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Response of a parasitoid fly, Gymnosoma rotundatum (Linnaeus) (Diptera: Tachinidae) to the aggregation pheromone of Plautia stali Scott (Hemiptera: Pentatomidae) and its parasitism of hosts under field conditions
Biological Control 58 (2011) 215–221
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Response of a parasitoid fly, Gymnosoma rotundatum (Linnaeus) (Diptera: Tachinidae) to the aggregation pheromone of Plautia stali Scott (Hemiptera: Pentatomidae) and its parasitism of hosts under field conditions Morio Higaki ⇑, Ishizue Adachi Apple Research Station, National Institute of Fruit Tree Science, 92-24 Nabeyashiki, Shimokuriyagawa, Morioka, Iwate 020-0123, Japan
a r t i c l e
i n f o
Article history: Received 29 July 2010 Accepted 17 May 2011 Available online 23 May 2011 Keywords: Tachinidae Pentatomidae Parasitoid Pheromone Kairomone Life cycle
a b s t r a c t We studied the attraction of a tachinid fly, Gymnosoma rotundatum (Linnaeus) to the male-produced aggregation pheromone of the brown-winged green bug, Plautia stali Scott, its parasitism on the bug, and its seasonal occurrence in the field. The tachinid fly was continuously attracted to the aggregation pheromone from spring to autumn and utilized the bugs as hosts. Our field experiment to clarify the effect of the pheromone on parasitism demonstrated that parasitism occurred only in female bugs baited with synthetic aggregation pheromone and did not occur in females without the pheromone. The parasitoid flies therefore appeared to use the bug’s pheromone as a host-finding kairomone. The pheromone attracted not only female flies but also males. Male flies may increase their chance of encountering pheromone-attracted females by waiting near pheromone sources. The tachinid develops multiple generations in active hosts from spring to autumn and overwinters in dormant hosts. Thus, G. rotundatum seems to be highly adapted to using P. stali as its host, and it is a potentially important biological control agent of P. stali populations in the field. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Approximately 10,000 species of tachinid flies have been recorded worldwide, and all tachinid species are endoparasitoids of arthropods (Stireman et al., 2006). The larvae develop in the body cavities of live host insects, causing harmful effects on the hosts such as the inhibition of development and the atrophy of reproductive organs (Clausen, 1940). Tachinids parasitize insects belonging to all of the main orders, including orders that contain major agricultural pests, and are considered to be potentially important biological control agents (Belshaw, 1994; Greathead, 1986; Grenier, 1988). The mechanisms by which most tachinids locate and select hosts are poorly understood. The information available on the few tachinid species that have been studied indicates that tachinids are capable of using a wide diversity of olfactory, visual, auditory, and tactile-chemosensory cues to locate their hosts (Stireman et al., 2006). In a host-restricted tachinid subfamily, the Phasiinae, flies exclusively parasitize Heteroptera: the adults are especially drawn to attractant pheromones produced by the heteropterans and appear to use such pheromones to find these potential hosts
⇑ Corresponding author. Fax: +81 19 641 3819. E-mail address: [email protected] (M. Higaki). 1049-9644/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2011.05.009
(Aldrich et al., 1991; Harris and Todd, 1980; Mitchell and Mau, 1971). The phasiine tachinid Gymnosoma rotundatum (Linnaeus) parasitizes live adults of the brown-winged green bug, Plautia stali Scott (formerly Plautia crossota stali), a fruit-piercing bug that is a serious pest in Japan and Korea (Higaki, 2003; Jang and Park, 2010; Mishiro and Ohira, 2002; Moriya et al., 1993; Yamada and Miyahara, 1979). Because P. stali is polyphagous and the adults are highly mobile, adults invade fruit orchards from woodland or uncultivated areas, requiring frequent insecticide applications to prevent infestations (Adachi et al., 2007). G. rotundatum is a potentially important biological control agent because its parasitism causes considerable reductions in the longevity and reproductive ability of P. stali (Higaki, 2003; Yamada and Miyahara, 1979). Adult males of P. stali are known to emit an aggregation pheromone that attracts both males and females of the same species (Adachi et al., 2007; Moriya and Shiga, 1984). G. rotundatum probably uses the male-produced aggregation pheromone of P. stali as a host-finding kairomone, because large numbers of this tachinid fly have been captured in traps baited with adult males of P. stali or a synthetic version of the aggregation pheromone (Jang and Park, 2010; Mishiro and Ohira, 2002; Moriya et al., 1993). However, the field responses of G. rotundatum to P. stali aggregation pheromones have not been well studied. In addition, researchers have a poor understanding of the ecology of the fly, including aspects such as its seasonal life cycle. In the present study, we investigated
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the response of G. rotundatum to P. stali aggregation pheromone, its parasitism of the host bugs, and its seasonal occurrence in the field.
2. Materials and methods 2.1. Seasonal abundance of parasitoid flies and host bugs caught by pheromone traps The male-produced aggregation pheromone of P. stali was identified as methyl (E,E,Z)-2,4,6-decatrienoate (Sugie et al., 1996), and synthetic pheromone synthesized by Shin-Etsu Chemical Co. (Tokyo, Japan) was used in our study. To track the seasonal occurrence of G. rotundatum and P. stali, we used a 3.0-L basin trap (yellow type of Kogane-call, Sankei Chemical Co., Kagoshima, Japan) baited with synthetic aggregation pheromone. A small amount of detergent was added to the water in the basin. The pheromone was supplied in lures, each of which contained 42 mg of pheromone, and the lures were replaced every 20 days. From April to November in 2000 to 2005, the traps were established in open areas of the National Institute of Fruit Tree Science (NIFTS), Tsukuba, Ibaraki Prefecture, Japan (36°030 N, 140°080 E, 20 m a.s.l.), where several kinds of deciduous trees are planted. Traps were suspended at a height of 160 cm, and the distance between traps was more than 100 m. We established two traps in 2000, five each year from 2001 to 2003, four in 2004, and three in 2005. We used five lures per trap in 2001 and two lures per trap in the other years of this study. Traps were checked daily for the number of G. rotundatum and P. stali caught. 2.2. Diel rhythm in parasitoid flies and host bugs caught by the pheromone traps We established two basin traps baited with a lure containing the synthetic aggregation pheromone (85 mg per lure) at NIFTS from May to November 1999. The traps were located more than 300 m apart. We replaced the lures every 20 days. Traps were checked for catches of P. stali and G. rotundatum five times per day (09:00, 12:00, 15:00, 18:00, and 21:00). The experiment was carried out on 10–17 days per month. 2.3. Effect of host pheromone on tachinid parasitism To reveal the effect of the aggregation pheromone on parasitism, we compared the parasitism by G. rotundatum between two groups of P. stali adults: one group was baited with synthetic aggregation pheromone and the other was not. To strictly control for the effect of the pheromone, we used only female bugs, which do not release the aggregation pheromone. P. stali adults reared in the laboratory according to the method described by Moriya et al. (1985) were provided as potential hosts. In each group, 10 live female bugs were attached at their abdomen to the tips of 10 cm tall poles using an adhesive (G17, Konishi Co., Osaka, Japan), and these 10 bugs were fixed on a frame at 7 cm intervals (Fig. 1). The two groups of potential hosts were placed at 160 cm above ground under the trees at NIFTS, separated by about 30 m. The potential hosts were exposed for 24 h (from 12:00 to 12:00 on the next day). We used three pheromone lures (each 42 mg) in the bated frame and replaced them after every trial. G. rotundatum lays its eggs primarily underneath the wings (abdominal tergum) of its hosts (Higaki, 2003), and we judged parasitism by checking for such eggs. When eggs are laid on other parts of the host body, parasitism never succeeds (Yamada and Miyahara, 1979), so we did not examine other body parts for the presence of eggs. We repeated the field experiments six times from June to July 1999.
