This diet can therefore optimize the larval development time and adult reproductive potential in industrially cultivated beneficial insects.

[0100] This is not clear from the literature. Svoboda, a world-leading researcher of 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 essential nutrients 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: - 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; and pollen or algae consuming animals such as ladybugs, soldier flies, hoverflies, oysters, shrimp, and fish larvae.

[0102] Nutritionally effective amount 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 represents 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 14 day period 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% a 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 insect's body weight consuming the meal to 0.087% (formula = 20 / 14 / 1650 x 100) of the insect's body weight 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 composition and dose range applies to other pollen-feeding insects.

[0108] In another embodiment, some or all of these sterol moieties can be delivered as prodrugs such as water-soluble sterol glycosides or sterol sulfates or similar salts, or lipid-soluble forms such as 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 conjugated sterol molecule.

[0109] The various methods of administration described above for isofucosterol can be applied to isofucosterol and 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, use the following formula: -[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 nurse bees and / or larvae; @30000 bees in a hive, 50%: half of which are nurse bees and / or larvae=1650 grams). -["dose of a given sterol or group of sterols delivered over a 14-day period for a colony of 30,000 bees" / 14] = "daily amount of sterol administered" - Daily dose (grams) / Weight (grams) of non-pollen supplement consuming insects (brood bees and larvae) in 30,000 hives x 100 = Percentage of body weight of sterol administered per gram of insect body weight per day -Dose of sterols administered to bumblebee colonies: "Percentage of body weight of sterols administered per gram of insect body weight per day (%)" x "Body weight of insects (brood wasps and larvae) fed" On average - see table above; 2 / 3 of the pollen consumed in a colony is consumed by nurse bees and 1 / 3 by the larvae present (see table above).

[0111] In a preferred embodiment, 1.5 to 3 pounds of patties are administered per 14 days to 30,000 bees with a 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, - 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 selected from the following group (concentration as % of the feed dry weight); resulting in a multiplicity of isofucosterols or fucosterols, 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-methylene cholesterol 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 Bombus, 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 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 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 aspect, 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, especially Thalassiosira pseudonana, Thalassiosira rotula, or Chaetoceros muelleri; and Extracts, oils or purified products of fungi, especially 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 a mixture 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 a mixture thereof, and 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 are provided to eusocial or proto-eusocial bee colonies 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 of 20% to 90% by weight, preferably 30% to 70% by weight, optionally vitamins, and Optionally, minerals wherein the total amount of the components and optional further components sums to 100% by weight, the weight percentage being relative to the total dry weight of the composition.

[0122] Liquid or semi-liquid experimental administration: In another aspect, 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 total amount of the components and optional further components sums to 100% by weight, the weight percentage being 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 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 bee-collected pollen. Cholesterol was purchased from commercial sources. β-Sitosterol and other sterols were purified from commercially available soybean 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, and 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 acid, stearic acid, linoleic acid, oleic acid, stearic acid, linolenic acid, etc.), - linked to fatty alcohols by alcohol groups (sterol alkyl ethers), Esterification with acids such as sulfuric acid (sterol sulfates), - Attached to carbohydrates (leading to 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 sterol group. esterified to g-trans-hydroxycinnamate, ferulate (4-hydroxy-3-methoxycinnamate), and p-coumarate (see the 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:

[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), and (C) acyl steryl glycosides (ASG), having the following structures:

[0147] [ka]

[0148] [ka]

[0149] [ka] The R side chain can be, in this patent, 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 term "pollen substitute" means that the composition 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, relative to the dry weight of the composition. The terms pollen substitute and non-pollen are used interchangeably.

[0170] Alternative pollen source of isofucosterol Isofucosterol is, for example, Synthetic chemical synthesis, or Any pollen substitute plant source, Pollen can be obtained from alternative 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 invertebrates, Arthropods, such as insects, arachnids, crustaceans, and myriapods; Mollusks, such as chitons, snails, clams, squids, and octopuses, Annelids, such as 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, Apis granulosa, or Bumblebees of the genus Bombus, and in particular Bombus terrestris, B. impatiens, B. ignites, Stingless bees of the genus Stingless Bee and other animals of the Apidae 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 feeding a 30,000 colony containing 1.5 to 3 pounds of bee patties over a 14-day period. The patty dose can be adapted to the size of the colony, and the dose per 14 days can be repeated as desired by the beekeeper. 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 different formulations, methods of administration or bee colony size, 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 bumblebee.

