Differential Oviposition by Aedes hendersoni and Aedes triseriatus (Diptera: Culicidae) in Response to Chemical Cues Associated with Treehole Water ROBERT S. COPELAND1 AND GEORGE B. CRAIG, JR.2 Vector Biology Laboratory, Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
KEY WORDS
Insecta, treehole mosquitoes, oviposition, chemical cues
with Aedes triseriatus (Say) inhibits development of Aedes hendersoni Cockerell in artificial treeholes (Copeland & Craig 1992). When density is held constant, substitution of conspecific larvae with Ae. triseriatus larvae can increase mortality, slow female developmental rate, and reduce weights of emerging Ae. hendersoni males and females. When density is varied, increases in the number of Ae. triseriatus larvae have a stronger negative effect on development of Ae. hendersoni than do increases in the number of conspecific larvae (Copeland & Craig 1992). Fitness of Ae. hendersoni also is reduced as a result of infection with the protozoan parasite Ascogregarina barretti (Vavra) (Copeland & Craig 1992), whereas infection apparently is benign in the natural host Ae. triseriatus (Beier 1983, Beier & Craig 1985, Walker et al. 1987a, Copeland & Craig 1992). If Ascogregarina infection and interspecific competition with Ae. triseriatus regulate natural populations of Ae. hendersoni, then the local distributions of treehole Aedes species should be disjunct. Field-collected larvae of Ae. triseriatus and Ae. hendersoni were distributed disjunctly in northern Indiana (Copeland & Craig 1990). Most Ae. triseriatus were found in clear water "pans," depressions with horizontal openings created where root buttresses grow together or where a branch joins INTERSPECIFIC COMPETITION
'Current address: USAMRU-Kenya, Box 401, APO N.Y. 09675 or Medical Research Unit, Box 30137, Nairobi, Kenya. 2 To whom reprint requests should be sent.
the trunk (see Copeland & Craig [1990] for details of treehole classification). Densities of immature stages of Ae. triseriatus in basal and elevated pans, respectively, were 351 and 336 per liter, whereas the corresponding densities of Ae. hendersoni were 0 and 18 immatures per liter. In contrast, most Ae. hendersoni were found in the dark reddish-brown water of "deep rotholes," cavities that usually have vertical openings formed by fungal and bacterial action at the site of a wound, often the result of a broken branch. The density of Ae. hendersoni in deep rotholes was 80 individuals per liter compared with only 4 Ae. triseriatus per liter. Substantial overlap of the two treehole Aedes occurred only in "shallow rotholes," which apppeared to be ecologically intermediate between pans and deep rotholes. Aedes species also were partially segregated according to treehole height, as a result of the absence of Ae. hendersoni from basal treeholes, a common microhabitat of Ae. triseriatus. Such differences in the local distributions of mosquito larvae could result either from differences in female oviposition behavior related to the physical or chemical attractiveness of treeholes, or from differential mortality of the immature stages among treehole classes. Preliminary observations indicated that larvae of the two Aedes species developed to adults without appreciable mortality regardless of the type of treehole water in which they were reared (R.S.C., unpublished data). Therefore, we decided to test the hypothesis that the relative attractiveness of water from different treehole types or the physical conformation of treeholes them-
0022-2585/92/0033-0036$02.00/0 © 1992 Entomological Society of America
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J. Med. Entomol. 29(1): 33-36 (1992) ABSTRACT The oviposition behavior of Aedes hendersoni Cockerell and Aedes triseriatus (Say) was examined in the laboratory by offering gravid females oviposition sites containing water from different treehole types and by varying the physical characteristics of oviposition sites. Ae. hendersoni females oviposited more frequently in containers with water from treeholes in which they are found commonly in nature (maple "deep rotholes") than in containers with water from holes in which they are found rarely (beech "pans"). In contrast, Ae. triseriatus eggs were distributed uniformly in containers holding the two types of treehole water. There were no differences between species in response to oviposition container height or orientation of the entrance hole (vertical or horizontal). Females of both species laid nearly all their eggs in containers with horizontal openings, and most were deposited at the higher of two levels. We suggest that the adaptive value of the oviposition behavior of Ae. hendersoni is to maximize fitness by reducing interspecific contact with Ae. triseriatus.