Fig. 1. Illustration of the experimental device used to reveal the effect of synthetic Plautia stali aggregation pheromone on parasitism by Gymnosoma rotundatum.
2.4. Parasitism under field conditions To estimate the percentage of tachinid parasitism in the field, we examined P. stali adults that had been collected at NIFTS by beating the branches of the Japanese Paulownia (Paulownia tomentosa Steudel) or by using water-basin traps baited with 85 mg of synthetic aggregation pheromone or light traps (100 W mercury vapor lamps). Collection by beating branches was done two to six times per month from July to October 1999. Collection using basin and light traps was carried out continuously from April to November 1999. 2.5. Overwintering of tachinid flies under semi-outdoor conditions Parasitized bugs collected on P. tomentosa trees at NIFTS from September through October 1999 were reared in Petri dishes (9 cm in diameter). Raw peanuts were provided to the bugs as food, and water was supplied in water-soaked cotton wool. The Petri dishes were kept in a non-air-conditioned room that was open to the ambient atmosphere on two sides but was screened to exclude small animals such as mice. G. rotundatum individuals that emerged from the hosts and that pupariated were kept on layers of moist tissue paper in plastic cups (4 cm high by 5 cm in diameter) until they emerged as adults. Pupariation and adult emergence were checked daily. 2.6. Statistical analysis We compared the parasitism percentages between groups and the male-to-female ratio of the flies attracted to the pheromone using Fisher’s exact probability test. All statistical analyses were performed with Stat View-J version 5.0 (SAS Institute Inc., 1998). 3. Results 3.1. Seasonal abundance of parasitoid flies and host bugs caught by pheromone traps G. rotundatum and P. stali were continuously attracted to the aggregation pheromone of P. stali from April to October (or November), but many fewer flies than bugs were attracted (Fig. 2). We studied the populations over 6 years, and the trend in seasonal
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Fig. 2. Seasonal abundance of Gymnosoma rotundatum and Plautia stali captured by pheromone traps in Tsukuba, Japan from 2000 to 2005.
abundance changed considerably from year to year for both G. rotundatum and P. stali and differed between the parasitoid and the host bug. In P. stali, one or more large peaks were observed in the seasonal prevalence, whereas in G. rotundatum no clear peaks were observed and the number caught tended to remain high from spring to summer and then decreased until autumn. The P. stali aggregation pheromone attracted both female and male flies, although more males than females tended to be attracted. The male-to-female ratio was 128:90 (1.4) in 2000 (Fisher’s exact probability test, p = 0.083), 708:363 (2.0) in 2001 (p < 0.001), 144:137 (1.1) in 2002 (p = 0.800), 276:154 (1.8) in 2003 (p < 0.001), 193:162 (1.2) in 2004 (p = 0.260), and 90:41 (2.2) in 2005 (p = 0.003). 3.2. Diel rhythm in parasitoid flies and host bugs caught by the pheromone traps Responsiveness to the aggregation pheromone clearly differed among times of day in both P. stali and G. rotundatum (Table 1). The daily rhythm of responsiveness of P. stali changed from month to month. From June to September, the largest numbers of bugs were attracted during the night, whereas in May, October, and November more individuals were attracted during the day. In G. rotundatum, we did not detect such a seasonal change in the daily rhythm of responsiveness, and most individuals were attracted to the pheromone during the day. 3.3. Effect of host pheromone on tachinid parasitism Parasitism by G. rotundatum was only observed in the bugs that had been baited with the aggregation pheromone, although the
parasitism percentage varied across the six observation periods (Table 2). The parasitism percentage did not differ significantly between the two groups in any period except July 1–2, although it was significantly higher in bugs with the pheromone when the data for all six periods were pooled (p < 0.001).
3.4. Parasitism under field conditions G. rotundatum females most often laid one egg per host and rarely two or more eggs. The proportions of male and female hosts parasitized with one egg were 92.6% and 99.1%, respectively in the light traps, 100% and 96.5% in the pheromone traps, and 94.5% and 96.2% in the sample collected by branch beating. The parasitism percentage was significantly higher in bugs captured by the pheromone traps than in those captured by the light traps (p < 0.001; Table 3). We also observed a significantly higher parasitism percentage in males than in females in the pheromone traps (male: 73 of 1004, 7.3%; female: 105 of 1968, 5.3%; p = 0.041), but no significant differences between the sexes in the light traps (male: 68 of 2441, 2.8%; female: 117 of 4398, 2.7%; p = 0.756) or in branch-beating sample (male: 53 of 532, 10.0%; female: 57 of 585, 9.7%; p = 0.920). The parasitism results for the light and pheromone traps showed that G. rotundatum attacked and utilized P. stali as its host from spring to autumn, although the parasitism percentage was not high (Table 3). The parasitism percentage in the bugs captured by beating branches on the P. tomentosa trees changed seasonally and gradually rose in autumn (Fig. 3). A similar autumn increase was also observed in the pheromone traps.
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Table 1 Seasonal changes in the responsiveness of Gymnosoma rotundatum and Plautia stali to the aggregation pheromone of Plautia stali. Month
May June July August September October November
No. of observation days
10 13 17 14 16 15 17
Total number of individuals per month captured at each time of day G. rotundatum
P. stali
21:00– 09:00
09:00– 12:00
12:00– 15:00
15:00– 18:00
18:00– 21:00
21:00– 09:00
09:00– 12:00
12:00– 15:00
15:00– 18:00
18:00– 21:00
13 17 5 2 3 16 0
28 16 18 9 13 17 12
37 28 22 6 10 15 24
21 28 23 9 8 3 1
0 1 0 2 0 0 0
11 78 155 161 287 36 0
7 16 24 17 27 28 2
19 13 13 19 20 68 12
19 10 28 9 61 66 1
15 113 201 13 189 16 0
Two basin traps with a lure containing the synthetic aggregation pheromone were established at NIFTS from May to November 1999. Traps were located more than 300 m apart. Traps were checked five times per day (09:00, 12:00, 15:00, 18:00, 21:00) for the number of G. rotundatum and P. stali caught.
3.5. Overwintering of tachinid flies under semi-outdoor conditions In all parasitized P. stali adults collected before September 27, mature larvae of G. rotundatum exited from the hosts, pupariated soon afterward, and emerged as adults in October or November (Table 4). In most of the parasitized bugs collected after September 30, however, parasitoid larvae did not emerge but overwintered within their hosts. The bugs and their parasitoids were dormant and inactive during the winter. After overwintering, the parasitoid larvae left their hosts and pupariated in April, and adults emerged in May.
