Pesticidal proteins and uses thereof

Abstract

Provided herein are methods of reducing a small hive beetle pest population comprising contacting the small hive beetle with a bacterial pesticidal protein.

Description

PESTICIDAL PROTEINS AND USES THEREOFGOVERNMENT SUPPORT CLAUSE

[0001] This invention was made with government support under Grant No. 2021-67013- 33559 awarded by The United States Department of Agriculture, National Institutes of Food & Agriculture. The Government has certain rights in the invention.BACKGROUND

[0002] The small hive beetle (SHB, Aethina tumida, Murray) native to sub-Saharan Africa, has spread over the course of the past two decades as a result of international trade of honey bees and hive-related products. SHB is a parasite of social bee colonies, feeding primarily on pollen, honey and brood of the European honey bee, Apis mellifera. This beetle has had a significant deleterious economic impact on the apiculture industry in North America: While often considered a secondary pest compared to Varroa mites, SHB pressure can cause the deaths of strong colonies within two weeks and can pose even greater problems for small colonies and apicultural storage facilities. SHB has been detected in all 48 contiguous U.S. states and Hawaii, with the most damage occurring in climates well- suited to SHB reproduction (e.g., Hawaii and the southeastern states), although SHB pressure appears to be increasing. Current management in the U.S. relies on the use of chemical insecticides, which are also toxic to honey bees, or mechanical traps that only affect adults and require additional labor. Alternative strategies for SHB management are urgently required to minimize losses to the apiculture industry.SUMMARY

[0003] In one aspect, described herein is a method of reducing a small hive beetle pest population comprising contacting the small hive beetle with a bacterial pesticidal protein selected from App6Aa2, Mpp51Aa1 and Cry2Ab.BRIEF DESCRIPTION OF THE FIGURES

[0004] Figure 1 . Representative survivorship curves of small hive beetle larval feeding assays. Five cups of larvae, each containing 10 larvae (n = 50), were given 3g of MegaBee diet treated with either carbonate buffer or dimethoate. The assay was conducted for 6 days, and all larvae in the dimethoate treatment died by Day 2. The bar with three asterisks “***” denotes a significant p-value of less than 0.001 in the differences of hazard ratios (HR) between the two treatments.

[0005] Figure 2. Initial screening assay with Cry2Aa, Cry2Ab, Cry2Ac, App6Aa2, and Mpp51Aa1 at 3ppm. 5 cups of larvae, each containing 10 larvae (n = 50), were given 3g of MegaBee diet treated with one of the pesticidal proteins or carbonate buffer. The assay wasconducted for 6 days. The bar with two asterisks “**” and three asterisks “***” denotes a significant p-value less than 0.01 and 0.001 , respectively, in the differences of hazard ratios (HR) between the treatment and the carbonate buffer control.

[0006] Figure 3. Initial screening assay with Tpp78Aa1 , Tpp78Ba1 , Tpp80Aa1 , and Xpp37Aa1 at 4ppm. 5 cups of larvae, each containing 10 larvae (n = 50), were given 3g of MegaBee diet treated with one of the pesticidal proteins or carbonate buffer. The assay was conducted for 6 days. There were no significant (p < 0.05) differences of hazard ratios (HR) between the treatments and the carbonate buffer control.

[0007] Figure 4. Initial screening assay with Mpp23Aa1 and Xpp55Aa1 at 3ppm. 5 cups of larvae, each containing 10 larvae (n = 50), were given 3g of MegaBee diet treated with one of the pesticidal proteins or carbonate buffer. The assay was conducted for 7 days. There were no significant (p < 0.05) differences of hazard ratios (HR) between the treatments and the carbonate buffer control.

[0008] Figure 5. Step-wise dose-response feeding assay with App6Aa2 at concentrations ranging from 0.04ppm to 2.0ppm. 5 cups of larvae, each containing 10 larvae (n = 50), were given 3g of MegaBee diet treated with one of the concentrations of App6Aa2 or carbonate buffer. The assay was conducted for 5 days. The bars with two asterisks “**” and three asterisks “***” denote a significant p-value less than 0.01 and 0.001 , respectively, in the differences of hazard ratios (HR) between the treatment concentration and the carbonate buffer control.

[0009] Figure 6. Step-wise dose-response feeding assay with Mpp51 Aa1 at concentrations ranging from 0.04ppm to 2.0ppm. 5 cups of larvae, each containing 10 larvae (n = 50), were given 3g of MegaBee diet treated with one of the concentrations of Mpp51 Aa1 or carbonate buffer. The assay was conducted for 5 days. The bar with three asterisks “***” denotes a significant p-value less than 0.001 in the differences of hazard ratios (HR) between the treatment concentration and the carbonate buffer control.

