Efficiency of Fruit Juice Feeding in Morpho Peleides (Nymphalidae

P1: GDX Journal of Insect Behavior [joib] pp806-joir-462148 March 10, 2003 10:31 Style ﬁle version Feb 08, 2000

Journal of Insect Behavior, Vol. 16, No. 1, January 2003 (C 2003)

Efﬁciency of Fruit Juice Feeding in Morpho peleides (Nymphalidae, Lepidoptera)

M. C. N. Knopp1 and H. W. Krenn1,2

Accepted August 9, 2002; revised October 22, 2002

We have described the feeding behavior of the frugivorous butterfly Morpho peleides (Butler 1872) under various conditions and tested its ability to take up fluid from selected natural and artificial food sources in comparison with the nectarvorous Vanessa cardui (Linnaeus 1758). Both nymphalids showed similar probing behavior except for one particular proboscis movement and the fact that M. peleides was unable to feed from Lantana flowers. In 2-min feeding trials, M. peleides imbibed a greater amount of fluid from the food sources, with the most conspicuous difference on rotting banana. Without time restriction, M. peleides gained a significantly greater percentage of body weight from soaked plotting paper, whereas no significant difference occurred from tubular artificial flowers. The ability of M. peleides to feed more efficiently from wet surfaces than V. cardui is discussed in context with proboscis morphology.

KEY WORDS: feeding behavior; fruit feeding; mouthparts; proboscis; butterﬂy; Morpho; Nymphalidae.

INTRODUCTION

Any tropical forest butterﬂy community can be divided into two adult feeding guilds. Members of the nectarvorous guild obtain the majority of their nutritional requirements from ﬂoral nectar and those of the frugivorous guild

1Institute of Zoology,Department of Evolutionary Biology,University of Vienna, Althanstrae 14, A-1090 Vienna, Austria. 2To whom correspondence should be addressed. e-mail: [email protected]. Fax: +43 1 42779544.

0892-7553/03/0100-0067/0 C 2003 Plenum Publishing Corporation P1: GDX Journal of Insect Behavior [joib] pp806-joir-462148 March 10, 2003 10:31 Style ﬁle version Feb 08, 2000

68 Knopp and Krenn

feed primarily on juices of rotting fruit or fermenting tree sap (DeVries, 1987, 1988; DeVries et al., 1997). Species of the fruit-feeding guild all belong to the Nymphalidae, and in this context Morpho peleides is one of the best-studied representatives. This neotropical butterfly is a well-known fruit-feeder and observations clearly indicate that it virtually never visits flowers for nectar (Young, 1975; DeVries, 1983, 1987). Its food preferences and its resource exploitation in the habitat have been studied (Young, 1975, 1979; DeVries, 1983, 1988), however, detailed knowledge of the behavior during feeding and the amount of fluid imbibed is lacking. The proboscis of butterflies is adapted primarily for the uptake of nectar from flowers. A number of proboscis features and the patterns of proboscis movements could be functionally interpreted in context with the extraction of nectar from blossoms (Krenn, 1990, 1998; Paulus and Krenn, 1996). A comparison of the mouthparts between nymphalids of both feeding guilds revealed a characteristic proboscis morphology for the fruit-feeders including Morpho species. It was hypothesized that features of the proboscis tip might enhance the performance of fluid uptake from surfaces such as squashed fruit (Krenn et al., 2001). The aim of this paper is to describe the feeding behavior of Morpho peleides on rotting fruit and to compare this with the flower handling capa- bility of the proboscis in a nectarvorous nymphalid butterfly. A characteristic pattern of probing movements of the uncoiled proboscis and a distinct bend at one-third of the proboscis length have been described in nectarvorous butterflies (Bänziger, 1971; Krenn, 1990, 1998; Penz and Krenn 2000). The analysis of the behavioral characters in fruit-feeding butterflies addresses the question if there is a specialized pattern of proboscis movements which could be interpreted in context with fluid uptake from a wet surface of rotting fruit. To investigate the efficiency of fluid uptake we experimentally tested the amount of fluid that can be imbibed from a variety of artifical nectar sources which mimic different conditions of fluid accessibility.