4. Discussion Numerous studies have found that many tachinid species belonging to the subfamily Phasiinae are attracted to the pheromone emitted by heteropterans (Aldrich and Zhang, 2002; Aldrich et al., 1987; Aldrich et al., 2006; Harris and Todd, 1980; Mishiro and Ohira, 2002; Mitchell and Mau, 1971; Moriya et al., 1993). However, few detailed studies have examined the ecology and
responsiveness of the flies, such as their seasonal prevalence and annual fluctuations in parasitism levels (e.g., Mishiro and Ohira, 2002). The present study showed that adults of G. rotundatum were attracted to the aggregation pheromone of P. stali in the field from spring to autumn (Fig. 2). Higaki (2003) conducted a series of laboratory experiments on G. rotundatum and reported that development proceeded without any delay under conditions of long-day photoperiod: the total larval and puparial duration was about 30 days and adult longevity was about 20 days at 21–22 °C, and adults could lay eggs on the day following emergence. These observations suggest that the fly is multivoltine and repeats several generations per year. The number of flies captured by the pheromone traps was generally large from spring to summer and decreased thereafter, although the seasonal abundance differed among years (Fig. 2). When the flies parasitized the bugs in late September or later, the larvae did not emerge but overwintered within the hosts (Table 4). Thus, P. stali is utilized as a host by G. rotundatum continuously throughout the year. The number of G. rotundatum caught by pheromone traps tended to remain high from spring to summer and then decreased until autumn, although the trend in seasonal abundance changed
Table 2 Effect of the aggregation pheromone of Plautia stali on parasitism by Gymnosoma rotundatum. Observation period
Treatment
Parasitized bugsa
Unparasitized bugs
June 15–16
Pheromone Control
4 0
6 10
June 16–17
Pheromone Control
3 0
7 10
June 21–22
Pheromone Control
2 0
8 10
June 29–30
Pheromone Control
0 0
10 10
July 1–2
Pheromone Control
7 0
3 10
July 2–3
Pheromone Control
1 0
9 10
Total
Pheromone Control
17 0
43 60
p-valueb
0.087
0.211
0.474
>0.999
0.003
>0.999
<0.001 Percentages of parasitism between two groups of P. stali adults were compared: the pheromone group was baited with synthetic aggregation pheromone and the control group was not. To strictly control for the effect of the pheromone, we used only female bugs, which do not release the aggregation pheromone. a Only bugs with one or more parasitoid eggs underneath their wings were considered to be parasitized. b The percentage of parasitism was compared between the two treatments using Fisher’s exact probability test.
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M. Higaki, I. Adachi / Biological Control 58 (2011) 215–221 Table 3 Seasonal changes in tachinid parasitism of Plautia stali collected by the light and pheromone traps. Observation period
Light traps No. of P.stali captured
Apr. 21–30 May 1–10 May 11–20 May 21–31 June 1–10 June 11–20 June 21–30 July 1–10 July 11–20 July 21–31 Aug. 1–10 Aug. 11–20 Aug. 21–31 Sept. 1–10 Sept.11–20 Sept.21–30 Oct. 1–10 Oct. 11–20 Oct. 21–31 Nov. 1–10 Nov. 11–20 Nov. 21–30
0 18 13 285 237 163 542 132 256 1074 931 810 470 665 726 384 125 8 0 0 0 0
Total
6839
p-Valueb
Pheromone traps a
Parasitized individuals (%) 5.6 0 5.6 6.8 2.5 4.2 6.8 0.8 1.3 3.1 2.8 0.6 1.5 2.9 2.3 3.2 12.5
2.7
No. of P.stali captured
Parasitized individuals (%)
3 33 15 184 165 82 116 77 381 125 173 156 183 214 604 224 71 98 46 11 7 4
0 3.0 20.0 7.1 6.1 6.1 6.9 6.5 3.7 4.8 4.6 3.8 0 1.9 5.0 6.7 18.3 23.4 21.7 18.2 0 50.0
2972
6.0
>0.999 0.226 0.559 0.839 0.166 0.228 >0.999 0.021 0.013 0.354 0.449 0.563 0.754 0.062 0.010 0.001 0.680
<0.001
Tachinid parasitism was checked in Plautia stali adults that had been collected by light traps and basin traps baited with synthetic aggregation pheromone in 1999. a Parasitism was defined as the presence of one or more parasitoid eggs underneath the wings of the host bugs. b Percentages of parasitism were compared between the two groups using Fisher’s exact probability test.
from year to year (Fig. 2). Mishiro and Ohira (2002) also obtained similar results in seasonal abundance for this fly. They focused on the emergence of new adults of P. stali in the summer, and hypothesized that the reduction in G. rotundatum captured by pheromone traps after the summer was caused by an increase in the numbers of pheromone releasers in natural populations of the host. Thus, it is possible that the number of flies captured by pheromone traps would be influenced by the number of male bugs near the traps. The ecological significance of the attractiveness of the pheromones emitted by male bugs to individuals of the same species may vary among species. For example, in Nezara viridula (Linnaeus), Mitchell and Mau (1971) and Brennan et al. (1977) regarded the male-emitted pheromone as a sex pheromone, and Harris and Todd (1980) hypothesized that the resultant aggregation serves as a forerunner and facilitator of mate-finding. According to Aldrich et al. (1987), males of Podisus maculiventris (Say) often search for food first and then call females with their pheromone. In P. stali, the attracted bugs are in poor nutritional condition and mating among them is not observed (Moriya and Shiga, 1984; Moriya et al., 1993). Shiga and Moriya (1989) hypothesized that the male-emitted pheromone is an aggregation pheromone and that the males often play the role of an exploiter of temporally and spatially limited food resources. For bugs searching for a food resource, orientation to a pheromone source would have an adaptive value. Both P. stali and its parasitoid, G. rotundatum, were attracted to the bug’s aggregation pheromone (Fig. 2). However, the time of day when attraction to the pheromone was highest clearly differed between P. stali and G. rotundatum (Table 1). The former was mainly attracted during the night and the latter during the day. For the bugs, flying to the pheromone source at night probably lowers the risk of parasitism. However, most individuals of the bug were attracted during the day in May, October, and November. Moriya and Shiga (1984) suggested that a shift of flying time from nighttime to daytime is caused by the suppression of flight activity under the low temperatures that occur at night in the spring and late
autumn. There was a clear seasonal change in temperature conditions in Tsukuba in 1999, and mean temperature was higher than 20 °C in June (21.1 °C), July (24.6 °C), August (27.1 °C), and September (24.4 °C), but lower in May (17.8 °C), October (16.9 °C), and November (10.8 °C) (Japan Meteorological Agency, 2010). Orientation to the pheromone source during the day would increase the probability of encountering the parasitoid fly. This may be one of the factors that raised the parasitism percentage in autumn of 1999 (Table 3, Fig. 3). To avoid parasitism by tachinids, nonproduction of the pheromone at certain times of the day seems to be evolutionarily more advantageous than rejection of the attraction. However, the pheromone gland of P. stali has not yet been found, and there is no information about mechanisms controlling pheromone release. The percentage of parasitism was very low in adult bugs caught by light traps and pheromone traps, but the percentage was significantly larger in bugs from pheromone traps than those from light traps. A greater probability of encountering the tachinids near the pheromone traps likely increased the parasitism percentage. However, the parasitism percentage observed in trapped bugs was much lower than that in bugs caught by beating branches (Table 3,
Fig. 3. Seasonal change in tachinid parasitism of Plautia stali collected on the host plant Paulownia tomentosa in 1999. Sample sizes ranged from 29 to 123 across the dates.
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Table 4 Dates of pupariation and adult emergence of Gymnosoma rotundatum that parasitized Plautia stali in autumna. Date of collection
Date of pupariation
Date of adult emergence
Date of collection
Date of pupariation
Date of adult emergence
Sept. 11
Sept. 20 Sept. 20 Sept. 21 Sept. 23 Sept. 26 Oct. 15 Sept. 30 Oct. 4 Oct. 4 Oct. 9 Oct. 11 Oct. 12 Oct. 13 Oct. 14 Oct. 15 Oct. 15 Oct. 14 Apr. 17 Apr. 22
Oct. 1 Oct. 2 Oct. 4 Deadb Oct. 8 Nov. 11 Dead Oct. 18 Oct. 21 Oct. 29 Oct. 31 Nov. 3 Nov. 4 Nov. 7 Nov. 7 Nov. 10 Dead May 8 May 10
Oct. 2
Oct. 18 Apr. 11 Apr. 14 Apr. 16 Nov. 1 Nov. 4 Apr. 15 Apr. 17 Apr. 17 Apr. 18 Apr. 19 Apr. 19 Apr. 21 Apr. 13 Apr. 16 Apr. 21 Apr. 23 Apr. 24 Apr. 26
Nov. 13 May 3 May 6 May 6 Dead Dead May 8 May 8 May 9 May 8 May 9 May 9 Dead May 4 Dead May 10 May 11 May 13 May 14
Sept. 19 Sept. 27
Sept. 30
Oct. 9 Oct. 13
Oct. 29
Parasitized bugs collected in the field from September through October 1999 were reared in a non-air-conditioned room, and pupariation and adult emergence were checked daily. a Parasitism was defined as the presence of one or more parasitoid eggs underneath the wings of the hosts. b Flies died during the pupal stage.
