AU2018279230A1 - Method for selecting bee colonies having the VSH trait and composition and kit for implementing same - Google Patents
Method for selecting bee colonies having the VSH trait and composition and kit for implementing same Download PDFInfo
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- AU2018279230A1 AU2018279230A1 AU2018279230A AU2018279230A AU2018279230A1 AU 2018279230 A1 AU2018279230 A1 AU 2018279230A1 AU 2018279230 A AU2018279230 A AU 2018279230A AU 2018279230 A AU2018279230 A AU 2018279230A AU 2018279230 A1 AU2018279230 A1 AU 2018279230A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
- A01N35/02—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aliphatically bound aldehyde or keto groups, or thio analogues thereof; Derivatives thereof, e.g. acetals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
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Abstract
The invention concerns a method for determining if a colony of bees of the Apis mellifera species has the
Description
METHOD FOR SELECTING BEE COLONIES HAVING THE VSH TRAIT AND COMPOSITION AND KIT FOR IMPLEMENTING SAME
The present invention lies in the field of beekeeping, and more particularly of the protection of bees against the Varroa destructor parasite and the selection of colonies resistant to this parasite.
More particularly, the present invention relates to a method for 5 determining whether a bee colony exhibits the Varroa destructor sensitive hygiene trait as well as the use of particular substances for such a determination. The invention also relates to a composition suitable for the implementation of such a method, as well as a kit for this implementation.
For some years, the Varroa destructor parasite has become the main 10 pathogenic threat of insects of the Apis mellifera species, commonly known, and designated in the present description, for more convenience, by the terms honeybees, domestic bees, or merely bees. This parasite, that will also be designated in the present description by the mere term Varroa, breeds on developing broods, transmitting to the latter several viral species. This causes significant harm in bee colonies, as the broods then generate dysfunctional adults with very short lifetimes, disrupting the social equilibrium of the colony. When this parasite is not controlled, infested bee colonies generally disappear in 2 to 3 years.
However, there are some honeybee colonies that exhibit the ability to naturally survive Varroa destructor parasite infestation. This ability is strongly associated with the expression of two interlinked traits. Resistant colonies are capable of detecting the presence of Varroa through the cap of the developing brood and of cleaning the parasite-infested brood. This form of hygiene behaviour is known as VSH (Varroa Sensitive Hygiene) behaviour. The cells containing female Varroa accompanied by immature Varroa are preferentially targeted by bees carrying the VSH trait. This VSH behaviour enables the bees to remove immature varroas. While it does not generally kill the founding female Varroa infesting the targeted cells, it nonetheless disrupts the reproduction of these females. Consequently, female Varroa located in colonies of bees expressing the VSH trait frequently encounter failure in their reproductive cycles. This reproductive failure of the Varroa is known as SMR (Suppressed mite reproduction).
Based on this discovery, the prior art has sought to develop methods for selecting colonies of bees resistant to Varroa, so as to favour the breeding of these colonies, that are less difficult to keep alive than the others, or to avoid treating unnecessarily, against Varroa parasite infestation, colonies that are naturally resistant thereto.
According to one of these methods, described in particular in the publication by Villa et al., 2009, in Journal of Apicultural Research 48(3):162167, the evaluation of the VSH trait of bee colonies is carried out by means of a procedure involving placing a brood frame highly infested with Varroa in the colony under test. After an incubation time, the frame is retrieved and the infestation reviewed, to determine whether the bees of the colony have cleaned the parasite-infested cells or not. Such a method is however constraining to carry out, and it requires having a large number of Va/roa-infested donor colonies.
According to a further method proposed by the prior art, described in particular in the publication by Villa et al., 2016, in Apidologie 47(6):771-778, the Varroa resistance potential of bee colonies is rather evaluated using a test for evaluating the SMR trait of the bees. This test consists of evaluating, on a capped brood frame, the proportion of female Varroa subject to reproductive failure. However, even though this test is simpler to carry out than the method described above, it is very time-consuming and in practice impossible to carry out on a large number of colonies in the field.
At the present time, no convenient-to-use tool is therefore available to beekeepers to estimate the Varroa resistance potential of their honeybee colonies.
The present invention aims to remedy the drawbacks of the solutions proposed by the prior art to evaluate whether a bee colony is resistant to the Varroa parasite, particularly the drawbacks described hereinabove, by proposing a method for determining in vivo, directly in the hive, whether a colony of bees of the Apis mellifera species exhibits the Varroa destructor sensitive hygiene trait, this method being easy to carry out, preferably by the beekeepers themselves, and moreover at low-cost.
Quite surprisingly, it was discovered by the present inventors that certain specific compounds trigger the VSH behaviour of bees carrying this trait, by contacting the bees with these compounds. The analysis of the degree of response of the bees to this contacting then advantageously makes it possible to determine whether the colony in question carries the VSH trait or not.
Thus, according to a first aspect, there is proposed according to the present invention a method for determining whether a colony of bees of the
Apis mellifera species of a hive has the Varroa destructor sensitive hygiene trait, called VSH trait. This method comprises:
- applying in the hive at least one substance chosen among tricosan2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate, nonacosan-2-one, methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate, or any one of the mixtures thereof, so as to expose the bee colony to said substance,
- determining a response level of the bee colony to the exposure to said substance,
- and comparing the response level determined in this way with a reference response level indicative that the bee colony exhibits the Varroa destructor sensitive hygiene trait.