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JOURNAL OF MEDICAL ENTOMOLOGY
selves influenced oviposition behavior of females of these species. Materials and Methods
Species Ae. hendersoni Ae. triseriatus
No. eggs laid in
Oviposition events" occurring in
Pan
Rothole
Pan
Rothole
155 1,161
2,820 2,325
2 10
28 20
Species were tested separately. Treehole water types were beech pan and maple deep-rothole water. " Based on 99.2 (2,975/30) eggs per Ae. hendersoni female and 116.2 (3,486/30) eggs per Ae. triseriatus female. Values were rounded to the nearest whole number before analysis.
the eggs to embryonate. To induce hatching, paddles and oviposition cans were flooded overnight with nitrogen-bubbled 0.1% nutrient broth in tap water. Larvae were reared to the second instar and identified to species according to characters described in Grimstad et al. (1974) and Lunt (1977). Paddles and cans were reflooded a week later. Effect of Hole Orientation and Height on Oviposition by Ae. triseriatus and Ae. hendersoni. TRISUKEN F5 and HENUKEN F5 adults were tested separately in a cage (0.6 by 0.6 by 1.8 m). A vertically oriented length of 1.8 cm (inside diameter) PVC pipe was attached to the center of the motorized turntable. The top of the pipe passed through a plastic ring suspended 1.57 m above the revolving disk by strings secured at the four corners of the cage. Four cylindrical, covered cardboard cartons were painted brown with metal primer (Rustoleum brand), and a hole 5.5 cm in diameter was cut into either the top or the side of each carton. A pair of cartons (one with a vertical and one with a horizontal hole) was attached on opposite sides of the pipe at heights of both 0.05 and 1.27 m. Black plastic oviposition cups were filled with tap water. The inside of each cup was lined with brown blotting paper, so that eggs could not be laid on the plastic cup itself. A single balsa paddle was cut into equal pieces and divided among the four cups, which then were placed inside the cartons. Nine gravid females were added each day for 5 d. Water, oviposition paddles, and blotter lining were changed daily. Data Analysis. In many studies of oviposition behavior, the measure considered for statistical analysis has been the number of eggs. Such an approach inflates the chances of detecting differences between sets of distributional data, because the analysis of such data is sensitive to changes in sample size. Because we were asking questions about the behavior of adult females and not that of eggs, we compared female oviposition events, not absolute egg numbers. Therefore, before analysis, egg numbers were divided by the mean number of eggs laid per female. Statistical methods followed those described by Sokal & Rohlf (1981) for the x2 tests of independence and goodness of fit.
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Strains of Aedes and Colony Maintenance. Two strains each of Ae. triseriatus and Ae. hendersoni were used for experimentation. Names of Ae. triseriatus strains begin with TRIS, whereas those of Ae. hendersoni begin with HEN. TRISPOT2 and HENPOT2 were collected as eggs at Potato Creek State Park, St. Joseph County, Ind., in July 1985. TRISUKEN and HENUKEN were collected as eggs near Lexington, Ky., in 1983. Strains of both species were maintained in the laboratory by induced copulation and rearing procedures described in detail for Ae. triseriatus by Munstermann & Wasmuth (1985). Effect of Treehole Water Type on Oviposition. Treehole water was collected from three basal beech "pans" (Fagus grandifolia Ehrh.), a common larval microhabitat of Ae. triseriatus but not of Ae. hendersoni, and three maple "deep rotholes" (Acer saccharum Marsh.), in which Ae. hendersoni larvae are common and Ae. triseriatus larvae are not. Collections from homologous habitats were pooled. Water was passed through a 0.14-mm mesh sieve and stored at 4°C until used. Experiments were conducted in a 0.6-m3 cage in an insectary at 21°C, «85% relative humidity, with an 18:6 (L:D) photoperiod. To eliminate position effects, oviposition containers were placed equidistant from each other around the edge of a brown masonite disk («40 cm diameter) which rested on a motorized turntable («0.5 rpm). Containers with water from the same treehole type were placed opposite each other on the disk, with two for each treehole-water type. Experiment 1. TRISPOT2 F2 and HENPOT2 F2 adults were tested alone. A 50-ml amount of each treehole water type was poured into each of two dixie cups. Because the maple rothole water was dark and the beech pan water was clear, five drops of india ink were added to each cup to eliminate background color effects (Wilton 1968). A single balsa wood paddle was cut into four equal pieces and distributed among the four cups. Six gravid females were added to the cage on days 1-5, and were left in the cage until the conclusion of the experiment on day 6. Oviposition paddles were replaced daily. Water was changed on days 2 and 4. Eggs were counted under a dissecting microscope. Experiment 2. TRISPOT2 F 3 and HENPOT2 F3 adults were tested together. Six gravid females of each species were added on days 1-5. The design was as in experiment 1, except that cans painted dull black were used as oviposition containers. Because the black paint eliminated background color effects, it was not necessary to add India ink. Balsa paddles were attached to the cans with paper clips. At the conclusion of the experiment, paddles and cans were held for 2 wk in the insectary to allow
Table 1. Response of ovipositing Ae. hendersoni and Ae. triseriatus females to different treehole water types (experiment 1)
January 1992
COPELAND
& CRAIG:
DIFFERENTIAL OVIPOSITION BY TREEHOLE
Table 2. Response of ovipositing Ae. hendersoni and Ae. triseriatus females to different treehole water types (experiment 2)
Species
Ae. hendersoni Ae. triseriatus
Oviposition events" occurring in
No. eggs laid in Pan
Rothole
462 (825) 918 (2,284)
1,206 (2,150) 483 (1,202)
Pan
Rothole
5 (8) 8 (20)
12 (22) 4 (10)
Species were tested together. Treehole water types were beech pan and maple deep-rothole water. Numbers in parentheses represent data corrected to include unhatched eggs (see text). " Based on 99.2 eggs per Ae. hendersoni female and 116.2 eggs per Ae. triseriatus female, as in experiment 1. Values were rounded to the nearest whole number before analysis.
