WAGENINGEN UNIVERSITY LABORATORY OF ENTOMOLOGY

Egg parasitoid community on Heliconiini butterflies in a Panamanian rainforest

No: 08.21 Name: Joop Woelke Period: February 2008 – June 2008 Thesis: ENT-80424 Supervisor: Ties Huigens 2nd examinator: Marcel Dicke Abstract

Egg parasitoids use insect eggs as food for their young by laying there own eggs inside their host eggs. They can find their host eggs in many different ways. One strategy includes the chemical espionage on anti-aphrodisiac pheromones of cabbage white butterflies that are transferred from males to females to render females less attractive to other males. Minute Trichogramma wasps exploit these pheromones by specifically hitch-hiking with mated female butterflies to an oviposition site. Anti-aphrodisiac pheromones are also known from neotropical Heliconius butterflies like Heliconius erato and Heliconius melpomene . In a tropical lowland rainforest around the Pipeline road area of the Soberania National Park (Panama) Heliconiini eggs are known to be parasitized by parasitoid wasps.

This study includes a field survey that represents a first step to understand if hitch-hiking parasitoid wasps constrain the use of anti-aphrodisiac pheromones by Heliconiini butterflies in nature. I describe a survey of the egg parasitoid community on Heliconiini butterflies in the area around Pipeline road from February to April 2008 in which not only parasitism rates of Heliconiini eggs on different Passiflora plant species were determined but also adult Heliconiini butterflies were monitored for the presence of different families of egg parasitoid wasps. In total 317 Heliconiini eggs were found on 6 Passiflora plant species and 51 of them were parasitized by egg parasitoids (parasitism rate = 16.1%). Three egg parasitoid families were collected from the eggs, being respectively Encyrtidae , Scelionidae and Trichogrammatidae wasps. Besides this, 133 adult Heliconiini butterflies were caught. In total 18 parasitoid wasps were found on the butterflies (hitch-hiking rate = 13.5%). These hitch- hiking wasps are not only belonged to the families Encyrtidae, Scelionidae and Trichogrammatidae but also to the Aphelinidae , Ceraphronidae , Eulophidae and Mymaridae . All families are known to contain egg parasitoids of Lepidoptera except the Ceraphronidae .

Overall it can be concluded that Heliconiini butterflies are attacked by a rather stable community of egg parasitoids belonging to three different families and that some egg parasitoid species probably hitch-hike with adult butterflies to find their eggs. In future work the species identity of these collected wasps needs to be determined and laboratory bioassays need to reveal whether these wasps exploit an anti-aphrodisiac by specifically hitch-hiking with mated female Heliconiini butterflies.

Key words: Host finding, anti-aphrodisiac pheromone, phoresy, egg parasitoids, Trichogramma tidae wasps, Encyrtidae wasp, Scelionidae wasps, Heliconiini butterflies, Heliconiini eggs, Passiflora plants.

2 Table of Contents

1 Introduction 4

2 Material and methods 7 2.1 Field work 7 2.1.1 Research locations 7 2.1.2 Passiflora plants 7 2.1.3 Egg collection 7 2.1.4 Butterfly catches 8 2.2 Laboratory 8 2.2.1 Butterflies 8 2.2.2 Eggs 8 2.2.3 Wasp rearing 9

3 Results 10 3.1 Egg parasitism 10 3.2 Hitch-hiking wasps 13

4 Discussion 15

Acknowledgements 17

References 18

Appendix 1: Research locations 21

Appendix 2: Research Passiflora species 22

Appendix 3: Research Heliconiini species 23

Appendix 4: Research data egg parasitism 26

3 1 Introduction

Insect eggs are a nutrient-rich source and are used by egg parasitoids for the development of their young by laying their own eggs into other insect eggs (Fatouros et al ., 2008). Egg parasitoids are also used world wide as biological control agents against pest insects (Smith, 1996). They can cause high mortality rates in their hosts. For example the egg parasite Oomyzus gallerucea can parasitize egg masses of the elm leaf beetle (Xanthogaleruca luteola ) ranging from 26.3 to 51.7% (Puttler & Bailey, 2003). Also from Trichogramma species it is known that they can cause parasitism levels up to 80% in the field (Smith, 1996).

Egg parasitoids use a wide variety of strategies to find their hosts (Fatouros et al ., 2008). One method is to recognize chemical plant cues induced by the deposition of eggs by their host insects (Fatouros et al ., 2005a; Hilker & Meiners, 2002). This benefits the plant as well as the parasitoid (Stowe et al ., 1995). The egg parasitoid Trichogramma brassicae can detect plant cues of the host plant (Brussels sprouts) of the butterfly Pieris brassicae . After laying their eggs, Brussels sprouts leaves release chemical cues that indicate that there are butterfly eggs on the leaves. Trichogramma brassicae wasps can detect those chemical cues and use them to find the P. brassicae eggs (Fatouros et al ., 2005a).

Another method is the recognition of chemical cues from the adult host stage (Vet & Dicke, 1992). For example, it is known from laboratory olfactometer experiments that female Trichogramma evanescens wasps respond to sex pheromones emitted by females of the moth Mamestra brassicae (Noldus & Van Lenteren, 1985). This seems surprising because the wasps are only active during day time and the M. brassicae moths release the sex pheromones at night (Noldus & Van Lenteren, 1985). Probably, the wasps can detect traces of the sex pheromone that are adsorbed onto the leaf surfaces (Noldus & Van Lenteren, 1985). The wasps respond to the pheromone odour and, after landing, probably rely on visual and short-range chemical cues to indicate where the eggs can be found. Recognizing sex pheromone scents could then be useful cues to find moth eggs (Stowe et al ., 1995).

Female wasps of Telenomus calvus parasitize eggs of the bug Podisus maculiventris . The wasps are attracted to an aggregation pheromone emitted by male bugs and, when the bugs start mating, they climb onto female bugs and subsequently hitch-hike with them. Telenomus calvus wasps need for their development freshly laid eggs that are not older than 12 hours. By hitch-hiking with just mated female bugs the female wasps can access to freshly laid eggs that are not yet discovered by other competing parasitoids (Aldrich, 1995; Aldrich et al ., 1985; Orr, et al ., 1986).