Said substance is particularly chosen among:
- tricosan-2-one,
- pentacosan-2-one,
- tetracosyl acetate,
- heptacosan-2-one,
- hexacosyl acetate,
- nonacosan-2-one
- and a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate.
Exposure of the bee colony to said substance herein means that the 10 substance is applied in the hive in such a way that it can be detected by the bee, for example in an olfactory way. In particular, the application of the substance(s) in the hive is not restricted to conditions ensuring that the bee can come into contact with the substance(s).
As stated above, it was discovered by the present inventors that 15 exposing a colony of bees to each of the substances listed above triggers the VSH behaviour of the bees when the colony carries this trait. Analysing the behaviour of the colony, following exposure to one or a plurality of these substances, hence makes it advantageously possible to determine in a relatively simple way whether it exhibits this VSH trait or not.
For this purpose, the analysis of any type of response that is suitable for reflecting a cleaning behaviour by the bee colony may be carried out. The response of the bee colony to exposure to the substance(s), the level of which is evaluated according to the present invention, may thus be determined in several manners. It falls within the skills of the person skilled in the art to choose the type of response to be analysed when implementing the method according to the invention, particularly according to the conditions chosen for the application of the substance(s) in the hive. Examples of types of response falling within the scope of the invention are described further in the present description.
The reference response level is dependent on several parameters, particularly on the type of response analysed, the specific substance(s) used, the concentration thereof applied in the hive, the application method, etc.
For a given set of operating parameters, it falls within the skills of the person skilled in the art to determine, for a given type of response, the reference response level which is indicative of the fact that the tested colony of bees exhibits the Varroa destructor sensitive hygiene trait. For this purpose, the person skilled in the art may for example perform, prior to carrying out the method according to the invention, comparative tests on guinea-pig bee colonies, more particularly carry out on these bee colonies, on one hand, the method according to the invention under the chosen operating conditions, and, on the other hand, a method according to the prior art, for example the method for measuring the SMR trait, as described above and in the publication by Villa et al., 2016, so as to determine, for colonies of bees classified as resistant to Varroa by the test according to the prior art, the reference response level for the method according to the invention, according to the particular type of response selected.
Once the reference response level has been obtained for a given type of response, the method according to the invention proves to be particularly simple to carry out, in vivo and directly in the hive, rendering it quite suitable for use by beekeepers themselves.
Preferably, the type of response that is analysed is chosen so as not to require complex and costly analysis equipment, or a time-consuming and restrictive analysis procedure. The method according to the invention thus represents a particularly convenient tool for estimating the resistance potential of domestic bee colonies to the Varroa destructor parasite, by identifying the colonies having a behaviour for detecting and cleaning parasite-infested brood cells. The method according to the invention represents in particular, as such, an effective tool for aiding the selection of bee colonies to be preferred for breeding.
The method according to the invention may further have one or a plurality of the features described hereinafter, embodied separately or in each of their technically operating combinations.
In particular embodiments of the invention, applying the substance in the hive is carried out by applying this substance on an element in the hive, and the response level of the bee colony to exposure to this substance is a level of destruction of this element by the bee colony. The analysis of such a type of response, i.e. the level of destruction of a given element, which reflects particularly well the natural VSH behaviour of the bees, the natural reaction whereof in the presence of the Varroa parasite being that of uncapping and cleaning the infested broods, makes it possible to obtain a particularly reliable evaluation of the VSH trait of the bee colony.
The element carrying the substance(s) according to the invention may equally well be a constituent element of the hive, such as a capped brood frame, or an external object relative to the hive, which is introduced into the hive for the needs of the method according to the invention.
The application of the substance(s) on this element may be carried out using any conventional manner per se, for example by spraying, steeping, injection, etc.
For this purpose, the substance(s) may in particular be used in a composition comprising them in a physiologically compatible vehicle, i.e. safe for bees, and preferably having no significant effect on the behaviour thereof.
This vehicle may equally well be in liquid form, wherein the substance(s) are solubilised and/or suspended, or in solid form, wherein the substance(s) are dispersed in powder form.
Preferentially, the reference response level is a numerical reference value. The method according to the invention then comprises the quantification of a response level of the bee colony to exposure to said substance according to the invention, followed by comparison of the value obtained to a reference threshold value, which has been predetermined to be indicative of the fact that the bee colony exhibits the Varroa destructor sensitive hygiene trait, particularly as described above.
The reference response level may alternatively be of any type, and in particular consist of a reference gauge, which is intended to be compared, for example visually, to the element having served by way of test in the hive, and the destruction level of which is analysed according to the invention.
In particular embodiments of the invention, determining the response level of the bee colony following the exposure thereof to the substance is carried out after at least 1 hour, preferably after at least 2 hours, or after at least 8 hours, or even at least 24 hours, and for example after at least 48 hours, of exposure of the bee colony to said substance. Such a feature advantageously ensures high reliability of the method according to the invention, in terms of evaluating the presence of the VSH trait for the bee colony tested.
In particular embodiments of the invention, applying the substance in the hive is carried out on a capped brood frame of the hive. This application may be carried out by injecting the substance(s) into cells of this frame, or further, and preferentially, by spraying the substance(s) onto this frame. The latter method is particularly preferred in the context of the invention, as it is simple and quick to carry out.
This step may be carried out away from the hive, the frame being removed from the latter beforehand, then reintroduced into the hive once treated. Preferentially, it is carried out in situ, if needed by removing the frame from the hive entirely, or only partially.
By way of example, applying the substance(s) on the hive may consist of spraying the substance(s) onto a surface of the frame equivalent to 100 cells.
