Touch-responsive targeted miticides delivery system, methods and composition for treating varroa mites, other pests and pathogenic microbes in honeybee hives

Abstract

This disclosure relates to the inventive concept of a pesticide / miticide / acaricide delivery device, Varr-Gate technology, that is touch-responsive and can precisely deliver miticide formulation in aqueous or viscous form directly targeting Varroa mites on the body of honeybees as they enter the hive. In particular aspects, the inventive concept provides a touch-responsive miticidal delivery device, wherein the device is a plastic structure comprising of hollow microtubular bristles hanging from the ceiling of the entrance tunnel comprising a liquid miticide droplet composition comprising NoPest-Ag5 in aqueous or viscous form and derivatives having pesticidal utility against Varroa mites, and pathogenic bee microbes such as viruses, bacteria and fungi, methods to produce such system, miticidal compositions containing such molecules, and processes of using such miticidal compositions against such pests and microbes. The device in combination with such miticidal compositions may be used to manage honeybee colonies against such pests and microbes.

Description

Attorney Docket No. 190412-00036WOTouch-Responsive Targeted Miticides Delivery System, Methods and Composition for Treating Varroa Mites, Other Pests and Pathogenic Microbes in Honeybee HivesCopyright

[0001] This disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.Field

[0002] The present inventive concept provides a touch-responsive targeted pesticide delivery system, methods and nanotechnology-based pesticide / miticide / acaricide composition that is effective against Varroa mites and pathogenic honeybee viruses and bacteria.Background

[0003] The European honeybee, Apis mellifera L. (Hymenoptera: Apidae), is a pollinator that exists on all continents except Antarctica (Chesapeake Bay Program, 2022). Although popularized for their production of honey for human consumption, honeybees are also responsible for plant pollination, including popular food staples such as apples, cucumbers, and sweet potatoes (FDA, 2018). In fact, the United States Food and Drug Administration (FDA) (2018) estimates that approximately 1 / 3 of the food that Americans consume is a direct or indirect result of honeybee pollination. These pollinators also produce useful byproducts including propolis, beeswax, and venom, all of which have a variety of cosmetic and household uses (Kurek-Gorecka et al., 2020).

[0004] Recent years have been marked by a general decline in honeybee populations worldwide, as well as an increase in colony losses reported by beekeepers (Woods, 2021 ). In the United States, a 43% colony loss was reported from April 2019 to April 2020 with Varna mites playing a major role in the loss (Insolia et al., 2022). The 2023-2024 US Beekeeping Survey indicated a 55.1% loss of managed honeybee colonies from April 2023 to April 2024 (Giacobino et al., 2024). The US entomologists have projected even higher losses for 2025, with commercial colony losses potentially reaching 60% to 70% (Sarnoff, 2025), a significant jump from the typical 40% to 50% range in previous years (Giacobino et al., 2025). These high colony losses translate to significant economic impacts for beekeepers and the agricultural industry. The decline of honeybees, as observed in recent years, has prompted increased awareness and conservationAttorney Docket No. 190412-00036WO efforts to ensure their survival and the vital role they play to maintain ecological balance of various ecosystems (Bartlett, 2022; Insolia et al., 2022). As ecosystems become disrupted due to honeybee loss, there may be unintended consequences, such as an increase in pests and invasive plant species (Hristov et al., 2020; Bartlett et al., 2022). Although many factors (e.g., habitat loss and pesticide-related exposures) have contributed to the global decline in honeybee populations, the most threatening stressor is the Varroa mite, Varroa destructor Anderson and Trueman (Parasitiformes: Varroidae) (Smitley et aL, 2019). Varroa destructor is an ectoparasite that targets adult honeybees and their brood. It infests honeybee colonies and feeds on fat body tissues of adult and larval bees, thus weakening their immune system. Infestation can lead to significant negative consequences in bees, including reduced lifespan, compromised metabolism, suppressed reproductive capabilities, and increased susceptibility to viral infections such as deformed winged virus (DWV) (Family Iflaviridae) and acute honeybee paralysis virus (ABPV) (Family Dicistroviridae) (Martin et al., 2012; Moore et al., 2014). Moreover, Varroa mites can cause indirect harm to honeybees by: (i) reducing the colony's overall population, which impacts foraging and pollination capabilities, (ii) decreasing honey production due to depleted foraging efforts and compromised health of worker bees, and (iii) weakening colonies and making them more susceptible to additional environmental stressors. These mites reproduce quickly and can overwhelm a honeybee colony in about six months (Page, 1998).

[0005] Apis mellifera is vulnerable to Varroa mite infestation (Traynor et al., 2020). Control of Varroa mites is challenging as they are resistant to commonly used acaricides (Underwood & Lopez-Uribe, 2019). Although mechanical and cultural control methods exist, research on the efficacy of such methods is limited and varied. Researchers globally have screened compounds for mite toxicity, yielding promising candidates for field tests. However, high costs hinder further testing, raising doubts about exploring new chemistries and efficacy. Many chemicals targeting other pests exist in the market but are untested on Varroa or honeybees. Despite this, chemical screenings remain valuable, but more targeted approaches are needed to increase the likelihood of adding new treatments for beekeepers (Fassbinder et aL, 2002; Lin et aL, 2020; Jack and Ellis, 2021 ).

[0006] The global pesticide market was valued at US $104.7 Billion in 2021 and is forecasted to show a lucrative 12.1% CAGR over 2022-2030, reaching a valuation of US $291 .4 Billion by 2030 (RND, 2022). Potential customers for NoPest-Ag5 include small- and large-scale honeybee farmers or apiarists, ranchers, individual honeybee hobbyists / enthusiasts, bee researchers, pesticide contractors / applicators, and state and federal agencies interested in protecting declining honeybee populations.Attorney Docket No. 190412-00036WO

[0007] Imperative is to effectively manage Varroa mite infestations to safeguard the diminishing honeybee populations in the US and globally. The value of pollinated crops stands at approximately $577 billion annually; however, the escalating decline in honeybees poses a significant threat to global food security and could result in widespread food scarcity and malnutrition. Additionally, Varroa mites serve as vectors for many pathogenic viruses, including DWV, SBV, ABPV, beeMLV, IAPV, BQCV, KBV, among others, further contributing to managed colony losses. Moreover, current miticides have significant limitations because they are either less effective as Varroa mites have developed resistance (e.g., amitraz, fluvalinate and coumaphos) or miticides such as formic acid and thymol are ineffective outside certain environmental conditions, while other miticides (hop beta acid) are toxic to brood and adult bees and can also contaminate honey posing health risk to humans.

[0008] In addition to the aforementioned limitations of the current product offerings, there are no touch- or contact-responsive hive gate / entrance technology in the market that would precisely deliver a liquid droplet of miticide(s) / acaricide(s) into the hive for the control of Varroa mites, other bee pests (e.g., black beetles, wax moth), and pathogenic microbes (bacteria, viruses, and fungi).Summary

[0009] To address this imminent need for safeguarding globally declining honeybee populations, an innovative bee touch-responsive delivery system, known as Varr-Gate technology, has been developed, which includes a gate, or an entrance / entrance tunnel designed (with uniformly spaced orifices and / or microtubular bristles) to precisely release a honeybee-safe Va / roa-active nanopesticide upon a bee’s touch directly onto the body, e.g., onto the dorsum or ventral portion of the body, of the bees, such as honeybees, where varroa mites are often located. A representative schematic rendition of the bee touch-responsive delivery system, suitable for installation / disposal at the entrance of any standard beehive, is presented in Figure 1 (top panel), and representative photographs of the prototype are presented in Figure 1 (bottom panel).

