Formic Pro™ Efficacy Trial

In the fall of 2017, the efficacy of Formic Pro™ to reduce infestations of varroa mite (Varroa destructor Anderson and Trueman) in colonies of honey bees (Apis mellifera L.) was tested. A total of 36 colonies were divided into three treatment groups. One treatment group (n = 12) received five separate doses of liquid formic acid at a concentration of 65% across a twenty day period, while the other two treatment groups were treated with strips of Formic Pro™ either with two strips for 14 days (n = 12) or two consecutive rounds of a single strip, each for 10 days (n= 12). The 65% liquid formic acid was used as a standard to compare the efficacy of Formic Pro™ pads. All three treatments reduced mite infestations, but the Formic Pro™ treatments resulted in the greatest mite mortality during the treatment period. Percent efficacies for the treatments were 55%, 90%, and 85% for the liquid formic acid, 14-day Formic Pro™, and 20-day Formic Pro™ treatments, respectively. Based on this trial, Formic Pro™ appears to be an effective tool for the control of varroa mite in Nova Scotia.

1.0 Introduction

Several chemical products designed to reduce infestations of the invasive varroa mite (Varroa destructor Anderson and Trueman) are registered for use in Canada. Canadian beekeepers typically rotate between the use of organic acid treatment (i.e. formic acid and oxalic acid) and synthetic miticide treatment (i.e. amitraz and fluvalinate)1. Beekeepers rely on alternating synthetic miticide applications with organic acids to reduce the risk of miticide resistance developing as a result of frequent and widespread application1. Formic acid is commercially available in Canada in bulk liquid form with a 65% concentration which can be soaked into cotton pads and placed on the top bars of frames within hives. The acid is highly volatile so several rounds of application are necessary to effectively treat an infested colony. This requires a beekeeper to frequently return to and open their hives which is labor intensive, time consuming, and disruptive to the colonies.

Formic acid as a varroa treatment is also commercially available in Canada as Mite Away Quick Strips® (MAQS®) (NOD Apiary Products, Frankford, ON). MAQS® are pre-divided strips which contain a formulation of 46.7% formic acid. These strips can be applied in one or two rounds over a shorter treatment period than liquid formic acid which requires four to six rounds of application2. A new product, Formic Pro™ (NOD Apiary Products, Frankford, ON) is an amended version of MAQS® that features an extended 24 month shelf life, and is commercially

1 Lebeouf, Anne, Medhat Nasr, Julie Ferland, Geoff Wilson, Chris Jordan, Melanie Kempers, Paul Kozak, Rheal Lafreniere, Chris Maund, Steve Pernal, Jason Sproule, and Paul Van Westendorp. "Canadian Association of Professional Apiculturists Statement on Honey Bee Wintering Losses in Canada (2016). http://www.capabees.com/shared/2015/07/2016-CAPA-Statement-on-Colony-Losses-July-19.pdf. 2 Health Canada Pest Management Regulatory Agency. “65% Formic Acid” http://pr-rp.hc-sc.gc.ca/1_1/view_label?p_ukid=94278586.

available in the United States (U.S.). Initial testing has shown Formic Pro™ to be 83%-97% effective at controlling both phoretic mites parasitizing adult honey bees and developing mites underneath brood cell cappings3. Due to its slow release formula, both MAQS® and Formic Pro™ are permitted for use while honey supers are in place and are appropriate late-summer/ early-fall treatments while ambient temperatures are typically within the recommended application range4,5. Applications of liquid 65% formic acid, however, are not permitted while supers are in place.2 Formic Pro™ is not yet commercially available in Canada. The objective of this trial was to evaluate the efficacy of Formic Pro™ as an alternative treatment for fall varroa management in Nova Scotia.

2.0 Materials and Methods 2.1 Test Colonies The trial took place in the fall of 2017 in five separate apiaries managed by the same beekeepers in Colchester County, Nova Scotia. Colonies of western honey bees (Apis mellifera L.) were housed in wooden Langstroth hive boxes. These test colonies were not managed separately from the rest of the colonies owned and operated by the hosting beekeepers apart from the specific treatments applied in this trial. The test colonies were not homogenized in terms of population prior to the initiation of the trial to reflect the efficacy of Formic Pro™ among apiaries with a diversity of colony strength and health profiles. Following the U.S. Formic Pro™ product label, only colonies whose populations of adult bees cover a minimum of six frames were included in the trial5. The number of seams of bees (i.e. spaces between frames lined with adult bees) were counted as an assessment of this colony strength threshold. The orientation and position of test colonies within each apiary were not adjusted.

