Investigaciones sobre el conocimiento del contenido de

To determine the impact of learning, experiments investigating LTM formation were conducted. It has been shown, that memory dynamics in parasitoid are different to, for example, memory dynamics in honeybees, and so far, in certain parasitoid species LTM formation is completed after already 1 d (Collatz et al., 2006; Smid et al., 2007). In C. vestalis, however, the memory trace began to wane after only 1 h (in the sugar group). Although the response rate was still around 60%, no significant differences were found compared to responses to the control odour (hexane) at any of the time points tested. Additionally, responses to linalool at day 2 and 3 were only 25% and 15%, respectively, which is similar to the response rate of parasitoids experiencing a non-associative

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exposure (ODOUR ONLY) to an odour (Section 2.3.2). These results indicate that no LTM formation occurred. This is different to other studies of the related specialist species, C. rubecula, where LTM was formed despite a decline of responses to the learned stimulus after 1 day (Bleeker et al., 2006a). Responses of

C. vestalis to the odour at 1 h,1 d, 2 d and 3 d could be due to a combination of

non-associative and associative learning (Bleeker et al., 2006a).

Learning and the influence of the inter-trial interval (ITI)

One explanation for this result could be the ITI. In the experiment investigating LTM formation, females were placed with the conditioned odour for 20 min during the HP treatment. This relates to a training protocol classified as massed learning (Smid et al., 2007), in which the female has a number of oviposition experiences in sequence, but is not removed from the leaf in between the trials. This resembles a natural situation in which the parasitoid would encounter a cluster of caterpillars on a plant. However, it has been shown that in some species, spaced learning trials are needed to establish LTM (Menzel, 2001; Giurfa et al., 2009).

Generalist parasitoid species, such as Eupelmus vuillet, C. glomerata and

Lariophagus distinguendus, however, showed a memory stable trace up to 7 d

after training, regardless of the training procedure (Cortesero et al., 1995; Müller et al., 2006; Smid et al., 2007). In the specialist Leptopilina boulardi (Kaiser et al., 2003) massed conditioning lead to a memory trace lasting about 48 h and in

Microplitis croceipes memory lasted about 24 h despite spaced conditioning

(Drost et al., 1986). In a few specialist species, on the other hand, spaced learning was needed for memory formation. In C. rubecula, for example, three massed learning trials failed to induce learning after 24 h (Smid et al., 2007), which is similar to results in C. vestalis. Thus, massed learning in C. vestalis might not induce LTM formation and spaced learning trials could be needed for LTM formation.

Memory formation and host distribution pattern

In the natural environment insects will not encounter a controlled ITI. Spaced trials could reflect a longer interval between encounters of scarce hosts or food sources and therefore require memory formation. Massed learning, on the

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other hand, could reflect a situation where the parasitoid encounters the host on a regular basis in bigger batches. Hence a memory lasting more than 24 h would be of little use for a species that experiences regular encounters with a host (Kaiser et al., 2003). Bleeker (Bleeker et al., 2006) argued that differences in the host distribution led to differences in memory dynamics between solitary parasitoids (foraging on a solitary feeding host) and gregarious parasitoids (foraging on a gregariously feeding host). A solitary parasitoid would encounter many single CT over a period of time. A gregarious one however, is more likely to encounter one massed CT, as it will encounter the host in high number patches (Hoedjes et al., 2011).

Host specialisation and the variability of odour cues

The lack of memory formation in C. vestalis could also be related to a high innate preference for certain HIPVs. Although a learning experience enhances the parasitoid’s performance over a brief period, memory formation over a long time would be too costly. Stephens (Stephens, 1991; Stephens, 1993) argued that the variability of the environment can influence the value of an innate response. If between generations variability is low, as it would be for a specialist species, the value of an innate response would be high. This would result in a low learning rate, which has also been shown in bumblebees (Ings et al., 2009). Furthermore, if the innate preference to an odour is important it would be too costly to maintain LTM formation for a specialist, as the variability in olfactory cues will remain low.

Costs involved in memory formation have been demonstrated in Drosophila, where animals producing LTM died earlier than ones that only produced ARM (Mery and Kawecki, 2005). Likewise, it has been argued that learning has less impact on initially strong responses (Vet and Dicke, 1992). Therefore, the attractiveness of initially preferred odours may be enhanced through learning; while it will be neglected for “ecological irrelevant” odours (which are not part of volatiles of the host-plant complex).

