Journal of Chemical Ecology. Vol+ 21, No. 11, 1995

EVALUATION OF ORGANIC INFUSIONS AND SYNTHETIC COMPOUNDS MEDIATING OVIPOSITION IN Aedes albopictus AND Aedes aegypti (Diptera: Culicidae)

SANDRA

A,

ALLAN*

and

DANIEL

L.

KLINE

United States Departnwnt of Agriculture Agriculture Research Service Medical and Veterinary Entomology Research L~lhorato O` P.O. Boa 14565, Gainexrille, Florida 32604 (Received March 17, 1995; accepted July I0, 1995)

Abstract--Oviposition responses of gravid Aedes albopictus and Ae. m'g3pti were evaluated to natural organic infusions (hay infusitm, larval rearing water, and field-collected larval water) its well as compounds isolated from hay intusion (3-methylindote, 4-methylphenol, 4-ethylphenol. indole, and phenol) known to elicit oviposition in Cuh~r mosquitoes. In laboratory bioassays. significant oviposition responses were obtained I?om Ae. albopietus, hut not from Ae, aegypti, to dilutions of hay infuskm and field water. Oviposition responses ¢~1"both species were moderate to the synthetic compounds tested in the laborato~ (0,01- I(10 ,ag/liler). Only 3-methylindole (0. I p.g/liter) and 4-elhylphenol (1+0 ,ag/liter) elicited significantly more oviposition by ,4e. albopictus than did well water. Of the synthetic compounds tested with At'. aegypti, only phenol (I .0 p.g/liter) and 4-ethylphenol (0. I /zg/liter) elicited significantly more ovipt)sition than did well water. Significant repellency or oviposition deterrence Ibr hc,th species occurred in response to at least one high concentration ~+1+most of the compounds tested. In field cage evaluations, oviposition responses by Ae, albopictus were strongest to larval water and field water, moderate to hay infusion and 3-methylindole ( |00 #g/liter), and low to, well water+ A mixture of five synthetic compounds min+ieking hay infusion was no more effective than 3+methylindole alone. For At'. aegypli, oviposition responses were greatest to larval water and least to 3-methylindole. In an olfactc,meter+ gravid females of both species oriented more to field water than to well water and only Ae. albopictus oriented more to larval water c,r hay infusion than well water. In general gravid Ae. albopictus responded more strongly to oviposition stimuli than did Ae. aeg3pti+

*To whom correspondence should be addressed, 1847 t~lqX4)331 ]tJ5/I I IX)- It'M75[)7.5nl(l ~ I t;t+5 P l e n u m Publi~,hing C o . r o l l , o i l

1848

ALLAN AND KLINE Key Words- --lnsecta, Diptcra, Culicidae, Aedes athopicms, Aedes aegypti, oviposition, attrac~ants, behavior,

INTRODUCTION

The selection of oviposition sites by many mosquito species is greatly influenced by chemical stimuli (Bentley and Day, 1989). Chemical factors mediating oviposition may result from the previous presence of larvae or pupae (reviewed in Bentley and Day, 1989) or from eggs, which in several Cul~:~- species contain an apical droplet of pheromone (Laurence and Pickett, 1985; Starratt and Osgood, 1973). In addition, water from natural breeding sites such as ponds (Qjullin et al., 1965) or infusions of organic material (such as hay or grass infusions) are attractive to gravid female mosquitoes (Ritchie, 1984: Millar et al., 1992). Different chemical cues may elicit oviposition behaviours beginning with the initial orientation towards a potential oviposition site and culminating in the deposition of eggs, A wide range of mosquito species, including Aedes albopictus (Hein, 1976; Holck et al., 1988), Ae. aeg3pti (Gjullin et al., 1965; Chadee, 1993), Ae. triseriatus (Holck et al., 1988), Culex nigripalpus (Ritchie, 1984), Cr. pipiens (Madder et al., 1980: Reiter, 1986), C~-. quinquc:fitsciatus (Gjullen et al., 1965: Reisen and Meyer, 1990: Millar et al., 1992), C~. restuans (Madder et al., 1980: Reiter, 1986), and Cx. tarsalis (Reisen and Meyer, 1990) have been reported to be attracted to hay and grass infusions for oviposition. For this reason, infusions have been used to enhance collections in CDC light traps (Reiter et al., 1991), gravid female traps (Reisen and Meyer, 1990: Reiter, 1983), and ovitraps (Holck et al., t988: Kitron el al., 1989). Recently Millar et al. (1992) isolated and identifed five compounds (3methyiindole, 4-methylphenol, 4-ethylphenol, phenol, and indole) from fermented Bermuda grass infusions that strongly stimulated oviposition by Cx. quinque{fasciatus, both individually and in a blend of similar concentrations as isolated from the grass infusion. The five-compound blend and 3-methylindole alone were equally attractive to ovipositing C~. quinquefasciatus in laboratory bioassays. In addition, 3-methylindole also mediated oviposition of C.r. tarsalis and Cr. stigmatosoma under field conditions (Beehler et al., 1994). It is not known if these compounds have similar effects on the oviposition behavior of other mosquito species known to be attracted to grass or hay infusions. The objectives of our study were to: (1) evaluate oviposition responses of Ae. albopictus and Ae. aegypti to various organic infusions and solutions: and (2) to evaluate the effect of the recently identified components from Bemauda hay infusion (Millar et al., 1992) on their oviposition.

