Abstract

Bumble bees are conspicuous and appealing insects. Their widespread distribution and visible nature make them a well-studied creature. Aspects of bumble bee behavior have been considered since Darwin's time. The male bumble bees' flight path behavior is just one investigated habit of these animals. The flight path is laid out in the morning and then patrolled throughout the day by the male bumble bee. The male will scent mark objects along the flight path during the early morning. Throughout the day he will then stop at these marked locations as he patrols his route. Different species of bumble bees will mark different types of objects along a path. Also, the different species will choose different heights from the ground at which to fly their route. Another variation between bumble bee species is how they mark the objects along the flight path. Some place small amounts of scent, or pheromone, on many objects marking these objects in a random fashion. Another species may mark few objects along the flight path in a slow and deliberate manner with large amounts of pheromone. However, some species had little or no differences between their flight paths. These similarities pose a problem for the species if a female bumble bee is unsure of the male with which to mate. This behavioral problem prompted scientists to delve more deeply into the chemical differences between bumble bees. Through chemical analyses scientists have been able to locate the gland where the pheromones originate. Further analysis of the labial gland has led to specific identification of many species of bumble bees as well as grouping of bees originally thought to be different species. Presently behavior, morphology, and chemistry are all used together to differentiate between bumble bee species.

Introduction

Bumble bees are intriguing creatures with their bright yellow stripes making them well known to nearly all. "Bumble bees are widely distributed with about 300 species . . . centered on the north temperate zone, extending through Europe, Asia and North America" (Prys-Jones & Corbet 1987). The bumble bee's life cycle begins in the spring when the overwintering queen emerges from hibernation. She will begin a nest by initially laying eggs that give rise to workers. These workers look after the nest, defend it and collect food for it (Prys-Jones & Corbet 1987). After her nest is established, the queen will begin to produce males and young queens for the next generation. "The males produced leave the nest and seldom or never return . . . . Their sole function is to mate with young queens" (Prys-Jones & Corbet 1987).

While the queen and her workers have most studies emphasis devoted to them, the male bumble bee's behavior is just as engaging. Males of many bumble bee species exhibit a conspicuous premating behavior with two distinct behavioral components: scent marking and patrol flying (Bergman & Bergström 1997). In addition to the behavioral aspects of a patrol flight, each species has its own method of differentiation from the mixture of compounds that make up their characteristic scents (Prys-Jones & Corbet 1987).
 

Species Differentiation

    Time of year

A bumble bee could differentiate itself from other species simply by emerging earlier or later in a season. If in the spring a queen awakes from hibernation and begins a nest after other species, she would consequently produce males and new queens later. This method of isolation would separate the species temporally making interspecific mating nearly impossible. "For the bumble bee, the easiest method of isolation of the species would be time distribution of sexual activity . . . [to] deviate from all other species by the late start of their flight period" (Svensson 1980). This does not seem to be the most effective method of differentiation as Svensson (1980) noted only two species employing this method. Bombus pratorum and Bombus lapidaruis, for instance, were not noted to differ in the time of year that they emerge from hibernation.
 

    Morphology

Morphology is yet another piece of the puzzle in species specificity. A bumble bee's physical appearance is another area in which it can separate its species from those with similar behavior. Bringer (1973) noticed that species with very similar behavior such as Bombus hortarum and Bombus hypnorum have a different appearance. In contrast, B. pratorum and B. lapidaruis do not appear the same. The males of the two species differ in body size and coloration. Svensson (1980) also observed that species exhibiting similar structural plans of the genitalia do not coexist spatially. This may not be enough by itself to separate the species from one another but it could play a role in species definition.
 

    Patrol Flying and Scent Marking

The patrol flight of the male bumble bee begins each morning. He begins by flying a route and scent marking particular objects. The type of objects chosen is species specific and can vary by position within the habitat, height above the ground, or kind of vegetation. After repeated scent marking for 2-3 hr, an individual male-specific flight route has been established (Svensson 1980). The male will continue "flying characteristic routes, circuiting the same flight path, pausing on the wing" (Prys-Jones & Corbet 1987) at the scent marked objects.

Flight routes of males of different bumble bees species will often overlap and it is therefore necessary for diversity in the behavior of males on patrol flights. Bringer (1973) noted that there seem to be many differences in the choice of habitat and behaviour in territorial flight among the Bombus species, which may be factors of importance for the maintenance of the isolation between species.
 