MATERIALS AND METHODS

Pupae from M. peleides were purchased from Worldwide Butterflies and Lullingstone Silk Farm (Dorset, UK) and reared in laboratories at the University of Vienna. A second generation was reared in the laboratory on Medicago x varia (Fabaceae). Eggs from V. cardui were purchased from Carolina Biological Supply Company (Burlington, N. C.). Larvae of the first generation were reared on artificial food (Carolina Biological Supply Company); larvae of the succeed- ing generations, on Cirsium arvense (Asteraceae). P1: GDX Journal of Insect Behavior [joib] pp806-joir-462148 March 10, 2003 10:31 Style file version Feb 08, 2000

Fruit Feeding in Morpho peleides 69

The experimental and behavioral studies were conducted in a greenhouse at the University of Vienna. Both natural and artificial food sources were used. Natural food sources included inflorescences of Lantana camara (Verbenaceae) and small amounts of squashed, rotting banana, presented on a petri dish. Inflorescences of L. camara are well known for their attrac- tiveness for a great variety of butterflies and have repeatedly been used in experimental observation of butterfly feeding behavior (Weiss, 1991; Penz and Krenn, 2000). Bananas were shown to be an attractive food for M. peleides in natural environments (Young, 1975). As artificial food sources we used cotton wool and plotting paper, both saturated with nectar mimics of L. camara, which contains a variety of sugars and amino acids (Alm et al., 1990), and presented them on a petri dish. In addition, for the experimental studies we used an artifical flower which consisted of a glass tube with a diameter of 1.5 mm, fixed in a polystyrene panel to serve as a landing site for the butterflies. The bottom of the tube was immersed in a glass of liquid nectar mimics, and by capillary action the glass tube became filled. Feeding behavior was recorded with a videocamera (Canon Canovision EX Hi8); the videotapes were analyzed on a TV monitor using the computer program The Observer Version 3.0. The butterflies were set on the food source and filmed until they left it or until 7 min had elapsed. More than 80% of the butterflies left the food source within 7 min. As the food source we used rotting squashed banana (for both species N = 7). The movements of the proboscis (coiled or uncoiled position, probing, and sucking) were measured as percentages of the observed time; the sequences of up-and- down movement were measured as events per minute. To study the efficiency of fluid uptake we measured the weight of the butterflies before and after feeding tests. For this purpose the butterflies were narcotized with CO2 and their weight was measured on a high-performance balance with a reading accuracy of 0.0001 g (Sartorius MC-1 Analytic AC 120S). The weight difference between before and after feeding represented the amount of fluid the butterflies had taken in. In the first feeding trial we tested the amount of fluid uptake during a 2-min period. The feeding sequence started with inflorescences of L. camara on the third day after emerging from pupae. The next feeding tests were carried out after a day of “rest” during which the butterflies were given only water to ensure a suffi- cient level of hunger. Tests proceeded in the following order: cotton wool, plotting paper, rotting banana, and tubular artificial flower (M. peleides, N = 9; V. cardui, N = 11). Two days after this feeding sequence one group of butterflies from each species was tested on plotting paper (M. peleides, N = 5; V. cardui, N = 16), and a second group on tubular artifical flower (M. peleides, N = 6; V. cardui, N = 8), without any time restriction (ad libitum). When the individuals P1: GDX Journal of Insect Behavior [joib] pp806-joir-462148 March 10, 2003 10:31 Style file version Feb 08, 2000

70 Knopp and Krenn

ceased feeding they were stimulated with a drop of nectar on the midtarsi, which led to uncoiling of the proboscis and further feeding. The tests were stopped when the butterﬂies did not recommence feeding after having been given a stimulus twice. The proboscis length was measured in narcotized specimens using a Vernier caliper with a reading accuracy of 0.1 mm. Horizontal widths of the proboscis and of the food canal were measured in semithin cross sections at various distances from the base (M. peleides, N = 7; V. cardui, N = 5). For the preparation of semithin sections, heads were ﬁxed in alcoholic Bouin solution and embedded in epoxy resin (Pernstich et al., 2003).