Fatouros et al . (2005b) recently discovered a new strategy in which the parasitic wasp T. brassicae exploits the anti-aphrodisiac pheromone, benzyl cyanide, of the large cabbage white butterfly P. brassicae , that is passed from male to female butterflies to render females less attractive to other males. By specifically climbing onto a mated P. brassicae female and hitch-hiking with her to a cruciferous host plant, the wasp has direct access to freshly laid butterfly eggs and parasitizes them by laying its own eggs into the butterfly eggs at the cost of caterpillar development (Fatouros et al ., 2005b).

This strategy of hitch-hiking with adult hosts is known for more parasitoid wasps. Until now this is know for more than 30 egg parasitoid wasp species belonging to 6 families (Fatouros, 2006; Woelke, 2008). For most of those egg parasitoid species it is unknown how the parasitoid finds its adult host.

Hitch-hiking with an adult mated female host and thereby exploiting a host’s anti-aphrodisiac pheromone is expected to be a common strategy among egg parasitoids, especially among those that have limited flight control, suffer from competition with other parasitoids, and need to parasitize freshly laid eggs (Fatouros et al ., 2005b). Besides cabbage white butterflies,

4 neotropical Heliconius butterflies have also been described to use anti-aphrodisiac pheromones.

One of the subfamilies of Nymphalid butterflies is the subfamily Heliconiinae . Adults of this subfamily have a forewing length between 23 and 54 mm (Chacon & Montero, 2007). In the butterflies of the Heliconiinae is a great biological interest. They are especially used in studies on mimetism, evolution and ecology of lepidopterans (Chacon & Montero, 2007). Heliconiinae are found in the tropics of the Old World, mainly in Africa and in the Neotropics. Only some species live in the temperate zones of North America. The subfamily Heliconiinae exists of 400 species in 40 genera (Ackery et al ., 1999). The host plants where the caterpillars of this subfamily feed on are Urticaceae , Violaceae , Asteraceae , Turneraceae , Flacourtiaceae and Passifloraceae (DeVries, 1987; Janzen & Hallwachs, 2005). One of the tribes of the subfamily Heliconiinae is the tribe Heliconiini. Heliconiini butterflies are found in the Neotropics and comprise of 72 species (Lamas, 2004). The butterflies of this tribe have a forewing length of 27 till 54 mm. They have attractive colourful colours. The forewings are extended and the head is large with long antennae. Females lay the eggs singly or in groups. The eggs are generally yellow or orange and have an oval shape. The caterpillars have a pair of horns on the head and six rows of spines on the body. They feed on plants of the families Turneraceae and Passifloraceae ( Passiflora plants) (DeVries, 1987; Janzen & Hallwachs, 2005). Some of the Passiflora plant species produce glands that mimic eggs laid by female butterflies of the genus Heliconius (Chacon & Montero, 2007). Adults feed on nectar and pollen. Heliconiini butterflies spent the night together in big groups in places with climbing vines, normally near to banks of creeks (Chacon & Montero, 2007).

When handled, all Heliconius species and related genera release strong odours that can even be detected by humans (Eltringham, 1925). The males display odoriferous glands between the eighth and ninth abdominal segment (Chacon & Montero, 2007). The glands are inside two highly chitinized claspers. With this, the male grabs the female abdomen during copulation (Eltringham, 1925; Emsley, 1963; Simonsen, 2006). In the female, on the other hand, the strong odours are produced in a large yellow gland. When extruded, the odours are probably distributed by exposing a pair of cub-shaped structures (Eltringham, 1925; Emsley, 1963; Simonsen, 2006). Probably, those strong odours act as an anti-aphrodisiac for Heliconius erato butterflies (Gilbert (1976). Gilbert (1976) suggested that males of H. erato transport an anti-aphrodisiac pheromone to female H. erato butterflies during mating thereby probably rendering the female less attractive to other males. However, he did not identify the chemical compound(s) (Gilbert, 1976). More recently, Schulz et al. (2007) also found evidence that this is likely to be the case in more Heliconiini species and this year they identified the first anti-aphrodisiac, β-ocimene, for Heliconius melpomene (Schulz et al ., 2008).

Interestingly, eggs of Heliconius butterflies, and other members of the Heliconiini tribe, are known to be attacked by parasitoids (Almeida de, 2004; Fleming et al ., 2005; Longino, 1984; Naisbit, 2001; Queroni & Zucchi, 2002; Queroni & Zucchi, 2003a; Queroni & Zucchi, 2003b). In a tropical lowland rainforest around the Pipeline road area of the Soberania National Park (Panama) even up to 33.3% of their eggs can be killed by parasitoid wasps (Naisbit, 2001; table 1.1A, B).

5 Table 1.1 Rate of egg parasitism (A) and number of eggs (B) found by Naisbit (2001) on different Passiflora plants. (A) Passiflora species N˚ of eggs N˚ of parasitised eggs Egg parasitism (%) Passiflora auriculata 45 7 15.6 Passiflora ambigua 10 0 0 Passiflora biflora 15 1 6.7 Passiflora coriacea 16 1 6.3 Passiflora menispermifolia 195 17 8.7 Passiflora tryphostemmatoides 2 1 50 Passiflora nitida 15 5 33.3 Passiflora vitifolia 215 34 15.8

(B) Butterfly species P. auriculata P. biflora P. coriacea P. foetida P. menispermifolia P. vitifolia Agraulis vanillae 1 Dione juno 70 Dryas iulia 33 6 8 1 Eueides spp. 1 11 Heliconius cydno 19 14 15 Heliconius erato 3 12 13 Heliconius hecale 1 4 126 Heliconius melpomene 1 146 1 Heliconius sara 46 Philaethria dido 1 19

This natural system of Heliconiini butterflies and egg parasitoids in Panama may offer an ideal opportunity to investigate to what extent hitch-hiking egg parasitoids select against the use of an anti-aphrodisiac by butterflies. This first needs an extensive follow-up field survey that expands the work by Naisbit (2001), with a more detailed description of the egg parasitoid families. Here, I describe such a follow up survey of the egg parasitoid community on Heliconiini butterflies in the area around Pipeline road. Not only parasitism rates of Heliconiini eggs on different Passiflora plant species were determined but also adult Heliconiini butterflies were monitored for the presence of different families of egg parasitoid wasps.

