NL2031802B1

NL2031802B1 - Ovipositioning in insect breeding

Info

Publication number: NL2031802B1
Application number: NL2031802A
Authority: NL
Inventors: Ali Hosseini Seyed
Original assignee: Protix Bv
Priority date: 2022-05-09
Filing date: 2022-05-09
Publication date: 2023-11-16
Also published as: WO2023219494A1

Abstract

The present invention in general relates to the field of large scale breeding of insects. More in particular, the invention relates to an olfactory attractant that can be used for ovipositioning in insect breeding facilities. Egg collection is a major 5 bottleneck in industrial production of insects such as the black soldier fly. It is an objective of the present invention to provide improved modalities of oviposition that can beneficially be applied in mass scale insect breeding facilities. To this end, the use of a gaseous attractant composition comprising a carrier gas, such as air, enriched with one or more olfactory compounds selected from ethyl acetate, mono- 10 alcohols, and 002, is provided. The present invention provides the gaseous attractant compositions per se, oviposition methods employing them, intermediate (liquid) compositions for making them and uses of the gaseous attractant compositions.

Description

OVIPOSITIONING IN INSECT BREEDING

Field of the Invention

The present invention in general relates to the field of large scale breeding of insects, such as black soldier flies, which select their location for egg laying according to olfactory cues. More in particular, the invention relates to an olfactory attractant that can be used for ovipositioning in insect breeding facilities.

Furthermore, the present invention relates to a method for stimulating ovipositioning of insects at a predetermined location, such as in a device designed to collect eggs.

The invention also relates to the use of an olfactory attractant for stimulating ovipositioning of insects.

Background of the Invention

Insects are considered one of the most promising means for protein recovery and for organic residual recovery. Numerous species proposed for applications in human food and animal feed and pet food rely on olfactory cues to select the location for oviposition. Prominent examples include the black soldier fly (Hermetia iflucens), the house fly (Musca domestica), and the mealworm (Tenebrio molitor L.). Methods improving the efficiency of egg collection from these species are particularly valuable for large scale production because the enormous quantity, delicacy, small size and stickiness of eggs. These challenges make it beneficial to collect them in a specific location since this simplifies collection operations and allows for efficient subsequent handlings. In the event that this location is a device designed to collect eggs, it will henceforth be referred to as an ovisite throughout this application.

Egg collection is a major bottleneck in industrial production of the black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae) (Pastor et al. 2015).

Currently the odor of different organic materials (EP3606339) is used to attract gravid BSF females for oviposition on egg collection sites, so-called ovisites.

One known approach is to provide a location with physical properties desirable to the insect species being cultivated. Egg collection interfaces with a mesh, slits or honey combs are popular because many insects seek out such places to lay eggs. Alternatively or additionally, a food source for adults or their larvae may be placed to attract gravid females to lay. Approaches that make use of an attractant may attempt to entice ovipositioning at a location that has desirable physical properties, like those described above. One approach is to place an ovisite above a container containing the attractant within the cage. More specifically, within the breeding environment, such as a cage, a dose of attractant is placed beneath the ovisite, and the odor that it produces entices gravid females to lay eggs in the ovisite above it. When the ovisite is ready for harvest, it is removed from the breeding environment.

WO 2016/011541, for example, explains that female black soldier flies (‘BSF’) are attracted by pungent odours or biochemical signals derived from BSF eggs or gravid BSF females, which usually indicates a potential food source for BSF offspring. WO 2016/011541 provides a specific ovipositing chamber comprising a rectangular tray with risers that suspend an ovipositioning substrate (such as a honeycomb structure) above an odoriferous attractant that resides in the bottom of the tray, which attractant, according to WO 2016/011541 may be a saturated 1:1 mixture of Gainesville diet mixed with BSF larvae leachate and BSF castings, a mixture of brewery grains and/or wheat bran, fermenting grain, such as corn brewery grain, manure, decomposing food waste, BSF larvae and/or eggs, and fatty acids such as lauric acid.

Systems wherein this kind of attractant composition is placed directly in the breeding environment (or even directly outside it) have a number of disadvantages.

For one thing, these approaches are only suitable for relatively small scale operations. Dosing attractant per cage is feasible when the number of cages is limited, but in production facilities ran at the scales needed, the number of cages can be in the hundreds or thousands. This necessitates extensive dosing, cleaning and replacement of the individual attractant sources.

Additionally, the direct or near direct interaction of attractant sources with breeding insects presents risks of contamination since all attractant sources disclosed in the literature (chicken feed, fly eggs, etc.) are suitable substrates for bacteria and mold and are also attractive to other species of insects and pests.

Furthermore, as is apparent from WO 2016/011541, many attractants described in the literature make use of fermentation to produce the desired attractive odors. After placing such attractant in individual cages, the control over this fermentation process is significantly hampered. This has two negative repercussions. Firstly, the chances of a failure are increased with the number of doses since more doses can potentially fail. Secondly, because of the potential of doses to fail proper monitoring would be required, which is highly effort intensive, especially as the scale of insect production increases.

An advantage of the kind of attractants described in the prior art, such as WO 2016/011541, is that they are easy to use and locally available for BSF production all around the world. On the other hand, variation in physical, chemical, and microbiological properties of such attractants may affect overall productivity in ways that are difficult to predict and control.

Finally, methods based on the kind of attractants described in the prior art, such as WO 2016/011541, are extremely labour intensive as the source of olfactory attractant needs regular replacement with fresh materials. The materials may also be a source of mold and disease in the insect culture.

It is an objective of the present invention to provide improved modalities of oviposition that overcome one or more or all of the above mentioned disadvantages of the state of the art and are therefore (better) suited for application in mass scale insect breeding facilities.

Summary of the Invention

The present inventors have found that the aforementioned objective(s) can be realized by the use of a gaseous attractant composition comprising a carrier gas, such as air, enriched with one or more olfactory compounds selected from ethyl acetate, mono-alcohols, and CO».

