A danaid butterfly, Ideopsis similis, overcomes parasitization by a tachinid fly, Sturmia bella

Authors


Norio Hirai, Entomological Laboratory, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, 599-8531 Japan. Email: n_hirai@envi.osakafu-u.ac.jp

Abstract

The danaid butterfly Ideopsis similis, which occurs in the Ryukyu Islands, Japan, is a sedentary and monophagous species, whose larval food plant, Tylophora tanakae (Asclepiadaceae), is shared with the migratory and polyphagous danaid Parantica sita. The tachinid Sturmia bella is known to be a principal parasitoid of larval P. sita, whereas there have been no records of parasitization of I. similis by this tachinid. We conducted laboratory experiments and field surveys at seven sites in the Ryukyu Islands to determine how I. similis evades parasitization by this tachinid. We found eggs of S. bella at four sites and eggs and/or larvae of I. similis on the leaves of T. tanakae at all seven sites in the field survey. Out of 40 I. similis allowed to ingest S. bella eggs, 28 (70%) emerged as normal host adults and nine (22.5%) emerged as host adults with crippled wings. Only three (7.5%) died of parasitization in the pupal stage, whereas 27 (87%) of 31 P. sita given S. bella eggs yielded parasitoid flies and eventually died. Melanized dead larvae of S. bella were found in the abdomens of the I. similes adults that had been parasitized but did not yield parasitoid flies. When I. similis females parasitized by S. bella were released in a greenhouse with males, one of them laid fertile eggs. Out of 125 field-collected I. similis adults, nine had melanized dipteran larvae within their abdomens, which were considered to be S. bella. These results demonstrate that I. similis has the ability to overcome parasitization by S. bella and develop into a fertile adult. It is possible that I. similis remains sedentary and monophagous because it has a strong defense mechanism against S. bella, whereas P. sita escapes from parasitization using its migratory and polyphagous capability.

INTRODUCTION

Host suitability is an important issue for insect parasitoids to complete their development, as are other host selection processes such as host habitat location, host location and host acceptance (e.g. Salt 1935, 1938; Vinson 1984). Host suitability may also influence host range. Endoparasitoids are subject to the physiological defenses of their hosts, that is, host immune responses, including hemocytic encapsulation by hosts, and have evolved a variety of strategies to overcome host defenses (Salt 1963, 1968; Vinson 1990). The Tachinidae are one of the most species-rich groups of Diptera (e.g. Tschorsnig & Richiter 1998). Although there have been great advances in understanding the relationships between host immune responses and parasitoid countermeasures in parasitoid wasps (e.g. Stoltz & Vinson 1979; Edson et al. 1981; Godfray 1994), physiological interactions between tachinid flies and their hosts have been neglected.

The tachinid Sturmia bella (Meigen, 1824) parasitizes more than 20 lepidopterous species, mostly nymphalids and danaids (Shima 1999). This species is known to be a principal larval parasitoid of a migratory danaid, Parantica sita (Koller, 1844) in Japan (Hirai & Ishii 1995, 1997, 2002; Shima 1999). Sturmia bella females deposit microtype eggs on foliage of the host’s food plant, and P. sita larvae are parasitized by the hatched fly larvae after they ingest the eggs along with leaf tissue. The parasitoid larvae emerge from the host after host pupates (Hirai & Ishii 1995).

The danaid Ideopsis similis (Linnaeus, 1758) is widely distributed in South-East Asia, including the Ryukyu Islands, Japan, where immature individuals share milkweed, Tylophora tanakae (Asclepiadaceae), with P. sita as a larval food plant. However, parasitism of I. similis by S. bella has not been recorded. In the present study, we conducted field surveys and laboratory experiments to determine how I. similis evades parasitization by S. bella, and discuss the host–parasitoid relationships between the two danaids and this tachinid.