[0010] Figure 7. Step-wise dose-response feeding assay with Cry2Ab at concentrations ranging from 0.04ppm to 2.0ppm. 5 cups of larvae, each containing 10 larvae (n = 50), were given 3g of MegaBee diet treated with one of the concentrations of Cry2Ab or carbonate buffer. The assay was conducted for 5 days. The bar with three asterisks “***” denotes a significant p-value less than 0.001 in the differences of hazard ratios (HR) between the treatment concentration and the carbonate buffer control.

[0011] Figure 8. Honey bee cage assays with App6Aa2, Cry2Ab, and Mpp51 Aa1 at3ppm. Five cages of 15 honey bees each (n = 75) were given a 33% sucrose solution adlibitum treated with one of the pesticidal proteins or carbonate buffer. The assay was conducted for 14 days. Any honey bees that died during the experiment were removed from their cage. There were no significant (p < 0.05) differences of hazard ratios (HR) between the treatments and the carbonate buffer control.DETAILED DESCRIPTION

[0012] The present disclosure is based on the discovery of several bacterial pesticidal proteins being toxic to the small hive beetle (SHB, Aethina tumida). The data provided herein show that of the eleven pesticidal proteins tested, three proteins (App6Aa2, Mpp51 Aa1 , Cry2Ab) were shown to be honey bee-safe, small hive beetle control agents.

[0013] The small hive beetle (SHB, Aethina tumida, Murray; Coleoptera; Nitidulidae) is an opportunistic parasite of social bees (19). SHB has spread to all habitable continents in large part due to migratory beekeeping, movement of package bees and equipment (6, 20, 21), and the beeswax trade (22). While SHB is a minor pest in its endemic range of sub-Saharan Africa, it can have a significant impact on honey bee populations elsewhere (6, 23, 24). SHB also impacts bumble bee species, although these impacts are harder to assess (25).

[0014] While often considered a pest of secondary importance after the mite, Varroa destructor, problems associated with SHB pressure are widespread, with all European honey bee colonies - even strong colonies, susceptible to beetle-induced death (26). SHB infestation is an even greater problem for weak colonies such as those suffering from exposure to other stresses (Varroa / virus, nutritional problems, pesticide exposure) as well as colonies managed for queen rearing, which are typically small (21). Further, while SHB- driven losses are difficult to track, many beekeepers must adapt management practices for other issues with consideration of SHB. For example, given that SHB feed well on artificial pollen feed supplements (10), it can be difficult for beekeepers to provide nutritional supplementation to weak colonies without risking losses from SHB. Apicultural products, particularly combs containing honey before extraction, are also highly vulnerable to SHB; when honey is removed from the colony for storage before extraction, bees are no longer present to protect the resources. Therefore, to protect these products from SHB damage, beekeepers are unable to store honey for any length of time without specialized storage facilities (23, 24). In states for which SHB is prevalent but may not appear to be a problem (e.g. Florida, Virginia), beekeepers commonly use off-label insecticides for SHB management. Hence, reported losses underestimate the extent of the problem with the illegal use of off-label insecticides. Identification of bacteria-derived pesticidal proteins, including those derived from Bacillus thuringiensis (Bt) for effective SHB management will reduce or stop this practice by providing effective, safe and legal control options.

[0015] Damage associated with SHB results from larval feeding. Female beetles deposit eggs in the vicinity of the hive, or on pollen or brood combs or in brood cells. After 2 to 4 days, the eggs hatch and larvae feed on pollen, honey, bee brood and eggs. Larval development is completed in 7 to 10 days at which stage the wandering larvae leave the hive and burrow into the soil to pupate.

[0016] SHB pressure is greatest in areas with soil conditions that support beetle pupation and overwintering, although adults can overwinter in winter clusters, i.e., within the hive (57) making eradication of beetles between years difficult. To make matters worse, models of habitat suitable for SHB, in conjunction with increasing soil temperatures that contribute to SHB pupal development time and survival, predict high risk of further SHB invasion with global warming particularly in temperate regions of the northern hemisphere (28).Furthermore, SHB has the potential to spread pathogens between apiaries, as adult beetles can disperse >10 km to find new colonies for infestation (6, 21 ); the beetles have already been shown to vector American foulbrood (59) and potentially also viral pathogens (3, 4). At the same time, there have been reports of increased virulence of some honey bee viruses, which are carried by SHB and may be transmissible through trophallaxis (30, 31 ). There is also evidence that SHB can target and damage bumble bees (25), and there has been growing concern that failure to properly control honey bee pests could increase pathogen movement into native bee populations (32). With these contributing problems, there is a clear need for improved management approaches for this invasive pest (27).