As is illustrated in the experimental part of the present application, it has been established experimentally that the present gaseous attractant compositions are very effective as attractive oviposition stimulus (‘AOS’), while their use does not negatively affect egg production and survival in any way, and thus can advantageously be used in (mass-scale) insect breeding facilities. In particular, using the gaseous attractant compositions of the invention, it becomes much easier to control the AOS and to maintain a consistent AOS across large numbers of ovisites and over time. Production of the gaseous attractant composition and its distribution within the breeding system can be fully automated and will, overall, require much less labour than the prior art methods, relying on the use of solid attractant compositions. Altogether the use of the present gaseous attractant compositions in mass scale insect breeding systems will contribute to high and/or consistent (harvestable) egg production levels, while reducing labour, reducing consumption of energy, reducing consumption of organic materials, and/or significantly reducing risks of contaminating the insects and/or infecting them with pathogens and/or (micro)organisms that could negatively affect productivity.

Hence, the present invention, in a first aspect concerns a gaseous attractant composition comprising a carrier gas, such as (ambient) air, enriched with one or more olfactory compounds selected from the group consisting of ethyl acetate, mono-alcohols, and CO:.

A further aspect of the invention concerns an ovipositioning method in an insect breeding operation, comprising the steps of: a) providing a breeding system comprising one or more chambers equipped with an ovisite and an inlet; b) introducing and/or keeping gravid insects in the breeding system;

Cc) introducing into the one or more chambers equipped with an ovisite, through said inlet, a gaseous attractant composition of the present invention, wherein said inlet is positioned such that the gaseous attractant composition passes the ovisite before it reaches the gravid insects; and d) allowing the gravid insects to reach the ovisite(s) and deposit their eggs at the ovisite.

Yet a further aspect of the present invention concerns the use of an olfactory component selected from ethyl acetate, mono-alcohols and CO: and/or a gaseous attractant composition comprising such olfactory component for any one or more of the following: - as a gaseous attractant composition in an insect breeding system; - as an attractive oviposition stimulus in an insect breeding system; - as an olfactory cue or stimulus for gravid insects in an insect breeding system; - for attracting gravid insects to an ovisite in an insect breeding system; - for increasing the production of (harvestable) eggs in insect breeding; - for increasing egg yield in an insect breeding system; and/or - in a method of ovipositioning in an insect breeding system.

These and other aspects of the invention, including exemplary and preferred embodiments thereof, will become apparent to those skilled in the art, based on the following description and examples.

Detailed description of the Invention

As will be apparent to those skilled in the art, based on the present teachings, the present invention has broad utility in the area of insect production, which may also be referred to as insect farming, insect rearing, insect breeding, etc., which is 5 typically done with the primary aim of providing an alternative, sustainable source of nutrients for human and/or veterinary and/or pet and/or fish consumption, such as protein and lipids, but it will be understood that the invention is not particularly limited in this respect.

In the context of the present invention, the term ‘insect’ refers to any edible species of insect or worm. More preferably, the insects are flies, bugs, mosquitos, butterflies, moths, cicadas, termites, bees, ants, wasps, beetles, grasshoppers, or crickets. More preferably, the insects belong to the species from the group consisting of black soldier fly (Hermetia illucens), house fly (Musca domestica), mealworm beetle (Tenebrio molitor), migratory locust (Locusta migratoria) and house cricket (Acheta domestica). In a particularly preferred embodiment of the invention, the insect belongs to the species selected from house fly, black soldier fly, morio worm, mealworm or cricket, more preferably the insect is black soldier fly (Hermetia illucens), which may also be abbreviated herein as ‘BSF’. As is generally known, insects go through certain development stages during their life, including larval stage, pupal stage and adult stage. Whether and which specific stage is meant whenever insects are referred to in this document in general, will be apparent to one skilled in the art based on the specific context.

Gaseous attractant composition

In accordance with the invention, a gaseous attractant composition is provided, which typically comprises a suitable carrier gas, such as (ambient) air, enriched with one or more olfactory compounds selected from the group consisting of ethyl acetate, mono-alcohols, and CO:2.

As used herein, the term ‘enriched’ means that the (relative) content of the respective component, such as the respective olfactory compound, is higher than the level or range that is considered normal for the selected carrier gas, as will be understood by those skilled in the art.

Hence, in embodiments of the invention, the gaseous attractant composition comprises a carrier gas, such as (ambient) air, and one or more olfactory compounds selected from the group consisting of ethyl acetate and mono-alcohols.

In further embodiments of the invention, the gaseous attractant composition comprises a carrier gas, such as (ambient) air enriched with CO, and further comprising one or more olfactory compounds selected from the group consisting of ethyl acetate and mono-alcohols.

In relation to the attractant composition, the term ‘gaseous’ is used to indicate that said attractant compositions essentially behave as a gas, while the presence of components that are not, strictly speaking, in the form of a gas, mixed with and/or carried by the gas, is not excluded. In particular, in accordance with the invention, the gaseous attractant composition, can be a combination of one or more gases, one or more vapours and one or more aerosols. In preferred embodiments of the invention, the gaseous attractant composition is produced by introducing into a carrier gas, a quantity of one or more olfactory compounds of the present invention in the form of a gas, a vapour, an aerosol, spray or any combination thereof.

Particularly suitable methods comprise introducing one or more olfactory compound into the carrier gas in gaseous or vaporized form or introducing one or more olfactory compounds to the carrier gas as a spray or aerosol. As will be understood by those skilled in the art, based on the present teachings, such processes will typically result in a gaseous attractant composition comprising the carrier gas in combination with the one or more olfactory compounds that may be in a gaseous state, in a liquid state, typically in the form of very fine droplets or aerosols, or a combination thereof.

In certain embodiments of the invention at least 10 % of (each of) the one or more olfactory compounds is present in the form of a gas or vapour, more preferably at least 25 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 97.5%, at least 98 %, at least 99 %, at least 99.5 %, at least 99.9 %. In certain embodiments of the invention the one or more olfactory compounds present in the gaseous attractant composition are essentially or completely in the form of a gas or vapour.

Furthermore, as will be understood by those skilled in the art based on the present teachings, it is preferred that the gaseous attractant composition of the invention is a homogeneously or essentially homogeneously mixed combination of gases and any component that is not (strictly speaking) a gas or vapour, such as any component that is (partially or completely) contained in the carrier gas in the form of small aerosols or droplets.