35
laid more eggs in cans with beech pan water than in those with maple rothole water, but the difference was not significant (x2 — 3.33, df = 1, P > 0.05). As in the first experiment, Ae. hendersoni was more likely than Ae. triseriatus to lay its eggs in containers with deep maple rothole water (x2 = 9.64, df = 1, P < 0.01). Effect of Hole Orientation and Height on Oviposition. Both Aedes species laid nearly all their eggs in containers with horizontal holes (Table 3). A significantly greater proportion of eggs were laid at the upper level by both Ae. hendersoni (x2 = 24.20, df = 1, P < 0.001) and Ae. triseriatus (x2 = 12.52, df = 1, P < 0.001). Ae. triseriatus females deposited a higher percentage (24%) of their eggs at the lower level than did Ae. hendersoni (13%), but the difference was not significant (x2 = 168, df = 1, P > 0.05). Discussion Females of both Aedes species rest in the understory while converting a blood meal into eggs (Nasci 1985, Walker et al. 1987b). Because pans are found at both ground level and higher elevations, gravid Ae. triseriatus females approach ovipostion sites from above and below. In contrast, nearly all holes containing immature stages of Ae. hendersoni occur above the height of understory vegetation. Consequently, Ae. hendersoni females probably approach these sites from below. In our study, when testing the effects of height and hole orientation on oviposition behavior, we provided sites which could be approached from above, below, or the side. Nonetheless, we were unable to detect any significant differences between the species with respect to physical cues. Both species used sites with horizontal holes almost exclusively and both laid most eggs at the higher level. These results suggest that our experimental oviposition sites with vertical openings were not perceived by gravid Ae. hendersoni as similar enough to natural vertical treeholes to elicit entering behavior, or that some other combination of factors in nature overrides the tendency of Ae. hendersoni to lay eggs in holes with horizontal openings. Among these factors are chemical cues associated with particular treehole classes. The responses
Table 3. Effect of hole orientation and height on oviposition site selection by Ae. triseriatus and Ae. hendersoni Oviposition events0 occurring in
No. eggs deposited a
Ae. hendersoni Ae. triseriatus 0
Lower le>/el
Upper level
Species
fc
Lower level
Upper level
Up
Side
Up
Side
Up
Side
Up
Side
4,065 3,954
148 99
638 1,234
0 0
38 34
1 1
6 11
0 0
Horizontal hole. Vertical hole. No. of oviposition events. Based on 107.8 (4,851/45) eggs per Ae. hendersoni female and 117.5 (5,287/45) eggs per Ae. triseriatus female; figures rounded to the nearest whole number before analysis. b
c
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Results Effect of Treehole Water Type on Oviposition. Experiment 1. Ae. hendersoni females preferred deep maple rothole water to beech pan water (x2 = 22.5, df = 1, P < 0.001) (Table 1). Ae. triseriatus females also used deep maple rothole water more than beech pan water, but the difference was not significant (x2 = 3.33, df = 1, P > 0.05). Ae. hendersoni females were more likely to oviposit in cups with deep maple rothole water than were Ae. triseriatus females (x2 = 6.67, df = 1, P < 0.01). Experiment 2. The hatch rate of eggs in this experiment was low, probably because of low insemination success during induced copulation. Therefore, before analysis, original larval counts were corrected for differences in detectable oviposition caused by reduced fertility. The proportion laid by each species in each hole type was multiplied by the total number of eggs laid by the same number of females of each species in experiment 1. The conversion rests on the assumption that egg-laying behavior of uninseminated females was not different from inseminated females with respect to choice of treehole water type. The converted data are shown in parentheses in Table 2. In this experiment, Ae. hendersoni again preferred deep maple rothole water over beech pan water (x2 = 6.53, df = 1, P < 0.05). Ae. triseriatus females
Aedes
36
JOURNAL OF MEDICAL ENTOMOLOGY
