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October 1997

Toxicity of Seven Monoterpenoids to Tracheal (: ) and Their Honey (Hymenoptera: Apidae) Hosts When Applied as Fumigants

Marion D. Ellis University of Nebraska-Lincoln, [email protected]

Frederick P. Baxendale University of Nebraska-Lincoln, [email protected]

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Ellis, Marion D. and Baxendale, Frederick P., "Toxicity of Seven Monoterpenoids to Tracheal Mites (Acari: Tarsonemidae) and Their (Hymenoptera: Apidae) Hosts When Applied as Fumigants" (1997). Faculty Publications: Department of Entomology. 147. https://digitalcommons.unl.edu/entomologyfacpub/147

This Article is brought to you for free and open access by the Entomology, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications: Department of Entomology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. APICULTURE AND SOCIAL INSECTS Toxicity of Seven Monoterpenoids to Tracheal Mites (Acari: Tarsonemidae) and Their Honey Bee (Hymenoptera: Apidae) Hosts When Applied as Fumigants

MARION D. ELLIS AND FREDERICK P. BAXENDALE

Department of Entomology, University of Nebraska, 202 Plant Industries Building, Lincoln, NE 68583-0816

J. Econ. Entomol. 90(5): 1087-1091 (1997) ABSTRACT Laboratory bioassays were conducted to characterize the acute toxicity of 7 monoterpenoids to tracheal mites, woodi (Rennie), and their honey bee, Apis mellifera L., hosts. Citral, thymol, carvacrol, a-terpineol, pulegone, d-limonene, and menthol were applied as fumigants to -infested honey . Thymol and menthol were the most toxic compounds to honey bees, and a-terpineol was the least toxic. Menthol, citral, thymol, and carvacrol were more toxic to tracheal mites than to honey bees. Pulegone, d-limonene, and a-terpineol were more toxic to honey bees than to tracheal mites. Menthol was 18.9 times more toxic to tracheal mites than to honey bees at the LCso concentrations; however, as the concentration increased, bee mortality increased more rapidly than mite mortality, and men­ thol was only 5.7 times more toxic at the LCgo concentrations. Probit regressions for bee and mite mortality were parallel for citral and thymol. Citral and thymol were 2.9 (2.5-3.3) and 2.0 (1.0-3.6) times more toxic to tracheal mites, respectively, at all concentrations estimated.

KEY WORDS , Apis mellifera, essential oils, monoterpenoids

MONOTERPENOIDS ARE THE principal components of (1991) examined 7 naturally occurring monoterpe­ essential oils and provide the essence of many noids in topical application and feeding tests for A. higher plants including mint, pine, cedar, citrus, woodi controL They applied 1 dosage level of each and eucalyptus (Templeton 1969). They are impor­ compound to infested bees and reported acaricidal tant allelochemicals in plant- interactions activity for a-terpinene, terpineol, citronellal, and (Banthorpe and Charlwood 1980). also clove oil. synthesize monoterpenoids, and many function as Citral, a monoterpenoid investigated in our semiochemicals in interactions between arthro­ study, serves as an alarm pheromone in several pods, as pheromones (Blum 1969), kairomones species of gregarious acarid mites (Kuwahara et al. (Trouiller et al. 1991), and allomones (Ghent 1980, Baker and Krantz 1984, Tuma et al. 1990). 1961) . Baker and Krantz (1984) used citral as a control Monoterpenoids can have both lethal and suble­ adjuvant in treating lily bulbs with dicofol thal effects on mites (Acarina). Cook (1992) found (Kelthane) to control bulb mites, Rhizoglyphus ro­ that d-limonene, beta-pinene, alpha-pinene, and bini Claparede. Citral is an alarm pheromone for delta 3-carene were toxic to the spruce mite, this mite, and when used as an adjuvant, caused an Oligonychus ununguis (Jacobi). Furthermore, at increase in mite activity and contact with the aca­ concentrations below the calculated LCsos, all 4 ricide. Citral also serves as a defensive chemical in compounds reduced oviposition, and 3 of the com­ some ant and bee species including the leafcutter pounds increased movement. Lee et al. (1997) re­ ant, Atta sexdens L. (Blum et al. 1968). ported that most of the 30 monoterpenoids they Currently, the only acaricide registered for tra­ tested for activity against the twospotted spider cheal mite control in the United States is a mono­ mite, Tetranychus urlicae Koch, exhibited some de­ terpenoid compound, menthoL Tracheal mite con­ gree of acaricidal activity, especially the alcohols, trol with menthol was first demonstrated in Italy by phertols, and ketones. Larson and Berry (1984) ex­ Giavarini and Giordani (1966). Herbert et aL amined T. urlicae fecundity and development on (1987), Moffett et al. (1987), Ellis and Simonds peppermint, Mentha piperita, leaves that differed in (1987), Cox et al. (1989), Moffett et al. (1989), monoterpene content, but were unable to correlate Wilson et al. (1990), and Duff and Furgala (1991) monoterpene content of leaves with either fecun­ have confirmed that menthol is effective against dity or development. However, in tests conducted tracheal mite populations in the United States. in unventilated petri dishes, the vapors from 5% The target sites and modes of action have not solutions of menthol and pulegone reduced ovipo­ been elucidated for the monoterpenoids, and only sition and increased mortality. Calderone et al. a few studies have examined this question (Lorber