6 2 Material and methods

2.1 Field work

2.1.1 Research locations Field work was done in a tropical lowland rainforest around Gamboa during a period of 10 weeks (from 3 February to 10 April 2008). Gamboa is a small village about 35 km north of Panama City (Republic of Panama) that is boarded by the Soberania National Park. For collecting eggs and butterflies of the tribe Heliconiini, five research locations were chosen (see appendix 1). The research locations were Pipeline road, Oil bunkers, Gamboa, Sendro la laguna and Camino de plantación. Pipeline road is a road that goes deep into the rainforest of the Soberania National Park. Alongside this road, there are a lot of open spaces where Passiflora plants have the opportunity to grow. Oil bunkers is a trail parallel to the Panama Canal that goes into the rainforest and that begins at the oil bunkers outside Gamboa. The location Gamboa consisted of several places in Gamboa where Passiflora plants were growing. Sendro la laguna is a trail between Gamboa and the Gamboa rainforest resort that is located just outside Gamboa. Camino de plantación is a trail in a rainforest about 6 km outside Gamboa on the way to Panama City. The trail is in a more dry area containing lots of rocks. For all research locations a collecting permit was given by the Autoridad Nacional del Ambiente (Permit no. SE/AP-3-08).

2.1.2 Passiflora plants In Gamboa and surroundings 6 Passiflora plant species were found. Those species are Passiflora auriculata , Passiflora biflora , Passiflora coriacea , Passiflora foetida , Passiflora menispermifolia and Passiflora vitifolia (see appendix 2). At each research location Passiflora plants were numbered and labelled with a red flag (figure 2.1).

Figure 2.1 Labelled Passiflora coriacea plant.

In total 122 plants were found and labelled. At Pipeline road 84 Passiflora plants were found, at Oil bunkers 11 plants, at Gamboa 7 plants, at Sendro la laguna 12 plants and at Camino de plantación 8 plants. In total 17 P. auriculata plants, 16 P. biflora plants, 17 P. coriacea plants, 5 P. foetida plants, 13 P. menispermifolia plants and 54 P. vitifolia plants were found. After the research all labels were removed from the plants.

2.1.3 Egg collection Each labelled Passiflora plant was checked once (and sometimes twice) a week for eggs of Heliconiini butterflies. Unfortunately, it was impossible to identify the butterfly eggs to genus or species level. Most of the time eggs can be found on the young shoots of the plants or on the tendrils (figure 2.2) and only sometimes on the older parts. Eggs are laid individually but sometimes multiple eggs can be found on one plant.

7

Figure 2.2 Young leaf of Passiflora foetida with two Heliconiini eggs.

When an egg was found on a plant, a small plant part containing the egg was carefully removed from the plant and put separately in a small glass vial with lid. All vials were taken to the laboratory in the school building of the Smithsonian Tropical Research Institute in Gamboa and left until caterpillar or wasp emergence.

2.1.4 Butterfly catches At all locations, adult butterflies of the tribe Heliconiini were collected. The species that were collected around Gamboa were Agraulis vanillae , Dione juno , Dryas iulia , Eueides aliphera gracilis , Eueides lybia olympia , Heliconius cydno , Heliconius doris (three colour variants: red, blue and green), Heliconius erato , Heliconius hecale (two different colour variants), Heliconius ismenius , Heliconius melpomene , Heliconius sapho , Heliconius sara and Philaethria dido (see appendix 3). The butterflies were collected with a butterfly net with a plastic pot inside (attached with a rubber band). The plastic pot inside the butterfly net should avoid wasps to escape through the net. When a butterfly was caught it was put as soon as possible in a cool box with a cool element inside to decrease their activity. After collection, the pots were placed overnight in a refrigerator (4˚C).

2.2 Laboratory The collected eggs and butterflies from the field were further treated in the laboratory under standard room conditions (21°C and 60% humidity).

2.2.1 Butterflies After a night in the refrigerator, the butterflies and pots were monitored for parasitoid wasps under a microscope. All egg parasitoid wasps were identified to family level. Encyrtid , Scelionid and Trichogrammatid wasps were sexed and then kept alive to start cultures on Heliconius eggs or they were put in 95% alcohol. After monitoring, the butterflies were fed with honey water and released in the same area where they were caught.

2.2.2 Eggs After collecting eggs in the field, eggs were stripped of the small plant parts to avoid fungal growth in the vials. Each day the small vials were opened once to decrease the humidity. When a caterpillar was found in the vial, it was released back into the field on the Passiflora plant species where the egg was found on. When a wasp was found in the vial and it was not belonging to the families Encyrtidae , Scelionidae or Trichogrammatidae , the wasp was put on alcohol (95%) after sexing it first. When it was an Encyrtid or Scelionid wasp, it was sexed under a microscope and after that put on 95% alcohol or a culture was started. With the Trichogrammatid wasps a culture was started. Eggs from which no caterpillar or wasp(s) emerged were not taken into account during this research.

8 2.2.3 Wasp rearing To start Trichogramma cultures, wasps were offered the opportunity to parasitize 10 up to 24 hours old eggs of Heliconius erato or Heliconius melpomene (eggs from the butterfly rearing of the Smithsonian Tropical Research Institute) for up to 5 hours in a large glass vial closed with a wet cotton lid. Also some honey was added as food for the wasps. The wasps were reared under a lamp with a light period of 9 hours and 15 hours of darkness. When the light was off the conditions were the same as the previously mentioned room conditions. When the light was on the temperature was 26°C (with a h umidity of 50%). After four days the eggs that were parasitized became black and after 10 to 14 days the wasps emerged. After parasitizing fresh Heliconius eggs the wasps were put in a freezer for 10 minutes. After 10 minutes they were sexed under a microscope and put in 95% alcohol. The rearing of the Encyrtid and Scelionid wasps was the same as described above, only they were put on 5 freshly laid Heliconius eggs.