RESULTS

Feeding from Rotting Banana

During food uptake the uncoiled proboscis bends distinctly at about one-third of its length. At this point, the proboscis is flexed downward and the tip region is sharply bent toward the body (Fig. 1). This results in a posture of the proboscis in which the proximal region is nearly horizontal, the distal region after the bending is held in a more or less vertical position, and the dorsal side of the tip region is positioned over the fruit (Fig. 1). Probing movements of the proboscis comprised (1) an up-and-down motion of the whole proboscis at its connection to the head, (2) an up-and-down motion of the whole proboscis produced by elevation and lowering of the head, (3) to-and-fro movements of the proboscis tip produced by flexion and extension of the bend, (4) a sideways motion produced by the turning head. These movements permit a range of action that included (a) maximal lateral movements in which the tip extends approximately to the tarsi of the legs, and (b) maximal forward movements in which the bend region is slightly stretched and the proboscis is fully extended in a forward sloping position so that only the apex touches the fruit. The proboscis tip region accumulated fluid when in the upside-down position. A droplet was formed from the juice of the rotting banana around the tip region, probably due to adhesive properties of structures at the tip region (Figs. 2 and 3). These structures include the proboscis tip sensilla, which are arranged in a dense row of flat ellipsoid structures on each side (Fig. 2), and cuticular ridges, which form grooves leading into the food canal (Fig. 3). The butterflies were able to dissolve dried sugar by discharging a fluid out of the tip region. M. peleides (N = 7) sucked juice from the wet surface of a rotten banana during 81% (SD, 7.85%) of the observation time; during 17.5% (SD, P1: GDX Journal of Insect Behavior [joib] pp806-joir-462148 March 10, 2003 10:31 Style file version Feb 08, 2000

Fruit Feeding in Morpho peleides 71

Figs. 1–3. Fig. 1. Uptake of fruit juice in Morpho peleides from the surface of a rotting banana. Note the ﬂexed position of the uncoiled proboscis and that the dorsal side of the tip region (arrow) touches the fruit surface. Fig. 2. The proboscis tip region in Morpho peleides has a brush-shaped appearance formed by the long sensilla styloconica (arrow). Fig. 3. Detail of the proboscis tip region in Morpho peleides (SEM micrograph) shows slits into the food canal and sensilla styloconica (sst). The sensilla and cuticle structures of the proboscis wall increase the tip surface. P1: GDX Journal of Insect Behavior [joib] pp806-joir-462148 March 10, 2003 10:31 Style ﬁle version Feb 08, 2000

72 Knopp and Krenn

8.02%) of the time they probed the fruit surface. In the remaining time the proboscis was coiled (0.6%; SD, 0.37%) or the exact position could not be detected (0.9%; SD, 0.37%). In general, nectarvorous V. cardui (N = 7) had a similar time partition and its sucking time (89.4%; SD, 11.29%) did not differ significantly (P = 0.11; Mann–Whitney U test, U = 12). Probing occurred significantly less often, at 4.8% (SD, 6.17%; P = 0.009; U = 4), and the proboscis remained coiled for a longer period (5.4%; SD, 8.57%). In M. peleides juice sucking was frequently interrupted (9.26 times/min SD, 2.6 times/min) by rapid elevation and lowering of the proboscis tip off and onto the fruit surface. This sequence of proboscis movements differs from typical probing movements on the fruit surface in terms of the velocity and the fact that the tip region is lowered to the same point from which it was elevated. M. peleides showed this behavior significantly more often than V. cardui (2.54 times/min; SD, 2.25 times/min; P = 0.004; U = 2).

