Download STINGLESS BEES: THE NEUROBIOLOGY OF FORAGING Abstract

STINGLESS BEES: THE NEUROBIOLOGY OF FORAGING
Abstract
Bees are social insects and great models in neurobiology for learning research and
memory. Although they have small brains, they can perform several different
behaviors such as foraging, food source recognition and route guidance. Bees
perform a foraging cycle that comprises a flight up to the source, the collection of
nectar, the return to the colony, food discharge, and dancing to reveal the food
source location. For this, the bees perceive various environmental stimuli through
receptors that are transformed into nervous impulses in the brain; they then transmit
the food source information to other foragers. The behavior of each bee is important
for success of the colony, for guaranteeing its survival and, moreover, it is important
for the environment, guaranteeing pollination and consequently food production for
humans and biodiversity conservation.
Foraging behavior
In social insects like bees, each individual behavior is performed to promote colony
development and procreation. Such behaviors are related to the needs of the colony
and environment conditions, and these parameters could accelerate, delay or
reverse these behavioral patterns (Wilson, 1971; Robinson, 1992).
Foraging is one of the greatest characteristics of social insects and concerns
practically all aspects of animal biology (Robinson and Page, 1989). The ecologists
who study foraging behavior have expanded such mechanisms to include situations
where there is a risk of predation while foraging (Stephens, 2008).
Bees perform a foraging cycle that comprises the flight to the source, the collection
of nectar, the return to the colony, food discharge and then dancing to reveal the
food source or resting location to other bees in a time scale of minutes, but the
recruitment of new workers happens in a time scale of hours (Cox and Myerscough,
2003).
The ability of social bees to recruit nest mates for group foraging is an important
factor in their capacity in dominating rich environment sources, such as trees with
dense inflorescences (Nieh et al., 2003; Lichtenberg et al., 2010). The foraging
success is determined by the habitat size and by the quantity and variety of food
sources that the bees use (Pasquet et al., 2008).
Foraging behavior studies have taken into consideration a variety of factors; among
them are the energetic gain rate and energetic efficiency, defined as a proportion of
the obtained energy and the energy used in foraging (Ydenberg et al., 1994). In
general, pollinator insects are sensitive to floral rewards and only forage in flowers
from which they can maximize the liquid energy profit. For honey bees collecting
nectar, energy serves as an appropriate currency in the assessment of behavioral
patterns. Flowers that offer a lower amount of energy are relatively uninteresting to
foraging bees, which could have promoted the coevolution between plants and bees,
resulting in the selection of plants with flowers that offer a great reward, according to
the foraging behavior pattern of these insects (Abrol, 2006).
Social insects have evolved information transfer systems of remarkable complexity
(Wilson, 1971). Bees use sophisticated methods to explore pollen, nectar, water,
resin, nesting sites, excrement, animal carcasses, gums, fruit juice, leaves,
trichomes, mud, and saline solutions, amongst others (Roubik, 1989; Kerr, 1969;
Schwarz, 1948; Wille, 1983; Roubik, 1983). Honeybees are important tropical
pollinators. They are among the most common pollinators in this environment, and
are the dominant species in different plant cultures (Roubik et al., 1986; Macõ´asMacõ´as et al. 2009).
The mechanisms that enable bees to inform others of a food source location have
been studied intensively, especially in Apis mellifera, which is able to communicate
polar coordinates (distance and direction) for the food source through a “round
dance” (von Frisch, 1967; Dyer, 2002).
Bees use dance as a means of communication, allowing foragers to inform their nest
mates about a food source. Apis mellifera bees are recruited through both odor and
through the dance. The latter is performed by the workers who had successful
foraging with the purpose of recruiting other nest mates to collect food from this
source. The dance can contain information about the food location, water, and about
a new habitat (Seeley, 1995; Esch and Burns, 1996; Von Frisch, 1967).
Upon returning to the hive, foragers perform highly specific movements to signal the
place and the floral richness of the source to the nest mates (Seeley, 1995; Barron et
al., 2009). The main function of the dance is to announce the sources found, making
the foraging colony more efficient (Barron et al., 2009).
Researchers have tried to understand the evolution of the complex recruitment
communication systems and have focused their attention on honeybees, exploring
possible homologies between Meliponini and Apini recruitment communication.
Research on stingless bees revealed the intricate components of communication
and the mechanisms of information transfer in foraging (Kerr, 1969; Kerr and Esch,
1965; Esch, 1967; Nieh et al., 2003c).
Foragers have a set of multiple senses such as touch, smell, vision and hearing that
allow the exchange of information about the existence of a food source (Nieh, 2004).
Foragers live in a complex three-dimensional environment (Cartwright and Collett
1982; Lehrer et al. 1988; Srinivasan 2001; Egelhaaf and Kern 2002; Nieh, 2004)
and, in some cases, communicate the specific location in three dimensions, without
the needing odor to indicate the way (Nieh and Roubik, 1995).