Only colonies whose varroa mite levels met the August economic threshold for treatment defined by OMAFRA6 (i.e. minimum 3 mites per 100 bees) were included in the trial. Pre-treatment varroa mite levels were quantified using the alcohol wash method described by The Canadian Association of Professional Apiculturists Best Management Practices for Honey Bee Health7 three days prior to treatment (D-3). At this time, colonies were confirmed to have a laying queen and brood present. Colonies were verified for suitability until 36 colonies were found across the smallest number of apiaries in the operation possible. The mean distance

3 http://nodglobal.com/formic-pro/. 4 Health Canada Pest Management Regulatory Agency. “Mite Away Quick Strips” http://pr-rp.hc-sc.gc.ca/1_1/view_label?p_ukid=93186931. 5 NOD Apiary Products. “Formic Pro FAQS” http://nodglobal.com/wp-content/uploads/2017/0®3/2017-Formic-Pro-FAQS-North-America.pdf. 6 Ontario Ministry of Agriculture, Food and Rural Affairs. "Varroa Mite - Sampling and Monitoring Infestation Levels." http://www.omafra.gov.on.ca/english/food/inspection/bees/varroa-sampling.htm. 7 Canadian Association of Professional Apiculturists. "Canadian Best Management Practices for Honey Bee Health." http://www.honeycouncil.ca/images2/pdfs/BMP_manual_-_Les_Eccles_Pub_22920_-_FINAL_-_low-res_web_-_English.pdf.

between apiaries was 11.83 km with the shortest distance being 2.78 km and the longest being 21.03 km. All but two apiaries were greater than 5 km apart (Appendix).

The selected treatment colonies were labeled 1 to 36. Each apiary contained several additional colonies that were not included in the trial whose varroa mite levels were unknown. These colonies were treated with 65% liquid formic acid by the hosting beekeepers in approximately the same treatment window as the colonies in the trial.

2.2 Temperature Monitoring

At each apiary, temperature probes (Watchdog B-series Button Loggers, Spectrum Technologies, Aurora IL) were placed in small plastic containers (Glad® To Go Snack containers, Glad, Oakland, CA) out of direct sunlight to record ambient temperature. Small holes were perforated into the containers to allow for air flow. Readings were recorded once per hour, 24 hours per day, from D-2 to D+34.

Figure 1. An example of temperature probe setup used to monitor ambient temperature throughout the treatment period at each apiary.

2.2 Experimental Design

The trial was set up using a completely randomized design. Each of the 36 test colonies were randomly assigned to one of three equally proportioned treatment groups regardless of strength and location: 14-day Formic Pro™ treatment, 20-day Formic Pro™ treatment, and 65% liquid formic acid treatment. Colonies in the first group were each treated with two strips of Formic Pro™ on Day 0 which were left in their respective hives for 14 days. Colonies in the second group were each treated with one strip Formic Pro™ on Day 0 for ten days, which was replaced by another strip for nine additional days. A full ten days for the second round of

treatment could not be reached due to time conflicts. The third group was treated with five rounds of 30 mL of 65% formic acid as per OMAFRA treatment recommendations8. The colonies in this group with two brood boxes (i.e. doubles) received two cotton pads (Dry Loc® Pads, Cryovac®, Sealed Air, Duncan, SC) soaked with 15 mL of 65% formic acid (Formic Acid 65%, Medivet Pharmaceuticals, High River, AB) and colonies spanning one brood box (i.e. singles) received five rounds of 15 mL pad applications every four days (except for the fifth application which remained for three days) each over a 19 day period (D0 – D+18). No untreated control group was included in the trial due to the risk of mite populations reaching dangerous levels in these hives leading to mite drift9, which could have potentially comprised the trial and the overall health of the host beekeeping operation.