0022-0493/97/1087-1091$02.00/0 © 1997 Entomological Society of America 1088 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 90, no. 5 and Muller 1976, Andrews et al. 1980, Watanabe et Table 1. Honey bee responses to monoterpenoids fumigants al. 1990, and Ikawa et al. 1992). Lee et al. (1997) and Rice and Coats (1994) are currently investigat­ Monoterpenoid n Slope:t SEM LCso (95% CL) ing structure-activity relationships for monoterpe­ Carvacrol 470 5.53:t 0.42 11.4 (10.0-12.8) noids applied to various arthropods. Citral 440 6.49 :t 0.55 10.1 (8.9-11.4) d-Limonene 360 16.18:t 1.79 10.4 (9.9-10.9) Although several monoterpenoids have shown Menthol 480 1.82 :t 0.23 5.3 (3.3-7.1) toxicity to arthropods, most are not acutely toxic to Pulegone 360 6.73:t 0.67 6.6 (5.4-7.9) mammals. Monoterpenoids are used widely as fra­ Thymol 400 1.81 :t 0.22 1.7 (0.5-2.9) grance and flavoring ingredients (Croteau 1980) a-Terpineol 420 13.77 :t 1.61 17.1 (16.0-18.2) and in over-the-counter formulations of expecto­ LCsos are expressed in micrograms of monoterpenoid per mil­ rants, decongestants, external analgesics, and anti­ liliter. septics (Klocke 1987). Characteristics ofthese sim­ ple compounds that make them attractive for development as environmentally sound arthropod suspended in the middle of the jars by a wire control agents include their selective arthropod tether. Bees were given water immediately before toxicity, low mammalian toxicity, and biodegrad­ and after all bioassays and also received water ability. twice daily after treatments until evaluations were The objectives of this study were to characterize completed. Bees from colonies with 30-80% mite the acute fumigant toxicities of pulegone, carva­ prevalence were used in all bioassays. After treat­ crol, d-limonene, citral, menthol, thymol, and a­ ment, bees were held in a dark storage area main­ terpineol to honey bees and their tracheal mite tained at 21°C. Relative humidity was not con­ parasites. These compounds represent a range of trolled during the holding period. monoterpenoids and include 1 terpene hydrocar­ Bee mortality was based on no response to prob­ bon, 1 terpene aldehyde, 1 terpene ketone, and 4 ing with minutin pins and was recorded at 24, 48, terpene alcohols. 72, and 96 h after treatment. Mite mortality was measured 96-144 h after treatment. Live and dead mites were differentiated using methods described Materials and Methods by Eischen et al. (1987). Prothoracic tracheae were Technical grade samples of monoterpenoids removed laterally from the spiracle and medially at were obtained from Aldrich (Milwaukee, WI). Va­ the bifurcations leading to the head and air sacs and por exposures were conducted in 1.9-liter glass jars. were transferred to glass slides coated with glyc­ Monoterpenoids were weighed and dispensed into erol. Tracheae were then teased open using minu­ 3.7-ml (I-dram) vials. The contents of the vials tin pins mounted in wooden probes. Adult and im­ were dispersed on the inside surface of each jar; mature mites were scored as alive if they walked or the vials and any remaining compound were then moved within 15 s of observation. If no movement deposited in the jars. The jars were sealed and was observed, but they had the normal appearance placed in a Precision Low Temperature Incubator of living mites (turgid and grey to pearl colored), (Model 816, GCA, Chicago, IL). All exposures were they also were scored as alive. Mites were scored as for a single 24-h period at 35°C and a photoperiod dead if they were discolored with colors ranging of 12:12 (L:D) h. Humidity was not controlled. from yellow to dark brown or if they had append­ Preliminary studies were performed with each ages appressed to their bodies and were desiccated compound to determine the appropriate concen­ in appearance. In this study, eggs were scored as tration ranges for testing. Six to 8 concentrations alive if turgid and as dead if flat or shriveled. (micrograms of compound per milliliter volume of Data were analyzed using POLO (LeOra Soft­ the jar) were selected and prepared for bioassay. A ware 1991), for probit analyses and SAS (SAS In­ control group, which received no monoterpenoid, stitute 1985) for analyses of variance. The arcsine was included in each replication. Each treatment transformation was applied to all percentage mor­ was repeated 3-6 times. tality data for life stage effects that were analyzed Honey bees infested with A. woodi were ob­ by analysis of variance. Monoterpenoids were se­ tained from 9 colonies located on the University of lected based on a review of the literature and rec­ Nebraska campus. All colonies were headed by ommendations by Joel Coats (Iowa State Univer­ Carniolan queens obtained from Wenner Apiaries sity) who is currently investigating this class of (Glenn, CA). All colonies were treated with terra­ compounds. mycin (Pfizer, New York, NY), fumidil-b (Mid­ Con, Olathe, KS), and apistan (Zoecon, Dallas, TX) Results to control brood and adult bee diseases and para­ sites not ofinterest in this study. Adult worker bees Honey Bee Bioassays. All probit regression esti­ were collected from the combs of the brood nest mates for bee mortality were significant (Table 1). and caged in Benton mailing cages (Kelley, Clark­ Compounds were significantly different (a = 0.05) son, KY) supplied with queen cage candy (Laidlaw in toxicity at any lethal concentration value if their 1979). One cage of 10 worker bees was placed in 95% CL did not overlap. Thymol was the most toxic each jar and constituted a replication. Cages were compound. Surprisingly, menthol, the monoterpe- October 1997 ELLIS AND BAXENDALE: TRACHEAL MITE CONTROL WITH MONOTERPENOIDS 1089

