Regulation of Age Polyethism in Bees and Wasps by Juvenile Hormone

ADVANCES IN THE STUDY OF BEHAVIOR, VOL. 26

Regulation of Age Polyethism in Bees and Wasps by Juvenile Hormone SUSANE. FAHRBACH DEPARTMENT OF ENTOMOLOGY UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN URBANA, ILLINOIS

The function of JH in the sterile female caste of honeybees cannot be compared with the function in fertile female insects. Its function consists apparently in the control of a number of physiological processes which are probably in close relation to behavioural changes associated with the division of labour. It seems likely that the increasing JH titre is responsible for inducing the transformation of hive bees into field bees. (Rutz, Gerig, Wille, and LUscher, 1976, p. 1490) The hypothesis of polyphenism of honeybees being controlled by juvenile hormone has still a deficient base. (Fluri, Liischer, Wille, and Gerig, 1982, p. 65) As a bee ages, she may undergo a programmed change in central nervous system (CNS) response thresholds to task-associated stimuli, mediated by changes in JH titre. (Page and Robinson, 1991, p. 133)

I. INTRODUCTION A N D THE DIVISION OF LABOR A. EUSOCIALITY

A long human appreciation of the division of labor characteristic of insect societies has in the twentieth century matured into an understanding rooted in evolutionary biology. The “advantage” of social life is recognized to lie in the efficiencies of task specialization and cooperative defense of the nest. The ecological success of the termites, ants, bees, and wasps results in large part from the division of labor that is at the heart of eusociality (Wilson, 1971; Oster and Wilson, 1978). Among the social insects, the primary division of labor is reproductive versus nonreproductive. Societies comprise reproductive and nonreproductive castes, with the reproductives being accorded “royal” status. Nonrepro285

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ductives (workers) are in some species also divided into distinct physical castes, such as the major and minor workers of some ants and termites. The mechanisms of physical caste determination have been investigated, and are, depending on the group studied, possibly under genetic control or susceptible to extrinsic factors, such as larval nutritional status and larval endocrine profiles (Wilde and Beetsma, 1982; Nijhout and Wheeler, 1982; Hardie and Lees, 1985). For the social bees and wasps that form the subject of this review, caste polymorphism is restricted to females. Depending on the species, differences between reproductives (queens) and nonreproductives (workers) may be based on differences in physiology, size, external morphology, behavior, or a combination of these features (reviewed by Hardie and Lees, 1985). In some groups, queens may be distinguished from workers primarily on the basis of their reproductive function, and may be as competent as workers to build a nest, forage, and to rear offspring. Such a description characterizes the queens of many of the primitively eusocial bees, such as the bumble bees, as well as those of temperate zone social wasps that found colonies in the spring after solitary hibernation. Some halictine species exhibit varying degrees of behavioral and morphological differentiation between the queens and the worker castes (Wilson, 1971). By contrast, the queens of the honey bees (Apini) and stingless honey bees (Meliponini) are unable to function as workers under any circumstances. B. AGEPOLYETHISM Even where differences between queens and workers are notable, workers are often very nearly monomorphic. This is particularly true for workers of the well-studied European honey bee, Apis mellifera (Wilson, 1971). This means that division of labor is often not associated with distinctive external morphologies. Age polyethism, one of the most common forms of behavioral polymorphism found in bee and wasp societies, is of this type (Wilson, 1971; Hardie and Lees, 1985; Robinson, 1992). Age polyethism refers simply to division of labor on the basis of worker age. Phrases such as “temporal castes” and “temporal polyethism” are also used to indicate this phenomenon. Scholars of the social insects have recognized that division of labor on the basis of behavioral characteristics alone is not well described by the term “polymorphism,” and many writers have followed the proposal of Michener (1961) that age polyethism be described instead as a form of “polyphenism.” Although Liischer (1976) subsequently used “polyphenism” to include division of labor on the basis of morphological differences, the sense intended by Michener remains highly useful. More recently, polyphenism has been defined as “the occurrence of two or more

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distinct phenotypes which can be induced in individuals of the same genotype by extrinsic factors” (Hardie and Lees, 1985, p. 443). This definition is highly appropriate for a consideration of age polyethism. Age polyethism is itself a neutral term indicating no fixed sequence of tasks, but almost without exception younger workers perform tasks in the nest, while older workers forage (Wilson, 1971; Page and Robinson, 1991; Robinson, 1992). Although few other species have been studied in as much detail as the European honey bee, all of the existing evidence demonstrates that this pattern is the rule among species of highly evolved social insects. Consistent and predictable differences in behavior must reflect underlying or “covert” differences in physiology or anatomy. Particularly in the social bees, age polyethism refers to a set of characters rather than a single feature (Hardie and Lees, 1985; Robinson, 1992). Hive bees not only spend almost all of their time within the nest, but also have better developed brood food glands (hypopharyngeal glands) than their foraging sisters (Gast, 1967; Imboden and Liischer, 1975; Rutz et af., 1976; Wilde and Beetsma, 1982; Fluri el al., 1982). Hive bees also have significantly lower titers of the sesquiterpenoid juvenile hormone than field bees. As I discuss later in this review, it is juvenile hormone that holds the key to understanding the mechanisms of strong age polyethism in the bees and wasps. As the quotations that open this chapter indicate, this is not a particularly new idea. Yet, despite significant recent improvements in juvenile hormone measurements that addressed the “deficient base” of Fluri ef al. (1982), this literature still has, if not a deficient base, a hollow core. This is because of our utter lack of understanding of how juvenile hormone regulates behavior in adult insects. However, recent demonstrations of striking structural changes in the brains of adult honey bees correlated with age polyethism suggest that it may be possible to link juvenile hormone and neuroanatomical plasticity in the bee brain. This provides a focus for future investigations of juvenile hormone regulation of behavioral plasticity that can be conducted at the cellular and molecular as well as the ethological and ecological levels. OF AGEPOLYETHISM C. OTHERASPECTS

A comprehensive review of the details of age polyethism, its function in insect societies, and likely evolutionary scenarios are not provided here. There is a large, excellent, and highly accessible literature available on this topic (e.g., Wilson, 1971; Michener, 1974; Alford, 1975; Seeley, 1985; Winston, 1987; Ross and Matthews, 1991; Robinson, 1992). Honey bees have also been put to excellent use in investigations of insect visual and olfactory learning; these studies are not reviewed here, as they have by and large emphasized the capabilities of the forager and have ignored the

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fact of age polyethism (Hammer and Menzel, 1995). This review is instead from the perspective of a neurobiologist interested directly in the neuroendocrine mechanisms that control the behavior of individual animals. 11. AGEPOLYETHISM A N D JUVENILE HORMONE IN THE EUROPEAN HONEY BEE,APISMELLIFERA