Acknowledgment We thank K. A. Taylor for reviewing an earlier version of this manuscript. S. L. Paulson provided strains of Aedes. We also thank two anonymous reviewers for their careful reading of the manuscript and helpful comments. This study was supported by NIH grant AI-02753. References Cited Beier, J. C. 1983. Effects of gregarine parasites on the development of Dirofilaria immitis in Aedes triseriatus (Diptera: Culicidae). J. Med. Entomol. 20: 7075. Beier, J. C. & G. B. Craig, Jr. 1985. Gregarine parasites of mosquitoes, pp. 167-184. In M. Laird & J. W. Miles, [eds.], Integrated mosquito control methodologies, vol. 2. Biocontrol and other innovative components and future directions. Academic, London. Bentley, M. D., I. IN. Mcdaniel, H. P. Lee, B. Stiehl & M. Yatagai. 1976. Studies of Aedes triseriatus ovi-
position attractants produced by larvae of Aedes triseriatus and Aedes atropalpus. J. Med. Entomol. 13: 112-115. Bradshaw, W. E. & C. M. Holzapfel. 1988. Drought and the organization of tree-hole mosquito communities. Oecologia (Berl.) 74: 507-514. Copeland, B. S. & G. B. Craig, Jr. 1990. Habitat segregation among treehole mosquitoes in the Great Lakes region of the United States. Ann. Entomol. Soc. Am. 83: 1063-1073. 1992. Interspecific competition, parasitism, and predation affect development of Aedes hendersoni and Aedes triseriatus (Diptera: Culicidae) in artificial treeholes. Ann. Entomol. Soc. Am. (in press.) Grimstad, P. R., C. E. Garry & G. R. DeFoliart. 1974. Aedes hendersoni and Aedes triseriatus (Diptera: Culicidae). Characterization of larvae, larval hybrids, and comparison of adult and hybrid mesoscutal patterns. Ann. Entomol. Soc. Am. 67: 795-804. Lunt, S. R. 1977. Morphological characteristics of the larvae of Aedes triseriatus and Aedes hendersoni in Nebraska. Mosq. News 37: 654-656. McDaniel, I. N., M. D. Bentley, H. P. Lee & M. Yatagai. 1976. Effects of color and larval-produced oviposition attractants on oviposition of Aedes triseriatus. Environ. Entomol. 5: 553-556. Munstermann, L. E. & L. M. Wasmuth. 1985. Aedes triseriatus, pp. 15-24. In P. Singh and R. F. Moore [eds.], Handbook of insect rearing, vol. 2. Elsevier Science, Amsterdam. INasci, R. S. 1985. Local variation in blood feeding by Aedes triseriatus and Aedes hendersoni (Diptera: Culicidae). J. Med. Entomol. 22: 619-623. Sokal, P. R. & F. J. Rohlf. 1981. Biometry, the principles and practice of statistics in biological research, 2nd ed. Freeman, San Francisco. Walker, E. D., S. J. Poirier & W. T. Veldman. 1987a. Effects of Ascogregarina barretti (Eugregarinida: Lecudinidae) infection on emergence success, development time, and size of Aedes triseriatus (Diptera: Culicidae) in microcosms and tires. J. Med. Entomol. 24: 303-309. Walker E. D., R. S. Copeland, S. L. Paulson & L. E. Munstermann. 1987b. Adult survivorship, population density, and body size in sympatric populations of Aedes triseriatus and Aedes hendersoni (Diptera: Culicidae) J. Med. Entomol. 24: 485-493. Wilton, D. P. 1968. Oviposition site selection by the tree-hole mosquito, Aedes triseriatus (Say). J. Med. Entomol. 5: 189-194. Received for publication 11 March 1991; accepted 1 July 1991.
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of the two treehole Aedes differed significantly when offered oviposition sites containing water from different types of treeholes. Ae. hendersoni females were more likely to lay eggs in containers with deep rothole water than those with pan water, behavior that followed the pattern of larval distribution of this species in nature. Egg laying is probably a complex event that depends upon the integration and processing of several external stimuli. Both physical and chemical properties of treeholes influence oviposition by treehole Aedes (Wilton 1968, Bentley et al. 1976, Bradshaw & Holzapfel 1988). Our data provide evidence that chemical cues associated with the water from particular types of treeholes play a role in mediating the segregation of treehole Aedes eggs in nature. These cues may relate to the intrinsic chemical properties of treehole water or to compounds evolved by larvae or pupae that have developed previously in that water (Bentley et al. 1976, McDaniel et al. 1976). For Ae. hendersoni, deep rotholes may serve as a refuge from the negative effects of parasitism and interspecific competition with Ae. triseriatus. In this context, the adaptive value of the oviposition behavior of Ae. hendersoni probably is expressed as increased fitness of progeny.