The response level of the bee colony to exposure to the substance, which is determined according to the invention, may then be a rate of cells of the frame having been uncapped by the bee colony following the application of the substance, with respect to the total number of cells to which the substance was applied.
In such an embodiment, the reference response level is a threshold rate, above which any result is indicative of a bee colony carrying the VSH trait, and for which and below which any result is indicative of a bee colony not carrying the VSH trait. It was established by the present inventors that this threshold rate is about 55% of cells uncapped by the bees, with respect to the total number of cells to which the substance was applied. It may vary according to a plurality of parameters, particularly according to the particular substance used, the concentration thereof applied in the hive, the application conditions, etc.
In other particular embodiments of the invention, applying the substance in the hive is carried out by introducing into the hive an external element which is destructible by the bees, carrying this substance in such a way that the bees of the colony can detect the presence thereof in the hive, for example in an olfactory way. Preferentially, the element is impregnated with the substance.
Of the elements falling within the scope of such a definition, mention may for example be made of objects made of soft material, such as paraffin, cardboard, etc., which further offer the advantage of being capable of being readily impregnated by the substance to be applied in the hive.
In such a configuration, the response level of the bee colony to exposure to the substance is then the proportion of the element which has been destroyed by the bee colony following the introduction into the hive of the element carrying the substance(s) according to the invention.
This proportion may in particular be determined by calculating a difference in mass between the initial known mass of the element and the final mass thereof, after a given time of presence in the hive.
As a general rule, the quantity of substance applied in the hive for carrying out the method according to the invention is dependent on the particular substance and the application method chosen.
In particular embodiments of the invention, a single one of the substances chosen among tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate, nonacosan-2-one, methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate, is applied in the hive.
In further particular embodiments of the invention, the method comprises the simultaneous application of at least two of these substances, or more, and even of all, of these substances.
More generally, the method may comprise the simultaneous application of a plurality of these substances, any number and any combination of these substances falling within the scope of the invention.
When a plurality of substances from the list above are used simultaneously, they may be present in any proportions in relation to one another.
In particular embodiments of the invention, the method comprises the application in the hive of at least one substance chosen among tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate and nonacosan-2-one; in particular at least two, at least three, at least four or at least five of these substances, or else all of these six substances. This or these substance(s) may be applied in the hive in combination with one or a plurality of the substances chosen among methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate; in particular in combination with at least two, at least three, at least four, at least five, at least six or at least seven of these substances; or else in combination with a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate.
The method according to the invention may further, or alternatively, comprise applying in the hive at least two substances chosen among methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate; in particular at least three, at least four, at least five, at least six or at least seven of these substances; or else a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate.
Combinations of particular substances suitable for use according to the invention are for example as follows:
- a mixture of tetracosyl acetate and hexacosyl acetate, excluding any other of the substances according to the invention;
- the following substances, in the following proportions by weight, with respect to the total weight of these substances: tricosan-2-one (4.8%), pentacosan-2-one (11.9%), tetracosyl acetate (11.9%), heptacosan-2-one (47.6%), hexacosyl acetate (9.5%) and nonacosan-2-one (14.3%); excluding any other of the substances according to the invention;
- the following substances, in the following proportions by weight, with respect to the total weight of these substances: methyl palmitate (8.1%), ethyl palmitate (27.0%), methyl stearate (8.1%), methyl oleate (8.1%), ethyl stearate (21.6%), ethyl oleate (5.4%), ethyl linoleate (19.0%) and methyl linolenate (2.7%); excluding any other of the substances according to the invention.
The substance(s) may be used alone, on a substrate, in particular a solid substrate, or in a composition containing same in a vehicle physiologically compatible with use in the presence of bees. Preferentially, this vehicle is chosen so as solubilise the substance(s) according to the invention. It is further preferably chosen so as not to have a substantial impact on the behaviour of the bees.
The method according to the invention may be applied to one or a plurality of colonies from the same apiary. The results that it allows to obtain for each of the colonies tested, in terms of response level to exposure to the substance(s), may be compared to one another, so as to determine which colonies exhibit the most significant Varroa destructor sensitive hygiene trait.
According to a further aspect, the present invention relates to the use of at least one substance chosen among tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate, nonacosan-2-one, methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate, or any of the mixtures thereof, to determine whether a colony of bees of the Apis mellifera species exhibits the Varroa destructor sensitive hygiene trait. This substance may in particular be a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate.
This use may respond to one or a plurality of the features described above with reference to the method proposed by the present invention to determine whether a colony of bees of the Apis mellifera species of a hive exhibits the Varroa destructor sensitive hygiene trait.
A further aspect of the present invention is a composition, in particular presented in solid form or in liquid form, particularly suitable for carrying out a method according to the invention. This composition contains at least one substance chosen among tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate, nonacosan-2-one, methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate, or any of the mixtures thereof, in a vehicle physiologically compatible with use in the presence of bees of the Apis mellifera species.
It contains for example at least one substance chosen among:
- tricosan-2-one,
- pentacosan-2-one,
- tetracosyl acetate,
- heptacosan-2-one,
- hexacosyl acetate,
- nonacosan-2-one
- and a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate, in a vehicle physiologically compatible with use in the presence of bees of the Apis mellifera species.
By “physiologically compatible” it is meant that the vehicle exhibits no 5 toxicity with respect to the bees, and preferably no substantial impact on the behaviour thereof.
This vehicle may equally well be in liquid form or in solid form, for example as a powder.