Fig. 3). Such rare parasitism in trapped bugs may be explained by the following: adult bugs parasitized by G. rotundatum gradually become inactive as the tachinid larvae mature (M. Higaki, unpublished data), which would reduce their ability to fly to traps. Therefore, it is likely that parasitism percentages based on bugs caught by light and pheromone traps underestimate the actual parasitism in the field, and collecting bugs by beating branches of host trees is the preferable sampling method. Tachinid flies belonging to the Phasiinae are considered to use pheromones to find their heteropteran hosts (Aldrich et al., 1987; Harris and Todd, 1980; Jang and Park, 2010; Mishiro and Ohira, 2002; Mitchell and Mau, 1971). No previous study, however, has provided direct evidence for utilization of a bug’s pheromone as a kairomone. In the present study, we investigated the effects of the aggregation pheromone of P. stali on parasitism by G. rotundatum. We did not obtain a statistically significant difference between parasitism percentages of bugs baited with pheromone and those without the pheromone on specific dates, although when data from all the observation periods were pooled significantly more parasitoids were attracted by the baited bugs (Table 2). Nevertheless, the flies certainly seemed to search for hosts by utilizing the bug’s pheromone as a host-finding kairomone: in five of the six observation periods, parasitism was only observed in bugs baited with the pheromone and not in bugs without the pheromone. To clarify the kairomonal effect of the pheromone, a future experiment with a larger number of bugs baited with pheromone should be conducted. Until now, studies of the attraction of parasitoid flies to bug pheromones have mainly focused on the relationship between the host and the parasitoid. However, tachinids seem to utilize the pheromone for reasons other than searching for hosts. Our results showed that the pheromone of P. stali attracted both female and male G. rotundatum. The number of male flies captured by the pheromone traps tended to be larger, although difference in attractiveness to male and female flies was not clear because the sex ratio of the natural population is unknown. Attractiveness of the male-bug-emitted pheromone to male flies was also reported in the green stink bug, N. viridula and its parasitoid fly, Trichopoda pennipes (Fabricius) (Harris and Todd, 1980; Mitchell and Mau, 1971). We often found G. rotundatum males perched on top of basin traps baited with pheromone and displaying territorial behav-
ior. Therefore, it is possible that male flies increase the chance of encountering pheromone-attracted females by waiting near pheromone sources. Thus, it appears that female G. rotundatum utilize the bug’s pheromone to find hosts, whereas males use it to find mates. The present study demonstrated that G. rotundatum uses the pheromone of P. stali to find both hosts and mates. The tachinid develops multiple generations in active hosts from spring to autumn and overwinters in dormant hosts. The parasitoid fly shortens the host’s longevity and causes a considerable reduction in its reproductive ability (Higaki, 2003). Ovaries gradually shrink and oviposition is suppressed in parasitized female bugs, and fertility rates decrease in parasitized male bugs. Thus, G. rotundatum seems to be highly adapted to using P. stali as its host and therefore may be an important biological control agent for P. stali, which is a serious agricultural pest. To estimate the potential of G. rotundatum as a natural enemy, future research should examine the parasitism percentage in the bugs on host plants throughout the year, the effect of the pheromone on the parasitism rate, and mechanisms for the regulation of the parasitoid’s life cycle. Acknowledgments We thank Dr. Hidenari Kishimoto (Citrus Research Station of NIFTS) and Dr. Yasuki Kitashima (Ibaraki University) for their kind support. Thanks are also due to Ms. Hisako Okano and Ms. Kumiko Ebihara for laboratory assistance. References Adachi, I., Uchino, K., Mochizuki, F., 2007. Development of a pyramidal trap for monitoring fruit-piercing stink bugs baited with Plautia crossota stali (Hemiptera: Pentatomidae) aggregation pheromone. Applied Entomology and Zoology 42, 425–431. Aldrich, J.R., Zhang, A., 2002. Kairomone strains of Euclytia flava (Townsend), a parasitoid of stink bugs. Journal of Chemical Ecology 28, 1565–1582. Aldrich, J.R., Oliver, J.E., Lusby, W.R., Kochansky, J.P., Lockwood, J.A., 1987. Pheromone strains of the cosmopolitan pest, Nezara viridula (Heteroptera: Pentatomidae). Journal of Experimental Zoology 244, 171–175. Aldrich, J.R., Hoffmann, M.P., Kochansky, J.P., Lusby, W.R., Eger, J.E., Payne, J.A., 1991. Identification and attractiveness of a major pheromone component for Nearctic Euschistus spp. Stink bugs (Heteroptera: Pentatomidae). Environmental Entomology 20, 477–483.
M. Higaki, I. Adachi / Biological Control 58 (2011) 215–221 Aldrich, J.R., Khrimian, A., Zhang, A., Shearer, P.W., 2006. Bug pheromones (Hemiptera, Heteroptera) and tachinid fly host-finding. Denisia 19, 1015– 1031. Belshaw, R., 1994. Life history characteristics of Tachinidae (Diptera) and their effect on polyphagy. In: Hawkins, B.A., Sheehan, W. (Eds.), Parasitoid Community Ecology. Oxford University Press, New York, pp. 145–162. Brennan, B.M., Chang, F., Mitchell, W.C., 1977. Physiological effects on sex pheromone communication in the southern green stink bug, Nezara viridula. Environmental Entomology 6, 169–173. Clausen, C.P., 1940. Entomophagous Insects. Hafner, New York. Greathead, D.J., 1986. Parasitoids in classical biological control. In: Waage, J.K., Greathead, D.J. (Eds.), Insect Parasitoids. Academic Press, London, pp. 289–318. Grenier, S., 1988. Applied biological control with tachinid flies (Diptera, Tachinidae): a review. Anzeiger Fur Schadlingskunde Pflanzenschutz Umweltschutz 61, 49–56. Harris, V.E., Todd, J.W., 1980. Male-mediated aggregation of male, female and 5thinstar southern green stink bugs and concomitant attraction of a tachinid parasite, Trichopoda pennipes. Entomologia Experimentalis et Applicata 27, 117–126. Higaki, M., 2003. Development of a tachinid parasitoid, Gymnosoma rotundatum (Diptera: Tachinidae) on Plautia crossota stali (Heteroptera: Pentatomidae), and its effects on host reproduction. Applied Entomology and Zoology 38, 215–223. Jang, S.A., Park, C.G., 2010. Gymnosoma rotundatum (Diptera: Tachinidae) attracted to the aggregation pheromone of Plautia stali (Hemiptera: Pentatomidae). Journal of Asia-Pacific Entomology 13, 73–75. Japan Meteorological Agency, 2010. Climate Statistics. Available at: (in Japanese). Mishiro, K., Ohira, Y., 2002. Attraction of a synthetic aggregation pheromone of the brown-winged green bug, Plautia crossota stali Scott to its parasitoids, Gymnosoma rotundata and Trissolcus plautiae. Kyushu Plant Protection Research 48, 76–80 (in Japanese with English summary).