9 3 Results

3.1 Egg parasitism In figure 3.1 the results of the egg parasitism rates (= (number of eggs from which wasps emerged / total number of eggs from which wasps or caterpillars emerged) x100) per Passiflora species by three different families of egg parasitoids are shown per research location (for more detailed information see appendix 4). In total 317 Heliconiini eggs were found and 51 of them were parasitized by egg parasitoids (parasitism rate = 16.1%).

A 2 50 B 50 45 45 12 15 40 40 35 50 35 30 4 30 25 25 157 20 13 55 20 15 15 13 % egg% parasitism

10 egg% parasitism 22 10 5 5 0 1 0 0 0 0 N = 17 N = 9 N = 9 N = 5 N = 5 N = 39 N = 84 N = 0 N = 1 N = 3 N = 0 N = 5 N = 2 N = 11 C D 50 50 45 45 40 40 35 10 35 30 30 25 25 35 20 20 15 54 15 64

% egg% parasitism 10 egg% parasitism 10 44 5 5 0 0 0 0 0 524 0 0 0 0 N = 0 N = 2 N = 0 N = 0 N = 0 N = 5 N = 7 N = 0 N = 3 N = 5 N = 0 N = 0 N = 4 N = 12 F E 2 50 50 45 45 40 40 35 35 30 23 34 30 25 25 9 20 13 55 145 317 b 20 27 15 a

15 egg% parasitism 10

% egg% parasitism 10 16 5 47 a 5 0 0 0 0 0 Passiflora Passiflora Passiflora Passiflora Passiflora Passiflora Total Passiflora Passiflora Passiflora Passiflora Passiflora Passiflora Total auriculata biflora coriacea foetida meni- vitifolia N = 122 auriculata biflora coriacea foetida meni- vitifolia N = 84 N = 17 N = 16 N = 17 N = 5 spermifolia N = 54 N = 17 N = 9 N = 9 N = 5 spermifolia N = 39 N = 13 % Scelionidae N = 5 plant species plant species % Trichogrammatidae % Encyrtidae

Figure 3.1 Egg parasitism rates per plant species per research location per egg parasitoid family. On the x-as the number of plants per Passiflora species is indicated. Above each bar the number of collected eggs is shown. The research locations are: A = Pipeline road, B = Oil bunkers, C = Gamboa, D = Sendro la laguna and E = Camino de plantación. The overall results are shown in F. In Figure 3.1F the last bar also shows that there is no difference between the parasitism rates by wasps of the families Encyrtidae and Trichogrammatidae but that there is a difference between the parasitism rates by wasps of the families Encyrtidae and Scelionidae and also between wasps of the families Trichogrammatidae and Scelionidae (indicated with a and b, α < 0.05, Chi-square test with contingency tables).

In total, 3 egg parasitoid families were collected, being respectively Encyrtidae (with most likely wasps of the genus Ooencyrtus ), Scelionidae (with most likely wasps of the genus Telenomus ) and Trichogrammatidae (with most likely wasps of the genera Trichogramma and Trichogrammatoidea ). Only one wasp of the families Encyrtidae and Scelionidae emerged per egg. The Trichogrammid wasps had an average of 9 wasps emerging per egg up to a maximum of 20 wasps per egg. Most eggs were parasitized by either Trichogrammatid or Encyrtid wasps and only one egg by a wasp of the family Scelionidae (figure 3.1F).

10 There was no significant difference between the parasitism rates by wasps of the families Encyrtidae and Trichogrammatidae (P=0.556, α < 0.05, Chi-square test with 2x2 contingency table), but there were significant differences between the parasitism rates by wasps of the families Encyrtidae and Scelionidae and also between wasps of the families Trichogrammatidae and Scelionidae (both P<0.0001, α < 0.05, Chi-square test with 2x2 contingency table).

During this research also clusters of eggs of Dione juno were found. They were only found at research location Gamboa on Passiflora vitifolia plants. In total 996 D. juno eggs were found (up to 243 eggs per cluster). None of the collected D. juno eggs were parasitized.

Not all plant species were found at all research locations. The egg parasitism rates by the different parasitoid families on the different plant species differed to large extent per research location (figure 3.1). For example Passiflora biflora is found in all research areas but in Pipeline road and Camino de plantación only Trichogrammatid wasps emerged from the parasitized eggs on that plant whereas in Oil bunkers and Gamboa only Encyrtid wasps emerged from the parasitized eggs on P. biflora . On P. vitifolia all three egg parasitoid families can be found in Pipeline road whereas in Gamboa only Encyrtidae and Trichogramma wasps, and in Sendro la laguna and Camino de plantación only Encyrtid wasps, can be found from parasitized eggs.

A B 50 50 45 45 40 40 35 35 30 30 a 25 25 a 20 a 20 a 15 15 a a a % egg% parasitism 10 egg% parasitism 10 ab ab 5 b b 5 b 0 0 a a ia a ea i da ol i lora c fol ora i if fol ia mifolia fl iace bif r r viti bi r foet rm a co pe ra co a pe is ra o or flor n flo fl l flora viti sif ssi me ssi ssi iflora s menis ssi a s a a Pa Passiflora foetida ra P Pa P ra P Passiflora o Pas o Passiflora auriculata % Encyrtidae Passiflora auriculata fl % Trichogrammatidae ssi a Passifl P plant species plant species

Figure 3.2 Egg parasitism rates per plant species by Encyrtid (A) and Trichogrammatid wasps (B). a and b indicate significant differences, α < 0.05, Chi-square test with contingency tables).

