Experimental & AppliedAcarology, 13 (1992) 295-301
295
Elsevier Science Publishers B.V., Amsterdam
Short Communication
Olfactory responses of adult Amblyomma hebraeum and A. variegatum ( Acari: Ixodidae) to attractant chemicals in laboratory tests C.E. Y u n k e r ~, T. P e t e r ~, R . A . I . N o r v a l b, D,E. S o n e n s h i n e c, M.J. B u r r i d g e b a n d J.F. B u t l e r d
aUniversity of FIorida/USAID/SADCC Heartwater Research Project, Veterinary Research Laboratory, Causeway, Zimbabwe bDepartment of lnfectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA CDepartment of Biological Sciences, Old Dominion University, Norfolk, VA, USA dDepartment of Entomology and Nematology, University of Florida, Gainesville, FL, USA (Accepted 14 November 1991 )
ABSTRACT Yunker, C.E., Peter, T., Norval, R.A.I., Sonenshine, D.E., Burridge, M.J. and Butler, J.F., 1992. Olfactory responses of adult Amblyomma hebraeum and A. variegatum (Acari: Ixodidae) to attraerant chemicals in laboratory tests. Exp. AppL Acarol., 13: 295-301. Unfed adults of the African ticks, Amblyomma hebraeum Koch and A. variegatum (Fabricius), important vectors of human and animal diseases, were exposed to volatile compounds in an olfactometer in efforts to identify both tick-produced or synthetic chemicals capable ofeliciting an attraction response. A formula, relative efficacy of attraction, was devised for comparison of responses between species and sexes to a particular test stimulus, or within a homogeneous population to different stimuli. Adults of both species responded strongly to known tick-pheromone constituents, nonanoic acid, methyl salicylate, 2.6-dichlorophenol and benzyl alcohol, as well as to a commercially produced antiseptic, TCP (Pfizer), and its major components, chlorinated and iodinated phenols. Benzaldehyde, a proposed tick-pheromone component, and heptadecane, not known from ticks, were markedly attractive to adults of A. hebraeum but not to those of A. variegatum. Males of the former species, but neither conspecific females not either sex of the latter species, responded significantly to salicylaldehyde (known from males of four species of ticks, includingA. variegatum), o-nitrophenol, a major component of the aggregation-attachment pheromone of males of both A. variegatum andA hebraeum and a proven long-range attractant for them in the field, was only partially attractive to either species in the olfactometer. Neither species was attracted to 2-methylpropanoic acid, previously identified in volatile effluents form feeding male A. hebraeum. It is concluded that these important disease vectors Correspondence to: Dr. C.E. Yunker, University of Florida/USAID/SADCC Heartwater Research Project, Veterinary Research Laboratory, P.O. Box 8101, Causeway, Zimbabwe.
296
C.E. YUNKERET AL.
respond positively to a variety of volatile chemicals, which may conceivably be used to attract them to traps, animals or acaricides in efforts to control ticks or the diseases they transmit.
INTRODUCTION
The potential for control of Amblyomma ticks parasitic on domestic ruminants through pheromone-mediated attraction to acaricides was first proposed by Gladney et al. (1974). Later, Rechav and Whitehead (1978) employed the technique to attract and kill the bont tick, A. hebraeum Koch, on cattle treated with an extract of the male-produced aggregation-attraction pheromone (AAP) and an acaricide. Some components of the AAPs of the tropical bont tick, A. variegatum (Fabricius), were identified by Sch/Sni et al. (1984). Apps et al. (1988) identified additional volatile compounds from feeding males of A. hebraeum, which they believed to be pheromones, but tests to confirm their biological activity were not done. The attractiveness for A. variegatum of three of these components was then tested in laboratory and field assays by Sch6ni et al. (1984) and Hess and de Castro (1986), respectively, and for A. hebraeum by Norval et al. (1989a). We now report results of olfactometry assays performed in the laboratory in efforts to identify a wider range of compounds potentially attractive to these ticks. Included are additional compounds recently identified in one or both species (Lusby et al., 1991 ), plus some additional volatiles not known to be produced by ticks. Results of these bioassays are applicable to improvements in control strategies aimed at these important vectors of human and animal diseases in Africa. MATERIALS A N D M E T H O D S
Ticks, from laboratory colonies maintained at the Veterinary Research Laboratory, Harare, Zimbabwe, were reared on rabbits, sheep or goats, and held at 27~ and 80% RH for oviposition and molting. Bioassays were performed in a four-choice olfactometer that consisted of a closed, circular plastic chamber containing four lateral inlet ports situated at 90 ~ from each other and 18 cm from an outlet port located centrally no the floor of the chamber (Butler and Katz, 1988a,b). Substances tested were applied in 5/A amounts and at various concentrations to small (0.5-2.0 cm) strips of filter paper housed in the inlet ports. Charcoal-filtered room air was drawn over the filter paper strips and evacuated by means of a battery-driven exhaust fan through the screened central outlet. The olfactometer was washed with detergent, multiply rinsed and dried between tests. Unfed ticks were tested in groups of 50 of the same species and sex. Two opposing inlets of the olfactometer were charged with different dilutions of the test substances; the remaining two, charged with solvent (see below),