[0017] In one aspect, described herein are methods of reducing a small hive beetle (SHB) population comprising contacting the SHB with a pesticidal protein. Exemplary pesticidal proteins include, but are not limited to, App6Aa2, Mpp51 Aa1 , Cry1 , such as members of the Cry1 A, Cry1 B, Cry1 C, Cry1 D, Cry1 E, and Cry1 F families; Cry2, Cry2Ab; Cry3, Cry8, Cry9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; etc. In some embodiments, the pesticidal protein is Cry2Ab, App6Aa2, or Mpp51Aa1.

[0018] Around 60 coleopteran-active Bt proteins have been identified to date (15-18). Following ingestion by the insect, the active toxins bind to the insect gut epithelium. Binding results in the creation of pores, disruption of gut function and ultimately death of the insect. These BPP are divided into different groups based on structure (2), including more than 45 Cry, two cytolytic (Cyt), 11 Vip and two Sip proteins toxic to one or more coleopteran species. The most extensively tested BPP and the protein with the broadest activity across coleopteran species is Cry3Aa with activity against 14 of 21 coleopteran species tested. Indeed, Cry3Aa and Cry3Bb have been used for production of transgenic Bt crops with resistance to Colorado potato beetle, and western corn rootworm (33).

[0019] The compositions and formulations may be fed to SHB in a variety of ways. In some embodiments, the pesticidal proteins may be formulated as a liquid, a patty (e.g., a pollen nutritional supplement patty), or a biscuit. In some embodiments, the pesticidal proteins are formulated in a composition and provided adjacent to a comb in the hive, e.g., on top of the comb. The compositions can be provided on a mesh through which the bees can pass. In certain embodiments, the compositions formulated as a liquid is provided in an inverted jar inserted into a hole in the roof of a hive. In certain embodiments, the compositions may be provided in an area surrounding a hive (e.g., within an apiary). In such embodiments, the composition may be formulated as a liquid or a powder.

[0020] In some embodiments, the composition may for example be provided via a frame feeder, or may be poured or sprayed.

[0021] In any of the foregoing, the compositions and formulations can be provided before or after detection of infestation of small hive beetles. This can be over any period of time, for example, minutes, hours, days, or weeks before or after infestation.

[0022] The compositions and formulations can be provided ad lib, i.e., the SHB can feed freely as desired, and / or via a time (e.g., slow) release device. For example, the compositions containing an effective amount of pesticidal protein can be formulated as a liquid (e.g., sugar syrup) that is placed in a bag or jar or strip in, on, or near the hive. The SHB can access and ingest the composition as desired over the period of time.

[0023] The SHB may ingest an effective amount of a composition comprising a pesticidal protein described herein on a single, repeated, or regular basis. For example, the pest may ingest an effective amount of a pesticidal protein composition one, two, three, or more times weekly, every other day, every day, or more than once every day (e.g., once, twice, three, or more times every day) during the performance of the disclosed methods or uses.

[0024] The amount of a composition administered to the SHB is typically enough to prevent, reduce, decrease, or inhibit one or more adverse effects associated with pest infestation. For example, in some embodiments, the amount of pesticidal protein in the compositions is effective to reduce, decrease, or inhibit establishment, and proliferation / expansion of SHB in the hive. In some embodiments, the compositions and methods reduce or prevent the SHB from damaging the habitat or food stores, for example, damage to the hive generally, or a comb(s), stored honey, and / or pollen specifically alone or in combination with reducing SHB pest-induced bee mortality. If an SHB infestation is sufficiently heavy, they may cause bees to abandon their hive. Thus, in some embodiments, the methods described herein additionally or alternatively prevent colony abandonment and / or colony collapse.

[0025] The compositions and methods are typically also carried out in a means that limits or prevents direct damage or injury to the honey bees or their hive. Thus, the combination of the amount of the pesticidal proteins described herein and its method of administration are typically balanced to provide maximum impact against the pest(s) with minimal impact against the honey bees.

[0026] The disclosure further provides a vector comprising one or more nucleotide sequences encoding the pesticidal proteins.

[0027] The term “vector” as used herein encompasses (but is not limited to) a phage, plasmid, viral or retroviral vector, as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below. The vector comprising the polynucleotides as described herein may comprise selectable markers for propagation and / or selection in a host. Further, the vector may be prepared from native (endogenous) and / or foreign (exogenous, heterologous) sequences with respect to the host.