Efﬁciency of Fluid Uptake

To investigate the fluid uptake capabilities, we compared the weights of individually marked butterflies of M. peleides and V. cardui before and after a 2-min period of fluid uptake from one of several possible natural and artifical food sources (Table I). The feeding trials were carried out in identical sequences in all tested individuals. The first 2-min trial was performed with flowers of L. camara. None of the tested individuals of M. peleides inserted their proboscis into the corolla tubes. Although all of them probed the surface of the inflorescences in the

Table I. Weight Gain During a 2-Min Feeding on Selected Natural and Artiﬁcial Sourcesa

Morpho peleides (N = 9) Vanessa cardui (N = 11) Food source [g] (SD) [%] (SD) [g] (SD) [%] (SD)

Lantana camara No nectar uptake <0.001 0.18 (0.94) Cotton wool 0.063 (0.01) 19.56 (7.96) 0.027 (0.005) 12.54 (2.97) Plotting paper 0.062 (0.01) 18.71 (7.35) 0.027 (0.004) 12.92 (2.29) Rotting banana 0.031 (0.01) 9.45 (4.86) 0.001 (0.001) 0.38 (0.54) Tubular artiﬁcial 0.079 (0.01) 23.6 (8.66) 0.029 (0.01) 15.23 (5.67) ﬂower

a The weight gain is the difference in weight before and after feeding for 2 min on the given food source. Due to the different initial weights of the butterflies, the percentage weight gain relative to the weight of the individuals before feeding was calculated [%]. M. peleides was able to imbibe more liquid from all food sources—except on the inflorescences of L. camara—in both absolute numbers [g] and percentage [%]. On L. camara, M. peleides did not insert its proboscis into the corolla tubes and therefore no nectar uptake took place. P1: GDX Journal of Insect Behavior [joib] pp806-joir-462148 March 10, 2003 10:31 Style file version Feb 08, 2000

Fruit Feeding in Morpho peleides 73

usual way, they were unable to take up nectar. On other tested food sources M. peleides sucked up fluid with a mean weight gain of 0.059 g per feeding trial; this amounts to a mean weight gain of 17.8% of the body weight within 2 min. The greatest body weight gain could be observed on the tubular artificial flower, where M. peleides imbibed about one-quarter of its body weight within 2 min. The lowest amount of fluid uptake (0.03 g), which is equivalent to 9.5% of the body weight, was obtained from rotting bananas. Rates of food uptake from soaked cotton wool and soaked plotting paper were equal, each leading to a weight gain of 0.06 g (Table I). We compared the results for M. peleides with those for V. cardui,a common nectar-feeding nymphalid butterfly (Table I). Since this species is smaller, it was reasonable only to compare the percentage body weight gain. V. cardui exploited all food sources; the smallest body weight gain occurred on L. camara and on rotting banana, both less than 0.5% within 2 min. The other food sources provided fluid amounts that led to a body weight increase between 12.5 and 15.2%. The amount of fluid that could be taken up by V. cardui (N = 11) was smaller than that for M. peleides (N = 9) for all food sources except L. camara. The mean weight gain of V.cardui per feeding trial for the four food sources (same as in M. peleides) was 0.02 g, corresponding to 10.3% of the body weight. This is significantly less than the 0.059 g of M. peleides (P = 0.007; U = 14). A linear discriminant analysis confirmed the strong separation between the results for the two tested species (coefficient of canonical correlation: 0.874); furthermore, this analysis showed that the greatest difference between M. peleides and V. cardui was manifested in the weight gain from rotting banana (structure matrix; pooled within-groups correlations between discriminating variables and canonical discriminant functions; rotting banana, 0.811; soaked cotton wool, 0.357; tubular artificial flower, 0.342; soaked plotting paper, 0.326). To estimate the maximal amounts of fluid which can be taken up, we compared two independent groups of M. peleides and V. cardui. Butterflies of the first group were allowed to feed ad libitum on soaked plotting paper; those of the second group fed on the tubular artificial flower. Soaked plotting paper was intended to simulate rotting fruit that have a thin layer of exposed fluid, while the artificial flower simulated a flower with a tubular corolla. The percentage body weight gain was significantly higher in M. peleides of the plotting paper group (P = 0.048; U = 15), with a mean gain of 51.4% in M. peleides (N = 5) versus 33.7% in V.cardui (N = 16). On the tubular artificial flower V. cardui (N = 8) gained a higher percentage of body weight (41.6%) than on the plotting paper (33.7%) but no significant differences from M. peleides (N = 6) were found by comparing the percentage body weight gain (P = 0.699; U = 21) (TableII). In both trials the time spent on the respective P1: GDX Journal of Insect Behavior [joib] pp806-joir-462148 March 10, 2003 10:31 Style file version Feb 08, 2000