Vision and foraging behavior are primordial functions that enable some
understanding of the social life of honeybees. Kerr (1969) reported that in some
forager species of bees, the whitish reflective abdominal hairs may facilitate
orientation as they leave the nest. An important tool used to control the course and
speed of flight is vision. Speed is controlled by altering the angular velocity of the
environment’s image on the retina, and the speed of the flight slows down when
bees pass through a narrow opening. This phenomenon, known as optical flow, not
only helps mediate a direct course of travel, but it can also avoid collisions with
obstacles (Srinivasan and Zhang, 2000).
Egellhaf and Kern (2002) and Hrncir et al. (2003) showed that Melipona seminigra is
able to use optical flow as an information source to manage the course and speed of
flight. For bees, visual flow (lateral and ventral) may be advantageous when foraging
in a tropical forest with dense vegetation, as demonstrated by Melipona seminigra
foragers, which can estimate flight distance using the number of motion pictures in
their foraging trips (Hrncir et al., 2003).
Moreover, visual flow, foraging information and the recruitment of new bees may be
directed by trophallaxis (Hrncir et al., 2006b), substrate vibrations (Lindauer and
Kerr, 1958) and the production of sound pulses (Esch, 1967; Nieh, 1998; Nieh et al.,
2003).
Foragers of honey bees, stingless bees and bumblebees generate sound pulses
from thoracic vibrations when they return from a rich food source (Hrncir et al.,
2006). Eighteen species of stingless bees: Frieseomelitta silvestrii, F. flavicornis, F.
freiremaiai, Leurotrigona muelleri, Melipona bicolor, M. costaricensis, M. mandacaia,
M. seminigra merrillae, M. panamica, M. scutellaris, M. seminigra, Nannotrigona
testaceicornis, Plebeia droryana, Cephalotrigona capitata, S. postica, Tetragonisca
angustula, Trigona (Hetetrotrigona) carbonaria, and Meliponula (Axestotrigona)
ferruginea tescorum produce pulsed sounds upon their return to the nest with
samples of food from the environment (Lindauer and Kerr, 1958; Esch, 1967; Kerr,
1969, 1994; Nieh Roubik, 1998; Hrncir et al., 2000; Aguilar and Briceño, 2002; Hrncir
et al., 2002; Nieh et al., 2003c).
The sounds are generated by thoracic, rhythmic oscillations and dorsoventral
vibrations of the wings (Michelsen et al., 1987; Kirchner et al., 1988), and they are
used by stingless bees to communicate the distance and height of food sources
(Esch et al., 1965; Nieh and Roubik, 1998). The thoracic vibrations and sound pulses
produced by foragers provide an intriguing and possible explanation of how spatial
information can be transferred to nest mates (Dyer, 2002).
A signal with a low variability is indispensable for preventing the transmission of
mistaken information to a receiver and for reducing the probability of a
misunderstanding and a false alarm (Hrncir et al., 2004). Assuming that Meliponini
thoracic vibrations contain information about the location of a food source (Nieh and
Roubik 1998; Nieh et al., 2003), there must be a low degree of change for a signal to
have a high communicative value. However, the temporal pattern of vibrations of the
Melipona thorax is highly variable (Hrncir et al., 2004). The success of recruitment of
different Melipona species is also influenced by the concentration of sugar in the
food source and its relationship to the duration of pulses (Jarau et al., 2000; Hrncir et
al., 2004).
In Melipona quadrifasciata and Melipona scutellaris (Lindauer and Kerr, 1958; Jarau
et al., 2000; Hrncir et al., 2000), Melipona panama (Nieh and Roubik 1998),
Scaptotrigona av. depilis (Schmidt et al., 2006a) and Nannotrigona testaceicornis
(Schmidt et al., 2008) a behavior was observed upon their return to the nest, called
"jostling", which was correlated with recruitment behavior.
In M. quadrifasciata and M. scutellaris, the success of alerting against intruders in
the nest was clearly correlated with the number of jostles per unit of time (Barth et
al., 2008). In this Melipona species (Hrncir et al., 2000), as well as N. testaceicornis
(Schmidt et al., 2008), the number of jostles per unit of time was positively correlated
with the concentration of the sugar solution offered in a feeder. The jostles seemed
to indicate the existence of a rich food source and stimulate the bees to leave the
nest and search for it; however, there was no correlation between foraging
movements (including the number of jostles) in the nest and distance or direction of
the food source (Barth et al., 2008).
The spatial information exchanged between Melipona bees about a food source can
only be sustained if three basic requirements are met. First, the bees should be able
to measure spatial parameters such as direction, distance and height of a food
source. Second, the bees must code the parameters measured in a signal and, third,
the receiving bees must be able to extract the spatial information signal and use it to
search for the food source (Hrncir et al., 2003). The dance language of honeybees
does not appear to encode the visual flow or inform other bees about the distance of
a food source (Srinivasan et al., 1997, 2000; Esch et al., 2001).
Odor is also an important factor of communication among bees and it fulfills an
essential role in the recruitment of social insects, assisting in the orientation of
foraging bees and new recruits (Nieh et al., 2003b). There are several behavioral
strategies for leaving scent marks at a food source, such as long odor trails that
begin in the vicinity of the nest and extend to the food source, short odor trails at a
short distance away from the food source in the direction of the nest (Nieh et al.,
2003a), and odor marking of the food source alone (Nieh, 1998b; Hrncir et al., 2004).