A. AGEPOLYETHISM The division of labor prevailing in honey bee colonies has been remarkably well described. Both marked cohorts of bees and individually tagged bees have been studied. A single worker bee in an undisturbed colony will perform a stereotyped sequence of tasks as she ages. As outlined by Seeley (1982), the major honey bee age castes are (in order of occurrence within the life of a single worker bee) cell cleaning, brood and queen care, food storagehest maintenance, and foraging. Tasks within the hive are typically performed by workers 1-3 weeks of age, while bees older than 3 weeks forage outside the hive or engage in colony defense. Careful studies have revealed that the transitions between age castes prior to the switch to foraging (e.g., the change from care of larvae to food storage) involve age-correlated changes in the relative frequencies with which different categories of tasks are performed (Robinson, 1992). By contrast, the final shift to foraging is more emphatic. The end of adult behavioral development is a behavioral phase devoted to specific tasks associated with foraging (alternatively, a subset of bees this age may become soldiers, engaged mainly in colony defense rather than resource collection). The world of the forager is quite unlike that of the younger hive bees. Younger bees take only short defecation and orientation flights; foragers take long-distance flights covering hundreds of meters and lasting as long as an hour (Winston, 1987). It has also been widely recognized that foraging bees display capacities for rapid learning, sun compass navigation, longterm memory formation, and symbolic communication remarkable for an invertebrate (Frisch, 1967; Winston, 1987; Menzel, 1985, 1990). Only rare idiosyncratic individuals fall outside of this analysis by exhibiting extreme task specialization, such as dedication to water carrying or social grooming (Robinson, Underwood, and Henderson, 1984; Moore, Angel, Cheeseman, Robinson, and Fahrbach, 1995). Two influences have been identified that regulate age polyethism in the honey bee. The first is a set of yet-undescribed genetic factors (Calderone and Page, 1991; Robinson and Page, 1988, 1989; Robinson, Page, Strambi, and Strambi, 1989; Page and Robinson, 1991; Robinson, 1992). The second is change in the titers of juvenile hormone during adult behavioral development.

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INFLUENCES ON AGEPOLYETHISM B. GENETIC The genetic basis of the division of labor in honey bees is being actively investigated (Page, Waddington, Hunt, and Fondrk, 1995; Hunt, Page, Fondrk, and Dullum, 1995; Dreller, Fondrk, and Page, 1995; Robinson and Page, 1995). A honey bee colony consists of different “subfamilies” of workers because diploid queens mate with multiple haploid drones (reviewed by Page and Robinson, 1991). The sperm obtained at these matings (which typically occur during a small number of “nuptial” flights taken during the first 10 days of the queen’s life) are then stored in the spermatheca, a sperm storage organ adjacent to the reproductive tract (reviewed by Winston, 1987). The sperm from different drones are mixed in this storage organ and used by the queen throughout the remainder of her life to fertilize eggs to produce female offspring (in the Hymenoptera, haploid eggs become males, while diploid eggs become females). Females that share both a mother and a father are referred to as super sisters (see Page and Laidlaw, 1988, for a full discussion). Assuming random mating, super sisters will have on average 75% of their genes in common. Individuals within a colony whose fathers are unrelated drones are called half sisters. Members of different subfamilies have on average only 25% of their genes in common. It has been shown that, within individual colonies, honey bee subfamilies show genetic variation in the probability of performing different tasks (Robinson and Page, 1988, 1989). Subfamilies vary in their propensity to perform tasks commonly done by older workers, such as nectar foraging and pollen foraging, as well as the more specialized tasks of nest site scouting, guarding the hive entrance, and undertaking. There also appear to be significant genotypic differences in the rate of development of foraging behavior (Giray and Robinson, 1994). INFLUENCES ON AGEPOLYETHISM C . HORMONAL The second factor, developmental changes in titers of juvenile hormone, is the topic of this review. This factor is related not only to the normal progression of an individual worker through the age castes but also to the behavioral flexibility that is a normal part of the age polyethism displayed by honey bee colonies. It has long been appreciated, despite the inevitable march from within-hive tasks to field tasks, that the age of the transitions differs among colonies (reviewed by Michener, 1974; Seeley, 1985;Winston, 1987; Robinson, 1992). This plasticity in the behavior of adult workers permits colonies to make rapid adjustments to new situations. This plasticity ranges from accelerated behavioral development (premature foraging in response to a shortage of older workers) and temporary slowdowns in the

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progress of age polyethism (often seen in times of bad weather), to delayed behavioral development (older bees rearing brood) in response to a shortage of younger workers. In extreme cases, it is even possible for foragers to revert to rearing brood in response to an extreme shortage of younger bees. Although this plasticity is often revealed by experimental manipulations of colony population demographics, there is no doubt that it exists in nature and can be elicited by natural situations including weather catastrophes, severe predation, and reproductive swarming. It is now well established that changes in juvenile hormone titer accompany all of these transitions, including accelerated development (Robinson ef al., 1989) and behavioral reversions (Robinson, Page, Strambi, and Strambi, 1992; Farris, Robinson, and Fahrbach, submitted). D. JUVENILE HORMONE A N D AGEPOLYETHISM

Juvenile hormone (Fig. 1) is an insect sesquiterpenoid hormone produced by the paired corpora allata glands located in the head on either side of the esophagus (Snodgrass, 1984). The Hymenoptera produce the form of this hormone referred to as JHIII (Hagenguth and Rembold, 1978), and hemolymph titers of juvenile hormone in the honey bee are closely related to rate of synthesis, rather than to variations in rate of metabolism (see Fahrbach and Robinson, 1996, for a recent review of juvenile hormone in honey bees). Juvenile hormone was first identified on the basis of its central role in the regulation of metamorphosis and maturation of the ovaries in adult females, but is now known to influence many aspects of insect life histories including diapause, migration, coordination of reproduction with environmental cues, and caste determination (Wigglesworth, 1934, 1936; Riddiford, 1994). It may even act as a pheromone in some species of A OCH3

B

FIG.1. The structures of compounds with juvenoid activity in the honey bee. ( A ) JHIII, the native form of juvenile hormone in the honey bee. (B) Methoprene, a potent juvenile hormone analogue widely used in behavioral studies of honey bee behavioral development.

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291

parasitic wasp (Holler, Bargen, Vinson, and Witt, 1994). The production of juvenile hormone by the corpora allata is regulated both by circulating factors and by direct innervation by neurosecretory cells located in the brain. These peptidergic signals are referred to as allatotropins and allatostatins. They are produced by both the central nervous system and peripheral tissues such as the ovaries, and are currently best described in terms of the reproductive function of the cockroach, Diploptera punctatu (Woodhead, Stay, Seidel, Tobe, and Khan, 1989; Yu, Stay, Joshi, and Tobe, 1993; Stay ef al., 1994). The molecules regulating juvenile hormone production in the honey bee are likely to be similar, but are currently undescribed. Additionally, little is known about the cellular and molecular mechanisms of juvenile hormone actions. Progress in this field has been hampered by a lack of information on the juvenile hormone receptor as well as by the difficulties of measuring juvenile hormone titers in the hemolymph of individual insects (reviewed by Fahrbach and Robinson, 1996; Jones, 1995; Riddiford, 1994). For example, the heroic microsurgeries required to remove the glandular source of juvenile hormone, the corpora allata, have until very recently been compromised by an inability to assess the results of the surgery in terms of juvenile hormone titers. On the other hand, progress has been greatly assisted by academic and commercial interest in developing juvenile hormone analogues to serve as insect growth regulators for pest management (Williams, 1967).This has resulted in the development of potent compounds that mimic the effects of juvenile hormones (juvenoids) such as methoprene, which was developed by the Zoecon Corporation (the development of artificial insect growth regulators is described by Djerassi, 1992), and has proved extremely useful in research on polyethism among the social Hymenoptera. The idea that juvenile hormone is associated with the transition of hive bees to field bees can be traced in the literature back to the late 1960s. The idea reflected accumulating evidence for the association of juvenile hormone with polymorphisms and polyphenisms (see, for example, Liischer’s work on the differentiation of termite castes: Liischer, 1960, 1972). Also influential were observations that the size of the corpora allata changes as worker bees age, with greatest corpora allata volumes attained by foragers (see references given by Jaycox, Skrwronek, and Gwynn, 1974). Gast (1967) demonstrated that implantation of corpora allata from foraging bees or the injection of farnesyl methyl ester, a juvenile hormone mimic, induced a physiological correlate of this behavioral transition, the degeneration of the hypopharyngeal glands. It was subsequently shown that a similar inhibitory effect on the hypopharyngeal glands could be obtained by application of JHIII, the native hormone, as well as by application of the juvenile hormone analogue, triprene (Rutz et al., 1976). Additionally, allatectomy