There was substantial variation in egg parasitism on the different Passiflora plant species (figure 3.1F; 3.2A, B). The highest egg parasitism rate occurred on P. biflora (26.1%) and the lowest on Passiflora coriacea (2.1%). On P. coriacea only wasps of the family Encyrtidae were found and on Passiflora foetida and on Passiflora menispermifolia only wasps of the family Trichogrammatidae were found. On P. vitifolia all three egg parasitoid families were found emerging from the Heliconiini eggs (figure 3.1F). Figure 3.2 shows the significant differences between parasitism rates of Encyrtid (figure 3.2A) and Trichogrammatid wasps (figure 3.2B) on the Passiflora plant species.

During the research the number of collected eggs is increasing slowly with a peak in week 4 and 5 (figure 3.3). The parasitism rates (figure 3.4F) over the ten weeks seem to be quite stable for all the three egg parasitoid families. But as shown in figure 3.4 the parasitizing rates per research area are different over the time and different between each other.

11 100 90 80 70 60 Nr. of eggs 50 Nr. of parasitized eggs 40 30

Nr. Nr. of collected eggs 20 10 0 Week Week Week Week Week Week Week Week Week Week 1 2 3 4 5 6 7 8 9 10 collection date

Figure 3.3 Number of collected eggs per research week. In total there were 10 research weeks between the period of February 3 rd to April 10 th 2008.

A 100 B 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 % egg% parasitism % egg% parasitism 20 20 10 10 0 0 C 100 D 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 % egg % parasitism % egg% parasitism 20 20 10 10 0 0 E 100 F 100 90 90 % Encyrtidae % Scelionidae 80 80 % Trichogrammatidae 70 70 60 60 50 50 40 40 30 30 % egg% parasitism % egg% parasitism 20 20 10 10 0 0 Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week Week 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 collection date collection date

Figure 3.4 Egg parasitism rate over the research weeks by the three different egg parasitoid families. The research locations are: A = Pipeline road, B = Oil bunkers, C = Gamboa, D = Sendro la laguna and E = Camino de plantación. The overview of all research locations is shown in F.

12 3.2 Hitch-hiking wasps During this research, in total 133 adult butterflies of the tribe Heliconiini were collected. The butterflies are not easy to catch because they fly rather fast and, in some cases, at several meters above ground. In total 18 hitch-hiking parasitoid wasps were found and both on male and female butterflies (figure 3.5). Only one individual parasitoid was found on one individual butterfly. The total percentage of butterflies containing hitch-hiking parasitoid wasps was 13.5%. Heliconius hecale contained most hitch-hiking parasitoid wasps, 33.3% (butterfly sex not included). The wasps belonged to the families Aphelinidae , Ceraphronidae , Eulophidae , Mymaridae , Encyrtidae, Scelionidae and Trichogrammatidae . All families are known to consist of egg parasitoids of Lepidoptera except the Ceraphronidae . Two Ceraphronidae wasps were both found on female Dryas iulia butterflies. One Encyrtid wasp was found on a male or female D. iulia butterfly. This is unknown because both sexes were caught together in a vial. All butterflies containing a wasp were collected at Pipeline road except one female H. hecale butterfly containing an Eulophidae wasp was caught at Camino de plantación. When looking at the three different egg parasitoid families that emerged from the collected Heliconiini eggs, it is obvious that the Scelionidae and Trichogrammatidae can be found on 3 different Heliconiini species and the Encyrtidae on one Heliconiini species. One Encyrtid wasp was found on D. iulia (hitch-hiking rate = 1/24x100= 4.2%). Three wasps belonging to the Trichogrammatidae were found: one on a Heliconius doris male (5.6%), one on a H. hecale female (11.1%) and one on a Heliconius sara female (16.7%). Besides this three Scelionid wasps were found: one on a Eueides lybia olympia female (100%), one on a H. hecale male (16.7%) and one on a H. sara female (16.7%).

1/1 Trichogrammatidae 100 Scelionidae 90 80 Mymaridae

70 Eulophidae

60 2/4 Encyrtidae 50 4/9 Braconidae 40 (%) Aphelinidae

30 2/14 3/10 2/6 1/5 20 1/6 1/11 10 1/18 1/16 0/4 0/3 0/4 0/0 0 ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀/ ♂

Proportion with hitch-hiking wasps

Dryas iulia Eueides Heliconius Heliconius Heliconius Heliconius Parides Anartia lybia olympia doris hecale sapho sara arcas mylotes fatima

Heliconiini Other

Caught butterfly species with a wasp

Figure 3.5 Proportion of butterfly species with a hitch-hiking wasps belonging to 7 different families. Above each butterfly sex the number of hitch-hiking wasp(s) and the number of caught butterflies are indicated. Not only hitch- hiking wasps were found on the research butterflies of the tribe Heliconiini but also on two other butterfly species (Parides arcas mylotes and Anartia fatima ).

As can be seen in figure 3.5, hitch-hiking egg parasitoids were not only found on Heliconiini butterflies. Two other butterfly species, Anartia fatima and Parides arcas mylotes , can also contain parasitoids. On A. fatima a female Trichogrammatid wasp, most likely belonging to the genus Trichogramma , was found. Unfortunately, it is impossible to distinguish between A. fatima sexes by eye under a microscope. There were also other butterflies caught during the

13 field work but none of them contained an egg parasitoid. Those species were: Castniomera atymnius futilis (1 female), Fountainea halice chrysophana (1 female), Marpesia petreus petrus (1 male), Memphis eurypyle confusa (1 female), Parides eurimedes mylotes (1 male) and Parides sesostris zestos (1 male).

14 4 Discussion

This field study represents a second more detailed attempt to describe the egg parasitoid community on Heliconiini butterflies in a Panamanian tropical lowland rainforest. The parasitism rates found on six different Passiflora species ranged between 2.1% (on Passiflora coriacea ) and 26.1% (on Passiflora biflora ) in a dry season period from February 2008 to April 2008. This is in accordance with Naisbit (2001), who found similar parasitism rates in a period ranging from August 1998 to March 2000 in which he routinely checked eight species of Passiflora plants between January and July 1999 (dry and mostly wet season). Naisbit (2001) only used Pipeline road as a research location. In this research Passiflora plants were monitored for eggs at 5 locations in and around Gamboa, including Pipeline road. During the dry period there are fewer butterflies active than during the wet period (Naisbit, 2001). Maybe in the other periods other and/or more egg parasitoids are active. A comparison of the results in this study with those of Naisbit (2001) suggests parasitism in the dry and wet period to be quite stable. However, it is unknown whether there are differences in parasitism on the species level between the two periods.