Stingless bees produce an odor trail that consists of odorized droplets periodically
deposited on vegetation from the nest and food source (Lindauer and Kerr, 1958).
When depositing scent marks on plants, the foragers generally land briefly and rub
their mandibles and tongue against the substrate (Kerr and Rocha, 1988; Nieh et al.,
2003a). Foragers may also use grooming to deposit scent marks (Kerr, 1994; Nieh et
al., 2003d).
Stingless bees can deposit odors in many ways: mandibular gland secretions are
deposited by rubbing the substrate with their mandibles and tarsal gland secretions
are left by walking on the substrate (Jarau et al., 2002; Hrncir et al., 2003; Schmidt et
al., 2003). Besides these glands, the Dufour gland, found in N. testaceicornis as well
in honeybees, contains geranyl acetate, used for territorial marking (Cross-L_pez et
al., 2001).
It is possible that the odor marks of foragers of some species are composed of
droppings, without the additional glandular products produced for communication
purposes (cues) (Nieh et al., 2003).
The mandibular glands of stingless worker bees produce semi-chemicals and most
of these components are volatile, encouraging communication and serving as
immediate defense compounds. Their high volatility, however, seems to be
inadequate for leaving odor trails that convey bees to a food source as the
recruitment of newcomers takes some time and the most highly volatile substances
will have evaporated. In T. spinipes and S. aff. Depilis, mandibular gland volatiles are
used for defensive/aggressive actions both close to the nest and at the food source
(Schorkopf et al., 2009).
In M. favosa, scent marks were observed emerging in the form of anal droplets and
were deposited near to the food source (Aguilar and Sommeijer 1996, 2001). Anal
scent droplets (Nieh et al., 2003b) are signs that contribute to foraging as attractants
to rich food sources and repellents to prevent food sources becoming scarce (Stout
and Goulson, 2001).
Regarding the use of olfactory information in stingless bee species, several studies
demonstrated that when the bees are alerted to a rich food source by nestmates,
some species do not inform on the location of the food source, such as Trigona
iridipennis (Lindauer 1956), while others inform on the direction but not the distance,
such as M. scutellaris, M. quadrifasciata (Jarau et al., 2000) and Plebeia postica
(Aguilar et al., 2005). In both cases, however, when alerted, the bees fly searching
for the odor and land at any food source along the way that has the same scent.
However, these studies did not examine how specific the information was regarding
how the floral odor attracts recruits to join the search for the origin of the food source
or how previous experiences of scent affect the performance of individual learning
(McCabe and Farina, 2009).
The pheromones involved in foraging
Pheromones have an important role in bee’s lives, especially when it comes to
foraging. Bees are capable of flying long distances in the search for nectar and
pollen, indicating to other foragers through pheromones the exact location of the
food supply and its viability, as well as the way back to the hive (Jarau, 2009).
Most pheromones include volatile substances that differ in their properties and
functions in each species in which they are produced. These chemical substances
allow individuals of the same species to recognize gifts brought in antennas and they
are released by bees for different purposes such as sexual attraction, defense and
foraging behaviors.
The pheromones in general are produced by glands, such as the mandibular gland
located in the head, and in each caste of bees they have a different function. In the
honey bee, Apis mellifera, their function in queens is to attract males (drones), and
they also to prevent the development of ovaries in workers and stimulate foraging
(Carvalho et al., 2001; Pankiw, 2004).
In stingless bees, the product of the mandibular glands is associated with foraging,
recruiting workers to search for food and defensive behaviors, alerting the colony
against possible invaders (Lindauer and Kerr, 1958, 1960; Kerr and Cruz, 1961). The
workers of Scaptotrigona postica use the secretion of these glands to mark the
location of a food source; however, these brands are not restricted to their colony,
and can attract bees from other colonies (Lindauer and Kerr, 1960).
Kerr (1973) showed that Trigona spinipes workers etched flowers with mandibular
gland secretions, leaving a trail of scent so that other workers could find the food
source. However, Schorkopf et al. (2009) showed that the pheromones released by
the mandibular glands of T. spinipes and S. aff. depilis (postica) are more closely
related to defense behaviors than to marking scent trails for foraging.
Research carried out on pheromones produced in the labial gland, which is well
developed in forager bees, has demonstrated their strong links with foraging. Studies
using gas chromatography showed that the substances produced in the labial glands
of the stingless bees T. spinipes (Fabricius 1793), such as hexyl decanoate and octyl
octanoate, are related to the scent trails left by foragers for the location of food
sources (Jarau et al., 2004, 2006; Schorkopf et al., 2007).
In order to prevent the loss of time in foraging, honeybees use pheromones to mark
flowers that are not viable (Giurfa and Nfifiez, 1992). Studies on the molecular basis
of the action of pheromones have shown their importance in the regulation of gene
expression in the brains of the bees. Alaux et al. (2009) found that the brood
pheromone can act on the maturity of bees by making them ready to leave earlier for
foraging or by delaying maturation, depending on the needs of the colony.
