Methods and uses for black soldier flies

Abstract

Disclosed are methods for inducing quiescence in black soldier flies (BSF), enhancing synchronization in their breeding cycles, and optimizing egg production. The methods, systems, and compositions include inducing quiescence during the larval stage of the life cycle of a black soldier fly (BSF). Also disclosed herein are methods of inducing 1:1 mating to generate inbred lines of BSF. Environmental controls for quiescence induction, optimized oviposition attractants, and tailored feed formulations to improve larval and pre-pupal survival rates are provided. These techniques support efficient propagation and utilization of BSFs for animal feed and sustainable products.

Description

[0001] Attorney Docket No.: 55877-0011WO1

[0002] METHODS AND USES FOR BLACK SOLDIER FLIES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U. S. C. § 119(e) to U. S.

[0004] Application No, 63 / 738,433 filed on December 23, 2024, and U. S. Application No.

[0005] 63 / 845,040 filed on July 16, 2025.

[0006] FIELD OF THE DISCLOSURE

[0007] The present disclosure relates to methods for inducing quiescence in black soldier flies (BSFs) and generating black soldier fly eggs (BSFEs). The invention further pertains to optimizing the synchronization of life cycles, improving breeding efficiency, and providing feed formulations for the larvae and late instar stages of BSFs.

[0008] BACKGROUND

[0009] Despite the increase in rearing of black soldier fly larvae (BSFL; Hermetia illucens (L.)) for the purpose of producing animal feed ingredients, plant and soil products, or other applications, little is known about their genetics and mating structure under wild or commercial farming conditions. The development of methods for directing the genetics of the black soldier fly (BSF) populations can enable development of effective selective breeding strategies to maximize yields in commercial farms. A major bottleneck in the genetic selection of BSF is the inability to control the mating behavior and pairings, due to the natural biology of mate selection in the species. To date, the inability to direct reproductive pairings and manage controlled breeding environments has limited the application of genetic studies (e.g., BSF genetic selection, BSF population structure).

[0010] There remains a need to develop controlled mating in order to selectively breed BSF populations.

[0011] SUMMARY

[0012] The method developed and described below allows for controlled mating of one male and one female BSF, including but not limited to induction of quiescence to allow synchronization of lifecycles and breeding capabili ty, inducing reproductive behaviors withAttorney Docket No.: 55877-0011P01

[0013] one male and one female BSF, and collection and rearing of very small quantities of BSFL eggs.

[0014] Thus, in one instance, disclosed herein are methods of inducing quiescence during larval life stage in a population of black soldier flies, in some instances, the method includes: (a) providing the population of black soldier flies, wherein the black soldier flies of the population are at a prepupal developmental stage; and (b) subjecting the population of black soldier flies to an environment of about 14°C to 17°C at about 40% to about 80% humidity for at least 1 to 7 days.

[0015] In some instances, the environment is at about 14°C, 15°C, 16°C, or 17°C. In some instances, the humidity is at about 50%. In some instances, the black soldier flies are in the environment of about 14°C to 17°C at about 40% to about 60% humidity for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In some instances, after the at least 1 to 7 days, the black soldier flies are maintained at about 27°C and about 55% humidity. In some instances, after inducing quiescence, individuals in the population of black soldier flies have life cycles and breeding capability that are synchronized. In some instances, each indi vidual in the population of black soldier flies have not mated with another black soldier fly previously. In some instances, the population of black soldier flies are maintained in a container.

[0016] Also disclosed herein is a method of generating black soldier fly eggs (BSFEs) from one mating pair. In some instances, the method includes (a) transferring a pair of black soldier flies from the population of black soldier flies to a chamber comprising an egg collection apparatus for about three to seven days, wherein the pair is one male black soldier fly and one female black soldier fly; and (b) adding an oviposition attractant to the chamber, thereby producing BSFEs.

[0017] In some instances, also disclosed is a method of generating black soldier fly eggs (BSFEs) from one mating pair, the method comprising: (a) inducing quiescence according to any one of the methods disclosed herein; (b) transferring a pair of black soldier flies from the population of black soldier flies to a chamber comprising an egg collection apparatus for about three to seven days, wherein the pair is one male black soldier fly and one female black soldier fly; and (c) adding an oviposition attractant to the chamber, thereby producing BSFEs.

[0018] In some instances, about 200 to about 1000 BSFEs are produced. In some instances, the BSFEs mature into black soldier fly larvae with an average survival rate of at least about 70%. In some instances, the pair is transferred in ( b) to the chamber for about five days. In some instances, the oviposition attractant is added to the egg collection apparatus. In some instances, about 10 grams of the oviposition attractant is added to the chamber in (c).Attorney Docket No.: 55877-0011P01

[0019] In some instances, the oviposition attractant comprises distillers dried grains; bakery by-product meal; calcium carbonate; calcium chloride; BSFLs; and EMI microbial inoculant.

[0020] In some instances, the oviposition attractant comprises about 38 grams of distillers dried grains; about 38 grams of bakery by-product meal; about 0.8 grams of calcium carbonate; about 0.7 grams of calcium chloride; about 5.5 grams of about 1 mg BSFL; and about 75 ml of EM1 microbial inoculant.

[0021] In some instances, the methods also include collecting the BSFEs from the egg collection apparatus and providing the BSFEs with an early instar feed formulation comprising ground com; soybean meal; calcium carbonate; dicalcium phosphate; soybean oil; sodium chloride; and calcium propionate. In some instances, the early instar feed formulation comprises about 65.0% (e.g., 64.7%) ground corn; about 24.0% (e.g., 23.5%) soybean meal; about 8.5% calcium carbonate; about 1.5% dicalcium phosphate; about 1.0% soybean oil; about 0.50% sodium chloride; and about 0.33% calcium propionate.

[0022] In some instances, the method includes providing late instar feed to the BSFEs. In some instances, the late instar feed comprises a formulation comprising distillers dried grains; bakery' by-product meal; calcium carbonate; and calcium chloride. In some instances, the late instar feed comprises a formulation comprising about 49.0 (e.g., 49.1%) distillers dried grains; about 49.0% bakery by-product meal; about 1.0% calcium carbonate; and about 0.85% calcium chloride. In some instances, the methods include inducing quiescence does not substantively change the number of BSFEs produced compared to a pair of black soldier flies in which quiescence is not induced.

[0023] In some instances, the methods also include measuring one or more of the following metrics: the number of BSFEs produced, early instar black soldier fly larvae (BSFL) survival rate, BSFL weight, late instar BSFL weight, and the number of black soldier fly pre-pupae (BSFPP), BSFPP survival rate, and BSFPP weight.