Preferentially, the vehicle is in liquid form, and the substances(s) are 10 soluble therein. They may alternatively be suspended therein.
By way of example, the vehicle used is isohexane.
The composition according to the invention may contain a single substance chosen among tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate, nonacosan-2-one, methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate.
It may alternatively contain at least two, or more, and even all, of these substances, mixed with one another.
Any number and any combination of these substances falls within the 20 scope of the invention.
In particular embodiments of the invention, the composition contains at least nonacosan-2-one, alone or in a mixture with one or a plurality of tricosan2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate, methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate
The composition according to the invention contains for example at least nonacosan-2-one and a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate; optionally with one or a plurality of tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate.
It may for example contain at least nonacosan-2-one and at least one substance, preferably at least two substances, or at least three substances, at least four substances, or preferentially at least five substances, among tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one and hexacosyl acetate.
In particular embodiments of the invention, the composition does not contain, by way of substance according to the invention, a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate, methyl linolenate, excluding tricosan-2-one, pentacosan2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate and nonacosan-2-one.
The composition according to the invention may in particular contain at least one substance chosen from tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate and nonacosan-2-one. It may in particular contain at least two, at least three, at least four or at least five of these substances, or else all of these six substances.
The composition according to the invention may for example contain a mixture of tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2one, hexacosyl acetate and nonacosan-2-one, as well as one or a plurality of substances chosen among methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate.
The composition according to the invention may alternatively contain a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate, as well as one or a plurality of substances chosen among tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate and nonacosan-2one.
The composition according to the invention may alternatively for example contain, by way of substance according to the invention, only tetracosyl acetate and hexacosyl acetate, in a mixture. This or these substance(s) may be present in the composition according to the invention in combination with one or a plurality of the substances chosen among methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate; in particular in combination with at least two, at least three, at least four, at least five, at least six or at least seven of these substances; or else in combination with a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate.
The composition according to the invention may alternatively only contain, by way of substances according to the invention, substances chosen among tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2one, hexacosyl acetate and nonacosan-2-one, in particular only these six substances mixed with one another.
The composition according to the invention may further contain at least two substances chosen among methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate, for example at least three, at least four, at least five, at least six or at least seven, of these substances.
When the composition according to the invention contains a plurality of substances according to the invention, all the relative proportions of these substances in relation to one another fall within the scope of the invention.
By way of example, a composition according to the invention may contain the following substances, in the following respectively associated proportions, expressed by weight with respect to the total weight of these substances: tricosan-2-one (4.8%), pentacosan-2-one (11.9%), tetracosyl acetate (11.9%), heptacosan-2-one (47.6%), hexacosyl acetate (9.5%) and nonacosan-2-one (14.3%).
Another composition according to the invention may contain the following substances, in the following respectively associated proportions, expressed by weight with respect to the total weight of these substances: methyl palmitate (8.1%), ethyl palmitate (27.0%), methyl stearate (8.1%), methyl oleate (8.1%), ethyl stearate (21.6%), ethyl oleate (5.4%), ethyl linoleate (19.0%) and methyl linolenate (2.7%).
A further aspect of the invention relates to a kit for implementing a method according to the invention. This kit comprises:
- at least one substance chosen among tricosan-2-one, pentacosan-2one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate, nonacosan-2one, methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate, or any of the mixtures thereof; for example at least one substance chosen among tricosan-2one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate, nonacosan-2-one and a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate; preferably in the form of a composition according to the invention, complying with one or a plurality of the features described in the present description,
- and instructions for implementing this or these substance(s), and preferentially of this composition, to determine whether a colony of bees of the Apis mellifera species exhibits the Varroa destructor sensitive hygiene trait, according to the method according to the invention.
In particular, this kit preferably contains the reference response level indicative of the fact that the bee colony exhibits the Varroa destructor sensitive hygiene trait, associated with the particular application method of the substance(s) in the hive which corresponds to the instructions for use provided.
In particular embodiments of the invention, the kit comprises an external element that is destructible by the bees, and capable of carrying the substance(s) in such a way that the bees of the colony can detect the presence thereof when the element is introduced into the hive. This element is in particular suitable for being impregnated by the substance(s).
The element and/or the substance(s), in particular in the form of a composition containing same in a suitable vehicle, may be present in the kit separately, and intended to be assembled by the user himself, in particular the beekeeper, for example extemporaneously.
Alternatively, they may be present in the kit in a form wherein the element does already carry the substance, in such a way that the bees of the colony can detect the presence thereof when the element is introduced into the hive. In particular, the element may be present in the kit in a form preimpregnated by the substance(s) according to the invention.
The kit according to the invention may further comprise a reference element, also known as a gauge element, or reference gauge, indicative of the fact that the bee colony exhibits the Varroa destructor sensitive hygiene trait. This reference element is intended to be compared to the element carrying the substance(s) according to the invention that have been used according to the invention, i.e. that have been introduced into the hive, to determine, in particular visually or by comparative weighing, whether the bee colony tested carries the VSH trait or not. In particular, if it is observed that, after a defined residence time in the hive (this time being indicated in the instructions for use of the kit), the element tested has been destroyed by the bees in a proportion such that it is smaller, or lighter, than the reference element, it will be determined that the bee colony exhibits the VSH trait. If, on the other hand, it is observed that, after this residence time, the element tested has been destroyed by the bees in a proportion such that it is identical or larger, or heavier, than the reference element, it will be determined that the bee colony does not exhibit the VSH trait.