In accordance with the invention, it is preferred that air, in particular ambient air, is used as the carrier gas. As will be understood, the term ‘air’ generally denotes the gas(es) making up the atmosphere of Earth. As used herein, the term ‘ambient air’ refers to air at the insect breeding facility at which the current method is carried out, given that it is most practical to produce the gaseous attractant composition as defined herein ‘on-site’. As is generally known, the main components of air are oxygen, and nitrogen, which typically make up about 99 mol.% of (dry) air, i.e. usually around 21 mol.% of (dry) air is oxygen and approximately 78 mol.% of (dry) air is nitrogen. Argon and carbon dioxide are the two other ‘main’ components of dry air, at levels (in dry air) of around 0.93 mol.% and 0.04 mol.% respectively. The exact composition of ambient air may vary depending on geography, height, pressure, conditions, etc., but this is not relevant in the context of the invention.

In preferred embodiments of the invention, the carrier gas is (ambient) air that has been scrubbed prior to producing the gaseous attractant composition, so as to remove contaminants, infectious and/or pathogenic agents and/or olfactory compounds that may be present in the environment/atmosphere, e.g. at the insect breeding facility, in case the gaseous attractant composition is produced on-site. In a particularly preferred embodiment of the invention, the carrier gas is (ambient) air that has been subjected to treatment with activated carbon or a carbon filter.

In preferred embodiments of the invention, the carrier gas is (ambient) air and contains an increased level of CO2 As already mentioned herein, CO2 has been found to independently act as an attractive oviposition stimulus and/or to aid in the attractant effects of the (other) olfactory compound(s) present in the composition.

Hence, in preferred embodiments of the invention, the gaseous attractant composition comprises (ambient) air and has an increased CO: level, such as a level of at least 0.5 mol. %, at least 1 mol. %, at least 1.5 mol. %, at least 2 mol. %, at least 2.25 mol. %, at least 2.5 mol. % or at least 2.75 mol.% and/or a CO: level of below 5 mol. %, such as below 4.5 mol. %, below 4 mol. %, below 3.5 mol.%, below 3.25 mol.% or below 3 mol.%.

As explained and illustrated herein, ethyl acetate, the organic compound with the formula CH3-CO2-CH2-CHs3 (or C4HsO3), and which is synonymous with (amongst others) ethyl ethanoate, has been found to be particularly effective as an attractive oviposition stimulus. In preferred embodiments of the invention, a gaseous attractant composition as defined herein is provided, wherein the carrier gas comprises ethyl acetate at a level of at least 0.5 mg/L of gas, such as at least 1 mg/L, at least 2 mg/L, at least 3 mg/L, at least 4 mg/L, at least 5 mg/L, at least 6 mg/L, at least 7 mg/L, at least 8 mg/L at least 9 mg/L or at least 10 mg/L. The invention is not particularly limited with regard to the maximum level of ethyl acetate.

For practical purposes it is preferred that the level of ethyl acetate is below 35 mg/L of gas, such as below 25 mg/L, below 20 mg/L, below 15 mg/L or below 12.5 mg/L.

As explained and illustrated herein, mono-alcohols have also been found to be particularly effective as an attractive oviposition stimulus. In preferred embodiments of the invention, gaseous attractant compositions as defined herein are provided, comprising a monoalcohol, wherein the monoalcohol is selected from the group consisting of alcohols derived from linear or branched alkanes or alkenes, such as linear or branched C2-C+0 alkane or alkene, linear or branched C»-Cs alkane or alkene, linear or branched C2-Cs alkane or alkene, or linear or branched C2-Cs alkane or alkene. In certain preferred embodiments of the invention, gaseous attractant compositions as defined herein are provided, comprising a monoalcohol, wherein the monoalcohol is selected from the group consisting of ethanol, 3-methyl- 1-butanol, decanol and 1-octen-3-ol, preferably the monoalcohol is selected from the group consisting of ethanol and 3-methyl-1-butanol.

In preferred embodiments of the invention, a gaseous attractant composition as defined herein is provided, wherein the carrier gas comprises mono-alcohols at a total level of at least 1 mg/L of gas, such as at least 1.5 mg/L, as at least 2 mg/L, at least 2.5 mg/L, at least 3.5 mg/L, at least 4 mg/L, at least 4.5 mg/L, at least 5 mg/L, at least 5.5 mg/L, or at least 6 mg/L. The invention is not particularly limited with regard to the maximum level of mono-alcohols. For practical purposes it is preferred that the total level of mono-alcohols is below 20 mg/L of gas, such as below 17.5 mg/L, below 15 mg/L, below 12.5 mg/L or below 10 mg/L.

In preferred embodiments of the invention, gaseous attractant compositions as defined herein are provided wherein the monoalcohol comprises or is ethanol.

In preferred embodiments of the invention, a gaseous attractant composition as defined herein is provided, wherein the carrier gas comprises ethanol at a level of at least 1 mg/L of gas, such as at least 1.5 mg/L, as at least 2 mg/L, at least 2.5 mg/L, at least 3.5 mg/L, at least 4 mg/L, at least 4.5 mg/L, at least 5 mg/L, at least

5.5 mg/L, or at least 6 mg/L. The invention is not particularly limited with regard to the maximum level of ethanol. For practical purposes it is preferred that the total level of ethanol is below 20 mg/L of gas, such as below 17.5 mg/L, below 15 mg/L, below 12.5 mg/L or below 10 mg/L. In this context, the term ‘ethanol’ refers to the organic compound with the formula CH2-CH2-OH (i.e. ethyl alcohol) per se and not to compositions comprising mixtures of water and ethanol at various ratio’s, such as ‘ethanol 95%’, ‘ethanol absolute’, ‘ethanol USP’ and the like, which are sometime colloquially referred to as ‘ethanol’, which does not detract from the fact that such products or mixtures can of course suitably be used for making the gaseous attractant compositions.

In preferred embodiments of the invention, gaseous attractant compositions as defined herein are provided wherein the monoalcohol comprises or is 3-methyl- 1-butanol.

