Patent Publication Number: US-2020281206-A1

Title: Sexual attraction pheromone of tribolium castaneum and insect control device

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims priority of U.S. Provisional Application No. 62/815,536 filed on Mar. 8, 2019 under 35 U.S.C. 119(e), the entire contents of which are hereby incorporated by reference. 
    
    
     TECHNICAL FIELD 
     The present invention relates to an insect sexual attraction pheromone and to a device useful for controlling pests, in particular insects, in grain silos. The device comprises an outer shell, a membrane impregnated with a sexual attraction pheromone extracted from  Tribolium castaneum  males and a nucleus comprising insecticide formulation. 
     BACKGROUND 
     Insect control devices with a “lure and trap” mechanism often include a luring composition such as an aggregation pheromone to attract insects, as well as an insecticide formulation or other pest control compounds. Several of these compounds may require carefully controlled conditions in order to activate the biological mechanisms involved in the insecticidal effect. For example, bioinsecticides based on dormant forms of several entomopathogenic fungi may be activated under adequate humidity conditions. These conditions may be obtained through the aggregation of a certain number of insects in the vicinity of these dormant forms. When the adequate conditions are met, the dormant forms activate, and the insecticidal effect is achieved. A sexual attraction pheromone, which targets only insects of a certain sex, can be advantageously used to achieve these controlled conditions. 
     4,8-dimethyldecanal, also known as tribolure, is an aggregation pheromone of  Tribolium castaneum . Although it has been mistakenly reported as a “sexual pheromone”, it has been found that this molecule attracts both male and female insects, therefore not constituting a true sexual pheromone. 
     There is therefore a need to provide a sexual pheromone of  Tribolium castaneum  useful for insect control devices employing a luring composition. 
     SUMMARY OF THE INVENTION 
     In a first aspect, an object of the present invention is an isolated sexual attraction pheromone of  Tribolium castaneum  that is exclusively obtained from male insects. The pheromone has an average molecular weight of approximately 210 g/mol, a molecular formula of C 14 H 26 O and a chemical structure having only one degree of unsaturation and a terminal carbonyl group. 
     In a second aspect, an object of the present invention is a method to obtain an isolated sexual attraction pheromone from a plurality of male insects, comprising the steps of:
         placing the plurality of male insects in an airtight container comprising a solid support,   placing and maintaining the container on a stove to generate volatile organic compounds,   separating the plurality of male insects from the solid support, extracting the volatile organic compounds from the solid support using solid-phase microextraction fibers, and   isolating of the pheromone from the volatile organic compounds by capillary gas chromatography.       

     In a preferred embodiment of the second aspect, said solid support is selected from filter paper and a feed medium comprising flour. Preferably, the feed medium comprises about 30 g of flour. 
     In another preferred embodiment of the second aspect, the container is maintained on a stove for a period of approximately one month. 
     In a third aspect, it as an object of the present invention an insect control device comprising:
         an outer shell comprising a plurality of orifices,   a central membrane impregnated with a luring composition, and   an active nucleus comprising an insecticide formulation.       

     In a fourth aspect, it as an object of the present invention an insect control device comprising:
         an outer shell comprising a plurality of orifices,   a central membrane impregnated with the isolated sexual attraction pheromone of  Tribolium castaneum  of the first aspect of the invention, and   an active nucleus comprising an insecticide formulation.       