[0024] In some instances, the methods include propagating a second generation of black soldier flies from the BSFEs, In some instances, the methods include propagating at least 2, at least 3, at least 4, at least 5 or more generations of populations of black soldier flies from the one mating pair of black soldier flies. In some instances, inducing quiescence does not substantively change the number of propagated black soldier flies from the second generation compared to a pair of black soldier flies in which quiescence is not induced. In some instances, black solder fly larvae produced by the BSFEs from the one mating pair are used as an ingredient in animal feed, plant products, and / or soil products.Attorney Docket No.: 55877-0011P01

[0025] In some instances, also disclosed is a method of generating black soldier fly eggs (BSFEs) from one mating pair, the method comprising: (a) transferring a pair of black soldier flies from the population of black soldier flies to a chamber comprising an egg collection apparatus for about three to seven days, wherein the pair is one male black soldier fly and one female black soldier fly; and (b) adding an oviposition attractant to the chamber, thereby producing BSFEs,

[0026] Also disclosed herein is an oviposition attractant composition comprising distillers dried grains; bakery by-product meal; calcium carbonate; calcium chloride; BSFL; and EM1 microbial inoculant. In some instances, the distillers dried grains are present in an amount of from about 20 grams to about 60 grams (e.g., about 38 grams); the bakery by-product meal is present in an amount of from about 20 grams to about 60 grams (e.g., about 38 grams); the calcium carbonate is present in an amount of from about 0.1 grams to about 2.5 grams (e.g., about 0.8 grams); the calcium chloride is present in an amount of from about 0.1 grams to about 2.5 grams (e.g., about 0.7 grams); the BSFL is present in an amount of from about 1 gram to about 10 grams (e.g., about 5.5 grams); and the EM1 microbial inoculant is present in an amount of from about 50 ml to about 100 ml (e.g., about 75 ml).

[0027] Further disclosed herein is an oviposition attractant composition comprising about 38 grams of distillers dried grains; about 38 grams of bakery by-product meal; about 0.8 grams of calcium carbonate; about 0.7 grams of calcium chloride; about 5.5 grams of about 1 mg BSFL; and about 75 ml of EM1 microbial inoculant.

[0028] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0029] Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0030] The term “each,” w hen used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in theAttorney Docket No.: 55877-0011P01

[0031] collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.

[0032] Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodimen ts described herein are also wi thin the scope of this disclosure.

[0033] DESCRIPTION OF DRAWINGS

[0034] The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements, FIG. 1 shows a graph of the number of BSF larval (BSFL) eggs collected for each mating pair.

[0035] DETAILED DESCRIPTION

[0036] Introduction

[0037] Black soldier flies (BSF or BSFs), Hermetia illucens, are insects in the order Diptera. BSFs are ubiquitous throughout much of the world extending between roughly the equator and 45th degree latitude (Newton et al., J. Anim Sci., 44:395-400, 1977; Bondari and Sheppard, Aquaculture and Fisheries Management, 18:209-220, 1987; Sheppard et al., Bioresource Technology, 50:275-279, 1994; Tomberlin et al., Ann. Entomol. Soc. Am., 95:379-386, 2002; St-Hilaire et al., J. World Aquaculture Society, 38:59-67, 2007; St-Hilaire et al., J. World Aquaculture Society, 38:309-313, 2007). They are holometabolous insects that undergo a life cycle of complete metamorphosis. The life cycle progresses from the egg to hatching of the larva, typically 5-6 larval instars or stages, pre-pupa, pupation into the pupal stage, then ecdysis (emergence) into the adult fly. During the pre-pupa stage, between the larva and pupal stages, the larva enters the wandering stage, moves away from or out of the nutrient source to find a pupation site on a dry surface.

[0038] BSFs have emerged as a vital species for sustainable waste management and as a source of protein for animal feed. Efficient breeding and synchronization of life cycles in BSFs are critical for large-scale production. However, challenges such as variability in reproductive cycles and the survival rates of larvae necessitate novel methods to enhanceAttorney Docket No.: 55877-0011P01

[0039] breeding outcomes. This disclosure addresses these challenges by providing methods to induce quiescence during the larval stage, synchronize breeding capabilities, and optimize the production of BSFEs.

[0040] Disclosed here are methods of propagating BSF to generate inbred (or pure) genetic lines of BSFs. This is achieved at least in two ways. First, Applicant has identified that propagation is optimized when BSF mates (a male and a female) are at the same developmental stage. To this end, provided herein are methods of inducing quiescence during the larval stage of BSF development so that each pair matures into a BSF on about the same day. Second, to generate a pure line, either with or without inducing quiescence, single mates can be isolated and generations from this single mate can be propagated (e.g., to Fl, F2, F3, F4, generations and beyond). These “pure” lines can be analyzed for optimal characteristics, thereby generating a pure line that, for example but not limited to, can produce more protein for animal food.

[0041] Additional detail on the disclosed methods are provided.

[0042] Method of Inducing Quiescence

[0043] In some instances, disclosed herein are methods for inducing quiescence during the larval stage of BSF development. The method of inducing quiescence during the larval life stage in a population of Black Soldier Flies (BSFs) involves two main steps. First, a population of BSFs is provided, ensuring that the flies are at the prepupal developmental stage, which is crucial for initiating quiescence. At this stage, the larvae are ready to enter a period of dormancy, which is vital for the synchronization of their life cycle. Second, the population is exposed to a controlled environment with specific conditions: a temperature range of 14°C to 17°C and humidity levels between 40% and 80%. This environmental setup is maintained for a period of at least 1 to 7 days, depending on the desired outcomes, to induce quiescence. These conditions help trigger the developmental arrest necessary for the flies to enter a dormant phase, ensuring that they will later be able to resume normal development once re-exposed to optimal conditions. This method allows a user to regulate the timing of reproduction and ensuring the consistent production of BSFs.

[0044] The BSFs used in these methods can be in any container that allows for quiescence to be induced,

[0045] Tire method for inducing quiescence in BSFs represents a highly effective approach to synchronizing their lifecycle and enhancing their reproductive potential. Quiescence is a physiological state of arrested development triggered by environmental cues such asAttorney Docket No.: 55877-0011P01

[0046] temperature, humidity, and photoperiod, allowing insects to conserve energy during unfavorable conditions. In the context of BSF farming, quiescence induction is strategically employed to regulate the timing of their development and optimize breeding processes. This method has been developed to ensure consistency, high yield, and better management of BSF populations in both research and large-scale production systems.

[0047] The process includes identifying BSFs that have reached the prepupal developmental stage. At this stage, the insects are physiologically prepared for quiescence, making them highly responsive to environmental manipulations. Prepupae can exhibit distinct morphological and behavioral characteristics, including a darkened body color, reduced feeding activity, and movement toward drier substrates in preparation for pupation. These observable traits make it easier to isolate individuals at the optimal stage for quiescence induction. In some instances, the selected prepupae are healthy and free from infections or deformities, as these factors can negatively influence quiescence outcomes. Additionally, in some instances, the BSF used in the methods described herein are uniform in size and developmental stage among the prepupal population.