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Mitchell, W.C., Mau, R.F.L., 1971. Response of the female southern green stink bug and its parasite, Trichopoda pennipes, to male stink bug pheromones. Journal of Economic Entomology 64, 856–859. Moriya, S., Shiga, M., 1984. Attraction of the male brown-winged green bug, Plautia stali Scott (Heteroptera: Pentatomidae) for males and females of the same species. Applied Entomology and Zoology 19, 317–322. Moriya, S., Shiga, M., Mabuchi, M., 1985. A method for successive rearing of the brown-winged green bug, Plautia stali Scott (Hemiptera, Heteroptera, Pentatomidae). Bulletin of the Fruit Tree Research Station 12, 133–143 (in Japanese with English summary). Moriya, S., Mabuchi, M., Shiga, M., 1993. Seasonal and annual fluctuations of the numbers of the brown-winged stink bug, Plautia stali Scott (Heteroptera: Pentatomidae) and its parasitoid, Gymnosoma rotundata L. (Diptera: Phasiidae) attracted by adult males of the former species. Bulletin of the Fruit Tree Research Station 24, 73–90 (in Japanese with English summary). SAS Institute Inc., 1998. Stat View User’s Manual, Version 5. SAS Institute Inc., Cary, NC. Shiga, M., Moriya, S., 1989. Temporal and spatial differences in the conditions of the internal organs of adults of the brown-winged green bug, Plautia stali Scott (Heteroptera: Pentatomidae). Bulletin of the Fruit Tree Research Station 16, 133–168 (in Japanese with English summary). Stireman III, J.O., O’Hara, J.E., Wood, D.M., 2006. Tachinidae: evolution, behavior, and ecology. Annual Review of Entomology 51, 525–555. Sugie, H., Yoshida, M., Kawasaki, K., Noguchi, H., Moriya, S., Takagi, K., Fukuda, H., Fujiie, A., Yamanaka, M., Ohira, Y., Tsutsumi, T., Tsuda, K., Fukumoto, K., Yamashita, M., Suzuki, H., 1996. Identification of the aggregation pheromone of the brown-winged green bug, Plautia stali Scott (Heteroptera: Pentatomidae). Applied Entomology and Zoology 31, 427–431. Yamada, K., Miyahara, M., 1979. Studies on the ecology and control of some stink bugs infesting fruit II. Gymnosoma rotundatum as a natural enemy to the brownwinged green bug. Bulletin of the Fukuoka Horticultural Experiment Station 17, 54–62 (in Japanese with English summary).
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Response of a parasitoid fly, Gymnosoma rotundatum (Linnaeus) (Diptera: Tachinidae) to the aggregation pheromone of Plautia stali Scott (Hemiptera: Pentatomidae) and its parasitism of hosts under field conditions Morio Higaki ⇑, Ishizue Adachi Apple Research Station, National Institute of Fruit Tree Science, 92-24 Nabeyashiki, Shimokuriyagawa, Morioka, Iwate 020-0123, Japan
a r t i c l e
i n f o
Article history: Received 29 July 2010 Accepted 17 May 2011 Available online 23 May 2011 Keywords: Tachinidae Pentatomidae Parasitoid Pheromone Kairomone Life cycle
a b s t r a c t We studied the attraction of a tachinid fly, Gymnosoma rotundatum (Linnaeus) to the male-produced aggregation pheromone of the brown-winged green bug, Plautia stali Scott, its parasitism on the bug, and its seasonal occurrence in the field. The tachinid fly was continuously attracted to the aggregation pheromone from spring to autumn and utilized the bugs as hosts. Our field experiment to clarify the effect of the pheromone on parasitism demonstrated that parasitism occurred only in female bugs baited with synthetic aggregation pheromone and did not occur in females without the pheromone. The parasitoid flies therefore appeared to use the bug’s pheromone as a host-finding kairomone. The pheromone attracted not only female flies but also males. Male flies may increase their chance of encountering pheromone-attracted females by waiting near pheromone sources. The tachinid develops multiple generations in active hosts from spring to autumn and overwinters in dormant hosts. Thus, G. rotundatum seems to be highly adapted to using P. stali as its host, and it is a potentially important biological control agent of P. stali populations in the field. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Approximately 10,000 species of tachinid flies have been recorded worldwide, and all tachinid species are endoparasitoids of arthropods (Stireman et al., 2006). The larvae develop in the body cavities of live host insects, causing harmful effects on the hosts such as the inhibition of development and the atrophy of reproductive organs (Clausen, 1940). Tachinids parasitize insects belonging to all of the main orders, including orders that contain major agricultural pests, and are considered to be potentially important biological control agents (Belshaw, 1994; Greathead, 1986; Grenier, 1988). The mechanisms by which most tachinids locate and select hosts are poorly understood. The information available on the few tachinid species that have been studied indicates that tachinids are capable of using a wide diversity of olfactory, visual, auditory, and tactile-chemosensory cues to locate their hosts (Stireman et al., 2006). In a host-restricted tachinid subfamily, the Phasiinae, flies exclusively parasitize Heteroptera: the adults are especially drawn to attractant pheromones produced by the heteropterans and appear to use such pheromones to find these potential hosts
⇑ Corresponding author. Fax: +81 19 641 3819. E-mail address: [email protected] (M. Higaki). 1049-9644/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2011.05.009
(Aldrich et al., 1991; Harris and Todd, 1980; Mitchell and Mau, 1971). The phasiine tachinid Gymnosoma rotundatum (Linnaeus) parasitizes live adults of the brown-winged green bug, Plautia stali Scott (formerly Plautia crossota stali), a fruit-piercing bug that is a serious pest in Japan and Korea (Higaki, 2003; Jang and Park, 2010; Mishiro and Ohira, 2002; Moriya et al., 1993; Yamada and Miyahara, 1979). Because P. stali is polyphagous and the adults are highly mobile, adults invade fruit orchards from woodland or uncultivated areas, requiring frequent insecticide applications to prevent infestations (Adachi et al., 2007). G. rotundatum is a potentially important biological control agent because its parasitism causes considerable reductions in the longevity and reproductive ability of P. stali (Higaki, 2003; Yamada and Miyahara, 1979). Adult males of P. stali are known to emit an aggregation pheromone that attracts both males and females of the same species (Adachi et al., 2007; Moriya and Shiga, 1984). G. rotundatum probably uses the male-produced aggregation pheromone of P. stali as a host-finding kairomone, because large numbers of this tachinid fly have been captured in traps baited with adult males of P. stali or a synthetic version of the aggregation pheromone (Jang and Park, 2010; Mishiro and Ohira, 2002; Moriya et al., 1993). However, the field responses of G. rotundatum to P. stali aggregation pheromones have not been well studied. In addition, researchers have a poor understanding of the ecology of the fly, including aspects such as its seasonal life cycle. In the present study, we investigated
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the response of G. rotundatum to P. stali aggregation pheromone, its parasitism of the host bugs, and its seasonal occurrence in the field.