2.3 Treatment Protocol

From D-2 to D0, the natural rate of mite mortality in each test colony was monitored. A screened bottom board10 was placed underneath each hive to allow mites to fall through the screen and down onto a file folder which was able to be inserted and removed below the screen without disturbing the colony. The file folders used in this trial were legal sized (37.5 cm x 46.4 cm) (Staples® Recycled File Folder, 1/2-Cut, Legal Size, 11 pt., Manila, Framingham, MA) covered in a thin layer of petroleum jelly (Vaseline®, Unilever, London, UK/ Rotterdam, Netherlands) to ensure falling mites were immobilized upon landing on the folders. The inner dimensions of a Langstroth hive body are 37.5 cm x 46.3 cm so the legal sized file folders functioned as a suitable alternative to commercially available Integrated Pest Management (IPM) boards11. Prior to the application of petroleum jelly, grid lines with approximate dimensions of 4cm x 4cm were drawn onto the folders to provide greater ease in counting fallen mites. The file folders were replaced two days later (D0) from each test colony and wrapped in a thin layer of plastic wrap (Cling Wrap®, Glad, Oakland, CA) to prevent mites from falling off the boards. The file folders were labeled and returned to the lab for quantification of fallen mites.

8 http://www.omafra.gov.on.ca/english/food/inspection/bees/2014-treatment.htm#varroa. 9 Jay, S. Cameron. “Drifting of Honeybees in Commercial Apiaries 1. Effect of Various Environmental Factors.” Journal of Apicultural Research, vol. 4, no. 3, 1965, pp. 167–175., doi:10.1080/00218839.1965.11100119. 10 https://dancingbeeequipment.com/products/screened-bottom-board. 11 https://countryfields.ca/products/varroa-sticky-board.

Figure 2. Legal sized file folders covered in petroleum jelly slid underneath the screens of the bottom boards functioned as sticky IPM boards to monitor mite drop (A). Collected file folders were wrapped in thin plastic to preserve the fallen mites (B). Grid lines were drawn on the IPM boards to assist in counting fallen mites (C).

Formic Pro™ and 65% formic acid were applied to the respective colonies on D0 with the screens in all bottom boards closed off. File folders were replaced on D0, D+2, D+8, D+12, and D+16 during the period of formic acid treatment. Pads with 15 mL of 65% formic acid were replaced in the liquid formic hives on D+4, D+8, D+12, and D+16. Formic Pro™ strips were replaced in the 20-day treatment hives on D+10. Formic Pro™ strips were removed from 14-day hives on D+14 and the remaining Formic Pro™ strips and 65% formic acid pads were removed on D+19. Post-treatment alcohol wash samples were taken from each test colony on D+19. On D+27, Apivar® (amitraz, Véto-pharma, Villebon sur Yvette, France) – a miticide with a known high efficacy at killing varroa mites12 – was applied to each test colony as a “clean-up” treatment at the appropriate product label dosage for 14 days. File folders were replaced on D+27 and again on D+34 which remained until D+41 when Apivar® strips were removed from each test hive and all sticky boards were removed.

Colony feeding was carried out by the hosting beekeepers. Despite label recommendations to avoid in-hive feeding during the Formic Pro™ treatment period5, hive top feeders were placed on all test hives the week of October 16th (approximately D+11 – D+16).

12 https://www.dadant.com/wp-content/uploads/2012/04/2011/09/Apivar-Brochure-USA.pdf.

Figure 3. Formic Pro™ strips placed on the top bars of frames in hives as per label recommendations – two strips per colony for 14-day treatment group (A) and one strip per colony for 20-day treatment (B).

2.4 Statistics

Analyses for colony strength data measured by the number of seams of bees were carried out using the statistical program Minitab 1713 and the model assumptions of normal distribution and constant variance of the residuals were met. Analyses for mite count data obtained from alcohol washes and mite drop data obtained from IPM boards were carried out using the statistical program SAS version 9.414. Square root transformations were applied to data reported in mite infestation and mite drop data in Figures 5 and 8, respectively, and a cube root transformation was applied to mite drop data reported in Figure 6 to induce normality and constant variance. Means reported in all figures from transformed data were back transformed to their original values. All analyses of significance were conducted at the 95% confidence level (i.e. α = 0.05).

