J. Med Eat.
Vol. 13, no. 1: 112-115
29 May 1976
STUDIES OF AEDES TRISERIATUS OVIPOSITION ATTRACTANTS PRODUCED BY LARVAE OF AEDES TRISERIATUS AND AEDES ATROPALPUS (DIPTERA: CULIGIDAE) By Michael D. Bentley1, Ivan N. McDaniel2, Hai-Poong Lee2, Barbara Stiehl1 and Mitsuyoshi Yatagai1 Abstract: Holding waters of 4th-instar larvae of both Aedes mosquitoes. For example, Ikeshoji et al. (1967) triseriatus (Say) and Aedes atropalpus (Coquillett) are shown to demonstrated the presence of oviposition stimulants contain an oviposition attractant for Ae. triseriatus adults. It in Culex pipiens quinquefasciatus (Say) field water, is demonstrated that the attractants are contained in the volatile fraction of this material. Experiments in which physical while Dadd & Kleinjan (1974) have shown that contact with the larval holding water is prevented demonstrate larval, pupal and egg waters of Culex pipiens pipiens L. the importance of olfactory mechanisms in oviposition site selection. Acetone extracts of eggs of Ae. triseriatus do not contain attractive materials. The eggs of Culex contain an oviposition attractant for this species. tarsails (Coquillett) have been shown to contain an
'Department of Chemistry, University of Maine, Orono, Maine 04473, U.S.A. department of Entomology, University of Maine, Orono, Maine 04473, U.S.A.
oviposition attractant (Starratt & Osgood 1972) and chromatography of an egg extract yielded an active fraction containing a mixture of 1,3-diglycerides of monoacetoxy and diacetoxy fatty acids. These authors (Starratt & Osgood 1973) have also reported that egg rafts of both Cx. p. quinquefasciatus and Cx. p. pipiens contain a similar mixture of compounds, with the fatty acid composition of the diglycerides differing slightly from that in Cx. tarsalis. Recently Kalpage & Brust (1973) concluded that larval and pupal holding waters of Aedes atropalpus (Coquillett) contain oviposition attractants for that species. Wilton (1968) has examined a number of factors which influence the choice of oviposition site by Ae. triseriatus and has concluded that the presence of decayed organic matter in the oviposition site results in increased oviposition.
TABLE 1. Number of eggs produced by Aedes triseriatus on various test media and H 2 O references.* Ae. triseriatus LARVAL
Ae. atropalpus LARVAL
Ae. triseriatus
HOLDING WATER**
HOLDING WATER**
EGG EXTRACT***
Test 1858 2504 (61%) II 408 372 (66%) III 2740 3003 (67%) IV 2569 2239 (65%) V 421 1961 2878 1640 304 2589 (56%) (83%) (17%) •Percentages in parentheses refer to % of total **(P0.05).
EXP.
I
Test 3335 3165 (67%) 3945 3626 (65%) 5634 5487 (64%) 6627 (63%)
H3O 1445 1762 (33%) 1759 2300 (35%) 3129 3205 (36%) 3953 (37%)
Test H8O 1438 1659 1293 1690 (39%) (50%) 215 1809 181 1274 (34%) (49%) 1693 1946 1144 1423 (33%) (54%) 1698 887 (35%) 1800 2529 (44%) eggs in each experiment.