74 Knopp and Krenn

Table II. Weight Gain During “ad Libitum Feeding” (No Time Restriction)a

Morpho peleides Vanessa cardui Weight gain Weight gain Mann–Whitney Time Time U test Food [s] [g] [%] [s] [g] [%] source (SD) (SD) (SD) N (SD) (SD) (SD) N Time [%]

Plotting 1350.8 0.205 51.4 5 638.2 0.063 33.7 16 P = 0.563 P = 0.048 paper (1439.3) (0.08) (18) (214.2) (0.028) (13.1) Tubular 572.7 0.15 43.7 6 396.4 0.079 41.6 8 P = 0.439 P = 0.699 artiﬁcial (503.4) (0.035) (15) (224.1) (0.026) (9.8) ﬂower

a Weight gain was tested in different groups of individuals of each species on either soaked plotting paper or a tubular artificial flower. A significant difference (, at the 0.05 level) was found only in the percentage body weight gain on plotting paper, which means that M. peleides can take up nectar more efficiently from the wet surface than V. cardui.

food sources was longer for M. peleides (1350.8 s on plotting paper; 572.7 s on tubular artificial flower) than for V. cardui (638.2 s on plotting paper, 396.4 s on tubular artificial flower), but the Mann–Whitney U test showed no significant difference (P = 0.563 on plotting paper, U = 33; P = 0.439 on tubular artificial flower, U = 18) (Table II). Comparison of the relative diameter of the food canal (food canal width divided by proboscis length) did not reveal significant differences between the examined species (P = 0.64; U = 14), although the proboscis of M. peleides (N = 7) is significantly broader (P = 0.03; U = 4) than that of V. cardui (N = 5).

DISCUSSION

Exploitation of food sources such as rotting fruit, tree sap, or other decaying materials poses fundamentally different challenges for fluid acquisi- tion. Normally nectar is available in bowl-shaped nectaries or in floral tubes. However, squashed fruit present a large surface on which a thin layer of liquid is exposed. It is reasonable to assume that behavioral adaptations for an efficient exploitation of this food source should have evolved. However, the present behavioral results revealed only a slightly modified pattern of proboscis movements in M. peleides compared to flower-visiting nymphalid butterflies. Nevertheless, the experimental results indicated more efficient fluid uptake from moist surfaces in M. peleides. The only remarkable difference concerns the series of rapid elevation-lowering movements of the proboscis in M. peleides. Rapid elevation and lowering of the proboscis from the fruit surface occurred significantly more frequently in M. peleides. This behavior might serve to clean or unclog the intake slits from fruit fibers P1: GDX Journal of Insect Behavior [joib] pp806-joir-462148 March 10, 2003 10:31 Style file version Feb 08, 2000