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(surgical removal of the corpora allata) prevented hypopharyngeal gland degeneration in aging worker bees (Imboden & Liischer, 1975). Jaycox and colleagues (1974) performed the first published studies in which behavioral endpoints in worker honey bees were assessed subsequent to treatment with a juvenile hormone mimic, the so-called Law-Williams mixture, hydrochlorinated methyl farnesenate (Law, Yuan, and Williams, 1966). Worker bees were treated on the day of emergence with the mimic, establishing a paradigm that has been followed in every subsequent juvenile hormone treatment study. The results included inhibitory effects on hypopharyngeal gland development, but also demonstrated that bees treated with the mimic appeared to attain behavioral maturity earlier than controls housed in the same colony. This result has since been confirmed by other investigators, with the landmark study by Robinson (1987b) and subsequent reports by this investigator and his colleagues forming much of the basis for our current understanding of the relationship of juvenile hormone to age polyethism in honey bees. Three lines of evidence are essential for demonstrating naturally occurring hormonal regulation of a specific behavior. First, the behavior in question should be reliably induced by application of that hormone at physiological levels in animals at the appropriate stage of development and, in the case of social insects, housed in the appropriate social setting. It should be noted that a technical barrier to demonstrating some of the effects of juvenile hormone on age polyethism is the inability of workers of many species to survive as isolated individuals. Second, naturally occurring changes in the titer of the suspect hormone or in the sensitivity of the target tissues should be correlated with the changes in behavior. Until recently data of this type have been highly suggestive but not entirely persuasive because of a lack of juvenile hormone titer determinations on individual bees. As will be discussed later, this difficulty has now been forcefully addressed by the development of a sensitive radioimmunoassay for juvenile hormone in honey bee hemolymph. Finally, it should be possible to disrupt the performance of the behavior in question by removal of the source of the hormone, or by blocking its action at its receptors. The definitive experiments of this type awaited the development of the ability to assay juvenile hormone titers in the p1 hemolymph samples that can be obtained from individual bees. An additional form of powerful correlational data can be obtained from the use of colony manipulations to induce alterations in juvenile hormone titers and behavior. Such manipulations are entirely feasible when honey bees are reared in experimental apiaries using techniques developed for commercial apiculture.

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The next sections will summarize the current evidence for the relationship of juvenile hormone to age polyethism in the European honey bee. This will serve as the basis for a discussion of structural plasticity in the adult bee brain, and its relationhsip to behavioral maturation. 111. EFFECTS OF EXPERIMENTAL TREATMENT WITH JUVENILE HORMONE, MIMICS, A N D ANALOGUE ON BEHAVIORAL MATURATION IN THE HONEY BEE A. EFFECTS OF EXPERIMENTAL TREATMENTS

Table I summarizes the literature on the behavioral effects of treatment of worker honey bees with juvenile hormone, juvenile hormone mimics, and juvenile hormone analogue such as methoprene. Several points are clear. First, results are consistent across experiments. Treatment of newly emerged adult worker bees (0-24 hr postemergence) with an active juvenoid (at a time when juvenile hormone levels are naturally quite low) results in accelerated behavioral maturation. Dose-dependent effects can be seen regardless of whether marked treatment groups or marked individual bees are studied, regardless of whether the experimental bees are housed in large or small colonies (or in glass-sided observation hives), and regardless of the specific behavior sampling strategy used. Although the native hormone JHIII is most active when injected (dissolved in oil) into the hemocoel, the analogue methoprene is extremely active when applied topically (dissolved in acetone), and can even be fed to bees dissolved in sugar syrup and still be effective. The report of Robinson is noteworthy in that it is based on the study of individually number-tagged bees housed in a large observation hive (Robinson, 1987b). This permitted the effects of topical application of methoprene to be examined on within-hive behavior as well as on activity monitored at the hive entrance. In this study it was established clearly that not only is the mean age at first foraging reduced by treatment with methoprene, but also that methoprene treatment inhibits brood and queen care and reduces the food-storing phase. The impression is not so much one of an abnormal trajectory of development as it is one of a “compression of normal development” (Robinson, 1987b). Another important finding was that worker bees treated with methoprene on the first day of adult life displayed precocious but otherwise apparently normal foraging behavior (Robinson, 1985). B. QUESTIONS RAISED BY TREATMENT STUDIES These accumulated data raise the following questions. First, is methoprene a suitable replacement for juvenile hormone in behavioral studies

TABLE I

EFFECTS OF TREATMENT WITH JUVENILE HORMONE, ANALOGUES. AND MIMICS ON Subjects"

Treatment

Method

THE

BEHAVIOR OF ADULT WORKER HONEYBEES,MIS

Observed behavior

Results of treatment leave brood nest earlier; precocious guarding; precocious pollen foraging; increase in total number of Bights no response to topical JHI; weak enhancement of flight activity with topical Law- Williams Mixture flight away from the hive begins earlier; more bees fly in treated groups; increase in total number of flights highest dose lethal; 250-pg dose results in early shift to food storage, earlier orientation flights, earlier foraging

0-24 hr adults, small observation hives

Law-Williams Mixtureb, injected in oil 10-200 p g

distribution in hive, activity at entrance

0-24 hr adults, single frame observation hives

JHI, 0.1-10 pg Law-Williams Mixture, 100 pg

topical application in 95 :5 acetone : olive oil

activity at entrance

0-24 hr adults, single frame observation hives

JHI, 10 p g Law-Williams Mixture, 100 pg

injected in oil

activity at entrance

0-24 hr adults, typical colony with modified entrance

methoprene, 2.52500 Pg

topical application in acetone

number-tagged bees; daily 1 hr entrance observations

N

P

MELLIFERA

Reference Jaycox

et

al., 1974

Jaycox, 1976

Jaycox, 1976

Robinson, 1985

g vI

0-24 hr adults, typical colony

methoprene. 50 or 200 1Lg

topical application in acetone

0-24 hr adults. typical colony with modified entrance

hydroprene, 20 or 200 pg methoprene. 200 p g or 1 mJ.3

orally, dissolved in sugar syrup

0-24 hr adults, large observation hives

methoprene, 25-250 pg

topical application in acetone

8 hr observatiordday. frequency of 30

0-24 hr adults, typical colony

JHIII, 1 pg methoprene. 0.1-200 p g hydroprene, 2 or 20 pg

topical application in acetone O R injected in oil

activity at entrance; latency to forage

0-24 hr adults, singlecohort colony

methoprene, 200 p g

topical application in acetone

number-tagged bees; daily 1 hr entrance observations

a

Age of worker honey bee at time of treatment. Hydrochlorinated methyl farnesenate (Law er al., 1966).

