Behavioral response of the small hive beetle , Aethina tumida ( Coleoptera : Nitidulidae ) to volatiles of Apicure ® , a plant-based extract

The small hive beetle (SHB), is an invasive pest Background: Aethina tumida of the honey bee. Although no previous methods have led to its successful management, yeast inoculated pollen baited-traps have showed promise as quick monitoring tools. In this study, we evaluated the role of olfaction in SHB response to Apicure®, an essential oil-based biopesticide that has shown potential for the management of honey bee pests and diseases. Volatiles from Apicure® were collected using super Q adsorbent Methods: traps. Subsequent analysis was done using Gas chromatographymass spectrophotometry (GC-MS) to ascertain the components of Apicure®. The selectivity and sensitivity of antennal receptors of adults to the A. tumida volatile compounds were determined using behavioral assays and Gas Chromatography-Electroantennodetection (GC-EAD). GC-MS analysis showed that Apicure® consists of 40 compounds. Results: GC-EAD analysis isolated 11 compounds that elicited antennal response with the SHB. Of these, linalool, camphor, geraniol and α-terpineol were confirmed to be strongly repellant, while limonene was attractive to SHB in dual-choice olfactometer assays. Our results demonstrate that the major components in Apicure® Conclusion: are mainly repellants thus prospective in disrupting the host recognition by the SHB. The product therefore can be up-scaled for the management of SHB.


Introduction
Biological invasions pose a global threat to both food security and natural ecosystems 1,2 .One of such invasive organisms is the small hive beetle (SHB), Aethina tumida Murray, a pest of honey bees which has the capacity to significantly affect the health of both managed and feral eusocial bees 3 .Originally of sub-Saharan descent, this nitidulid (sap beetle) has now become established in honey bee colonies in North and Central America (USA, Canada, Mexico, Cuba, and Nicaragua), North Africa (Egypt), Europe (Italy), Australia and South East Asia 4 .As its spread is likely to have been facilitated by increased global connectivity via world trade, much of Europe and the rest of the world are at risk if its spread continues without appropriate intervention 5 .Many beekeepers have reported huge colony losses that were attributed to the SHB following its identification in the United States in 1998 6 .
Intra-colony destruction has been accredited to the feeding behavior of adult and larval beetles 7 .This pest infests bee colonies as either individuals or in swarms with both adult and larval stages known to cause damage as they feed on pollen, honey, brood and young worker bees 8,9 , causing fermentation of hive products and damage to combs reducing the brood area.During the migration of beetles from colony to colony, they transmit bee pathogens that stick to their bodies hence the possibility of horizontal pathogen transmission to other colonies or apiaries 10 .In addition, the beetles may act as vectors of viruses such as the deformed wing virus 11 and sacbrood virus, that increase the risk of colony collapse and hence lesser productivity in infected colonies.
Previous studies have also reported potential alternative hosts for the SHB such as stingless bee colonies 12,13 and bumble bees 14,15 .In addition to eusocial bees, the SHB has been shown to successfully develop under laboratory conditions on various fruits such as Kei apple, cantaloupe, pineapple, mango, banana, grapes, oranges and decaying meat [16][17][18][19] .Thus, it not only poses a threat to the honey bees but also to fruits as potential diets if not sustainably monitored.However, most of the tools developed are laborious to deploy and monitor.For example, bait like pollen is not cost effective to small-scale beekeepers in the tropics, while chemicals such as organophosphate acaricide coumaphos pose health risks to both beekeepers and consumers due to residues on products.This has led to the need for the development of affordable and more effective management tools 20 .
To date, several studies on non-chemical management strategies for A. tumida, are geared towards development of attractant compounds such as pollen dough inoculated with yeast 21 , beehive produced volatiles 8 , honey bee hive products that have been exposed to the SHB-associated yeast, Kodamaea ohmeri and small hive beetle larvae 22 , apple cider vinegar and yeastbased attractants 23 .None of the studies on SHB management has explored possible repellants that can be used to prevent invasion of SHB into honey bee colonies.Apicure ® , is a novel product developed by Lwande et al 24 .It is an essential oil-based product that comprises of a sponge as a slow release material.The essential oil in Apicure ® is extracted by hydrodistillation from the camphor basil, Ocimum kilimandscharicum a variety species of Ocimum basilicum that is native of Kilimanjaro, Kenya, highly foraged by honey bees 25 .Preliminary field trials illustrated the potential of this product to repel small hive beetles and other pests.
In this study, we hypothesized that Apicure ® comprises of specific compounds that are bioactive against the SHB.Previous studies indicate that some plant secondary metabolites possess biological activities with the potential to exert physiological and/or behavioral effect on insects 26 .We tested this hypothesis by chemical analyses of volatiles in Apicure ® using coupled gas chromatography-electroantennographic detection (GC-EAD) and GC-mass spectrometry (GC-MS).Electrophysiologically active compounds were tested on SHB in dose-response olfactory assays to demonstrate that olfactory cues play a major role in the social behavior of SHB.Our study demonstrated that Apicure ® is generally a repellant to the SHB and this is related to its chemical composition.The repellency is attributed to synergistic or antagonistic activities of its major compounds.This product and its constituent compounds are potential candidates for SHB control programs.