Vol. 29, no. 1
KEY WORDS
Insecta, treehole mosquitoes, oviposition, chemical cues
with Aedes triseriatus (Say) inhibits development of Aedes hendersoni Cockerell in artificial treeholes (Copeland & Craig 1992). When density is held constant, substitution of conspecific larvae with Ae. triseriatus larvae can increase mortality, slow female developmental rate, and reduce weights of emerging Ae. hendersoni males and females. When density is varied, increases in the number of Ae. triseriatus larvae have a stronger negative effect on development of Ae. hendersoni than do increases in the number of conspecific larvae (Copeland & Craig 1992). Fitness of Ae. hendersoni also is reduced as a result of infection with the protozoan parasite Ascogregarina barretti (Vavra) (Copeland & Craig 1992), whereas infection apparently is benign in the natural host Ae. triseriatus (Beier 1983, Beier & Craig 1985, Walker et al. 1987a, Copeland & Craig 1992). If Ascogregarina infection and interspecific competition with Ae. triseriatus regulate natural populations of Ae. hendersoni, then the local distributions of treehole Aedes species should be disjunct. Field-collected larvae of Ae. triseriatus and Ae. hendersoni were distributed disjunctly in northern Indiana (Copeland & Craig 1990). Most Ae. triseriatus were found in clear water "pans," depressions with horizontal openings created where root buttresses grow together or where a branch joins INTERSPECIFIC COMPETITION
'Current address: USAMRU-Kenya, Box 401, APO N.Y. 09675 or Medical Research Unit, Box 30137, Nairobi, Kenya. 2 To whom reprint requests should be sent.
the trunk (see Copeland & Craig [1990] for details of treehole classification). Densities of immature stages of Ae. triseriatus in basal and elevated pans, respectively, were 351 and 336 per liter, whereas the corresponding densities of Ae. hendersoni were 0 and 18 immatures per liter. In contrast, most Ae. hendersoni were found in the dark reddish-brown water of "deep rotholes," cavities that usually have vertical openings formed by fungal and bacterial action at the site of a wound, often the result of a broken branch. The density of Ae. hendersoni in deep rotholes was 80 individuals per liter compared with only 4 Ae. triseriatus per liter. Substantial overlap of the two treehole Aedes occurred only in "shallow rotholes," which apppeared to be ecologically intermediate between pans and deep rotholes. Aedes species also were partially segregated according to treehole height, as a result of the absence of Ae. hendersoni from basal treeholes, a common microhabitat of Ae. triseriatus. Such differences in the local distributions of mosquito larvae could result either from differences in female oviposition behavior related to the physical or chemical attractiveness of treeholes, or from differential mortality of the immature stages among treehole classes. Preliminary observations indicated that larvae of the two Aedes species developed to adults without appreciable mortality regardless of the type of treehole water in which they were reared (R.S.C., unpublished data). Therefore, we decided to test the hypothesis that the relative attractiveness of water from different treehole types or the physical conformation of treeholes them-
0022-2585/92/0033-0036$02.00/0 © 1992 Entomological Society of America
Downloaded from http://jme.oxfordjournals.org/ by guest on June 8, 2016
J. Med. Entomol. 29(1): 33-36 (1992) ABSTRACT The oviposition behavior of Aedes hendersoni Cockerell and Aedes triseriatus (Say) was examined in the laboratory by offering gravid females oviposition sites containing water from different treehole types and by varying the physical characteristics of oviposition sites. Ae. hendersoni females oviposited more frequently in containers with water from treeholes in which they are found commonly in nature (maple "deep rotholes") than in containers with water from holes in which they are found rarely (beech "pans"). In contrast, Ae. triseriatus eggs were distributed uniformly in containers holding the two types of treehole water. There were no differences between species in response to oviposition container height or orientation of the entrance hole (vertical or horizontal). Females of both species laid nearly all their eggs in containers with horizontal openings, and most were deposited at the higher of two levels. We suggest that the adaptive value of the oviposition behavior of Ae. hendersoni is to maximize fitness by reducing interspecific contact with Ae. triseriatus.