[0010] When installed at the entrance of a standard beehive, the delivery system, including an entrance / gate tunnel, or a plurality of entrance / gate tunnels, releases a microdroplet of a nanopesticide that can adhere to the body of the bees as they pass through the delivery system entrance / gate tunnel, and effectively kills both the Varroa mites and any associated bee viruses. Embedded in an aqueous or a viscous hydrogel formulation, l / arraa-active nanopesticide, NoPest-Ag5, or any other current miticides / acaricides (e.g., oxalic acid, formic acid, thymol, fluvalinate, etc.), is released through the end of the microcapillary tubules or bristles triggered byAttorney Docket No. 190412-00036WO bee’s touch. Such microcapillary tubules or bristles would hang from the ceiling of the entrance / gate tunnel. Alternately, aspects of the delivery system may include regularly spaced orifices directly on to the ceiling wall of the entrance tunnel may serve the same purpose. The pesticide can be supplied through a tank or multiple tanks into the entrance via suitable sized microtubings and the miticides can be replenished by the beekeeper as desired.

[0011] Furthermore, the miticide, such as NoPest-Ag5, i.e., amino-functionalized silver nanoparticles; NH2-AgNP; (US Patent Application No. 17 / 759,260, incorporated herein by reference in its entirety), is proven to be highly potent against the Varroa mite at a low-dose of 5 pg / mL, is nontoxic to honeybees (100% survival) and non-irritant to eyes (EPA Class IV Nonirritant). It can be delivered via the bee touch-responsive delivery system of the inventive concept in the form of an aqueous or a viscous hydrogel droplet as the bee touches the microtubular bristles while traveling through the entrance tunnel into the hive. The droplet release is triggered by the touch of the bee, while excess droplet release control is a function of several factors in play: surface tension of the droplet at the end of the microcapillary tube, gravitational force, internal and outer diameters of the capillary tube, including the viscosity of the pesticide formulation (aqueous, or viscous hydrogel, or viscous aerogel).

[0012] This bee touch-responsive delivery system of the inventive concept precisely delivers a l / a / roa-active nanopesticide, and is a breakthrough innovation that is sustainably advantageous as it allows for effective mite control, while avoiding unwanted environmental release of the nanopesticide and, unlike conventional miticides, the l / arraa-active nanopesticide used in aspects of the inventive concept is safe to brood cells (larvae and pupae) and adult bees and does not leave residue in honey and combs. Therefore, there is a significant market opportunity for this novel delivery system technology, that would not only protect the precipitously declining honeybee populations from the parasitic Varroa mites, but would also protect over 130 fruits and vegetables that directly depend on honeybee pollination.

[0013] Accordingly, in an aspect of the inventive concept, provided is a pesticide / miticide / acaricide delivery system comprising a hard or semi-hard structure configured to fit the entrance / exit of a beehive, wherein the structure comprises: at least one entrance tunnel; at least one exit tunnel, wherein each entrance tunnel comprises at least one microcapillary tube / bristle hanging from the ceiling of the entrance tunnel into the lumen of the entrance tunnel that is connected to a reservoir for the pesticide / miticide / acaricide disposed atop of the entrance tunnel.

[0014] In another aspect of the inventive concept, provided is a method delivering a pesticide / miticide / acaracide to a bee / bees comprising providing at least one entrance tunnel to aAttorney Docket No. 190412-00036WO beehive, wherein each entrance tunnel comprises at least one microcapillary tube / bristle hanging from the ceiling of the entrance tunnel into the lumen of the entrance tunnel that is connected to a reservoir for the pesticide / miticide / acaricide disposed atop of the entrance tunnel, and wherein the bee / bees must pass through the entrance tunnel to enter the beehive.Brief Description of the Drawings

[0015] FIG. 1. Schematic depiction of bee touch-responsive Varr-Gate technology to precisely deliver honeybee-safe l / arraa-active miticide (shown as dots on the bee’s dorsum) as the bee passes through the entrance tunnel into the hive (top panel). Images showing a working prototype of Varr-Gate technology placed at the entrance of the hive and bees actively entering and exiting the Varr-Gate technology (bottom panels).

[0016] FIG. 2. Transmission electron microscopy (TEM) micrographs of NoPest-Ag5 (panels A and B) showing spherical particle morphology with mean particle diameter of 5.8 nm ± 2.8 nm (N=807) (panels B and D), and EDS (Energy Dispersive X-Ray Spectroscopy) spectrum showing NoPest-Ag5 is primarily composed of elemental / metallic silver (Ag°) (panel C). The fuzzy fairly uniform capping layer (shown with paired white dashes) on the surface of Ag° core is primarily composed of amino (-NH2) functional group (panel B), with a trace amount of ‘intermediate state’ of amide (C=O-N) within the metal surface (ref. [Example 4]).

[0017] FIG. 3. NoPest-Ag5: X-ray photoelectron spectroscopy (XPS) high resolution scan of Ag (3d) and N (1s). Panel A. XPS high resolution scan of NH2-AgNPs shown: typical asymmetric well-separated peaks of elemental / metallic silver (Ag°) with primary Ag (3d) peak around 367.46 eV and loss features observed to higher binding energy side of each spin-orbit component for Ag metal (arrows). Panel B. Primary N (1 s) peak of amino (NH2-) functional group at 398.47 eV, with a minor secondary peak at 401 .2 eV indicative of trace level of 'intermediate state' of amide (C=O- N) on the AgNPs surface.

[0018] FIG. 4. Mean survival of Varroa destructor with NoPest-Ag5 treatments, showing 100% death by 24 hours and 60% death by 60 minutes with a single treatment of 5 pg / mL.

[0019] FIG. 5. NoPest-Ag5 mechanisms of action: NoPest-Ag5 downregulated several important genes in Varroa destructor mites with a single treatment of 5 pg / mL NoPest-Ag5 (5.0 NP). Higher Ct value denotes down regulation and lower Ct value denotes up regulation. NP denotes NoPest-Ag5 in pg / mL.

[0020] FIG. 6. NoPest-Ag5 completely neutralized deformed winged virus (DWV) and sacbrood virus (SBV) with no resurgence of the viruses occurred for 60-day study period with aAttorney Docket No. 190412-00036WO single treatment of 5 ig / mL NoPest-Ag5. Higher Ct value denotes down regulation and lower Ct value denotes up regulation. ND denotes not detected.

[0021] FIG. 7. Comparison of NoPest-Ag5 attributes with three widely used miticides for Varroa mite control.

[0022] FIG. 8. Schematic depiction of the bee touch-responsive pesticide / miticide / acaracide delivery system according to exemplary embodiments of the inventive concept.

[0023] FIG. 9. Schematic depiction of bee touch-activated miticide release in the pesticide / miticide / acaracide delivery system according to exemplary embodiments of the inventive concept.

[0024] FIG. 10. The left panel of FIG. 10 depicts an image showing placement of the bee touch-responsive pesticide / miticide / acaracide delivery system with entrance tunnels on the left. The right panel of FIG. 10 depicts an image showing placement of the bee touch-responsive pesticide / miticide / acaracide delivery system with entrance tunnels on the right.

[0025] FIG. 11 . Image of an embodiment of a pesticide / miticide / acaracide delivery system including two entrance tunnels on the left side and two entrance tunnels on the right side, while the exit tunnels are placed / disposed in the middle section. Also shown are oblique placements of endoscope cameras and UV lights, alongside a pesticide reservoir.