The features and advantages of the invention will emerge more clearly in the light of the examples of embodiments hereinafter, provided merely by way of illustration and in no way restrictive of the invention, with reference to figures 1 to 4, wherein:
- figure 1 shows a bar chart representing the response of bee colonies, expressed as the uncapping rate (plus or minus the standard error SE), by the bee colonies, after 48 hours of exposure to the following compositions, injected into the caps of hive frames: isohexane (Iso, negative control), dead pupa extract (NM, positive control), composition according to the invention (C1) at different dilution rates;
- figure 2 shows a bar chart representing the response of bee colonies, expressed as the uncapping rate (plus or minus the standard error SE), by the bee colonies, after 48 hours of exposure to the following compositions, injected into the caps of hive frames: isohexane (Iso, negative control), dead pupa extract (NM, positive control), and various compositions according to the invention, for different concentrations (3-fold higher concentrations of substances according to the invention in b/ than in a/);
- figure 3 shows a bar chart representing the response of bee colonies, expressed as the uncapping rate (plus or minus the standard error SE), by the bee colonies, after 48 hours of exposure to the following compositions, injected into the caps of hive frames: isohexane (Iso, negative control), dead pupa extract (NM1, positive control), and various compositions according to the invention (C1w, C22o, C3, CI30, C230, C4);
- and figure 4 shows graphs illustrating the results obtained for bee colonies tested in parallel for the SMR score, on one hand, and by the method according to the invention, on the other hand, the result then being expressed as the uncapping rate by the bee colonies after 48 hours of exposure to the composition according to the invention, injected into the caps of hive frames; for various compositions according to the invention: a/ CI10, b/ C220 and c/ C3.
MATERIALS AND METHODS
Method according to invention
Test design and preliminary validations
The substances / combinations of substances were solubilised in isohexane and exposed to the brood by injecting 1 pL of the composition obtained through the cap of cells containing white- to pinkish-eyed pupae using a Hamilton syringe.
Preliminary laboratory tests were used to check that injecting solvent through the cap (70 to 100 cells per sample) does not impede the survival of developing pupae. The survival rate was estimated by measuring the number of cells emerging at the end of metamorphosis. The frames were kept in a stove (34°C) after injection and until emergence. The control cells (with no injection) showed an emergence rate of 95.9% (± 4% - n = 6) and the perforated cells (control group wherein a hole was formed in the cap with the syringe) showed an emergence rate of 93% (± 7% - n = 3). 93.5% (± 6% - n =
6) of the cells injected with 1 pl_ of isohexane emerged, while merely 15% (± 12% - n = 3) of the cells injected with 5 pl_ of isohexane emerged.
The field tests were consequently carried out with the injection of 1 μΙ_ of composition into each cell.
This preliminary validation has also helped ensure that if a hygiene behaviour activity was recorded in the field test, it was not targeted on dead brood.
In order to check the age of the brood in the cells receiving the treatments and to ensure the homogeneity between the replicas of cells and colonies, on each brood frame sampled, some capped cells were opened to check that the brood was at the pupal stage, 3 to 6 days after capping. The tests were carried out on a total of 112 frames belonging to 46 different colonies.
On each frame, 15 or 30 capped cells were injected for each treatment condition. The position of each injected cell was marked on a transparent plastic sheet in order to track the uncapping/cleaning activity. 2, 4, 24 and 48 hours after reintroducing the frames into the original colonies thereof, the frames were checked for the uncapping or cleaning response. At the end of the 48-hour observation period, the total proportion of targeted cells was estimated.
Validation of the test with positive and negative controls
The first step consisted of the bio-test validation. The treatments were considered as negative controls if after injection they did not trigger more hygiene activity on the treated cells than on untreated cells. Three different negative controls were tested: perforated cells (cells injected with 1 pl_ of air), isohexane (cells injected with 1 pl_ of isohexane), non-parasite-infested pupa extracts (cells injected with 1 μΙ_ of non-parasite-infested purple-eyed stage pupa extract). None of the three controls triggered significant hygiene activity compared to the cells that had not been handled. The negative control using isohexane was chosen as the negative control for the subsequent experiments.
The validation of the behavioural field test also required identification of a biological extract containing odours to which bees may be naturally exposed in a colony, and which, once injected through the cap of the brood cells, would trigger a high level of hygiene activity. Preliminary tests demonstrated that injecting an extract obtained from 10 dead pupae prepared in isohexane triggered a quasi-systematic hygiene type response, with on average 85% of the treated cells being targeted. This response is significantly higher than that obtained for the negative control. Dead pupa extracts were therefore used as a positive control in the field test.
The proportion of injected cells targeted by the hygiene behaviour (uncapping and/or cleaning) was compared to the proportion of non-injected cells that were targeted by the behaviour during the same time frame. The negative controls included the injection of 1 μΙ_ of air, 1 μΙ_ of isohexane (Iso) or 1 μΙ_ of non-parasite-infested pupae (NP). The positive control consisted of 1 μΙ_ of dead pupa extract (NM). The NM extract was for this purpose prepared by immersing 10 dead pupae in 2 mL of isohexane for 10 min, retrieving the extract, then concentrating in a nitrogen flow and adding 1 pL of isohexane.
Preparation of compositions according to the invention
A plurality of compositions were prepared using different substances / mixtures of substances according to the invention. The compounds methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate were purchased from SigmaAldrich, and the compounds tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate and nonacosan-2-one were custom synthesised by Omega-Cat System (Rennes, France). The purity of the compounds varied between 94 and 99%. The identity of all the compounds was verified by gas chromatography coupled with mass spectrometry (GC-MS).
