J. Appl. Ent. 109 (1990), 268-273 0 1990 Verlag Paul Parey, Hamburg und Berlin ISSN 0931-2048

USDA, ARS, Southern Field Crop Insect Management Laboratory, Stoneville, MS, and Department of Entomology, Mississippi State University, Mississippi State, MS, USA

Learning in Microplitis croceipes Cresson (Hym., Braconidae) By J. P. KAAS,”~, G. W. ELZEN’, and S. B. RAMASWAMY~

Abstract Responses of experienced Microplitis croceipes Cresson females in a wind tunnel choice situation were tested at three periods. Training consisted of exposing females to cotton or soybean leaf material with host (Heliothis virescens) larvae for parasitization or without host larvae. Control parasitoids received no initial exposure. Testing periods were: within 1 hour after exposure; 3 days after exposure or after exposure to reverse training. Pairwise comparison of choices of trained wasps versus controls showed a si nificant difference in only one case. Pairwise comparison of cotton versus soybean exposed wasps w i t h each training level showed significance for “rewarded” wasps in periods 1 and 3 with highest numbers of wasps flying to the source-plant of initial training. Results suggest the occurrence of associative learning of M. croceipes on host-host plants, and indicate a time dependent component in the learning process and irreversibility of this conditioning. ,

1 Introduction

Associative conditioning has been demonstrated in several species of insect parasitoids (ARTHUR 1966, 1971; MONTEITH 1963; VET 1983; VET and OPZEELAND 1984, 1985; THORPE andJoNEs 1937). ARTHUR (1966, 1967) was able to condition Itoplectis conquisitor (Say) on shape and color whereas MONTEITH (1963) found that Drino bohemicu (Messn.) responded to movement as a conditioned stimulus. In the host finding process of parasitoids as described by VINSON (1976), olfactory cues generally are important and much research has been undertaken to determine the chemical and physiological nature of host plant-host-parasitoid relations (WILLIAMS et al. 1988).

Olfaction may play a role as a conditioning stimulus for several insect parasitoids (ARTHUR 1971; THORPE and JONES 1937). Learning ability is thought to play a greater role in oligophagous parasitoids than in monophagous parasitoids, enabling the animals to respond with more flexibility to their variable environment (VINSON 1976).

Microplitis croceipes Cresson is a hymenopterous parasitoid of several economically important Heliothis species. The parasitoid is thought to be of particular importance for biological control (HOPPER and KING 1984) and for this reason receives considerable attention. Recently LEWIS and TUMLINSON (1988) showed olfactory conditioning of M . croceipes by the odor of vanilla extract, using Hefiothis frass as an unconditioned stimulus. The aim of this paper is to demonstrate the potential importance of olfactory conditioning of M . croceipes in biological control using cotton and soybean as representative host plants.

2 Materials and methods

2.1 Insects Twenty-four to forty-eight hours prior to the training of wasps, lab reared Helzorhis vzrescezs larvae (third to fourth instar) were transferred from their soybean-wheatgerm diet in multicellular rearing trays (HARTLEY et al. 1982; KING and HARTLEY 1985) to shallow 20 ml vials containing field grown

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Learning in Microplitis croceipes 269

soybean (Gfycine rnax var. Forrest) or cotton (Gossypiurn hirsuturn var. DES 119) leaves. Each vial contained 6 larvae.

Two-day-old M. croceipes females (POWELL and HARTLEY 1987) from the laboratory colony were transferred individually to 30 ml sna cap vials. Females were provided a drop of 50 % honey solution daily throughout the experiment. AEer testing, females were transferred back to their original vial.

2.2 Conditioning Two treatments per plant source and a control were used; for the first treatment, the actual conditioning, females were placed under a shallow vial on a 16 cm2 leaf disk of the source plant to which frass and leaf fragments from the larval containers was added. Wasps were offered four successive Heliothis larvae obtained from the same source plant-containing vial on a Q-ti over a eriod of 3-5 min. In the second treatment, a test for sensitization, females were placed uncfer a vial

for 3-5 min containing a leaf disk of each source plant and were not offered any larvae. The control consisted of females not receiving any treatment prior to the experiment. The same control females were used throughout the ex eriment and thus were unexposed only at first flight. Next morning, as a brief reinforcement shortly {efore flight testing, the same training schedule as above was repeated except that only one larva was offered instead of four.

2.3 Testing Wasp responses were tested in a wind tunnel (ELZEN et al. 1986) 30 min to 1 hour after the first treatment. The second test was 3 days later without any pretreatment. For the third test, 4 days after the first flight, females received the same type of treatment as in the first test but on material of the plant species other than the one they were exposed to before (referred to as reverse exposure). The 3 day lag time between the first and second test was based on the observation of high was mortality beyond 3 days. The reverse experience treatment started also on the third day, thus aiowing for testing on the fourth day.