Pheromones are not just released by adult individuals within a colony; honeybee
larvae also release pheromones through their epidermis to induce foragers to search
for pollen (Carvalho et al., 2001).
Pheromones need an intermediate to be able to intercept and forward the message
to the brain for foraging success. Bees have an excellent neurological apparatus
capable of understanding and decoding these chemical messages (Faber and
Menzel, 2001).
Juvenile hormones and vitellogenin in foraging
In honeybees, the behavioral change from nursing activities to foraging represents a
major transitional step in the workers’ life cycle. The regulators of this change have
been reported to be juvenile hormones (JH) and, more recently, the vitellogenin (Vg)
protein (Marco Antonio et al., 2008).
The juvenile hormones represent a group of sesquiterpenoid hormones unique to
insects. They are synthesized and secreted from the corpora allata (CA), a pair of
small specialized endocrine organs (Hartfelder, 2000). Juvenile hormones are
involved in numerous regulatory functions in tissues, developmental stages,
physiological and reproductive processes and behavior (Bellés et al., 2005;
Applebaum et al., 1997; Goodman and Granger, 2005; Hartfelder, 2000; Verma,
2007; Bonetti, et al., 1995, Robinson and Vargo, 1997).
In the beehive, younger individuals working inside the hive take care of the brood
and food storage. They have low circulating levels of JH and large hypopharyngeal
glands (HPG). As bees age, they work outside the hive as foragers and express high
JH levels and a degeneration of the HPG (Winston, 1987; Elekonich, et al., 2001;
Fluri et al., 1982; Kubo et al., 1996). Moreover, the treatment of young workers with
JH, JH analogs and JH mimics causes premature degeneration of the HPG and
precocious foraging (Rutz et al., 1975; Jaycox et al., 1974; Sasagawa et al., 1986;
Beetsma and Houten, 1974). Workers lacking JH because of allatectomy still
become foragers, but they start this activity later (Robinson, 1992; Robinson et al.,
1989; Giray and Robinson 1996; O’Donnel and Jeanne 1993; Sullivan et al., 2000).
Juvenile hormones and ecdysteroids act in the processes of initiation, maintenance
and inhibition of vitellogenin synthesis in several insects (Raikel et al., 2005).
Vitellogenin is involved in many biological processes, such as reproduction,
longevity, immunity, the transport of zinc, the synthesis of larval food, foraging
activity, and regulation of the hemolymphatic juvenile hormone titer (Hartefelder and
Engels, 1998; Amdam and Omholt, 2002; Amdam et al., 2003; Amdam and Omholt,
2003; Amdam et al., 2004; Amdam et al., 2005; Guidugli et al., 2005; Nelson et al.,
2007; Denison and Raymond-Delpech, 2008).
In Apis mellifera, vitellogenin synthesis is high in nursing bees (30 to 40% of the total
protein) and low in newly emerged workers and foragers (Engels, 1974; Melody et
al., 1982; Engels et al., 1990).
Studies have demonstrated an inverse relationship between the titers of JH and Vg
in the hemolymph of workers; thus, in nurse bees the Vg levels are high and the JH
levels are low, and in foragers the reverse occurs (Engels et al., 1990; Hartfelder and
Engels, 1998; Jassim et al., 2000).
This inverse relationship was proposed by the double repressor hypothesis, in that
the high titer of Vg has an inhibitory influence on the synthesis of JH and foraging
behavior; however, a decline of the vitellogenin titer causes an increase in the JH
titer and activates the behavioral change resulting in foraging (Amdam and Omholt,
2003).
Researchers investigated the effect of depletion of Vg on the JH titer and the onset
of foraging and proposed a model with three steps for the development of this
behavior in workers. The first coincides with the maturation of young workers, which
is observed through a gradual increase in Vg and low concentrations of JH and
ecdysteroids in the hemolymph, preventing the onset of foraging flight. The second
stage is marked by the depression of the JH titer due to the high titer of Vg, and the
last stage, in the period of foraging, is a depression in the Vg titer and a high JH titer
(Marco Antonio et al., 2008).
In Scaptotrigona postica, Melipona quadrifasciata and Melipona scutellaris, newly
emerged workers do not have Vg in the hemolymph, whereas the nurse Vg protein
corresponds to 40 to 50% of the total proteins, and in the forages the Vg titer
decreases in the hemolymphatic stock. Only in the permanently sterile Frieseomelitta
varia workers was the Vg protein found to be consistently present throughout pupal
and adult stages (Engels and Engels, 1977; Dallacqua et al., 2007).
Recent studies have shown a direct relationship between the reproductive
physiology and foraging behavior. The onset of foraging and the foraging bias
towards either nectar or pollen can be influenced by the levels of Vg. When the Vg
gene was silenced early onset of foraging occurred and it also verified that the
workers who already were foraging left to collect pollen, passing collect nectar
(Nelson et al., 2007; Ihle et al., 2010).