     In preferred embodiments of the third and fourth aspects, said membrane is comprised in a capsule attached to the outer shell. 
     In preferred embodiments of the third and fourth aspects, said capsule further comprises a conical surface. Preferably, said conical surface has a smooth, polished and shining surface. 
     In further preferred embodiments of the third and fourth aspects, the insecticide formulation comprises an effective amount of isolated microsclerotia of  Beauvaria bassiana  and an agronomically acceptable carrier. Such microsclerotia are described in U.S. patent application No. 62/815,541, the contents of which are incorporated by reference herein. 
     In yet further preferred embodiments of the third and fourth aspects, the active nucleus is located in a lower portion of the device. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a chromatograph with the main components of male  Tribolium castaneum  extracts. 
         FIG. 2  shows the attraction of female insects to a paper impregnated with the sexual pheromone of the present invention. 
         FIGS. 3 to 10  show embodiments of the insect control device of the present invention as well as its individual components. 
         FIG. 11  shows the results of the use of the insect control device of the present invention in a laboratory-scale silo containing 5 kg of wheat. 
         FIG. 12  shows the results of the use of the insect control device of the present invention in a pilot-plant scale silo containing 20 kg of wheat. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention will be described in detail below, with reference to the figures and examples, which are included only for the purpose of illustrating the invention and are not to be construed as limiting thereof. 
     The term “approximately” as used herein when referring to a measurable value means that it comprises variations of ±10% from the specified amount. 
     As used herein, the terms “comprises”, “has” and “includes” and their conjugations mean “including but not limited to”. 
     The sexual pheromone of the present invention is biosynthesized exclusively by male  Tribolium castaneum  insects and thus constitutes a true sexual pheromone, as opposed to those of the prior art. 
     The insect control device of the present invention operates by a “lure and trap” mechanism. In an embodiment, insects are attracted and aggregate into the inside of the device, by crawling through the orifices of the outer shell. In an embodiment of the device, when a certain number of insects has entered the device, the resulting humidity conditions activate a bioinsecticide formulation. In the case of bioinsecticides comprising microsclerotia, adequate humidity conditions will cause the proliferation of conidia from the microsclerotia. 
     In this manner, the initial charge of bioinsecticide can be activated several times and in response to the entrance and exit of insects into the device. 
     Alternatively, when a single insect or a lower number of insects enter the device, they become impregnated with a bioinsecticide in dormant form (i.e. microsclerotia). This form will yield a large number of conidia when the humidity conditions are met, for example in the insects&#39; nests. 
     Examples 
     1) Extraction of  Tribolium castaneum  Sexual Pheromone 
     Insects 
     Specimens of pest insects of stored grains,  Tribolium castaneum , were employed. The insects were bred in 500 mL glass jars covered with a fine cloth net and kept in a breeding chamber at 27±2° C. and 70±5% relative humidity, with a light:darkness photoperiod of 12 h/12 h. 
     The insects were fed a mixed diet of wheat flour (85%), brewer&#39;s yeast (5%), wheat germ (5%) and skimmed milk powder (5%). 
     During the pupa stage, males and females were separated taking into account the morphological differences of both sexes and were kept in the diet until adult molting, thus obtaining virgin insects. 
     For the extraction of the pheromone of  Tribolium castaneum , pools of 200 male insects were employed, which were placed in individual 200 mL containers with 30 grs of flour and kept in the stove in the above-mentioned conditions for a period of 1 month. Finally, the insects were separated, and 90 grs of flour were placed in 250 mL Erlenmeyer flasks sealed with a Teflon lid with a septum to aid the extraction of volatile organic compounds (VOCs). 
     Extraction in Solid Phase 
     The VOCs released by  Tribolium castaneum  males were extracted from the head space of the Erlenmeyer flasks containing the flour using solid-phase microextraction fibers (SPME) of intermediate polarity composed of polydimethylsiloxane/divinylbenzene and the sample was taken for 60 minutes at room temperature (in a temperature range of 25 to 30° C.). 
       FIG. 1  shows the main components of the VOCs released by male and female  Tribolium castaneum , including defense pheromones (benzoquinones) and aggregate pheromones (4,8-dimethyldecanal). The detector signal corresponding to the sexual pheromone of the present invention is only present in male insects. 
     Isolation of the Sexual Pheromone by Capillary Gas Chromatography 
     The VOCs extracted by SPME were analyzed and isolated by capillary gas chromatography (CGC). A Hewlett Packard 6850 gas chromatograph with a DB-5 non-polar capillary column was used. The injector was operated in splitless mode at 250° C., He gas was used as a carrier at constant flow rate of 1.5 mL/min, the furnace temperature was programmed at 40° C. for 0 min, 50° C./min at 100° C., 4° C./min at 220° C., with a retention time of 5 min at the final temperature. The flame ionization detector (FID) temperature was set to 280° C. This equipment is fitted with a microcontrol valve that allowed part of the analyzed sample to be diverted to the outside of the chromatograph, which is coupled with a transfer line maintaining the temperature of the column at 220° C. The fraction of interest was collected in a 4 mL flask at 0° C. The purity of the compound was verified using a Hewlett Packard 6890 chromatograph equipped with an infernal Zebron-5HT capillary column (30 m×0.25 mm I.D., F.T. 0.25 μm, Phenomenex, USA), in the above-mentioned conditions. The CGC was operated coupled to an Agilent 5975C VL mass selective detector (MSD). The MSD was operated in SCAN mode with a mass range of 35 to 650 u.m.a.; in electronic impact mode at 70 eV; transfer line at 320° C.; ionization chamber temperature at 230° C. and quadrupole at 150° C. The MSD was operated in SCAN mode with a mass range of 35 to 650 u.m.a.; in electronic impact mode at 70 eV; transfer line at 320° C.; ionization chamber temperature at 230° C. and quadrupole at 150° C. 