Wasvs were flown individuallv in the wind tunnel and offered a choice between field grown cotton (var. DkS 119) and soybean (var. Forrest) lant terminals of similar sizes in waterpicks placed ca. 14 cm apart. Illumination in the wind tunnefwas ca. 1900 lux and wind speed was 12 cm/sec. Wasps were placed between the odor plumes, downwind at ca. 70 cm from the source in their container with the lid removed and observed for 10 min after exposure to the airflow or after the first choice was made. The experiment was conducted as a completely randomized design. Twenty wasps were tested per run. Six replicates of the experiment were made (in total 120 were tested in the first test). Wasp choice (number of first landings on the source) was recorded and data were analysed using a G test (SAS 1982).

3 Results

Wasp choices exhibited in each of the three wind tunnel test periods are given in the figure. For the first period (fig. A), pairwise testing of each of the four treatments versus the control showed n o significant differences. Pairwise testing of the two sensitization treatments (cotton only and soybean only) against each other showed no significant difference (P = 0.545). However, the two conditioning treatments (cotton + larvae vs soybean + larvae) tested against each other resulted in significantly ( P = 0.005) more wasps flying to their respective training sources. The second testing period (fig. B), 3 days after the initial exposure, gave no significant differences for tests of treatments versus control or for each treatment against the other. During the third testing period (fig. C), after reverse training or experience response of soybean trained females was significantly higher than that of “control” females (P = 0.030). The comparison of the t w o sensitization treatments was not significant. However, trained wasps showed a significant difference (P = 0.045) in response with the highest number of wasps flying to their host+hostplant of initial training. The second and third testing periods (fig. B, C) data were obtained on fewer wasps responding because of non-responsiveness o r mortality of wasps.

270 J . P. Kaas, G. W. Elzen, and S. B. Ramaswamy

p values vs. control

2 4 : 0.448 3-c : 0.368

1 4 : 0.894

4-C : 0.051

1 2 3 4 c

1 2 3 4 c

1-C : 0.479 2 4 : 0.732

4-C : 0.386 3-C : 0.286

p values vs. control

I-C : 0.916 2-C : 0.906

4-C : 0.030 3 4 : 0.780

1 2 3 4 c

TYPE OF EXPERIENCE

I SENSITKATKN I CONDm3NMj I

Female M . mceipes host plant choices in a wind tunnel. A pair of bars represents number flying to cotton or to soybean for each type of pretreatment in test period 1, immediately after first experience (A); test period 2, three days after first experience (B) and test period 3 , four days after first experience and immediately after reverse experience (first experience shown in fig. C). ':. Reverse experience is same as first experience but cotton and soy are interchanged. P values given are for 2 x 2 G test for cotton experience vs soybean experience (listed above treatment bars) and for each type of experience vs control. Legend: 1 = Exposure to cotton leaf only, prior to first test, 2 = Exposure to soybean leaf only, prior to first test, 3 = Exposure to cotton-leaf, frass, chewed leaf fragments and H. virescens larvae from cotton, prior to first test, 4 = Exposure to soybean-leaf, frass, chewed leaf fragments and H. virescens larvae from soybean, prior to first test, C = No exposure prior to first test, 'i Significance at a = 0.05

4 Discussion

The control wasps in our experiment were unexposed only in their first testing period. However, a control would be indispensible only in a single source testing procedure; the current study, testing the response to two different exposure sources increases the

Learning in Microplitis o-oceipes 271

sensitivity of the test. Wasps having received exactly the same treatment but on different sources may also be a better comparison than on each source vs “naive controls” (DROST et al. 1986). The current study differs from many other wind tunnel studies on M . croceipes in offering a two choice situation and therefore testing response quality under flight condi- tions. The results of the first testing period are in accordance with those of LEWIS and TUMLINSON (1 988), where M . croceipes showed a conditioned response to olfactory cues. Studies on learning in parasitoid wasps are numerous: for example DMOCH et al. (1985) studied the influence of prior experience on search behaviour of Cotesia rnarginiventris and found an increased response to stimuli and increased activity of wasps after previous contact with host material. DING et al. (in press) demonstrated increased response of Macrocentrtts grandii Goidanich to several host plants in an olfactometer after conditioning with host frass and found preference for plant material on which wasps had previously found hosts. DROST et al. (1986, 1988) and ELLER et al. (1988) studied responses of experienced and inexperienced M . croceipes in a wind tunnel and olfactometer respectively. Both authors found increased responsiveness to host plant sources after prestimulation with larvae or larval products.