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

[0180] In one embodiment, 24-methylene cholesterol is administered in a nutritionally effective amount to 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, particularly 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 a nutritionally effective amount to invertebrates, particularly 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 an amount that is nutritionally effective for invertebrates, particularly 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 wherein the sum of the total amount of the components and optional further components a) to e) is 100% by weight, and the weight % is 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-methylene cholesterol, 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 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.

[0187] A further aspect of the present invention is the use of a pollen replacement composition of the present invention for feeding invertebrates 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 wherein the sum of the total amount of the components and optional further components a) to e) is 100 wt. %, where wt. % is 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-methylene cholesterol, 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 invertebrates, particularly honeybees, 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 wt. %, where wt. % is 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-methylene cholesterol, 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] Example Comparative Example 1: Isofucosterol concentration in commercial honeybee feed Commercially available pollen replacement bee feed compositions do not contain isofucosterol, as shown in Table 1 below.

[0191] [Table 2]

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

[0193] Example 1 - Preference assay: Bees prefer specific concentrations 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, 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, carbohydrates were 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 (amount of treatment consumed - amount of 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 their diet, as shown in Figure 7.

[0197] Example 2 - Survival: Bees live longer on isofucosterol-containing foods Newly emerged adult worker honeybees 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, carbohydrates were maintained at 60% using sucrose, and fat was maintained at 8%. Consumption of each diet was measured daily throughout 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 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 on isofucosterol.

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

[0201] Colonies were placed in a closed greenhouse with ventilation to prevent bees from accessing nectar or pollen. Each treatment consisted of three to six 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 days 1 and 6, and the amount consumed was measured on days 6 and 15. The experiment was terminated if no larvae or eggs / queens were observed by day 6. The number of lidded larval cells was counted on day 15. The number of hives was estimated between each inspection. 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 over 75% carbohydrates.

[0203] Figure 9 shows the threshold values for Example 3 of 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 emerging adults was counted over the entire 10-week period.

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

[0205] Colonies were placed in closed, ventilated screen houses to prevent bees from accessing nectar or pollen. Each treatment consisted of three to six colonies. Each colony was offered solid food ad libitum in a top-mounted feeder with a mesh floor, and the amount consumed was monitored at each feeding. The number of lidded larval cells was monitored every 12 days. The number of hives was recorded between each inspection. When colonies reached the maximum Apidea box size, oversized 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 over 40% carbohydrates (Diet 1).

[0207] The experiment included the following treatments: Diet 1: No sterols added, diet 1 as above Feed 2 = Feed 1, to which isofucosterol, 24-methylenecholesterol, campesterol, β-sitosterol, stigmasterol, and cholesterol are added in proportions and concentrations according to the preferred embodiment described above. Feed 3 = Feed 2 without 24-methylene cholesterol Diet 4 = Diet 2 without isofucosterol Diet 5 = Pollen-based patty (pollen patty) consisting of 60.5% pollen (fresh frozen honeybee pollen collected 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 (cm) 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 (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 without isofucosterol Honeybees: Three to five miniframes housing adult worker bees and Apidea nucs of fully functional insulating Styrofoam containing one mated queen were inhabited with 300–400 mL of young adult worker bees (approximately N < 1000 bees (various ages)).

[0209] Colonies were placed in closed, ventilated screen houses to prevent bees from accessing nectar or pollen. Each treatment consisted of three to six colonies. Each colony was offered solid food ad libitum in a top-mounted feeder with a mesh floor, and the amount consumed was monitored at each feeding. The number of lidded larval cells was monitored every 12 days. The number of hives was recorded between each inspection. When colonies reached the maximum Apidea box size, oversized 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 over 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 more than 40% carbohydrates 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-methylene cholesterol 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, commercially available feed Figure 5 shows the results of Experiment 4B: the average area (cm) 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), or Diet 5 (asterisk, thin dashed-dotted line).

[0215] Example 4C: 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] Honeybees: Three to five miniframes housing adult worker bees and Apidea nucs of fully functional insulating Styrofoam containing one mated queen were inhabited with 300–400 mL of young adult worker bees (approximately N < 1000 bees (various ages)).

[0217] Colonies were placed in closed, ventilated screen houses to prevent bees from accessing nectar or pollen. Each treatment consisted of three to six colonies. Each colony was offered solid food ad libitum in a top-mounted feeder with a mesh floor, and the amount consumed was monitored at each feeding. The number of lidded larval cells was monitored every 12 days. The number of hives was recorded between each inspection. When colonies reached the maximum Apidea box size, oversized 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 more than 40% carbohydrates with no added sterols.