Insects
A colony of the small hive beetle, A. tumida was established from beetles collected at Karura forest (1° 14' 15.00" S; 36° 49' 14.99" E), Kenya in August 2017 using a modified standard protocol 27 .Briefly, female beetles were introduced into a plastic container (11 cm long × 11cm wide × 11 cm high) with a meshed insert (~1 mm mesh) in the middle of the lid to provide aeration.Sliced ripe bananas were added in the containers as feeding substrate and moistened cotton wool maintained humidity and provided water for the beetles.Emerged larvae were also fed on the same substrate (bananas) which was replenished when needed.One-week old larvae which had entered the wandering stage (i.e.crawled away from the food source in search of pupation substrate) were removed from the container and transferred to another plastic container (measuring, 18.5 cm × 14 cm × 9.5 cm) filled up 6 cm depth of autoclaved and moistened sandy loam soil.The larvae burrowed into the soil to pupate.After three weeks, adult beetles emerged from the soil and were transferred to 11 cm long × 11cm wide × 11 cm high plastic containers for feeding.The beetles were then used a week after when they were fully mature (when they turned black).Daily monitoring and collection of emerging SHB were done.

Headspace volatile collection
One sachet of Apicure ® , comprising a sponge with 3.5 g of essential oil, was obtained from the Environmental Health Department at the International Center for Insect Physiology and Ecology (Nairobi, Kenya; icipe).The sachet was cut open with a scalpel and the contents emptied into a 500-ml cylindrical glass flask (Sigma Scientific, Gainesville, FL, USA).Headspace volatiles were collected for 24 hours by aeration and adsorption on charcoal filter adsorbents (5 mg, Brechbuhler, Schlierensee, Switzerland).Each filter was connected by PVC tubing (Masteflex.06409-15 Tygon mfg by St. Gobain) to a mobile battery operated pump (PAS-500 Personal Air Sampler, Supelco, Bellefonte, PA, USA), which supplied a continuous flow of clean air through the sample and also pulled the volatiles to the filter at a flow rate of 348 ml min -1 .All the filters were eluted with 100 ml of GC-grade dichloromethane (Sigma Aldrich, Gillingham, UK) into vials.The eluates were kept at -80 °C in amber screw-capped glass vials until use.