    Marking Object and Flight Height

One method of species contrast is for the individual bumble bee species to scent mark different objects (such as small twigs, leaf edges, grass, tree trunks) at different heights from theground level up to tree tops (Bringer 1973). Table 3 from Prys-Jones & Corbet (1987) condenses Bringer's work with respect to flight height and shows the diversity of the flight levels of various species of bumble bees. The bumble bee males can differ in their flight altitude from ground level marking of grasses to leaf marking in the canopy. Haas (1949) tried to classify the species studied by him according to their choice of flight level or the height above the ground at which the bumble bee flies. From Table 3, B. lapidaruis flies at 0 - 3 and 6 -17 meters from the ground. B. pratorum flies at 0 - 3 meters above ground level. This shows that the two species have overlap in their flight behavior making flight height an unreliable means of differentiation between these bumble bees.

In Figure 2 Svensson (1980) shows the great diversity between species with respect to horizontal habitats (or type of cover chosen for the flight) and vertical habitats (or flight path height). B. lapidaruis and B. pratorum were not both present in this study area but the variation in habitat usage by the bumble bees studied is extensive.
 

    Scent Marking Method

The method by which a male bumble bee marks objects can be a key species identifier. O'Neill et al. (1991) noticed Bombus rufocinctus went along the margins of leaves, covering up to 10 leaves in a single bout of scent marking. Bergman and Bergström (1997) watched Bombus pratorum males and found them to be hasty in their marking behavior: "they were more likely to mark only a part (quarter or half) of a leaf edge before leaving it, either to choose another leaf nearby or to fly to one of the other birch branches." They also noted that "in B. lapidaruis, scent marking on birch leaves was much slower and the entire edge of the leaf was marked." Bergman and Bergström (1997) speculate that the amount of pheromone deposited while scent marking relates to the volatile nature of the chemical blend. A more volatile mixture of compounds would need a greater amount deposited in order for the scent trail to exist for an adequate period of time.

Chemistry

Another means of species differentiation in bumble bees is through chemistry. Bumble bee males use different marking pheromones or secretions to distinguish themselves from other species. The combinations of the compounds in each species make its secretions species-specific (Bergman et al. 1996). Pheromone marked places thus can be defined as the spatial dimension of the mating niche for bumble bee species (Svensson 1980).
 

    Scent Origin

Sladen (1912) proposed that the patrol-flying bumble bee males emitted a scent at the buzzing places and that "males from each species emit a species-specific scent that most probably originates from their heads." Kullenberg et al. (1973) was the first to identify the cephalic portion of the labial gland as the origin of the bumble bee pheromone. This was later confirmed by Bergman and Bergström in 1997. Their tests consisted of analysis of the labial glands of the bees as well as the pheromone collected from the leaves marked by the bumble bees. In these studies, the chemical composition was found to be the same on the leaves as well as the labial glands. Kullenberg et al. as well as Bergman and Bergström were able to conclude that the marking pheromone of bumble bees resides in the cephalic portion of the labial gland. Refined dissection and analysis techniques make it possible to state that the characteristic compounds of the marking scents are produced in the cephalic part of the labial gland (Kullenberg et al. 1973). The labial gland secretion or pheromone of the bumble bee is "of importance for species recognition and isolation" (Svensson 1980). "In addition . . . there seem to be interspecific quantitative differences in the amount of labial gland secretion produced" (Bergström et al. 1996).
 

    Types of compounds

Many of the compounds from labial gland dissection and analysis have been identified and found to be fatty acid derivatives and terpene alcohols and esters (Prys-Jones & Corbet 1987). Each species has a characteristic blend of straight-chain fatty acid derivatives, especially esters, and acyclic mono-, sesqui- and di-terpenes, which are alcohols, aldehydes and acetates (Appelgren et al. 1991). Species differ in relative amounts of isomers of the straight-chain monounsaturated aldehydes, alcohols and acetates (Appelgren et al. 1991). Svensson (1980) noted that the blends are usually made up of one very dominating compound and several minor/minute components. Table 2 from Prys-Jones & Corbet (1987) illustrates the vast numbers of chemical components of the marking secretions. Even in minute quantities chemicals can combine to give each species its own characteristic pheromone. This is highly useful as a species isolating mechanism. If two bumble bee species share the same morphology or spatial aspects, the species' can still avoid interspecific mating through a unique chemical blend.
 