OVIPOSITION MEDIATORS IN Aedes

1849

METHODS AND MATERIALS

Aedes albopictus and Ae. aegypti were reared and maintained at 27 _+ 2°C, 70-80% relative humidity, and a 16L: 8D-hr photoperiod. Larvae were reared by conventional methods using standardized density and nutrition (1000 immatures/3 liters of water and fed on a 1 : 1 mixture of liver powder and brewer's yeast). Female mosquitoes were blood-fed four to five days after emergence on defibrinated beef blood (Ae. aegypti) or chicks (Ae. albopictus). Blood-fed mosquitoes were held four to five days at 30 + 1°C under fluorescent lighting with a 14L: 10D photoperiod including 1 hr of simulated dawn and dusk period (provided by a 15-W incandescent bulb). A 10% sucrose solution was provided continuously to mosquitoes prior to bioassays. Laboratory Bioassays. Bioassay cages were cylindrical cardboard containers (25.4 cm high x 24.7 cm diana.) with clear plastic lids. A 2-cm-diam. plugged access hole in the side of the container allowed introduction of mosquitoes into the cage. Disposable 70-ml plastic cups were painted black and filled with either 60 ml of well water (nonchlorinated water used to rear rnosquitoes as controls) or the treatment preparations. One control and one treatment cup were placed in each bioassay cage ca. 6 cm apart, and a strip of seed germination paper (2 x 5 cm) (Steinley et al., 1991) was placed against the inside of each cup. A single gravid female was added to each cage at least 1 hr prior to the scotophase and mosquitoes were removed after 22 hr. Bioassays were conducted using individual females to reduce the potential influence of previously laid eggs on oviposition site preference (Chadee et al., 1990). Assays were run overnight in an environmental chamber at 30 + I°C with 14L: 10D photoperiod with 1 hr of simulated dawn and dusk. They were scored by counting the numbers of eggs on the germination paper from the control and treatment cups. Organic infusions used for bioassays included hay infusion (prepared after Reiter, 1986), larval rearing water, and water collected from a field site known to contain populations of Ae. albopictus. Preparation of the hay infusion involved adding 450 g Bermuda grass hay, 5 g brewer's yeast, and 20 g lactalbumin hydrolysate to 75 liters of well water, held at ambient temperatures (25-30°C) for 12 days. Two preparations of infusion were made and each was frozen in ca. 2-liter aliquots until needed. Hay infusion was diluted with well water as needed to produce dilutions of 10%, 25%, 50%, and 100% for testing in bioassays. Larval rearing water that had contained immature mosquitoes was obtained from the mosquito colony and stored at t0°C for a maximum of two days prior to use. Field-collected water originated from a natural oviposition site (an open container with oak leaves) that contained a continuous population of immature Ae. albopictus. Water was collected directly from the source prior to bioassays,

1850

ALLAN AND KLINE

Field water was diluted with well water to produce dilutions of 5%, 25%, 75%, and 100% for testing in bioassays. Synthetic compounds previously identified as components of hay infusion (3-methylindole, indote, phenol, 4-methylphenol, 4-ethylphenol) (Sigma Chemical Co.) (Millar et al., 1992) were prepared in hexane and added to well water in t0 p.l of solvent. Concentrations of test compounds ranged from 0.01 to 100 gg/liter, and reflected concentrations typical of hay infusions (Millar et al.. 1992). Control cups were treated with I0 /A hexane. All treatments consisted of 20-30 replicates. Oviposition activity was expressed using the oviposition activity index (OAI) of Kramer and Mulla (1979), which is calculated as follows: O A I = (N, -

N,.)I(N, + N,.)