response to alarm pheromone, tests at 1-23 days posttreatment entrance observations at 11. 12. 13, 15. 22, 26 days

behaviors; duration of task phase; daily entrance observations

dose-dependent strengthening of all responses

Robinson, 1987a

dose-dependent increase in number of flights during week 2 of study dose-dependent reduction in mean age at foraging; dosedependent inhibition of brood and queen care; reduction in food-storing phase precocious guarding, pollen foraging up to 5 days earlier than controls; weaker effects with topical applications 83% treated bees forage by 10 days of age, compared with 16% controls

Robinson and Ratnieks. 1987 Robinson, 1987b

Sasagawa, Sasaki, and Okada, 1989

Robinson er al., 1989

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SUSAN E. FAHRBACH

of honey bees? Second, are the effects of early treatment with juvenoid agents a result of effects exerted at the time of application (the first 24 hr of life), or do they reflect instead the persistence of unmetabolized hormone analogue, which then acts at a later time? Third, what is (are) the target(s) of juvenile hormone action? In response to the first question, methoprene has been widely used in studies of insect endocrinology, and is a potent juvenile hormone analogue, with activity in all insect species in which it has been tested (Staal, 1975). It has the advantage of being more active than JHIII when topically applied, which is an alternative to injection that minimizes handling of treated bees. There are no discrepancies in the behavioral actions of juvenile hormone and methoprene (see Table I). A comparison of physiological and anatomical effects of juvenile hormone and methoprene treatment of adult worker honey bees (such as effects on hypopharyngeal gland development) also reveals few discrepancies. It has been reported that wax secretion, which is higher in hive bees than in foragers, may not be under inhibitory control by JHIII, but may be sensitive to methoprene (Muller and Hepburn, 1994). This report is difficult to interpret in light of the lack of information concerning juvenile hormone .titers in manipulated bees and the lack of any other behavioral or physiological measures validating the effectiveness of the treatment. Discrepancies in this study between the effects of JHIII and methoprene may reflect the greater potency of methoprene at the doses selected to accelerate behavioral development, which led in turn to the aforementioned “compression” of the wax secreting period. The doses of JHIII may have been less effective in producing accelerated development. Therefore, at the present time we can be confident that data based on methoprene treatment are useful and reliable. Second, because studies have not systematically varied the time of juvenile hormone treatment, it is not possible to determine the time of action on the basis of these experiments alone. Methoprene is likely to persist longer in the body than JHIII, and may be absorbed through the cuticle at a different rate than JHIII. Whether or not this treatment is the honey bee equivalent of implantation of a hormone-filled Silastic capsule or osmotic minipump is not known. This issue is taken up again in the following discussion of the naturally occurring fluctuations in juvenile hormone titers that occur during adult life in the honey bee. What are the targets of juvenile hormone action with regard to behavior? Sites within the central nervous system are obvious candidates. Only one study has investigated potential peripheral sites of action. Application of methoprene on the first day of adult life was shown to induce a precocious behavioral response (wing flickering) to alarm pheromone under controlled conditions in the laboratory (Robinson, 1987a). This same dose had no

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297

effect on the electroantennogram response to these compounds, suggesting that the hormone-mediated changes in behavior occurred centrally rather than at the level of the olfactory receptors.

IV. CORRELATIONAL DATAINDICATING JUVENILE HORMONE TITERS INCREASE DURING BEHAVIORAL MATURATION A N D AREAT THEIR HIGHEST I N FORAGING BEES DEVELOPMENTAL CHANGES IN JUVENILE HORMONE PRODUCTION As discussed in the preceding section, premature exposure to high levels of juvenile hormone leads to accelerated behavioral development, with an earlier onset of foraging. These results gain significance when taken together with measurements showing that older, foraging bees typically have higher levels of juvenile hormone than younger hive bees. As Table I1 suggests, it has been appreciated for some time that foraging bees are characterized by high levels of juvenile hormone relative to hive bees. A major problem, however, has been to develop methods sensitive enough to measure titers in individual bees. The most widely used methods of juvenile hormone determination have been bioassay (the Galleria wax-wound test, in which a wound made in the pupal cuticle of a moth is sealed with wax containing juvenile hormone: see description in Nijhout, 1994), gas chromatography combined with mass spectrometry (Mauchamp, Lafont, and Krien, 1981), and radioimmunoassay (Granger and Goodman, 1988). The first radioimmunoassay used for juvenile hormone determinations in the honey bee required pooled hemolymph samples in order to achieve detectable amounts of hormone in the sample (Strambi, Strambi, deReggi, Hira, and Delaage, 1981). Researchers in this field, however, have dealt systematically with the problems of quantification of this small, lipophilic molecule, and recently new, more sensitive assays that can be used with simple but effective extraction procedures have been developed (Goodman et al., 1995). The development of a new radioimmunoassay for juvenile hormone that permits determinations on hemolymph samples from individual bees has added clarity to this field, and will be widely used in future behavioral studies (Hunnicutt, Toong, and Borst, 1989; Huang, Robinson, and Borst, 1994). This methodological development also permits the rigorous assessment of the effects of allatectomy. An alternative to radioimmunoassay that permits insight into the endocrine function of individual bees is the biosynthesis assay. In this assay, cultured corpora allata glands are provided with a radiolabeled precursor and the amount of juvenile hormone produced is determined by scintillation

TABLE I1 MEASUREMENTS OF JUVENILE HORMONE TITERS IN ADULT WORKER HONEYBEES.APlS Subjects h)

8

Method

Pooled or individual

MELLlFERA

Values"

Reference

0-24 hr adults (i) 12 day hive bees (ii) 24 day field bees (iii)

bioassay using Galleria wax test

pooled: 40 bees/sample

328 GUh/ml (i) 1807 GU/ml (ii) 3820 Gdml (iii)

Rutz. er al., 1976

0-24 hr adults (i) 3-13 day adults (ii)

gas chromatography with electron capture detection

pooled: 1-4 g bees

0.7 ng/g (i) 10 nglg (ii)

Hagenguth and Rembold, 1978

0-24 hr adults (i) 10-15 day adults (ii) field bees (iii)

bioassay using Galleria wax test

pooled: 40 bees/sample

very low (i) 1000-1500 GUlml (ii) >2000 GU/ml (iii)

Fluri ef al., 1982

hive bees guard bees foragers

HPLC-determination

pooled: 10 beeslsample: and individual

low until day 14; H. Sasagawa, 1988. unpublished observations intermediate until day 28: 2-3 ndp1 in older bees

swarm nurses (i) foragers (ii)

RIA (Strambi et af., 1984)

pooled: 4-16 beedgroup

t 5 pmo1/100 p1 (i) 20-65 pmo1/100 pI (ii)

workers from a single-cohort RIA (Strambi er al.. 1984) colony of known age and behavioral status

pooled: 4-16 bees/group

2-10pmo1/100pl, nurse bees Robinson, et al., 1989 20-75 pmo1/100 @I, foragers

Robinson, ef al., 1989

nurse bees (i) foragers of known age (ii)

radiochemical analysis of JHIII biosynthesis by cultured CC-CA complex

individual

nurse bees ( i ) foragers (ii)