[0026] FIGS. 12-14. Images of pesticide / miticide / acaricide distribution on the body of honeybees delivered by Varr-Gate technology. Oxalic acid (OA; 18.4%), a conventional miticide / acaricide, was used in aqueous formulation and was mixed with riboflavin (Vitamin B2) as an UV-responsive dye that fluoresces yellow green under UV illumination.Detailed Description

[0027] The present inventive concept will now be described with reference to the following embodiments. As is apparent by these descriptions, this inventive concept can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the inventive concept to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant inventive concept.Attorney Docket No. 190412-00036WO

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this inventive concept belongs. The terminology used in the description of the inventive concept herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the inventive concept.

[0029] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0030] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Additionally, as used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as 7".

[0031] The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0032] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).

[0033] The term "comprise," as used herein, in addition to its regular meaning, may also include, and, in some embodiments, may specifically refer to the expressions "consist essentially of" and / or "consist of." Thus, the expression "comprise" can also refer to, in some embodiments, the specifically listed elements of that which is claimed and does not include further elements, as well as embodiments in which the specifically listed elements of that which is claimed may and / or does encompass further elements, or embodiments in which the specifically listed elements of that which is claimed may encompass further elements that do not materially affect the basic and novel characteristic(s) of that which is claimed. For example, that which is claimed, such as a composition, construct, formulation, method, system, etc. "comprising" listed elements also encompasses, for example, a composition, construct, device, formulation, method, system, etc.Attorney Docket No. 190412-00036WO"consisting of," i.e., wherein that which is claimed does not include further elements, and a composition, construct, device, formulation, method, system, etc. "consisting essentially of," i.e., wherein that which is claimed may include further elements that do not materially affect the basic and novel characteristic(s) of that which is claimed.

[0034] The term "about" generally refers to a range of numeric values that one of skill in the art would consider equivalent to the recited numeric value or having the same function or result. For example, "about" may refer to a range that is within ± 1 %, ± 2%, ± 5%, ± 10%, ± 15%, or even ± 20% of the indicated value, depending upon the numeric values that one of skill in the art would consider equivalent to the recited numeric value or having the same function or result. Furthermore, in some embodiments, a numeric value modified by the term "about" may also include a numeric value that is "exactly" the recited numeric value. In addition, any numeric value presented without modification will be appreciated to include numeric values "about" the recited numeric value, as well as include "exactly" the recited numeric value. Similarly, the term "substantially" means largely, but not wholly, the same form, manner or degree and the particular element will have a range of configurations as a person of ordinary skill in the art would consider as having the same function or result. When a particular element is expressed as an approximation by use of the term "substantially," it will be understood that the particular element forms another embodiment.

[0035] The term "bee" or "honeybee" as used herein generally refers to pollinator bees, such as Apis mellifera, Apis cerana, Apis florea, Apis andreniformis, among others, but is not specifically limited thereto, and any other pollinator bees belonging to, for example, the families: Apidae, Halictidae, Megachilidae, Andrenidae, Colletidae, Melittidae, and Stenotritidae, may be considered as part of and under the scope of embodiments of the inventive concept without limitation.

[0036] The present inventive concept provides a pesticide / miticide / acaricide delivery system, called Varr-Gate technology, that upon triggering by a bee’s touch delivers a liquid microdroplet of miticide, such as NoPest-Ag5 (amino-funtionalized silver nanoparticles, NH2-AgNPs), on to the dorsal part of honeybee body, that precisely targets the Varroa mites and effectively killing them within one day.

[0037] Embodiments of the present inventive concept provide a bee touch-sensitive delivery system that offers the following competitive advantages against the current market offerings: i. Bee touch-responsive to precisely deliver novel highly effective miticide / acaricide, NoPest-Ag5, on to the body of honeybee where Varroa mites are located; ii. Can also precisely deliver any other miticides / acaricides in liquid formulation;Attorney Docket No. 190412-00036WO iii. Fits standard 10- or 8-frame hives and 7-frame flow hives; iv. Bee-safe and weather-proof materials ensure durability; v. Tank can be replenished by beekeeper as and when desired; vi. Can be fitted with endoscope camera(s) connected to cell phone via Bluetooth to observe bee activities in real time; vii. Can prevent the hive from swarming as it limits mated queen bee escape; viii. Ecofriendly as it avoids off-target environmental release of miticide; ix. Prevents other pests such as skunks, wasps, hornets and mice from robbing the hive; x. Treats Varroa mites and many pathogenic bee viruses such as deformed winged virus Type A-C (DWV A-C), sacbrood virus (SBV), acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), and Black queen cell virus (BQCV), among others, and possibly treats American foulbrood (Paenibacillus larvae), European foulbrood (Melissococcus plutonius), chalkbrood (Ascosphaera apis), and Nosema spp.

[0038] The dimensions of the touch-sensitive system s / devices of the inventive concept are not particularly limited. Nevertheless, in some embodiments, the systems / devices are configured to precisely fit the entrance / exit of any standard beehive as would be appreciated by one of skill in the art without limitation. Exemplary beehives include vertical and horizontal hives, such as, but not limited to, the Langstroth hive, the top-bar hive, the long box hive, the BeeCastle hive, and the Warre hive, etc. In some embodiments, traditional beehives may include, for example, 5- frame, 7-frame, 8-frame, and 10-frame beehives, such as, but not limited to, 5-frame, 7-frame, 8- frame, and 10-frame Langstroth beehives.

[0039] In some embodiments, exemplary construction and dimensions of the delivery system / devices of the inventive concept may include a hard or semi-hard plastic structure having a length between about 9” to 16”, width / depth between about 2” to 3”, a first half / portion having a height between about 1.5” to 4”, and a second half / portion having a height between 1 ” to 2”. In some embodiments, the first half / portion, e.g., a half / portion including entrance openings, may be on the left / lefthand side when looking at / viewing the system installed on a beehive, and the second half / portion, e.g., a half / portion including exit openings, may be on the right / righthand side when looking at / viewing the system installed on a beehive. In some embodiments, the first half / portion, e.g., a half / portion including entrance openings, may be on the right / righthand side, and the second half / portion, e.g., a half / portion including exit openings, may be on the left / lefthand side when looking at the system installed / disposed on a beehive. In some embodiments, the firstAttorney Docket No. 190412-00036WO half / portion and the second half / portion of the structure may be of the same height. In some embodiments, the structure may include a central portion, e.g., a portion including exit tunnels, and a left / lefthand portion and a right / righthand portion, e.g., portions that include entrance tunnels. In some embodiments, there may be, an entrance opening, or a plurality of entrance openings, e.g., one to four, but not limited thereto, entrance openings, each of about, e.g., 1 ” in diameter, wherein each entrance opening is tapered, e.g., conically tapered, to the tunnel ending of about, e.g., 7-8 mm diameter pore, i.e. , optimal for a honeybee to pass through and enter the hive. This configuration is reversed for the exit opening, or plurality of exit openings, e.g., one to four, but not limited thereto, exit openings, located adjacent to the entrance openings. In some embodiments, the entrance openings may be to the left of the exit openings. In some embodiments, the entrance openings may be to the right of the exit openings. In some embodiments, the number of entrance openings is equal to the number of exit openings, but the number of entrance and exit openings are not limited thereto. Each entrance opening may include microcapillary tubes / bristles / brushes made of, e.g., silicone, that are connected to a miticide / acaricide reservoir above and are disposed on / hang from the ceiling into the lumen of the entrance tunnel. In some embodiments, there are no microcapillary brushes / bristles disposed on the exit tunnels.