In preferred embodiments of the invention, a gaseous attractant composition as defined herein is provided, wherein the carrier gas comprises 3-methyl-1-butanol at a level of at least 0.05 mg/L of gas, such as at least 0.1 mg/L, at least 0.15 mg/L, at least 0.20 mg/L, or at least 0.25 mg/L. The invention is not particularly limited with regard to the maximum level of 3-methyl-1-butanol. For practical purposes it is preferred that the level of 3-methyl-1-butanol is below 1 mg/L of gas, such as below 0.75 mg/L, below 0.5 mg/L, below 0.4 mg/L or below 0.35 mg/L.

In preferred embodiments of the invention, a gaseous attractant composition as defined herein is provided, comprising ethyl acetate and ethanol, preferably at a ratio (w/w) of ethyl acetate : ethanol within the range of 0.2/1 to 5/1. For instance, said ratio may be at least 0.5/1, at least 0.751, at least 1/1, at least 1.25/1, at least 1.511 or at least 1.751 and/or said ratio may be less than 4/1, less than 3.5/1, less than 3/1, less than 2.751, less than 2.5/1 or less than 2.25/1, such as around 2:1.

In preferred embodiments of the invention, a gaseous attractant composition as defined herein is provided, comprising ethyl acetate and 3-methyl-1-butanol, preferably at a ratio (w/w) of ethyl acetate : 3-methyl-1-butanol within the range of 50/1 to 250/1. For instance, said ratio may be at least 60/1, at least 70/1, at least 75/1, at least 80/1, at least 85/1, at least 90/1 or at least 95/1 and/or said ratio may be less than 200/1, less than 150/1, less than 1251 or less than 110/1, such as around 100/1.

In preferred embodiments of the invention, gaseous attractant compositions as defined herein are provided wherein the composition comprises ethyl acetate, ethanol and 3-methyl-1-butanol. In particularly preferred embodiments of the invention, gaseous attractant compositions as defined herein are provided wherein the ratio (w/w) of ethyl acetate : ethanol : 3-methyl-1-butanol is about 100/50/1

In embodiments of the invention, gaseous attractant compositions as defined herein are provided wherein the composition does not comprise acetic acid, is completely free of acetic acid or is essentially free of acetic acid. In other embodiments of the present invention, the gaseous attractant composition comprises acetic acid at a level below or far below the odour threshold (for humans), which, according to literature is around 0.2 mg/L. Preferably, the gaseous attractant composition comprises acetic acid at a level below 0.1 mg/L, below 0.05 mg/L, below 0.01 mg/L, below 0.005 mg/L or below 0.001 mg/L.

In preferred embodiments of the invention, the gaseous attractant composition contains suitable amounts of water vapor. More in particular, in accordance with the invention, a gaseous attractant composition as defined herein is provided, preferably comprising (ambient) air as a carrier gas, having a relative humidity, RH, of above 25 %, such as a relative humidity of at least 50 % RH, at least 60 % RH, at least 65 % RH, at least 70 % RH, at least 75 % RH, at least 80 %

RH, or atleast 82.5 % RH and, typically, below 100 % RH, such as below 95 % RH, below 90 % RH or below 87.5 % RH, e.g. a relative humidity of 85 + 2 % RH or a relative humidity of around 85 % RH. The gaseous attractant composition having a

RH of above 25 %, preferably at least 70 % is advantageous since such a RH helps preventing drying out of the eggs laid in ovisites through which the gaseous attraction composition is flowing. That is to say, eggs in an ovisite which are in a stream of gaseous attractant composition may suffer from drying, when positioned in the stream for an extended period of time. Applying humidified gaseous attractant composition prevents eggs from drying while exposed to the stream of gaseous attractant composition. Therefore, application of humidified gaseous attractant composition, by applying humidified (ambient) air as the carrier gas is preferred.

In preferred embodiments of the invention, the gaseous attractant composition is provided at a temperature, which is increased compared to normal room temperature, such as a temperature of at least 22.5 °C, at least 25 °C, at least 26 °C, at least 27 °C, at least 27.5 °C or at least 28 °C and/or below 40 °C, such as below 38 °C, below 37 °C, below 36 °C, below 35 °C, or below 34 °C, e.g. a temperature within the range of 32 + 1 °C or a temperature or around 32 °C.

Method of producing gaseous attractant composition

A further aspect of the invention concerns a method of producing a gaseous attractant composition as defined herein. In preferred embodiments of the invention, a method is provided, comprising the steps of providing a stream of a carrier gas, as defined herein before, and introducing into said stream of a carrier gas, at least one olfactory compound, preferably ethyl acetate, typically in the form of an aerosol, in the form of a (fine) liquid spray, or in the form of a gas or vapour.

In other embodiments of the invention, a method is provided comprising providing a stream of a carrier gas, as defined herein before, and leading said stream of the carrier gas through one or more olfactory compound(s) in liquid form.

In preferred embodiments of the invention, the method comprises the initial step of capturing air from the atmosphere/environment, e.g. the environment of the insect breeding facility where the gaseous attractant composition is to be used.

However, the invention is not particularly limited in this regard and embodiments are also envisaged where the carrier gas is provided in other manners, e.g. as compressed air.

In preferred embodiments of the invention, the method comprises the step of scrubbing the carrier gas, typically the (ambient) air, e.g. by leading it over an (active) carbon filter so as to remove contaminants, infectious and/or pathogenic agents, prior to introducing the one or more olfactory compounds.

In preferred embodiments of the invention, the method comprises the step of heating the carrier gas, typically the (ambient) air, to a temperature of at least 22.5 °C, at least 25 °C, at least 26 °C, at least 27 °C, at least 27.5 °C or at least 28 °C and/or below 40 °C, such as below 38 °C, below 37 °C, below 36 °C, below 35 °C, or below 34 °C, e.g. a temperature within the range of 32 + 1 °C or a temperature or around 32 °C. The heating step may be carried out before or after the step of introducing the one or more olfactory compounds. Most preferably heating takes place before introducing the one or more olfactory compounds.