[0048] Once the prepupal BSFs are selected, they are placed in a carefully controlled environment designed to mimic signals for quiescence induction. In some instances, the temperature is maintained within a range of about 14°C to about 17°C. These temperatures are sufficiently low to signal the onset of quiescence without causing undue stress or mortality. In some instances, the temperature settings include precise values such as 14°C, 15°C, 16°C, or 17°C, selected based on the desired intensity of quiescence and the length of the dormancy period required for the specific application. At a temperature that is too low, mortality increases to unacceptable levels (e.g., about 40%, 50%, 60%, 70% or greater mortality), whereas at a temperature that is too high, the induction of quiescence decreases to unacceptable levels (e.g., about 70%, 60%, 50%, or 40% or less quiescence).

[0049] In some instances, BSF larvae are held at specific humidity ranges. In some instances, relative humidity (RH) is adjusted to a range of 40% to 80%, ensuring that the prepupae remain hydrated without creating conditions that promote mold growth or microbial infections. In some instances, the humidity level is approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% RH. In some instances, the RH is about 50%.

[0050] In some instances, the duration of exposure to these conditions typically ranges from 1 to 7 days, depending on the desired quiescence depth and synchronization requirements. Shorter exposure periods (1-3 days) may induce a mild quiescence, suitable for minimal developmental delays, whereas longer durations (4-7 days) result in deeper quiescence,Attorney Docket No.: 55877-0011P01

[0051] effectively halting development for a more extended period. In some instances, the duration of exposure is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In some instances, the duration of exposure does not exceed 7 days, since longer than 7 days can result in increasing mortality rates (e.g., about 40%, 50%, 60%, or 70% mortality). During this phase, metabolic activity in the prepupae is significantly reduced, The insects conserve energy, minimize physiological processes, and remain in a dormant state, awaiting more favorable environmental conditions. This dormancy allows for achieving lifecycle synchronization, particularly in large populations where individual variability in development can disrupt breeding schedules.

[0052] After the quiescence induction period, the BSFs are moved to an environment optimized for active development and breeding. The temperature is increased to approximately 27°C, which increases BSF metabolic activity, pupation, and adult emergence and promotes rapid and uniform development across the population. In some instances, the relative humidity is adjusted to around 55% RH, a level that prevents dehydration while ensuring optimal conditions for the emergence of adults. This humidity level also supports egg production and larval development once the adults begin breeding. By providing these post-quiescence conditions, the previously dormant BSFs resume synchronized development, resulting in a uniform adult population ready for breeding. Uniformity is essential for efficient production, as it simplifies resource allocation and management in large-scale farming operations.

[0053] In some instances, BSFs do not undergo quiescence induction prior to mating.

[0054] However, when they do, a critical component of this method is ensuring that BSFs do not mate prior to the quiescence induction process. By preventing mating, the insects’ reproductive systems remain in an unutilized state, preserving their full reproductive potential. Once quiescence ends, the adults are primed for peak reproductive performance, resulting in higher egg production. Mated BSFs emerging from quiescence demonstrate increased egg-laying consistency and yield, which is particularly important for industries relying on BSF larvae for waste management, feed production, or other applications.

[0055] Furthermore, synchronizing the reproductive cycles of the entire population reduces variability and increases the efficiency of breeding programs.

[0056] This method of inducing quiescence in BSFs provides several key benefits for farming and research applications. Induced quiescence ensures that all individuals within a population develop at the same rate, simplifying management and enabling precise production planning. The preservation of reproductive potential leads to higher-quality eggAttorney Docket No.: 55877-0011P01

[0057] production and improved breeding efficiency. Synchronization allows for more efficient use of resources such as food, space, and environmental controls, reducing operational costs. Quiescence-prepared BSFs are also more robust and better equipped to handle fluctuations in environmental conditions, improving survival rates during transport or storage. Furthermore, this method is easily scalable, making it suitable for large-scale BSF farming operations aimed at sustainable waste management, animal feed production, or other industrial applications.

[0058] The carefully controlled induction of quiescence in black soldier flies represents a powerful tool for optimizing lifecycle management and enhancing productivity in insect farming. By leveraging environmental cues to regulate development, farmers and researchers can achieve consistent and reliable outcomes, ensuring the success of BSF-based systems. This method not only improves breeding efficiency but also contributes to the broader goal of sustainable agriculture and waste reduction through the use of BSFs. In addition, in some instances, inducing quiescence does not substantively change the number of propagated black soldier flies from the second generation compared to a pair of black soldier flies in which quiescence is not induced. In some instances, inducing quiescence does not substantively change the number of BSFEs produced compared to a pair of black soldier flies in which quiescence is not induced.

[0059] Method of Generating Black Soldier Fly Eggs using 1:1 Mating

[0060] In some instances, BSFs do not undergo quiescence induction prior to mating.

[0061] However, in other cases, they do. Disclosed herein are methods of generating black solder fly eggs (BSFEs). To generate BSFEs, a precise and controlled method is employed, utilizing a population of BSFs that have undergone quiescence, ensuring their reproductive systems are primed for egg-laying.

[0062] In some instances, the methods include (a) transferring a pair of black soldier flies from the population of black soldier flies to a chamber comprising an egg collection apparatus for about three to seven days, wherein the pair is one male black soldier fly and one female black soldier fly; and (b) adding an oviposition attractant to the chamber, thereby producing BSFEs. In some instances, the methods include (a) inducing quiescence; (b) transferring a pair of black soldier flies from the population of black soldier flies to a chamber comprising an egg collection apparatus for about three to seven days, wherein the pair is one male black soldier fly and one female black soldier fly; and (c) adding an oviposition attractant to the chamber, thereby producing BSFEs.Attorney Docket No.: 55877-0011P01

[0063] The BSFs used in these methods can be in any container that allows for 1: 1 mating. In some instances, the process includes selecting a single mating pair, one male and one female BSF. In some instances, both mates have completed a quiescence stage. In some instances, neither mate has completed a quiescence stage. In some instances, only one mate (either the male mate or the female mate) has completed a quiescence stage.

[0064] In some instances, each individual in the population of black soldier flies have not mated with another black soldier fly previously.

[0065] In some instances, the population of black soldier flies are maintained in a container. In some instances, the mating pair is carefully transferred to a specialized egg collection chamber — a purpose-built environment designed to support and encourage egg¬ laying. Tlie chamber is equipped with an egg collection apparatus, which is a crucial feature for efficiently gathering the eggs once they are laid, ensuring minimal loss and contamination.

[0066] The egg-laying process also includes the addi tion of an oviposition attractant — a mixture of substances designed to draw the female BSF to the chamber and stimulate her natural egg-laying behaviors. This attractant contains a combination of organic materials that are highly appealing to the BSFs. For example, the oviposition attractant can include distillers dried grains; bakery by-product meal; calcium carbonate; calcium chloride; BSFLs; and / or EM1 microbial inoculant. Distillers dried grains (DDG) are included as a major component of this mixture, DDG is a by-product of the alcohol fermentation process and is rich in proteins, fiber, and other nutrients, making it an ideal food source for the BSFs, especially in the egg¬ laying phase. In some instances, the mixture includes bakery by-product meal, providing additional carbohydrates and fats, which support reproductive health and energy. In some instances, the mixture includes calcium carbonate and / or calcium chloride. BSFLs can be added to the mixture as a natural food source, which also stimulates the flies to lay eggs. Lastly, an EMI microbial inoculant is incorporated to maintain a healthy microbial environment within the chamber, promoting the breakdown of organic matter and preventing potential issues with mold or pathogens that could hinder the egg-laying process. This carefully balanced combination of ingredients ensures that the BSFs are provided with all the necessary nutrients and stimuli to encourage optimal egg-laying.