MATERIALS AND METHODS

Field survey of S. bella eggs on T. tanakae

A field survey for S. bella eggs and immature stages of I. similis on T. tanakae was conducted at seven sites, sites A–G, in Iriomote and Ishigaki Islands, Okinawa Prefecture, in the south-westernmost part of Japan during 1998–2001 (Fig. 1). Sites A–F were near the seashore, where vegetation developed on sandy soil, whereas site G was located near the roadside and was surrounded by evergreen broad-leaved forests. We visited sites A, B, and D during 14–18 March 1998, sites D, E, and F during 17–21 February 2000, and all seven sites during 5–10 October 2001, and randomly collected 10–53 T. tanakae leaves at each site to check for eggs of S. bella, and eggs and larvae of I. similis. Eggs of S. bella were identified on the basis of their surface structure under a stereomicroscope in the laboratory (see Hirai & Ishii 2002). To confirm the viability of the eggs, some of the eggs were given to final (fifth) instar larvae of P. sita with a piece of leaf on which the eggs were laid.

Figure 1.

Study sites in the Ryukyu Islands.

Host suitability experiment

We experimentally gave S. bella eggs to larvae of the two danaid species to compare their parasitism rates. Sturmia bella originated from eggs collected on leaves of Marsdenia tomentosa, one of the host plants of P. sita, at Susami Town, Wakayama Prefecture (33°30′N, 135°33′E) in 2000, and had been maintained continuously using P. sita from the Wakayama population as the host. Eggs of the two danaids were obtained from females collected in Iriomote and Ishigaki Islands in 2001 for I. similis and from those collected in Wakayama Prefecture in 2000 for P. sita. Larvae of each danaid species were divided into three groups, L4, L5 and the control, and reared individually in 5 cm plastic Petri dishes during the first to fourth instars, and 120 mL plastic cups during the fifth (final) instars on the artificial diet described by Hirai and Ishii (2001) under a photoregime of 16 h light : 8 h dark (LD 16:8) at 20°C. In groups L4 and L5, each larva was given 10 parasitoid eggs within the day of the third and fourth larval ecdyses, respectively, whereas no parasitoid eggs were given to the control group. In each experiment, S. bella eggs removed from the uteruses of fecund females were given to larval hosts by inoculating them onto the surface of a small amount of the artificial diet. The periods from ingesting the parasitoid eggs to pupation and adult emergence of the hosts were recorded.

Male hosts from which no parasitoids had emerged were dissected under a stereomicroscope immediately after adult emergence to examine parasitism. Individual female hosts were kept under a photoregime of 16 h light : 8 h dark (LD 16:8) at 25°C in a glassine envelope, and were fed daily on a 10% sugared water solution. The hosts were dissected 14 days after adult eclosion to check for both mature eggs and parasitoids in their abdomens. We defined mature eggs as those with a height of approximately 1.6 mm and longitudinal ribs on the surface, and mature females as individuals with at least one mature egg at the proximal end of each ovariole. Melanized objects found inside the host body were removed and examined after maceration in a 10% KOH solution for 1–2 days. When dead larvae of S. bella were found, their body lengths were determined by using an eyepiece micrometer.

Fertility of parasitized I. similis females

We investigated the fertility of I. similis adults obtained from the host suitability experiment. Twelve newly emerged I. similis adults, including six males and six females obtained from the experiment, were numbered on the ventral surfaces of both hindwings using a black felt-tip pen. We released them in a 600 m2 greenhouse insectarium kept at above 25°C that included approximately 1000 butterflies, including approximately 200 I. similis adults, between 30 July and 7 August 2001. The female adults were collected on 9 or 24 August 2001 and dissected after being reared inside a net with a potted host plant to obtain eggs.

Wild-caught I. similis adults

We dissected I. similis adults collected in the Ryukyu Islands between February 2001 and November 2002 to examine parasitism. The procedure was the same as that described above.

RESULTS

Field survey of S. bella eggs on T. tanakae

Eggs and/or larvae of I. similis were found on T. tanakae at all sites investigated in each survey in 1998, 2000, 2001 and 2002 (Table 1), whereas no eggs or larvae of P. sita were found on T. tanakae in any of the surveys. Leaves of T. tanakae with S. bella eggs were found at four sites, A, B, D and E. The number of S. bella eggs per leaf was one in 11 leaves or two in six leaves. The percentages of leaves with S. bella eggs were less than 20% in all surveys, but they were found in both autumn and spring.