RIA (Strambi er al., 1984)

pooled 5-9 bees/age group <10 pmoUl00 pl (i) 40-45 pmoUlOO pl (ii)

Huang et al., 1991

workers of known age: wax producers, food storers, guards, undertakers. soldiers, foragers

radiochemical analysis of JHIII biosynthesis by cultured CC-CA complex

individual

1-2 pmol/hr: queen attendants, nurses, wax producers, food storers 3-5 pmolhr: foragers. guards, undertakers

Huang, Robinson, and Borst. 1994

0-24 hr adults (i) foragers (ii)

radiochemical analysis of JHIII biosynthesis by cultured CC-CA complex

individual

approx. 2 pmoYhr (i) approx. 6 pmol/hr (ii)

Huang and Robinson, 1995

0-24 hr adults (i) foragers (ii)

RIA (Hunnicutt, Toong, and individual Borst, 1989)

(100 nglml (i)

Huang and Robinson, 1995

nurse bees (i)

RIA (Hunnicutt era[., 1989) individual

200 ng/ml (i) 600-800 ng/ml (ii)

Farris, Robinson, and Fahrbach, submitted

foragers (ii)

Expressed as concentration per hemolymph volume where possible. Galleria units.

1-2 pmol/hr (i) 4-6 pmol/hr (ii)

400 ng/ml (ii)

Huang et al., 1991

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counting of an extract of the medium. A biosynthesis assay developed for the honey bee (based on the successful Diploptera punctata assay developed by Tobe and co-workers: Tobe and Pratt, 1974; Pratt and Tobe, 1974) produced data in good agreement with subsequent radioimmunoassaydetermined titers, and confirmed that rate of synthesis is the primary determinant of juvenile hormone titer in the honey bee (Huang etaf.,1991,1994). Because it is labor intensive relative to radioimmunoassay, the usefulness of the biosynthesis assay has been superseded for large-scale behavioral studies, although it will be vital for the exploration of factors regulating juvenile hormone synthesis. As in the case of the effects of treatment with juvenile hormone and its analogue, there is striking agreement as to the overall profile of juvenile hormone titers during behavioral development. The correlations between low levels of juvenile hormone and nursing and high levels of juvenile hormone and foraging are very, very strong. One surprising discovery has been the finding that a few tasks not directly related to foraging are typically accompanied by foragerlike high levels of juvenile hormone. Examples of such activities are guarding the hive entrance and undertaking (Huang et al., 1994). An area for future investigation is the filling in of the day-byday details of the profile. It is possible that transient peaks in juvenile hormone titers are hidden in the current profiles, particularly early in adult life. This information will be vital to our interpretation of the timing of juvenile hormone action during development.

V. COLONY MANIPULATIONS THATINDUCE ALTERED JUVENILE HORMONE TITERS A N D ALTERED BEHAVIOR; SEASONAL CHANGES I N JUVENILE HORMONE TITERS A N D BEHAVIOR

A. SINGLE-COHORT COLONIES The predictable association of high levels of juvenile hormone with foraging in honey bees is no less strong when behavior is manipulated by alterations in colony population demography. Although the oldest workers are typically a colony’s foragers, age alone determines neither behavior nor endocrine status. An experimental single-cohort colony of honey bees can be founded with a queen and 1000-2000 day-old workers. In these experimental colonies, a subset of workers will begin to forage between 7 and 10 days of age, rather than at the more typical 3 weeks. It has been shown that precocious foragers in a single-cohort colony have levels of juvenile hormone equal to those of normal-aged foragers (Robinson et al., 1989). While these precocious foragers have high levels of juvenile hormone, the

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same-aged nurses from the same colony have the lower levels characteristic of hive bees. In the same way, delayed behavioral development (“overage” nursing) is associated with maintenance of relatively low juvenile hormone titers (Robinson et al., 1989). Behavioral reversion from foraging to nursing is accompanied by a drop in juvenile hormone titers (Robinson et af., 1992; Farris et al., submitted; Huang and Robinson, 1996).

B. SWARMING Natural scenarios can also lead to changes in behavior and changes in juvenile hormone production that are independent of age. For example, when a new colony is founded by a reproductive swarm, bees of the same age may perform age-inappropriate tasks. These bees have juvenile hormone titers that are predictable on the basis of their observed behavior, not their age (Robinson et al., 1989). C. SEASONAL CHANGES It has also been noted that, in temperate zones, those workers that overwinter have lower levels of juvenile hormone than do summer foragers (Fluri, Wille, Gerig, and Luscher, 1977; Fluri er al., 1982). Recently, both juvenile hormone titers and rates of biosynthesis were shown to be decreased in workers at times of the year when colony activity is reduced (Huang and Robinson, 1995). These responses strengthen the possibility that changes in juvenile hormone titer may mediate plasticity in age polyethism required by changing environmental conditions. VI. BRAIN CHANGES CORRELATED WITH BEHAVIORAL MATURATION HONEY BEE:A PROPOSED MECHANISM FOR JUVENILE HORMONE ACTIONON AGEPOLYETHISM

I N THE

A. PLASTICITY OF THE MUSHROOM BODIES

The capacity for behavioral change in adult worker bees is correlated with anatomical plasticity in the honey bee brain. The corpora pedunculata, or mushroom bodies, of the protocerebrum undergo significant volume changes during adult behavioral development. In this structure, a decrease in the volume occupied by the somata of the intrinsic neuronal population (the Kenyon cells) is accompanied by an increase in the volume of the associated neuropil (Withers, Fahrbach, and Robinson, 1993). One-dayold workers have a neuropil-to-neuronal somata ratio for the mushroom

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bodies of approximately 1: 1; in foragers, this ratio is approximately 2: 1. Equivalent volume changes are seen in normal-aged and precocious foragers (Withers et al., 1993). Volume changes are detected by application of the Cavalieri method to camera lucida drawings of the mushroom bodies. The drawings are typically based on 10-pm-thick Paraplast sections, which are stained with Luxol Fast Blue and cresyl violet. (For a detailed description of the methods, see Fahrbach, Giray, and Robinson, 1995.) The changes observed within the mushroom bodies are highly selective, as the overall volume of the brain, and the volumes of most other brain regions, remain stable during the period of behavioral maturation (Withers et ul., 1993). BODIES A N D LEARNING B. THEMUSHROOM The mushroom bodies are the insect brain center primarily responsible for the integration of olfactory information; in the Hymenoptera there is also a substantial input from visual centers (Mobbs, 1982, 1985). In all insects studied, the mushroom bodies have been shown to be critical for learning and memory (Erber, Masuhr, and Menzel, 1980; Menzel, 1990; Davis, 1993; Strausfeld, Buschbeck, and Gomex, 1995; Davis and Han, 1996). In addition to a well-established role in olfactory learning, the mushroom bodies may also function in spatial learning (Mizunami, Weibrecht, and Strausfeld, 1993), which is of signal importance to insects maintaining a fixed nest site. The anatomy of the mushroom bodies of the insect brain has been extensively described in honey bees and in several other insects, although with rare exceptions all studies have been conducted outside of a developmental context. Mutant Drosophilu melanogaster deficient in olfactory learning have both anatomical and molecular abnormalities of the mushroom bodies (reviewed by Davis, 1993), a finding that has heightened interest in this insect brain structure. Neuroanatomical analyses of enhancer trap lines of Drosophila have recently demonstrated the heterogeneity of the population of Kenyon cells, the intrinsic neurons of the mushroom bodies, in terms of patterns of gene expression (Yang, Armstrong, Vilinsky, Strausfeld, and Kaiser, 1995).