Table 2. Adult tracbeal mite responses to monoterpenoids 6.5 fumigants 6 Monoterpenoid n Slope + SEM LCso (95% CL) Carvacrol 551 1.76 :!: 0.35 7.8a 5.5 Citral 130 7.76 :!: 0.57 3.6 (3.3-3.8) S Menthol 999 1.04 :!: 0.17 0.3 (0.01-0.8) :c 5 Thymol 581 2.00:!: 0.29 0.9 (0.3-1.3) e a. 4.5 LCsos are expressed in micrograms of monoterpenoid per mil­ liliter. 4 aNot significant. 3.5 -2 -1.5 -1 -0.5 0 0.5 1.5 2 Log Concentration of Menthol (ug/ml) noid currently registered for tracheal mite control, was more toxic to honey bees than were most ofthe Fig. 2. Bee and mite mortality responses to menthol other compounds examined in this study. Slopes of when applied as a fumigant to mite-infested honey bees. the probit regression estimates were not equal for Bee mortality slope = 1.82 (1.58-2.06), intercept = -1.31 all compounds; therefore, relative toxicities could (-1.07 to -1.55). Mite mortality slope = 1.04 (0.87-1.21), only be compared at specific lethal concentration intercept = 0.58 (0.47-0.69). values. Mite Bioassays. Citral and menthol were more estimates for bee and adult mite responses to citral toxic to adult tracheal mites than to honey bees and menthol, respectively. Regression estimates (Table 2). Although thymol was numerically more were parallel for citral. Citral was 2.9 times more toxic to mites, the difference was not significant (0: toxic to mites at all concentrations. The regressions = 0.05). Carvacrol was significantly more toxic to were not parallel for menthol. Menthol was 52.0, mites than bees; however, although the regression 18.8, and 5.7 times more toxic to mites at the LCIO' was significant, the fit was not good enough to LCso, and LCgo, respectively. Thymol and carvacrol estimate 95% CL. Pulegone, d-limonene, and a­ were numerically more toxic to mites, although the terpineol were more toxic to host honey bees than differences were not significant statistically (0: = to tracheal mites, and probit regression estimates 0.05). could not be generated for these compounds. Relative Toxicities to Life Stages of A. woodi. Menthol was the most toxic compound to tra­ Citral was the only compound that gave significant cheal mites. The slopes for menthol, thymol, and control (P < 0.05) of all tracheal mite life stages at citral (the 3 most effective compounds) were not a concentration close to the LCso for their honey parallel (0: = 0.05); therefore, relative toxicities bee hosts (Table 3). Thymol gave significant con- could only be compared at specific lethal concen­ tration values. Lethal concentration values with nonoverlapping 95% CL (Table 2) were considered Table 3. Relative toxicities of monoterpenoids fumigants to 3 life stages of tracbeal mites significantly different (0: = 0.05). There were no significant differences in natural mortalities among Life % mortality Compound n Dose pa value untreated control groups (P = 0.270). stage :!: SEM Relative Toxicities to Bees and Mites. Figs. 1 and Carvacrol 514 10 Adults 0.625 :!: 0.085 0.010' 2 show combined plots of the probit regression Larvae 0.239 :!: 0.081 0.312 Eggs 0.156 :!: 0.103 0.355 Citral 688 10 Adults 1.00 :!: 0.031 0.0001' Larvae 0.972:!: 0.079 0.0001' 6.5 Eggs 0.967 :!: 0.084 0.0001' d-Limonene 719 10 Adults 0.114:!: 0.036 0.492 6 Larvae 0.077 :!: 0.073 0.653 Eggs 0.000 :!: 0.053 5.5 Menthol 976 5 Adults 0.937 :!: 0.032 0.0001' ~ Larvae 0.356 :!: 0.102 0.191 .a 5 Eggs 0.225 :!: 0.097 0.378 a.e Pulegone 671 7 Adults 