[0040] In some embodiments, the microcapillary tubes / bristles may have, e.g., an internal diameter of about 0.2 mm to about 0.4 mm, and have, e.g., an external diameter of about 0.5 mm to about 0.8 mm. The microcapillary tube / bristles, in some embodiments, form a pendant droplet of pesticide / miticide / acaricide of about 0.05 mm to about 0.8 mm, which remains at the end of the microcapillary tube / bristle unless triggered by bee’s touch. Upon a bee’s touch, the pesticide droplet is released from the microcapillary tube / bristle onto, e.g., the body / dorsum / ventral portion of the bee, precisely targeting Varroa mites, and which may also be transferred into the hive via social interactions of bees within the hive.

[0041] In some embodiments, a miticide / acaricide reservoir, or plurality of reservoirs, each of which is associated with a single entrance tunnel and placed within, atop, or partially within and atop of the delivery system / mainframe may vary in number. The number of reservoirs may be, e.g., between 1-4, but are not limited thereto, so long as there is a reservoir associated with each tunnel entrance. The volume of each reservoir may be in the range of about 3 mL to about 50 mL. In other embodiments, a single reservoir may be used to supply miticide / acaricide to all of the plurality of entrance tunnels. In the case where a single reservoir supplied the miticide / acaricide to all the entrance tunnels, the volume of the reservoir can be in the range of about 50 mL to about 250 mL. In some embodiments, the single reservoir dimension can also be about 10 cm xAttorney Docket No. 190412-00036WO4 cm (length x diameter) with a volume of e.g., about 125.66 cubic centimeters (cc), and the wall thickness can be about 0.5 mm to 2 mm. In some embodiments, the reservoir or reservoirs may be composed of any material that might be appreciated by one of skill in the art. The material of which the reservoir or reservoirs may be composed of any hard or semi-hard plastic, a rubber, or have an elastomeric balloon-like structure without limitation.

[0042] In some embodiments, the microcapillary tubes / bristles may be hollow, and there may be a microcapillary tube / bristle, or plurality of microcapillary tubes / bristles, that are associated with a single entrance tunnel. In some embodiments, there may be about 1-4 microcapillary tubes / bristles associated with each entrance tunnel.

[0043] Although exemplary embodiments of the microcapillary tubes / bristles in the delivery systems of the inventive concept have been described with respect to delivery of an aqueous or a viscous hydrogel droplet including the pesticide / miticide / acaricide that is released when a bee touches the microtubular bristles while traveling through the entrance tunnel into the hive, alternatively, in some embodiments, each hollow microcapillary tube / bristle, e.g., a silicone microcapillary tube / bristle having an inside diameter of about 0.5-0.6 mm and an about 0.8-1 .5 mm outside diameter, may include, e.g., an about 0.4 mm diameter elastomeric filament co-axially placed inside the silicone tube. The top / proximal end of the elastomeric filament is anchored to, e.g., glued to the reservoir junction, and the distal end terminates with a spheroid / oblate spheroid (ball) of diameter of about 1 mm to about 2 mm. The elastomeric filament is installed under tension, which causes the spheroid / ball to act as a plug at the end of silicon microcapillary tubing. When a bee passes through the tunnel and touches / brushes the silicon microtubing, distortions to the spheroid plug due to contact by the bee / bee-touch with the microcapillary tube / bristle allow for leakage of an amount of and delivery of the miticide / acaricide onto the body of the bee. After bee passage, the spheroid plug snaps back into place, resealing the silicon microcapillary tubing.

[0044] Accordingly, in some embodiments, the bee-touch actuated release of a micro-droplet of miticide / acaricide is a complex function of the various elastomeric properties of the co-axially placed filament, its spheroid plug, and the specific geometries of all three components: silicone tubing, elastomeric filament and spheroid plug. In some embodiments, when an elastomeric balloon-like reservoir is used, the micro-droplet release may also be influenced in part by atmospheric pressure. Fabrication of any of the micro-components included in the systems of the inventive concept may be performed according to any method within the grasp of one of skill in the art.

[0045] According to embodiments of the present inventive concept, and unlike current miticides / acaricide that are becoming less effective at least due to the growing resistance inAttorney Docket No. 190412-00036WOVarroa mites, the active ingredient of the present inventive concept has shown significant potential for Varroa mites and pathogenic bee diseases control applications. Specifically, the present inventive concept includes a novel, nanotechnology-based miticide / acaricide, NoPest-Ag5 (i.e., NH2-AgNPs), which possesses some, if not all, of the following properties: a. Water-based, hence sustainable; b. Easily scalable to meet commercial demands; c. Highly stable at room temperature with potential shelf life between 3-5 yrs. d. Acts via electrostatic cell-surface interactions, so the Varroa mites and pathogenic microbes may not develop resistance; e. Low dose application, hence economical to use and reduce overall pesticide burden in the environment; f. May be broad-spectrum, and may effectively work against other honeybee pests and microbial species; g. Non-toxic to honeybees and safer to human cells and crop plants at doses that are toxic to Varroa mites and pathogenic hive microbes; h. Antioxidative and non-irritant to human eyes; and i. Only persistent in the environment for less than two weeks.

[0046] Embodiments of the present inventive concept further provide preventively and / or curatively active ingredients in the field of honeybee pest control, even at low rates of application, which have a very favorable biocidal spectrum against the Varroa destructor and other Varroa species, including other honeybee pests, and various pathogenic bee viruses, bacteria and fungi.

[0047] As used herein, "honeybee pest" generally includes, but is not limited to, Varroa mites ( Varroa destructor and V. jacobsoni), honeybee tracheal mite (Acarapis woodi), small hive beetle (Aethina tumida), bee louse (Braula coeca), greater wax moth (Galleria mellonella), mice, skunks, bumble bees, hornets, wasps, and the like. The present inventive concept may be used to control pests at various stages of their life cycle, including eggs, larvae, nymphs and adults. The present inventive concept may be used to control major honeybee diseases, including American foulbrood bacteria, European foulbrood bacteria, chalkbrood, sacbrood, and Nosema apis, and Nosema ceranae. Along with parasitic pests, these diseases can have serious impacts on honeybee colony health and survival.

[0048] The present inventive concept may be also used to precisely deliver or apply any other miticides / acaracides in aqueous and / or viscous forms without limitation and as would be appreciated by one of skill in the art, including in the form of water-based formulations, oil- or organic solvent-based formulations, hydrogels, nanogels, microgels, aerogels, and the like.Attorney Docket No. 190412-00036WO

[0049] The compositions of the active ingredient can be generally formulated in various ways using formulation adjuvants, such as carriers, solvents and surface-active substances. The formulations can be in various physical forms; e.g., in the form of gels, water-dispersible granules, water-dispersible tablets, emulsifiable concentrates, microemulsifiable concentrates, oil-in-water emulsions, oil-flowables, aqueous dispersions, oily dispersions, suspo-emulsions, emulsifiable granules, soluble liquids, water-soluble concentrates (with water or a water-miscible organic solvent as carrier), impregnated polymer films or in other forms known; e.g., from the Manual on Development and Use of FAO and WHO Specifications for Pesticides, United Nations, First Edition, Second Revision (2010). Such formulations can either be used directly or diluted prior to use. The dilutions can be made, for example, with water, liquid antibiotics, micronutrients, biological organisms, oil or solvents.Examples