In preferred embodiments of the invention, the method comprises a step of humidifying the carrier gas, typically the (ambient) air to a relative humidity, RH, of above 25 %, such as a relative humidity of at least 50 % RH, at least 60 % RH, at least 65 % RH, at least 70 % RH, at least 75 % RH, at least 80 % RH, or at least 82.5 % RH and, typically, of below 100 % RH, such as below 95 % RH, below 90 %

RH or below 87.5 % RH, e.g. a relative humidity of 85 + 2 % RH or a relative humidity of around 85 % RH. The humidification step may be carried out before or after the step of introducing the one or more olfactory compounds. Most preferably humidification takes place before introducing the one or more olfactory compounds

In preferred embodiments of the invention, the method comprises the step of introducing a stream of gaseous CO: into the stream of the carrier gas, typically up to the levels defined herein above. This step may take place prior to or after the step of introducing the one or more olfactory compounds. Embodiments are envisaged wherein a gaseous attractant composition is first produced without CO: addition and streams of the said gaseous attractant composition and gaseous CO: are lead to the ovisite separately.

In preferred embodiments of the invention, the method comprises providing a flow or stream of the olfactory compound in gas or vapor form or, alternatively, in the form of an aerosol or spray, and leading said flow or aerosol or spray directly into the flow of the carrier gas. With a view to consistency and controllability of the gaseous attractant composition, it is preferred that the one or more olfactory compounds, in some embodiments comprising CO2, and carrier gas are combined in the form of separate flows using equipment that allows for constant monitoring and adjustment of the flow rates of the respective components. It is within the capabilities of the person skilled in the art to, based on the present teachings, determine appropriate ways of implementing this. As will be understood by those skilled in the art, based on the present teachings, during the step(s) of combining the carrier gas and the olfactory compound(s), the flow of the carrier gas, in volume (L) per time unit, and the flow(s) of the olfactory compound(s), in mg per time unit, should be adjusted so as to attain the respective olfactory compound level(s) (and ratios) that are defined herein above.

Quvipositioning method

A further aspect of the invention concerns an ovipositioning method in an insect breeding operation, comprising the steps of: a) providing a breeding system comprising one or more chambers equipped with an ovisite and an inlet;

b) introducing and/or keeping gravid insects in the breeding system; c) introducing into the one or more chambers equipped with an ovisite, through said inlet, a gaseous attractant composition of the present invention, wherein said inlet is positioned such that the gaseous attractant composition passes the ovisite before it reaches the gravid insects; and d) allowing the gravid insects to reach the ovisite(s) and deposit their eggs at the ovisite.

As used herein, the term ‘breeding system’ generally refers to systems designed/equipped to facilitate one or more stages of insect breeding processes.

As will be understood by those skilled in the art, the term ‘ovipositioning’ as used herein refers to the process of directing gravid insects, preferably BSF, to a specific location for depositing their eggs, which specific location is destined and designed for collecting the eggs in an efficient manner. Said location destined for deposition of the eggs is referred to herein as the ‘ovisite’. Suitable ovisites comprise or consist of an interface material with a mesh, one or more slits, perforations, honeycomb structure or the like. After ovipositioning, the ovisites can typically be taken from the breeding system chamber and therewith the eggs are readily collected while stuck to said interface material. Hence, in preferred embodiments of the invention, a method as defined herein is provided, wherein the ovisites comprises a plurality of openings, slits or perforations, typically having dimensions suitable for holding insect eggs deposited therein, and the gaseous attractant composition is made to flow through said openings, slits or perforations before reaching the gravid insects.

Step c. of the ovipositioning method defined herein, preferably comprises a step c1) of providing a gaseous attractant composition as defined herein before and a step c2) of leading a flow of the gaseous attractant composition into the chamber equipped with the ovisite. In preferred embodiments of the ovipositioning method, said step ¢1) comprises the method of producing gaseous attractant composition as defined here above. Methods are also envisaged wherein the gaseous attractant composition is created at the ovisite (i.e. in situ), e.g. by leading separate flows of carrier gas, CO: and/or one or more olfactory compounds into the chamber equipped with the ovisite concurrently.

In preferred embodiments of the invention, entrainment of gas takes place at a location within the breeding system or ovisite chamber that is distant from the location where the gaseous attractant composition of the invention is introduced.

In preferred embodiments of the invention, resulting in an optimal stimulus for gravid insects, preferably gravid BSF, to oviposition at the ovisite, the gaseous attractant composition is lead into the breeding chamber through an inlet positioned at the side of the ovisite interface opposite to where the gravid insects reside, so that the gaseous attractant composition has to pass the ovisite interface to reach the insects and the ovisite will be perceived as the source of the AOS. One approach is to provide a breeding chamber comprising a planar ovisite, comprising a plurality of openings, slits or perforations, said planar ovisite defining two spaces within the chamber, wherein the gaseous attractant composition is introduced through an inlet positioned in the space beneath the ovisite while the space above the ovisite is in open connection to the parts of the breeding system where the gravid insects (may) reside. In another embodiment, the breeding chamber comprises a planar ovisite, comprising a plurality of openings, slits or perforations, said planar ovisite defining two spaces within the chamber, wherein the gaseous attractant composition is introduced through an inlet positioned in the space located at one side of the planar ovisite while the space at the other side of the planar ovisite is in open connection to the parts of the breeding system wherein the gravid insects (may) reside. The

AOS that passes through the ovisite entices the gravid female insects to lay their eggs in the ovisite. When the ovisite is ready for harvest, it can be removed from the breeding environment for further processing.

In preferred embodiments of the invention, the ovipositioning method as defined herein comprises or is followed by a step e), which comprises removing the ovisite from the breeding chamber and subjecting it to further processing, such as to a process of collecting the eggs from the ovisite and/or a process of placing the ovisite containing the eggs in a system or chamber adapted and/or operated to facilitate breeding and/or hatching of the eggs.