Brain structures and learning
The bees are social insects and great models in neurobiology for learning research
and memory. Although they have small brains, they can perform several behaviors
(Menzel, 1999; Page and Erber, 2002). The volume of their brain components
depends on brain size; the central body is different to the other components, which
are placed below the mushroom calyx and show allometric growth. There is a
negative correlation between the relative volumes of the optic lobe and the antennal
lobe; the bees that have a large visual field show a small olfactory field and vice
versa. The progress in learning is linked to the body mushroom volume (Gronenberg
e Couvillon, 2010).
The body mushroom is a very important place in the brain for learning and
sensory integration; it is the central processing area of olfactory information in
insects (Erber et al., 1980; Faber et al., 1999; Straufeld, 2002), be paired, receive
sensory modal of antennal lobe to calyx and are compound of Kenyon cells
(neurons) (Technau and Heisenberg, 1982; Crittenden et al., 1998; Lee et al., 1999),
had high level of structure plasticity, according with age and sex (Robinson, 1997;
Withers, 1993).
The experience of foraging increases the volume of neuropils in the
mushroom body (Nyla et al., 2006), although, due to plasticity, the mushroom body
shows growth in the first day of life bees, even under conditions that prevent the
bees from interacting with the environment (Fahrbach et al., 1998).
Another example of the plasticity of the mushroom body starts when the bee
begins the process of foraging. Bees that foraged for two weeks without confinement
exhibited growth in the neuropils and those that foraged for only a week and were
confined during the following week showed no growth. No difference in relative
neuropil size was found between the confined bees and the bees that foraged for
only a day and then stayed in captivity for two weeks. Muscarinic cholinergic
receptor stimulation causes the increased volume of neuropils in the confined bees
(Nyla et al., 2006)
These social behaviors, such as foraging, have demonstrated the influence of
genes on neural plasticity under ecological and evolutionary conditions, contributing
to a greater understanding of the relationship between genes, brain and behavior
(Robinson, 1998).
Recently, many studies have discussed the pattern of gene expression in the
bee brain. Many researchers isolated genes encoding several different receptors
involved in neurotransmission such as the dopamine D1 receptor (Blenau et al.,
1998) and the recipient of tyramine (Blenau et al., 2000). In 2005, Mustard et al.
analyzed the expression profile of the tyramine receptor in the brain of Apis mellifera
during development and observed an increase correlated with age.
The Honey Bee Brain EST Project coordinated by Dr Gene Robinson,
University of Illinois (USA), conducted a study on the transcriptome of the brain of
Apis mellifera to accelerate molecular studies of neuroscience and behavior in bees.
We sequenced 20,256 cDNA clones, after processing programs that exclude
vectors, low-quality reads and sequences smaller than 200 bp, leaving 15,311 ESTs
(expressed sequence tags) that were grouped as 3136 contigs and 5830 singlets (
Whitfield et al., 2002).
Whitfield conducted a study on the expression profile of individual genes in
Apis mellifera brains and predicted the associated behavior. In this study, we
analyzed 5500 genes and found that 39% were associated with the transition of
young bees to foragers.
In 2004, Tsuchimoto et al. examined changes in gene expression in the brain
of Apis associated with age. These authors found that genes involved in the signal
transduction pathway, ion channels, neurotransmitter transporters, cell adhesion
proteins, transcription factors and membrane-associated proteins were more highly
expressed in foragers than in newborns.
In Melipona, there is no data on brain function. This organ is responsible for
controlling activities and the neurosecretion of biogenic amines and neuropeptides
that control the biosynthesis of JH by the corpora allata; therefore, it has an
important role in determining caste.
Social behavior requires an elaborate sensory system that enables learning
and memory (Tsuchimoto et al., 2004) and it is accompanied by changes in the
endocrine system and in the biochemistry of the brain and other associated
structures (Fahrbach and Robison, 1996; Wagener-Hulme et al., 1999).
The gene expression of the acetylcholinesterase decoder in the Kenyon cells
provides an indication of the importance of the cholinergic system for the operation
of the mushroom body (Shapira et al., 2001). The expression of this gene is reduced
in the brains of foragers in relation to the bees that play a role inside the hive
(Shapira, 2001), suggesting that the breakdown of acetylcholine is attenuated in
foragers.
In the cerebral lobes of bees, the visual neurons can be divided into two
groups according to the conditions to generate or not generate a spontaneous
response (Hertel, 1980).
The responses of visual neurons are similar when stimulated by different
colors (Erber and Menzel, 1977) When stimulated by flashing light 69% of them are
in a state of a transient short answer to this stimulus and another 57% respond to
colored flashes (Yang et al., 2004); in the medullary region about 52% respond to
these stimuli transiently and only 16% to the colored stimulus (Hertel, 1980).
Menzel (1979, 1985) suggested that insects have two patterns of color vision,
one related to light intensity due to broadband neurons and one related to color
vision due to neurons with a low bandwidth.
Issues related to social learning in bees have been studied by means of
classical conditioning using the proboscis extension response (PER) (Farina et al.,
2005; Gil and De Marco, 2005; Grüter et al., 2006). When bee antennae are placed
in contact with sucrose there is a stimulus that induces a reflex reaction (Kuwabara,
1957), which is a standardized procedure for studies on learning (Schorkopf et al.,
2009).