[0028] Preferably, the vector referred to herein is suitable as a cloning vector, i.e., replicable in microbial systems. Such vectors ensure efficient cloning in bacteria, yeasts or fungi.

[0029] Suitable vector backbones are, in some embodiments, derived from vectors known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia) or pSPORTI (GIBCO BRL). Further examples of typical fusion expression vectors are pGEX (Pharmacia Biotech Inc; Smith, D.B., and Johnson, K.S. (1988) Gene 67:31 -40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), where glutathione S transferase (GST), maltose E-binding protein and protein A, respectively, are fused with the nucleic acid of interest encoding a protein to be expressed.

[0030] In some embodiments, the vector comprising one or more nucleotide sequences described herein is propagated and amplified in a host cell. In some embodiments, one copy of the vector is propagated and amplified in a host cell. In some embodiments, two or more (e.g., 3, 4, 5, 6 7, 8 or more) copies of the vector are propagated and amplified in a host cell.

[0031] In some embodiments, the vector described herein comprises a promoter. The term "promoter" as used herein refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing therecognition site for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence comprised, in some cases, of a TATA box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for enhancement of expression."Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements and that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence, which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements, derived from different promoters found in nature, or even be comprised of synthetic DNA segments.

[0032] The terms “operably linked” or “functionally linked” refer to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0033] In some embodiments, an operable linkage comprises a sequential arrangement of a nucleotide sequence encoding a promoter, with a nucleic acid sequence to be expressed, and optionally, additional regulatory elements such as, for example, polyadenylation or transcription termination elements, enhancers, introns, etc., such that the nucleotide sequence of interest is expressed under the appropriate conditions (i.e., in a plant cell). Suitable arrangements include, e.g., those in which the nucleic acid sequence to be expressed is placed downstream (i.e., in 3’-direction) of the transcription regulating nucleotide sequence such that both sequences are covalently linked. Optionally, additional sequences may be inserted in-between the two sequences. Such sequences may be, for example, linker or multiple cloning sites. Furthermore, sequences can be inserted which encode parts of a fusion protein, in the event that a fusion protein comprising the product of the nucleic acid disclosed herein is desired. Preferably, the distance between thepolynucleotide to be expressed and the transcription regulating nucleotide sequence is not more than 200 base pairs, such as not more than 100 base pairs or not more than 50 base pairs.

[0034] In another aspect, described herein is a method for expressing a polynucleotide of interest in a host cell comprising introducing a vector described herein into the host cell and expressing the polynucleotide of interest in the host cell.

[0035] The term "expression" refers to the transcription and / or translation of an endogenous gene, ORF or portion thereof. The "expression pattern" of a promoter (with or without enhancer) is the pattern of expression levels, which shows where and in what developmental stage transcription is initiated by said promoter. Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter. The level of expression of a promoter can be determined by measuring the steady state concentration of a standard transcribed reporter mRNA. This measurement is indirect since the concentration of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded. Therefore, the steady state level is the product of synthesis rates and degradation rates. When promoters are compared in this way, techniques available to those skilled in the art are hybridization S1-RNAse analysis, northern blots and competitive RT-PCR. This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA. The analysis of transcription start points in practically all promoters has revealed that there is usually no single base at which transcription starts, but rather a more or less clustered set of initiation sites, each of which accounts for some start points of the mRNA. Since this distribution varies from promoter to promoter the sequences of the reporter mRNA in each of the populations would differ from each other. Since each mRNA species is more or less prone to degradation, no single degradation rate can be expected for different reporter mRNAs. It has been shown for various eukaryotic promoter sequences that the sequence surrounding the initiation site (“initiator") plays an important role in determining the level of RNA expression directed by that specific promoter. This includes also part of the transcribed sequences. The direct fusion of promoter to reporter sequences would therefore lead to suboptimal levels of transcription.

[0036] A commonly used procedure to analyze expression patterns and levels is through determination of the 'steady state' level of protein accumulation in a cell. For quantification and determination of localization a number of tools are suited. Detection systems can readily be created or are available which are based on, e.g., immunochemical, fluorescent detectionand quantification. Protein levels can be determined using in situ analysis of protein expression.

[0037] The expression of a polynucleotide of interest in a host cell can be determined by various techniques, e.g., immunological assays such as ELISAs and western blots.EXAMPLES

[0038] Materials and Methods

[0039] Bacterial strains, pesticidal proteins, and plasmids. A total of 11 Bt-derived pesticidal proteins from different structural groups were expressed and tested against SHB. These proteins were either synthesized in vitro, acquired from the Bacillus Genetic Stock Center (BGST), or provided by other laboratories (Table 1 ). Details of the plasmids in which genes were inserted, the bacterial expression system, bacterial strains, and protein purification methods used for each protein are shown in Table 1.