Table 1.  Numbers of eggs and larvae of Ideopsis similis, and eggs of Sturmia bella found on Tylophora tanakae at study sites in Iriomote and Ishigaki Islands in March 1998, February 2000, October 2001 and October 2002
DateSitesNo. individuals of T. tanakaeNo. eggs or larvae of I. similisNo. leaves examinedNo. T. tanakae leaves with S. bella eggs (%)No. S. bella eggs per leaf
12
14–18 March 1998A>10>10 533 (5.6)12
B  2  1 101 (10.0) 1
D  2  1 100 (0)  
17–21 February 2000D  2  1 101 (10.0)1 
E>10  7 102 (20.0)2 
F  2  2 100 (0)  
5–10 October 2001A>10>10 443 (6.8)21
B  3  2 160 (0)  
C>10>10 110 (0)  
D>10  2 160 (0)  
E>10  7 101 (10.0)1 
F  7  5 100 (0)  
G  1  1 140 (0)  
25 October 2002A>10  35606 (1.1)42

The inoculation experiment showed that the S. bella eggs collected on leaves of T. tanakae in February 2000 had the ability to parasitize P. sita: three larvae of S. bella emerged from two pupae of P. sita that had ingested the eggs during the final instar, and developed into a female and two male adults.

Host suitability experiment

Most L4 and L5 I. similis developed into adults, whereas most L4 and L5 P. sita were parasitized and killed. Mean periods from the intake of S. bella eggs to pupation of L4 and L5 I. similis were not significantly different from the mean periods of the fourth and/or fifth instars of the control for both males and females (P > 0.05), whereas mean pupal stages of both L4 and L5 were significantly shorter than those of the controls for both sexes (P < 0.05, Mann–Whitney U-test; Table 2) The mean forewing length of L4 males was significantly larger than that of the control males (P < 0.05), whereas there were no significant differences in the mean forewing lengths of L4 females and L5 males and control females (P > 0.05; Table 2). Although all of the L4 and L5 P. sita successfully pupated, round dark spots associated with the respiratory attachments of S. bella larvae appeared on the host integument around spiracles a few days after pupation (Fig. 2A). After the whole body of the host pupa turned dark, the larval emergence of S. bella from the host occurred. The number of S. bella larvae emerging from a single host ranged from one to six. Even in the case of gregarious parasitism, the S. bella larvae emerged almost simultaneously. The mean duration between host pupation and larval emergence of S. bella was 5.0 ± 0.17 (SE) days. The mean pupal stage of S. bella (i.e. the mean duration between larval emergence from the host and adult eclosion of the first individuals) was 15.0 ± 0.16 (SE) days. However, it should be noted that female adult eclosion usually occurred 1 day later than that of males.

Table 2.  Mean developmental time and forewing length of Ideopsis similis parasitized or unparasitized by Sturmia bella
Stage of parasitismSexnMean developmental time (days; mean ± SE)Forewing length
(mm; mean ± SE)
Fourth and fifth instarsFifth instarPupa
  1. Significance data given are between means for individuals that reached adult eclosion and those for the control of the corresponding sex: *P < 0.05,**P < 0.01,***P < 0.001; NS, not significant (Mann–Whitney U-test).

Fourth instar
(Group L4)

 4
 6
15.5 ± 1.3 NS
15.3 ± 0.7 NS

18.8 ± 0.3*
17.8 ± 0.2**
46.6 ± 0.4*
45.3 ± 0.5 NS
Fifth instar
(Group L5)

10
 7

10.2 ± 0.6 NS
8.4 ± 0.5 NS
19.0 ± 0.4**
17.3 ± 0.3***
44.7 ± 0.7 NS
45.0 ± 0.5 NS
Control
23
28
14.7 ± 0.3
15.5 ± 0.3
9.0 ± 0.2
10.1 ± 0.2
20.2 ± 0.4
20.5 ± 0.3
45.0 ± 0.3
44.8 ± 0.3
Figure 2.

Two-day-old pupae of Parantica sita (A) and Ideopsis similis (B) parasitized by Sturmia bella. Arrows indicate dark spots caused by parasitism (see text).

In I. similis, all the L4 and L5 individuals developed and pupated like the control. However, a few irregular shaped dark spots appeared on the integument of the pupal abdomen in some individuals approximately 2 days after pupation (Fig. 2B). Similar spots also appeared on the integument of the adult abdomen in some L4 and L5 individuals (Fig. 3A).