Like Naisbit (2001), three parasitoid families emerged from the collected Heliconiini eggs. The difference is that this study also described the relative importance of these three families: Encyrtidae and Trichogrammatidae wasps were the two most common parasitoid families, and only very few wasps from the family Scelionidae were found emerging from the butterfly eggs (all wasps identified to family level by Yde Jongema). Naisbit (2001) determined which Heliconiini species lay their eggs on which Passiflora species (see table 1.1). Unfortunately, it was impossible to determine the species identity of the parasitized eggs in this study. In the future, eggs may be recognized by taking good pictures, measuring egg size and determining egg colour and rear them until caterpillars or even adult butterflies to determine the species identity. Naisbit’s (2001) data show that several Heliconiini species can lay their eggs on one Passiflora species. This is the case for 5 Passiflora species that were also used in this research, except one. The data of Naisbit (2001) showed that only Agraulis vanillae lays its eggs on Passiflora foetida . However, in this study it was found that not all eggs on P. foetida were of the same colour, thereby suggesting that more Heliconiini species lay their eggs on this plant species.

The parasitism rate by Trichogrammatidae (7.3%) is quite low when comparing it with parasitism rates in other natural Trichogramma -host associations. Huigens (2003) found a parasitism percentage of 42.1% by Trichogramma wasps and a parasitism percentage of 42.1% by Trichogramma wasps was found by Simpson (2007). Also the parasitism rates of Encyrtidae (8.5%) and Scelionidae (0.3%) are low when comparing them to other studies. Hirose et al . (1996) found for Encyrtidae (Ooencyrtus ) wasps a parasitism percentage of 38.7% and for Scelionidae (Telenomus ) wasps a parasitism percentage of 42.9%. Chabi- Olaye et al . (2001) also showed a parasitism percentage of 56.7% for Scelionidae (Telenomus ).

In this study, we may have missed some members of the egg parasitoid community on Heliconiini butterflies in this area. Naisbit (2001) searched eggs on 9 different Passiflora species and during this research only eggs were collected on 6 Passiflora species. The data of Naisbit (2001; table 1.1) showed that for example eggs of Heliconius doris were only found on Passiflora ambiguna . This Passiflora species was not found in this study, so we missed egg parasitoids on this species. Besides this, egg-larval parasitoids were not taken into account in this research. It may be that some of the eggs that were now seen as unparasitized actually contained parasitoids that emerge from the larval stage. For such parasitoids hitch-hiking with adult hosts may be an efficient host location strategy.

15 In this study, it was also determined whether egg parasitoid wasps use hitch-hiking with adult butterflies as a means of finding butterfly eggs. In total 133 butterflies of the tribe Heliconiini were caught. In total 18 parasitoid wasps were found in the vials of the butterflies. From the 18 parasitoids, 16 belonged to families of which it is known that they contain members that are egg parasitoids of Lepidoptera (personal communication, Yde Jongema; Hanson & Gauld, 1995). It is unknown if the egg parasitoids other than the ones belonging to the Encyrtidae , Scelionidae and Trichogrammatidae parasitize eggs of the Heliconiini butterflies. The butterflies were not always caught in the air but also when they were feeding on flowers or sitting on a leave. So it is possible that the collected parasitoid wasps were not present on the butterfly. In future research those wasps have to be offered Heliconiini eggs to determine if they are egg parasitoids of Heliconiini butterflies.

The hitch-hiking rates of wasps of the family Trichogrammatidae are slightly higher when comparing them to rates found in previous studies. Woelke (2008) showed that the hitch- hiking percentage in the Netherlands on the moth Xestia c-nigrum was 1.5% by Trichogramma brassicae . Huigens and Fatouros (unpublished data) showed that the hitch- hiking percentage by Trichogramma evanescens wasps in the Netherlands on female Pieris brassicae butterflies was 6.3% and 1.1% on female Pieris rapae butterflies. Probably, for both Panama as well the Netherlands, the hitch-hiking percentages are an underestimation of the actual proportion of hitch-hiking wasps. Wasps probably only stay for a short time on the butterflies or fall of the butterflies when trying to catch them.

Overall it can be concluded that Heliconiini butterflies are attacked by a rather stable community of egg parasitoids and that some of the egg parasitoid species probably hitch- hike with adult butterflies to find their eggs. This goes for the wasps of the families Encyrtidae , Scelionidae and Trichogrammatidae . These families were found on Heliconiini butterflies as well emerging from the collected Heliconiini eggs. Now it is very important to determine the species identify of the different wasps and subsequently, to determine if hitch- hiking egg parasitoids exploit anti-aphrodisiacs of Heliconiini butterflies, behavioral tests as described in Fatouros et al . (2005b) have to be done (see for more information De Rijk, 2008). Because recently the anti-aphrodisiac is known from Heliconius melpomene (Schutz et al. , 2008), tests can be done with this butterfly species. Also tests can be done with Heliconius erato , because Gilbert (1976) already showed that the females smell different after mating with males.

16 Acknowledgments

The following people I want to thank for their support and help:
Ties Huigens for supervising and help with the research.
Marjolein de Rijk for help with the research.
Catalina Estrada for help with the research.
Nina Fatouros for help with the research.
Yde Jongema for identification of the collected egg parasitoid families (in some cases to genus level).
Richard Merrill for the butterfly rearing (Smithsonian Tropical Research Institute; STRI).
Moises Abanto Flores for the butterfly rearing (STRI).
Josephine Dessmann for the butterfly rearing and help with collecting butterflies (STRI).
Annette Aiello for help with the project and information about Anartia fatima butterflies (STRI).
Orelis Arosemena for the permits (STRI).
Raineldo Urriola for arranging the car and 95% alcohol (STRI).