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

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

[0221] Diet 4 = Similar to Diet 3 but without 24-methylenecholesterol and cholesterol, maintaining the % 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 without 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] Figure 6: Results from Example 4C: Results of a long-term feeding experiment in an Apidea colony 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 experiments 7 shows the results of Example 5A: Feeding adult black soldier flies a diet containing multiple sterols: effects on the hatchability of laid eggs and the weight of laid egg masses. Adult survival and number of egg masses laid by 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 2022 JARQ 56 (2), 211-217 (2022).

[0228] Figure 8 shows the results of Example 5B: Feeding adult black soldier flies a diet containing multiple sterols: effects on adult lifespan and larval survival to the prepupal stage. Black soldier fly (BSF) larvae were fed a diet containing 22% protein and 4% lipid 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 concentration 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] Figure 9 shows the results of Example 5C: Feeding adult black soldier flies various diets containing multiple sterols: sterols found in the 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.

[0230] Example 6: Experiments with black soldier flies The nutritional composition of BSF larvae relative to the target species (honeybees) was altered by incorporating multiple sterols into their diet. A diet formulation containing larvae was prepared and these larvae were fed the target species. Black soldier fly larvae were fed the diet shown 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). Some groups had no sterols added directly to the diet. Hexane extracts were made from freeze-dried BSF larvae. Hexane solvent containing the fat portion was added to the diet; after the hexane was evaporated, the diet was used to feed the larvae.

[0231] This example generally demonstrates a method for feeding multiple sterols tailored to meet the needs of a target species, which can benefit the performance of the target species. Because 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 their larvae, fucosterol, isofucosterol, and 24-methylenecholesterol (all enriched in algae but absent from current non-algae artificial diets for these species) are added to the BSF feed, and then the algae-feeding aquaculture organisms are fed lipids, proteins, or total extracts of these BSF larvae. This method improves the performance of aquaculture artificial diets 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 honeybee colonies in the Apidea experiment with a diet containing an extract of black soldier flies fed with 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 the long-term larval production of 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 varied 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 honeybee 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 it is more complete and balanced in nutrients, providing a superior nutritional foundation. 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 honeybee-sourced pollen, including β-sitosterol, campesterol, and stigmasterol (but excluding isofucosterol and 24-methylenecholesterol), resulting in phytosterol concentrations and ratios that, on average, reflect the natural composition of pollen. Semisynthetic 24-methylenecholesterol (97% pure) and semisynthetic isofucosterol (97% pure; 75% isofucosterol and 25% EZ isomer fucosterol; hereafter interchangeably referred to as "isofucosterol / fucosterol" or "isofucosterol") were obtained from Phytant NV, Belgium. The background diet for Diet A was a pollen-free, proprietary feed (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. To maintain a constant total added sterol concentration in the diet, the concentration of other sterols added to the diet was increased proportionally to the total sterol concentration of Diet A's 0.375% added sterol. This increased the concentrations of the other individually added sterols by a factor of 1.2, which places the concentrations of these sterols in the center of the range present in the pollen on which the bees successfully feed. Diet C: Pollen-free and 24-methylenecholesterol-free diet. This is Diet A with the 0.125% added 24-methylenecholesterol removed. To maintain a constant total added sterol concentration in the diet, the concentration of other sterols present in the diet was increased proportionally to the total sterol concentration of the 0.375% added sterols in Diet A. This placed the added sterol concentrations in the center of the range present in the pollen on which the bees successfully fed.

[0236] These three diets, along with a fourth commercial diet widely used in the United States (Diet E, see Figure 20 and Example 8), were examined for their ability 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). Six small hives per diet, each containing 800–850 mL (approximately 2000 bees) and one queen, were distributed into adjacent tent enclosures, each measuring 2 m × 4.5 m × 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 six days by placing patties on top of the frames (above the larval nests), following standard beekeeping practices (Figures 11A-11D). All three hives within 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 within the tent every six 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 six days. Photographs of each frame containing larvae in each hive were taken every 12 days over a 96-day period (Figure 11D). The number of operculated larvae in each photograph was manually counted by two to three different individuals (Figures 15, 16, and 17), and results were verified (quality assurance checked) by another individual. During the first 24 days (hive stability period), hives in which the queen stopped laying eggs and hives in which the queen was accidentally crushed during observation were removed from the experiment. This observation regime ensured that all bees born in the hive (all larvae that developed into operculated larvae) were photographed at least once, and only once. On day 84, a sample of 25 to 30 bees was collected 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, using a more complex and complete background diet, and investigating the effect of removing a single sterol 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 the artificial diet). It takes approximately 24 days, or 1.5 life cycles, for the nurse bees to deplete all pools of food stored in the hive and nutrients stored by the bees 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 artificially. Any operculated larvae observed after day 36 are expected to be completely fed by nurse bees, who themselves were reared on the artificial diet. The same applies to nutrients provided by nurse bees to worker bees.