C. SUMMARY OF THE STRUCTURE OF THE MUSHROOM BODIES The mushroom bodies are formed by the Kenyon cells and their processes and afferents (Fig. 2). As is typical in arthropods, the neuronal somata of the Kenyon cells are completely segregated from all regions of synaptic contact. The neuropil of the hymenopteran mushroom bodies can be divided into distinct regions: the medial and lateral calyces, the peduncle,

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'afferent axon

FIG.2. A highly simplified diagram of a single calyx of the mushroom bodies of the honey bee. The arborizations of the intrinsic neurons (Kenyon cells) form the walls of the cup that contains them. while the axons travel via the peduncle to form the alpha and beta lobes (not shown). The afferents to the Kenyon cells are segregated according to modality. The lip receives olfactory input from the antennal lobes of the brain: the collar receives visual input from the optic lobes; and the basal ring afferents arise in both the antennal and optic lobes (Mobbs. 1982. 1985). There are separate medial and lateral calyces in each hemisphere of the honey bee brain. The Kenyon cells are small and tightly packed: the total number of Kenyon cells in the brain of a worker honey bee is approximately 340,000 (Witthlift, 1967).

and the alpha and beta lobes. The calycal neuropils are the input regions of the mushroom bodies, receiving both olfactory and visual information from the antennal lobe and optic lobe, respectively. The dendrites of the Kenyon cells arborize extensively in the calyces. Kenyon cell axons form the peduncle. The Kenyon cell axons divide at the base of the peduncle so that each cell sends one branch into the alpha lobe and one branch into the beta lobe. The extrinsic neurons of the mushroom bodies are located in the lateral protocerebrum. A complex set of projections forms circuits within the mushroom bodies in addition to these connections with extrinsic neurons. In the honey bee, there are between 100,000 and 200,000 densely packed Kenyon cells in each brain hemisphere. D.

FURTHER

STUDIES OF NEUROANATOMICAL PLASTICITY

IN

WORKERS

Subsequent studies have confirmed the first reports that the mushroom bodies are reorganized in foragers, regardless of age (Durst, Eichmiiller, and Menzel, 1994), and have shown that the largest changes in neuropil volume may occur within the subregion of neuropil that receives visual input, the collar. It has been shown that these changes also occur in bees that have not had foraging experience (Withers, Fahrbach, and Robinson, 1995). In this experiment, a single-cohort colony was established. A subset

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of bees was treated with methoprene on the day of emergence, but were not allowed to fly because a plastic disk glued onto their dorsal thorax made the bee too large to squeeze through a modified hive exit. The structure of the mushroom bodies of these bees did not differ from that of methoprene-treated bees allowed to forage. More recently, the brain changes associated with foraging have been shown to be stable, being maintained in foragers that had reverted to nursing, even in the face of reduced levels of juvenile hormone (Farris et al., submitted). OF NEUROANATOMICAL PLASTICITY IN E. STUDIES

QUEENS

It appeared possible to test the association of foraging, changes in the structure of the brain, and juvenile hormone by studying brain development in honey bee queens. Mature queens (more than 2 months of age) have very low levels of juvenile hormone (Fluri, Sabatini, Vecchi, and Wille, 1981; Robinson, Strambi, Strambi, and Feldlaufer, 1992), and they do not take flights away from the hive except on the rare occasion of a reproductive swarm. Queen bees, however, were shown to undergo reorganization of the mushroom bodies, but the changes were seen earlier than in worker bees (during the first week of adult life). This result appeared to contradict the association of high levels of juvenile hormone with altered volumes in the mushroom bodies until radioimmunoassay analysis of juvenile hormone titers in queens (including the queens taken for brain analysis) revealed the surprising finding that 1-day-old queens, in dramatic contrast to 1-dayold workers, have high levels of juvenile hormone that are equal to those of foragers (Fahrbach et al., 1995).

F. ALLATECTOMY STUDIES There are currently no data inconsistent with the hypothesis that exposure to raised levels of juvenile hormone is responsible for the structural changes observed in the bee brain during the transition from hive bee to forager. Several key questions, however, remain unanswered. What is the effect of allatectomy on behavior and brain development? When must the brain be exposed to juvenile hormone before these changes occur? And what are the cellular mechanisms that drive these changes? Allatectomy verified by subsequent radioimmunoassay has recently been performed in collaboration with G. E. Robinson (J. P. Sullivan, G. E. Robinson, and S. E. Fahrbach, unpublished results). Workers allatectomized on the first day of adult life were returned to a large observation colony for several weeks until unmanipulated and sham-operated controls had completed normal development. Allatectomy produced a striking and

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selective change in worker behavior. Allatectomized bees appeared normal and were able to take orientation flights near the hive. They did not, however, make the transition to foraging that is a normal part of the age polyethism pattern in a honey bee colony. The brains of bees undergoing truncated behavioral development in the absence of juvenile hormone are currently being subjected to volume estimation. This study explicitly confirms the interpretation of earlier studies as indicating an activational effect for juvenile hormone on foraging behavior. We predict that these bees will not exhibit significant reorganization of the mushroom bodies. The critical time when exposure to juvenile hormone can have behavioral impact remains undetermined, in part because of the methodological uncertainties detailed earlier. It has recently been shown, however, that some of the volume changes seen within the honey bee mushroom bodies are detectable before workers begin foraging, suggesting that the differences seen between foragers and younger bees represent the culmination of a process begun earlier rather than an abrupt transition (Fahrbach, Farris, and Robinson, 1995). Such a temporal dissociation suggests that the high levels of juvenile hormone seen later in foragers could not have the sole or primary organizing effect on the mushroom bodies. One possibility is that the volume changes seen in the mushroom bodies are the result of both hormone-dependent and hormone-independent processes, with the hormone-dependent processes taking over after the onset of foraging. Another possibility is suggested by the study of juvenile hormone profiles in queens. Might there be an earlier period of hormone exposure critical for activating structural changes in the brain? This question will in part be answered by allatectomy studies. It can also be addressed by studies of juvenile hormone titers in workers with a finer temporal resolution than those previously published. Such studies are currently in progress in the Robinson laboratory, motivated in part by the detection of early changes in the structure of the mushroom bodies. One previous report suggests the possibility of a small peak around the second day of adult life. Radioimmunoassay determinations have suggested that this peak may be a consistent feature of worker endocrine development (0.Jassim, Z.-Y. Huang, and G. E. Robinson, unpublished results).

G. CELLULAR MECHANISMS The cellular mechanisms that may account for these changes are currently under active investigation, and have been recently reviewed (Fahrbach and Robinson, in press). They do not result from hormone-induced neurogenesis in the honey bee (Fahrbach, Strande, and Robinson, 1995), although there is evidence that juvenile hormone may regulate the birth of adult

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specific neurons in the brain of other insects such as crickets (Cayre, Strambi, and Strambi, 1994). The anatomical studies described here suggest, however, that it will be necessary and highly profitable to focus studies of juvenile hormone action on the brain on the mushroom bodies. It is in this brain region alone that the structural plasticity correlated with behavioral maturation and juvenile hormone titers converges with the neural circuitry essential for insect learning and memory.