Percent efficacies for each treatment group were calculated by dividing the average number of mites fallen from D+27 – D+41 by the average number of mites fallen from D0 – D+26, multiplying that value by 100, and subtracting that percent value from 100 (Table 1).

3.0 Results 3.1 Temperature Ambient temperature during the treatment period remained largely within the recommended range of 10°C and 29.5°C5 for all five test apiaries (Figure 4). One of the five apiaries (i.e. A1)

13 http://www.minitab.com/en-us/products/minitab/. 14 https://www.sas.com/en_ca/software/stat.html.

reached temperatures above 29.5°C on six separate occasions but remained above this threshold for only one hour each time (Appendix 1).

Figure 4. Daily high ambient temperatures for each of the five apiaries in the trial (A1 – A5). The upper limit to the daily high ambient temperature recommended for Formic Pro (i.e. 29.5°C) is indicated with a red line.

3.2 Colony Strength

Seams of bees in each test hive were counted prior to the trial initiation (i.e. D-3) and again at the end of the treatment period (i.e. D+18). Comparative colony strength between treatment groups did not differ significantly at the 95% confidence level both pre (F2, 33 = 0.82, P = 0.449) and post-treatment (F2, 33 = 0.99, P = 0.383). Average initial colony strength was 11.8, 11.1, and 9.88 seams and average final colony strength was 8.46, 6.75, and 6.46 seams for the liquid formic, 14-day, and 20-day treatment groups respectively. Colony strength differed marginally at the 95% confidence level in the liquid formic group pre vs. post-treatment (F1, 22 = 3.68, P = 0.068) and significantly in the 14-day and 20-day treatment (F1, 22 = 7.51, P = 0.012; F1, 22 = 7.34, P = 0.013, respectively). Average initial colony strength was greater in each treatment group than average final colony strength.

3.3 Varroa Mite Mortality

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Varroa mite infestation levels of each test colony were assessed pre-treatment (D-3) and again post-treatment (D+19) with alcohol washes. There was a significant difference between the pre and post-treatment mite washes for each of the treatments (F 5, 63 = 0.09, P < 0.0001) (Figure 5). Average initial varroa mite levels were 12.0%, 13.3%, and 12.3% and average final levels were 6.82%, 2.47%, and 3.67% for the liquid formic, 14-day, and 20-day treatments respectively (Figure 5).

Figure 5. Percent mite infestation levels for each treatment period pre and post formic treatment (obtained from alcohol washes). Letter groupings were generated using Fishers LSD and indicate significant difference at the 95% confidence level.

Separating the test colonies into double and single hives, there was a significant difference between the pre and post-treatment mite washes for both doubles and singles (F 3, 65 = 15.97, P < 0.0001) (Figure 6). Initial and final infestation levels between single and double colonies did not differ significantly.

Figure 6. Percent mite infestation levels in all treatment periods separated into single and double colonies pre and post formic treatment (obtained from alcohol washes). Letter groupings were generated using Fishers LSD and indicate significant difference at the 95% confidence level.

Mite drop was recorded over eight separate monitoring periods. Overall, there was a significant interaction between treatment and time period (F 14, 264 = 4.70, P < 0.0001) (Figure 7). The natural mite drop that was monitored over a two-day pre-treatment period (D-2 – D0) did not differ significantly across the three treatment groups (Figure 7). During the two-day monitoring period after the first round of treatment (D0 – D+2), a drastic increase in mite drop occurred in all colonies which differed significantly from the pre-treatment period as well as among the three treatment groups (Figure 7). The 14-day Formic Pro™ group resulted in the greatest increase in mite drop comparing the D0 – D+2 and D-2 – D0 periods (2,249%), followed by the 20-day Formic Pro™ (1,702%), and the liquid formic acid (1,169%). Mite drop did not differ significantly among the three treatment groups for the remainder of the formic treatment period (D0 – D+26).

Figure 7. Mean mite drop obtained from file folder collection for each treatment group monitored over one pre formic collection period, five during formic collection periods, and two post formic collection periods. Letter groupings were generated using Fishers LSD and indicate significant difference at the 95% confidence level.