The compounds were solubilised in isohexane, except for 25 heptacosanone and 2-nonacosanone which were solubilised in a first step in cyclohexane. All the dilutions were carried out with pure isohexane, for all the compounds. The compounds and the mixtures were tested at various concentrations.
Evaluation of SMR trait
For 18 of the colonies used for the field test, the SMR trait was measured concomitantly. In each colony, a frame containing capped brood at a late development stage was sampled then dissected in the laboratory. The cells were opened carefully, under a binocular magnifier and the presence of Varroa was tested. When a cell was parasite-infested, the content of the Varroa family was accurately described in order to determine whether the founding female could have produced at least one fertilised female at the time of the emergence of the bee, according to the protocol described in the publication by Villa et al., 2016, cited hereinabove. This procedure was repeated until at least 35 infested cells were described, and the proportion of infested cells containing effectively breeding Varroa was calculated, so as to obtain the SMR score.
Statistical analyses
All the statistical analyses and the figures were generated in the R environment (Version 3.3.1). In the behavioural field tests, the colonies were considered as the statistical unit. The analyses were carried out using mixed linear models (LMEM - package Ime4). In the bio-test, the proportion of cells targeted by hygiene behaviour was analysed according to the treatments. In order to account for the fact that different treated cells could belong to the same colony, the identity of the cells in the experimental colonies was included as a random factor, and the treatment effect as a fixed explanatory variable. The relationship between the response obtained in the SMR test and in the method according to the invention was analysed using linear regressions (LM).
EXAMPLE 1 - mixture of substances according to the invention
Healthy pupae were exposed to chemical stimulus by a composition 5 according to the present invention, so as to evaluate cell uncapping/cleaning behaviour.
This composition, referenced C1, contains the following compounds, in the following proportions by weight, with respect to the total weight of these compounds in the composition: tricosan-2-one (4.8%), pentacosan-2-one (11.9%), tetracosyl acetate (11.9%), heptacosan-2-one (47.6%), hexacosyl acetate (9.5%) and nonacosan-2-one (14.3%).
For the stock composition (C1so), the total quantity of compounds in isohexane is 21000 ng/pL.
This composition is used at the following different dilutions in 15 isohexane: undiluted (CI50), diluted 1.67 fold (CI30), diluted 2.5 fold (CI20), diluted 5 fold (CI10), diluted 10 fold (CI5), diluted 50 fold (C11), and diluted 100 fold (CI0.5).
The response to the exposure of bee colonies to these different dilutions of the composition C1 was analysed. Each dilution was tested on 12 unrelated colonies.
By way of validation, isohexane alone (Iso), and the dead pupa extract positive control (NM) were also tested.
The injection of the composition triggers a dose-dependent response, as can be observed in figure 1, which shows the uncapping activity measured according to the dose of compounds injected (n = 12 colonies) (LMEM: χ2 = 87.08, p < 10-15).
The comparison of the response obtained for each dose of composition injected indicates that a significant response is obtained when dilution rates of 10 fold or less of the composition C1 are used. The highest tested dose triggers a similar response to that obtained with the positive control (“NM”).
In the subsequent tests, the composition C1 at dilution rates of 5 fold (C1w) and 1.67 fold (CI30) was used.
EXAMPLE 2 - Compounds according to the invention tested alone or in pairs
The following compounds: tricosan-2-one (TrCO), pentacosan-2-one (PCO), tetracosyl acetate (TCA), heptacosan-2-one (HPCO), hexacosyl acetate (HCA) and nonacosan-2-one (NCO), were tested separately. The tetracosyl acetate (TCA) I hexacosyl acetate (HCA) compound combination was also tested.
By way of comparison, the following were also tested: the composition C1 of Example 1 diluted 5 fold (CI10) or 1.67 fold (CI30), isohexane (Iso) and a dead pupa extract (NM).
The different compounds were placed in solution in isohexane, at the following concentrations:
- experiment a/ (33 colonies): 200 mg/l tricosan-2-one; 500 mg/l pentacosan-2-one; 500 mg/l tetracosyl acetate; 2000 mg/l heptacosan-2-one; 400 mg/l hexacosyl acetate; 600 mg/l nonacosan-2-one; combination of
500 mg/l tetracosyl acetate and 400 mg/l hexacosyl acetate (compared with the composition C110);
- experiment b/ (22 colonies): 600 mg/l tricosan-2-one; 1500 mg/l pentacosan-2-one; 1500 mg/l tetracosyl acetate; 6000 mg/l heptacosan-2-one; 1200 mg/l hexacosyl acetate; 1800 mg/l nonacosan-2-one; combination of
1500 mg/l tetracosyl acetate and 1200 mg/l hexacosyl acetate (compared with the composition CI30).
The results obtained, in terms of rate of uncapping by the bees are shown in figure 2, in a/ for experiment a/, and in b/ for experiment b/.
It is observed therein that relatively similar results were obtained in both experiments. There is a significant treatment effect (LMEM: x2ioeq = 366.75, p < 10-15; x23oeq = 247.28, ρ < 10’15) for all the compounds tested. This indicates that all of these compounds according to the invention may be used to evaluate the VSH trait of domestic bee colonies.