[0050] Some aspects of the present inventive concept are described in more detail in the following non-limiting Examples. These are not intended to restrict the present inventive concept and may be modified within the range not deviating from the scope of this invention.Varr-Gate Technology

[0051] An example of the touch-sensitive delivery system / device of the inventive concept includes a hard plastic block that may be adapted to fit / connect to the entrance / exit of a typical / traditional beehive. Exemplary dimensions of the system / device include a hard plastic block with a length of about 14.625”, width of about 2”, and height of about 2” on the left part and height of 1 ” on the right part, although the dimensions and the material of the device are not limited thereto. There may be a plurality of entrance openings, such as, but not limited to, for example, four entrance openings, the dimensions of the openings being about, e.g., about 1 ” diameter, that lead into a tapering, in some embodiments, a conically tapering, tunnel ending, e.g., at an about 7-8 mm diameter pore, i.e., a dimension / size that is optimal for the honeybee to pass through and enter the hive. This configuration is reversed for the plurality of exit openings, such as, but not limited to, for example, four exit openings, located, for example, adjacent to, for example, to the right of the entrance openings. A plurality, for example, but not limited to, two to three, microcapillary bristles / brushes made of silicone, that are connected to the miticide / acaricide reservoir above, hang from the ceiling into the lumen of the entrance tunnel. There are no microcapillary bristles / brushes on the exit tunnels (FIG. 1 ). The dimensions and materials noted are provided as exemplary in describing the device of the inventive concept. The dimensionsAttorney Docket No. 190412-00036WO and / or the materials of the system / device are by no means intended to limit the dimensions, and any potential variants thereof, e.g., dimensions varied to fit various beehives, and / or materials, such as, but not limited to plastic and / or wood, to form the system / device, are envisioned to fall within the scope of the inventive concept. For example, entry and exit openings are based on the physical dimensions of bees, and the device dimensions are designed to fit any standard beehive entrances as would be appreciated by one of skill in the art.

[0052] The exemplary bee touch-responsive delivery system can precisely deliver a Varroa- active nanopesticide, and allows for effective mite control, while avoiding unwanted environmental release of the nanopesticide and, unlike conventional miticides, the l / a / roa-active nanopesticide of the inventive concept used is safe to brood cells (larvae and pupae) and adult bees and does not leave residual contamination in honey and combs.

[0053] An exemplary touch-responsive delivery system / device has demonstrated that having capillary tubes of e.g., about 0.3 mm (300 pm) internal diameter and e.g., about 0.6 mm (600 pm) external diameter forms a pendant droplet of a formulation including a pesticide / miticide / acaricide, e.g., a formulation of NoPest-Ag5, of about, e.g., 0.6 mm (600 pm), which will stay at the end of the capillary tube unless triggered by bee’s touch. Upon a bee’s touch, the pesticide / miticide / acaricide droplet is released onto the dorsum of the bee that specifically / precisely targets Varroa mites, and which may also be further transferred into the hive via social interactions of bees (FIG. 1 top panel). Upon testing in the field, the Varr-Gate technology was efficiently utilized by the bees within 1 -2 hours and droplets were released, upon triggered by bee’s touch, directly on to the body of the bees as they travel through the entrance tunnel (FIG. 1 bottom panel).Physicochemical Properties of NoPest-Ag5State: Pure CrystallinePrimary phase: Elemental or Metallic Silver (Ag°) Solubility: Water solubleTEM diameter: 5.8 nm (average) (S.D.=2.8 nm) Hydrodynamic diameter: 4.3 nm (average) (S.D.=1 .3 nm) Polydispersity Index (PDI): "0.3 Shape: Spherical / substantially sphericalZeta potential / Surface charge ( ): +39.4 mV to +47.8 mV Electrophoretic mobility (EPM): "3.157 pm-cm I V-s Solution conductivity: -122 pS-c r1Attorney Docket No. 190412-00036WOLocalized plasmon resonance peak (Amax): 416.5 nm (Absorbance=3.5 a.u.) Surface ligand: amino (-NH2)Thickness of surface NH2layer around nanoparticle: 0.5-1 .5 nmStability: over 3 years at STP

[0054] Transmission electron microscopy (TEM) showing NoPest-Ag5 particle morphology, energy dispersive X-ray spectroscopy (EDS) showing NoPest-Ag5 composition, and size distribution of NoPest-Ag5 particles are depicted in FIG. 2, panels A-D.

[0055] X-ray photoelectron spectroscopic (XPS) high resolution scan analysis shown below confirmed that chemically NoPest-Ag5 is primarily composed of amino (NH2)-functional group surface bound to the elemental / metallic silver (Ag°) nanoparticle core, with a trace level of ‘intermediate state’ of amide (C=O-N) (ref.

[0029] ) within the core Ag° metal surface (FIG. 3).Foundational Research

[0056] Effectiveness: NoPest-Ag5 Effectively Controls Varroa Mites. The device of the present inventive concept delivers NoPest-Ag5 to bees and significant inhibitory effects at 5 pg / mL or higher concentration are observed against the Varroa destructor miles (n=30 mites per group). 100% death occurred with an application of 5 pg / mL of NoPest-Ag5 within 24 hours of application, and over 60% deaths occurred by 60 minutes (FIG. 4).

[0057] Mechanism of Action: NoPest-Ag5 Significantly Downregulated Various Important Genes in Varroa Mites. The present inventive concept has demonstrated to significantly downregulate several biologically important genes in Varroa mites. Molecular analyses by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) revealed several genes were downregulated in Varroa mites (n = 10 mites per group) upon treatment with 5 pg / mL or higher concentration of NoPest-Ag5 compared to untreated controls. These downregulated genes were related to: detoxification and contributing to pesticide resistance (FMO5), regulating cell cycle progression at G1 including apoptosis (Cdknl ), complex I (NADH dehydrogenase) enzyme involved in respiratory chain (NADH), anaphase-promoting complex dependent catabolic process (Cdc27), metabolic clearance of toxic compounds such as pesticides and drugs (CYP3A56), and ubiquitin ligase genes involved in apoptosis (E3). These results hold significance as the treatment of NoPest-Ag5 against Varroa mite involves downregulation of multiple pathways related to key metabolic / catabolic processes meant to confer resistance and survival in mites. Thus, the use of NoPest-Ag5 would be advantageous overAttorney Docket No. 190412-00036WO current miticide offerings considering that the mites will be unable to develop resistance against NoPest-Ag5 (FIG. 5).

[0058] Effective Against Pathogenic Bee Viruses: NoPest-Ag5 is Significantly Inhibitory against Multiple Pathogenic Bee Viruses. At a single low-dose spray of 5 pg / mL of NoPest-Ag5 (5 mL per hive; n = 2 hives), a complete (100%) inhibition of two key pathogenic bee viruses: deformed winged virus (DWV) and sacbrood virus (SBV), were observed and that there was no resurgence of these viruses in the hives for 60 days (study period) based on RT-qPCR analysis (FIG. 6). These results suggest that NoPest-Ag5 acts both as a miticide / acaricide and viricidal agent in honeybee colonies.