Finally, I would like to thank STRI for funding me through a Short-Term Fellowship.

17 References

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18 Hirose, Y., Takasu, K. & Takagi, M. (1996). Egg parasitoids of phytophagous bugs in soybean: Mobile natural enemies as naturally occurring biological control agents of mobile pests. Biological Control 7, 84-94. Huigens, M.E. (2003). On the evolution on Wolbachia -induced parthenogenesis in Trichogramma wasps. (PhD thesis, Laboratory of Entomology, Wageningen University). Janzen, D.H. & Hallwachs, W. (2005). Caterpillars, pupae, butterflies & moths of the ACG. San José. Consultado el 14 de junio de 2005. Disponible en: http://Janzen.sas.upenn.edu/caterpillars/database.lasso. Kukuk, P. (1985). Evidence for an antiaphrodisiac in the Sweat bee Lasioglossum (Dialictus) zephyrum . Science 227 , 656-666. Labine, P.A. (1968). The population biology of the butterfly Euphydryas editha . VIII. Oviposition and its relation to patterns of oviposition in other butterflies. Evolution 22 , 799-805. Lamas, G. (2004). Checklist: part 4a. Hesperioidae-Papilionoidae. In Atlas of Neotropical Lepidoptera 5, ed. Heppner, J.B. (Scientific Publishers, Gainsville), p 439. Law, R.H. & Regnier, F.E. (1971). Pheromones. Annual Review of Biochemistry 40 , 533-548. Longino, J.T. (1984). Shoots, parasitoids, and ants as forces in the population dynamics of Heliconius hewitsoni in Costa Rica (PhD thesis, University of Texas, Austin). Naisbit, R.E. (2001). Ecological divergence and speciation in Heliconius cydno and H. melpomene . (PhD thesis, University of London, London), pp. 45-83. Noldus, L.P.J.J. & Van Lenteren, J.C. (1985). Kairomones for the egg parasite Trichogramma evanescens Westwood I. Effect of volatile substances released by two of its hosts, Pieris brassicae L. and Mamestra brassicae L. Journal of Chemical Ecology 11 , 781-791. Nordlund, D.A. (1981). Semiochemicals: a review of the terminology. In Semiochemicals: their role in pest control , ed. Nordlund, D.A., Jones, R.L. & Lewis, W.J. (Wiley, New York), pp. 13-28. Orr, D.B., Russin, J.S. & Boethel, D.J. (1986). Reproductive biology behavior of Telenomus calvus (Hymenoptera: Scelionidae), a phoretic egg parasitoid of Podisus maculiventris (Hemiptera: Pentatomidae). Canadian Entomologist 118 , 1063-1072. Puttler, B. and Bailey, W.C. (2003). Establishment of Oomyzus gallerucae (Hymenoptera: Eulophidae), an egg parasite of the elm leaf beetle (Coleoptera: Chrysomelidae), in Missouri and adjacent states. Biological Control 27 , 20-24. Queroni, R.B. & Zucchi, R.A. (2002). Intraspecific variation in Trichogramma bruni Nagaraja, 1983 (Hymenoptera: Trichogrammatidae) associated with different hosts. Brazilian Journal of Biology 62 , 665-679. Querino, R.B. & Zucchi, R.A. (2003a). Six new species of Trichogramma Westwood (Hymenoptera: Trichogrammatidae) from a Brazilian forest reserve. Zootaxa 134 , 1-11. Querino, R.B. & Zucchi, R.A. (2003b). New species of Trichogramma Westwood (Hymenoptera: Trichogrammatidae) associated with lepidopterous eggs in Brazil. Zootaxa 163 , 1-10. Rijk, M. de (2008). Hitch-hiking behaviour of egg parasitoids on Heliconiini butterflies in a tropical lowland rainforest. (MSc thesis, Laboratory of Entomology, Wageningen University), pp 1-39. Schulz, S., Estrada, C., Yildizhan, S., Boppré, M. & Gilbert, L.E. (2008). An antiaphrodisiac in Heliconius melpomene butterflies. Journal of Chemical Ecology 34 , 82-93. Schulz, S., Yildizhan, S., Stritzke, K., Estrada, C. & Gilbert, L.E. (2007). Macrolides from the scent glands of the tropical butterflies Heliconius cydno and Heliconius pachinus . Organic and Biomolecular Chemistry 5, 3434-3441.

19 Silberglied, R., Aiello, A. & Lamas, G. (1979). Neotropical butterflies of the genus Anartia : systematics, life histories, and general bioogy ( Lepidoptera : Nymphalicae). Psyche 82 , 219-260. Simonsen, T.J. (2006). Glands, muscles and genitalia. Morphological and phylogenetic implications of histological characters in the male genitalia of fritillary butterflies (Lepidoptera : Nymphalidae : Argynnini ). Zoologica Scripta 35, 231-241. Simpson, L. (2007). Trichogramma wasps of Kauai: A survey of Trichogramma species and the presence of Wolbachia. (MSc thesis, Laboratory of Entomology, Wageningen University). Smith, S.M. (1996). Biological control with Trichogramma : advances, successes, and potential of their use. Annual Review of Entomology 41 , 375-406. Stowe, M.K, Turlings, T.C.J., Loughrin, J.H., Lewis, W.J. & Tumlinson, J.H. (1995). The chemistry of eavesdropping, alarm, and deceit. Proceedings of the National Academy of Sciences 92 , 23-28. Vet, L.E.M. & Dicke, M. (1992). Ecology of infochemical use by natural enemies in a tritrophic context. Annual Review of Entomology 37 , 141-172. Vinson, S.B. (1984). Parasitoid-host relationships. Chemical Ecology of Insects , eds. Bell, W.J. & Cardé, R.T. (Sinauer, Sunderland), pp. 111-124. Woelke, J.B. (2008). Hitch-hiking behaviour of Trichogramma wasps on cabbage white butterflies and moths. (MSc thesis, Laboratory of Entomology, Wageningen University), pp. 1-49. Wyatt, T.D. (2003). Pheromones and animal behaviour, communication by smell and taste. (Cambridge), pp1-4.