where N, = number of eggs deposited in treatment cups and N, = the total number of eggs deposited in control cups. Responses using this system range from 1 (attraction) to - 1 (repellency) with zero denoting no preference. In this context, attraction refers to the deposition of eggs as the end result of a complex sequence of behaviors including orientation towards the source and stimulation to oviposit. These behaviors were not differentiated under the current bioassay conditions and only the endpoint of the pre- and oviposition behaviors (i.e.. oviposition) was used to determine attraction or repellency. Treatment means were subjected to ANOVA to determine if significant differences were present (P < 0,05). Mean numbers of eggs laid under each treatment were compared by chi-square analysis to the number of eggs. Statistical comparisons were made by chi-square analysis between the mean numbers of eggs deposited in Ireatment cups (that were paired with well water control cups) compared to the expected number of eggs it" oviposition was random between the treatment and control. F i e l d B i o a s s a y s . Bioassays were conducted in a screened field cage (ca. 5 × 5 x 5 m). Large potted plants were placed along the perimeter and in the center of the cage to provide resting sites li)r mosquitoes. Black glass oviposition jars (500 ml), each with 400 ml of water or solution and a strip of seed germination paper (4.5 × 9.6 cm) fastened with a paper clip to the inside upper edge of the jar, were used to evaluate oviposition responses. Jars were spaced evenly around the interior of the cage about 0.5 m from the cage wall. Each day, one jar containing each treatment was randomly assigned to one of five positions (experiment 1) or four positions (experiment 2) in the cage and treatments rotated in position from one day to the next to avoid positional bias. Each day, 50 gravid females were released from a central point in the cage in midafternoon. Treatments placed in the cage in mid-afternoon and oviposition strips with eggs were collected 24 hr later. Temperature and climatic conditions were

OVIPOSITION MEDIATORS IN Aedes

1851

noted for each day. The numbers of eggs present on seed germination paper from each day were counted to determine oviposition responses. Two experiments were conducted for each species. For experiment 1, treatments consisted of well water (nonchlorinated), a 25% solution of hay infusion, 3-methylindole (100 ~g/liter), larval rearing water, and field water. Immediately before the test, hay infusion and 3-methylindole solutions were prepared in well water and field water and larval rearing water were obtained. Aliquots of thawed hay infusion and rearing water were kept at 10°C for up to two days. Treatments were repeated for 15 days (replicates). For experiment 2, treatments consisted of 25% hay infusion, 3-methylindole (100/~g/liter), well water, and a mixture of synthetic compounds combined in concentrations similar to those found in hay infusion as described by Millar et al. (1992). This mixture consisted of phenol (0.27 mg/liter), 4-methylphenol (1.99 mg/liter), 4-ethylphenol (0.07 mg/liter), indole (0.01 mg/liter), and 3methylindole (0.25 rig/liter). Synthetic compounds and mixtures were added to jars 10-15 rain prior to placement in the field cage. Treatments were repeated for 10 days (replicates). For each day, the percentage of eggs deposited in response to each treatment was determined. Means for each treatment were transformed to arcsine values and subjected to ANOVA (P < 0.05) to determine if significant differences were present in each experiment. Differences between means were determined using Tukey's studentized t test (P < 0.05). Data are presented as untransformed means. Olfiwtometer Bioassays. To determine if organic infusions elicited an upwind orientation response, gravid Ae. albopictus and Ae. aegypti were tested in a dual-port olfactometer similar to that described by Schreck et al. (1967), but consisting of three stacked chambers (35 cm high x 90 cm long x 48 cm wide). Only one chamber at a time was used for assays. Outside air was conditioned prior to entry through the choice ports, the mosquito trap, and the olfactometer by passing through a series of charcoal filters and then heated and humidified. Conditions in the olfactometers were 27°C, 80% relative humidity with an air flow of 1 liter/sec. One hour before initiation of tests (ca. 14 : 00 hr EST), females were aspirated into the olfactometer chamber and allowed to acclimate. For each of the 10 replicates, 50 gravid females were used. Treatments were placed into two test ports upwind of the traps and olfactometer chamber. For each olfactometer test, one port contained well water and the other contained a test solution. Treatments (field water, larval water, and 50% hay infusion) were presented in glass Petri dishes that were cleaned and handled with gloves to minimize contamination and were alternated between ports from one day to the next. After 24 hr, the relative numbers caught in the treatment and control traps upwind of each port were compared. Treatments were added to each port and tests initiated at 15:00 hr EST.

1852

ALLAN AND KLINE

Means were transformed to arcsine values and differences between means of treatments and controls determined by ANOVA (P < 0.05). Data are presented as untransformed means.