VII. COMPARATIVE STUDIES A. EUSOCIAL BEESA N D WASPS Within the order Hymenoptera, there are more than 20,000 identified species in the superfamily, Apoidea (Michener, 1974). Although primitive eusocial or quasi-social behavior may be found in some members of the families Xylocopinae (stem- or wood-boring bees) and Halictinae (sweat bees), advanced social behavior is restricted to the three tribes of the family Apidae: the Bombini (bumble bees), the Meliponini (stingless honey bees), and the Apini (true honey bees of the genus Apis). Since the seventeenth century, there have been excellent descriptions available of the temporal polyethism characteristic of honey bee colonies (Butler, 1609, cited in Wilson, 1971); the study of the division of labor in other advanced insect societies is more recent. It has been noted that the social bees and wasps (discussed later) differ from the ants and termites in their sole reliance on age polyethism to provide an efficient division of labor, rather than on differentiation of morphologically discrete worker castes (Wilson, 1971). This means that there should be identifiable other species of bees and wasps that would be predicted to show similar links between juvenile hormone, behavior, and brain structure. It also means that there are many closely related species available in which these relationships ought not to hold, given significant differences in worker life histories. Five members of the genus Apis, of which the European honey bee Apis mellifera is by far the best studied, have been defined, although the specieslevel taxonomy of Apis is currently subject to revision (Michener, 1974; Winston, 1987; Sheppard and Berlocher, 1984). Other Apis spp. are found in Asia: Apis florea, Apis dorsata, Apis cerana, and Apis laboriosa. Little is known of the endocrinology and detailed neuroanatomy of the species other than A. mellifera. Like A . mellifera, A . cerana is a medium-sized bee that builds comb within enclosed cavities, while A . florea, A . dorsata, and A . laboriosa all build single combs in the open (Michener, 1974; Winston, 1987). A. florea is a dwarf honey bee that builds its nest amid dense vegeta-

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tion, while A . dorsata and A . laboriosa are large and aggressive. These species provide testing grounds for hypotheses generated by the study of A . mellifera. Strong age polyethism is well documented in the Meliponini, the stingless bees, and is similar to that seen in Apis mellifera, although the life-span is longer and foraging typically begins at a later age (Kerr and Netos, 1956; Hebling, Kerr, and Kerr, 1964, cited in Wilson, 1971). A recent study based on observations of paint-dot-marked individuals of the species Plebeiu rernotu, a stingless bee that nests in cavities in tree trunks in South America, documented that building activities occurred between 6 and 30 days of age, activities on the royal chambers were performed between 23 and 85 days of age, and foraging was performed by bees thirty days of age or older (Benthem, Imperatriz-Fonseca, and Velthuis, 1995). Little is known of the endocrinology and neuroanatomy of the several hundred species of social stingless bees. The stingless bees constitute a group in which patterns of juvenile hormone production and plasticity of the mushroom bodies are predicted to parallel relationships described for honey bees. The bumble bees (genus Bombus) exhibit a weak version of typical age polyethism, with worker size a confounding factor, as smaller workers are less likely to forage outside the nest than the largest workers (Wilson, 1971). Their brains and patterns of juvenile hormone production in the adults will also be of great interest, given the apparent variability of their behavior relative to Apis spp. and the stingless bees. There seems to be little published information available on the neuroanatomy of bumble bees, with only general descriptions of gross brain anatomy in the literature (Alford, 1975). The eusocial wasps are members of a different superfamily within the Hymenoptera, superfamily Vespoidea (Wilson, 1971). Many degrees of social behavior can be found within the family Vespidae, which includes the subfamilies Stenogastrinae (hover wasps), Polistinae (paper wasps), and Vespinae (hornets and yellowjackets). Examples of eusocial behavior are also found within the family Sphecidae (superfamily Sphecidea), although most sphecid wasps are solitary. Interestingly, the Sphecidae and the Vespidae are not phylogenetically closely related, with the sphecid wasps more closely allied to the bees than to all other social wasps. The phylogenetic relationships of the Vespidae have been discussed in light of the origins of social behavior by Carpenter (1991), in a notable volume devoted to the social biology of wasps (Ross and Matthews, 1991). A comprehensive review of age polyethism among workers in social wasps has been provided by Jeanne (1991). For example, in the paper wasp Polistes dominulus, there is a juvenile phase of 1-5 days, during which the young female works exclusively on the nest. This is followed by a “construction

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phase,” which lasts from 6 to 20 days of age. During this phase, nest construction activity peaks and foraging begins. At approximately 20 days of age a final phase begins, during which construction activity declines and foraging predominates. (Another Polistes species, Polistes fadwigae, provides the only reported example of reverse age polyethism, in which older workers have a greater tendency to participate in nest work: Yoshikawa, 1963.) Examples of age polyethism are also clear in the advanced swarm-founding wasps. In the vespid Polybia occidentalis, there are at least two clear-cut age castes: nest workers, which engage in building, nest maintenance, defense of the colony, and brood care, and foragers, which collect pulp, prey, water, or nectar. Although the ages at which individual workers switched to foraging ranged from 6 to 40 days, the typical individual made the transition relatively abruptly (Jeanne, 1991). B. EFFECTS OF JUVENILE HORMONE TREATMENT IN SPECIES OTHER THAN A P I S MELLIFERA

Juvenile hormone, or more typically, methoprene, treatment has been tested on several representatives of non-Apis groups. Juvenile hormone and its analogues were found to have no effect on the division of labor when applied to primitively eusocial bumble bees: Bombus terrestris (Roseler and Roseler, 1978), Bombus impatiens (Cameron and Robinson, 1990), and Bombus bimaculatus (Cameron and Robinson, 1990). Juvenile hormone and/or methoprene also had no effect on age polyethism when applied to several paper wasps: Polistes metricus (Bohm, 1972), Polistes annularis (Barth, Lester, Sroka, Kessler, and Hearn, 1975), and Polistes gallicus (Roseler, 1985). Unlike the paper wasps just described, in which reproductive females found new colonies independently, the advanced social wasps are characterized by swarm founding of new colonies, as in Apis mellifera. As described above, the advanced social wasps also have well-developed temporal castes. In Polybia occidentalis, methoprene accelerates age polyethism with effects highly reminiscent of methoprene effects on honey bees (O’Donnell and Jeanne, 1993). It is obviously a question of great interest regarding the juvenile hormone profiles and configuration of the mushroom bodies of these wasps. A comparative neuroendocrinology of the Hymenoptera does not currently exist, but its outlines can be glimpsed in present studies of the honey bee. C. SUMMARY To summarize, the social bees and wasps provide numerous examples of age polyethism, ranging from strong expression in honey bees, some

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stingless bees, and the advanced social wasps to more variable expression in bumble bees and primitively eusocial wasps. It is a testable hypothesis that all cases of strong age polyethism will be associated with high levels of juvenile hormone in individuals working outside the hive and in associated changes in the brain space devoted to the mushroom bodies. Conversely, such changes in the structure of the adult nervous system are unlikely to be seen where age polyethism is weak and the youngest workers can both work in the nest and forage.