OLFACTORY RESPONSES A M B L Y O M M A TO ATTRACTANT CHEMICALS
297
served as controls. Ticks were placed on the central screen after a waiting period of approximately 3 min, during which time the solvent evaporated. When they sensed the current of air containing an attractive substance, they migrated to its source where they either aggregated or wandered away. The number of ticks remaining at each outlet at the end of 1 h was recorded. Multiple tests were performed for each substance or dilution of substance, as indicated in the tables, and results were combined and compared with corresponding control values. A formula was devised to assess the relative attractiveness of test substances for a single sex or species of ticks, or of a single substance between different species or sexes of a single species. This formula, termed relative efficacy of attraction (REA), is calculated as: REA-
(% unattracted to control ) - (% unattracted to test) • 100 (% unattracted to control )
Substances and mixtures of substances tested included volatiles already identified from either or both A. variegatum or A. hebraeum (SchiSni et al., 1984; Apps et al., 1988; Lusby et al., 1991 ). These were o-nitrophenol, methyl salicylate, nonanoic ( = pelargonic) acid, 2-methylpropanoic acid, benzaldehyde, 2,6-dichlorophenol, salicylaldehyde and benzyl alcohol (Sigma, St. Louis, MO, USA). Also tested were certain phenols not identified from these ticks: TCP | (Pfizer, Harare), and chlorinated and iodinated phenols, the classes of compounds that comprise TCP. In addition, heptadecane (Sigma), previously suspected to be a volatile component of fed bont ticks males (D. Sonenshine, unpublished results), but not confirmed, was included in the assays. Solids were dissolved at 1-M concentrations in diethylether:hexane ( 1 : 1 ); these and stock liquids, were used neat or serially diluted in ten-fold increments (Tables 1-4). RESULTS AND DISCUSSION
Both sexes ofA. hebraeum and A. variegatum were strongly attracted to one or more dilutions of nonanoic acid, methyl salicylate, 2,6-dichlorophenol, benzyl alcohol, TCP, and chlorinated and iodinated phenols (Tables 1-4). All of these substances except the commercial antiseptic TCP and its components (chlorinated and iodinated phenols) have been identified as present in fed males of both species of ticks (Sch/Sni et al., 1984; Apps et al., 1988 ). TCP was chosen for assay because its odor strongly resembles that of feeding bont ticks. The attraction of both species of ticks to this pharmaceutical preparation may indicate a non-specific response to halogenated phenols in general or a specific response to one or more halogenated phenols (e.g. 2,6-dichlorophenol) possibly present in the mixtures. Additional work on this aspect is warranted. Benzaldehyde and heptadecane were markedly attractive to both sexes of
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C.E. YUNKER ET AL.
TABLE1 Relative efficacy of attractants for Amblyomma hebraeum males Material tested
Benzaldehyde Benzyl alcohol 2,6-dichlorophenol Heptadecane 2-methylpropanoic acid Methyl salicylate o-nitrophenol Nonanoic acid Salicylaldehyde TCP | Chlorinated phenols Iodinated phenols
Concentration b No. tested c
Relative efficacy a
Number attracted a Experimental %
Control
%
M/100 M/100 M~ 100
150 100 100 100 100 100 200 100 150
48 44 24 35 8 47 17 43 71
32.0 44.0 24.0 35.0 8.0 47.0 8.5 43.0 47.3
4.7 5.0 3.5 2.0 7.0 1.0 11.0 4.0 2.8
M~ 1000 M/1000 M/IO00
1O0
41
41.0
4.5
100 148
38 38
38.0 38.8
5.5 1.3
1.6 5.0 3.5 2.0 7.0 1.0 5.5 4.0 1.9 4.5 5.5 0.9
M/100
M/1000 M/100 M/100
M/1000 M/100
28.7 41.1 21.2 33.7 1.1 46.5 3.1 40.6 46.2 38.9 34.4 37.4
"Fractional number indicates average of multiple tests or controls. bFinal concentration expressed as 1 M/100, 1 M/1000, etc. c 100 = total of two tests, 148-150 = total of three tests, 200 = total of four tests. dSee text for formula. TABLE 2 Relative efficacy of attractants for Amblyomma hebraeum females a Material tested
Concentration No. tested Number attracted Experimental %
Benzaldehyde Benzyl alcohol 2,6-dichlorophenol Heptadecane 2-methylpropanoic acid Methyl salicylate o-nitrophenol Nonanoic acid Salicylaldehyde TCP | Chlorinated phenols Iodinated phenols
M~ 1000 M/1000 M/IO 000 M/IO0 M/1000 M/100 M/100
M/IO M~ 100 M/I000 M~ 100 M/1000
100 100 100 100 100 100 150 100 100 100 150 200
45 37 34 39 8 41 12 44 7 28 35 75
Relative efficacy Control %
45.0 2.0 37.0 1.5 34.0 4.0 39.0 7.0 8.0 7.0 41.0 5.5 8.0 16.0 44.0 2.5 7.0 6.0 28.0 4.5 23.3 4.0 37.5 4.8
2.0 1.5 4.0 7.0 7.0 5.5 10.7 2.5 6.0 4.5 2.7 3.2
43.9 36.0 31.3 34.1 1.1 37.6 - 3.0 42.6 1.1 24.6 21.2 35.4
aSee footnotes for Table 1.