2. Materials and methods 2.1. Seasonal abundance of parasitoid flies and host bugs caught by pheromone traps The male-produced aggregation pheromone of P. stali was identified as methyl (E,E,Z)-2,4,6-decatrienoate (Sugie et al., 1996), and synthetic pheromone synthesized by Shin-Etsu Chemical Co. (Tokyo, Japan) was used in our study. To track the seasonal occurrence of G. rotundatum and P. stali, we used a 3.0-L basin trap (yellow type of Kogane-call, Sankei Chemical Co., Kagoshima, Japan) baited with synthetic aggregation pheromone. A small amount of detergent was added to the water in the basin. The pheromone was supplied in lures, each of which contained 42 mg of pheromone, and the lures were replaced every 20 days. From April to November in 2000 to 2005, the traps were established in open areas of the National Institute of Fruit Tree Science (NIFTS), Tsukuba, Ibaraki Prefecture, Japan (36°030 N, 140°080 E, 20 m a.s.l.), where several kinds of deciduous trees are planted. Traps were suspended at a height of 160 cm, and the distance between traps was more than 100 m. We established two traps in 2000, five each year from 2001 to 2003, four in 2004, and three in 2005. We used five lures per trap in 2001 and two lures per trap in the other years of this study. Traps were checked daily for the number of G. rotundatum and P. stali caught. 2.2. Diel rhythm in parasitoid flies and host bugs caught by the pheromone traps We established two basin traps baited with a lure containing the synthetic aggregation pheromone (85 mg per lure) at NIFTS from May to November 1999. The traps were located more than 300 m apart. We replaced the lures every 20 days. Traps were checked for catches of P. stali and G. rotundatum five times per day (09:00, 12:00, 15:00, 18:00, and 21:00). The experiment was carried out on 10–17 days per month. 2.3. Effect of host pheromone on tachinid parasitism To reveal the effect of the aggregation pheromone on parasitism, we compared the parasitism by G. rotundatum between two groups of P. stali adults: one group was baited with synthetic aggregation pheromone and the other was not. To strictly control for the effect of the pheromone, we used only female bugs, which do not release the aggregation pheromone. P. stali adults reared in the laboratory according to the method described by Moriya et al. (1985) were provided as potential hosts. In each group, 10 live female bugs were attached at their abdomen to the tips of 10 cm tall poles using an adhesive (G17, Konishi Co., Osaka, Japan), and these 10 bugs were fixed on a frame at 7 cm intervals (Fig. 1). The two groups of potential hosts were placed at 160 cm above ground under the trees at NIFTS, separated by about 30 m. The potential hosts were exposed for 24 h (from 12:00 to 12:00 on the next day). We used three pheromone lures (each 42 mg) in the bated frame and replaced them after every trial. G. rotundatum lays its eggs primarily underneath the wings (abdominal tergum) of its hosts (Higaki, 2003), and we judged parasitism by checking for such eggs. When eggs are laid on other parts of the host body, parasitism never succeeds (Yamada and Miyahara, 1979), so we did not examine other body parts for the presence of eggs. We repeated the field experiments six times from June to July 1999.
Fig. 1. Illustration of the experimental device used to reveal the effect of synthetic Plautia stali aggregation pheromone on parasitism by Gymnosoma rotundatum.
2.4. Parasitism under field conditions To estimate the percentage of tachinid parasitism in the field, we examined P. stali adults that had been collected at NIFTS by beating the branches of the Japanese Paulownia (Paulownia tomentosa Steudel) or by using water-basin traps baited with 85 mg of synthetic aggregation pheromone or light traps (100 W mercury vapor lamps). Collection by beating branches was done two to six times per month from July to October 1999. Collection using basin and light traps was carried out continuously from April to November 1999. 2.5. Overwintering of tachinid flies under semi-outdoor conditions Parasitized bugs collected on P. tomentosa trees at NIFTS from September through October 1999 were reared in Petri dishes (9 cm in diameter). Raw peanuts were provided to the bugs as food, and water was supplied in water-soaked cotton wool. The Petri dishes were kept in a non-air-conditioned room that was open to the ambient atmosphere on two sides but was screened to exclude small animals such as mice. G. rotundatum individuals that emerged from the hosts and that pupariated were kept on layers of moist tissue paper in plastic cups (4 cm high by 5 cm in diameter) until they emerged as adults. Pupariation and adult emergence were checked daily. 2.6. Statistical analysis We compared the parasitism percentages between groups and the male-to-female ratio of the flies attracted to the pheromone using Fisher’s exact probability test. All statistical analyses were performed with Stat View-J version 5.0 (SAS Institute Inc., 1998). 3. Results 3.1. Seasonal abundance of parasitoid flies and host bugs caught by pheromone traps G. rotundatum and P. stali were continuously attracted to the aggregation pheromone of P. stali from April to October (or November), but many fewer flies than bugs were attracted (Fig. 2). We studied the populations over 6 years, and the trend in seasonal
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Fig. 2. Seasonal abundance of Gymnosoma rotundatum and Plautia stali captured by pheromone traps in Tsukuba, Japan from 2000 to 2005.
abundance changed considerably from year to year for both G. rotundatum and P. stali and differed between the parasitoid and the host bug. In P. stali, one or more large peaks were observed in the seasonal prevalence, whereas in G. rotundatum no clear peaks were observed and the number caught tended to remain high from spring to summer and then decreased until autumn. The P. stali aggregation pheromone attracted both female and male flies, although more males than females tended to be attracted. The male-to-female ratio was 128:90 (1.4) in 2000 (Fisher’s exact probability test, p = 0.083), 708:363 (2.0) in 2001 (p < 0.001), 144:137 (1.1) in 2002 (p = 0.800), 276:154 (1.8) in 2003 (p < 0.001), 193:162 (1.2) in 2004 (p = 0.260), and 90:41 (2.2) in 2005 (p = 0.003). 3.2. Diel rhythm in parasitoid flies and host bugs caught by the pheromone traps Responsiveness to the aggregation pheromone clearly differed among times of day in both P. stali and G. rotundatum (Table 1). The daily rhythm of responsiveness of P. stali changed from month to month. From June to September, the largest numbers of bugs were attracted during the night, whereas in May, October, and November more individuals were attracted during the day. In G. rotundatum, we did not detect such a seasonal change in the daily rhythm of responsiveness, and most individuals were attracted to the pheromone during the day. 3.3. Effect of host pheromone on tachinid parasitism Parasitism by G. rotundatum was only observed in the bugs that had been baited with the aggregation pheromone, although the
parasitism percentage varied across the six observation periods (Table 2). The parasitism percentage did not differ significantly between the two groups in any period except July 1–2, although it was significantly higher in bugs with the pheromone when the data for all six periods were pooled (p < 0.001).
3.4. Parasitism under field conditions G. rotundatum females most often laid one egg per host and rarely two or more eggs. The proportions of male and female hosts parasitized with one egg were 92.6% and 99.1%, respectively in the light traps, 100% and 96.5% in the pheromone traps, and 94.5% and 96.2% in the sample collected by branch beating. The parasitism percentage was significantly higher in bugs captured by the pheromone traps than in those captured by the light traps (p < 0.001; Table 3). We also observed a significantly higher parasitism percentage in males than in females in the pheromone traps (male: 73 of 1004, 7.3%; female: 105 of 1968, 5.3%; p = 0.041), but no significant differences between the sexes in the light traps (male: 68 of 2441, 2.8%; female: 117 of 4398, 2.7%; p = 0.756) or in branch-beating sample (male: 53 of 532, 10.0%; female: 57 of 585, 9.7%; p = 0.920). The parasitism results for the light and pheromone traps showed that G. rotundatum attacked and utilized P. stali as its host from spring to autumn, although the parasitism percentage was not high (Table 3). The parasitism percentage in the bugs captured by beating branches on the P. tomentosa trees changed seasonally and gradually rose in autumn (Fig. 3). A similar autumn increase was also observed in the pheromone traps.