Analysis of volatiles
Coupled GC-EAD analyses was carried out on an Agilent 7890A gas chromatograph equipped with an HP-1 column (30 m × 0.32-mm diameter × 0.25 mm thickness; Agilent, Palo Alto, California, USA).Nitrogen was the carrier gas at a flow rate of 1.2 ml min -1 .The injection was splitless at 280 °C with a split valve delay of 3 min.The oven temperature was held at 35 °C for 5 min, increased to 280 °C at 10 °C/min, and then held at this temperature for 10 min.The column effluent was split 1:1 for simultaneous detection by flame ionization detection (FID) and EAD.The column effluent was mixed with humidified air (200 ml min -1 ) and then passed over the EAD preparation 28,29 .For EAD, silver wires in glass capillary electrodes filled with ringer solution served as the reference and recording electrodes.
To prepare the antennae, the whole head of SHB was cut using a scalpel; the basal segment of the head of SHB was placed in contact with the reference microelectrode and the recording electrode was connected to the distal end of the antenna.The antennal signal was detected through an amplifier (INR-II, Syntech, Hilversum, The Netherlands), which was acquired and processed by a data Acquisition controller (IDAC-4, Syntech, Hilversum, The Netherlands) and later analyzed with GC/ EAD 2000 software (Syntech).An aliquot (3 µl) of the charcoal filter-adsorbed volatile extract of Apicure ® was analyzed with either fresh male or female antenna in four replicates.
GC/MS analyses of the charcoal filter-adsorbed volatile extract of Apicure ® was carried out on an Agilent 7890A gas chromatograph linked to a 5795C mass spectrometry, equipped with MSD Chemstation E.02.00.493, and Wiley 9th/NIST 2008 MS library.Similar GC-MS column and temperature conditions were used as described above in GC-EAD analyses.
The identification of compounds in Apicure ® was done by comparing their retention time and mass spectral fragmentation of corresponding authentic standards in the library.

Bioassays with Apicure ® and synthetic components
To test the behavioral response of SHB to Apicure ® and its components, a Pyrex glass Y-tube olfactometer (Internal diameter 10 mm; stem 85 mm; arms 75 mm at a 60° angle to the stem; Analytical Research System INC, Gainesville FL, USA) was used 28 .The Y-arms of the olfactometer were connected with PVC tubing (Masteflex.06409-15 Tygon mfg.by St. Gobain, Paris, France) to a sealed glass odor source chamber (internal volume 50 ml) supplied with charcoal-filtered and humidified air (90% RH).The airflow through each arm of the Y-tube was maintained at 30 ml min -1 by a battery-powered pump (USDA/ARS-CMAVE, Gainesville, FL, USA).A PVC tube was connected at the base of the Y-tube to the vacuum source of the pump at 60 ml min -1 to avoid volatiles build-up in the test arena.Prior to behavioral tests with Apicure ® and the synthetic standards, positional bias of the Y-tube was done (blank vs blank).Subsequently, about 40 µl of the extracts was applied onto 2.5 cm × 2.5 cm filter papers (No.1 Whatman Int Ltd.Maidstone, England) using a micropipette.The solvent was allowed to evaporate for 2 min before placing into the holding chambers of the Y-tube.An adult SHB was individually introduced at the entrance of the main vertical arm of the Y-tube and considered to make a choice after walking beyond the Y-tube intersection in 1 min.SHB were prevented from escaping through the arms of the olfactometer by a screen mesh barrier at the openings of each arm.In total, 25 adult females were used for each trial which was replicated three times (N = 75).Each individual was used once during the bioassays.The position of test and control odor sources was reversed after every three tests to avoid positional bias.After the experiments, glassware was washed with Teepol ® (multipurpose detergent; Teepol ® products, Kent, UK), rinsed with acetone and then with distilled water and baked in an oven at 80 °C for 2 hours.Apicure ® and the synthetic standards of the electrophysiologically active compounds were tested at three concentrations at 10 ng/µl, 100 ng/µl and 1000 ng/µl.

Chemical standards and reagents
Synthetic standards of camphor, α-terpineol, limonene geraniol and linalool were obtained from Fluka Analytical (>98% purity).All chemical standards were prepared in dichloromethane (Sigma Aldrich) and kept in amber screw-capped glass vials at -20 °C prior to analysis.

Statistical analysis
The response of the beetle to the treatment, i.e., Apicure ® volatiles and synthetic standards of the electrophysiologically active compounds compared to the control (solvent/blank), was analyzed using Chi-square (χ 2 ) goodness-of-fit tests, assuming a distribution ratio of 1:1 to compare responses of the test individual to odor sources and control.Non-responders were not included in the analysis.Data analysis was done using R version 3.4.1 software (R Core Team, 2017).