[0240] The Importance of Isofucosterol / Fucosterol The bees and hives behaved normally in the tents, as evidenced by a normal diurnal cycle of flight from and back to the hive at dusk or during rain, foraging for sugar syrup and water, orientation adjustments, and defecation flights. However, these conditions subject the hives to significant stress: (1) the hives are surrounded and fly against the mesh, and (2) more importantly, the hives are opened and all frames are removed every 12 days for photography, a procedure that exposes larvae to cold temperatures and substantially disturbs the hives. The latter procedure will have a significant negative impact on hive health and reproductive performance. 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 are less able to heat and cool themselves as effectively as larger colonies of, for example, 4,000 bees. During the heatwave, spraying from a single nozzle reduced the temperature inside the tent, maintaining it at a maximum of 37.5°C.

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

[0242] To date, no artificial pollen-free complete diets for honeybees have 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, 2,353 operculated larvae were produced in hives fed diet A between days 12 and 96, compared with 890 operculated larvae produced in hives fed diet B during the same period. Figure 12 also shows that after 60 days, the amount of larvae produced in all but one hive fed diet B (hive 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 honeybee hives to produce larvae, and that other sterols cannot compensate for this deficit. Larval production is an essential function for the survival of the hive.

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

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

[0251] Photographs of the frames containing the larvae clearly show the difference in the number and pattern of operculated larvae in hive number 29, which was fed diet A (Figure 15), and hive number 26, which was 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 adding isofucosterol-fucosterol to an incomplete diet containing cholesterol, campesterol, β-sitosterol, stigmasterol, and 24-methylenecholesterol at concentrations similar to those of pollen without isofucosterol-fucosterol provides a diet that will sustain a hive for 4.5 larval cycles (generations) of nurse bees (who have no access to pollen or other nutrients except sugar solution). Conversely, the embodiments show that removing isofucosterol-fucosterol from a complete diet reduces hive and bee fitness, and that this removal is not compensated for by proportionally compensating for isofucosterol-fucosterol removal with other sterols.

[0254] This is the first clear demonstration that isofucosterol-fucosterol is functionally important and essential in honeybee hive diet. The data indicate that isofucosterol-fucosterol is required for honeybee larval production, despite the presence of all other major sterols present in pollen and honeybees, and that its physiological role cannot be assumed by other sterols present in the diet (cholesterol, 24-methylenecholesterol, isofucosterol, stigmasterol, and β-sitosterol, which typically 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) began to show increasing differences in hatch yields from day 48 onwards (Figures 18 and 19, Tables 2, 3 and 4). Between days 36 and 96, hives fed diet C cumulatively hatched an average of 1126 operculated larvae, while hives fed diet A cumulatively hatched 1588 operculated larvae. Note that the difference in 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 manifestation of 24-methylenecholesterol removal observed in Diet C compared to the effect of isofucosterol removal in Diet B can be explained by the observation by Sbovoda et al. (Table 7) that honeybees have mechanisms to maintain (some of) the 24-methylenecholesterol pool within the hive by recycling it to larvae and nurse bees. The data reported here further emphasize the increased importance of isofucosterol-fucosterol in artificial honeybee diets, as well as 24-methylenecholesterol, since the effects of its removal become apparent sooner.

[0256] These data provide the first technical evidence that 24-methylenecholesterol is functionally required in honeybee diets 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 crops.

[0259] Honeybees have to perform complex behavioral functions inside the hive (cleaning, larval feeding, cell construction, defense, ventilation, heating…) and outside the hive (long-distance flying – up to 2 km from the hive, homing, 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 a hive can gather to maintain and grow the hive. From a commercial point of view, many more active and well-fed hives (more fit, 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 a hive is important.