Figure 3.

(A) Lateral view of a male adult of Ideopsis similis with deformity due to parasitism by Sturmia bella on its abdomen (arrow). (B, C) Melanized larvae of S. bella (arrows) in the abdomen of a female adult of I. similis. (D) A melanized S. bella larva dissected from a host. (E, F) Sturmia bella larvae dissected from a host and exposed to 10% KOH.

In most L4 and L5 I. similis adults dissected, melanized S. bella larvae were found inside the abdomen (Fig. 3B–D). They were often found around the spiracles of the host, and some of these were attached to the tracheoles of the host (Fig. 3C). These melanized S. bella larvae were identified based on their morphology by dissolving the clot adhering to the body surface in a KOH solution (Fig. 3E,F).

Adults of L4 and L5 I. similis were classified into four categories according to the extent of parasitization by S. bella: category 0, host adults without any indications of parasitization; category I, normal with melanized S. bella larvae inside the body; category II, with crippled wings and melanized S. bella larvae inside the body; category III, those killed by S. bella that completed development successfully. The sex of hosts in category III was also determined by dissecting the pupae after parasitoid emergence. Out of the 40 I. similis examined, four (10%), 24 (60%), nine (23%) and three (7%) fell into categories 0, I, II and III, respectively (Table 3). Categories I, II, and III were recorded in both L4 and L5, whereas category 0 was only found in L4. All nine adults of category II were females. In category III, only one female S. bella emerged from each host pupa in all three cases. The durations between host pupation and larval emergence of S. bella were 12, 16 and 22 days, which were much longer than that on P. sita mentioned above, and the durations of the pupal stages of S. bella were 16, 17 and 17 days for each case, respectively.

Table 3.  Numbers (%) of adults in categories 0, I, II and III (see text) for Ideopsis similis and Parantica sita parasitized by Sturmia bella as fourth and/or fifth instar larva
HostStage of parasitism nCategories
0IIIIII
I. similisFourth instar 42 (50) 2 (50)0 (0)0 (0)
(Group L4)102 (20) 4 (40)3 (30) 1 (10)
Fifth instar120 (0)11 (92)0 (0)1 (8)
(Group L5)140 (0) 7 (50)6 (43)1 (7)
Total 404 (10)24 (60)9 (23)3 (7)
P. sitaFifth instar 314 (13)0 (0)0 (0)27 (87)

The number of S. bella larvae per I. similis adult ranged from one to six, with means of 2.5 and 1.7 in categories I and II, respectively, which were not significantly different (χ2 = 0.96, d.f. = 2, P > 0.05, Kruskal–Wallis test; Table 4). The body lengths of S. bella larvae found inside the host bodies ranged from 0.5 to 3.5 mm, with the mean being significantly longer in category II females than in both category I males and females (χ2 = 10.4, d.f. = 2, P < 0.01; Table 4).

Table 4.  Mean number of Sturmia bella larvae found per an adult of Ideopsis similis and mean body length of the larvae
Host category No. hosts
examined
No. S. bella larvae per
host (mean ± SE)
No. larvae
examined
Body length of larvae
(mm; mean ± SE)
  • Including individuals of both L4 and L5 groups.

  • Means followed by the same letter are not significantly different at the P = 0.05 level as assessed by non-parametric multiple comparison.

I132.4 ± 0.37311.0 ± 0.10b
112.5 ± 0.49221.0 ± 0.07b
II 0
 91.7 ± 0.22
 χ2 = 0.96
171.8 ± 0.24aχ2 = 10.4
Kruskal–Wallis test  d.f. = 2
P = 0.62
 d.f. = 2
P = 0.0055

The proportion of L4 and L5 females with mature eggs 14 days after adult eclosion was 83% (n = 12) in categories 0 and I, which was not significantly different from that of the control (75%, n = 20; P > 0.05, Fisher’s exact probability test; Table 5). The mean number of mature eggs per mature female was 5.1 ± 1.75 (SE) in categories 0 and I, which was not significantly different from that of the control (6.8 ± 1.17; Mann–Whitney U-test; P > 0.05; Table 5).