H3O 1586 1783 (50%) 1781 1470 (51%) 1392 1519 (46%)
Downloaded from http://jme.oxfordjournals.org/ by guest on June 8, 2016
Aedes triseriatus (Say), a common tree-hole mosquito whose range includes the deciduous forests of the eastern United States, is a species of particular interest since it has been identified as the principal vector of LaCrosse virus (California encephalitis group). A recent important study (Watts et al. 1973) has proven that transovarial transmission of this virus by Ae. triseriatus is possible. It also has potential as a vector of eastern equine encephalitis, dog heartworm and bird malaria (Zavortink 1972). Within recent years, it has become increasingly evident that chemical attractancy and stimulation are important components of the web of factors influencing the choice of oviposition sites by gravid
113
Bentley et al.: Ae. triseriatus oviposition attractants
1976
acetone was then removed using a rotary evaporator We have investigated the influence of chemicals present in larval water of both Ae. triseriatus and Ae. under water aspirator pressure at room temperature atropalpus on oviposition of Ae. triseriatus and have to yield 10 mg of a yellow oil which was rediluted also examined Ae. triseriatus egg extracts for pos- with 10 ml of aceione. Samples for bioassay were prepared by evaporating 1 ml of the latter solution sible attractancy. We report the results of these (3000 egg equivalents) onto a sheet of 9-cm S&S studies in the present paper. Blue Ribbon no. 589 filter paper. MATERIALS AND METHODS
Bioassay methods
All samples were bioassayed in the 13.5-m3 cage Our Ae. triseriatus colony, started from eggs with environmental conditions as described in the section on colony maintenance. A sample for collected near Old Town, Maine, was maintained bioassay was prepared by placing 150 ml of test in our laboratory for about 2 years prior to the solution in the amber glass cylindrical container present study. Larvae were reared in covered (8.5 cm diam. by 10 cm high). To prepare the plastic trays on a diet of Fleischmann's dry yeast, 3 oviposition substrate, a 9-cm S&S Blue Ribbon and pupae were transferred daily to a 13.5-m filter paper disc was moistened with the test solucage for emergence. Adults were maintained at tion and caused to adhere smoothly to the inner 25 ± 3°C, 58 ± 12% RH, and at a photoperiod of wall of the glass vessel, with 1/2 the paper subL:D 16:8 hr by means of an overhead 60-watt lamp merged, and with the lines on the paper horizontal. controlled by a timer. Cotton pads saturated with aqueous honey solution were available at all times Test solutions were prepared in duplicate for each experiment and duplicate 150-ml distilled water and 2 guinea pigs were presented for blood-feeding references were prepared in a similar manner. for 2-hr periods on alternate days. The unsexed adult population in the cage was maintained at When assaying egg extracts, the test solution was deposited on the paper substrate as described in the about 5000. The Ae. atropalpus colony was started with eggs section on preparation of test samples. The 4 oviposition sites were alternately placed obtained from Professor George Craig of Notre on the perimeter of a 100-cm diam., 1/4 rpm, Dame and was maintained in our laboratory for about 2 months prior to the present study. This plywood turntable at 5:00 PM EDT and oviposition bioassays were terminated at 10:00 PM, which ended stock was originally colonized from rock pools near the 16-hr photophase. Eggs were counted under Belleville, Ontario, during the summer of 1968 by magnification with the exception of those correspondAnne Hudson. Rearing procedures were the same ing to the data in TABLE 2. The latter were counted as those described for Ae. triseriatus, except adults 3 with an egg-counting device recently constructed in were maintained in a small cage (0.22-m ) at a our laboratory (Bentley et al. 1974). population of several hundred and, since the females To demonstrate the importance of olfaction in were autogenous, honey was the only food presented.
Colony maintenance
Solutions were
normally prepared by washing 600 fourth-instar larvae with several portions of distilled water, then placing them in a covered beaker containing 600 ml of distilled water for a 48-hr period. Larvae were removed by filtration and the solution was stored in a refrigerator until used. LHW distillate was prepared by distillation of LHW in a rotary evaporator at water aspirator pressure while heating with a water bath at 70 °C. Distillate was trapped at 0°C. The distillation residue was diluted to the original LHW volume with distilled water for bioassay; LHW distillate was subjected to bioassay without further dilution. Egg extracts: Thirty thousand eggs of Ae. triseriatus
were extracted with acetone (20 ml, Fisher A.C.S., redistilled) in a Soxhlet apparatus for 15 hr. The
TABLE 2. Oviposition at sites containing larval holding water (LHW) in screened containers vs distilled water in screened containers.* TEST EXPERIMENT
I II III IV
V »(P0.05).