[0260] Sterols are known to have different effects on membrane structure within cells. The role of sterols in animal cell membranes is to regulate membrane fluidity, the organization of proteins in membrane rafts, which are important for proper functioning, among other things, in muscle cells and neurons, 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 will produce bees of equal behavioral fitness.

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

[0263] 1. Outside hives: These hives initially had the same sister queens and 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 nest - Diet A: Diet A see above (same nest and time point as in Example 7). Diet A' = Diet A with the same composition and proportions as Diet A but with twice the concentration of added sterols (0.75%). Diets A and A' have the same phenotype. 3. Tent nest - Diet B: Diet A - isofucosterol / fucosterol (concentrations of other sterols added adjusted proportionally upwards as described above; same nests and time points as in Example 1) 4. Tent nests - Diet C: Diet A - 24-methylene cholesterol (concentrations of other sterols added proportionally adjusted upwards as described above; same nests and time points as in Example 1) 5. Nests in tents - Diet D: Diet A lacking isofucosterol / fucosterol and 24-methylenecholesterol (the concentration of added sterols (0.75%) was adjusted upward to that of Diet A'; carried out in a parallel tent at the same time points as in Example 1; feeding and nest box investigation procedures were generally the same) 6. Nests in tents - Diet E: Commercial diet (completely different diet; conducted at the same time points as in Example 1, in a parallel tent; feeding and nest box disturbance procedures were the same overall)

[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 day 98-99, the hives were opened and visually scored for several phenotypes (score: 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 an open to a 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 feeder and / or at the hive entrance Responsiveness: This is an integrated score of 1-5 (low to high) that describes the overall activity of the 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] The bees on diets A and A' were equally active, responsive, and mobile as the bees in the natural food-gathering hive placed next to the tent enclosure.

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

[0273] Nests fed Diet B (Diet A-Isofucosterol / Fucosterol) showed lethargy (++), trembling / lack of coordination (++); weak response / flying (++), and slow movement (++) phenotypes, lowering the reactivity score to 2.

[0274] Nests fed diet D (diet A-24 methylene cholesterol and -isofucosterol) showed a much greater trembling / lack of coordination (+++) phenotype than diet B, but no lethargy phenotype. Because the lethargy phenotype was absent in diet D, nests fed diet D received a reactivity score of 3. The poor reactivity / flying (++) and slow movement phenotype (++) phenotypes were similar to those in 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 perform complex hive tasks efficiently and exhibit a number of phenotypes that likely affect physiological fitness.

[0276] It is further shown that the phenotypes resulting from the lack of isofucosterol-fucosterol or 24-methylenecholesterol, or both, cannot be physiologically compensated for by increasing the other sterol, and that removing both isofucosterol and 24-methylenecholesterol has a more severe effect on muscle nerves (lack of coordination) and fitness than removing either of these sterols at once 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 in the field, as 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, as both isofucosterol and 24-methylenecholesterol, either alone or in combination, are removed and functionally irreplaceable 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 production in the hive, but also in terms of producing high-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 pollen. The same was shown for 24-methylenecholesterol, but to a lesser extent. Figure 20 and Table 5 demonstrate the superior performance of Complete Diet A compared to a commercially available 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. A method of feeding an invertebrate selected from a honeybee, a bumblebee, a stingless bee, an American soldier fly, or a hoverfly, 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; Including, 1. A method according to claim 1, wherein 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 an invertebrate of the family Apidae, 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.

4. 2. The method of claim 1, wherein 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 of the pollen replacement composition.

5. 10. The method of claim 1, wherein the isofucosterol, fucosterol, or mixture thereof is administered at a rate of 0.14 g to 12 g of isofucosterol, fucosterol, or mixture thereof per 30,000 of the invertebrates per two week period.

6. 6. The method of claim 5, wherein the isofucosterol, fucosterol, or mixture thereof is administered in an amount of 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 of the pollen replacement composition.

7. the administration rate of the nutritionally effective amount of isofucosterol, fucosterol, or a mixture thereof, and the at least one additional sterol is 0.2 to 48 grams per 30,000 of the invertebrates 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 invertebrate or of the pollen replacement composition; The method of claim 1.

8. the isofucosterol, fucosterol, or mixture thereof, and the at least one further sterol are administered at a rate of 0.4 g to 36 g per 30,000 invertebrates 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 invertebrate or of the pollen replacement composition; The method of claim 1.

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 of the invertebrates 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 invertebrate or of the pollen replacement composition; The method of claim 1.