Table 5.  Numbers (%) of Ideopsis similis females with mature eggs and the mean number of mature eggs per mature female 14 days after adult emergence at 25°C and with a 16 h light : 8 h dark photoperiod
 No. femalesNo. (%) individuals with mature eggsNo. mature eggs per mature female (mean ± SE)
  • Including individuals of both groups L4 and L5.

  • Fisher’s exact probability test.

  • §

    Mann–Whitney U-test.

0 and I1210 (83%)5.1 ± 1.75
Control2015 (75%)6.8 ± 1.17
P 0.680.33§

Fertility of parasitized I. similis females

Three males and one female out of 12 I. similis adults (six males and six females) released in the greenhouse were recaptured on both 9 and 24 August 2001. Two of the three males were recaptured again on 24 August 2001. The female recaptured on 9 August, which had been released on 31 July, laid unfertilized eggs, and showed no signs of parasitism at dissection on 2 September 2001. The female recaptured on 24 August, which had been released on 3 August, laid approximately 50 fertile eggs, although it had six dead S. bella larvae in its abdomen when dissected on September 2, 2001.

Wild-caught I. similis adults

A total of nine (eight males and a female) out of 125 I. similis adults collected in Iriomote, Ishigaki, Okinawa and Tokunoshima Islands contained dead dipteran larvae, which were considered to be S. bella on the basis of a morphological inspection (Table 6, Fig. 4). The I. similis adults with dipteran larvae were collected in February, March, August, September and October. The mean proportion of I. similis adults with dipteran larvae was 10% in males and 3% in females, although the difference was not significant (Fisher’s exact probability test: P > 0.05; Table 6). The number of dipteran larvae per I. similis adult ranged from one to four, and the body lengths of dipteran larvae ranged from 0.3 to 0.7 mm.

Table 6.  Number of Ideopsis similis adults with tachinid larvae in their bodies collected in Ishigaki, Iriomote and Okinawa islands between February 2001 and November 2002
Collection siteDate No. I. similis
adults
collected
No. I. similis
adults with
S. bella larvae
No. S. bella
larvae per
host
Range of body
length of S. bella
larvae (mm)
  • Fisher’s exact probability test for the proportion of I. similis adults with and without S. bella larvae in males and females.

Ishigaki Island4 February 2001
10
 1
1
0
1
0.45
5–10 October 2001
 8
 8
0
0

 
22–24 February 2002
 5
 3
1
0
2
0.30–0.40
26–31 March 2002
 3
 3
0
0

 
14–16 May 2002
26–27 October 2002


 2
10
 2
0
1
0

1
0.30
Iriomote Island9–11 March 2002
5–10 October 2001


 8
18
10
1
1
0
3
1
0.40–0.65
0.50
26–31 March 2002
 7
 3
0
1

2
0.40–0.50
14–16 May 2002 10 
23 October 2002
10
 1
0
0

 
Okinawa Island3 October 2001 2140.40–0.70
17 September 2002
 2
 1
1
0
1
0.40
17 November 2002 10 
Amamioshima Island12 September 2002 10 
Nakanoshima Island9 October 2002
 1
 1
0
0

 
Tokunoshima Island15 October 2002
 2
 1
1
0
1
0.50
Total
90
35
8
1
P = 0.443 
Figure 4.

Photomicrograph of a tachinid larva dissected from the field collected adults of I. similis collected in Iriomote Island in March 2002 and exposed to 10% KOH.

DISCUSSION

In the field survey of this study, viable eggs of S. bella were found on leaves of T. tanakae, the larval food plant of I. similis, and the larvae of no herbivorous insect except I. similis was found on the leaves, indicating that I. similis is one of the potential hosts of S. bella in the field. The physiological defense of a host species is an important factor determining the host range of parasitoids (Alleyne & Wiedenmann 2001). However, few studies have addressed the defense mechanisms of non-hosts against parasitic flies. The present study demonstrates that unlike P. sita, which is a natural host of S. bella, I. similis is unsuitable for parasitism by S. bella.