RESULTS

Oviposition responses of Ae. albopictus females differed significantly across the range of concentrations of hay infusion (ANOVA: F = 6.58; df = 3,76: P < 0.001) and field water tested (ANOVA; F = 3.94; df = 3,76; P < 0.01) (Figure 1). Only the 25% dilution of hay infusion elicited significantly more oviposition than expected (if oviposition was random between the paired treatments and well water controls) (chi square, P < 0.001). In response to 10% and 100% hay infusion, significantly lower levels of oviposition occurred (chi square, P < 0.001). Oviposition responses were strongly in response to solutions of 25% field water and 75% field water (chi square, P < 0.001). Oviposition responses to 100% field water were significantly lower than expected (chi square, P < 0.001). Oviposition responses of Ae. aeg3pti did not differ across the same range of concentrations of hay infusion (ANOVA; F = 0.94; df = 3.76; P = 0.42) or field water (ANOVA, F = 0.54; df = 3,76, P = 0.65) (Figure 1). In general, OAI values for hay infusion were negative with significantly lower oviposition than expected in response to a 25% solution of hay infusion (chi square, P < 0.001). OAI values were moderately positive in response to field water with significantly greater oviposition than expected with solutions of 75% and 100% field water (chi square, P < 0.001). Responses of gravid female Ae. albopictus to the synthetic compounds tested were moderate. Oviposition responses differed significantly across the range of concentrations of 3-methylindole (ANOVA: F = 2.48; df = 4,145; P < 0.05) and phenol (ANOVA: F = 2.45; df = 4,95; P < 0.05) but not indole (ANOVA; F = 0.83; df = 4,95; P = 0.50), 4-ethylphenol (ANOVA, F = 0.55, df = 4,145; P = 0.69) and 4-methylphenol (ANOVA; F = 0.64; df = 4, 95; P = 0.63) (Figure 2). Of the treatments with positive OAf values, only one concentration of 4-methylphenol (0.01 #g/liter) (chi square, P < 0.001) and 3-methylindole (0.1 #g/liter) (chi-square, P < 0.05) elicited significantly more oviposition than expected. Significantly fewer eggs were deposited in treatment cups than expected for 10 ~g/liter (chi square, P < 0.05) and 100 /zg/liter (chi square, P < 0.001) of 3-methylindole, 0.01 ~g/liter (chi square, P < 0.001) and 100 p.g/liter (chi square, P < 0.00t) of phenol, 0.01 /zg/liter (chi square, P < 0.001) of 4-ethylphenol and 100 p.g/liter (chi square, P < 0.001) of indole. Oviposition responses to synthetic compounds by Ae. aegypti females were

Aedes

OVIPOS1T[ON MEDIATORS IN

~d

e~

1853

1.0

H a y infusion

z

~.

o.5

Z

~.

0----

Ae. a l b o p ~ t u s ~ ~

--

Ae. aegypti

-1.0 !

0

20

40

60

80

100

f~

z

Field w a t e r 0.5

0

0"----

Ae. albopictus

-

Ae. aegypti

i 20

i 40

i 60

i 80

i 100

CONCENTRATION (%)

FIG. 1. Oviposition responses of gravid Ae. albopictus and Ae. aeg3pti to organic infusions in a laboratory bioassay. Data are presented as means (+ SE) of oviposition activity indices (OAI) f¥om 20 replicates. Positive OAI values indicated attractiveness and negative values repellency. *Significantly different at P < 0.05: **Significantly different at P < 0.001. also moderate and only increased significantly across the range of concentrations of 4-ethylphenol ( A N O V A ; F = 3.82; df = 4,95; P < 0.01) and phenol ( A N O V A ; F = 6.68; df = 4,95; P < 0.001) but not 3-methylindole ( A N O V A , F = 0.943; df = 4,95; P = 0.44), indole ( A N O V A , F = 1.19, df = 2,57; P = 0.31), or 4-methylphenot ( A N O V A ; F = 0.59; d f = 4,95; P = 0 . 2 7 ) ( F i g u r e 2). O f the treatments with positive OAI values, only 1.0 p.g/liter of phenol and 0.1 #g/liter o f 4-ethylphenol were significantly attractive (chi square, P < 0.001). Significantly lower oviposition than expected was noted for 3-methylindote (0.1 #g/liter) (chi square, P < 0.001), phenol (0.01 ~g/liter, 100 #g/liter)

t854

AI+I.ANAND KL! Aedesalbop~tus

Aedes aegypti

3-methylindole ~5

&

i

0"0I -LO

J

",

l

~henol

0.5 0+0 -0.5

-1.0 I+01 0.++

4"ethylphen°i I

< Z

~

f~

4-ethylphenolI

r~ 0+5" 0.0" -0.~" -1,0 1.0

-LO! .01

4-methylphenol

.1 I 10 lOG CONCENTRATION(ug/l)