[0067] In some instances, about 10 grams of the attractant is added to the egg collection chamber. This quantity has been optimized to provide sufficient appeal for the female BSFs, encouraging them to lay their eggs within the designated collection area. The precise amount ensures that the chamber is not overwhelmed with attractant, which could lead to waste or anAttorney Docket No.: 55877-0011P01

[0068] imbalanced environment. The 10-gram amount is carefully balanced to provide enough nutrients and stimuli for effective egg-laying while maintaining optimal conditions for the mating pair. This quantity is suitable for small-scale operations or as a baseline for adjusting the attractant quantity in larger-scale BSF farming environments. By using a measured amount, the method also ensures the sustainability' and cost-efficiency of the attractant, which is vital for both commercial and research purposes.

[0069] In some instances, an oviposition attractant is used during 1: 1 mating. The attractant can include a mixture of several key ingredients, each serving a specific purpose in the reproductive process. Distillers dried grains (DDG) are included in this mixture due to their high nutritional content, which supports the overall health and energy requirements of the BSFs during the egg-laying phase. The mixture can include bakery by-product meal, calcium carbonate, calcium chloride, BSFLs and / or an EM-1 microbial inoculant. A representative oviposition attractant includes the following: distiller’s dried grains are measured at about 35 to about 40 grams (e.g., 38.2 grams). Bakery by-product meal is added at about 35 to about 40 grams (e.g., 38.15 grams), complementing the DDG with additional carbohydrates and fats. In some instances, amounts of calcium carbonate (e.g., about 0.5 to about 1.0 grams; e.g., 0.79 grams) and calcium chloride (e.g., about 0.4 to about 0.9 grams; e.g., 0.66 grams) are included to ensure the proper mineral balance needed for healthy egg formation and optimal metabolic functioning during the egg-laying process, lire addition of about 2.5 to about 7.5 grams (e.g., 5.44 grams) of BSFLs, or approximately 1 mg per BSFLI, provides a natural, highly digestible food source for the BSFs, which encourages them to lay eggs by mimicking their natural environment. The inclusion of about 50 to 100 ml (e.g., 75 ml) of EM-1 microbial inoculant (EMRO USA Effective Microorganisms, Tucson, AZ) ensures a balanced microbial environment that aids in decomposing organic material and maintaining the health of both the adult flies and the eggs. Finally, about 15 to about 35 ml (e.g., 22.3 ml) of water is added to the attractant mixture to ensure the proper hy dration of the ingredients and to create a consistency that facilitates effective egg-laying.

[0070] In addition to optimizing the egg-laying process, feed formulations are tailored to support the growth and development of BSFL during both their early and late instar stages. For the early instar larvae, the feed is specifically designed to provide the necessary nutrients for rapid development and to support the larvae’s growth in the initial stages of their life cycle. In some instances, early instar feed includes ground com; soybean meal; calcium carbonate; dicalcium phosphate; soybean oil; sodium chloride; and / or calcium propionate. A representative early instar feed formulation includes 64.7% ground corn, which serves as theAttorney Docket No.: 55877-0011P01

[0071] primary energy source, providing the carbohydrates necessary for early growth. 23.5% soybean meal is added to supply high-quality proteins, which are essential for the development of tire larvae's body mass and muscle growth. 8.5% calcium carbonate is included to ensure the larvae have adequate calcium for development and to help maintain metabolic functions. 1.5% dicalcium phosphate is included as a phosphate source, vital for cellular energy processes. The feed also contains 1.0% soybean oil, which supplies the necessary' fats for energy storage, and 0.50% sodium chloride, which supports the larvae's osmotic balance and fluid regulation. Finally, 0.33% calcium propionate is added as a preservative to prevent mold growth and ensure feed quality. This early instar feed formulation promotes strong, healthy larvae, ensuring that they are well-prepared for their transition to the late instar stage.

[0072] For the late instar lan ae, the feed formulation shifts to support the larvae's increased size and energy demands as they prepare for the transition to the prepupal stage. In some instances, late instar feed comprises a formulation comprising distillers dried grains; bakery' by-product meal; calcium carbonate; and calcium chloride. In some instances, tire late instar feed includes primarily of 49.1% distillers dried grains (DDG), which provides a high level of protein and fiber, critical for sustained growth. 49,0% bakery by-product meal is used to provide carbohydrates and fats, which help maintain energy levels and support fat storage in the larvae. 1.0% calcium carbonate is again included to ensure proper calcium levels for shell development as the larvae approach pupation, and 0.85% calcium chloride ensures that the larvae can maintain optimal ion balance as they prepare to enter quiescence. This late instar feed formulation ensures that the BSFLs are fully nourished, promoting optimal weight gain and health as they prepare for their next life stage.

[0073] The methods for propagating BSFs are designed to ensure efficient reproduction, with the ability to produce 2 to 5 ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore) inbred / pure generations from a single mating pair. In some instances, one or more of the following metrics can be measured: the number of BSFEs produced, early instar BSFL survival rate, BSFL weight, late instar BSFL weight, and the number of BSF pre-pupae (BSFPP), BSFPP survival rate, and BSFPP weight. Metrics such as egg production, survival rates, and larval weights are monitored to maintain consistency and quality throughout the process. In some instances, the BSFEs mature into black soldier fly larvae with an average survival rate of at least about 70% (e.g., about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%). In some instances, about 200 to about 1000 (about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000)Attorney Docket No.: 55877-0011P01

[0074] BSFEs are produced. In some instances, more than 1000 BSFEs are produced. By tracking pre-pupal weights, the development of larvae can be assessed, ensuring they are healthy and ready for the next stage. The ability to propagate multiple generations from one pair allows for scalable and cost-effective BSF farming, reducing the need for external stock and ensuring sustainability.

[0075] BSFL have a wide range of applications. In some instances, BSFL produced by the BSFEs from the one mating pair are used as an ingredient in animal feed, plant products, and / or soil products. In some instances, one of its uses is in animal feed, where BSFL provide a sustainable alternative to traditional feed sources like soy and fishmeal. The larvae are rich in proteins and fats, making them a highly nutritious and environmentally friendly option. The increasing demand for alternative protein sources is further driven by the environmental benefits of BSFL farming, such as reducing deforestation and overfishing. This application not only provides nutritional benefits for animals but also addresses the global protein gap, offering a sustainable solution to the growing demand for animal-based protein.

