What can larvae do?

Drosophila Fruifly

Drosophila Melanogaster

State of the art: Drosophila melanogaster is a small fly that has been used as one of the major biological research tools for around 100 years; much of what we know about genetics, neuroscience and development, and now learning, comes from this animal. The lifecycle of Drosophila
melanogaster comprises four stages: egg, larva, pupa, and adult. At the larval stage, flies centuplicate their weight in only 6 days.

Thus, larvae are motivated by a single objective: localizing substrates rich in proteins and sugars to permit their development through to the adult stage. This includes a range of
innate orienting responses to gustatory and olfactory cues, as well to temperature, gravity, light and tactile signals. For odour cues the orientation response is not ultimately about odour but about finding or avoiding odour sources; as a consequence, olfactory processing includes pathways with an enormous potential to discriminate odours and to flexibly attach acquired meaning to them, leading to altered behaviour. LIN recently established a robust paradigm for odour learning in larval Drosophila [27] reviewed in [28]. The paradigm presents an odour with a sugar reward and measures the consequential change in preference for the odour (Figure 1); alternatively, low-concentration salt can be used as gustatory reward [29]. Larvae have also been shown to learn with aversive tastants such as high-concentration salt [29]or quinine [30] with alternative reinforcers such as shock [31] or vibration [32], and to learn about the association of visual cues with gustatory reinforcement [33]. The odour-sugar paradigm is the main focus of the current proposal because it has already been analysed in some detail with respect to its neurogenetic underpinnings e.g. [34][10]. Importantly in the current context, we have shown that the association between the odour and internal reward signal happens in the socalled mushroom bodies (MB, see section 1.2.4), in line with a rich experimental and modeling literature on learning in adult flies as well as other insects[35] [36] [37]. Also, the paradigm has been used to test generalisation between different odours as well as between different concentrations of the same odour, and for characterizing the learning of odour mixtures [38].

Associative learning assay for larval Drosophila.

Figure 1. Associative learning assay for larval Drosophila.
A group of 30 larvae is trained in a petri dish by pairing sugar (green) with n-amylacetate (AM), and a pure, tasteless substrate(white) with either a
second odor (X) (‘two-odor paradigm’) or no odor (‘one-odor paradigm’). In the subsequent test, the AM preference (Pref 1) is calculated as number of larvae on the AM side minus the number of larvae on the other side, divided by the total number. A second group of 30 animals is trained reciprocally (Pref 2). The performance index as measurement of conditioned behaviour is calculated as the difference of Pref 1 and Pref 2,
divided by 2. Appetitive memory results in positive performance index scores (rightmost plot); negative scores would indicate aversive memory. From Schleyer et al (in press).

(A) Larvae are trained such that in the reciprocal groups n-amylacetate (AM) is either paired with or not paired with a 0.2mol–1 fructose  reward. Subsequently, larvae are tested for their AM preference on either a tasteless, plain agarose substrate, or in the presence of 0.02, 0.2 or 2mol–1 fructose. (B) Neither the training concentration of fructose alone, nor the testing concentration of fructose alone can  account for conditioned behaviour. Rather, appetitive conditioned behaviour is expressed only when the trained fructose concentration is higher than the fructose concentration during the test. (C)Innate, experimentally naive olfactory preference towards AM is not influenced by the presence of fructose. (D)Innate, experimentally naive gustatory preference towards fructose is not influenced by the presence of AM. Thus, the learnt odour is tracked down only ʻin search for moreʼ, i.e. when doing so promises a positive outcome or ʻgainʼ. For the aversive case (not shown), the situation is inverse: aversive memory traces are behaviourally expressed only when the testing situation is as ʻbadʼ as or worse than the punishment used in training. From Diegelmann et al, 2013.

(A) Larvae are trained such that in the reciprocal groups n-amylacetate (AM) is either paired with or not paired with a 0.2mol–1 fructose reward. Subsequently, larvae are tested for their AM preference on either a tasteless, plain agarose substrate, or in the presence of 0.02, 0.2 or 2mol–1 fructose. (B) Neither the training
concentration of fructose alone, nor the testing concentration of fructose alone can account for conditioned behaviour. Rather, appetitive conditioned
behaviour is expressed only when the trained fructose concentration is higher than the fructose concentration
during the test. (C)Innate, experimentally naive
olfactory preference towards AM is not influenced by
the presence of fructose. (D)Innate, experimentally naive gustatory preference towards fructose is not
influenced by the presence of AM. Thus, the learnt odour is tracked down only ʻin search for moreʼ, i.e.
when doing so promises a positive outcome or ʻgainʼ.
For the aversive case (not shown), the situation is inverse: aversive memory traces are behaviourally expressed only when the testing situation is as ʻbadʼ as or worse than the punishment used in training. From Diegelmann et al, 2013.

Baseline: There is clear evidence that the change in the larval response to odours after pairing with appetitive or aversive tastants is not simply assimilation of the unconditioned response to the conditioned stimulus, and that the increased attraction towards the odour represents an action in search for food, rather a mere response towards an appetitively ‘charged’ odour. LIN and UEDIN have shown a dramatic dissociation between learnt behaviour towards the odour versus innate behaviour towards the reinforcer [29][39][40]. Larvae find some high concentrations of salt aversive; however, pairing such a repulsive salt concentration with an odour can lead to increased attraction to the odour.

A similar effect was demonstrated with sugar, which becomes aversive at very high concentrations, but still acts as reward [41]. For quinine, on the other hand, low concentrations which produce no innate avoidance response can still be effective reinforcers leading to aversion to the odour [42].
Further results from LIN (Fig 2) show larvae with identical training (A) show different behaviour (B) depending on the test conditions: they show increased attraction to the odour only if the level of sugar in the test condition is less than that paired with the odour in training. This effect is not due to direct effects of sugar presence (C) on behaviour to odour, or vice versa (D). On the other hand if test conditions are the same but training differs (B, far right group), larvae again only show the response to odour if the test level is less than that paired with the odour in training. It appears outcome expectation, and not the value of the memory trace, is the immediate cause of conditioned behaviour [40], as reviewed in [28].

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