A further aspect of the invention concerns an ovipositioning method in an insect breeding operation, comprising the steps of: a) providing a breeding system comprising one or more chambers equipped with an ovisite; b) introducing and/or keeping gravid insects in the breeding system;

C) introducing into the one or more chambers equipped with an ovisite a gaseous attractant composition, by placing a receptacle filled with a liquid comprising the one or more olfactory compounds selected from ethyl acetate and mono-alcohols, as defined herein, preferably one or more olfactory compounds selected from ethyl acetate, ethanol and 3-methyl-1-butanol, wherein said receptacle has an open top, so as to allow the one or more olfactory compounds to evaporate and form a gaseous attractant composition, and wherein said receptacle is positioned in the chamber such that the gaseous attractant composition passes the ovisite before it reaches the gravid insects; d) allowing the gravid insects to reach the ovisite(s) and deposit their eggs at the ovisite.

Cc) introducing into the one or more chambers equipped with an ovisite a gaseous attractant composition of the present invention by applying a liquid comprising the one or more olfactory compounds selected from ethyl acetate and mono-alcohols as defined herein, preferably one or more olfactory compounds selected from ethyl acetate, ethanol and 3-methyl-1-butanol, on the ovisites, e.g. by pouring or spraying said liquid on the ovisites or by dipping the ovisite in said liquid, and allowing the one or more olfactory compounds to evaporate and form a gaseous attractant composition; d) allowing the gravid insects to reach the ovisite(s) and deposit their eggs at the ovisite.

Liquid pre-mix composition

Embodiments of the invention are also envisaged and encompassed, wherein the AOS is produced using a liquid mixture of the one or more olfactory compounds selected from ethyl acetate and mono-alcohols, preferably one or more olfactory compounds selected from ethyl acetate, ethanol and 3-methyl-1-butanol, as will be apparent to those skilled in the art, based on the present teachings. This liquid mixture, which is referred to herein as ‘the pre-mix’, constitutes yet another aspect of the present invention.

In preferred embodiments of the invention, a liquid pre-mix as defined herein is provided, comprising ethyl acetate and ethanol, preferably at a ratio (w/w) of ethyl acetate : ethanol within the range of 0.2/1 to 5/1. For instance, said ratio may be at least 0.5/1, at least 0.75/1, at least 1/1, at least 1.25/1, at least 1.5/1 or at least 1.75/1 and/or said ratio may be less than 4/1, less than 3.5/1, less than 3/1, less than 2.75/1, less than 2.5/1 or less than 2.25/1, such as around 2:1.

In preferred embodiments of the invention, a liquid pre-mix as defined herein is provided, comprising ethyl acetate and 3-methyl-1-butanol, preferably at a ratio (w/w) of ethyl acetate : 3-methyl-1-butanol within the range of 50/1 to 250/1. For instance, said ratio may be at least 60/1, at least 70/1, at least 75/1, at least 80/1, at least 85/1, at least 90/1 or at least 95/1 and/or said ratio may be less than 200/1, less than 150/1, less than 125/1 or less than 110/1, such as around 100/1.

In preferred embodiments of the invention, liquid-premix compositions as defined herein are provided, comprising ethyl acetate, ethanol and 3-methyl-1- butanol. In particularly preferred embodiments of the invention, (gaseous) attractant compositions, e.g. the pre-mix, as defined herein are provided wherein the ratio (w/w) of ethyl acetate : ethanol : 3-methyl-1-butanol is about 100/50/1

In preferred embodiments of the invention the total combined amount of the ethyl acetate and the one or more mono-alcohols make up at least 50 wt.% of the total weight of the liquid pre-mix composition, the balance being selected from suitable carrier liquids, such as water, and/or further olfactory compounds. In more preferred embodiments, the ethyl acetate and the one or more mono-alcohols make up at least 60 wt.%, at least 70 wt.%, at least 75 wt.%, at least 80 wt.%, at least 85 wt.%, at least 90 wt.%, at least 95 wt.%, at least 97.5 wt.%, at least 98 wt.%, at least 98.5 wt.%, at least 99 wt.%, at least 99.5 wt.% or at least 99.5 wt.% of the total weight of the liquid pre-mix composition.

In embodiments of the invention, liquid pre-mix compositions as defined herein are provided wherein the composition does not comprise acetic acid, is completely free of acetic acid or is essentially free of acetic acid. In other embodiments of the present invention, the liquid pre-mix composition comprises acetic acid at a level below 5 wt.%, below 2.5 wt.%, below 1 wt.%, below 0.5 wt.%, below 0.25 wt.%, below 0.1 wt.%, below 0.05 wt.% or below 0.01 wt. %.

Uses of the gaseous attractant composition and/or olfactory compounds

A further aspect of the present invention concerns the use of the gaseous attractant composition as defined herein before for any one or more of the following: - as a gaseous attractant composition in an insect breeding system; - as an attractive oviposition stimulus in an insect breeding system; - as an olfactory cue or stimulus for gravid insects in an insect breeding system; - for attracting gravid insects to an ovisite in an insect breeding system; - for increasing the production of (harvestable) eggs in insect breeding; - for increasing egg yield in an insect breeding system; and/or - in a method of ovipositioning in an insect breeding system.

Yet a further aspect of the present invention concerns the use of one or more olfactory compounds selected from the group consisting of ethyl acetate and mono- alcohols as defined herein, preferably one or more olfactory compounds selected from the group consisting of ethyl acetate, ethanol and 3-methyl-1-butanol, for any one or more of the following: - as a gaseous attractant composition in an insect breeding system; - as an attractive oviposition stimulus in an insect breeding system; - as an olfactory cue or stimulus for gravid insects in an insect breeding system; - for attracting gravid insects to an ovisite in an insect breeding system; - for increasing the production of (harvestable) eggs in insect breeding; - for increasing egg yield in an insect breeding system; and/or - in a method of ovipositioning in an insect breeding system.

It will be understood by those skilled in the art that these uses correspond to the methods defined here above and, more in particular, that the purposes defined in relation to said uses equally apply to said methods and that the specific details of said methods equally apply to said uses. Preferably, the insect is BSF.

Miscellaneous

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

"A", "an", and "the" as used herein refer to both singular and plural forms unless the context clearly dictates otherwise. By way of example, "a compartment” refers to one or more than one compartment. "About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-10% or less, more preferably +/-5% or less, even more preferably +/-1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed. "Comprise", "comprising", "comprises" and "comprised of" as used herein are synonymous with “include”, "including", "includes" or “contain”, “containing”, "contains" and are inclusive or open-ended terms that specify the presence of what follows, e.g. a component, and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints. The skilled person will appreciate that the present invention can incorporate any number of the specific features described above.