A. hebraeum (Tables 1 and 2) but not to either sex ofA. variegatum (Tables 3 and 4). The former compound occurs naturally in both species of ticks (Apps et al., 1988; Lusby et al., 1991 ) but the latter has not been reported for either species.
OLFACTORYRESPONSESAMBLYOMMA TO ATTRACTANTCHEMICALS
299
TABLE 3 Relative efficacy of attractants for Amblyomrna variegatum males a Material tested
Concentration No. tested Number attracted Experimental %
Benzaldehyde Benzyl alcohol 2,6-dichlorophenol Heptadecane 2-methylpropanoicacid Methyl salicylate o-nitrophenol Nonanoic acid Salicylaldehyde TCP | Chlorinated phenols Iodinated phenols
M/100 M/1000 M/100 M/100 M/1000 M/1000 M/100 M/100 M/IO 000 M ! 1000 M/1000 M/100
100 150 100 100 100 100 100 150 150 100 100 200
12 47 34 8 8 39 9 36 18 31 42 65
Relative efficacy Control %
12.0 "10.0 31.3 6.0 34.0 4.0 8.0 8.0 8.0 12.0 39.0 6.0 9.0 9.0 24.0 6.0 12.0 13.0 31.0 8.0 42.0 0.0 32.5 7.5
10.0 2.2 4.0 28.5 4.0 31.3 0.0 0.0 12.0 - 4.6 6.0 35.1 14.0 - 5.8 4.0 20.8 8.7 3.7 8.0 25.0 0.0 42.0 1.9 31.2
aSee footnotes for Table 1. TABLE 4 Relative efficacy of attractants for Amblyomma variegatum females a Material tested
Concentration No. tested Number attracted Experimental %
Benzaldehyde Benzyl alcohol 2,6-dichlorophenol Heptadecane 2-methylpropanoicacid Methyl salicylate o-nitrophenol Nonanoic acid Salicylaldehyde TCP | Chlorinated phenols Iodinated phenols
M~ 1O0
1O0
9
3///1000
150 100 100 100 100 100 100
48 31 11 9 40 14 40
M/100 M/100 M/10000
M/1000 M/100 M/1000
M/1 O0 M/IO M/1000
M/1000
9.0
Relative efficacy Control %
6.0
32.0 7.0 31.0 4.0 11.0 8.0 9.0 11.0 40.0 4.0 14.0 5.0 40.0 8.0
6.0
3.2
4.7 28.7 4.0 28.1 8.0 3.3 11.0 - 2.3 4.0 37.5 5.0 9.5 8.0 34.8
100
7
7.0
6.0
6.0
1.1
100 200 100
35 56 47
35.0 28.0 47.0
6.0 5.5 4.5
6.0 1.4 9.0
30.5 27.0 41.8
aSee footnotes for Table 1.
Salicylaldehyde, known to be produced by females of four species of ticks, includingA, variegatum (Wood et al., 1975 ), was strongly attractive to males ofA. hebraeum (Table 1 ). However, neither females of this species (Table 2 ), nor either sex ofA. variegatum (Tables 3 and 4) responded significantly to this substance. o-nitrophenol, the most abundant constituent of AAPs of both tick species
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C.E, YUNKER ET AL.