Discussion
Exploitation of natural products that are effective and are environmentally safe is a promising alternative to synthetic chemicals.Among these potential products, essential oils from several species of plants have been extensively researched to ascertain their repellent activities as a prospective natural resource 30 .In this paper, we demonstrate the phytochemical and bioactivity studies of Apicure ® , an essential oil-based bio pesticide as a potential non-chemical resource to manage the small hive beetle (SHB).While previous reports 24 showed the potential of this product in field experiments to significantly repel beetles, our study provided the chemical basis of the observed bioactivity against the SHB.
Response to Apicure ® .Essential oils have demonstrated a wide range of activity against pests and pathogens ranging from, repellent, antifeedant, oviposition deterrent, insecticidal, growth regulatory and antivector activities 31 .From our olfactometer bioassays, it was ascertained that Apicure ® is a repellant to the SHB and repellency increased with concentration.This results concurs with preliminary field results 24 ., that reported significant repellency of beetles from bee hives when one sachet of Apicure ® was applied.
Analysis of volatiles.GC-MS analyses of volatile organic compounds of Apicure ® revealed that this product is a complex of 40 compounds belonging to different chemical classes.
Essential oils such as Eucalyptus hybrida are described by either two or three major compounds 32 .Our results suggest that Apicure ® is an essential oil-based product that is majorly camphor based.Camphor has been reported for its toxicity and repellency of up to 80-100% to four stored grain beetles 33 .Moreover, it has been incorporated into a push-pull strategy in integrated pest management, developed for multicolored Asian lady beetle, Harmonia axyridiis Pallas 34 .In addition, camphor oil solutions have been reported for treating microbial diseases in bees like sacbrood and bee chronic paralysis viruses 35 .
Bioassays with synthetics standards.Bioactivity of essential oils may be attributed to their major constituents and the minor compounds present in the oil 36 .They may either act either synergistically or else antagonistically to contribute to some activity of the tested oil.This was evident in our behavioral bioassays with the major compounds that generally elicited repellant activity and other attractants signifying that these compounds contribute to the general repellant activity of Apicure ® against the SHB.It will therefore be interesting to study how these compounds blend to contribute to the general bioactivity of this product.The electrophysiologically active compounds identified in this study have been reported to be important in a number of insects and arthropods, especially in host recognition.For instance, camphor which is a repellant to the SHB has also been shown to repel Asian lady beetles 34 .A previous study documented repellency of p-cymene and camphor in oil of tansy (Tanacetun vulgare) against the Colorado potato beetle,Leptinotarsa decemlineata (Say) 37 .Camphor, and α-terpineol have been studied individually on toxicity and mosquito repellency and were also detected in this product and elicited repellency to the SHB significantly.Geraniol has been shown to strongly repel ticks (Ixodes ricinus) 38 .Geraniol, which is highly abundant in nurse bees has been shown to impair the ability of varroa mites to infest nurse bees 39 because of its repellant activity against the varroa mite.Hence this compound can be used to repel varroa mites and SHB at colony level.Some monoterpenes such as linalool has been reported to repel mosquitoes 40,41 and the pollen beetle, Meligethes aeneus Fab. 42.Geraniol and caryophyllene oxide have been reported to repel the Afrotropical malaria mosquito, Anopheles gambiae Giles 43,44 .
Noteworthy, limonene attracted the SHB, though its activity in Apicure ® is suppressed and it will be interesting to evaluate the effect of limonene relative to the activity of other compounds.
Further studies could also evaluate limonene in comparison to reported attractants and its use as a lure in trapping programs.Small hive beetles use semiochemicals to locate honey bee colonies.Limonene in this study was found to be an attractant suggesting that it could be one of the contributing in hive semiochemicals that attracts SHB since it has been reported in bee propolis 2 .Additional, previous studies have highlighted limonene as an attractant to white pine cone beetle, Conophthorus coniperda (Schwarz) 45 .Further studies may evaluate the role of limonene in host recognition by the SHB.
In addition, other studies should evaluate the contribution of individual components in the overall repellency of Apicure ® , and the development of an optimum synthetic blend that can effectively repel the SHB.The interruption of SHB communication is a prospective milestone towards development of a semiochemical-based tool to manage this invasive honey bee pest.This study suggests that Apicure ® repellent components could be utilized as the 'push' and limonene as the 'pull' as a strategy in the management of the SHB and minimize toxic synthetic pesticides in hive products.
Introduction, last sentence in the first paragraph: Please quantify losses instead of being alarmist.
Methods, Headspace volatile collection: (a) Why didn't the authors replicate sachets from which volatiles were collected?This comprises the validity of the findings, (b) Use "of" instead of "for" in the name "International Center for Insect Physiology and Ecology".
Methods, Analysis of volatiles: (a) Use "Ringer's" not "ringer", (b) "SBH was beheaded" could be better than "the whole head of SHB was cut", (c) It is indicated that "volatile extract of Apicure was analyzed with either fresh male or female antenna" but the results, discussion and conclusions never reflect the sex of the insect.
Methods, Bioassays with Apicure and synthetic components: The air flow rates of 30 and 60 ml min are far below the minimum reading of 85 ml min of flow meters used at could these be icipe, scale readings rather than flow rates?And, why was the inlet 'flow rate' half that of the outlet?Why were males not tested, as it was done in GC-EAD assays?Results, Figure 1: Where are the peaks for compounds 38-41 that are shown in Table 1? Figures 2  and 3: Specify which SHB sex are represented since both sexes were tested.Indicate whether all the four replicate antennae generated consistent EADs or only some, which should be specified.The statement "but an avoidance behavior was observed at 1000 ng/µl (χ2 = 15.413,d.f = 1, P = 0.1659)" is not backed up by data on Figure 5.
There are also several minor grammatical errors in the manuscript.