10. 10. The method of claim 1, wherein the isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, beta-stigmasterol, stigmasterol, and / or campesterol is administered in an amount of 0.001% to 0.087% by weight of the live weight of the invertebrate 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-methylene cholesterol 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 of claim 1, wherein

12. 2. The method of claim 1, wherein the total concentration of sterols is from 0.01% to 4% by weight compared to the total weight of the pollen replacement composition.

13. The concentration of sterols is cholesterol in an amount of 0.001% to 2% by weight compared to the total weight of the pollen replacement composition; 24-methylene cholesterol in an amount of 0.001% to 2% by weight compared to the total weight of the pollen replacement composition; sitosterol in an amount of 0.001% to 2% by weight compared to the total weight of the pollen replacement composition; isofucosterol in an amount of 0.01% to 5% by weight compared to the total weight of the pollen replacement composition, campesterol in an amount of 0.001% to 2% by weight compared to the total weight of the pollen replacement composition, stigmasterol in an amount of 0.001% to 2% by weight compared to the total weight of the pollen replacement composition, or Any combination of these The method of claim 1, wherein the compound is selected from the group consisting of:

14. 10. The method of claim 1, 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 additional sterol or combination of additional sterols is from 10:1 to 1:

1.

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

16. 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 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; The method of claim 1.

17. 2. The method of claim 1, wherein 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, is an extract, oil, or purified product of non-pollen tissue of one or more plant species or combinations thereof.

18. 2. The method of claim 1, wherein 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, stigmasterol, is pollen substitute tissue of one or more plant species selected from the group consisting of Solanaceae, Poaceae, Ranunculaceae, Fabaceae, and Bacillus subtilis.

19. 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, - Marine or freshwater algae; Marine diatoms; and ・Fungi 2. The method of claim 1, wherein the pollen alternative source is selected from the group consisting of:

20. 2. The method of claim 1, wherein the isofucosterol or fucosterol and the 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, bacterium, or algae.

21. 2. The method of claim 1, wherein the source of isofucosterol, fucosterol, or a mixture thereof, or the 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.

22. 10. The method of claim 1, wherein the isofucosterol, fucosterol, or mixture thereof, and the at least one additional 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.

23. The isofucosterol, fucosterol, or mixture thereof, and one or more additional sterols selected from the group consisting of cholesterol, 24-methylenecholesterol, campesterol, stigmasterol, and β-sitosterol, are provided as part of the pollen replacement composition, which pollen replacement composition comprises: - protein in an amount between 10% and 50% by weight, fatty acids in an amount between 1% and 20% by weight, carbohydrates in an amount between 20% and 90% by weight, Optionally, vitamins, and Optionally, minerals Including, 10. The method of claim 1, wherein the sum of the total amounts of the components is 100% by weight, the weight percentages being relative to the total dry weight of the composition.

24. 10. The method of claim 1, wherein the pollen replacement composition is substantially free of pollen.

25. A honeybee, bumblebee, American soldier fly, or hoverfly, 10. Use of the pollen replacement composition of claim 1 for feeding organisms selected from the group consisting of:

26. 26. Use of the pollen replacement composition according to claim 25 as a concentrated patty, wherein the concentration of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol is from 10% to 33% by weight compared to the total weight of the pollen replacement composition.

27. 27. Use of a pollen replacement composition according to claim 26, 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.

28. 28. Use of a pollen replacement composition according to claim 27, wherein the concentration of the bee-ingesting / appetite-inducing component is from 1% to 20% by weight compared to the total weight of the pollen replacement composition.

29. 26. Use of a pollen replacement composition according to claim 25, wherein the pollen replacement composition is in liquid or powder form.

30. 30. The use according to claim 29, wherein the concentration of isofucosterol, fucosterol, cholesterol, 24-methylenecholesterol, campesterol, stigmasterol and β-sitosterol is from 0.01% to 99% by weight compared to the total dry weight of the pollen replacement composition.

31. - 0.01% to 1% 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 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 compared 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 compared to the total weight of the pollen replacement composition to increase feed consumption; or - 0.01% to 1% by weight of isofucosterol / fucosterol 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 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-methylene cholesterol 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; 1. A pollen-free feeding composition or pollen replacement composition for bees, comprising:

32. A pollen-free feeding composition or pollen replacement composition for bees, comprising 0.02% to 3% by weight of a sterol compared to the total weight of the 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-methylene cholesterol 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 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 consisting of 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 pollen replacement composition for bees.

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