    Extraction & Identification Methods

Compounds in the labial glands of the bumble bees were identified using Gas Chromatography-Mass Spectrometry (GC-MS). Prior to this analysis the bumble bee's heads were dissected and the labial glands removed. Each gland was placed in a vial and extracted with 150 l of diethyl ether (Bergman & Bergström 1997). For each extraction the same GC-MS analysis was run on HP 5890 gas chromatograph. Bergman & Bergström (1997) ran the chemical analysis with a CP-Sil-5 (Chrompack) column or an OV-351column (GeneTec) both 30 m X 0.25 mm with Helium as the carrier gas running at a constant flow of 0.7 ml/min. In their analysis the GC oven was kept at 50 C for 2 minutes and the temperature increased at a rate of 10 C/min to 240 C where it was held for 40 minutes. The resulting gas chromatograms for two species are shown in Figure 1 (Bergman & Bergström 1997).

An interesting relationship has been noted between the species B. pratorum and B. lapidaruis. The two species share the same pheromone components with only one difference, B. pratorum uses two different biosynthetic pathways to produce the labial compounds and B. lapidaries uses only one of them (see Figure 5) (Bergman & Bergström 1997). Because of this, the difference between the two species is simply a presence of three isoprenoids in the labial gland secretion of B. pratorum males that is absent in B. lapidarius. In an evolutionary sense, the presence of terpenoids implies simply the expression of an already existing enzyme system, rather than the evolution of a new one (Bergman et al. 1996)

All three of these factors, behavior, morphology, and chemistry, come in to play to distinguish species of bumble bees from each another in order to preserve individual species and counteract hybridization. For instance, species with indistinct segregation along the other niche dimensions examined may use largely dissimilar compositions of the marking pheromones (Svensson 1980). This would mean that if several bumble bees share the same habitat and perhaps look similar, their difference would lie in the chemical makeup of their marking pheromone. B. pratorum and B. lapidarius employ several of these methods to maintain species specificity without needing to use them all. Both species' queens emerge from hibernation at similar times and both fly at similar heights above the ground. In order to avoid interspecific mating, the two bumble bees posses different morphology and different chemistry. These factors are enough to maintain species integrity.
 

Bioassays

To date, the chemicals isolated from the labial gland secretions have not been tested in a bioassay. The main focus of all the studies lies either in the identification of the compounds present or the flight behavior of the male bumble bee. Compounds identified from the labial glands secretions are identified reliably by Gas Chromatography-Mass Spectrometry but they have not then been synthesized. If the compounds were synthesized, they could then be tested in a bioassay to see if the male bumble bees respond the same to the synthetic blend as their original marking compounds.

In order to study this problem, male bumble bees would have to be acclimated to an artificial environment. This would need to be done so that the synthetic compounds and actual compounds could be presented in identical environments. A method was implemented by Bergman and Bergström (1997) whereby greenhouses were equipped with artificial "trees" or bottles of water containing birch branches. Once the bumble bees acclimated to the habitat, they behaved the same as they did in a natural environment.

After the acclimation period, the bees would then need to be allowed to establish a normal flight path. After the males had established the path and were in the patrolling phase of their day, some of the marked objects would be removed. The removed objects would then be replaced with identical objects marked with the synthetic components. Control tests would need to be run to compare objects replaced with synthetic components with objects replaced with actual pheromone marking. This would remove any artifact associated with the removal and replacement of articles. Once the new objects were in place, the bumble bees could be observed to see if they reacted to the synthetic materials in the same manner as the pheromone. In this way, the active components of the marking blend could be identified. Identifying the active compounds would show which component or combinations of components present in the labial gland will actually cause the male bumble bee's response. At present, knowledge is limited to the entire multicomponent blend of labial gland secretions.
 

Conclusions

Behavior, morphology, and chemistry all combine to make each bumble bee species unique. Species can differ behaviorally in their flight height, choice of marking objects, or even method of marking chosen objects. Morphologically, species can appear different. Another trait a species can employ is to emerge from hibernation at a different time than other species. Sympatric species also differ in the types of chemicals used or in the proportions within multicomponent blends (Svensson 1980). Svensson (1980) suggests that the combination of these factors promotes premating isolation among sympatric circuit flying Bombus.
 

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