(Lusby et al., 1991 ) and markedly attractive in field assays (Norval et al., 1989a, 1991 ), was only weakly attractive in the olfactometer (Tables 1 and 4). Females of A. variegatum and males of A. hebraeum responded to this substance at 1/ 100 molar (M! 100) concentration only slightly more than to the controls, while the opposite sexes failed to respond or responded only briefly. Undiluted o-nitrophenol and serial dilutions made at M/100, M! 1000 and M! l0 000 failed to elicit an aggregation response that endured for 1-h test (data not shown ). Similarly, Sch/Sni et al. ( 1984 ) reported an incomplete aggregation response ofA. variegatum to o-nitrophenol. However, it has been adequately demonstrated that this substance is an effective long-range attractant for both species of ticks, especially when combined with CO2 vapors (Norval et al., 1989a, 1991 ). Thus, other substances, such as CO2, may participate in the aggregation response of these species to o-nitrophenol. This possibility warrants further study. Neither A. hebraeum nor A. variegatum adults were attracted to 2-methylpropanoic acid. This compound was identified in volatile effluents collected from feeding males ofA. hebraeum (Apps et al., 1988 ) but its biological role remains unknown. It could possibly function to repel adults of other species of Amblyomma, inasmuch as a negative REA was seen for both sexes of A. variegatum when exposed to this compound, while adults ofA. hebraeum were indifferent to it. It is concluded that both the bont tick and the tropical bont tick respond positively to a variety of synthetic volatile substances (in this study, principally phenols), which could conceivably be used to attract them to traps or acaricides. That such attractants might also be used in endemic areas of heartwater in order to increase transmission rates of the disease to young ruminants, which are temporarily protected by an age-related resistance, has also been suggested (Norval et al., 1989b). ACKNOWLEDGEMENTS
This study was supported by Cooperative Agreement No. AFR-0435-A-009084-00 between the U.S. Agency for International Development and the University of Florida. We thank Mr. Lameck Chakurungama for rearing the ticks used in this study.
REFERENCES Apps, P.J., Viljoen, H.W. and Pretorius, V., 1988. Aggregation pheromones of the bont tick Amblyomma hebraeum: Identification of candidates for bioassay. Onderstepoort J. Vet. Res., 55: 135-137. Butler, J.F. and Katz, I., 1988a. Apparatus for determination of insect repellency and attraction. U.S. Patent No. 4 748 860. Date of patent: 7 June 1988. Inventors: Jerry F. Butler, Gaines-
OLFACTORYRESPONSESAMBLYOMMATOATTRACTANTCHEMICALS
301
ville, FL, and Ira Katz, West Long Branch, NJ. Assignees: International Flavors and Fragrances Inc., New York, NY; The University of Florida, Gainesville, FL. Butler, J.F. and Katz, I., 1988b. Process for determination of repellency and attractancy. U.S. Patent No. 4 759 228. Date of patent: 26 July 1988. Inventors: Jerry F. Butler, Gainesville, FL, and Ira Katz, West Long Branch, NJ. Assignees: International Flavors and Fragrances Inc., New York, NY; The University of Florida, Gainesville, FL. Gladney, W.J., Ernst, S.E. and Oehler, D.D., 1974. The Gulf Coast tick; evidence of a pheromone produced by males. J. Med. Entomol, 11: 303-306. Hess, E, and de Castro, J.J., 1986. Field tests of the response ofAmblyomma variegatum (Acari: Ixodidae) to the synthetic aggregation-attachment pheromone and its components. Exp. Appl. Acarol., 2: 249-255. Lusby, W.R., Sonenshine, D.E., Yunker, C.E., Norval, R.A.I. and Burridge, M.J., 1991. Comparison of known and suspected pheromonal constituents in males of the African ticks, A mblyomma hebraeum Koch and Amblyomma variegatum (Fabricius). Exp. Appl. Acarol., 13: 143-152. Norval, R.A.I., Butler, J.F. and Yunker, C.E., 1989a. Use of carbon dioxide and natural or synthetic aggregation-attachment pheromone of the bont tick, Amblyornma hebraeum, to attract and trap unfed adults in the field. Exp. Appl. Entomol., 7:171-180. Norval, R.A.I., Andrew, H.R. and Yunker, C.E, 1989b. Pheromone-mediation of host selection in bont ticks (A. hebraeum) Koch. Science, 243: 364-365. Norval, R.A.I., Peter, T.F., Yunker, C.E., Sonenshine, D.E. and Burridge, M.J., 1991. Response of the ticks Amblyomma hebraeum and A. variegatum to known or potential components of the aggregation-attachment pheromone. I. Long-range attraction. Exp. Appl. Acarol., 13:1118. Rechav, Y. and Whitehead, G.B., 1978. Field trials with pheromone-acaricide mixtures for control ofArnblyomma hebraeum J. Econ. Entomol., 71: 149-151. SchSni, R., Hess, E,, Blum, W. and Ramstein, K., 1984. The aggregation-attachment pheromone of the tropical bont tick Amblyomma variegatum Fabricius (Acari, Ixodidae): Isolation, identification and action of its components. J. Insect. Physiol., 30:613-618. Wood, W.F., Leahy, Sister M.G., Galun, R., Prestwich, G.D., Meinwald, J., Purnell, R.E. and Payne, R.C., 1975. Phenols as pheromones ofixodid ticks: A general phenomenon? J. Chem. Ecol., 1: 501-509.