EXAMPLE 3 - mixtures of substances according to the invention
The following compositions were tested:
- composition C1 described in Example 1, at the dilution rates of 5 fold (Clio) and 1.67 fold (C1so);
- composition C2 containing, in isohexane: 900 mg/l methyl palmitate,
3000 mg/l ethyl palmitate, 900 mg/l methyl stearate, 900 mg/l methyl oleate, 2400 mg/l ethyl stearate, 600 mg/l ethyl oleate, 2100 mg/l ethyl linoleate and 300 mg/l methyl linolenate; undiluted (C23o) or diluted 1.5 fold (C22o);
- composition C3 containing, in isohexane: 200 mg/l tricosan-2-one,
500 mg/l pentacosan-2-one, 500 mg/l tetracosyl acetate, 2000 mg/l heptacosan-2-one, 400 mg/l hexacosyl acetate, 600 mg/l nonacosan-2-one, 600 mg/l methyl palmitate, 2000 mg/l ethyl palmitate, 600 mg/l methyl stearate, 600 mg/l methyl oleate, 1600 mg/l ethyl stearate, 400 mg/l ethyl oleate, 1400 mg/l ethyl linoleate and 200 mg/l methyl linolenate; undiluted;
- composition C4 containing, in isohexane: 600 mg/l tricosan-2-one,
1500 mg/l pentacosan-2-one, 1500 mg/l tetracosyl acetate, 6000 mg/l heptacosan-2-one, 1200 mg/l hexacosyl acetate, 1800 mg/l nonacosan-2-one, 900 mg/l methyl palmitate, 3000 mg/l ethyl palmitate, 900 mg/l methyl stearate, 900 mg/l methyl oleate, 2400 mg/l ethyl stearate, 600 mg/l ethyl oleate,
2100 mg/l ethyl linoleate and 300 mg/l methyl linolenate; undiluted.
For the compositions C1 w, C22o and C3, the number of colonies tested was 9. For the compositions CI30, C230 and C4, the number of colonies tested was 17.
By way of comparison, isohexane alone (Iso) and the dead pupa extract as described in Example 1 (NM) were also tested.
The results obtained, in terms of rate of uncapping by the bee colonies, are shown in figure 3.
As can be observed in this figure, for all the tested compositions 5 according to the invention, the treatment effect is significant (LMEM: χ2 =
204.25, p < IO’15).
EXAMPLE 4 - Comparison with the SMR score of the colonies tested
The method according to the invention, and the SMR test described by the prior art, were carried out in parallel on the same bee colonies.
The correlation between the response obtained in the reference SMR test (SMR score) and the response obtained by the method according to the invention (uncapping rate) was analysed.
For this purpose, the compositions C1w (18 colonies), C22o (17 colonies), and C3 (9 colonies) according to the invention, described in
Example 3, were used.
For each tested colony, there were calculated, on one hand, the SMR score obtained using the protocol described in the prior art, in the publication by Villa et al., 2016, cited hereinabove, and, on the other hand, the uncapping rate obtained according to the invention. The results obtained are shown in figure 4, in a/ for the composition C1w, in b/ for the composition C22o and in c/ for the composition C3.
It is observed that when the composition according to the invention C1w is applied, the association between the results of the method according to the invention and the SMR score is significant (LM: F = 14.49, p = 0.00155) and the degree of correlation is relatively high (r2 = 0.44) (Figure 4, a/).
When the composition C22o is applied, a significant linear relationship is also obtained (LM: F = 8.93, p = 0.008681) (Figure 4, b/).
The highest degree of correlation with the SMR score is detected for the composition C3 (LM: F = 14.29, p = 0.0069, r2 = 0.62, n = 9) (Figure 4, c/).
For each of the compositions according to the invention that were tested, it is observed that a reference value may be established, expressed in the form of a threshold uncapping rate, above which it can be determined, by comparison with the SMR score (of which it is known from the prior art that it is greater than 0.5 for bees carrying the VSH trait), that the tested bee colony carries the VSH trait.
For the composition C1w, this threshold rate is 63%, for the composition C22o, it is 55%, and for the composition C3, it is 62%.
Claims (16)
1. Method for determining whether a colony of bees of the Apis mellifera species of a hive has the Varroa destructor sensitive hygiene trait, characterised in that it comprises:
CLAIMS applying in the hive at least one substance chosen among: o tricosan-
2-one, o pentacosan-2-one, o tetracosyl acetate, o heptacosan-2-one, o hexacosyl acetate, o nonacosan-2-one o and a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate, so as to expose said bee colony to said substance,
15 - determining a response level of said bee colony to the exposure to said substance,
- and comparing the response level determined in this way with a reference response level indicative that said bee colony exhibits the Varroa destructor sensitive hygiene trait.
20 2. Method according to claim 1, wherein applying said substance in the hive is carried out by applying said substance on an element in the hive, and the response level of said bee colony to exposure to said substance is a level of destruction of said element by said bee colony.
3. Method according to any one of claims 1 to 2, wherein determining a response level of said bee colony to exposure to said substance is carried out after at least 1 hour, preferably at least 8 hours, of exposure of the bee colony to said substance.
4. Method according to any one of claims 1 to 3, wherein applying 5 said substance in the hive is carried out on a capped brood frame of the hive.
5. Method according to claim 4, wherein applying said substance in the hive is carried out by spraying said substance on said capped brood frame.
6. Method according to any one of claims 4 to 5, wherein the response level of said bee colony to exposure to said substance is a rate of
10 cells of the frame having been uncapped by the bee colony following said application, with respect to the total number of cells to which the substance was applied.