[0040] Expression and purification of Bt proteins from crystals. The proteins Cry2Aa, Cry2Ab, Cry2Ac, Mpp51 Aa1 and App6Aa2 were expressed in Bt sporulation medium as previously described (34, 35) with modifications. Briefly, overnight 5 mL cultures were used to inoculate 200 mL of Bt sporulation medium, and sporulation was induced with sodium phosphate buffer after overnight growth. Spores and crystals were harvested by centrifugation at 10,000 rpm (Beckman J2-21 centrifuge), suspended in crystal washing buffer (0.1 M NaCI, 2% Triton-X 100, 2% sodium deoxycholate, 20mM Bis-tris pH 6.5), and sonicated on ice. The pellet was then washed three times in the same buffer without deoxycholate, two times in 1 M NaCI, and two times in distilled water. The protein was then solubilized with 50 mM sodium hydroxide (NaOH) for one hour at 37°C and 120 rpm shaking. The samples were then dialyzed against 6 L of 20 mM carbonate buffer (2 L overnight and 2 L every 2 h) using 3,500 MWCO dialysis cassettes (PIERCE, Rockford, IL). Protein concentration was determined via Bradford assay, and protein was resolved in a 4- 12% SDS PAGE gel following staining with Coomassie brilliant blue R-250.

[0041] Expression and His-tag affinity purification of Bt proteins. A total of eight Bt proteins were expressed in E. coli and purified using His-tag affinity chromatography. DNA sequences for six of these proteins were cloned into pET30 for optimized protein yield. The conditions for expression varied depending on the protein. For Xpp37Aa1 , Xpp55Aa1 and Tpp80Aa1 , E. coli cells harboring the recombinant plasmid were grown in LB medium supplemented with 10 pg / mL of kanamycin at 37 °C and 220 rpm until GD600 reached 0.5. Gene transcription was induced with 1 mM of IPTG. The cultures were grown overnight at 20 °C with 150 rpm shaking. For Tpp78Aa1 , Tpp78Ba1 , and Mpp23Aa1 , cells were grown for 4h at 37°C and 220 rpm after IPTG induction. Cells were then harvested by centrifugation (2,600 x g, 20 min, 4°C) and lysed using xTractorTM buffer (Takara, Kusatsu, Japan) supplemented with 1 mg / mL of lysozyme plus 1 mM phenylmethylsulphonyl fluoride (PMSF) and 1x protease inhibitor (PI) followed by sonication on ice. All proteins were purified using HisPurTM Ni-NTA agarose (Fisher Scientific, Rockford, IL, USA). Briefly, lysates were centrifuged [10,000 rpm (Beckman J2-21 centrifuge), 30 min, 4°C], equilibrated with an equal volume of equilibration buffer (10 mM imidazole, 300 mM NaCI, and 50 mM Tris pH 8), and passed through columns packed with Ni-NTA agarose beads. Beads were previously equilibrated with six-column volumes of equilibration buffer. Columns were then washed with six-column volumes of wash buffer (25 mM Imidazole, 300 mM NaCI, and 50 mM Tris pH 8), and protein was eluted with a gradient concentration of imidazole (50, 100, 250, and 500 mM) in 300 mM NaCI and 50 mM Tris pH 8. Protein concentration was determined (36), and protein integrity was assessed in 4-12% SDS PAGE gels following Coomassie brilliant blue staining and further corrected with bovine serum albumin (BSA) in SDS-PAGE gel (37). Before bioassays were performed, imidazole was removed by dialysis against 6 L of 20 mM carbonate buffer in 3,500 MWCO dialysis cassettes (PIERCE, Rockford, IL, USA).

[0042] Pesticidal Proteins: Pesticidal proteins were solubilized in a 20mM carbonate buffer (except for Mpp51 Aa1 , which used 50mM carbonate buffer), each placed into a 1 .5mL microcentrifuge tube. Pesticidal proteins were placed in a -80°C freezer for future use. The 11 pesticidal proteins, the bioassays that were performed, and the testing status for the organisms tested are listed in Table 1 .

[0043] Table 1 . Eleven pesticidal proteins for SHB larval activity in initial screenings. If activity was shown via increased larval mortality, an additional step-wise bioassay was run with a range of concentrations based on the initial screening results. Honey bee cage assays were also used with pesticidal proteins that passed initial screening to investigate protein activity in honey bees at high concentrations.