295
Elsevier Science Publishers B.V., Amsterdam
Short Communication
Olfactory responses of adult Amblyomma hebraeum and A. variegatum ( Acari: Ixodidae) to attractant chemicals in laboratory tests C.E. Y u n k e r ~, T. P e t e r ~, R . A . I . N o r v a l b, D,E. S o n e n s h i n e c, M.J. B u r r i d g e b a n d J.F. B u t l e r d
aUniversity of FIorida/USAID/SADCC Heartwater Research Project, Veterinary Research Laboratory, Causeway, Zimbabwe bDepartment of lnfectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA CDepartment of Biological Sciences, Old Dominion University, Norfolk, VA, USA dDepartment of Entomology and Nematology, University of Florida, Gainesville, FL, USA (Accepted 14 November 1991 )
ABSTRACT Yunker, C.E., Peter, T., Norval, R.A.I., Sonenshine, D.E., Burridge, M.J. and Butler, J.F., 1992. Olfactory responses of adult Amblyomma hebraeum and A. variegatum (Acari: Ixodidae) to attraerant chemicals in laboratory tests. Exp. AppL Acarol., 13: 295-301. Unfed adults of the African ticks, Amblyomma hebraeum Koch and A. variegatum (Fabricius), important vectors of human and animal diseases, were exposed to volatile compounds in an olfactometer in efforts to identify both tick-produced or synthetic chemicals capable ofeliciting an attraction response. A formula, relative efficacy of attraction, was devised for comparison of responses between species and sexes to a particular test stimulus, or within a homogeneous population to different stimuli. Adults of both species responded strongly to known tick-pheromone constituents, nonanoic acid, methyl salicylate, 2.6-dichlorophenol and benzyl alcohol, as well as to a commercially produced antiseptic, TCP (Pfizer), and its major components, chlorinated and iodinated phenols. Benzaldehyde, a proposed tick-pheromone component, and heptadecane, not known from ticks, were markedly attractive to adults of A. hebraeum but not to those of A. variegatum. Males of the former species, but neither conspecific females not either sex of the latter species, responded significantly to salicylaldehyde (known from males of four species of ticks, includingA. variegatum), o-nitrophenol, a major component of the aggregation-attachment pheromone of males of both A. variegatum andA hebraeum and a proven long-range attractant for them in the field, was only partially attractive to either species in the olfactometer. Neither species was attracted to 2-methylpropanoic acid, previously identified in volatile effluents form feeding male A. hebraeum. It is concluded that these important disease vectors Correspondence to: Dr. C.E. Yunker, University of Florida/USAID/SADCC Heartwater Research Project, Veterinary Research Laboratory, P.O. Box 8101, Causeway, Zimbabwe.
296
C.E. YUNKERET AL.
respond positively to a variety of volatile chemicals, which may conceivably be used to attract them to traps, animals or acaricides in efforts to control ticks or the diseases they transmit.
INTRODUCTION
The potential for control of Amblyomma ticks parasitic on domestic ruminants through pheromone-mediated attraction to acaricides was first proposed by Gladney et al. (1974). Later, Rechav and Whitehead (1978) employed the technique to attract and kill the bont tick, A. hebraeum Koch, on cattle treated with an extract of the male-produced aggregation-attraction pheromone (AAP) and an acaricide. Some components of the AAPs of the tropical bont tick, A. variegatum (Fabricius), were identified by Sch/Sni et al. (1984). Apps et al. (1988) identified additional volatile compounds from feeding males of A. hebraeum, which they believed to be pheromones, but tests to confirm their biological activity were not done. The attractiveness for A. variegatum of three of these components was then tested in laboratory and field assays by Sch6ni et al. (1984) and Hess and de Castro (1986), respectively, and for A. hebraeum by Norval et al. (1989a). We now report results of olfactometry assays performed in the laboratory in efforts to identify a wider range of compounds potentially attractive to these ticks. Included are additional compounds recently identified in one or both species (Lusby et al., 1991 ), plus some additional volatiles not known to be produced by ticks. Results of these bioassays are applicable to improvements in control strategies aimed at these important vectors of human and animal diseases in Africa. MATERIALS A N D M E T H O D S
Ticks, from laboratory colonies maintained at the Veterinary Research Laboratory, Harare, Zimbabwe, were reared on rabbits, sheep or goats, and held at 27~ and 80% RH for oviposition and molting. Bioassays were performed in a four-choice olfactometer that consisted of a closed, circular plastic chamber containing four lateral inlet ports situated at 90 ~ from each other and 18 cm from an outlet port located centrally no the floor of the chamber (Butler and Katz, 1988a,b). Substances tested were applied in 5/A amounts and at various concentrations to small (0.5-2.0 cm) strips of filter paper housed in the inlet ports. Charcoal-filtered room air was drawn over the filter paper strips and evacuated by means of a battery-driven exhaust fan through the screened central outlet. The olfactometer was washed with detergent, multiply rinsed and dried between tests. Unfed ticks were tested in groups of 50 of the same species and sex. Two opposing inlets of the olfactometer were charged with different dilutions of the test substances; the remaining two, charged with solvent (see below),