[0076] In addition to their use in animal feed, BSFL are valuable in plant and soil applications. The larvae are excellent for composting organic waste and enriching soil with nutrients. Their frass (larvae excrement) acts as a natural, nutrient-rich fertilizer, improving soil fertility and promoting plant growth. This aligns with sustainable agricultural practices, reducing the need for chemical fertilizers and supporting soil health. BSFL also contribute to a circular economy, turning waste into valuable resources for agricultural production, reducing waste while boosting productivity.

[0077] BSFL also play a role in waste management, consuming organic waste such as food scraps and agricultural by-products. This conversion of organic material into high-value products is a key solution for addressing global waste management challenges. The larvae help reduce the volume of waste, turning it into biomass that can be used as animal feed or fertilizer. This process of waste-to-resource conversion highlights the environmental benefits of BSF farming, offering a sustainable way to address food waste and environmental sustainability.

[0078] Furthermore, BSFL show potential as a feedstock for biofuels, especially in biodiesel production due to their high lipid content. This presents an alternative energy source, reducing reliance on fossil fuels. The role of BSFL in biofuels underscores the broader environmental benefits of BSF farming, contributing to a more sustainable energy future and supporting the bioeconomy.Attorney Docket No.: 55877-0011P01

[0079] The environmental benefits of BSF farming are further emphasized through its role in sustainable waste management. By using organic waste as feed for BSFL, farms can reduce waste, cut down on landfill use, and lower the carbon footprint associated with feed production. This contributes to the broader goal of creating a more sustainable agricultural system while minimizing tire impact of waste on the environment.

[0080] BSF farming also offers opportunities to integrate with other sustainable farming techniques, such as integrated pest management (IPM) and crop rotation. The larvae provide protein-rich feed that supports regenerative farming practices. By incorporating BSF production into these farming systems, farmers can diversify income, improve soil health, and contribute to more resilient food systems.

[0081] Embodiments

[0082] Embodiment 1 is a method of inducing quiescence during larval life stage in a population of black soldier flies, the me thod comprising: (a) providing the population of black soldier flies, wherein the black soldier flies of the population are at a prepupal developmental stage; and (b) subjecting the population of black soldier flies to an environment of about 14°C to 17°C at about 40% to about 80% humidity for at least 1 to 7 days.

[0083] Embodiment 2 is the method of Embodiment 1, wherein the environment is at about 14°C, 15°C, 16°C, or 17°C.

[0084] Embodiment 3 is the method of Embodiment 1 or 2, wherein the humidity is at about 50%.

[0085] Embodiment 4 is the method of any one of the preceding Embodiments, wherein the black soldier flies are in the environment of about 14°C to 17°C at about 40% to about 60% humidity for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0086] Embodiment 5 is the method of any one of the preceding Embodiments, w'herein, after the at least 1 to 7 days, the black soldier flies are maintained at about 27°C and about 55% humidity.

[0087] Embodiment 6 is the method of any one of the preceding Embodiments, wherein, after inducing quiescence, individuals in the population of black soldier flies have life cycles and breeding capability’ that are synchronized.

[0088] Embodiment 7 is the method of any one of the preceding Embodiments, w'herein each individual in the population of black soldier flies has not mated with another black soldier fly previously.Attorney Docket No.: 55877-0011P01

[0089] Embodiment 8 is the method of any one of the preceding Embodiments, wherein the population of black soldier flies are maintained in a container.

[0090] Embodiment 9 is a method of generating black soldier fly eggs (BSFEs) from one mating pair, the method comprising: (a) transferring a pair of black soldier flies from the population of black soldier flies to a chamber comprising an egg collection apparatus for about three to seven days, wherein the pair is one male black soldier fly and one female black soldier fly; and (b) adding an oviposition atractant to the chamber, thereby producing BSFEs.

[0091] Embodiment 10 is a method of generating black soldier fly eggs (BSFEs) from one mating pain the method comprising: (a) inducing quiescence according to the method of any one of Embodimen ts 1-8; (b) transferring a pair of black soldier flies from the population of black soldier flies to a chamber comprising an egg collection apparatus for about three to seven days, wherein the pair is one male black soldier fly and one female black soldier fly; and (c) adding an oviposition attractant to the chamber, thereby producing BSFEs.

[0092] Embodiment 11 is the method of Embodiment 9 or 10, wherein about 200 to about 1000 BSFEs are produced.

[0093] Embodiment 12 is the method of any one of Embodiments 9-11, wherein the BSFEs mature into black soldier fly larvae with an average survival rate of at least about 70%.

[0094] Embodiment 13 is the method of any one of Embodiments 9-12, wherein the pair is transferred in (b) to the chamber for about five days.

[0095] Embodiment 14 is the method of any one of Embodiments 9-13, wherein the oviposition attractant is added to the egg collection apparatus.

[0096] Embodiment 15 is the method of Embodiment 14, wherein about 10 grams of the oviposition attractant is added to the chamber in (c).

[0097] Embodiment 16 is the method of any one of Embodiments 9-15, wherein the oviposition attractant comprises distillers dried grains; bakery by-product meal; calcium carbonate; calcium chloride; BSFLs; and EM1 microbial inoculant.

[0098] Embodiment 17 is the method of any one of Embodiments 9-16, wherein the oviposition attractant comprises: about 38 grams of distillers dried grains; about 38 grams of bakery by-product meal; about 0.8 grams of calcium carbonate; about 0.7 grams of calcium chloride; about 5.5 grams of about 1 mg BSFL; and about 75 ml of EM1 microbial inoculant.

[0099] Embodiment 18 is the method of any one of Embodiments 9-17, further comprising collecting the BSFEs from the egg collection apparatus and providing the BSFEs with an early instar feed formulation comprising ground corn; soybean meal; calcium carbonate; dicalcium phosphate; soybean oil; sodium chloride; and calcium propionate.Attorney Docket No.: 55877-0011P01

[0100] Embodiment 19 is the method of Embodiment 18, wherein the early instar feed formulation comprises about 65.0% ground corn; about 24.0% soybean meal; about 8.5% calcium carbonate; about 1.5% dicalcium phosphate; about 1.0% soybean oil; about 0.50% sodium chloride; and about 0.33% calcium propionate.

[0101] Embodiment 20 is the method of any one of Embodiments 9-19, further comprising providing late instar feed to the BSFEs.

[0102] Embodiment 21 is the method of Embodiment 20, wherein the late instar feed formulation comprises distillers dried grains; bakery by-product meal; calcium carbonate; and calcium chloride.

[0103] Embodiment 22 is the method of Embodiment 20 or 21, wherein the late instar feed formulation comprises about 49.0% distillers dried grains; about 49.0% bakery by-product meal; about 1.0% calcium carbonate; and about 0.85% calcium chloride.

[0104] Embodiment 23 is the method of any one of Embodiments 9-22, wherein inducing quiescence does not substantively change the number of BSFEs produced compared to a pair of black soldier flies in which quiescence is not induced.

[0105] Embodiment 24 is the method of any one of Embodiments 9-23, further comprising measuring one or more of the following metrics: the number of BSFEs produced, early instar BSFL survival rate, BSFL weight, late instar BSFL weight, and the number of black soldier fly pre-pupae (BSFPP), BSFPP survival rate, and BSFPP weight.