34
Vol. 29, no. 1
JOURNAL OF MEDICAL ENTOMOLOGY
selves influenced oviposition behavior of females of these species. Materials and Methods
Species Ae. hendersoni Ae. triseriatus
No. eggs laid in
Oviposition events" occurring in
Pan
Rothole
Pan
Rothole
155 1,161
2,820 2,325
2 10
28 20
Species were tested separately. Treehole water types were beech pan and maple deep-rothole water. " Based on 99.2 (2,975/30) eggs per Ae. hendersoni female and 116.2 (3,486/30) eggs per Ae. triseriatus female. Values were rounded to the nearest whole number before analysis.
the eggs to embryonate. To induce hatching, paddles and oviposition cans were flooded overnight with nitrogen-bubbled 0.1% nutrient broth in tap water. Larvae were reared to the second instar and identified to species according to characters described in Grimstad et al. (1974) and Lunt (1977). Paddles and cans were reflooded a week later. Effect of Hole Orientation and Height on Oviposition by Ae. triseriatus and Ae. hendersoni. TRISUKEN F5 and HENUKEN F5 adults were tested separately in a cage (0.6 by 0.6 by 1.8 m). A vertically oriented length of 1.8 cm (inside diameter) PVC pipe was attached to the center of the motorized turntable. The top of the pipe passed through a plastic ring suspended 1.57 m above the revolving disk by strings secured at the four corners of the cage. Four cylindrical, covered cardboard cartons were painted brown with metal primer (Rustoleum brand), and a hole 5.5 cm in diameter was cut into either the top or the side of each carton. A pair of cartons (one with a vertical and one with a horizontal hole) was attached on opposite sides of the pipe at heights of both 0.05 and 1.27 m. Black plastic oviposition cups were filled with tap water. The inside of each cup was lined with brown blotting paper, so that eggs could not be laid on the plastic cup itself. A single balsa paddle was cut into equal pieces and divided among the four cups, which then were placed inside the cartons. Nine gravid females were added each day for 5 d. Water, oviposition paddles, and blotter lining were changed daily. Data Analysis. In many studies of oviposition behavior, the measure considered for statistical analysis has been the number of eggs. Such an approach inflates the chances of detecting differences between sets of distributional data, because the analysis of such data is sensitive to changes in sample size. Because we were asking questions about the behavior of adult females and not that of eggs, we compared female oviposition events, not absolute egg numbers. Therefore, before analysis, egg numbers were divided by the mean number of eggs laid per female. Statistical methods followed those described by Sokal & Rohlf (1981) for the x2 tests of independence and goodness of fit.
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Strains of Aedes and Colony Maintenance. Two strains each of Ae. triseriatus and Ae. hendersoni were used for experimentation. Names of Ae. triseriatus strains begin with TRIS, whereas those of Ae. hendersoni begin with HEN. TRISPOT2 and HENPOT2 were collected as eggs at Potato Creek State Park, St. Joseph County, Ind., in July 1985. TRISUKEN and HENUKEN were collected as eggs near Lexington, Ky., in 1983. Strains of both species were maintained in the laboratory by induced copulation and rearing procedures described in detail for Ae. triseriatus by Munstermann & Wasmuth (1985). Effect of Treehole Water Type on Oviposition. Treehole water was collected from three basal beech "pans" (Fagus grandifolia Ehrh.), a common larval microhabitat of Ae. triseriatus but not of Ae. hendersoni, and three maple "deep rotholes" (Acer saccharum Marsh.), in which Ae. hendersoni larvae are common and Ae. triseriatus larvae are not. Collections from homologous habitats were pooled. Water was passed through a 0.14-mm mesh sieve and stored at 4°C until used. Experiments were conducted in a 0.6-m3 cage in an insectary at 21°C, «85% relative humidity, with an 18:6 (L:D) photoperiod. To eliminate position effects, oviposition containers were placed equidistant from each other around the edge of a brown masonite disk («40 cm diameter) which rested on a motorized turntable («0.5 rpm). Containers with water from the same treehole type were placed opposite each other on the disk, with two for each treehole-water type. Experiment 1. TRISPOT2 F2 and HENPOT2 F2 adults were tested alone. A 50-ml amount of each treehole water type was poured into each of two dixie cups. Because the maple rothole water was dark and the beech pan water was clear, five drops of india ink were added to each cup to eliminate background color effects (Wilton 1968). A single balsa wood paddle was cut into four equal pieces and distributed among the four cups. Six gravid females were added to the cage on days 1-5, and were left in the cage until the conclusion of the experiment on day 6. Oviposition paddles were replaced daily. Water was changed on days 2 and 4. Eggs were counted under a dissecting microscope. Experiment 2. TRISPOT2 F 3 and HENPOT2 F3 adults were tested together. Six gravid females of each species were added on days 1-5. The design was as in experiment 1, except that cans painted dull black were used as oviposition containers. Because the black paint eliminated background color effects, it was not necessary to add India ink. Balsa paddles were attached to the cans with paper clips. At the conclusion of the experiment, paddles and cans were held for 2 wk in the insectary to allow
Table 1. Response of ovipositing Ae. hendersoni and Ae. triseriatus females to different treehole water types (experiment 1)
January 1992
COPELAND
& CRAIG:
DIFFERENTIAL OVIPOSITION BY TREEHOLE
Table 2. Response of ovipositing Ae. hendersoni and Ae. triseriatus females to different treehole water types (experiment 2)
Species
Ae. hendersoni Ae. triseriatus
Oviposition events" occurring in
No. eggs laid in Pan
Rothole
462 (825) 918 (2,284)
1,206 (2,150) 483 (1,202)
Pan
Rothole
5 (8) 8 (20)
12 (22) 4 (10)
Species were tested together. Treehole water types were beech pan and maple deep-rothole water. Numbers in parentheses represent data corrected to include unhatched eggs (see text). " Based on 99.2 eggs per Ae. hendersoni female and 116.2 eggs per Ae. triseriatus female, as in experiment 1. Values were rounded to the nearest whole number before analysis.