The PER protocol has been widely used to study the dynamics of learning in
several species of insects, including stinging bees (Takeda, 1961), bumble bees
(Lalo et al., 1999), Drosophila (Chabaud et al., 2006), and mosquitoes (Tomberlin et
al., 2006), and recently it was used for the stingless bee Melipona quadrifasciata
(McCabe et al., 2007).
In olfactory learning in the neural structures involved in this process, as well
as in the antennal lobe, the primary neuropils and mushroom bodies receive
information from multiple sensory modalities that have integral functions (Erber et al.,
1980, 1987; Menzel et al., 1994) and the lateral protocerebral lobe (lpl) receives
olfactory information from the antennal lobe and later the mushroom body processes
this information (Mauelshagen, 1993; Rybak and Menzel, 1993), resulting in the
brain controlling motor centers in the ventral nerve cord. Reward information related
to these signals is transmitted to the neuropils by a neuron identified as VUMmx1
(Hammer, 1993), which belongs to a particular class of neurons in the ventral nerve
cord in insects, which in turn has a remarkable morphology: unpaired symmetrical
bilateral dorsal median neurons and ventral unpaired median neurons (DUM and
VUM neurons) and octopamine immunoreactivity, and an excitatory neurotransmitter
(Bräuniger, 1991) Ganglion VUM neurons subesofagico soaring to the region of the
brain are probably octopaminergic (Kreissl et al., 1994) Excitation of the neuron
VUMmx1 followed by an olfactory stimulus is sufficient to initiate a behavior
associated with olfactory learning. However, if an odor is present during excitation a
behavioral change occurs that is not associated with learning (Hammer, 1993). This
suggests that the molecular and cellular mechanisms involved in the continuous
detection and temporal VUMmx1 in the neural activity related to odor should be
sensitive to time (Heisenberg et al., 1985; Bele and Heisenberg, 1994).
The three sites of convergence of the processing of conditioned stimuli and
unconditioned stimuli, i.e. the antennal lobe, the lateral protocerebral neuropil and
mushroom bodies express high levels of cAMP-dependent protein kinase.
In the antennal lobe the cAMP cascade is involved in the processing pathway
of an unconditioned stimulus. The application of an unconditioned stimulus causes a
rapid increase in the transient protein kinase activity, whereas application of the
conditioned stimulus has no effect (Hildebrandt and Müller, 1995a, b) This temporary
increase in protein kinase is mediated via the cAMP system and octopamine. The
immunoreactivity to neuron VUMmx1 octopamine, which is also wooded antennal
lobe, is a likely mediator of unconditioned stimuli, thereby increasing the activity of
protein kinase (Muller and Hildebrandt, 1995b). The application of paired conditioned
and unconditioned stimuli produces a prolonged activation of protein kinase in the
antennal lobe, whereas a conditioned stimulus alone does not affect protein kinase
activity and the unconditioned stimulus alone only causes a transient increase in
protein activity. The prolonged activation of the protein kinase substrate can be
associated with olfactory learning at the antennal lobe level (Menzel, 1996).
The neurotransmitters involved in learning and memory
The honeybee queen controls the physiology and behavior of her partners, and this
determines societal functioning. In order to wield this influence, she produces a
cocktail of pheromones known as queen mandibular pheromones (QMP) (Galizia,
2007).
The effect of this pheromone tends to influence the neurons that are being
modulated and can therefore cause behavioral changes. With maturity, honeybees
spend a lot of time absent from the colony, such as flying to forage and performing
tasks outside the nest, including distinguishing not only the sweet smell of nectar but
also dangerous and unpleasant odors (Galizia, 2007).
Biogenic amines are organic nitrogen compounds that are among the best
studied compounds in neurochemistry, produced by life’s metabolism and
fundamental to the existence and maintenance of life. They perform an important
role
in
controlling
physiological
and
behavioral
modifications,
acting
as
neurotransmitters, neuromodulators and neurohormones (Burrows, 1996; Kravitz,
2000; Roeder, 1999) and they are also involved in learning and memory (Hammer
The amines may cause physiological changes and consequently
and Menzel, 1998).
influence animal behavior. Octopamines have a great influence on the physiology of
insects (Roeder, 1999). It has been speculated that octopamine acts as a
neuromodulator to influence the behavioral development of bees (Schulz and
Robinson, 2001). This neurotransmitter acts on the behavioral development of
foraging honeybees. Treatment with octopamine changed the ability of bees to
incubate pheromones (present in the epidermis of bees at the larval stage, who,
when in contact with adult bees, induce foraging, regardless of age), increasing the
numbers of foragers entering and leaving the colony, demonstrating its performance
as a neuromodulator (Barron et al., 2002).
Brain levels of octopamine are high in foragers and low in brood nurses
(Wagener-Hulme et al., 1999). The relationship between the level of octopamine and
foraging behavior is most marked in the antenna lobes (Schulz and Robinson, 1999).