[0106] Embodiment 25 is the method of any one of Embodiments 9-24, further comprising propagating a second generation of black soldier flies from the BSFEs.

[0107] Embodiment 26 is the method of any one of Embodiments 9-21, further comprising propagating at least 2, at least 3, at least 4, at least 5 or more generations of populations of black soldier flies from the one mating pair of black soldier flies.

[0108] Embodiment 27 is the method of any one of the preceding Embodiments, wherein inducing quiescence does not substantively change the number of propagated black soldier flies from the second generation compared to a pair of black soldier flies in which quiescence is not induced.

[0109] Embodiment 28 is the method of any one of Embodiments 9-27, wherein black solder fly larvae produced by the BSFEs from the one mating pair are used as an ingredient in animal feed, plant products, and / or soil products.

[0110] Embodiment 29 is a method of generating black soldier fly eggs (BSFEs) from one mating pair, the method comprising: (a) transferring a pair of black soldier flies from the population of black soldier flies to a chamber comprising an egg collection apparatus forAttorney Docket No.: 55877-0011P01

[0111] about three to seven days, wherein the pair is one male black soldier fly and one female black soldier fly; and (b) adding an oviposition attractant to the chamber, thereby producing BSFEs.

[0112] Embodiment 30 is an oviposition attractant composition comprising: distillers dried grains; bakery by-product meal; calcium carbonate; calcium chloride; BSFL; and EMI microbial inoculant.

[0113] Embodiment 31 is the oviposition attractant composition of Embodiment 30, wherein: the distillers dried grains are present in an amount of from about 20 grams to about 60 grams; the bakery by-product meal is present in an amount of from about 20 grams to about 60 grams; the calcium carbonate is present in an amount of from about 0.1 grams to about 2.5 grams; the calcium chloride is present in an amount of from about 0.1 grams to about 2.5 grams; the BSFL is present in an amount of from about 1 gram to about 10 grams; and the EM1 microbial inoculant is present in an amount of from about 50 ml to about 100 ml.

[0114] Embodiment 32 is the oviposition attractant composition of Embodiment 31, wherein: the distillers dried grains are present in an amount of about 38 grams; the bakery' by-product meal is present in an amount of about 38 grams; the calcium carbonate is present in an amount of about 0.8 grams; the calcium chloride is present in an amount of about 0.7 grams; the about 1 mg BSFL is present in an amount of about 5.5 grams; and the EM1 microbial inoculant is present in an amount of about 75 ml.

[0115] Embodiment 33 is an oviposition attractant composition comprising: about 38 grams of distillers dried grains; about 38 grams of bakery by-product meal; about 0.8 grams of calcium carbonate; about 0.7 grams of calcium chloride; about 5.5 grams of about 1 mg BSFL; and about 75 ml of EM1 microbial inoculant.

[0116] EXAMPLES

[0117] Black soldier fly populations were obtained from populations maintained year-round in the Genetics R& D laboratory, EnviroFlight, Apex, North Carolina. All BSF were housed in containers proven suitable for their harborage and feeding. The populations have been maintained at 27°C and 55% relative humidity.

[0118] Example 1 — Method for 1x1 Mating Pair Establishment (F0 Generation)

[0119] To begin the rearing process, cages (appropriate for 1,000 - 100,000 flies) containing approximately equal number of virgin (non-mated) BSF males of selected population ‘“A” and virgin BSF females of selected population ‘" B” w ere established to control for the targeted mating. These may also be termed the F0 individuals as they are producing the BSFAttorney Docket No.: 55877-0011P01

[0120] family. Individual mating containers (each being a 16-ounce clear square jar with 89 mm neck, 9.14 cm x 9.14 cm x 7.62 cm) were prepared to receive one male and one female, the mating pair (MP). Each MP was carefully collected from the source cage to prevent separation during transfer. Using gentle techniques, the mating pair was coaxed or scooped into the prepared container, ensuring that no other flies were included. Once tire pair was successfully transferred, the container was carefully removed from the cage, maintaining close observation to ensure the pair remained together. After all the MPs were collected, the containers were moved to the environmental chamber rearing area. An egg collection apparatus (ECA) containing 10 g of a novel oviposition attractant w as placed inside each container 24 hours after set-up or once mating separation had occurred. The novel oviposition attractant recipe is provided in Table 1. The individual mating containers were checked daily for the presence of black soldier fly eggs (BSFE).

[0121] Table 1. Oviposition attractant formulation

[0122] Distillers dried grains 38.20 g

[0123] Bakery by-product meal 38.15 g

[0124] Calcium carbonate 0.79 g

[0125] Calcium chloride 0.66 g

[0126] ~ 1 mg BSFL (2nd instar) 5.44 g

[0127] EMI microbial inoculant175 ml

[0128]

[0129] Water 22.3 ml

[0130] 1From TeraGanix (USA)

[0131] 2 — Method for Collecting Eggs and

[0132]

[0133] Larvae from FO Generation

[0134]

[0135] in BSF for Breeding in Fl Generation

[0136] When BSFEs were oviposited on the ECA, the ECA was carefully removed from tire container. Eggs were scraped directly into a 2-ounce (oz.) plastic container with lid con taining early instar feed to minimize handling, avoiding intermediate transfer to weigh boats. On the day of egg collection, typical production was approximately 0.015 g BSFE, and all BSFE produced by each female were launched into 8 g (±0.4 g) of prepared early instar feed in the con tainer. Based on visual observation of BSFE on the ECA, the amount of feed required for each container was determined with 5% accuracy. Feed-to-egg guidelines were as follows: 0.02 g (±0.001 g) BSFE required 8 g (±0.4 g) early instar feed, while 0.1 g (±0.005 g) BSFE required 15 g (±0.75 g) early instar feed (Table 2), The containers were incubated until the BSFL reached 2 mg weight.Attorney Docket No.: 55877-0011P01

[0137] Larvae from the containers were transferred into 32 oz. plastic container with ventilated lid containing 240 g of prepared early instar feed. U sing a careful transfer method, the 2 oz. container contents were gently laid on top of the prepared feed in the 32 oz. container, allowing larvae to migrate into the feed over the course of a few hours in an isolated area. Ihe 32 oz. container with feed and BSFL was incubated until the BSFL reached 130-150 mg weight.

[0138] Table 2. Early instar feed formulation

[0139] Ingredient

[0140] Ground com 64.7

[0141] Soybean meal 23.5

[0142] Calcium carbonate 8.5

[0143] Dicalcium phosphate 1.5

[0144] Soybean oil 1.0

[0145] Salt 0.50

[0146]

[0147] Calcium propionate 0.33

[0148] Example 3 — Assessment of Larval Development

[0149] Once larvae reach 130-150 mg, larvae in the 32 oz. container were assessed. Total counts, total weight, and 10-count weights of the BSFL were recorded. Larvae were then launched into a 32 oz, container, containing 100 g of late instar feed (Table 3) and 60 g water. Daily watering of the container followed, with 30 g water applied each day until the larva reach 150 mg. At 150 mg a 10-count weight of BSFL, black soldier fly pre-pupae (BSFPP), and black soldier fly pupae (BSFP) was recorded.