OLFACTORY RESPONSES A M B L Y O M M A TO ATTRACTANT CHEMICALS
297
served as controls. Ticks were placed on the central screen after a waiting period of approximately 3 min, during which time the solvent evaporated. When they sensed the current of air containing an attractive substance, they migrated to its source where they either aggregated or wandered away. The number of ticks remaining at each outlet at the end of 1 h was recorded. Multiple tests were performed for each substance or dilution of substance, as indicated in the tables, and results were combined and compared with corresponding control values. A formula was devised to assess the relative attractiveness of test substances for a single sex or species of ticks, or of a single substance between different species or sexes of a single species. This formula, termed relative efficacy of attraction (REA), is calculated as: REA-
(% unattracted to control ) - (% unattracted to test) • 100 (% unattracted to control )
Substances and mixtures of substances tested included volatiles already identified from either or both A. variegatum or A. hebraeum (SchiSni et al., 1984; Apps et al., 1988; Lusby et al., 1991 ). These were o-nitrophenol, methyl salicylate, nonanoic ( = pelargonic) acid, 2-methylpropanoic acid, benzaldehyde, 2,6-dichlorophenol, salicylaldehyde and benzyl alcohol (Sigma, St. Louis, MO, USA). Also tested were certain phenols not identified from these ticks: TCP | (Pfizer, Harare), and chlorinated and iodinated phenols, the classes of compounds that comprise TCP. In addition, heptadecane (Sigma), previously suspected to be a volatile component of fed bont ticks males (D. Sonenshine, unpublished results), but not confirmed, was included in the assays. Solids were dissolved at 1-M concentrations in diethylether:hexane ( 1 : 1 ); these and stock liquids, were used neat or serially diluted in ten-fold increments (Tables 1-4). RESULTS AND DISCUSSION
Both sexes ofA. hebraeum and A. variegatum were strongly attracted to one or more dilutions of nonanoic acid, methyl salicylate, 2,6-dichlorophenol, benzyl alcohol, TCP, and chlorinated and iodinated phenols (Tables 1-4). All of these substances except the commercial antiseptic TCP and its components (chlorinated and iodinated phenols) have been identified as present in fed males of both species of ticks (Sch/Sni et al., 1984; Apps et al., 1988 ). TCP was chosen for assay because its odor strongly resembles that of feeding bont ticks. The attraction of both species of ticks to this pharmaceutical preparation may indicate a non-specific response to halogenated phenols in general or a specific response to one or more halogenated phenols (e.g. 2,6-dichlorophenol) possibly present in the mixtures. Additional work on this aspect is warranted. Benzaldehyde and heptadecane were markedly attractive to both sexes of
298
C.E. YUNKER ET AL.
TABLE1 Relative efficacy of attractants for Amblyomma hebraeum males Material tested
Benzaldehyde Benzyl alcohol 2,6-dichlorophenol Heptadecane 2-methylpropanoic acid Methyl salicylate o-nitrophenol Nonanoic acid Salicylaldehyde TCP | Chlorinated phenols Iodinated phenols
Concentration b No. tested c
Relative efficacy a
Number attracted a Experimental %
Control
%
M/100 M/100 M~ 100
150 100 100 100 100 100 200 100 150
48 44 24 35 8 47 17 43 71
32.0 44.0 24.0 35.0 8.0 47.0 8.5 43.0 47.3
4.7 5.0 3.5 2.0 7.0 1.0 11.0 4.0 2.8
M~ 1000 M/1000 M/IO00
1O0
41
41.0
4.5
100 148
38 38
38.0 38.8
5.5 1.3
1.6 5.0 3.5 2.0 7.0 1.0 5.5 4.0 1.9 4.5 5.5 0.9
M/100
M/1000 M/100 M/100
M/1000 M/100
28.7 41.1 21.2 33.7 1.1 46.5 3.1 40.6 46.2 38.9 34.4 37.4
"Fractional number indicates average of multiple tests or controls. bFinal concentration expressed as 1 M/100, 1 M/1000, etc. c 100 = total of two tests, 148-150 = total of three tests, 200 = total of four tests. dSee text for formula. TABLE 2 Relative efficacy of attractants for Amblyomma hebraeum females a Material tested
Concentration No. tested Number attracted Experimental %
Benzaldehyde Benzyl alcohol 2,6-dichlorophenol Heptadecane 2-methylpropanoic acid Methyl salicylate o-nitrophenol Nonanoic acid Salicylaldehyde TCP | Chlorinated phenols Iodinated phenols
M~ 1000 M/1000 M/IO 000 M/IO0 M/1000 M/100 M/100
M/IO M~ 100 M/I000 M~ 100 M/1000
100 100 100 100 100 100 150 100 100 100 150 200
45 37 34 39 8 41 12 44 7 28 35 75
Relative efficacy Control %
45.0 2.0 37.0 1.5 34.0 4.0 39.0 7.0 8.0 7.0 41.0 5.5 8.0 16.0 44.0 2.5 7.0 6.0 28.0 4.5 23.3 4.0 37.5 4.8
2.0 1.5 4.0 7.0 7.0 5.5 10.7 2.5 6.0 4.5 2.7 3.2
43.9 36.0 31.3 34.1 1.1 37.6 - 3.0 42.6 1.1 24.6 21.2 35.4
aSee footnotes for Table 1.