4-methylphenol

.01

,1J

Ir

•

10 I0@ CONCENTRATION(ug/l)

OVIPOSITION MEDIATORS IN Aedes

1855

TABLE 1. OVIPOSITIONAL RESPONSES IN FIELD CAGES OF GRAVID Ae. albopictus AND Ae. aegypti TO OVITRAPS BAITED WITH ORGANIC INFUSIONS, 3-METHYLINDOLE, OR A MIXTURE OF SYNTHETIC COMPOUNDS REPRESENTING MAJOR COMPONENTS OE HAY INFUSION

Aedes aibopictus Treatment Experiment I Well water Hay infusion 3-Methylindote (100 rag/liter) Larval water Field water Experiment 2 Well water 3-Methylindole(100mgtliter) Mixture Hay infusion

% of eggs" 3.5 20.4 14.9 34.9 31.4

+ + ± ± ±

1,2 a 4~6 bc 3.5 bc 6.6 b 5.7 bc

18.2 24. t 21.1 36.1

_+ 2.8 a ± 3.2 a + 2.4a + 3.9 b

Aedes aegypti N

% of eggs

N

10 10 10 I0 10

16.0 15.9 13.5 28.9 25.5

+ + + ± +

4.2 2.4 3.6 3.3 4.6

ab ab b a ab

10 10 10 10 10

15 15 15 15

16.2 21,1 29.1 30.8

+ ± ± ±

0.8 a 2.1 ab 4.0b 4,1 b

I0 10 10 10

"Mean +_ SE of 10-15 replicates of 50 mosquitoes. Means in the same column and experiment lbllowed different letter are significantly different ITukey's studentized t test, P < 0.05).

(chi s q u a r e , P < 0.001 ), 4 - e t h y l p h e n o l (0.01 # g / l i t e r , 100 #g/liter) (chi s q u a r e , P < 0 . 0 0 1 ) , and 4 - m e t h y l p h e n o l (0.01 # g / l i t e r , 1.0 /xg/liter) (chi s q u a r e , P < 0.0011. In field c a g e s , o v i p o s i t i o n a l r e s p o n s e s by gravid Ae. albopictus differed significantly b e t w e e n well w a t e r , hay i n f u s i o n , 3 - m e t h y l i n d o l e (100 # g / l i t e r ) , larval water, and field w a t e r ( A N O V A ; F = 9 . 2 3 : d f = 4 , 4 5 , P < 0 . 0 0 1 ) (Table I). In c o m p a r i s o n to well water, significantly m o r e e g g s w e r e d e p o s i t e d in r e s p o n s e to hay i n f u s i o n , 3 - m e t h y l i n d o [ e , larval water, and field water. Ovipositional r e s p o n s e s to larval w a t e r w e r e g r e a t e r than to 3 - m e t h y l i n d o l e but s i m i l a r to hay i n f u s i o n and field water. O v i p o s i t i o n a l r e s p o n s e s by gravid Ae. aegypti were also significantly different b e t w e e n t r e a t m e n t s in e x p e r i m e n t 1 ( A N O V A ; F = 3.35; d f = 4 , 4 5 ; P < 0.011 (Table 11. O v i p o s i t i o n a l r e s p o n s e s to o v i t r a p s with larval w a t e r w e r e significantly g r e a t e r than t h o s e with 3 - m e t h -

FIG. 2. Oviposition responses of gravid Ae. albopictus and Ae. aeg3pti to concentrations of synthetic chemical cornpounds. Data are presented as means ( ± S E ) of oviposition activity indices (OAf) from 20 to 30 replicates. Positive OAI values indicated attractiveness and negative values repellency, *Significantly different at P < 0.05; **Significantly different at P < 0.001.

1856

ALLAN AND KLINE

TABLE 2. RESPONSES OF GRAVID Ae. albopicms AND Ae. aegypti FEMALES TO FIELD WATER, LARVAL WATER AND HA'r" INFUSION (50%) COMPARED TO WELL WATER IN DUAL-PORT OLFACTOMETER

% responding"

Ae. albopiclus

Ae. ae~ypti

Field water Well water

83.9 + 6.3** 17.0 + 6.2

63.8 + 8.7* 35.9 _+ 8.6

Laval water Well water

74.5 + 6.6** 18.2 + 5.1

54.5 + 5.9 40.2 _+ 6.4

Hay infusion Well water

69,4 +_ 8.7* 30,5 _+ 9.2

59.9 + 5.5 40.1 +_ 5.5

"Mean + SE of 10 replicates of 50 mosquitoes. Paired comparisons of means of well water and paired treatment significantly different by ANOVA and F statistics, *P < 0.05, **P < 0.001.