[0044] SHB Larval Rearing: To provide larvae for conducting bioassays, adult small hive beetles were placed into larval rearing containers. Adequate MegaBee diet was provided, and all adults were sexed to ensure the presence of at least one adult male and female. Containers were placed in a dark incubator room at 31 °C and 50-70% humidity for 4 days. All adults used for each pesticidal protein treatment were age-matched siblings to further control any age or genetic factors that could affect the results of the bioassays.

[0045] After the fourth day since placing adults in the larval rearing containers, all containers were inspected daily to observe larval development. Third instar larvae were used in these bioassays, usually reaching this stage of development after 5 days of the adults being introduced to the rearing chamber. If containers developed at different rates (i.e. some adults were slower to lay eggs), only containers with third instar larvae were used. If an individual container contained a mixture of instars, especially common when more than one adult female is present, then only third instar larvae were used and separated from the other larvae via straining or picking up the larvae gently with forceps. Larvae were separated from contaminants the morning of each bioassay, placed in a 25mL plastic cup, and a paper towel was placed over the lid to reduce stress by providing a dark environment.

[0046] Bioassays of SHB Larvae with Pesticidal Proteins: These bioassays consisted of 5 x 25mL plastic cups of 10 third-instar SHB larvae per treatment concentration (n = 50). In each cup, 3g of MegaBee diet mixture was added to the center. In carbonate-bicarbonate buffer control treatments, a volume equivalent to the volume of the lowest treatment concentration of the corresponding pesticidal protein was dispensed via pipet to 25mL of a 33% sucrose solution in a 100mL glass beaker and mixed with MegaBee powder using a ScoopulaTM in a 38:30 mass ratio, respectively. In all other pesticidal protein treatments, the desired volume of the solubilized pesticidal protein was removed from the 1 .5mL microcentrifuge tube and dispensed into 25mL of 33% sucrose solution in a 100mL glass beaker to acquire the desired pesticidal protein concentration in the sucrose solution. The MegaBee powder was added and mixed in the same manner as the carbonate buffer solution.

[0047] Once the larvae and MegaBee diet had been added, lids were placed on the plastic cups and 5 holes were poked in each lid using a thumbtack to provide ventilation. All cups in each bioassay were placed randomly on plastic trays. The trays were filled a quarter of the way with water, paper towels were placed on the edges, and another plastic tray was placed upside down on top of the bottom tray to create a high humidity (+80%) environment. Mortality was checked daily, and water was refilled each day for 5-7days. Larvae were considered dead if, upon probing, they did not respond. Dead larvae were not removed from their cups.

[0048] Initial screening assays were conducted with all pesticidal proteins at relatively high concentrations of 2-4ppm. If a protein caused significant mortality in SHB larvae, another trial was conducted using a range of lethal to sub-lethal concentrations to provide further understanding of the optimal lethal concentration for SHB larvae.

[0049] Honey Bee Cage Assays with Pesticidal Proteins: Honey bee cage assays were conducted on Cry2Ab, App6Aa2, and Mpp51Aa1 proteins (Table 1); these were conducted in a dark incubator room kept at 34°C set to 50% humidity. A total of 15 one-day-old honey bees were placed in each cage with 5 cages per protein treatment (n = 75). Each cage was supplied with a sucrose feeder on top of the cage made from a 15mL conical tube with two small holes poked at the bottom for sucrose accessibility. Sucrose feeders were filled ad libitum with 33% sucrose solutions. Each protein treatment was made by adding the pesticidal protein to 100mL of sucrose solution to make 3ppm protein treatments and kept in a glass Pyrex bottle at 4°C on ice for the duration of the experiment. Bee mortality was checked daily for 14 days. Bees were considered dead if unresponsive to external stimuli and were removed each day.

[0050] Results:

[0051] Initial screening SHB Bioassays: The first bioassay (Fig. 2) was conducted using the proteins Cry2Aa, Cry2Ab, Cry2Ac, App6Aa2, and Mpp51 Aa1 at 3ppm. There were no significant differences between control and treatment survival for Cry2Aa (HR=0.47, p=0.055) and Cry2Ac (HR=0.77, p=0.45). There were, however, significant effects of treatment on SHB larval survival for App6Aa2 (HR=3.74, p=6.4e-6), Mpp51Aa1 (HR=1.55, p=0.02), and Cry2Ab (HR=3.81 , p=1 .2e-5). A second bioassay (Fig. 3) showed that neither Xpp37Aa1 (HR=0.78, p=0.83), Tpp78Aa1 (HR=0.46, p=0.39), Tpp78Ba1 (HR=0.99, p=0.99), norTpp80Aa1 (HR=1.13, p=0.81 ) affected SHB survival at 4ppm compared to the carbonate buffer control. A third bioassay (Fig. 4) showed that neither Xpp55Aa1 (HR=1 .05, p=0.96) nor Mpp23Aa1 (HR=1.20, p=0.95) fed at 3ppm resulted in significant differences in mortality from the buffer control.