Testing period 2 shows that this conditioned response decreases with time, thus meeting the requirements of reversibility for learning (MCGUIRE 1984). ARTHUR (1966, 1967) and WARDLE and BORDEN (1985) also noticed a decline in learned response with time in I . conqttisitor and Exeristes roborator (F.), respectively. Test period 3 shows that wasps not only fail to respond to training on the reverse source, as might be explained by an age dependency of learning ability (WARDLE and BORDEN 1985), but the experience seems to trigger the wasp’s initial preferences or makes them “remember” their first training. This might indicate a sensitive period in the development of the animal, often referred to as “critical period” (EIBL-EIBESFELDT 1970) or “sensitive phase”. This characteristic of M . croceipes wasps remembering their first training resembles the process of imprinting in higher animals; such behavior has been referred to as imprinting in parasitoid wasps also (HERARD et al. 1988). Characteristics that distinguish imprinting from other, related, processes are (EIBL-EIBESFELDT 1970): 1 . presence of a sensitive period; 2. a lifelong irreversible retention; 3. based on supraindividual species characters; 4. eliciting specific reactions; 5 . ability to take place before manifestation and 6. better in massed trials than in distributed trials. Study of imprinting has undergone its historical development in warm- blooded vertebrates and is placed in a context of social interaction (e.g., BATESON 1966). Consequently, use of the term imprinting may not be appropriate to the phenomena observed in M . croceipes in the current study. Learning in parasitoid wasps may, however, show some characteristics in common with the process of imprinting. Indication of an age dependent component in conditioning capability (WARDLE and BORDEN 1985; VET 1983) may resemble a “sensitive period” but only superficially because normally a sensitive period is a well defined, relatively short, period in the life of an animal (EIBL-EIBESFELDT 1970). Indication of irreversibility, as shown by our results agrees with the second characteristic of imprinting listed by EIBL-EIBESFELDT (1970). Additionally, wasps exhi- bited a specific reaction to the choice offered; a “latent learning” component would be difficult to demonstrate in our situation because of the conditioned nature of the process. The advantages of this learning process for a searching wasp are apparent for the first test result, i.e. having parasitized a host on a host plant species the parasitoid not only shows increased response (DROST et al. 1986) but also limits its search to the rewarding host plant species, thereby optimizing chances of parasitization. This response decreases in intensity with time if n o reinforcement is received and thus, females with little success in finding hosts may broaden their choice. The fall back to acceptance of the source of initial conditioning after a lagtime and subsequent reward is inexplicable at this time. However, host switching would seem to be the most rewarding strategy and reverting back to a host plant-host situation that may not even exist any more may be counteradaptive.

2 72 J. P. Kaas, G. W. Elzen, and S. B. Ramaswamy

Acknowledgements Thanks are due to S. HARTLEY and Staff of the SFCIML for rearing the insects, to R. NAVASERO for maintenance of wasp colonies and to W. LANGHOLFF for statistical advice. Funding for this project was provided by a USDA Competitive Grant #87-CRCR-1-2473. Mention of a trade name, proprietary product, or specific equi ment does not constitute a guarantee or warranty by the USDA and does not imply its approval to tKe exclusion of other products that may be suitable.

Zusammenfassung Zum Lernverhalten von Microplitis croceipes Cresson (Hym., Braconidae)

Mittels Wahlversuchen in einem WindkanaI wurden die Reaktionen von gepragten Microplitis croceipes-Weibchen in drei verschiedenen Zeitabschnitten nach der Pragungsphase getestet. Die Pragung bestand darin, dad den Weibchen Baumwoll- oder Sojabohnenblatter mit und ohne Wirtslar- ven (Heliothis virescens) angeboten wurden. Die Kontrolltiere wurden keiner Pragung unterzogen. Die Windkanalversuche wurden wie folgt durchgefuhrt: 1 Stunde nach der Pragung, 3 Tage danach oder nachdem die Weibchen nicht der Pragungs flanze, sondern der anderen Art ausgesetzt wurden. Der paarweise Vergleich der Ergebnisse der Wahversuche bei den gepragten und den Kontrolltieren ergab nur in einem Fall einen signifikanten Unterschied. Der paarweise Vergleich von Parasitoiden- weibchen, die auf Baumwolle bzw. Sojabohne gepragt waren, ergab in den Uberpriifungsperioden 1 und 3 einen signifikant hoheren Anflug an die Pflanze, auf welche die Weibchen gepragt wurden. Die Ergebnisse lassen darauf schlieden, dad bei M. crocei es eine Pragung auf Wirt und Wirtspflanze vorlie t, dai3 diese Pragung zeitabhangig ist und d a l e s sich dabei um eine irreversible Pragung handeft.

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Authors’ addresses: J. P. KAAS and G. W. ELZEN, Southern Field Crop Insect Management Labora- tory, P.O. Box 346, Stoneville, MS 38776, USA; S. B. RAMASWAMY, Department of Entomology, Mississippi State University, Mississippi State, MS 39762, USA

968-971.