ylindole, No differences were present in ovipositional response between well water, hay infusion, larval water, and field water, or between well water, hay infusion, 3-methylindole, and field water. Oviposition responses o f Ae. albopictus in experirnent 2 differed significantly between treatments ( A N O V A ; F = 5.04, df = 3. 56; P < 0,01) (Table 1). Ovipositional responses to well water, 3-methylindole, and the mixture of c o m p o u n d s were similar and significantly lower than to hay infusion. Ovipositional responses of Ae, aegypti also differed significantly between treatments ( A N O V A , F = 4.79, df = 3, 36: P < 0.05). Ovipositional responses to well water and 3-methylindole were similar. Responses to hay infusion and the mixture of synthetic c o m p o u n d s were significantly greater than to well water, but not significantly different from 3-methylindole. Significantly more gravid Ae. albopictus and Ae. aegypti were collected in the olfactometer port containing field water than in the one containing well water (Table 2). The preference for field water o v e r well water appeared stronger for Ae, albopictus (4.9-fold increase) than for Ae. aeg3pti ( l . 8 - t b t d increase). Significantly more gravid Ae. albopictus were collected in the ports with larval water and hay infusion than in the ports with well water. Gravid Ae. aeg3pti, however, were collected equally in olfactometer ports with well water and either of these solutions.

DISCUSSION Organic infusions such as hay infusion, larval rearing water, and field water clearly elicited strong oviposition responses from gravid Ae, albopictus, whereas

O V I P O S I T I O N M E D I A FORS IN

Aedes

1857

oviposition by Ae. aeg3pti was moderate to larval rearing water and field water and low in response to hay infusion even over a wide range of concentrations. These results are in agreement with previous reports that gravid Ae. albopictus oviposit readily in grass or hay infusions (Gubler, 1971; Hein, 1976: Holck et al., 1988) and in water containing immatures (Gubler, 1971). Our data support previous reports of the low response of gravid Ae. aeg3pti to grass or hay infusions (Hazard et al., 1967: Holck et al., 1988: Chadee, 1993), and the lack of orientation of gravid Ae. aeg~pti to hay infusion in an olfactometer is consistent with previous reports (Hazard et al., 1967). In contrast, Reiter et al. (1991) reported enhanced oviposition by Ae. aegypti in ovitraps with hay infusion. Responses of both species to organic infusions were lower than those reported for C~-. quinqu~:[~sciatus (Millar et al. 1992), and this may reflect that these Aedes species characteristically deposit eggs from a single gonotrophic cycle in several sites and that both species readily oviposit in clean water (Gub[er, 1971; Holck et al., 1988). Organic infusions such as those tested in our study consist of complex mixtures of compounds that vary over time. At least some of the oviposition attractants from such organic infusions are products of bacterial degradation (Hazard et al., 1967: Ikeshoji et al., 1979: Benzon and Apperson, t988). Larval water and field water, both of which contained mosquito larvae, elicited more oviposition by both Aedes species than hay infusion, and the production of more volatiles by these infusions related to attraction of females and/or stimulation of oviposition could possibly be related to the presence of larvae. Compounds isolated and identified from Bermuda grass infusions known to elicit oviposition from Cx. quinque['asciatus (Millar et al., 1992) clearly play a lesser role in mediation of oviposition for both Ae. albopictus and Ae. aeg3pti. In C~. quinquefasciatus, a species generally associated with polluted water, oviposition responses to 3-methylindole were strong across the entire range of concentrations tested (0,01-100 p~g/liter), with up to 3.5 x more egg rafts deposited in treatment cups compared to control cups (Millar et al., 1992). In our study, oviposition responses of female Ae. albopictus were significantly greater than to well water at only one concentration of 3-methylindole (0,1 p,g/liter) and one of 4-methylphenol (0.01 #g/liter). Gravid Ae. aegypti did not appear to differentiate between well water and 3-methylindole at any dose for selection of oviposition sites. Millar et al. (t992) also reported moderate oviposition responses by gravid Cr. quinquefasciatus to one concentration of 4-ethylphenol (10 #g/liter) and two concentrations of indole (10 and 100 ~g/liter). However, the latter responses were possibly due to trace amounts of 3-methylindole present. In contrast, 3-methylindole did not increase oviposition by either Ae, albopictus or Ae. aegypti. Ae. aegypti also exhibited a moderate increase in oviposition to 4-ethylphenol and phenol, but only at one of the concentrations tested. Exposure of gravid females to several concentrations of the synthetic compounds and organic infusions clearly resulted in lower oviposition responses than to well