Lukasz L Stelinski
Entomology and Nematology Department, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA This manuscript describes the investigation of a commercially available product named Apicure, which releases an essential oil as a biopesticide, and that is intended for management of the small hive beetle (SHB).This formulation is known to affect SHB negatively when deployed in honeybee hives and the investigators tested the hypothesis that the mode of action of this product may include a behavioral effect on SHB, such as repellency.The investigation employed standard techniques, including gas chromatography-mass spectrophotometry gas-chromatography coupled with electroantennographic detection to narrow down potentially behaviorally active compounds released by the essential oil active ingredient followed by behavioral testing using a two choice olfactometer.Chemical and electrophysiological analyses isolated 11 compounds (mostly terpenes) that elicited electrophysiological responses from SHB antennae.Subsequent behavioral testing revealed that linalool, camphor, genaniol, and alpha-terpeniol (known insect repellents) affected SHB responses in a manner consistent with the and alpha-terpeniol (known insect repellents) affected SHB responses in a manner consistent with the hypothesis that they acted as repellents.An ancillary finding was that limonene attracted SHB in the two choice behavioral assays tested.Based on the results, the authors suggest that the mode of action of Apicure against SHB may be repellency caused by the specifically identified terpinoid compounds.The authors suggest investigating these specific compounds (optimizing blend) to improve activity against SHB and potentially other hive pests.
Overall the conclusions appear justified by the results suggested.I would suggest conducting additional toxicological work with the essential oil and its constituents in future studies, as well as, more sophisticated behavioral testing in behavioral assays that better replicate the hive environment than the two-choice arenas.Perhaps this could be elaborated/discussed further in the manuscript.Finally, there are minor grammatical errors throughout the manuscript that should be corrected.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Is the work clearly and accurately presented and does it cite the current literature?PartlyIs the study design appropriate and is the work technically sound?PartlyAre sufficient details of methods and analysis provided to allow replication by others?Yes If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?PartlyAre the conclusions drawn adequately supported by the results?PartlyNo competing interests were disclosed.Competing Interests:Reviewer Expertise: entomology; chemical ecology I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.27 February 2019 Reviewer Report https://doi.org/10.21956/aasopenres.14023.r26793© 2019 Stelinski L. This is an open access peer review report distributed under the terms of the Creative Commons , which permits unrestricted use, distribution, and reproduction in any medium, provided the original Attribution Licence work is properly cited.