sites having the characteristic odor of larval holding water even though contact chemoreception is not possible. In agreement with these data, it was The results shown in TABLE 1 provide strong (TABLE 3) that a distillate of LHW serves as found evidence that a substance is produced by Ae. triseriatus an oviposition attractant. Bioassay of aqueous larvae which influences oviposition by this species. the solid residue remaining after vacuum solutions of The substance appears to be reasonably stable since distillation yielded data with considerable fluctuait retains activity after distillation involving temtion and which showed no significant attractancy at peratures up to 70 °C and after storage in a re(TABLE 4). the 95% probability level frigerator for several weeks. In order to determine TABLE 1 which Evidence is also presented in if olfactory detection of volatile compounds is inproves that larvae of Ae. atropalpus, a rock pool volved, experiments were conducted in which larval breeder, produce a substance which can serve as an holding water was placed in a screened vial within oviposition attractant for gravid Ae. triseriatus adults. the usual oviposition container and the ovipositional response compared with that of distilled water placed We have not observed any cases in the field where in a like container. The data in TABLE 2 demon- Ae. triseriatus and Ae. atropalpus have utilized a common oviposition site. Although this has been strate that gravid adults oviposit preferentially in reported to occur rarely (Zavortink 1972), we feel that the presence of oviposition attractants in TABLE 3. Oviposition at sites containing LHW distillate vs larval water is only one of an apparent multitude of distilled water.* physical and chemical factors affecting oviposition TEST CONTROL (LHW site choice and would not necessarily result in DISTILLATE) (H,O) oviposition site crossover in cases where common EXPERIMENT (NO. OF EGGS) (NO. OF EGGS) attractants were produced by related species. In 2505 3305 I 3410 our work with Ae. triseriatus we have found, for 3610 (46%) (54%) example, that simple physical factors such as oviposi3020 2720 II tion site color can have a much greater influence 2215 3620 on site choice than the presence of larval-produced (45%) (55%) 2210 3010 chemicals. Also, we have observed that aqueous III 2510 3320 extracts, which contain many of the same wood (43%) (57%) degradation products found in natural tree holes, 3520 3820 IV attract Ae. triseriatus to oviposition sites more fre3210 4115 (54%) (46%) quently than do larval holding waters. 1605 2105 V Since other workers have shown that eggs of Cx. 1560 2210 tarsalis (Osgood 1971) and Cx. pipiens (Dadd & (42%) (58%) Kleinjan 1974) contain oviposition attractants, we '(P
Vol. 13, no. 1: 112-115
29 May 1976
STUDIES OF AEDES TRISERIATUS OVIPOSITION ATTRACTANTS PRODUCED BY LARVAE OF AEDES TRISERIATUS AND AEDES ATROPALPUS (DIPTERA: CULIGIDAE) By Michael D. Bentley1, Ivan N. McDaniel2, Hai-Poong Lee2, Barbara Stiehl1 and Mitsuyoshi Yatagai1 Abstract: Holding waters of 4th-instar larvae of both Aedes mosquitoes. For example, Ikeshoji et al. (1967) triseriatus (Say) and Aedes atropalpus (Coquillett) are shown to demonstrated the presence of oviposition stimulants contain an oviposition attractant for Ae. triseriatus adults. It in Culex pipiens quinquefasciatus (Say) field water, is demonstrated that the attractants are contained in the volatile fraction of this material. Experiments in which physical while Dadd & Kleinjan (1974) have shown that contact with the larval holding water is prevented demonstrate larval, pupal and egg waters of Culex pipiens pipiens L. the importance of olfactory mechanisms in oviposition site selection. Acetone extracts of eggs of Ae. triseriatus do not contain attractive materials. The eggs of Culex contain an oviposition attractant for this species. tarsails (Coquillett) have been shown to contain an
'Department of Chemistry, University of Maine, Orono, Maine 04473, U.S.A. department of Entomology, University of Maine, Orono, Maine 04473, U.S.A.
oviposition attractant (Starratt & Osgood 1972) and chromatography of an egg extract yielded an active fraction containing a mixture of 1,3-diglycerides of monoacetoxy and diacetoxy fatty acids. These authors (Starratt & Osgood 1973) have also reported that egg rafts of both Cx. p. quinquefasciatus and Cx. p. pipiens contain a similar mixture of compounds, with the fatty acid composition of the diglycerides differing slightly from that in Cx. tarsalis. Recently Kalpage & Brust (1973) concluded that larval and pupal holding waters of Aedes atropalpus (Coquillett) contain oviposition attractants for that species. Wilton (1968) has examined a number of factors which influence the choice of oviposition site by Ae. triseriatus and has concluded that the presence of decayed organic matter in the oviposition site results in increased oviposition.