A. hebraeum (Tables 1 and 2) but not to either sex ofA. variegatum (Tables 3 and 4). The former compound occurs naturally in both species of ticks (Apps et al., 1988; Lusby et al., 1991 ) but the latter has not been reported for either species.
OLFACTORYRESPONSESAMBLYOMMA TO ATTRACTANTCHEMICALS
299
TABLE 3 Relative efficacy of attractants for Amblyomrna variegatum males a Material tested
Concentration No. tested Number attracted Experimental %
Benzaldehyde Benzyl alcohol 2,6-dichlorophenol Heptadecane 2-methylpropanoicacid Methyl salicylate o-nitrophenol Nonanoic acid Salicylaldehyde TCP | Chlorinated phenols Iodinated phenols
M/100 M/1000 M/100 M/100 M/1000 M/1000 M/100 M/100 M/IO 000 M ! 1000 M/1000 M/100
100 150 100 100 100 100 100 150 150 100 100 200
12 47 34 8 8 39 9 36 18 31 42 65
Relative efficacy Control %
12.0 "10.0 31.3 6.0 34.0 4.0 8.0 8.0 8.0 12.0 39.0 6.0 9.0 9.0 24.0 6.0 12.0 13.0 31.0 8.0 42.0 0.0 32.5 7.5
10.0 2.2 4.0 28.5 4.0 31.3 0.0 0.0 12.0 - 4.6 6.0 35.1 14.0 - 5.8 4.0 20.8 8.7 3.7 8.0 25.0 0.0 42.0 1.9 31.2
aSee footnotes for Table 1. TABLE 4 Relative efficacy of attractants for Amblyomma variegatum females a Material tested
Concentration No. tested Number attracted Experimental %
Benzaldehyde Benzyl alcohol 2,6-dichlorophenol Heptadecane 2-methylpropanoicacid Methyl salicylate o-nitrophenol Nonanoic acid Salicylaldehyde TCP | Chlorinated phenols Iodinated phenols
M~ 1O0
1O0
9
3///1000
150 100 100 100 100 100 100
48 31 11 9 40 14 40
M/100 M/100 M/10000
M/1000 M/100 M/1000
M/1 O0 M/IO M/1000
M/1000
9.0
Relative efficacy Control %
6.0
32.0 7.0 31.0 4.0 11.0 8.0 9.0 11.0 40.0 4.0 14.0 5.0 40.0 8.0
6.0
3.2
4.7 28.7 4.0 28.1 8.0 3.3 11.0 - 2.3 4.0 37.5 5.0 9.5 8.0 34.8
100
7
7.0
6.0
6.0
1.1
100 200 100
35 56 47
35.0 28.0 47.0
6.0 5.5 4.5
6.0 1.4 9.0
30.5 27.0 41.8
aSee footnotes for Table 1.
Salicylaldehyde, known to be produced by females of four species of ticks, includingA, variegatum (Wood et al., 1975 ), was strongly attractive to males ofA. hebraeum (Table 1 ). However, neither females of this species (Table 2 ), nor either sex ofA. variegatum (Tables 3 and 4) responded significantly to this substance. o-nitrophenol, the most abundant constituent of AAPs of both tick species
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C.E, YUNKER ET AL.