1858

ALLAN AND KLINE

water controls. These decreased responses may reflect repellency at these concentrations. The presence of the compounds identified by Millar et al. (1992) in concentrations found in this study to elicit low oviposition responses may contribute to the apparent repellent/deterrent effects to these species. Results from the field cage and olfactometer studies clearly indicate that larval water and field water contain attractants and/or stimulants for gravid females o f both Aedes species. More studies are needed to isolate and identify compounds from these sources and to evaluate the behavioral roles of these compounds. Compounds of infusions that attract gravid Ae. albopictus and Ae. aegypti females to potential oviposition sites have considerable potential for both increasing the sensitivity for monitoring populations o f these species and for the potential deliver3' of pathogens or pesticides into larval populations (Schlein and Pener, 1990; Itoh et al., 1994). Surveillance of Ae. albopictus and Ae. aeg3pti is difficult, as adults are not readily attracted to light traps (Service, 1977; Hawley, 1988) and larval sampling is time-consuming. Ovitraps are often used for population monitoring and surveillance (Chadee et al., 1988: McHugh and Hanny, 1990), and the use o f oviposition attractants/stimulants to increase the sensitivity of these traps as well as gravid female traps (Reiter, 1983; Freier and Francy, 1991) has considerable potential. O f particular importance for this application are infusions or compounds that both elicit long-range orientation of gravid females to potential oviposition sites and stimulate oviposition once at the oviposition site. The use of infusions such as hay infusions in conjunction with ovitraps or gravid females traps may, however, bias trap collections towards Ae. albopictus, as our results indicated a greater attraction of Ae. albopictus than Ae. aegypti to hay infusion. This is currently being examined under field conditions. Acknowledgments--The authors thank Edward Lavagnino, Gisette Seferina. Mike Miller, and Hank McKeithen lor technical assistance. This research was supported in part by the State of Florida, Department of Agriculture and Consumer Services. Bureau of Entomology and Pest Control, Department of Regulation, Solid Waste Management and Trust Fund, and by USDA.

REFERENCES BEEHLER, J.W., MILLAR,J.G., and MULLA,M.S. 1994. Field evaluation of synthetic compounds

mediating oviposition in Culex mosquitoes (Diptera: Culicidae). J. Chem. Ecol. 20:291-299. BENTLEY, M.D., and DAY, J.F. 1989. Chemical ecology and behavioral aspects of mosquito ovi-

position. Annu, Rev. Entomol~ 34:40t-421. BENZON, G.L,, and C.S. APPERSON. 1988. Reexamination of chemically mediated oviposition behavior in Aedes aegypti (L.) (Diptera: Culicidae). J. Med. Entomol. 25: 158-164. CHADEE, D.D. 1993. Laboratory observations on the oviposition response of Aedes aegypti mosquitoes to different concentrations of hay-infusion in Trinidad. J. t71. Mosq. Control Assoc. 64:22-23,