[0059] Competitive Advantages of NoPest-Ag5 Against the Gold Standards. Unlike current miticides / acaricides in the market, NoPest-Ag5 effectively controls both the Varroa mites and multiple pathogenic bee viruses, and prevents them from resurgence. NoPest-Ag5 is low cost, eco-friendly, non-ecopersistent, human- and bee-safe, and does not lead to honey contamination (FIG. 7).Value Propositions of Varr-Gate Technology

[0060] The bee touch-responsive pesticide / miticide / acaracide delivery system as described herein has the following advantages as a solution for Varroa mite control:Precision targeting of Varroa mites on the body of bees, improving the efficacy of current miticides;Plug-n-play; Bee-touch-activated miticide release mechanism;100% Bee-safe, ecofriendly, weather-proof and low cost;80-90% reduction in miticide volume and application duration;Reduces labor, eliminates colony contamination;Seamlessly integrates with standard hives;Prevents robbing of hives by other pests (e.g., skunks, wasps, hornets, and mice); and Permits real-time remote monitoring of bee activities via an integrated light and camera.Further Exemplary Details of Varr-Gate Delivery System

[0061] A schematic depiction is shown in FIG. 8 of an exemplary bee touch-responsive pesticide / miticide / acaracide delivery system, including a single reservoir connected to four (4) silicone microcapillary tubes, each leading into the ceiling of a conical entrance tunnel to precisely deliver pesticide / miticide / acaracide onto the bee’s body as the bee passes through the entrance tunnel into the hive.Attorney Docket No. 190412-00036WO

[0062] A schematic depiction is shown in FIG. 9 of bee touch -activated pesticide / miticide / acaracide release in the bee touch-responsive pesticide / miticide / acaracide delivery system including a silicone microcapillary tube running down from a reservoir into the ceiling of an entrance tunnel and precisely delivering a microdroplet of a miticide (shown as a dot at the end of the microcapillary tubing / bristle) directly on to the bee’s body as the bee passes through the entrance tunnel into the hive interior.

[0063] The left panel of FIG. 10 shows a configuration of the Varr-Gate delivery system placed at the entrance of the 10-frame hive, including entrance tunnels on the left side and exit holes / tunnels on the right side of the delivery system, with four individual reservoirs, each 3 mL volume, disposed atop the Varr-Gate delivery system on the left side. The right panel of FIG. 10 shows a configuration of the Varr-Gate delivery system placed at the entrance of the 10-frame hive, including entrance tunnels on the right side and exit holes / tunnels on the left side of the delivery system, with four individual reservoirs, each 3 mL volume, disposed atop the Varr-Gate delivery system on the right side.

[0064] FIG. 11 shows another configuration of the bee touch-responsive pesticide / miticide / acaracide delivery system, including two entrance tunnels on each (left and right) side, and four exit tunnels located in the middle of the system. Also included are oblique placements of endoscope cameras on the far left and far right entrance tunnels along with an UV light alongside a pesticide reservoir / tank.

[0065] FIGS. 12-14 show images of the distribution of pesticide / miticide / acaracide on the bodies of honeybees under UV light and delivered by the bee touch-responsive delivery system shown in FIG. 11. Oxalic acid (OA; 18.4%) was used in an aqueous formulation and was mixed with riboflavin (Vitamin B2) as an UV-responsive dye that fluoresces yellow green under UV illumination. These images show that the bee touch-responsive pesticide / miticide / acaracide delivery system can effectively deliver a pesticide / miticide / acaracide onto the body of bees, thereby avoiding any off-target environmental release and exposure.References Cited1. Chesapeake Bay Program. (2022). European Honey Bee. https: / / www.chesapeakebay.net / discover / field-guide / entry / european-honey-bee2. FDA. (2018). Helping Agriculture’s Helpful Honey Bees. U.S. Food and Drug Administration. https: / / www.fda.gov / animal-veterinary / animal-health-literacy / helping-agricultures-helpful- honey-beesAttorney Docket No. 190412-00036WO3. Kurek-Gorecka, A., Gorecki, M., Rzepecka-Stojko, A., Balwierz, R., & Stojko, J. (2020). Bee products in dermatology and skin care. Molecules, 25(3), 556. https: / / doi.org / 10.3390 / molecules250305564. Woods, J. (2021 ). US beekeepers continue to report high colony loss rates, no clear progression toward improvement. Auburn University. https: / / ocm.auburn.edu / newsroom / news_articles / 2021 / 06 / 241121 -honey-bee-annual-loss- survey-results.php5. Insolia, L., Molinari, R., Rogers, S. R., Williams G. R., Chiromonte, F., Calovi, M. (2022). Honey bee colony loss linked to parasites, pesticides and extreme weather across the United States. Scientific Reports 12, 20787. https: / / doi.org / 10.1038 / s41598-022-24946-46. Giacobino, A., Steinhauer N., Brunner, S., Garcia-Andersen, N., Aurell, D., Rogers, S. & Williams, G. (2024). Preliminary Results from the 2023-2024 US Beekeeping Survey: Colony Loss and Management. Available at: https: / / apiaryinspectors.org / US-beekeeping-survey-23- 247. Sarnoff, L. (2025). Honeybee colonies could face 70% losses in 2025, impacting agriculture. ABC News, March 25, 2025. Available at: https: / / abcnews.go.com / amp / US / honey-bee- colonies-face-70-losses-2025-impacting / story?id=1201917208. Giacobino, A., Steinhauer N., Brunner, S., Garcia-Andersen, N., Aurell, D., Rogers, S. & Williams, G. (2025). Preliminary Results from the 2024-2025 US Beekeeping Survey: Colony Loss and Management. Available at: https: / / apiaryinspectors.org / US-beekeeping-survey-24- 259. Bartlett, L. (2022). Frontiers in effective control of problem parasites in beekeeping. International Journal for Parasitology: Parasites and Wildlife, 17:263-272. https: / / doi.org / 10.1016 / j-ijppaw.2022.03.003.10. Hristov, P., Neov, B., Shumkova, R., Palova, N. (2020). Significance of Apoidea as main pollinators. Ecological and economic impact and implications for human nutrition. Diversity, 12(7):280. https: / / doi.Org / 10.3390 / d120702801 1. Smitley, D., Brown, D., Finneran, R., Elsner, E., Landis, J., Shrewsbury, P., Herms, D., & Palmer, C. (2019). Factors that threaten pollinator health. Michigan State University Extension, https: / / www.canr.msu.edu / news / factors-that-threaten-pollinator-health12. Martin, S. J., Highfield, A. C., Brettell, L., Villalobos, E. M., Budge, G. E., & Powell, M. (2012). Global honey bee viral landscape altered by a parasitic mite. Science, 336(6086), 1304-1306.13. Moore, P. A., Wilson, M. E., & Skinner, J. A. (2014). Honey Bee Viruses, the Deadly Varroa Mite Associates. University of Tennessee, https: / / bee-health.extension.org / honey-bee- viruses-the-deadly- arroa-mite-associates / 14. Page R. 1998. Blessing or curse? Varroa mite impacts Africanized bee spread and beekeeping. Calif Agr 52(2) :9- 13. https: / / d0i.0rg / l 0.3733 / ca.v052n02p9.15. Traynor, K. S., Mondet, F., de Miranda, J. R., Techer, M., Kowallik, V., Oddie, M. A.,Chantawannakul, P., & McAfee, A. (2020). Varroa destructor. A complex parasite, crippling honey bees worldwide. Trends in Parasitology, 36(7), 592-606. https: / / doi.Org / 10.1016 / j. pt.2020.04.00416. Underwood, R., & Lopez-Uribe, M. (2019). Methods to Control Varroa Mites: An Integrated Pest Management Approach. Retrieved October 3, 2022. https: / / extension.psu.edu / methods- to-control- Va / roa-mites-an-integrated-pest-management-approachAttorney Docket No. 190412-00036WO17. Lin, Z., Su, X., Wang, S., Ji, T., Hu, F. L., Zheng, H. Q. (2020). Fumigant toxicity of eleven Chinese herbal essential oils against an ectoparasitic mite ( Varroa destructor1) of the honey bee (Apis mellifera). J. Apicult. Res. 59 (2): 204-210.18. Fassbinder, C., Grodnitzky, J., Coats, J. (2002). Monoterpenoids as possible control agents for Varroa destructor. J. Apicult. Res. 41 :83-88.19. Jack, C. J., & Ellis, J. D. (2021 ). Integrated Pest Management Control of Varroa destructor (Acari: Varroidae), the Most Damaging Pest of (Apis mellifera L. (Hymenoptera: Apidae)) Colonies. Journal of Insect Science, 21(5), 6. https: / / doi.org / 10.1093 / jisesa / ieab05820. RND (Reports and Data), 2022. Materials and Chemicals - Pesticides Market. https: / / www.reportsanddata.com / report-detail / pesticides-marketAdditional aspects and implementations of the inventive concept are described in the following clauses.