(Lusby et al., 1991 ) and markedly attractive in field assays (Norval et al., 1989a, 1991 ), was only weakly attractive in the olfactometer (Tables 1 and 4). Females of A. variegatum and males of A. hebraeum responded to this substance at 1/ 100 molar (M! 100) concentration only slightly more than to the controls, while the opposite sexes failed to respond or responded only briefly. Undiluted o-nitrophenol and serial dilutions made at M/100, M! 1000 and M! l0 000 failed to elicit an aggregation response that endured for 1-h test (data not shown ). Similarly, Sch/Sni et al. ( 1984 ) reported an incomplete aggregation response ofA. variegatum to o-nitrophenol. However, it has been adequately demonstrated that this substance is an effective long-range attractant for both species of ticks, especially when combined with CO2 vapors (Norval et al., 1989a, 1991 ). Thus, other substances, such as CO2, may participate in the aggregation response of these species to o-nitrophenol. This possibility warrants further study. Neither A. hebraeum nor A. variegatum adults were attracted to 2-methylpropanoic acid. This compound was identified in volatile effluents collected from feeding males ofA. hebraeum (Apps et al., 1988 ) but its biological role remains unknown. It could possibly function to repel adults of other species of Amblyomma, inasmuch as a negative REA was seen for both sexes of A. variegatum when exposed to this compound, while adults ofA. hebraeum were indifferent to it. It is concluded that both the bont tick and the tropical bont tick respond positively to a variety of synthetic volatile substances (in this study, principally phenols), which could conceivably be used to attract them to traps or acaricides. That such attractants might also be used in endemic areas of heartwater in order to increase transmission rates of the disease to young ruminants, which are temporarily protected by an age-related resistance, has also been suggested (Norval et al., 1989b). ACKNOWLEDGEMENTS
This study was supported by Cooperative Agreement No. AFR-0435-A-009084-00 between the U.S. Agency for International Development and the University of Florida. We thank Mr. Lameck Chakurungama for rearing the ticks used in this study.
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OLFACTORYRESPONSESAMBLYOMMATOATTRACTANTCHEMICALS
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ville, FL, and Ira Katz, West Long Branch, NJ. Assignees: International Flavors and Fragrances Inc., New York, NY; The University of Florida, Gainesville, FL. Butler, J.F. and Katz, I., 1988b. Process for determination of repellency and attractancy. U.S. Patent No. 4 759 228. Date of patent: 26 July 1988. Inventors: Jerry F. Butler, Gainesville, FL, and Ira Katz, West Long Branch, NJ. Assignees: International Flavors and Fragrances Inc., New York, NY; The University of Florida, Gainesville, FL. Gladney, W.J., Ernst, S.E. and Oehler, D.D., 1974. The Gulf Coast tick; evidence of a pheromone produced by males. J. Med. Entomol, 11: 303-306. Hess, E, and de Castro, J.J., 1986. Field tests of the response ofAmblyomma variegatum (Acari: Ixodidae) to the synthetic aggregation-attachment pheromone and its components. Exp. Appl. Acarol., 2: 249-255. Lusby, W.R., Sonenshine, D.E., Yunker, C.E., Norval, R.A.I. and Burridge, M.J., 1991. Comparison of known and suspected pheromonal constituents in males of the African ticks, A mblyomma hebraeum Koch and Amblyomma variegatum (Fabricius). Exp. Appl. Acarol., 13: 143-152. Norval, R.A.I., Butler, J.F. and Yunker, C.E., 1989a. Use of carbon dioxide and natural or synthetic aggregation-attachment pheromone of the bont tick, Amblyornma hebraeum, to attract and trap unfed adults in the field. Exp. Appl. Entomol., 7:171-180. Norval, R.A.I., Andrew, H.R. and Yunker, C.E, 1989b. Pheromone-mediation of host selection in bont ticks (A. hebraeum) Koch. Science, 243: 364-365. Norval, R.A.I., Peter, T.F., Yunker, C.E., Sonenshine, D.E. and Burridge, M.J., 1991. Response of the ticks Amblyomma hebraeum and A. variegatum to known or potential components of the aggregation-attachment pheromone. I. Long-range attraction. Exp. Appl. Acarol., 13:1118. Rechav, Y. and Whitehead, G.B., 1978. Field trials with pheromone-acaricide mixtures for control ofArnblyomma hebraeum J. Econ. Entomol., 71: 149-151. SchSni, R., Hess, E,, Blum, W. and Ramstein, K., 1984. The aggregation-attachment pheromone of the tropical bont tick Amblyomma variegatum Fabricius (Acari, Ixodidae): Isolation, identification and action of its components. J. Insect. Physiol., 30:613-618. Wood, W.F., Leahy, Sister M.G., Galun, R., Prestwich, G.D., Meinwald, J., Purnell, R.E. and Payne, R.C., 1975. Phenols as pheromones ofixodid ticks: A general phenomenon? J. Chem. Ecol., 1: 501-509.