OVIPOSITION MEDIATORSIN Aedes

1859

CHADEE, D.D., CONNELL, N.K., LEMAITRE, A., and FERREiRA, S.B. 1988. Surveillance forAedes aegypfi in Tobago, West Indies (1980-82). Mosq. News 44:490-492, CHADEE D.D., CORBET, P.S., and GREENWOOD,J.J.D. 1990. Egg-laying yellow fever mosquitoes avoid sites containing eggs laid by themselves or by conspecifics. Entomol. Exp. Appl. 57:295298. FREIER, J,E,, and FRANC¥, D.B. 1991. A duplex cone trap for the collection of adult Aedes atbopictus. J. Ant. Mosq. Control Assoc. 7:73-79. GJULLIN. C.M., JOHNSON,J.O., and PLAPP, F.W.. Jr, 1965. The effect of odors released by various waters on the oviposition sites selected by Culex. Mosq. News 25:268-271. GUBLER, D.J, 1971. Studies on the comparative oviposition behavior of Aedes (Stegomyia) albopictus Skuse and Aedes (Stegomyia) polynesiensis Marks, J. Meal Entmnol. 6: 675-682, HAWLEY, W.A. 1988, The biology of Aeries albopietus. J. Am. Mosq, C~mtrol Assoc. 4:1-40. HAZARD, E.I., MAYER, M.S., and SAVAOE, KE. 1967. Attraction and oviposition stimulation of gravid female mosquitoes by bacteria from hay infusion. Mosq. News 27: 133-136, HEIN, D.S. 1976. Biology ofAedes aegypti (L.. 17621 and Aedes albopictus (Skuse 18657 (Diptera, Culicidae). V. The gonotrophic cycle and oviposition. Acta Parasiml. Pol. 24:37-55. HOLC~., A.R., MI:~EK,C.L,, and MEL:K,J.C, 1988. Attraclant enhanced ovitraps for the surveillance of container breeding mosquitoes. J. Am. Mosq, Control Assoc. 4:97-98. IKESHO.II.T., ICHIMO'ro,I.. KONISHI,J.. NAOSHIMA,Y,. and UEDa, H, 1979. 7, I I-Dimethyl octadecane-an oviposition attractant for Aedes aegypti produced by Pseudomonas aertlgenosa on capric acid substrate. J, Pestic. SoL 187:t94, [TC)H, T,, KAWADA,H., ABE, A., ESHITA. Y., RONGSRYAM.Y., and IGARASHI,A. 1994. Utilization of bloodfed females of Aedes aegypti as a vehicle for transfer of the insect growth regulator pyriproxyfen to larval habitats. J. Am. Mosq. Control Asst)c. 10:344-347. KrrR()N, U.D.. WEBB. D.W., and NOVAK, R.J. 1989. Oviposition behavior of Aedes triseriatus (Diptera: Culicidae): Prevalence, intensity and aggregation of eggs in oviposition traps. J. Med. Enmmol. 26:462-467. KRAMER, W.L., and Mt)LLA, M.S. I979. Oviposition attractants and repellents of mosquitoes: oviposition responses of Cuh,x mosquitoes to organic inlusions. Em'ir~m. Entomol. 8: I 1I 11117. L.aURI~NCE, B.R., and PICKETT, J.A. 1985. An oviposition attractant pheromone in Culex quinquefilsciatus Say (Diptera: Cuiicidae) Bull. Emmn~)l. Res, 75:283-290, MADDER, D.J., MACDON?,LD, R.S., SURG't~ONER,G,A., and HELSON, B.V, 1980. The use of oviposition activity to monitor populations of Cldex pipiens and Cn/e,r rt'stuans (Diptera: Culicidae). Can. Entmnol. 112:1013-1017. McHtlc;H C.P.. and P.A. HANNV. 1990. Records ofAedes albopictus, Ae. aegypti and ,4e. triseriatus from the U.S, Air Force ovitrapping program 1989. J. Am. Mosq. Ctmtrol A,~soc. 6:549-551, MILLAR, J,G., CHANEY, J.D,, and MULLA, M.S. 1992. Identification of oviposition attractants for Cnh~ quinqueftlsciatus from Jem~ented Bermuda grass infusions, J. Am, Mosq. Control Assoc. 8:11-17. REISEN, W.K., and M~-~VER,R.P. 1990. Attractiveness of selected oviposition substrates lot gravid Culler tarsulis and Culex qttinquefttsciatus in Calilbrnia. J. Am, Mosq. Control Assoc. 6:244250, REITI~a, P. 1983. A portable battery-powered trap for collecting gravid Cule.r mosquitoes. Mosq. News 43:496-498. REITER. P. 1986. A standardized procedure for the quantitative surveillance of certain Cult:r mosquitoes by egg raft collection. J, Am. Mosq. Control Assoc. 2:219-221. REITER, P., AMADOR, M.A., and COLON, N, 1991. Enhancement of the CDC ovitrap with hay infusion tot daily monitoring of Aedes ueg3pti populations. J. Ant. Mosq. Control Assoc. 7:5255.

1860

ALLAN AND KLINE

RITCmE, S,A. 1984. Hay infusion and isopropyl alcohol-baited CDC light trap; a simple effective trap lot gravid Culex mosquitoes. Mosq. News 44:404-407. SCHLEm, Y., and PENER, H, 1990. Bait-fed Culex pipiens carry the larvicide Bacillus sphaericus to larval habitat. Med, Vet. Entomol. 4:283-288. SCHRECK, C.E., GOUCK, H,K., and SMITH, N, 1967. An improved olfactometer for use in studying mosquito attractants and repellents. J. Econ. EnmmoL 60:1188-1190. SERVICe, M.W. 1977. A critical review of procedures for sampling populations of adult mosquitoes. Bull. Entomol. Res. 67:343-382. STARRA3-r, A,N., and OSGOOD, C.E. 1973. 1,3-diglycerides from egg'~ of Culex pipiens quinquefasciutus and C~dex pipiens pipiens. Comp. Biochem, Physiol, 46B:857-859. STEINLEY, B.A., NOVAK, R.J.. and WEBB, D,W. 1991. A new method for monitoring mosquito oviposition in artificial and natural containers. J. Am. Mosq. Control Assoc. 7:649-650,