Study of Melipona quadrifasciata brain under operant learning using proteomic and phosphoproteomic analysis.

Learning to anticipate events based on the predictive relationship between an action and an outcome (operant conditioning) is a form of associative learning shared by humans and most of other living beings, including invertebrates. Several behavioral studies on the mechanisms of operant conditioning have included Melipona quadrifasciata, a honey bee that is easily manipulated due to lack of sting. In this work, brain proteomes of Melipona bees trained using operant conditioning and untrained (control) bees were compared by two-dimensional gel electrophoresis analysis within pI range of 3-10 and 4-7; in order to find proteins specifically related to this type of associative learning.One protein was detected with differential protein abundance in the brains of trained bees, when compared to not trained ones, through computational gel imaging and statistical analysis. This protein was identified by peptide mass fingerprinting and MS/MS peptide fragmentation using a MALDI-TOF/TOF mass spectrometer as one isoform of arginine kinase monomer, apparently dephosphorylated. Brain protein maps were obtained by 2-DE (Two-dimensional gel electrophoresis) from a total proteins and phosphoproteins extract of the bee Melipona quadrifasciata. One isoform of arginine kinase, probably a dephosphorylated isoform, was significantly more abundant in the brain of trained bees using operant conditioning. Arginine kinase has been reported as an important enzyme of the energy releasing process in the visual system of the bee, but it may carry out additional and unexpected functions in the bee brain for learning process.

Simple discrimination in stingless bees (Melipona quadrifasciata): Probing for select- and reject-stimulus control.

Simple and conditional discrimination training may produce various types of controlling relations. Responses may be controlled primarily by the positive stimulus (select-control relation) or by the negative stimulus (reject-control relation; the subject excludes the negative stimulus and chooses the positive). Bees learn to respond in simple and conditional discriminations. However, no study has searched for reject-control responding in Melipona bees. We trained Melipona quadrifasciata on a simple discrimination task (S+ vs. S-; e.g., blue vs. yellow) and then probed for stimulus control with two types of probe trials, S+ versus a new stimulus (Select-control probes) and S- versus a new stimulus (Reject-control probes). For Group Different, a new-stimulus color (e.g., white) was used in one type of probe and another color (e.g., black) was used in the other type. For Group Same, a single new-stimulus color was used in both types of probes. On Select probes, the bees always preferred S+ to the new stimulus. On Reject probes, results were mixed. Depending on the colors used in training and probing, bees responded to both stimuli, and even preferred the S-. The data suggest no control by the negative function of the S- and support the select-stimulus control hypothesis of responding.

A comparative study of relational learning capacity in honeybees (Apis mellifera) and stingless bees (Melipona rufiventris).

BACKGROUND: Learning of arbitrary relations is the capacity to acquire knowledge about associations between events or stimuli that do not share any similarities, and use this knowledge to make behavioural choices. This capacity is well documented in humans and vertebrates, and there is some evidence it exists in the honeybee (Apis mellifera). However, little is known about whether the ability for relational learning extends to other invertebrates, although many insects have been shown to possess excellent learning capacities in spite of their small brains. METHODOLOGY/PRINCIPAL FINDINGS: Using a symbolic matching-to-sample procedure, we show that the honeybee Apis mellifera rapidly learns arbitrary relations between colours and patterns, reaching 68.2% correct choice for pattern-colour relations and 73.3% for colour-pattern relations. However, Apis mellifera does not transfer this knowledge to the symmetrical relations when the stimulus order is reversed. A second bee species, the stingless bee Melipona rufiventris from Brazil, seems unable to learn the same arbitrary relations between colours and patterns, although it exhibits excellent discrimination learning. CONCLUSIONS/SIGNIFICANCE: Our results confirm that the capacity for learning arbitrary relations is not limited to vertebrates, but even insects with small brains can perform this learning task. Interestingly, it seems to be a species-specific ability. The disparity in relational learning performance between the two bee species we tested may be linked to their specific foraging and recruitment strategies, which evolved in adaptation to different environments.

Discrimações condicionais entre estímulos visuais e testes de simetria em meliponas / Conditional visual discriminations and symmetry probes in meliponas

Oito melíponas (Melipona quadrifasciata) foram expostas a uma versão adaptada do procedimento de emparelhamento arbitrário com o modelo (matching-to-sample) em que a presença ou ausência de luz era empregada como estímulo condicional e as cores azul e amarelo como estímulos discriminativos. As cores eram apresentadas por semicírculos de material emborrachado sustentados por duas caixas retangulares de madeira, cada uma posicionada sobre uma caixa experimental a uma distância de 15 cm uma da outra. Cada lâmpada estava localizada sobre uma das caixas. As caixas experimentais eram de material acrílico com um painel superior de alumínio. Cada caixa era equipada com um alimentador operado externamente por uma haste e possuía um conjunto de pequenos orifícios no painel de alumínio que davam acesso ao xarope. Uma abelha podia voar da colméia para o aparato experimental e obter o xarope condicionalmente ao pouso direto sobre o estímulo de comparação definido como S+ em uma tentativa particular. Depois de instalada a linha de base, foram conduzidas sondas de simetria em extinção, intercaladas com tentativas de linha de base. Nas sondas, os discos coloridos tinham a função de estímulos condicionais (as duas caixas mostravam os discos de uma mesma cor, azul ou amarelo) e a presença e a ausência de luz eram usadas como estímulos discriminativos. As discriminações condicionais foram estabelecidas com todas as abelhas e seis entre as oito abelhas alcançaram o critério de simetria emergente (ao menos cinco tentativas entre seis). Estes resultados estendem achados prévios sobre discriminações condicionais e emparelhamentode identidade em abelhas. Contudo, no presente procedimento os estímulos tinham posição fixa, o que permite questionar se os comportamentos observados revelam propriedades simétricas (emergentes) ou apenas o controle por componentes de estímulos compostos (diretamente aprendidos).(AU)

Eight adult, worker bees (Melipona quadrifasciata) were exposed to an adapted version of the matching-to-sample procedure in which the presence or absence of lights was used as the conditional stimuli and yellow and blue colors were the discriminative stimuli. The color stimuli were presented as semi-circles of opaque rubber material located on top of two rectangular boxes, located 15 cm apart from each other. The lights were located one above each box. The experimental boxes were custom-made, constructed of gray plexiglass with an aluminum top panel. Each box was equipped with a feeder operated from outside by a handle and small holes on a circumscribed area on the top gave access to syrup. The bee would fly from the hive to the experimental setting and would obtain syrup conditional upon flying to the box holding the comparison stimulus defined as S+ on a particular trial. After the baseline was established, symmetry probes interspersed with baseline trials were conducted inextinction. During the symmetry probes the colored disks had the function of conditional stimuli (the two boxes held disks of the same color, either blue or yellow) and the presence and absence of light were used as discriminative stimuli. The conditional discriminations were established with all bees and six out of eight reached the criterion foremergent symmetry (at least five trials out of six). These results extended previous findings for conditional discriminations and identity matching with bees. For symmetry tests, however, in the present procedure the stimuli had fixed positions, an issue which raises questions as to whether the observed behavior reveals symmetric(emergent) properties or just (learned) joint-control by components of a compound stimulus.(AU)