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Table 1 Seasonal changes in the responsiveness of Gymnosoma rotundatum and Plautia stali to the aggregation pheromone of Plautia stali. Month
May June July August September October November
No. of observation days
10 13 17 14 16 15 17
Total number of individuals per month captured at each time of day G. rotundatum
P. stali
21:00– 09:00
09:00– 12:00
12:00– 15:00
15:00– 18:00
18:00– 21:00
21:00– 09:00
09:00– 12:00
12:00– 15:00
15:00– 18:00
18:00– 21:00
13 17 5 2 3 16 0
28 16 18 9 13 17 12
37 28 22 6 10 15 24
21 28 23 9 8 3 1
0 1 0 2 0 0 0
11 78 155 161 287 36 0
7 16 24 17 27 28 2
19 13 13 19 20 68 12
19 10 28 9 61 66 1
15 113 201 13 189 16 0
Two basin traps with a lure containing the synthetic aggregation pheromone were established at NIFTS from May to November 1999. Traps were located more than 300 m apart. Traps were checked five times per day (09:00, 12:00, 15:00, 18:00, 21:00) for the number of G. rotundatum and P. stali caught.
3.5. Overwintering of tachinid flies under semi-outdoor conditions In all parasitized P. stali adults collected before September 27, mature larvae of G. rotundatum exited from the hosts, pupariated soon afterward, and emerged as adults in October or November (Table 4). In most of the parasitized bugs collected after September 30, however, parasitoid larvae did not emerge but overwintered within their hosts. The bugs and their parasitoids were dormant and inactive during the winter. After overwintering, the parasitoid larvae left their hosts and pupariated in April, and adults emerged in May.
4. Discussion Numerous studies have found that many tachinid species belonging to the subfamily Phasiinae are attracted to the pheromone emitted by heteropterans (Aldrich and Zhang, 2002; Aldrich et al., 1987; Aldrich et al., 2006; Harris and Todd, 1980; Mishiro and Ohira, 2002; Mitchell and Mau, 1971; Moriya et al., 1993). However, few detailed studies have examined the ecology and
responsiveness of the flies, such as their seasonal prevalence and annual fluctuations in parasitism levels (e.g., Mishiro and Ohira, 2002). The present study showed that adults of G. rotundatum were attracted to the aggregation pheromone of P. stali in the field from spring to autumn (Fig. 2). Higaki (2003) conducted a series of laboratory experiments on G. rotundatum and reported that development proceeded without any delay under conditions of long-day photoperiod: the total larval and puparial duration was about 30 days and adult longevity was about 20 days at 21–22 °C, and adults could lay eggs on the day following emergence. These observations suggest that the fly is multivoltine and repeats several generations per year. The number of flies captured by the pheromone traps was generally large from spring to summer and decreased thereafter, although the seasonal abundance differed among years (Fig. 2). When the flies parasitized the bugs in late September or later, the larvae did not emerge but overwintered within the hosts (Table 4). Thus, P. stali is utilized as a host by G. rotundatum continuously throughout the year. The number of G. rotundatum caught by pheromone traps tended to remain high from spring to summer and then decreased until autumn, although the trend in seasonal abundance changed
Table 2 Effect of the aggregation pheromone of Plautia stali on parasitism by Gymnosoma rotundatum. Observation period
Treatment
Parasitized bugsa
Unparasitized bugs
June 15–16
Pheromone Control
4 0
6 10
June 16–17
Pheromone Control
3 0
7 10
June 21–22
Pheromone Control
2 0
8 10
June 29–30
Pheromone Control
0 0
10 10
July 1–2
Pheromone Control
7 0
3 10
July 2–3
Pheromone Control
1 0
9 10
Total
Pheromone Control
17 0
43 60
p-valueb
0.087
0.211
0.474
>0.999
0.003
>0.999
<0.001 Percentages of parasitism between two groups of P. stali adults were compared: the pheromone group was baited with synthetic aggregation pheromone and the control group was not. To strictly control for the effect of the pheromone, we used only female bugs, which do not release the aggregation pheromone. a Only bugs with one or more parasitoid eggs underneath their wings were considered to be parasitized. b The percentage of parasitism was compared between the two treatments using Fisher’s exact probability test.
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M. Higaki, I. Adachi / Biological Control 58 (2011) 215–221 Table 3 Seasonal changes in tachinid parasitism of Plautia stali collected by the light and pheromone traps. Observation period
Light traps No. of P.stali captured
Apr. 21–30 May 1–10 May 11–20 May 21–31 June 1–10 June 11–20 June 21–30 July 1–10 July 11–20 July 21–31 Aug. 1–10 Aug. 11–20 Aug. 21–31 Sept. 1–10 Sept.11–20 Sept.21–30 Oct. 1–10 Oct. 11–20 Oct. 21–31 Nov. 1–10 Nov. 11–20 Nov. 21–30
0 18 13 285 237 163 542 132 256 1074 931 810 470 665 726 384 125 8 0 0 0 0
Total
6839
p-Valueb
Pheromone traps a
Parasitized individuals (%) 5.6 0 5.6 6.8 2.5 4.2 6.8 0.8 1.3 3.1 2.8 0.6 1.5 2.9 2.3 3.2 12.5
2.7
No. of P.stali captured
Parasitized individuals (%)
3 33 15 184 165 82 116 77 381 125 173 156 183 214 604 224 71 98 46 11 7 4
0 3.0 20.0 7.1 6.1 6.1 6.9 6.5 3.7 4.8 4.6 3.8 0 1.9 5.0 6.7 18.3 23.4 21.7 18.2 0 50.0
2972
6.0
>0.999 0.226 0.559 0.839 0.166 0.228 >0.999 0.021 0.013 0.354 0.449 0.563 0.754 0.062 0.010 0.001 0.680
<0.001
Tachinid parasitism was checked in Plautia stali adults that had been collected by light traps and basin traps baited with synthetic aggregation pheromone in 1999. a Parasitism was defined as the presence of one or more parasitoid eggs underneath the wings of the host bugs. b Percentages of parasitism were compared between the two groups using Fisher’s exact probability test.
from year to year (Fig. 2). Mishiro and Ohira (2002) also obtained similar results in seasonal abundance for this fly. They focused on the emergence of new adults of P. stali in the summer, and hypothesized that the reduction in G. rotundatum captured by pheromone traps after the summer was caused by an increase in the numbers of pheromone releasers in natural populations of the host. Thus, it is possible that the number of flies captured by pheromone traps would be influenced by the number of male bugs near the traps. The ecological significance of the attractiveness of the pheromones emitted by male bugs to individuals of the same species may vary among species. For example, in Nezara viridula (Linnaeus), Mitchell and Mau (1971) and Brennan et al. (1977) regarded the male-emitted pheromone as a sex pheromone, and Harris and Todd (1980) hypothesized that the resultant aggregation serves as a forerunner and facilitator of mate-finding. According to Aldrich et al. (1987), males of Podisus maculiventris (Say) often search for food first and then call females with their pheromone. In P. stali, the attracted bugs are in poor nutritional condition and mating among them is not observed (Moriya and Shiga, 1984; Moriya et al., 1993). Shiga and Moriya (1989) hypothesized that the male-emitted pheromone is an aggregation pheromone and that the males often play the role of an exploiter of temporally and spatially limited food resources. For bugs searching for a food resource, orientation to a pheromone source would have an adaptive value. Both P. stali and its parasitoid, G. rotundatum, were attracted to the bug’s aggregation pheromone (Fig. 2). However, the time of day when attraction to the pheromone was highest clearly differed between P. stali and G. rotundatum (Table 1). The former was mainly attracted during the night and the latter during the day. For the bugs, flying to the pheromone source at night probably lowers the risk of parasitism. However, most individuals of the bug were attracted during the day in May, October, and November. Moriya and Shiga (1984) suggested that a shift of flying time from nighttime to daytime is caused by the suppression of flight activity under the low temperatures that occur at night in the spring and late
autumn. There was a clear seasonal change in temperature conditions in Tsukuba in 1999, and mean temperature was higher than 20 °C in June (21.1 °C), July (24.6 °C), August (27.1 °C), and September (24.4 °C), but lower in May (17.8 °C), October (16.9 °C), and November (10.8 °C) (Japan Meteorological Agency, 2010). Orientation to the pheromone source during the day would increase the probability of encountering the parasitoid fly. This may be one of the factors that raised the parasitism percentage in autumn of 1999 (Table 3, Fig. 3). To avoid parasitism by tachinids, nonproduction of the pheromone at certain times of the day seems to be evolutionarily more advantageous than rejection of the attraction. However, the pheromone gland of P. stali has not yet been found, and there is no information about mechanisms controlling pheromone release. The percentage of parasitism was very low in adult bugs caught by light traps and pheromone traps, but the percentage was significantly larger in bugs from pheromone traps than those from light traps. A greater probability of encountering the tachinids near the pheromone traps likely increased the parasitism percentage. However, the parasitism percentage observed in trapped bugs was much lower than that in bugs caught by beating branches (Table 3,
Fig. 3. Seasonal change in tachinid parasitism of Plautia stali collected on the host plant Paulownia tomentosa in 1999. Sample sizes ranged from 29 to 123 across the dates.