20 Appendix 1: Research locations

Area map of all research locations

1. Pipeline road (Camino del oleoducto) 3. Gamboa 5. Camino de plantación 2. Oil bunkers 4. Sendro la laguna

Area map of the research locations in Gamboa

3. Gamboa 4. Sendro la laguna

21 Appendix 2: Research Passiflora species All the reseach Passiflora plant species that were used for the egg collection in and around Gamboa.

Passiflora auriculata Passiflora biflora

Passiflora coriacea Passiflora foetida

Passiflora menispermifolia Passiflora vitifolia

22 Appendix 3: Research Heliconiini species All the butterfly species of the tribe Heliconiini that were used for the butterfly catches and egg collection in and around Gamboa.

Agraulis vanillae Dione juno

Dryas iulia Eueides aliphera gracilis

Eueides lybia olympia Heliconius cydno

23

Heliconius doris (red variant) Heliconius doris (blue variant)

Heliconius doris (green variant) Heliconius erato

Heliconius hecale (two variants)

24

Heliconius ismenius Heliconius melpomene

Heliconius sara Heliconius sapho

Philaethria dido

25 Appendix 4: Research data egg parasitism In tables 1 to 6 raw research data are shown per research location about the parasitism percentages of the Heliconiini eggs per Passiflora species.

Table 1 Parasitism rates of the Heliconiini eggs per Passiflora species from all research locations without Dione juno eggs. In total 996 D. juno eggs were found in clusters, none of them was parasitized. Overall Nr. of Nr. of Nr. of % eggs % by % by % by plants eggs parasitised parasitised Encyrtidae Scelionidae Trichogrammatidae eggs by wasps wasps waps wasps Passiflora auriculata 17 13 2 15.4 7.7 0 7.7 Passiflora biflora 16 23 6 26.1 17.4 0 8.7 Passiflora coriacea 17 47 1 2.1 2.1 0 0 Passiflora foetida 5 55 9 16.4 0 0 16.4 Passiflora menispermifolia 13 34 8 23.5 0 0 23.5 Passiflora vitifolia 54 145 25 17.2 14.4 0.7 2.1 Total 122 317 51 16.1 8.5 0.3 7.3

Table 2 Parasitism rates of the Heliconiini eggs per Passiflora species at research location Pipeline road. Pipeline road Nr. of Nr. of Nr. of % eggs % by % by % by plants eggs parasitised parasitised Encyrtidae Scelionidae Trichogrammatidae eggs by wasps wasps waps wasps Passiflora auriculata 17 13 2 15.4 7.7 0 7.7 Passiflora biflora 9 4 1 25 0 0 25 Passiflora coriacea 9 22 1 4.5 4.5 0 0 Passiflora foetida 5 55 9 16.4 0 0 16.4 Passiflora menispermifolia 5 13 1 7.7 0 0 7.7 Passiflora vitifolia 39 50 15 30 24 2 4 Total 84 157 29 18.5 8.9 0.6 8.9

Table 3 Parasitism rates of the Heliconiini eggs per Passiflora species at research location Oil bunkers. Oil bunkers Nr. of Nr. of Nr. of % eggs % by % by % by plants eggs parasitised parasitised Encyrtidae Scelionidae Trichogrammatidae eggs by wasps wasps waps wasps Passiflora auriculata 0 0 0 0 0 0 0 Passiflora biflora 1 2 1 50 50 0 0 Passiflora coriacea 3 1 0 0 0 0 0 Passiflora foetida 0 0 0 0 0 0 0 Passiflora menispermifolia 5 12 5 41.6 0 0 41.6 Passiflora vitifolia 2 0 0 0 0 0 0 Total 11 15 6 40 13.3 0 26.7

Table 4 Parasitism rates of the Heliconiini eggs per Passiflora species at research location Gamboa without Dione juno eggs. In total 996 D. juno eggs were found in clusters, none of them was parasitized. Gamboa Nr. of Nr. of Nr. of % eggs % by % by % by plants eggs parasitised parasitised Encyrtidae Scelionidae Trichogrammatidae eggs by wasps wasps waps wasps Passiflora auriculata 0 0 0 0 0 0 0 Passiflora biflora 2 10 3 30 30 0 0 Passiflora coriacea 0 0 0 0 0 0 0 Passiflora foetida 0 0 0 0 0 0 0 Passiflora menispermifolia 0 0 0 0 0 0 0 Passiflora vitifolia 5 44 2 4.5 2.25 0 2.25 Total 7 54 5 9.3 7.4 0 1.9

26 Table 5 Parasitism rates of the Heliconiini eggs per Passiflora species at research location Sendro la laguna. Sendro la laguna Nr. of Nr. of Nr. of % eggs % by % by % by plants eggs parasitised parasitised Encyrtidae Scelionidae Trichogrammatidae eggs by wasps wasps waps wasps Passiflora auriculata 0 0 0 0 0 0 0 Passiflora biflora 3 5 0 0 0 0 0 Passiflora coriacea 5 24 0 0 0 0 0 Passiflora foetida 0 0 0 0 0 0 0 Passiflora menispermifolia 0 0 0 0 0 0 0 Passiflora vitifolia 4 35 7 20 20 0 0 Total 12 64 7 10.9 10.9 0 0

Table 6 Parasitism rates of the Heliconiini eggs per Passiflora species at research location Camino de plantación. Camino de Nr. of Nr. of Nr. of % eggs % by % by % by plantación plants eggs parasitised parasitised Encyrtidae Scelionidae Trichogrammatidae eggs by wasps wasps waps wasps Passiflora auriculata 0 0 0 0 0 0 0 Passiflora biflora 1 2 1 50 0 0 50 Passiflora coriacea 0 0 0 0 0 0 0 Passiflora foetida 0 0 0 0 0 0 0 Passiflora menispermifolia 3 9 2 22.2 0 0 22.2 Passiflora vitifolia 4 16 1 6.3 6.3 0 0 Total 8 27 4 14.8 3.7 0 11.1

27