[0052] Step-wise Dose-response SHB Bioassays: Based on the results from the initial screening bioassays, App6Aa2 (Fig. 5), Mpp51Aa1 (Fig. 6), and Cry2Ab (Fig. 7) were selected for additional bioassay testing with a distribution of lower concentrations (ranging from 0.04ppm to 2.0ppm) to gather dose-response data to find the optimal concentrations for SHB larval mortality.

[0053] All concentrations of App6Aa2 showed significant effects on SHB larval survival compared to controls (Table 2).

[0054] Table 2. App6Aa2 concentrations from a step-wise dose-response SHB larval bioassay.Each concentration (n = 50) is listed with its corresponding hazard ratio (HR) and p-value in comparison to the carbonate buffer control treatment. All p-values were corrected manually using a Benjamini-Hochberg correction to decrease the false discovery rate at a < 0.05.

[0055] As shown in Table 2, the pesticidal effects were not significantly different from one another in pairwise comparisons from 0.04ppm to 1 .Oppm. At 2.0ppm, however, the pesticidal effects, though significantly different from the carbonate buffer control treatment, were reduced. Notably, feeding aversion (abandoning diet) in cups was observed at 2.0ppm.

[0056] Mpp51 Aa1 (Table 3) and Cry2Ab (Table 4) treatment concentrations all had significant impacts on SHB larval mortality. Similar to App6Aa2, none of the concentrations significantly differed from each other. Despite this similarity, at 2.0ppm, feeding aversion was not observed to the same extent in both Mpp51 Aa1 and Cry2Ab.

[0057] Table 3. Mpp51 Aa1 concentrations from a step-wise dose-response SHB larval bioassay.

[0058] Table 4. Cry2Ab concentrations from a step-wise dose-response SHB larval bioassay.

[0059] Honey Bee Cage Assays: After 14 days of exposure to 33% sucrose solutions treated with App6Aa2, Mpp51 Aa1 , or Cry2Ab, there were no significant differences in honey bee survival in any of these treatments when compared to the carbonate buffer control (Fig. 8). Mpp51Aa1 (HR=1.22, p=0.66) had the numerically highest mortality of all treatments with 11 of 75 honey bees dying by the end of the 2-week trial. App6Aa2 (HR=0.80, p=0.67) and Cry2Ab (HR=0.66, p=0.46) both had numerically lower honey bee mortality than the carbonate buffer control with 9 of 75 honey bees dying.

[0060] Discussion:

[0061] From the initial screening bioassays, SHB larval survival was not affected by the ingestion of diet containing Cry2Aa, Cry2Ac, Mpp23Aa1 , Tpp78Aa1 , Tpp78Ba1 , Tpp80Aa1 , Xpp37Aa1 , and Xpp55Aa1 at doses between 2-3ppm.

[0062] Cry2Aa, Cry2Ab, and Cry2Ac were chosen for these bioassays due to their activity in a variety of insect larvae. Each of these proteins promote colloidosmotic lysis by binding to the midgut epithelial cells of lepidopteran larvae, with Cry2Aa and Cry2Ac also affecting dipteran larvae. That Cry2Aa and Cry2Ac have no significant impact on SHB larval mortality is unsurprising as they are effective primarily against lepidopteran larvae, not coleopteran larvae (8). Cry2Ab increased mortality in SHB larvae relative to the carbonate buffer control,affecting beetles after only a few days. Cry2Ab is often used in combination with Cry1 Ac to delay resistance evolution in lepidopteran species, such as the cotton bollworm (Helicoverpa. armigera) (13). Similarly, the Tpp class proteins tested in these bioassays did not cause significant mortality in small hive beetle larvae; Tpp proteins are p pore-forming proteins and are highly toxic to hemipterans is toxic to Laodelphax striatelus (1), and Tpp78Aa1 is toxic to L. striatelus and Nilaparvata lugens (18). No significant effects on small hive beetle mortality from this class of pesticidal proteins was observed.

[0063] Mpp51 Aa1 , Mpp23Aa1 , and Xpp37Aa1 , however, are toxic to coleopterans.Mpp51 Aa1 is effective against the Colorado potato beetle (Leptinotarsa decemlineata) (14) and Mpp23Aa1 and Xpp37Aa1 are toxic to beetle grubs in the family Scarabaeidae (5). Mpps are aerolysin-type pore-forming pesticidal proteins with unique specificity for target species due to the diversified head region of the protein involved in receptor binding (12). This specificity of Mpp proteins may explain why only one of the Mpp proteins had significant toxic effects on SHB larvae. Additionally, Mpp23 and Xpp37 proteins are considered binary toxins, meaning they require both proteins together to cause toxicity in organisms (7).