Oral treatment with octopamine induces precocious foraging in young honeybees
(Schulz and Robinson, 2001), providing evidence of the role of octopamine in the
development of this behavior. The effect of octopamine is only seen in bees that are
capable of foraging, suggesting that it has control over the activation of foraging
(Schulz and Robinson, 2001).
Harris and Woodring (1992) showed that dopamine, serotonin and
octopamine
levels
in
the
brain
of
A.
mellifera
increase
with age, and during the summer, when foraging activity is more expressive.
Foragers have significantly elevated levels of dopamine in the brain compared to
younger bees. Foraging is associated with higher levels of biogenic amines in the
brain (Taylor et al., 1992). Before foraging occurs, an increase in the level of
octopamine in the lobes of the antenna suggests that octopamine is a foraging agent
(Wagener-Hulme et al., 1999); this does not happen with serotonin or dopamine
levels during the
same
period,
providing
evidence
to
show that
these
neurotransmitters are not causative agents of such activity (Schulz and Robinson,
2001).
The inveigling of neurotransmitters and ion exchange through the plasma
membrane in neurons and glial cells is more enhanced in the brains of foragers than
in younger bees. All gene components of the transduction pathway, such as the
genes of the phosphatidylinositol-specific phospholipase C (PI-PLC) epsilon (Song et
al., 2001) and diacylglycerol kinase (DGK) (Harden et al., 1993), are expressed at
higher levels in the brains of foragers when compared to newborn bees. This
suggests that the signal transduction mediated by intracellular Ca2+ and secondary
phospholipid messengers is more intense in foragers than in bees that do not forage.
The activities of cell signaling mediated by protein kinases and phosphatases are
also more expressed at a higher level in foragers than in newborn honeybee brains
(Tsuchimoto et al., 2004).
The neurotransmitter transporter and ion channel genes are expressed at a
higher level in foragers compared to newborn, for example the genes of the choline
transporter (Okuda et al., 2000), glutamate transporter (Besson et al., 1999)
transporter of betaine/GABA (Borden et al., 1995), the channel subunit voltagedependent calcium (Klugbauer et al., 1999), Na + / K + transporting ATPase α
subunit (Sun et al., 1998).
The foraging activity of honeybees is also responsible for the pollination of
cultivars. Several pesticides have affected foragers, causing changes in the
captivation dynamics of sources and impairing colony fitness (De la Ru'a et al.,
2009). The insecticides used to protect crops can induce behavioral alterations, such
as disorientation, and the reduction of foraging activity, which affects both
honeybees and pollination and consequently damages food production and the
environment. Bortolotti et al. (2003) observed the effects of sublethal doses of the
insecticide imidacloprid on foraging behavior and the return of bees to the nest.
Honeybees were trained to forage on an artificial field and the results showed that
the control honeybees returned to the nest and the feeder within 5 hours, whereas
those treated with the chemical solution at 100 ppb returned to the feeder after 24
hours, and bees that foraged on the samples treated with 500 ppb and 1000 ppb did
not return to the nest or the feeder.
Medrzycki et al. (2003) also evaluated the effect of sublethal doses of
imidacloprid at concentrations of 100 ppb and 500 ppb on the behavior of A.
Mellifera, and observed a decrease in mobility and communication. Another study
showed an influence on the activity of adult bees, with a decrease in the frequency of
pollen conduction during foraging and changes in the comb (Faucon et al., 2005).
Molecular communication pathways
Through receptors, bees perceive various environmental stimuli that are transformed
into nervous impulses in the brain. The receptors are formed by the sensilla, the
small cuticular sensory organs. The main sensilla identified so far are sensilla
placodae (odor receptors) (Halberg and Hansson, 1999; Cruz-Landim, 2009), the
ocelli (luminous intensity photoreceptor) (Goodman, 1981) and compound eyes
(visual organs) (Carminda Cruz Landim, 2009).
The receptors are found in many parts of the body of insects. These
structures are classified according to the nature of the stimulus perceived, such as
chemoreceptors that detect the presence of chemicals in air or in a substrate,
photoreceptors that detect the presence of light and mechanoreceptors that detect
motion, vibration and touch (Halberg and Hansson, 1999).
Insects have two chemosensory systems: gustatory and olfactory (Dethier,
1976). The antenna, parts of the mouth and the leg tarsus are the main structures
quimiossensorial of honeybees (Goodman, 2003).
Specific receptors on nerve cells for odor and taste are important for
honeybees to discriminate between the diverse chemical cues found in nature
(Goodman, 2003). Robertson and Wanner (2006) identified these receptors in the
honeybee Apis mellifera with the publication of its genome sequence. They identified
163 olfactory receptors (Ors) and only 10 gustatory receptors (Grs) in this honeybee.
The Ors and Grs receptors belong to the same insect chemoreceptor
superfamily (Scott et al., 2001; Hill et al., 2002; Robertson et al., 2003) and they are
recognized as a highly divergent family of proteins that have seven transmembrane
domain G-protein-coupled receptors (GPCR).