Comparing the final during-formic (D+16 – D+26) and first “clean-up” mite drop monitoring periods, there was a significant difference between treatment and period (F 2, 66 = 3.58, P = 0.0334) (Figure 8). Mean mite drop in both Formic Pro™ treatment groups was significantly greater than the liquid formic treatment in the first post-formic “clean up” monitoring period (D+27 – D+33).

Figure 8. A comparison of mean mite drop obtained from file folder collection for each treatment group between the final during-formic monitoring period (D+16 – D+26) and the initial post-formic, “clean-up” monitoring period (D+27 – D+33).Letter groupings were generated by Fishers LSD and indicate significant difference at the 95% confidence level.

3.4 Percent Efficacy The total mite drop of each colony over the formic treatment period (D0 – D+26) was summed and means were calculated for each treatment group (Table 1). The aforementioned was also done for the post-formic Apivar® “clean-up” treatment (Table 1). Table 1. Percent efficacies for each treatment group calculated from mite drop data averaged across all colonies in each treatment group both during the formic acid treatment and during the Apivar® “clean-up” treatment.

D0 - D+26 D+27 - D+34 % efficacy Liquid formic 2493 1125 54.86 14-day Formic Pro™ 3785 390.8 89.68 20-day Formic Pro™ 3209 487.4 84.81

4.0 Discussion

Despite the late-season initiation of the trial, temperatures remained relatively warm and daytime temperatures remained within the recommended temperature guidelines for Formic Pro™. The late-season warm temperatures allowed colonies in Nova Scotia to continue brood rearing later in the fall than normal. All test colonies contained brood at the beginning of the trial. Due to the warm temperatures, adult bees in all treatment colonies remained active as foragers were regularly seen departing and returning to their hives. In order for formic acid to effectively reduce varroa infestations, adult bees must remain active enough to fan the fumes produced as the formic acid impregnated in Formic Pro™ strips and soaked into the cotton pads evaporates.3

As expected with the seasonal timing of the trial, almost every colony decreased in strength over the course of the treatment period. Due to the lack of an untreated control group, it is difficult to discern whether the decrease in colony strength can be fully attributed to a natural autumnal decrease in colony population15 or whether a treatment effect on colony strength occurred. No statistical difference was observed among treatment groups both before and after the treatment period but the final colony strength was significantly smaller than the initial strength in all three treatment groups. Therefore, if Formic Pro™ did have an effect on colony strength in this trial, its effect was not significantly different than that of liquid formic acid.

Due to its slow release formula, Formic Pro™ is suitable to use during the honey flow while supers remain on hives5. At the request of the hosting beekeepers, the trial was initiated after the removal of the honey supers, which remained on the hives relatively late in the season due to an autumn honey flow. Mite levels in test colonies were relatively high at the initiation of the trial and on average were well above the late-summer economic threshold6. It is possible these mite levels reached these high levels due to a delay in mite treatment. Drift from foraging bees from neighbouring colonies carrying phoretic mites can also contribute to high varroa populations in the fall16. Phoretic varroa mites shift their preference to parasitize older forager bees as opposed to nurse bees in the fall as a possible means of dispersal17.

As brood rearing decreases in the fall, so does the proportion of developing varroa mites under brood cappings compared to phoretic mites18. Alcohol washes only sample a colony’s

15 Harris, J L. “Development of Honey Bee Colonies Initiated from Package Bees on the Northern Great Plains of North America.” Journal of Apicultural Research, Jan. 2008, pp. 141–150., doi:10.3896/ibra.1.47.2.10. 16 Degrandi-Hoffman, Gloria, et al. “Population Growth of Varroa Destructor (Acari: Varroidae) in Honey Bee Colonies Is Affected by the Number of Foragers with Mites.” Experimental and Applied Acarology, vol. 69, no. 1, 2016, pp. 21–34., doi:10.1007/s10493-016-0022-9. 17 Kuenen, L. P. S., and N. W. Calderone. “Transfers of Varroa Mites from Newly Emerged Bees: Preferences for Age- and Function-Specific Adult Bees (Hymenoptera: Apidae).” Journal of Insect Behavior, vol. 10, no. 2, 1997, pp. 213–228., doi:10.1007/bf02765554. 18 Sakofski, F., et al. “Seasonality of Honey Bee Colony Invasion by Varroa Jacobsoni Oud.”Apidologie, vol. 21, no. 6, 1990, pp. 547–550., doi:10.1051/apido:19900608.