0.192 :!: 0.125 0.902 4.5 Larvae 0.147 :!: 0.117 0.937 Eggs 0.046 :!: 0.028 0.289 4 ",-Terpineol 677 15 Adults 0.363 :!: 0.067 0.051 Larvae 0.159 :!: 0.074 0.402 3.5 -2 -1.5 -1 -0.5 0 0.5 1.5 2 Eggs 0.000 :!: 0.077 Thymol 486 3 Adults 0.854 :!: 0.048 0.0001' Log Concentration of Citral (ug/ml) Larvae 0.647 :!: 0.078 0.018' Eggs 0.274 :!: 0.086 0.074 Fig. 1. Bee and mite mortality responses to citral when applied as a fumigant to mite-infested honey bees. Concentrations are expressed in micrograms of monoterpenoid Bee mortality slope = 6.49 (5.94-7.04), intercept = -6.54 per milliliter. "P ,;; 0.05 represent tests in which mite mortality (-5.96 to -7.12). Mite mortality slope = 7.76 (7.19-8.33), was significantly greater than natural mortality. intercept = -4.28 (-3.95 to -4.61). aprobability that mortality was greater than natural mortality. 1090 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 90, no. 5 trol of adults and larvae. Menthol and carvacrol lapped at the LClO and LCso concentrations. It may gave significant control of adults only (Table 3). be difficult to use thymol at brood nest tempera­ tures without inducing unacceptable levels of bee mortality. Chiesa (1991) was able to control V. Discussion jacobsoni in honey bee colonies with thymol with­ Honey bee mortality responses to thymol and out noticeable damage to the colonies at tempera­ menthol extended over a wider range of concen­ tures ranging from 5 to 9°C. However, Gal et al. trations than did responses to the other 5 com­ (1992) found thymol applications deleterious to pounds bioassayed. With the other 5 compounds, bees when temperatures reached 27-28°C. Overlap once the response threshold concentration was in the LCso confidence limits for bee and mite reached, small increases in concentration increased mortality found in this study supports the findings bee mortality. All monoterpenoid LCso values fell of Gal and his colleagues that thymol is too toxic to in a narrow range (1.7-17.1 Itg/ml). Rice and Coats bees for use at high temperatures. Further studies (1994) found LCsos in the same range for menthol, of concentration-temperature interactions are pulegone, and citral when applied to Musca domes­ needed to determine optimum conditions for thy­ tica L. In their study, menthol, pulegone, and citral mol application. If the relative toxicities to bees had LCsos of 3.6, 9.2, and 13 Itg/ml, respectively. and mites change at cooler temperatures, a satis­ Interestingly, although thymol was the most toxic factory treatment window may be found. compound to honey bees examined in this study Although carvacrol did not provide a good fit to (LCso of 1. 7 Itg/ml), thymol was not highly toxic to the probit regression model, it has acaricidal prop­ M. domestica in Rice and Coats' study (LCso of 142 erties and should be examined in future studies. Gal Itg/ml). Some compounds assayed in this study et al. (1992) obtained 81-92% control of V. jacob­ were more toxic to honey bees than to their A. soni under what they described as "subtropical" woodi parasites, although others were more toxic to conditions with origanum oil (carvacrol); however, A. woodi. Results obtained in this study support they also reported a high degree of robbing in Rice and Coats' (1994) observations that monoter­ treated colonies. Pulegone, d-limonene, and a-ter­ penoid potencies vary considerably, and that minor pineol were more toxic to honey bees than to tra­ structural differences can elicit major differences cheal mites and should be eliminated as candidate in toxicity. compounds for A. woodi control. Citral, which has not been reported previously to The dose-response relationships reported in this be toxic to tracheal mites, killed all life stages of study provide a foundation for future investigations tracheal mites at concentrations below those re­ of monoterpenoid fumigant use to control A. woodi quired to kill their honey bee hosts. The margin of in honey bee colonies. Length of exposure, temper­ safety was 2.9, and this relationship was constant ature, and humidity are factors that can influence for all concentrations. Citral was the only com­ the activity of monoterpenoid fumigants and merit pound tested that exhibited both uniform margins further investigation. Likewise, mode of action of safety at all concentrations and no overlap in the studies can provide insight into how best to use confidence limits for bee and mite mortality at any monoterpenoids. The potential use of these selec­ concentration. The performance of citral in our tive and fully biodegradable compounds in the bioassays makes it a candidate for development as a management of parasitic bee mites is encouraging. control agent for A. woodi. Citral is an alarm pher­ omone for some species of gregarious acarid mites References Cited (Kuwahara et al. 1980) and its effect on A. woodi behavior merits investigation. Citral may be useful Andrews, R. E., L. W. Park, and K. D. Spence. 1980. as an adjuvant at low concentrations if found to Some effects of Douglas fir terpenes on certain micro­ affect the behavior of A. woodi making them more organisms. Appl. Environ. Microbiol. 40: 301-304. vulnerable to acaricides. Baker, G. T., and G. W. Krantz. 1984. Alarm phero­ Menthol was the most toxic compound to A. mone production in the bulb mite, Rhizoglyphus robini Claparede, and its possible use as a control adjuvant in woodi in our studies. It also had the largest margin lily bulbs, pp. 686-692. In D. A. Griffiths and C. E. ofsafety (18.9) at the LCso; however, the margin of Bowman [eds.], Acarology VI. Horwood, New York. safety declined to 5.7 at the LCgo' The low toxicity Banthorpe, D. V., and B. V. Charlwood. 1979. Second­ of menthol to mite eggs and larvae and relatively ary plant products, pp. 185-220. In E. A. Bell and B. V. high toxicity to honey bees are undesirable at­ Charlwood [eds. J, Encyclopedia of plant physiology. tributes. Menthol is currently registered as a fumi­ Springer, New York. gant for use in honey bee colonies. Beekeepers Blum, M. S. 1969. larm pheromones. Annu. Rev. En­ purchased 5.7 metric tons of menthol in 1993 tomol. 14: 57-80. (Mann Lake Supply, Hackensack, MN) to control Blum, M. S., F. Padovani, and E. Amante. 1968. AI­ kanones and terpenes in the mandibular glands ofAtta tracheal mites. species. Compo Biochem. Physiol. 26: 291-299. Thymol controlled A. woodi adults and larvae, Calderone, N. W., W. A. Bruce, G. Allen-Wardell, and H. but it also was the most toxic compound to bees in Shiminuki. 1991. Evaluation of botanical com­ this study. The margin of safety (2.0) was not large pounds for control of the honey bee tracheal mite, and the 95% CL for bee and mite toxicity over- Acarapis woodi. Am. Bee J. 131: 589-591. October 1997 ELLIS AND BAXENDALE: TRACHEAL MITE CONTROL WITH MONOTERPENOIDS 1091

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