An important physiological mechanism used by lepidopteran hosts as a defense against endoparasitoids is the encapsulation response, in which host hemocytes recognize the invader as non-self, attach, and form a capsule around the parasitoid, thereby killing it (e.g. Salt 1963, 1968; Ratcliffe & Rowley 1979; Pech & Strand 1996). In the present study, most S. bella larvae were killed inside the adult bodies of I. similis. It is possible that unsuccessful development of S. bella within the I. similis body was caused by the strong hemocytic reaction of the host, resulting in encapsulation. Although there are some instances of lepidopteran hosts being parasitized by tachinids and developing into adults, there have been no reports of lepidopteran adults with dead tachinid larvae inside the body. DeVries (1984) reported that the larvae of a nymphalid butterfly, Cissia confusa Staudinger (1887), recover from attack by the tachind Eumasiscera sp. without any reduction of adult fecundity, although both hosts and parasitoids emerge as adults. English-Loeb et al. (1990) also found a similar relationship between the host arctiid moth Platyprepia virginalis (Boisduval, 1852) and the tachind parasitoid Thelairia bryanti Curran, 1925. When the tachinid Archytas marmoratus (Townsend, 1915) attacks the noctuid moth Helicoverpa zea (Boddie, 1850), most tachinids fail to develop to late final instars, because of insufficient time to complete larval development before host pupation (Bratti et al. 1992).

It has been suggested that there is a trade off between defense against invaders and other physiological processes in host insects parasitized by parasitic wasps. For example, Drosophila hosts that encapsulated cynipid wasps took a longer time to complete development, and developed into smaller female adults, which laid fewer eggs compared with unparasitized hosts (Carton & David 1983; Fellowes et al. 1999). In the present study, the mean pupal stage of I. similis females parasitized by S. bella was less than that of unparasitized individuals, and the size and ability to produce mature eggs were not different from the controls. In addition, I. similis females parasitized by S. bella produced fertile eggs. Thus, no trade-off was found, at least between defense against S. bella and physiological processes such as development and reproduction in I. similis.

Ideopsis similis uses only one plant species as a host in each habitat (Honda et al. 1995, 2001; Abe et al. 1998), whereas P. sita uses several out of approximately 20 plant species known to be hosts in each habitat (e.g. Fukuda 2003). Furthermore, adults of I. similis have been seen almost all year round in Iriomote Island, whereas no adults of P. sita were found there between July and September (Nagamine 1997, 1999). Thus I. similis is a sedentary butterfly with monophagaous feeding habits, whereas P. sita is a migrant using a large variety of milkweeds as larval food plants. Ohsaki and Sato (1990, 1999) and Sato (1976) studied the relationship between habitat utilization of Pieris butterflies and parasitism of a braconid wasp, Cotesia (=Apanteles) glomeratus (Linnaeus, 1758), and pointed out that Pieris melete (Menetries, 1857), with a physiological defense ability to encapsulate eggs of the braconid, is sedentary and uses permanent habitats, whereas Pieris rapae (Linnaeus, 1758), with no physiological mechanism to avoid the braconid, always disperses and uses temporary habitats to avoid parasitization. Although there is no direct evidence of seasonal migration in Japan for P. rapae, there are many records of long-distance migration and seasonal alternation of habitats in P. sita (Fukuda 1991; Hirai & Ishii 1997). Thus, it is possible that P. sita, an important host of S. bella, may escape from parasitization by using their migratory and polyphagous capacities, whereas I. similis remains sedentary and monophagous because of its ability to overcome parasitization physiologically. Field collection of I. similis females with dead dipteran larvae in August on Iriomote Island may support this hypothesis.

ACKNOWLEDGMENTS

We express our sincere thanks to Mr M. Ushirokita and other members of the Itami City Museum of Insects for their technical assistance with experiments in the greenhouse insectarium. We are grateful to Dr Y. Nakatani of the National Institute for Agro-Environmental Sciences and Mr Y. Tanaka of Itami City Parks and Greenery Association for their help with collection of insects. Our thanks are also due to the Susami Town Board of Education, and the Iriomote station of the Tropical Biosphere Research Center of the University of the Ryukyus for allowing the use of their fields and facilities. We are indebted to Dr T. Hirowatari, Mr Y. Miyamoto, Dr Y. Sawada, Dr Y. Nishinaka, Mr S. Miyazaki, Dr N. Ahn, Dr B. Lee and other members of the Entomological Laboratory, Osaka Prefecture University for collecting insects and their helpful comments. This study was partly supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (nos. 11896001 and 12308028).

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