7. Method according to any one of claims 1 to 3, wherein applying said substance in the hive is carried out by introducing into the hive an external
15 element destructible by said bees and carrying said substance in such a way that the bees of said bee colony can detect the presence thereof in the hive.
8. Method according to claim 7, wherein the response level of said bee colony to exposure to said substance is a proportion of said element destroyed by said bee colony following said introduction.
20
9. Use of a substance chosen among:
- tricosan-2-one,
- pentacosan-2-one,
- tetracosyl acetate,
- heptacosan-2-one,
25 - hexacosyl acetate,
- nonacosan-2-one
- and a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate, to determine whether a colony of bees of the Apis mellifera species 5 has the Varroa destructor sensitive hygiene trait.
10. Composition characterised in that it contains at least one substance chosen among:
- tricosan-2-one,
- pentacosan-2-one,
10 - tetracosyl acetate,
- heptacosan-2-one,
- hexacosyl acetate,
- nonacosan-2-one
- and a mixture of methyl palmitate, ethyl palmitate, methyl stearate,
15 methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate, in a vehicle physiologically compatible with use in the presence of bees of the Apis mellifera species.
11. Composition according to claim 10, containing at least two 20 substances chosen among:
- tricosan-2-one,
- pentacosan-2-one,
- tetracosyl acetate,
- heptacosan-2-one,
25 - hexacosyl acetate,
- nonacosan-2-one
- and a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate.
12. Composition according to claim 10, containing at least one 5 substance chosen among tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate and nonacosan-2-one.
13. Kit for implementing a method according to any one of claims 1 to 8, characterised in that it comprises:
- at least one substance chosen among:
• tricosan-2-one, • pentacosan-2-one, • tetracosyl acetate, • heptacosan-2-one, • hexacosyl acetate, • nonacosan-2-one • and a mixture of methyl palmitate, ethyl palmitate, methyl stearate, methyl oleate, ethyl stearate, ethyl oleate, ethyl linoleate and methyl linolenate,
- and instructions for using said substance for determining whether a 20 colony of bees of the Apis mellifera species from a hive has the Varroa destructor sensitive hygiene trait.
14. Kit according to claim 13, comprising an element which is destructible by said bees and which is capable of carrying said substance in such a way that the bees of said bee colony can detect the presence thereof.
25
15. Kit according to claim 14, wherein said element does carry said substance in such a way that the bees of said bee colony can detect the presence thereof.
16. Kit according to any one of claims 14 to 15, comprising a reference element indicative that said bee colony exhibits the Varroa destructor sensitive hygiene trait.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR1754978A FR3066884B1 (en) | 2017-06-06 | 2017-06-06 | PROCESS FOR SELECTING COLONIES OF BEES PRESENTING THE VSH CHARACTER AND COMPOSITION AND KIT FOR ITS IMPLEMENTATION |
| FR1754978 | 2017-06-06 | ||
| PCT/FR2018/051295 WO2018224765A1 (en) | 2017-06-06 | 2018-06-05 | Method for selecting bee colonies having the vsh trait and composition and kit for implementing same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2018279230A1 true AU2018279230A1 (en) | 2020-01-16 |
Family
ID=59930473
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2018279230A Abandoned AU2018279230A1 (en) | 2017-06-06 | 2018-06-05 | Method for selecting bee colonies having the VSH trait and composition and kit for implementing same |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP3634133A1 (en) |
| AU (1) | AU2018279230A1 (en) |
| FR (1) | FR3066884B1 (en) |
| WO (1) | WO2018224765A1 (en) |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2638326B1 (en) * | 1988-11-03 | 1991-01-25 | Agronomique Inst Nat Rech | PROCESS FOR THE BIOLOGICAL CONTROL OF VARROATOSIS AND DEVICES FOR CARRYING OUT SAID METHOD |
| FR2692106B1 (en) * | 1992-06-16 | 1994-09-02 | Agronomique Inst Nat Rech | Process for modulating the behavior of worker bees by brood pheromones. |
| US6595828B2 (en) * | 2001-02-02 | 2003-07-22 | The Regents Of The University Of California | Synthetic bee pollen foraging pheromone and uses thereof |
| US7727517B2 (en) * | 2006-09-07 | 2010-06-01 | Contech Enterprises Inc. | Stabilized brood pheromone for manipulating the behavior and physiology of honey bees |
| WO2009059415A1 (en) * | 2007-11-07 | 2009-05-14 | Contech Enterprises Inc. | Slow-release device for delivering stabilized honey bee brood pheromone within the hive |
| EP2695517B1 (en) * | 2012-08-09 | 2019-02-06 | Interbran Nature GmbH | Pheromone composition for treating Varroa mite infestation |
| WO2017139261A1 (en) * | 2016-02-12 | 2017-08-17 | Bayer Cropscience Lp | Method for controlling parasitic mites |
-
2017
- 2017-06-06 FR FR1754978A patent/FR3066884B1/en not_active Expired - Fee Related
-
2018
- 2018-06-05 AU AU2018279230A patent/AU2018279230A1/en not_active Abandoned
- 2018-06-05 EP EP18749422.4A patent/EP3634133A1/en not_active Withdrawn
- 2018-06-05 WO PCT/FR2018/051295 patent/WO2018224765A1/en unknown
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| Publication number | Publication date |
|---|---|
| EP3634133A1 (en) | 2020-04-15 |
| FR3066884B1 (en) | 2020-12-18 |
| FR3066884A1 (en) | 2018-12-07 |
| WO2018224765A1 (en) | 2018-12-13 |
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