[0064] In honey bee cage assays, no SHB-active pesticidal proteins (App6Aa2, Mpp51 Aa1 , Cry2Ab) caused significant mortality in honey bees during these bioassays. These assays were conducted with 3ppm pesticidal proteins (an extremely high dose) and diet containing these high doses were the only food sources for these honey bees for 14 days, so results provided herein demonstrate that even in a worst case scenario the honey bee survival is not affected by these pesticidal proteins. Of 11 pesticidal proteins tested, three proteins (App6Aa2, Mpp51Aa1 , Cry2Ab) were shown to be honey bee-safe, small hive beetle control agents.

[0065] References:1 . Cao, et al., Journal of Agricultural and Food Chemistry, 68(8): 2539-2546, 2020.2. Crickmore et al., Journal of Invertebrate Pathology, 186: 107438, 2021 .3. Eyer, et al., Journal of Apicultural Research and Bee World 48(4): 296-297, 2009a.4. Eyer, et al., Apidologie 40(4): 419-428, 2009b.5. Naveenarani, et al., Genomics, 114(1): 185-195, 2022.6. Neumann, et al., Apidologie 35: 229-247, 2004.7. Palma, Toxins, 6(12): 3296-3325, 2014.8. Saleem, et al., Toxins, 9(11 ): 358, 2017.9. Schafer, et al., Biological Invasions, 21 : 1451-1459, 2019.10. Stuhl, C., Journal of Apicultural Research, 62(3): 502-506, 2023.11 . Stuhl CJ. Fla Entomol 100(4): 693-697, 2017.12. Tavares et al., Journal of Invertebrate Pathology, 195: 107845, 202213. Tay, et al., PLoS Genetics, 11 (11): e1005534, 2015.14. Xu et al., Biochemical and Biophysical Research Communications, 462(3): 184-189, 2015.15. Dominguez-Arrizabalaga et al., Toxins, (Basel) 12, 2020.16. Elgaizawy et al., J. Biol. Pest Contr. 29:94, 2019.van Frankenhuyzen et al., J. Invertebr. Pathol. 101 :1 -6, 2009. Wang et al., Arch. Insect. Biochem. Physiol., 102:e21558, 2019 Gonthier et al., Ecol Evol 9:6422-32, 2019 Gordon et al., Australian Veterinary Journal 92:283-90, 2014. Neumann, et al., Apidologie 47:427-466, 2016. Idrissou et al., Scientific Reports 9:10665, 2019. Hood et al., In Proceedings of the 8th IBRA International Conference on Tropical Bees and VI Encontro sobre Abelhas, Ribeirao Preto, Brasil, 6-10 September, 2004, ed. KH Hartfelder, Dd Jong, RA Pereira, Ad Santos Cristino, MM Morais, et al:106-10. Number of 106-10 pp, 2004. Spiewok et al., Apidologie 38:595-605, 2007. Hoffmann et al., Insectes Sociaux 55:153-62, 2008. Neumann et al., Journal of Apicultural Research 49:1 -138, 2010. Schafer et al., Annals of the Entomological Society of America 104:476-82, 201 1 Cornelissen et al., Glob Chang Biol 25:3642-55, 2019. Schafer et al., Apidologie 41 :14-20, 2010. Amiri et al., Insects 10:9, 2019 Grozinger et al., Annual Review of Entomology 64:205-26, 2019. Manly et al., Molecular Ecology 29:380-93, 2020. Siegfried et al., Journal of Economic Entomology 98:1320-4, 2005. Tavares, et al., Journal of Invertebrate Pathology, 95: p. 107845, 2022. Zhang, R., et al., Biochemistry, 47(43): p. 1 1263-11272, 2008. Bradford, M.M., Analytical biochemistry, 72(1 -2): p. 248-254, 1976. Wang, Y., et al., Journal of invertebrate pathology, 158: p. 1 -5, 2018.

Claims

CLAIMSWhat is claimed is:1 . A method of reducing a small hive beetle pest population comprising contacting the small hive beetle with one or more bacterial pesticidal proteins selected from App6Aa2, Mpp51Aa1 and Cry2Ab.

2. The method of claim 2, wherein the contacting step comprises ingestion of the bacterial pesticidal protein.

3. The method of claim 1 or claim 2, wherein the bacterial pesticidal protein is formulated in a honey bee nutritional supplement patty.

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