TABLE 1. Number of eggs produced by Aedes triseriatus on various test media and H 2 O references.* Ae. triseriatus LARVAL
Ae. atropalpus LARVAL
Ae. triseriatus
HOLDING WATER**
HOLDING WATER**
EGG EXTRACT***
Test 1858 2504 (61%) II 408 372 (66%) III 2740 3003 (67%) IV 2569 2239 (65%) V 421 1961 2878 1640 304 2589 (56%) (83%) (17%) •Percentages in parentheses refer to % of total **(P0.05).
EXP.
I
Test 3335 3165 (67%) 3945 3626 (65%) 5634 5487 (64%) 6627 (63%)
H3O 1445 1762 (33%) 1759 2300 (35%) 3129 3205 (36%) 3953 (37%)
Test H8O 1438 1659 1293 1690 (39%) (50%) 215 1809 181 1274 (34%) (49%) 1693 1946 1144 1423 (33%) (54%) 1698 887 (35%) 1800 2529 (44%) eggs in each experiment.
H3O 1586 1783 (50%) 1781 1470 (51%) 1392 1519 (46%)
Downloaded from http://jme.oxfordjournals.org/ by guest on June 8, 2016
Aedes triseriatus (Say), a common tree-hole mosquito whose range includes the deciduous forests of the eastern United States, is a species of particular interest since it has been identified as the principal vector of LaCrosse virus (California encephalitis group). A recent important study (Watts et al. 1973) has proven that transovarial transmission of this virus by Ae. triseriatus is possible. It also has potential as a vector of eastern equine encephalitis, dog heartworm and bird malaria (Zavortink 1972). Within recent years, it has become increasingly evident that chemical attractancy and stimulation are important components of the web of factors influencing the choice of oviposition sites by gravid
113
Bentley et al.: Ae. triseriatus oviposition attractants
1976
acetone was then removed using a rotary evaporator We have investigated the influence of chemicals present in larval water of both Ae. triseriatus and Ae. under water aspirator pressure at room temperature atropalpus on oviposition of Ae. triseriatus and have to yield 10 mg of a yellow oil which was rediluted also examined Ae. triseriatus egg extracts for pos- with 10 ml of aceione. Samples for bioassay were prepared by evaporating 1 ml of the latter solution sible attractancy. We report the results of these (3000 egg equivalents) onto a sheet of 9-cm S&S studies in the present paper. Blue Ribbon no. 589 filter paper. MATERIALS AND METHODS
Bioassay methods
All samples were bioassayed in the 13.5-m3 cage Our Ae. triseriatus colony, started from eggs with environmental conditions as described in the section on colony maintenance. A sample for collected near Old Town, Maine, was maintained bioassay was prepared by placing 150 ml of test in our laboratory for about 2 years prior to the solution in the amber glass cylindrical container present study. Larvae were reared in covered (8.5 cm diam. by 10 cm high). To prepare the plastic trays on a diet of Fleischmann's dry yeast, 3 oviposition substrate, a 9-cm S&S Blue Ribbon and pupae were transferred daily to a 13.5-m filter paper disc was moistened with the test solucage for emergence. Adults were maintained at tion and caused to adhere smoothly to the inner 25 ± 3°C, 58 ± 12% RH, and at a photoperiod of wall of the glass vessel, with 1/2 the paper subL:D 16:8 hr by means of an overhead 60-watt lamp merged, and with the lines on the paper horizontal. controlled by a timer. Cotton pads saturated with aqueous honey solution were available at all times Test solutions were prepared in duplicate for each experiment and duplicate 150-ml distilled water and 2 guinea pigs were presented for blood-feeding references were prepared in a similar manner. for 2-hr periods on alternate days. The unsexed adult population in the cage was maintained at When assaying egg extracts, the test solution was deposited on the paper substrate as described in the about 5000. The Ae. atropalpus colony was started with eggs section on preparation of test samples. The 4 oviposition sites were alternately placed obtained from Professor George Craig of Notre on the perimeter of a 100-cm diam., 1/4 rpm, Dame and was maintained in our laboratory for about 2 months prior to the present study. This plywood turntable at 5:00 PM EDT and oviposition bioassays were terminated at 10:00 PM, which ended stock was originally colonized from rock pools near the 16-hr photophase. Eggs were counted under Belleville, Ontario, during the summer of 1968 by magnification with the exception of those correspondAnne Hudson. Rearing procedures were the same ing to the data in TABLE 2. The latter were counted as those described for Ae. triseriatus, except adults 3 with an egg-counting device recently constructed in were maintained in a small cage (0.22-m ) at a our laboratory (Bentley et al. 1974). population of several hundred and, since the females To demonstrate the importance of olfaction in were autogenous, honey was the only food presented.