[0066] 1. A pesticide / miticide / acaricide delivery system / device comprising a hard or semi-hard structure, in some aspects, a plastic structure, aspects of which have a length of about 9-16", a width / depth of about 2-3", a first half / portion having a height of about 1 .5-4", and a second half / portion having a height of about 1-2", more particular aspects of which have a length of about 14.625”, width of about 2”, and height of about 2” on one side, for example, the left half or part, and height of about 1 ” on the other side, for example the right half or part. The structure may have a plurality, e.g., four entrance openings of about, but not limited to, 1 ” diameter that lead into a tapering, e.g., conically tapering, tunnel ending at e.g., an about 7-8 mm diameter pore, i.e., a pore having a diameter optimal for the honeybee to pass through and enter the hive. This configuration is reversed for the four exit openings, located, besides the entrance openings, for example, to the right of the entrance openings. A plurality of, e.g., two to three, microcapillary bristles / brushes, in some aspects, made of silicone, connected to a miticide / acaricide reservoir above, hang from the ceiling of the entrance openings into the lumen of the entrance tunnel. In some aspects, there are no microcapillary bristles / brushes on the exit tunnels (ref. Fig. 1).

[0067] 2. The system / device of Clause 1 , comprising microcapillary tubings / bristles, in some aspects, comprising microcapillary tubings / bristles of about 0.3 mm (300 pm) internal diameter and about 0.6 mm (600 pm) external diameter, the microcapillary tubings / bristles capable of forming a pendant droplet, in some aspects, a pendant droplet of around 0.6 mm (600 pm), in which the pendant droplet will stay at the end of the capillary tube unless triggered by bee’s touch. Upon a bee’s touch, the pesticide / miticide / acaricide droplet is released onto the body, e.g., onto the dorsum or ventral portion of the body, of the bees, for example, honeybees, wherein the pesticide / miticide / acaricide targets specifically Varroa mites, and wherein theAttorney Docket No. 190412-00036WO pesticide / miticide / acaricide may also be transferred into the hive via social interactions of bees (Fig. 1 top panel) (Fig. 1 bottom panel).

[0068] 3. The system / device of Clause 1 , wherein the pesticide / miticide / acaricide reservoir(s) placed within or atop of the plastic structure can vary in number between about 1 -4, and the volume of each reservoir can be in the range about 2 mL to about 30 mL.

[0069] 4. The system / device of Clause 1 , wherein the microcapillary tubings / bristles are hollow and vary in number in the range of 1 to 5 within each entrance tunnel.

[0070] 5. The system / device of any one of Clauses 1-4, wherein the device is used to deliver an aqueous or viscous formulation of, for example, NoPest-Ag5 (as described in U.S. Patent Application No. 17 / 759,260, incorporated herein by reference in its entirety), a honeybee safe and Varroa-active miticide, directly on to the dorsal body of each bee as they enter the hive.

[0071] 6. The system / device of Clause 5, wherein the viscous formulation of NoPest-Ag5 comprises NHs-AgNPs mixed, in some aspects mixed overnight, and in some aspects under gentle shaking, with alginate / alginic acid and / or cyclodextrin, whereby the cyclodextrin can be alpha, beta, or gamma-cyclodextrin, and / or glycerol, and / or ethylene imine.

[0072] 7. The system / device of Clause 6, wherein the NH2-AgNP concentration can be in the range of about 0.1 pg / mL to 1.0 g / mL; alginate / alginic acid concentration can be in the range of about 0.1 mg / mL to 10 g / mL; alpha, beta, or gamma-cyclodextrin concentration can be in the range of about 0.1 mg / mL to 10 g / mL; glycerol concentration can be in the range of about 0.1 % to 100 %; and ethylene imine concentration can be in the range of about 0.1 mg / mL to 10 g / mL.

[0073] 8. The system / device of any one of Clauses 1-7, wherein the device can also be used to precisely deliver other miticides targeting Varroa mites, including but not limited to, aqueous or viscous formulations of oxalic acid, tau-fluvalinate, coumaphos, amitraz, formic acid, hop beta acids, thymol, and the like.

[0074] 9. The device / system of any one of Clauses 1-8, wherein the system / device is configured to be attached to and / or fit on a typical beehive.

[0075] 10 A method of controlling honeybee pests comprising applying a pesticide / miticide / acaricide to a pest, subject, substrate or an environment using the device / system of any one of Clauses 1-9.

[0076] 1 1. The method of Clause 10, wherein the honeybee pest is selected from the group consisting of Varroa mites (Varroa destructor and V. Jacobson / ), honeybee tracheal mite (Acarapis wood), small hive beetle (Aethina tumida), bee louse (Braula coeca), greater wax moth (Galleria mellonella), mice, skunks, bumble bees, hornets, wasps, and the like.Attorney Docket No. 190412-00036WO

[0077] 12. The method of Clause 1 1 , wherein the pest is a stage of its life cycle selected from the group consisting of eggs, larvae, pupae / nymphs and / or adults.

[0078] 13. A method of reducing honeybee disease transmission comprising exposing a pest, a subject, a substrate or an environment, using the method of any one of Clauses 1-9.

[0079] 14. The method of Clause 13, wherein the honeybee disease can be deformed winged virus (DWV A-C), sacbrood virus (SBV), acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), and Kashmir bee virus (KBV), Black queen cell virus (BQCV), American foulbrood, European foulbrood, chalkbrood, Nosema spp., and the like.

[0080] While specific embodiments of the present inventive concept have been shown and described, it will be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the inventive concept, which should be determined from the appended claims.

Claims

Attorney Docket No. 190412-00036WOTHAT WHICH IS CLAIMED:1 . A pesticide / miticide / acaricide delivery system comprising a hard or semi-hard structure configured to fit the entrance / exit of a beehive, wherein the structure comprises: at least one entrance tunnel; at least one exit tunnel, wherein each entrance tunnel comprises at least one microcapillary tube / bristle hanging from the ceiling of the entrance tunnel into the lumen of the entrance tunnel that is connected to a reservoir for the pesticide / miticide / acaricide installed / disposed atop of the entrance tunnel.

2. The pesticide / miticide / acaricide delivery system of claim 1 , wherein the structure comprises a plurality of entrance tunnels and / or exit tunnels.

3. The pesticide / miticide / acaricide delivery system of claim 1 or 2, wherein each entrance tunnel comprises a plurality of microcapillary tubes / bristles.