The gustatory receptors are so called because they express themselves
primarily in gustatory organs such as the mouthparts (Clyne et al., 2000), although
some Grs can function as olfactory receptors by expressing themselves in olfactory
structures (Scott et al., 2001).
There is a relationship between juvenile hormones and the gustatory system.
The first peak of JH during the adult worker life is detected a few days after their
emergence (Jassim et al., 2000). Interestingly, a related study found high gustatory
response scores for the older honeybees (eight days old) after a topical application
of a JH analogue (methoprene), but this effect was not found in newborns
honeybees treated with the same compound (Pankiw and Page, 2003).
Physiological modifications in the nervous system and non-neural tissues can
cause differences in sucrose response and memory retention between young (0-1
days old) and old (4-9 and 12-16 days) pre-forager bees. These differences were
observed in hormones and biogenic amines, such as juvenile hormones (JH) and
octopamine (OA) throughout the workers’ life (Robinson and Vargo, 1997; Bloch et
al., 2002). Octopamine and other biogenic amines, such as tyramine and dopamine,
were identified as modulators of bee behavior during learning (Hammer, 1993;
Vergoz et al., 2007) and gustatory responses (Pankiw and Page, 2003).
The insects use pheromones to communicate, which are interpreted in the
brain; however, to elicit a response, such compounds need to cross the cuticle; they
are then detected by small sensory organs and distributed in various body sites
insect, called sensile (Hallberg, Hansson, 1999).
The olfactory sensile sites have a varied morphology, although they do share
one characteristic: they have multiple pores, facilitating the access of odor molecules
to olfactory neurons that project their axons into the olfactory lobe in the insect’s
brain, where they are then processed as information (Chapman, 1998; Vogt, 2005;
Letzkus et al., 2006).
The scent trail biochemistry left by honeybees to locate a viable food source
involves three types of proteins: odor receptors (ORs), present in the dendrites
membrane, ready to plug in the odor molecule; odorant binding proteins (OBPs),
which assist the molecules, and odor degrading enzymes (ODEs), which degrade
the odor molecules after signage.
When an odor molecule enters the iolfactory sensilla the OBPs lead in to the
ORs, forming a complex, and thus the activation of a signaling cascade via inositol
triphosphate (IP3) occurs, leading to open channels for sodium/potassium and local
depolarization. In this manner, changes occur in the action potential of the neuron,
and this electric signal is transferred to the central nervous system, which is
connected to other systems and generates a physiological or behavioral
characteristic of each odor (Xu et al., 2005; Vogt, 2003, 2005).
The photoreceptors are arranged into two structural forms: the ocelli and
compound eyes. The ocelli are three small eyes situated on the top of the head,
forming a triangle. Each ocellus has an external convex lens placed over a layer of
vitreous cells derived from the epidermis, the cornea cells. In Apis mellifera, beneath
these structures is the retinal layer (Cruz-Landim, 2009). The retina is one of the
most demanding body tissues from a metabolic viewpoint. To ensure the adequate
furnishing of oxygen and nutrients, the retina is supplied by two separate vascular
beds: the inner retina vascularization and the external choroidal circulation (SaintGeniez, 2008). The retinal layer consists of modified dendrites of neuron receptors
(Cruz-Landim, 2009).
The bee's eye consists of ommatidia, with each one containing nine
photoreceptor cells, and three photoreceptors with a spectral sensitivity for
ultraviolet, blue and green. The receptors feed into lamina neurons that amplify the
signal modulation and cut out the persistent signal from constant light. In terms of
visual pathways in insects, the photoreceptors provide information to lamina cells,
which send their projections to the medulla (Strausfeld, 1976; Ribi and Scheel, 1981;
Paulk et al., 2009). The medulla, which is the structure that contains the largest
amount of neurons in the visual system, connects to the central brain and, in parallel,
to a third order visual processing center, o lobulo visual, which then sends inputs via
several tracts into the central brain (Hertel and Marondera , 1987; Yang et al., 2004;
Gronenberg Paulk, 2008). As with the medulla, the lobula is a structure that is
sensitive to motion and color (Ribi and Scheel, 1981; Paulk et al., 2008). The
projection patterns of color-sensitive neurons in the lobula also suggest the
segregation of color processing in downstream regions of the brain center,
particularly in the lateral protocerebrum (Dyer et al., 2010). This part of the brain is a
multimodal structure that receives olfactory, visual, and mechanosensory information
(Marondera, 1991; Kirschner et al., 2006).
The lateral protocerebrum is a central brain area sensitive to the orientation of
visual stimuli (Strausfeld and Okamura, 2007), which involves well-defined
anatomical structures of the mushroom bodies and the central complex and receives
tracts from the antennal lobe and the region of the optic lobe (medulla and lobula)
(Schildberger, 1984). Color and visual motion information appears to be segregated
along the anatomical routes based on the posterior (motion) and anterior (color) axes
of the brain. Most of the lobe neurons projecting into or residing in the anterior lateral
protocerebrum were sensitive to color, suggesting that the site along the anteriorposterior axis is the best predictor of color sensitivity, and thus visual information
(Paulk et al., 2009).