The embodiments of the invention described herein can operate in combination and cooperation, unless specified otherwise.

Description of Figures

Figure 1. Experimental AOS delivery system. ‘FM’ refers to a Flow Meter.

Figure 2. Egg yield per hour by different volatile compounds, clean air, and standard attractant in black soldier fly breeding cages. Differences between the odor stimuli and clean air are depicted using the following codes: **** =P < 0.001, *** =

P<0.01,™=P<0.05, *=P <£0.1 (Dunnett's post-hoc test). The relative amount of the compounds in each blend is shown in the parenthesis following the name of the compounds. The percentage of the headspace volatiles of each compound/blend in total airstream is indicated after the parenthesis. The central line shows the median, and the whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box. The large dot shows the average egg yield by each treatment.

Figure 3. Total egg yield by different chemical blends over a complete black soldier fly breed run. The central line shows the median, and the whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box. The large dot shows the average egg yield by each treatment. The relative amount of the compounds in each blend is shown in the parenthesis following the name of the compounds. The percentage of the headspace volatiles of each blend in total airstream is indicated after the parenthesis.

Figure 4: Egg yield by different chemical blends at different harvests during a complete breed run. Solid lines are fitted to egg yield and the shaded areas represent 95% confidence intervals. The relative amount of the compounds in each blend is shown in the parenthesis following the name of the compounds. The percentage of the headspace volatiles of each blend in total airstream is indicated after the parenthesis.

Figure 5. Number of black soldier fly larvae survived to day 4 from one gram egg collected by different odor stimuli. The central line shows the median, and the whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box. The large dot shows the average egg yield by each treatment.

Examples

Example 1: Screening for AOS activity

Experiments were conducted in order to screen the AOS activity of the following compounds: 1-octen-3-ol, 2-phenylethanol, ammonia, 3-methyl-1-butanol, 2-methyl-3-buten-2-ol, ethyl acetate (anhydrous, = 99.8%), ethyl acetate (99.5%), ethanol, n-hexane, acetic acid, linalool, methyl eugenol, methyl salicylate, decanol, and CO:.

Materials and Methods

The BSF population of the breeding facility of Protix {Noord Brabant, the

Netherlands) was used in all bioassays. The oviposition experiments were conducted in breed cages of ~1 m3 volume using ~13000 last-stage BSF pupae per cage. The insects were let to eclose and mate for 3 days (day 0-3). On day 3, the ovisites were placed in the cages for a 4 days (day 3-7) egg collection. In all experiments, the breed room was kept at 32 °C, 85 % RH, and a light-dark cycle of

12L:12D. AOS delivery and egg collection was performed during the light cycle (photophase).

Several volatile compounds and blends (Figure 2) were screened for their

AOS activity.

Figure 1 shows a schematical representation of the system that was designed and build to deliver the odor stimuli to the ovisites of the breed cages. The compound or blend was placed in a 500 ml glass bottle which provided ~ 50.26 cm? surface area for evaporation. The headspace volatile of the compound(s) was carried to the ovisite by a humidified and purified airstream. Air was first purified and humidified by passing through 1 L charcoal and 1 L demineralized water, respectively (Figure 1). CO: gas was also tested as an AOS, and it was added to the outlet of the compound bottle (Figure 1) with a constant flow of 50 pl/min per cage which was determined as the most efficient flow for egg collection in preliminary experiments. A total airflow of 1.75 L/min was provided for each ovisite using an electric air pump. The relative amount of the compounds in each blend as well as the percentage of the headspace volatiles of each compound or blend in total airstream are shown in Figure 2. Release rates of the airstream, volatile compound, and CO; were controlled by flow meters.

Each oviposition experiment was started with 100 ml of headspace gas/volatiles. Blends were used for a 12 hours photophase, after which it was replaced with 100 ml fresh headspace gas/volatiles to continue an experiment. For single compound treatments, 100 ml of headspace gas/volatiles was introduced every 12 hours of egg collection.

As an arbitrary positive control, the headspace volatiles of 6 kg nursery diet with 22000 8-d-old BSF larvae in a container with a surface area of 1134 m?, were distributed to 32 cages. A purified and humidified airstream was used as a negative control.

In the screening experiments, each odor stimulus was tested for several hours during different breed runs. Each stimulus was tested in at least 4 different cages and eggs were collected on a minimum of 8 ovisites.

The ovisite was a perforated rectangular plastic plate of 20 x 25 cm with 1 cm thickness. One ovisite per cage was used at a time, and it was mounted as a window on the wall of the cage.

In the screening experiments, egg vield per hour was analyzed by a generalized linear mixed model with a gamma error distribution. Odor stimulus and breed run were included as fixed factors. Egg collection day in a breed run and the duration (hours) of egg collection were set as random factors. All odor stimuli were compared to clean air using Dunnett's post-hoc test. Generalized linear models (GLM) were used to analyze total egg yield data with the odor stimulus as the explanatory variable. Gaussian and gamma error distributions were used for normal and non-normal distributions, respectively. The Shapiro test was used to determine the normality of data. All analyses were performed in R, version 4.0.3 (R Core Team 2020).

Results

Ethyl acetate, Ethanol, and CO: displayed significant AOS activity (Figure 2,

Table 1) compared with the negative control (clean air) in BSF. Combinations of these components with 3-methyl-1-butanol improved egg yield (Figure 2, Table 1). 1-Octen-3-ol, 2-Phenylethanol, Ammonia, 3-Methyl-1-butanol, 2-Methyl-3-buten-2- ol, n-Hexane, and Decanol did not show AOS activity (Figure 2, Table 1). Acetic acid inhibited oviposition (Figure 2, Table 1). Linalool, methyl eugenol, and Methyl salicylate yielded less eggs compared with clean air (Figure 2, Table 1), although the statistical analyses did not find a significant difference between them.

Table 1. Egg vield per hour (average, median, and standard deviation (SD)) by different volatile compounds (blends and single compounds), clean air, and standard AOS in black soldier fly breeding cages.