35
laid more eggs in cans with beech pan water than in those with maple rothole water, but the difference was not significant (x2 — 3.33, df = 1, P > 0.05). As in the first experiment, Ae. hendersoni was more likely than Ae. triseriatus to lay its eggs in containers with deep maple rothole water (x2 = 9.64, df = 1, P < 0.01). Effect of Hole Orientation and Height on Oviposition. Both Aedes species laid nearly all their eggs in containers with horizontal holes (Table 3). A significantly greater proportion of eggs were laid at the upper level by both Ae. hendersoni (x2 = 24.20, df = 1, P < 0.001) and Ae. triseriatus (x2 = 12.52, df = 1, P < 0.001). Ae. triseriatus females deposited a higher percentage (24%) of their eggs at the lower level than did Ae. hendersoni (13%), but the difference was not significant (x2 = 168, df = 1, P > 0.05). Discussion Females of both Aedes species rest in the understory while converting a blood meal into eggs (Nasci 1985, Walker et al. 1987b). Because pans are found at both ground level and higher elevations, gravid Ae. triseriatus females approach ovipostion sites from above and below. In contrast, nearly all holes containing immature stages of Ae. hendersoni occur above the height of understory vegetation. Consequently, Ae. hendersoni females probably approach these sites from below. In our study, when testing the effects of height and hole orientation on oviposition behavior, we provided sites which could be approached from above, below, or the side. Nonetheless, we were unable to detect any significant differences between the species with respect to physical cues. Both species used sites with horizontal holes almost exclusively and both laid most eggs at the higher level. These results suggest that our experimental oviposition sites with vertical openings were not perceived by gravid Ae. hendersoni as similar enough to natural vertical treeholes to elicit entering behavior, or that some other combination of factors in nature overrides the tendency of Ae. hendersoni to lay eggs in holes with horizontal openings. Among these factors are chemical cues associated with particular treehole classes. The responses
Table 3. Effect of hole orientation and height on oviposition site selection by Ae. triseriatus and Ae. hendersoni Oviposition events0 occurring in
No. eggs deposited a
Ae. hendersoni Ae. triseriatus 0
Lower le>/el
Upper level
Species
fc
Lower level
Upper level
Up
Side
Up
Side
Up
Side
Up
Side
4,065 3,954
148 99
638 1,234
0 0
38 34
1 1
6 11
0 0
Horizontal hole. Vertical hole. No. of oviposition events. Based on 107.8 (4,851/45) eggs per Ae. hendersoni female and 117.5 (5,287/45) eggs per Ae. triseriatus female; figures rounded to the nearest whole number before analysis. b
c
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Results Effect of Treehole Water Type on Oviposition. Experiment 1. Ae. hendersoni females preferred deep maple rothole water to beech pan water (x2 = 22.5, df = 1, P < 0.001) (Table 1). Ae. triseriatus females also used deep maple rothole water more than beech pan water, but the difference was not significant (x2 = 3.33, df = 1, P > 0.05). Ae. hendersoni females were more likely to oviposit in cups with deep maple rothole water than were Ae. triseriatus females (x2 = 6.67, df = 1, P < 0.01). Experiment 2. The hatch rate of eggs in this experiment was low, probably because of low insemination success during induced copulation. Therefore, before analysis, original larval counts were corrected for differences in detectable oviposition caused by reduced fertility. The proportion laid by each species in each hole type was multiplied by the total number of eggs laid by the same number of females of each species in experiment 1. The conversion rests on the assumption that egg-laying behavior of uninseminated females was not different from inseminated females with respect to choice of treehole water type. The converted data are shown in parentheses in Table 2. In this experiment, Ae. hendersoni again preferred deep maple rothole water over beech pan water (x2 = 6.53, df = 1, P < 0.05). Ae. triseriatus females
Aedes
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JOURNAL OF MEDICAL ENTOMOLOGY