Colony maintenance
Solutions were
normally prepared by washing 600 fourth-instar larvae with several portions of distilled water, then placing them in a covered beaker containing 600 ml of distilled water for a 48-hr period. Larvae were removed by filtration and the solution was stored in a refrigerator until used. LHW distillate was prepared by distillation of LHW in a rotary evaporator at water aspirator pressure while heating with a water bath at 70 °C. Distillate was trapped at 0°C. The distillation residue was diluted to the original LHW volume with distilled water for bioassay; LHW distillate was subjected to bioassay without further dilution. Egg extracts: Thirty thousand eggs of Ae. triseriatus
were extracted with acetone (20 ml, Fisher A.C.S., redistilled) in a Soxhlet apparatus for 15 hr. The
TABLE 2. Oviposition at sites containing larval holding water (LHW) in screened containers vs distilled water in screened containers.* TEST EXPERIMENT
I II III IV
V »(P0.05).
sites having the characteristic odor of larval holding water even though contact chemoreception is not possible. In agreement with these data, it was The results shown in TABLE 1 provide strong (TABLE 3) that a distillate of LHW serves as found evidence that a substance is produced by Ae. triseriatus an oviposition attractant. Bioassay of aqueous larvae which influences oviposition by this species. the solid residue remaining after vacuum solutions of The substance appears to be reasonably stable since distillation yielded data with considerable fluctuait retains activity after distillation involving temtion and which showed no significant attractancy at peratures up to 70 °C and after storage in a re(TABLE 4). the 95% probability level frigerator for several weeks. In order to determine TABLE 1 which Evidence is also presented in if olfactory detection of volatile compounds is inproves that larvae of Ae. atropalpus, a rock pool volved, experiments were conducted in which larval breeder, produce a substance which can serve as an holding water was placed in a screened vial within oviposition attractant for gravid Ae. triseriatus adults. the usual oviposition container and the ovipositional response compared with that of distilled water placed We have not observed any cases in the field where in a like container. The data in TABLE 2 demon- Ae. triseriatus and Ae. atropalpus have utilized a common oviposition site. Although this has been strate that gravid adults oviposit preferentially in reported to occur rarely (Zavortink 1972), we feel that the presence of oviposition attractants in TABLE 3. Oviposition at sites containing LHW distillate vs larval water is only one of an apparent multitude of distilled water.* physical and chemical factors affecting oviposition TEST CONTROL (LHW site choice and would not necessarily result in DISTILLATE) (H,O) oviposition site crossover in cases where common EXPERIMENT (NO. OF EGGS) (NO. OF EGGS) attractants were produced by related species. In 2505 3305 I 3410 our work with Ae. triseriatus we have found, for 3610 (46%) (54%) example, that simple physical factors such as oviposi3020 2720 II tion site color can have a much greater influence 2215 3620 on site choice than the presence of larval-produced (45%) (55%) 2210 3010 chemicals. Also, we have observed that aqueous III 2510 3320 extracts, which contain many of the same wood (43%) (57%) degradation products found in natural tree holes, 3520 3820 IV attract Ae. triseriatus to oviposition sites more fre3210 4115 (54%) (46%) quently than do larval holding waters. 1605 2105 V Since other workers have shown that eggs of Cx. 1560 2210 tarsalis (Osgood 1971) and Cx. pipiens (Dadd & (42%) (58%) Kleinjan 1974) contain oviposition attractants, we '(P