[0150] Once tire larvae are 170-200 mg, the con tainers were further assessed by sieving to remove frass. Ihe total weight of BSFs, including all life stages, was recorded, along with 10-count weights for BSFL, BSFPP, and BSFP separately. Data collected at this stage were used to estimate total counts, individual weights, and survival rates for the various growth periods. Selected containers were loaded into breeding cages (appropriate for 100 - 5,000 individuals) for continued rearing.Attorney Docket No.: 55877-0011P01

[0151] Table 3. Late instar feed formulation

[0152] Ingredients %

[0153] Distillers dried grains 49.1

[0154] Bakery' by-product meal 49.0

[0155] Calcium carbonate 1.0

[0156]

[0157] Calcium chloride 0.85

[0158] Example 4 — Method for Breeding Fl Generation

[0159] To initiate the first generation (Fl), an estimated 500 BSFs were homogenized based on a 100-count weight and loaded into breeding cages. In cases where fewer than 500 BSFs were produced, all available BSFs were loaded into cage. Daily monitoring of brood emergence continued until 20% emergence was achieved, corresponding to 100 BSFs in the case of a 500-BSF brood load. At this point, an ECA containing attractant was placed in the SBC to collect BSFE. The first collection or occurred two days after the ECA was introduced (ECA Launch Date +2), with egg production during this period ranging from 0.0-0.3 g.

[0160] BSFE collection continued over multiple scrapes, targeting the production of at least 10 egg containers (i.e. 2 oz. containers with early instar feed) from each family. Husbandry protocols for egg density, feed ratio and rearing conditions followed F0 generation description.

[0161] Example 5 — Phase 2 and Subsequent Generations

[0162] On ED 12, larvae in DCUP-NF wrere assessed to determine whether they met performance targets. Larvae from DC UP high-performing target scores were launched into shoeboxes (SBs) with 3200 BSFL doses. Daily watering and incubation in SB-PF continued through ED 20, followed by a series of 50-count weight assessments at ED 15, 18, and 28 to monitor developmental progress. On ED 30, a homogenized sample of 1200 BSFs was loaded into SBCs, initiating the next generation (F3).

[0163] Example 6 — Method for Induced Quiescence

[0164] Induced quiescence allows for synchronization ofBSF emergence such that targeted breeding populations can be established from two or more BSF populations or individuals. The method for this occurs at the prepupal (BSFPP) life stage. To induce quiescence in prepupae, a population of BSFPP were homogenized and placed in a controlled environment 14-17 °C, at ambient humidity conditions (generally ~ 50%) for up to seven days. BSFPP can beAttorney Docket No.: 55877-0011P01

[0165] maintained in this induced quiescence state for longer than 7 days but with negative impact to the survival and performance of the population. Following induction of quiescence, the BSFPP was transferred to standard rearing environments appropriate for the density of anticipated fly populations. BSF emergence typically occurred within five days of transfer under normal rearing conditions.

[0166] Example 7 — Establishing BSF Families Via Mating Pairs

[0167] The present example demonstrates the methods used for establishing the BSF families via a one by one cross of male and female breeding were successful. The oviposition attractant formulated to be placed in the ECA increased the likelihood of target oviposition and BSFE collection from the FO pair when bias towards BSFE reabsorption and oviposition avoidance is most problematic. BSFE yields using oviposition attractant created a 65% oviposition rate in testing trials with FO BSFE production averaging 0.01439 g (estimated 540 BSFE, using conservative estimate 1 g = 37500 BSFE) (FIG. 1).

[0168] After mating, BSFEs were produced, and performance metrics (Table 4) were recorded during the rearing of the Fl population. Performance metrics measured included: the number of BSFE produced, early instar BSFL survival rate and weight, late instar BSFL weight, and the number of BSFPP and corresponding survival rate and weight. The F1 population produced sufficient individuals to establish a cage for producing the next generation of the family.

[0169] Table 4. number of BSFL, average survival rate, and average BSFL weight

[0170] at different rearing stages

[0171] Rearing stage Average BSFL # Average Survival Rate Average BSFL Weight Egg 540

[0172] Early Instar 422 91 % from egg 138 mg

[0173] Late Instar 183 mg

[0174]

[0175] Prepupae 400 94% from early instar 141 mg

[0176] Example 8 — Delayed Emergence in BSF Families with Induced Quiescence

[0177] The present example demonstrates the methods used for delaying the emergence of BSF via induced quiescence did not impact the emergence (Table 5) and BSFE production (Table 6) of the treated populations. Populations that underwent the induced quiescence treatment began producing eggs 7-10 days after non-treated populations. Additionally, the BSFE produced by the treated and non-treated populations were similar in overall yield.Attorney Docket No.: 55877-0011P01

[0178] Table 5. Number of Emerged BSF in Six Different BSF Populations; Quiescence was Induced in Three Populations (Populations 1-3)

[0179] BSFE day 1 BSFE day 2 BSFE day 3 BSFE day 4 BSFE Total Treatment

[0180] (g) (g) (g) (g) (g) Induced

[0181] 0.5069 0.8239 0.0000 0.0000 1.3308 Quiescence

[0182] Induced

[0183] 0.4151 0.3615 0.0000 0.0000 0.7776 Quiescence

[0184] Induced

[0185] 0.2347 0.4176 0.7163 0.0000 1.3686 Quiescence

[0186] Non-induced 0.2130 0.5567 0.5005 0.0000 1.2702 Non-induced 0.0736 0.6509 0.5001 0.2210 1.4456

[0187]

[0188] Non-induced 0.0000 0.3399 0.5705 0.4237 1.3341

[0189] Table 6. BSFE Production in Six Populations of BSF; Quiescence was Induced in Three Populations (Populations 1-3)

[0190] BSFE BSFE BSFE Total BSF BSFE g

[0191] Treatment CageID BSF ED BSFE g Day 1 BSFE g Day 2 BSFE g Day 3 BSFE g Day 4 Total BSFE (g) Induced Population

[0192] 46 0.5069 0.8239 1.3308 Quiescence 1

[0193] Induced Population

[0194] 44 0.4161 0.3615 0.7776 Quiescence 2

[0195] Induced Population

[0196] 43 0.2347 0.4176 0.7163 1.3686 Quiescence 3

[0197] Population

[0198] Non-induced 36 0.2130 0.5567 0.5005 1.2702

[0199] 4

[0200] Population

[0201] Non-induced 36 0.0736 0.6509 0.5001 0.2210 1.4456

[0202] 5

[0203] Population

[0204] Non-induced 36 0.0000 0.3399 0.5705 0.4237 1.3341

[0205]

[0206] 6

Claims

Attorney Docket No.: 55877-0011WO1WHAT IS CLAIMED IS:

1. A method of inducing quiescence during larval life stage in a population of black soldier flies, the method comprising:(a) providing the population of black soldier flies, wherein the black soldier flies of the population are at a prepupal developmental stage; and(b) subjecting the population of black soldier flies to an environment of about 14°C to 17°C at about 40% to about 80% humidity for at least 1 to 7 days.