Acstcaadioh Jo oo 100%

: 1) 100% + CO: acid : Linalool (1: 1: 1:1) 100% :1) 10% + CO2 100% + CO2

Ethyl acetate : Ethanol (1 : 1) 10% + CO: 50:1) 10% + CO»

Clean air 100% (negative control)

Ethyl acetate was the major component of the most effective blends. A high- purity (2 99.8%) ethyl acetate which is an expensive compound was used in the screening test. To lower the cost of a chemically defined AOS, a cheaper Ethyl acetate with 99.5% purity was used in the Complete breed run experiment.

Example 2: Complete breed run experiment

Complete breed run experiments were conducted with the blends that showed significant AOS activity during screening. In the Complete breed run experiments, chemical blends were compared to arbitrary positive control AOS (as described in example 1) in terms of total egg yield and survival (number larvae per gram egg, analysed using a GLM with a gaussian error distribution).

Two chemical blends performed better than the arbitrary positive control AOS in terms of total egg yield. With 1.49 ml of the mix of Ethyl acetate : Ethanol : 3-

Methyl-1-butanol (100 : 50: 1) and 72 ml CO: per cage per day the highest total egg yield was achieved by these compounds (Figure 3).

It seems that the egg yield pattern is affected by different breed batches as well as the type of AOS (Figure 4). The most stable egg yield over a breed run was achieved by Ethyl acetate 10% + CO:2.

None of the AOS compounds and blends affected the survival of the eggs (Figure 5).

Claims

AMENDED CONCLUSIONS — (clean copy) — October 30, 2023

A method for oviposition in an insect farm, comprising the following steps: a) providing a culture system comprising one or more chambers equipped with an oviposition and an inlet; b) introducing and/or keeping pregnant insects in the breeding system; c) introducing a gaseous attractant composition via said inlet into the one or more chambers equipped with an ovisite, wherein said inlet is positioned such that the gaseous attractant composition passes the ovisite before reaching the pregnant insects; and (d) allow the pregnant insects to reach the ovivite and lay their eggs on the ovivite; wherein the gaseous attractant composition comprises a carrier gas, such as (ambient) air, enriched with CO: wherein the gaseous attractant composition comprises CO: in a content in the range of 1-5 mol.% and wherein the gaseous attractant composition has a humidity level in the range of 60 - 90% RH.

Method for ovipositioning according to claim 1, wherein the gaseous attractant composition comprises ethyl acetate, preferably in an amount of at least 0.5 mg/L.

The method of ovipositioning according to claim 1 or 2, wherein the gaseous attractant composition comprises one or more monoalcohols.

A method of ovipositioning according to claim 3, wherein the one or more monoalcohols are selected from the group consisting of ethanol, 3-methyl-1-butanol, decanol and 1-octen-3-ol, more preferably wherein the one or more monoalcohols are selected from the group consisting of ethanol and 3-methyl-1-butanol.

Method for ovipositioning according to claim 4, wherein the gaseous attractant composition comprises a combination of ethyl acetate, ethanol and 3-methyl-1-butanol, preferably wherein ethyl acetate and ethanol are present in a ratio (w/w) within the range of 1 /1 — 3/1 and/or where ethyl acetate and 3-methyl-1-butanol are present in a ratio (w/w) within the range 50/1 — 2001.

Method for ovipositioning according to any one of the preceding claims, wherein the gaseous attractant composition has a temperature in the range of 25-40°C, preferably 30-34°C, more preferably 32°C +1°C.

Method for ovipositioning according to any one of the preceding claims, wherein the gaseous attractant composition has a humidity in the range of 60-87.5% RH, more preferably 85% RH + 2% RH.

Method for oviposition according to any one of the preceding claims, wherein the insects belong to a species selected from the group consisting of black soldier fly (Hermetia ilucens), housefly (Musca domestica), mealworm beetle (Tenebrio molitor), migratory locust (Locusta migratoria) and house cricket (Acheta domestica).

The method of ovipositioning according to claim 8, wherein the insects belong to the species black soldier fly (Hermetia illucens).

10. Gaseous attractant composition comprising a carrier gas, such as (ambient) air, enriched with CO, wherein the gaseous attractant composition comprises CO: in a content in the range of 1-5 mol.% and wherein the gaseous attractant composition has a humidity level in the range from 60 – 90% RH.

11. Gaseous attractant composition according to claim 10, wherein the gaseous attractant composition comprises ethyl acetate, preferably in an amount of at least 0.5 mg/L.

12. Gaseous attractant composition according to claim 10 or 11, wherein the gaseous attractant composition comprises one or more monoalcohols, preferably one or more monoalcohols selected from the group consisting of ethanol, 3-methyl-1-butanol, decanol and 1-octene-3- ol, more preferably one or more monoalcohols selected from the group consisting of ethanol and 3-methyl-1-butanol.

13. Gaseous attractant composition according to claim 12, wherein the gaseous attractant composition comprises a combination of ethyl acetate, ethanol and 3-methyl-1-butanol, preferably wherein ethyl acetate and ethanol are present in a ratio (w/w) within the range of 1/ 1 — 3/1 and/or in which ethyl acetate and 3-methyl-1-butanol are present in a ratio (w/w) within the range 50/1 — 200/1.

A gaseous attractant composition according to any one of claims 10-13, wherein the temperature of the gaseous attractant composition is within the range of 25-40°C, preferably 30-34°C, more preferably 32°C +1°C.

A gaseous attractant composition according to any one of claims 10-14, wherein the humidity of the gaseous attractant composition is within the range of 60-87.5% RH, more preferably 85% RH + 2% RH.

Use of a gaseous attractant composition as defined in any of claims 10-15 comprising such an olfactory component, for one or more of the following: - as a gaseous attractant composition in an insect breeding system; - as an attractive oviposition stimulus in an insect breeding system; - as an olfactory cue or stimulus for pregnant insects in an insect breeding system; - for attracting pregnant insects to an insect breeding system; - to increase the production of (harvestable) eggs in insect farming; - to increase egg yield in an insect breeding system; and/or - in a method for ovipositioning in an insect breeding system.