4. The pesticide / miticide / acaricide delivery system of any one of claims 1-3, wherein the structure has a length of about 9-16" a width / depth of about 2-3", a first half / portion having a height of about 1 .5-4", and a second half / portion having a height of about 1-2".

5. The pesticide / miticide / acaricide delivery system of any one of claims 1-4, wherein the at least one entrance tunnel is tapered to the entrance of the beehive and / or the at least one exit tunnel is tapered to the exit of the beehive.

6. The pesticide / miticide / acaricide delivery system of claim 5, wherein the at least one entrance tunnel is conically tapered to the entrance of the beehive, and / or the at least one exit tunnel is conically tapered to the exit of the beehive.

7. The pesticide / miticide / acaricide delivery system of any one of claims 1-6, wherein the microcapillary tubes / bristles associated with a single entrance tunnel are connected to a single reservoir.

8. The pesticide / miticide / acaricide delivery system of any one of claims 1-6, wherein all the microcapillary tubes / bristles associated with all the entrance tunnels are connected to a single reservoir.Attorney Docket No. 190412-00036WO9. The pesticide / miticide / acaricide delivery system of any one of claims 1-8, wherein the microcapillary tubes / bristles are configured to form a pendant droplet of pesticide / miticide / acaracide that remains on a distal end of the microcapillary tubes / bristles, and wherein contact by a bee with the distal end of the microcapillary tubes / bristles causes release of the pendant droplet onto the body of the bee.

10. The pesticide / miticide / acaricide delivery system of any one of claims 1-8, wherein each microcapillary tube / bristle comprises an elastomeric filament disposed coaxially inside the microcapillary tube / bristle and anchored to the reservoir junction and terminates at the distal end with a spheroid / oblate spheroid ball that acts as a plug of the microcapillary tube / bristle.1 1 . The pesticide / miticide / acaricide delivery system of claim 10, wherein contact by a bee with the spheroid / oblate spheroid ball causes the pesticide / miticide / acaricide to be released onto the body of the bee.

12. The pesticide / miticide / acaricide delivery system of any one of claims 1-11 , wherein the system further comprises at least one endoscopic camera associated with an entrance tunnel for monitoring delivery of the pesticide / miticide / acaricide to bees.

13. The pesticide / miticide / acaricide delivery system of any one of claims 1-12, wherein the pesticide / miticide / acaricide comprises a formulation comprising nanoparticles having the following physicochemical properties: a primary phase of elemental or metallic silver (Ag°); a solubility in water; a mean diameter of about 5.8 nm (S.D. = 2.8 nm); a mean hydrodynamic diameter of about 4.3 nm (S.D. = 1 .3 nm); a polydispersity index (PDI) of about 0.3; a spherical / substantially spherical shape; a zeta potential / surface charge ( ) of about +39.4 mV to about +47.8 mV; an electrophoretic mobility (EPM) of about 3.157 pm-cm / V-s; a solution conductivity of about 122 pS-cmr1; a localized plasmon resonance peak (Amax) at 416.5 nm (Absorbance=3.5 a.u.);Attorney Docket No. 190412-00036WO having amino (-NH2) surface ligand(s); a surface -NH2layer thickness around nanoparticle of about 0.5-1 .5 nm; and a stability / shelf life of over 3 years at STP.

14. A method of delivering a pesticide / miticide / acaracide to a bee or bees comprising disposing the pesticide / miticide / acaracide delivery system of any one of claims 1-13 at the entrance / exit of a beehive, wherein the bee / bees must pass through an entrance tunnel of the pesticide / miticide / acaracide delivery system to enter the beehive.

15. A method of controlling / reducing honeybee pests comprising applying / exposing a pesticide / miticide / acaricide to a subject / honeybee, pest, substrate, or environment using the delivery system of any one of claims 1-13.

16. The method of claims 15, wherein the honeybee pest is selected from the group consisting of Varroa mites (Varroa destructor and V. jacobsoni), honeybee tracheal mites (Acarapis woodi , small hive beetles (Aethina tumida), bee lice (Braula coeca), and greater wax moths (Galleria mellonella).

17. The method of claim 16, wherein the pest is at a stage in its life cycle selected from the group consisting of eggs, larvae, pupae / nymphs and / or adults.

18. A method of reducing / controlling honeybee disease transmission comprising applying / exposing a pesticide / miticide / acaricide to a subject / honeybee, pest, substrate, or environment using the delivery system of any one of claims 1-13.

19. The method of claim 18, wherein the honeybee disease is selected from the group consisting of deformed winged virus (DWV A-C), sacbrood virus (SBV), acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), and Kashmir bee virus (KBV), Black queen cell virus (BQCV), American foulbrood, European foulbrood, chalkbrood, and Nosema spp.

20. A method delivering a pesticide / miticide / acaracide to a bee / bees comprising providing at least one entrance tunnel to a beehive, wherein each entrance tunnel comprises at least one microcapillary tube / bristle hanging from the ceiling of the entrance tunnel into theAttorney Docket No. 190412-00036WO lumen of the entrance tunnel that is connected to a reservoir for the pesticide / miticide / acaricide disposed atop of the entrance tunnel, and wherein the bee / bees must pass through the entrance tunnel to enter the beehive.21 . The method of claim 20, wherein the at least one entrance tunnel comprises a plurality of microcapillary tubes / bristles.

22. The method of claim 20 or 21 , wherein the at least one entrance tunnel is tapered to the entrance of the beehive.

23. The method of claim 22, wherein the at least one entrance tunnel is conically tapered to the entrance of the beehive.

24. The method of any one of claims 20-23, wherein the microcapillary tubes / bristles associated with a single entrance tunnel are connected to a single reservoir.

25. The method of any one of claims 20-23, wherein all the microcapillary tubes / bristles associated with all the entrance tunnels are connected to a single reservoir.

26. The method of any one of claims 20-25, wherein the microcapillary tubes / bristles are configured to form a pendant droplet of pesticide / miticide / acaracide that remains on a distal end of the microcapillary tubes / bristles, and wherein contact by a bee with the distal end of the microcapillary tubes / bristles causes the pendant droplet to be released onto the body of the bee.

27. The method of any one of claims 20-25, wherein each microcapillary tube / bristle comprises an elastomeric filament disposed coaxially inside the microcapillary tube / bristle that is anchored to the reservoir junction and terminates at the distal end with a spheroid / oblate spheroid ball that acts as a plug of the microcapillary tube / bristle.

28. The method of claim 27, wherein contact by a bee with the spheroid / oblate spheroid ball causes the pesticide / miticide / acaricide to be released onto the body of the bee.Attorney Docket No. 190412-00036WO29. The method of any one of claims 20-28, wherein the pesticide / miticide / acaricide comprises a formulation comprising nanoparticles having the following physicochemical properties: a primary phase of elemental or metallic silver (Ag°); water solubility; a mean diameter of about 5.8 nm (S.D. = 2.8 nm); a mean hydrodynamic diameter of about 4.3 nm (S.D. = 1 .3 nm); a polydispersity index (PDI) of about 0.3; a spherical / substantially spherical shape; a zeta potential / surface charge (Q of about +39.4 mV to about +47.8 mV; an electrophoretic mobility (EPM) of about 3.157 pnrcm / V-s; a solution conductivity of about 122 pS-cm-1; a localized plasmon resonance peak (Amax) at 416.5 nm (Absorbance=3.5 a.u.); having amino (-NH2) surface ligand(s); a surface -NH2layer thickness around nanoparticle of about 0.5-1 .5 nm; and a stability / shelf life of over 3 years at STP.

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