phoretic mite population and so infestations measured with this monitoring technique may appear to be larger than if they were sampled earlier in the year. It is unknown what the mite infestation levels were in the test colonies in late summer prior to the initiation of the trial. Although ambient temperatures were too cold to inspect colonies for brood at the end of the trial, there was likely little to no brood present at this time and effectively all varroa mites were phoretic. The alcohol washes taken post-treatment would therefore have been reflective of the true total mite population, unlike the pre-treatment samples which excluded the proportion of developing mites under brood cappings. Regardless, a significant reduction in mite infestation level was sampled across all three treatment groups over the course of the trial. The liquid formic treatment, reduced mite levels to a lesser degree than both Formic Pro™ treatments.

The largest mite drop occurred in the first two days after treatment and mites continued to drop over the course of the treatment period. Evidently, both Formic Pro™ and liquid formic acid induce their greatest treatment effect in the first few days of application. Untreated control colonies should have been used to observe this effect but were unable to be used due to the magnitude of the infestation levels during treatment. All treatments reduced mite populations to some degree but some treatments performed better than others.

After the first application of formic acid and Formic Pro™, the 14-day treatment group experienced the largest initial mite drop. This is to be expected as two strips are placed in the hives simultaneously as opposed to two rounds of a single strip at a time. Mite drop then leveled off relatively equally across all three treatments as the treatment period progressed but the 14-day application was the only one to effectively bring mite levels down below the 3% late summer economic threshold, demonstrating its continuous miticidal activity over the course of the treatment period. The “clean up” treatment with Apivar® revealed that there were significantly more mites left in the liquid formic colonies than both groups of Formic Pro™ colonies. Disruptions to the colonies from hive-top feeding could have reduced the efficacy of the final liquid formic treatment. The second round of 20-day Formic Pro™ strips were also present at this time and mite infestation levels were relatively equal between both Formic Pro™ groups post-treatment despite the feeding disruptions.

Considering the alcohol wash and mite drop data, Formic Pro™ was more effective at controlling mite infestations in this trial than liquid formic acid. Considering the percent efficacy calculated from mite drop data, the 14-day treatment had the greatest treatment effect, although not significantly different than 20-day.

Mite levels during this trial were dangerously high in almost every single test colony and repeated testing earlier in the year (i.e. early fall) is necessary to deduce whether Formic Pro™ is an effective fall varroa mite treatment. Symptoms of deformed wing virus (DWV) – an RNA virus vectored by varroa mites that causes wing deformities in developing honey bees – were observed in several colonies across all three treatment groups. The observance of DWV itself indicates serious varroa infestation and suggests that the health of the population of winter

bees being reared is compromised. Correlations between DWV and overwinter colony loss have been observed19. Regardless of DWV, winter bees infested with varroa mites as developing pupae contain lower levels of vitellogenin, a storage protein necessary to sustain bees through the winter season20. Late-season mite treatments may therefore fail to prevent overwinter colony loss.

The treatment results in this trial should not be interpreted as an attempt to answer whether the tested formic acid products are effective at preventing overwinter loss; spring colony survival was not recorded in this trial. Instead, this trial demonstrates the high miticidal efficacy of Formic Pro™, particularly the 14-day treatment option in which mites are exposed to a relatively high initial dose of formic acid.

Prepared by Cameron Menzies, Robyn McCallum, and Sawyer Olmstead, 2018.

For more information, contact Atlantic Tech Transfer Team for Apiculture (http://www.perennia.ca/fieldservices/honey-bees-and-pollination/).

19 Highfield, A. C., et al. “Deformed Wing Virus Implicated in Overwintering Honeybee Colony Losses.” Applied and Environmental Microbiology, vol. 75, no. 22, 2009, pp. 7212–7220., doi:10.1128/aem.02227-09. 20 Amdam, Gro V., et al. “Altered Physiology in Worker Honey Bees (Hymenoptera: Apidae) Infested with the Mite Varroa Destructor (Acari: Varroidae): A Factor in Colony Loss During Overwintering?” Journal of Economic Entomology, vol. 97, no. 3, Jan. 2004, pp. 741–747., doi:10.1093/jee/97.3.741.