Fruit Feeding in Morpho peleides 75

by expelling a fluid. In M. peleides the expelled fluid was found to dissolve dried sucrose. Although the total time sucking from fruit juice did not differ between V. cardui and M. peleides, the sucking activity of M. peleides was often interrupted by short intervals of probing followed by continued sucking activity at another site. Comparison of the two species for the 2-min sessions of nectar uptake shows that M. peleides gained a higher percentage of body weight and that the greatest difference between the species was obtained on rotting banana. Comparison of the two species for the ad libitum feeding tests showed that, on one hand, M. peleides took up more fluid from plotting paper than from tubular artificial flowers, whereas V. cardui imbibed more fluid on tubular artificial flowers than from plotting paper. On the other hand, the percentage body weight gain in M. peleides on plotting paper was significantly higher than in V. cardui, whereas no significant differences were found on the tubular artificial flower. Since the time spent on the food source and the dimensions of the food canal did not differ significantly, these tests indicate that M. peleides can take up fluid from moist surfaces of squashed fruit more efficiently than the nectar-feeding V. cardui. There are no reports of flower-visiting behavior in M. peleides, but under laboratory conditions individuals of this species explored L. camara inflorescences with the proboscis tip. However, they were not able to insert it into the corolla. We cannot account for the fact that M. peleides never fed from the corolla tubes of L. camara. Maybe sensorial cues are absent for the recognition of these flowers as a food source. Many species of nymphalid butterflies only exceptionally visit flowers. They feed instead on tree sap, juice of rotting fruit, and other decaying substances. Feeding from nonfloral food sources might have several advan- tages in the tropics. A dense, tropical rainforest offers only a few flowers in the understory, whereas fruit fall from trees throughout the year, burst open, and ferment quite rapidly (Larsen, 1994). Once butterflies find the fruit, it provides a large amount of food which can be exploited over several days, and usually, additional fruit will be available in the close surroundings for a long period. Observations by Brakefield and Kesbeke (1995) on Bicyclus anynana led to the suggestion that the fruit-feeding habit may promote a longer adult life span and may potentially increase female reproductive out- put. They also supposed that the quality of nutrients available from fruit is likely to be high. However, detailed chemical, physiological, and microbio- logical analyses are missing for fruit juice at different stages of ripeness and decay. Braby and Jones (1995) tested the influence of adult diet with respect to reproduction. Females of Mycalesis terminus fed on rotting fruit could maintain a constant egg weight over their lifetime, in contrast to those fed on a 25% honey solution. P1: GDX Journal of Insect Behavior [joib] pp806-joir-462148 March 10, 2003 10:31 Style file version Feb 08, 2000

76 Knopp and Krenn

The principal morphology of the proboscis and its mechanism of movements (Krenn, 1990) restrict butterflies to liquid food, hence apparently constraining evolutionary changes in the mode of feeding. The comparative investigation of the proboscis in “non-flower-visiting” nymphalid butterflies showed that members of feeding guilds have structural adaptations corresponding to their specific diets. The proboscis tip sensilla are densely arranged and form a flat brush on the dorsal side near the tip (Krenn et al., 2001). The brush-tipped proboscis was interpreted as an adaptation to ex- ploit food resources such as rotting fruit, tree sap, and dung. The present results prove that, at least in Morpho peleides, the brush-tipped proboscis is more efficient in sucking fluids from wet surfaces, thus illustrating the adaptive value of this derived proboscis morphology. Another type of derived feeding preference has been found in Heliconius butterflies, in which the proboscis has acquired a new biological role as pollen collector beyond that of nectar sucking. These nymphalids supplement their diet with amino acids derived from pollen, thereby ob- taining fitness-related benefits (review by Gilbert, 1991). The proboscises of these species have significantly longer sensory bristles in the proximal region which appear to enhance adherence of pollen grains to the proboscis (Krenn and Penz, 1998). It was demonstrated in a comparative behavioral study that pollen-feeding species exhibited a modified time pattern in flower handling compared to non-pollen-feeding relatives (Penz and Krenn, 2000). In Heliconius butterflies modifications of the proboscis in concert with its modified movements created a new form–function relationship that allows access to a new food source. In M. peleides and probably in other fruit-feeding Nymphalidae, structural adaptations of the proboscis appear to be the key to understanding the evolution of their specialized feeding habit, while modified patterns of proboscis movements are of only minor importance for the efficient exploitation of the openly exposed food sources.

ACKNOWLEDGMENTS

We are grateful to the Institute of Ecology and Conservation Biology (University of Vienna) for granting permission to use their greenhouse and to J. Plant for linguistic help. This work was supported by the Austrian Science Fund (Project 13944 Bio).

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