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Table 4 Dates of pupariation and adult emergence of Gymnosoma rotundatum that parasitized Plautia stali in autumna. Date of collection
Date of pupariation
Date of adult emergence
Date of collection
Date of pupariation
Date of adult emergence
Sept. 11
Sept. 20 Sept. 20 Sept. 21 Sept. 23 Sept. 26 Oct. 15 Sept. 30 Oct. 4 Oct. 4 Oct. 9 Oct. 11 Oct. 12 Oct. 13 Oct. 14 Oct. 15 Oct. 15 Oct. 14 Apr. 17 Apr. 22
Oct. 1 Oct. 2 Oct. 4 Deadb Oct. 8 Nov. 11 Dead Oct. 18 Oct. 21 Oct. 29 Oct. 31 Nov. 3 Nov. 4 Nov. 7 Nov. 7 Nov. 10 Dead May 8 May 10
Oct. 2
Oct. 18 Apr. 11 Apr. 14 Apr. 16 Nov. 1 Nov. 4 Apr. 15 Apr. 17 Apr. 17 Apr. 18 Apr. 19 Apr. 19 Apr. 21 Apr. 13 Apr. 16 Apr. 21 Apr. 23 Apr. 24 Apr. 26
Nov. 13 May 3 May 6 May 6 Dead Dead May 8 May 8 May 9 May 8 May 9 May 9 Dead May 4 Dead May 10 May 11 May 13 May 14
Sept. 19 Sept. 27
Sept. 30
Oct. 9 Oct. 13
Oct. 29
Parasitized bugs collected in the field from September through October 1999 were reared in a non-air-conditioned room, and pupariation and adult emergence were checked daily. a Parasitism was defined as the presence of one or more parasitoid eggs underneath the wings of the hosts. b Flies died during the pupal stage.
Fig. 3). Such rare parasitism in trapped bugs may be explained by the following: adult bugs parasitized by G. rotundatum gradually become inactive as the tachinid larvae mature (M. Higaki, unpublished data), which would reduce their ability to fly to traps. Therefore, it is likely that parasitism percentages based on bugs caught by light and pheromone traps underestimate the actual parasitism in the field, and collecting bugs by beating branches of host trees is the preferable sampling method. Tachinid flies belonging to the Phasiinae are considered to use pheromones to find their heteropteran hosts (Aldrich et al., 1987; Harris and Todd, 1980; Jang and Park, 2010; Mishiro and Ohira, 2002; Mitchell and Mau, 1971). No previous study, however, has provided direct evidence for utilization of a bug’s pheromone as a kairomone. In the present study, we investigated the effects of the aggregation pheromone of P. stali on parasitism by G. rotundatum. We did not obtain a statistically significant difference between parasitism percentages of bugs baited with pheromone and those without the pheromone on specific dates, although when data from all the observation periods were pooled significantly more parasitoids were attracted by the baited bugs (Table 2). Nevertheless, the flies certainly seemed to search for hosts by utilizing the bug’s pheromone as a host-finding kairomone: in five of the six observation periods, parasitism was only observed in bugs baited with the pheromone and not in bugs without the pheromone. To clarify the kairomonal effect of the pheromone, a future experiment with a larger number of bugs baited with pheromone should be conducted. Until now, studies of the attraction of parasitoid flies to bug pheromones have mainly focused on the relationship between the host and the parasitoid. However, tachinids seem to utilize the pheromone for reasons other than searching for hosts. Our results showed that the pheromone of P. stali attracted both female and male G. rotundatum. The number of male flies captured by the pheromone traps tended to be larger, although difference in attractiveness to male and female flies was not clear because the sex ratio of the natural population is unknown. Attractiveness of the male-bug-emitted pheromone to male flies was also reported in the green stink bug, N. viridula and its parasitoid fly, Trichopoda pennipes (Fabricius) (Harris and Todd, 1980; Mitchell and Mau, 1971). We often found G. rotundatum males perched on top of basin traps baited with pheromone and displaying territorial behav-
ior. Therefore, it is possible that male flies increase the chance of encountering pheromone-attracted females by waiting near pheromone sources. Thus, it appears that female G. rotundatum utilize the bug’s pheromone to find hosts, whereas males use it to find mates. The present study demonstrated that G. rotundatum uses the pheromone of P. stali to find both hosts and mates. The tachinid develops multiple generations in active hosts from spring to autumn and overwinters in dormant hosts. The parasitoid fly shortens the host’s longevity and causes a considerable reduction in its reproductive ability (Higaki, 2003). Ovaries gradually shrink and oviposition is suppressed in parasitized female bugs, and fertility rates decrease in parasitized male bugs. Thus, G. rotundatum seems to be highly adapted to using P. stali as its host and therefore may be an important biological control agent for P. stali, which is a serious agricultural pest. To estimate the potential of G. rotundatum as a natural enemy, future research should examine the parasitism percentage in the bugs on host plants throughout the year, the effect of the pheromone on the parasitism rate, and mechanisms for the regulation of the parasitoid’s life cycle. Acknowledgments We thank Dr. Hidenari Kishimoto (Citrus Research Station of NIFTS) and Dr. Yasuki Kitashima (Ibaraki University) for their kind support. Thanks are also due to Ms. Hisako Okano and Ms. Kumiko Ebihara for laboratory assistance. References Adachi, I., Uchino, K., Mochizuki, F., 2007. Development of a pyramidal trap for monitoring fruit-piercing stink bugs baited with Plautia crossota stali (Hemiptera: Pentatomidae) aggregation pheromone. Applied Entomology and Zoology 42, 425–431. Aldrich, J.R., Zhang, A., 2002. Kairomone strains of Euclytia flava (Townsend), a parasitoid of stink bugs. Journal of Chemical Ecology 28, 1565–1582. Aldrich, J.R., Oliver, J.E., Lusby, W.R., Kochansky, J.P., Lockwood, J.A., 1987. Pheromone strains of the cosmopolitan pest, Nezara viridula (Heteroptera: Pentatomidae). Journal of Experimental Zoology 244, 171–175. Aldrich, J.R., Hoffmann, M.P., Kochansky, J.P., Lusby, W.R., Eger, J.E., Payne, J.A., 1991. Identification and attractiveness of a major pheromone component for Nearctic Euschistus spp. Stink bugs (Heteroptera: Pentatomidae). Environmental Entomology 20, 477–483.
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