2. The method of claim 1, wherein the environment is at about 14°C, 15°C, 16°C, or 17°C.

3. The method of claim 1, wherein the humidity is at about 50%.

4. The method of claim 1, wherein the black soldier flies are in the environment of about 14°C to 17°C at about 40% to about 60% humidity for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

5. The method of claim 1, wherein, after the at least 1 to 7 days, the black soldier flies are maintained at about 27°C and about 55% humidity,6. The method of claim 1, wherein, after inducing quiescence, individuals in the population of black soldier flies have life cycles and breeding capability that are synchronized.

7. The method of claim 1, wherein each individual in the population of black soldier flies has not mated with another black soldier fly previously.

8. The method of claim 1, wherein the population of black soldier flies are maintained in a container.

9. A method of generating black soldier fly eggs (BSFEs) from one mating pair, the method comprising:Attorney Docket No.: 55877-0011P01(a) transferring a pair of black soldier flies from the population of black soldier flies to a chamber comprising an egg collection apparatus for about three to seven days, wherein the pair is one male black soldier fly and one female black soldier fly; and (b) adding an oviposition attractant to the chamber,thereby producing BSFEs.

10. A method of generating black soldier fly eggs (BSFEs) from one mating pair, the method comprising:(a) inducing quiescence according to the method of claim 1;(b) transferring a pair of black soldier flies from the population of black soldier flies to a chamber comprising an egg collection apparatus for about three to seven days, wherein the pair is one male black soldier fly and one female black soldier fly; and (c) adding an oviposition attractant to the chamber,thereby producing BSFEs.

11. The method of claim 9 or 10, wherein about 200 to about 1000 BSFEs are produced.

12. The method of claim 9 or 10, wherein the BSFEs mature into black soldier fly larvae with an average survival rate of at least about 70%,13. The method of claim 9 or 10, wherein the pair is transferred to the chamber for about five days.

14. The method of claim 9 or 10, wherein the oviposition attractant is added to the egg collection apparatus.

15. The method of claim 14, wherein about 10 grams of the oviposition attractant is added to the chamber.

16. The method of claim 9 or 10, wherein the oviposition attractant comprises distillers dried grains; bakery by-product meal; calcium carbonate; calcium chloride; BSFLs; and EM1 microbial inoculant.Attorney Docket No.: 55877-0011P0117. The method of claim 9 or 10, wherein the oviposition attractant comprises:(a) about 38 grams of distillers dried grains;(b) about 38 grams of bakery by-product meal;(c) about 0.8 grams of calcium carbonate;(d) about 0.7 grams of calcium chloride;(e) about 5,5 grams of about 1 mg BSFL; and(f) about 75 ml of EM1 microbial inoculant.

18. The method of claim 9 or 10, further comprising collecting the BSFEs from the egg collection apparatus and providing the BSFEs with an early instar feed formulation comprising ground corn; soybean meal; calcium carbonate; dicalcium phosphate; soybean oil; sodium chloride; and calcium propionate.

19. The method of claim 18, wherein the early instar feed formulation comprises about 65.0% ground corn; about 24.0% soybean meal; about 8.5% calcium carbonate; about 1.5% dicalcium phosphate; about 1.0% soybean oil; about 0.50% sodium chloride; and about 0.33% calcium propionate.

20. The method of claim 9 or 10, further comprising providing late instar feed to the BSFEs.

21. The method of claim 20, wherein the late instar feed formulation comprises distillers dried grains; bakery by-product meal; calcium carbonate; and calcium chloride.

22. The method of claim 21, wherein the late instar feed formulation comprises about 49.0% distillers dried grains; about 49.0% bakery by-product meal; about 1.0% calcium carbonate; and about 0.85% calcium chloride.

23. The method of claim 9 or 10, wherein inducing quiescence does not substantively change the number of BSFEs produced compared to a pair of black soldier flies in which quiescence is not induced,24. The method of claim 9 or 10, further comprising measuring one or more of the following metrics: the number of BSFEs produced, early instar BSFL survival rate, BSFLAttorney Docket No.: 55877-0011P01weight, late instar BSFL weight, and the number of black soldier fly pre-pupae (BSFPP), BSFPP survival rate, and BSFPP weight.

25. The method of claim 9 or 10, further comprising propagating a second generation of black soldier flies from the BSFEs.

26. The method of claim 9 or 10, further comprising propagating at least 2, at least 3, at least 4, at least 5 or more generations of populations of black soldier flies from the one mating pair of black soldier flies.

27. The method of claim 9 or 10, wherein inducing quiescence does not substantively change the number of propagated black soldier flies from the second generation compared to a pair of black soldier flies in which quiescence is not induced.

28. The method of claim 9 or 10, wherein black solder fly larvae produced by the BSFEs from the one mating pair are used as an ingredient in animal feed, plant products, and / or soil products.

29. A method of generating black soldier fly eggs (BSFEs) from one mating pair, the method comprising:(a) transferring a pair of black soldier flies from the population of black soldier flies to a chamber comprising an egg collection apparatus for about three to seven days, wherein the pair is one male black soldier fly and one female black soldier fly; and (b) adding an oviposition attractant to the chamber,thereby producing BSFEs.

30. An oviposition attractant composition comprising:(a) distillers dried grains;(b) bakery by-product meal;(c) calcium carbonate;(d) calcium chloride;(e) BSFL; and(f) EMI microbial inoculant.Attorney Docket No.: 55877-0011P0131. The oviposition attractant composition of claim 30, wherein:(a) the distillers dried grains are present in an amount of from about 20 grams to about 60 grams;(b) the bakery by-product meal is present in an amount of from about 20 grams to about 60 grams;(c) the calcium carbonate is present in an amount of from about 0.1 grams to about 2.5 grams;(d) the calcium chloride is present in an amount of from about 0.1 grams to about 2.5 grams;(e) the BSFL is present in an amount of from about 1 gram to about 10 grams; and(f) the EMI microbial inoculant is present in an amount of from about 50 ml to about 100 ml.

32. The oviposition attractant composition of claim 31, wherein:(a) the distillers dried grains are present in an amount of about 38 grams; (b) the bakery by-product meal is present in an amount of about 38 grams; (c) the calcium carbonate is present in an amount of about 0.8 grams; (d) the calcium chloride is present in an amount of about 0.7 grams;(e) the about 1 mg BSFL is present in an amount of about 5.5 grams; and (f) the EMI microbial inoculant is present in an amount of about 75 ml.

33. An oviposition attractant composition comprising:(a) about 38 grams of distillers dried grains;(b) about 38 grams of bakery by-product meal;(c) about 0.8 grams of calcium carbonate;(d) about 0.7 grams of calcium chloride;(e) about 5.5 grams of about 1 mg BSFL; and(f) about 75 ml of EM1 microbial inoculant.

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