VIII. CONCLUSIONS AND SIGNIFICANCE ROLESFOR A SINGLE A. MULTIPLE HORMONE Pener points out in his consideration of the role of hormones in the control of insect flight and migration that preexisting chemical signals were very likely “captured” during arthropod evolution for new functions (Pener, 1985). The same endocrine factor may therefore have different roles in different species with different adaptive strategies. He gives the relationship of juvenile hormone and flight muscle as an illustrative example: in most insects juvenile hormone induces degeneration of the flight muscles, but in the Colorado potato beetle juvenile hormone induces flight muscle growth or regeneration (Kort, 1969). Developmental hormones may similarly have been “captured” for use by the nervous system (Truman and Riddiford, 1974). In its regulation of age polyethism in social insects, juvenile hormone appears to have been “captured” by advanced social insects more than once, with a possible independent evolution of this strategy in Apidae and Vespidae (O’Donnell and Jeanne, 1993). Juvenile hormone is predicted to share the same role in these advanced social insects: a mediator of joint brain and behavior development. It is likely to be limited to such a role in those insects in which age polyethism is so strong that there is a transition of consequence between the hive and the field. The cognitive demands of navigation and learning to handle floral resources are assumed to require adult development of the mushroom bodies. This development is coordinated with behavior by juvenile hormone. Bees without these endocrine-mediated changes in the structure of the mushroom bodies are predicted to perform less adeptly on tasks that reflect the cognitive demands of orientation to the nest site and foraging. This hypothesis can also be readily tested. Robinson initially hypothesized that changes in responses to environmental cues were a result of juvenile hormone action on the nervous system (Robinson, 1987a,b). More recent neuroanatomical investigations suggest that the effects of juvenile hormone may be more accurately described as

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leading to more brain space dedicated to complex information processing. Such changes are intriguingly analogous to changes in hippocampal volume detected in food-storing birds such as the marsh tit (Krebs, Sherry, Healey, Perry, and Vaccarino, 1989; Krebs, 1990), or in the song centers of the avian forebrain in many songbirds (Nottebohm, 1981; Konishi, 1994). By study of the relationships of mushroom body structure, juvenile hormone, and the ontogeny of foraging in the honey bee, we are able to study the control of behavior in individual bees. By future studies of the incidence of juvenile hormone regulation of age polyethism in the social insects, we can gain insight into the evolution of mechanisms for neural and behavioral plasticity. B. SUMMARY The regulation of the division of labor by juvenile hormone has been well established for honey bees. Recent studies suggest that juvenile hormone may control the volume of specific regions of the honey bee brain. Comparative neuroanatomical and neuroendocrinological studies of the eusocial and primitively social Hymenoptera are needed to reveal the relationships between juvenile hormone, brain structure, and behavioral maturation. Acknowledgments G . E. Robinson introduced me to the topic of juvenile hormone and age polyethism in the social Hymenoptera, and I thank him for many fruitful discussions of his intellectual and experimental contributions to this field. I also thank my graduate students past and present (G. S. Withers, S. M. Farris, and J. P. Sullivan) for their vigorous investigations of the juvenile hormone-brain-behavior relationship in honey bees. Recent research described here was supported by the National Science Foundation.

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Cameron, S. A., and Robinson, G . E. (1990). Juvenile hormone does not affect division of labor in bumble bee colonies (Hymenoptera: Apidae). Ann. Enfomol. Soc. Am. 83, 626-631. Carpenter, J. M. (1991). Phylogenetic relationships and the origin of the social behavior in the Vespidae. In “The Social Biology of Wasps” (K. G . Ross and R. W. Matthews, eds.), pp. 7-32. Comstock Publishing, Ithaca, New York. Cayre. M.. Strambi. C., and Strambi, A. (1994). Neurogenesis in an adult insect brain and its hormonal control. Nature (London) 368,57-59. Davis, R. L. (1993). Mushroom bodies and Drosophila learning. Neuron 11, 1-14. Davis, R. L., and Han, K-A. (1996). Mushrooming mushroom bodies. Curr. Biol. 6, 146-148. Djerassi, C. (1992). “The Pill, Pygmy Chimps, and Degas’ Horse.” HarperCollins. New York. Dreller. C., Fondrk, M. K.. and Page, R. E. (1995). Genetic variability affects the behavior of foragers in a feral honeybee colony. Naturwissenschafi. 82,243-245. Durst, C.. Eichmiiller. S..and Menzel, R. (1994). Development and experience lead to increased volume of subcompartments of the honeybee mushroom body. Behav. Neural. Biol. 62, 259-263. Erber, J., Masuhr, T., and Menzel, R. (1980). Localization of short-term memory in the brain of the bee, Apis mellifera. Physiol. Enromol. 5, 343-358. Fahrbach, S. E., Farris. S. M., and Robinson, G . E. (1995). Coincident maturation of flight behavior and the mushroom bodies in the honey bee. Soc. Neurosci. Absrr. 21,458. Fahrbach, S. E., Giray. T.. and Robinson, G . E. (1995). Volume changes in the mushroom bodies of adult honey bee queens. Neurobiol. Learn. Memory 63, 181-191. Fahrbach, S. E., and Robinson, G. E. (1996). Juvenile hormone, behavioral maturation, and brain structure in the honey bee. Dev. Neurosci. 18, 102-114. Fahrbach, S. E., Strande, J. L., and Robinson, G . E. (1995). Neurogenesis is absent in the brains of adult honey bees and does not explain behavioral neuroplasticity. Neurosci. Lrrr. 197, 145-148. Farris. S. M., Robinson, G . E., and Fahrbach, S. E. (submitted). Behavioral reversion affects juvenile hormone titers but not brain structure in the worker honey bee. J . Neurobiol. Fluri. P.. Liischer, M., Wille, H., and Gerig. L. (1982). Changes in the weight of the pharyngeal gland and haemolymph titres of juvenile hormone, protein and vitellogenin in worker honey bees. J . Insect Physiol. 28, 61-69. Fluri. P.. Sabatini, A. G . , Vecchi, M. A., and Wille, H. (1981). Blood juvenile hormone, protein, and vitellogenin titres in laying and non-laying queen honeybees. J. Apiculrural Res. 20, 221-225. Fluri. P.. Wille, H., Gerig, L., and Liischer, M. (1977). Juvenile hormone, vitellogenin, and haemocyte composition in winter worker honeybees (Apis mellifera). Experienria 33, 1240- 1241. Frisch, K. von (1967). “The Dance Language and Orientation of Bees.” Harvard University Press, Cambridge, Massachusetts. Cast. R. (1967). Untersuchungen iiber den Einfluss der Koniginnensubstanz auf den Entwicklung dcr endokrinen Driisen bei der Arbeiterin der Honigbiene (Apis mellifica). Insectes Sociaux 14, 1-12. Giray, T., and Robinson, G . E. (1994). Effects of intracolony variability in behavioral development on plasticity of division of labor in honey bee colonies. Behav. Ecol. Sociobiol. 35, 13-20. Goodman, W. G., Orth. A. P., Toong, Y. C., Ebersohl, R., Hiruma, K., and Granger, N. A. (1 995). Recent advances in radioimmunoassay technology for the juvenile hormones. Arch. Insect Biochem. Physiol. 30, 295-306. Granger, N. A., and Goodman, W. G . (1988). Radioimmunoassays: Juvenile hormones. In “Immunological Techniques in Insect Biology” (L. 1. Gilbert and T. A. Miller, eds.), pp. 215-251. Springer-Verlag. New York.

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