     Characterization of the Sexual Pheromone 
     Preliminary mass spectrometry analysis revealed a molecular weight of 210 g/mol, a twelve-carbon unsaturated backbone and at least one methyl-branch. 
     Further analysis provided a molecular formula of C 14 H 26 O and a chemical structure having only one degree of unsaturation and a terminal carbonyl group. 
       FIG. 2  shows the attraction of female insects to a paper impregnated with a solution comprising the extracted male pheromone. 
     2) Insect Control Device 
       FIGS. 3 to 10  show different configurations of the device of the present invention, representing exemplary and non-limiting embodiments of the device and/or individual components thereof. 
     The device ( 1 ) of the present invention comprises of the present invention an outer capsule ( 2 ) and an inner capsule ( 3 ). The capsules are coupled to each other, and an active nucleus ( 4 ) comprising an insecticide formulation is placed between the two capsules. The coupling of the capsules is by means of shape and are arranged concentrically. In this manner, the insect which is crawling in the interior of the device ( 1 ) is forced to go through the inner capsule ( 3 ) in order to reach the active nucleus ( 4 ) comprising the insecticide. 
     The outer capsule ( 2 ) defines the outer shell ( 5 ) of the device, which is provided with a plurality of orifices ( 6 ), so that insects may crawl into or out of the device. 
     The inner capsule ( 3 ) may be open or closed. Open capsules allow the insect to freely crawl about it, entering and exiting the capsules without retention. Closed capsules are provided with a trap ( 7 ) at the entrance zone ( 8 ) to the active nucleus ( 4 ). This trap allows the insect to remain in contact with the inner capsule and can be achieved by means of a conical surface ( 9 ) placed inside the inner capsule. This conical surface ( 9 ) allows insects crawling around the active nucleus, aided by the roughness of the external surfaces, until they reach the conical surface ( 9 ). Once inside the conical surface ( 9 ), the insect will remain trapped inside due not only to the shape and the complexity of the reverse path, but to the smooth, polished and shining surface of the conical surface ( 9 ). In this manner, insects remain in the interior of the inner capsule ( 3 ) until they are removed or liberated. 
     The capsules are provided with fit and lock mechanism ( 10 ) in their perimeters, allowing their coupling to each other. The closing of the device is ensured by flexible fasteners ( 11 ), thus obtaining semi-permanent couplings. 
     The device of the present invention comprises a membrane ( 12 ), located near the center of the interior of the device, impregnated with a substance acting as a lure mechanism. For example, the isolated sexual pheromone of the present invention may be used. Due to its central location, the membrane will generate an adequate concentration gradient of the substance in the surrounding air by diffusion and natural convection, thus promoting the luring effect. 
     Insecticides that may be used for the active nucleus comprise bioinsecticides, such as dormant forms as antipathogenic fungi, such as  Beauveria bassiana  microsclerotia. Since these dormant forms require controlling humidity conditions in order to ensure a controlled release, their combination with the sexual pheromone of the present invention, which only attracts female insects, is advantageous to achieve a controlled and efficient release of an insecticide formulation. 
     3) Use of the Insect Control Device in a Laboratory Scale Silo 
       FIG. 11  illustrates the attraction effect of a luring composition comprising the isolated sexual pheromone of the present invention obtained from  Tribolium castaneum  male insects in two laboratory-scale silo containing 5 kg of wheat of the  audaz  variety. 
     Two sets of experiments were carried out: each set consisted of a “control” silo, containing an insect control device without any luring composition, and an “attraction” silo, containing the insect control device of the present invention, i.e. with a luring composition comprising the isolated sexual attraction pheromone of male  Tribolium castaneum.    
     The left hand side of  FIG. 11  shows the results for the distributions of insects for different positions inside the control and model silos containing  Sitophilus  sp insects. It can be seen that for the control species  Sitophilus  sp., both the control and the attraction silos have equivalent insect distributions. 
     The right hand side  FIG. 11  shows the corresponding results for silos containing  Tribolium castaneum  insects. In this case, it is observed that this species is distributed such that the number of insects closer to the insect control device is higher, which illustrates the successful and specific attraction of  Tribolium castaneum  insects using the luring composition of the present invention. 
     4) Use of the Insect Control Device in a Pilot-Plant Scale Silo 
     The insect control device was further tested in pilot-plant scale silos containing 200 kg of wheat of the  audaz  variety. 
     Two experiments were carried out: the first in a “control” silo, containing six insect control devices, each one without any luring composition, and the second in an “attraction” silo, containing six insect control devices of the present invention, i.e. each one with a luring composition comprising the isolated sexual attraction pheromone of male  Tribolium castaneum.    
       FIG. 12  shows the results for the distributions of insects for different positions inside the control and model silos containing  Tribolium castaneum  insects. The graph in  FIG. 12  represents the percentage of insects found in different areas of the silo, as defined by three radial sections in the silo, corresponding to 0 cm, 15 cm and 25 measured from the center of the silo, as well as 8 longitudinal sections from the bottom of the silo to the loading surface. 
     When comparing the obtained results for the distribution of female  Tribolium castaneum  insects, it can be observed that the insects are attracted to the part of the “attraction” silo containing the insect control devices, as evidenced by the distribution in the right hand side of the graph in  FIG. 12 , and compared to the distribution in the left hand side of the graph in  FIG. 12 , corresponding to the experiment in the “control” silo (i.e. insect control devices without any luring composition).