Acknowledgment We thank K. A. Taylor for reviewing an earlier version of this manuscript. S. L. Paulson provided strains of Aedes. We also thank two anonymous reviewers for their careful reading of the manuscript and helpful comments. This study was supported by NIH grant AI-02753. References Cited Beier, J. C. 1983. Effects of gregarine parasites on the development of Dirofilaria immitis in Aedes triseriatus (Diptera: Culicidae). J. Med. Entomol. 20: 7075. Beier, J. C. & G. B. Craig, Jr. 1985. Gregarine parasites of mosquitoes, pp. 167-184. In M. Laird & J. W. Miles, [eds.], Integrated mosquito control methodologies, vol. 2. Biocontrol and other innovative components and future directions. Academic, London. Bentley, M. D., I. IN. Mcdaniel, H. P. Lee, B. Stiehl & M. Yatagai. 1976. Studies of Aedes triseriatus ovi-
position attractants produced by larvae of Aedes triseriatus and Aedes atropalpus. J. Med. Entomol. 13: 112-115. Bradshaw, W. E. & C. M. Holzapfel. 1988. Drought and the organization of tree-hole mosquito communities. Oecologia (Berl.) 74: 507-514. Copeland, B. S. & G. B. Craig, Jr. 1990. Habitat segregation among treehole mosquitoes in the Great Lakes region of the United States. Ann. Entomol. Soc. Am. 83: 1063-1073. 1992. Interspecific competition, parasitism, and predation affect development of Aedes hendersoni and Aedes triseriatus (Diptera: Culicidae) in artificial treeholes. Ann. Entomol. Soc. Am. (in press.) Grimstad, P. R., C. E. Garry & G. R. DeFoliart. 1974. Aedes hendersoni and Aedes triseriatus (Diptera: Culicidae). Characterization of larvae, larval hybrids, and comparison of adult and hybrid mesoscutal patterns. Ann. Entomol. Soc. Am. 67: 795-804. Lunt, S. R. 1977. Morphological characteristics of the larvae of Aedes triseriatus and Aedes hendersoni in Nebraska. Mosq. News 37: 654-656. McDaniel, I. N., M. D. Bentley, H. P. Lee & M. Yatagai. 1976. Effects of color and larval-produced oviposition attractants on oviposition of Aedes triseriatus. Environ. Entomol. 5: 553-556. Munstermann, L. E. & L. M. Wasmuth. 1985. Aedes triseriatus, pp. 15-24. In P. Singh and R. F. Moore [eds.], Handbook of insect rearing, vol. 2. Elsevier Science, Amsterdam. INasci, R. S. 1985. Local variation in blood feeding by Aedes triseriatus and Aedes hendersoni (Diptera: Culicidae). J. Med. Entomol. 22: 619-623. Sokal, P. R. & F. J. Rohlf. 1981. Biometry, the principles and practice of statistics in biological research, 2nd ed. Freeman, San Francisco. Walker, E. D., S. J. Poirier & W. T. Veldman. 1987a. Effects of Ascogregarina barretti (Eugregarinida: Lecudinidae) infection on emergence success, development time, and size of Aedes triseriatus (Diptera: Culicidae) in microcosms and tires. J. Med. Entomol. 24: 303-309. Walker E. D., R. S. Copeland, S. L. Paulson & L. E. Munstermann. 1987b. Adult survivorship, population density, and body size in sympatric populations of Aedes triseriatus and Aedes hendersoni (Diptera: Culicidae) J. Med. Entomol. 24: 485-493. Wilton, D. P. 1968. Oviposition site selection by the tree-hole mosquito, Aedes triseriatus (Say). J. Med. Entomol. 5: 189-194. Received for publication 11 March 1991; accepted 1 July 1991.
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of the two treehole Aedes differed significantly when offered oviposition sites containing water from different types of treeholes. Ae. hendersoni females were more likely to lay eggs in containers with deep rothole water than those with pan water, behavior that followed the pattern of larval distribution of this species in nature. Egg laying is probably a complex event that depends upon the integration and processing of several external stimuli. Both physical and chemical properties of treeholes influence oviposition by treehole Aedes (Wilton 1968, Bentley et al. 1976, Bradshaw & Holzapfel 1988). Our data provide evidence that chemical cues associated with the water from particular types of treeholes play a role in mediating the segregation of treehole Aedes eggs in nature. These cues may relate to the intrinsic chemical properties of treehole water or to compounds evolved by larvae or pupae that have developed previously in that water (Bentley et al. 1976, McDaniel et al. 1976). For Ae. hendersoni, deep rotholes may serve as a refuge from the negative effects of parasitism and interspecific competition with Ae. triseriatus. In this context, the adaptive value of the oviposition behavior of Ae. hendersoni probably is expressed as increased fitness of progeny.
Vol. 29, no. 1