For generalists on the other hand, environmental stimuli may vary or change. Therefore, learning different odour cues would increase their host searching efficiency and allow them to alter their behaviour if necessary. For generalists it would be more useful to store olfactory information. Depending on

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the variability of odour cues, information could be stored in a more accessible memory, in which information can easily be modified (such as STM or MTM). This would also suggest that LTM which has been observed under laboratory conditions (with a low variability) (Collatz et al., 2006; Smid et al., 2007) might not be formed under more natural conditions. The degree of foraging specialisation can therefore not solely be responsible for, but may nevertheless influence memory formation. It appears that due to its specialisation (and thus a lower variability in HIPVs), C. vestalis does not rely on memory formation, and it can be argued that massed learning does not lead to an associative LTM formation in this species.

Side effects of antibiotics blocking protein synthesis

Evidence suggests that LTM formation is dependent on the synthesis of new proteins (Giurfa et al., 2009; Guerrieri et al., 2011; Chen et al., 2012). The control experiment (sugar group) did not provide evidence for a LTM memory formation in C. vestalis, thus, the lack of response in the parasitoids in the ACD group tested 1 d after conditioning cannot be explained by a disruption of transcription and thus inhibition of memory formation.

The process of protein synthesis has been shown to occur in critical time- windows following conditioning (Fulton et al., 2005; Fulton et al., 2008). Inhibitors of transcription and translation are thus thought to be most effective in blocking LTM when administered just before or immediately after training (Davis and Squire, 1984). In the ACD group, animals were therefore fed ACD 24 h before the experiment, and 2 h after testing (1 h for the 1 h group), to ensure that the drug had been consumed in the time frame sensitive to protein synthesis disruption (Grünbaum and Müller, 1998; Wüstenberg et al., 1998; Igaz et al., 2002), following the protocol of other studies (Tully et al., 1994; Wüstenberg et al., 1998; Collatz et al., 2006).

With the exception of testing 1 d after training, responses to the learned stimulus were similar to the sugar group at other time points. After 1 d, however, animals showed an avoidance response to the learned odour. These results were unexpected for a number of reasons. The lack of memory formation after 1 d is similar to findings in other parasitoids (Collatz et al., 2006; Smid et al., 2007), but due to the decrease in response to the learned odour over the course of time, one

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would have expected no LTM memory formation and thus no dependency on RNA synthesis. Previous experiments in parasitoids (Collatz et al., 2006; Smid et al., 2007) did not test the effect of ACD at time points beyond 24 h, however, if ACD was blocking protein synthesis, responses after 2 d and 3 d should be affected as well, as the effects of this drug are irreversible (Igaz et al., 2002).

The fact that the memory impairment only took place at one time point could suggest that (ACD) had a toxic effect on the animal. Actinomycin D, like other protein synthesis inhibitors, is a highly toxic (Waksman and Woodruff, 1940, 1941; Philips et al., 1960) DNA binding molecule (Kersten et al., 1960), which has been shown to inhibit the growth of tumours (Sugiura, 1960; Malogolowkin et al., 2008; Ferrari et al., 2010), gram-positive bacteria (Waksman and Woodruff, 1942) and even reverse transcription in the HIV virus (Paramanathan et al., 2011) and fungi (Schoenian et al., 2011). Apart from blocking RNA synthesis (Kirk, 1960) by inhibiting polymerase A, an enzyme essential for DNA replication, it was further shown that ACD also inhibits cell growth, RNA loss, causes shrinking of cytoplasm and inhibits biosynthesis (Reich et al., 1962). ACD can intercalate (via a planar tricyclic phenoxazone ring) between double stranded DNA (dsDNA) base pairs (SOBELL et al., 1971) and bind to single-stranded DNA (ssDNA) (Zhou et al., 2007), thus preventing elongation by RNA polymerase. A recent study (Paramanathan et al., 2012) has found that by binding to pre-melted dsDNA, as found in transcription bubbles (destabilised duplexes, where duplex DNA is unwound at the forward end of RNA polymerase and rewound at its rear end) (Berg JM et al., 2002), ACD stops RNA synthesis and thus leads to cell death (Paramanathan et al., 2012).

Toxic effects of the drug after 1 d have been discussed in several papers (Squire and Barondes, 1970). Squire (Squire and Barondes, 1970) suggested that an impaired memory retention 1 d after training is not due to ACD inhibiting RNA synthesis needed for LTM formation, but caused by toxic effects of ACD. They have also been found to occur after 1 d in rat neurons (Rizzuto and Gambetti, 1976). Rizzuto (Rizzuto and Gambetti, 1976) demonstrated that intracranial injection of ACD resulted in a status spongiosus (fluid-filled spaces) of the white matter in the cerebrum, cerebellum, brain stem and optic nerve within 48 h after injection, caused by vacuole formation within the myelin sheath. Although it was suggested that this was a result of the inhibitiory effect of ACD, a

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direct toxic effect of the drug can nevertheless not be excluded. Barondes and Cohen (Barondes and Jarvik, 1964; Cohen and Barondes, 1966) argued that the effectivity of ACD as a protein synthesis inhibitor is difficult to interpret, as concentrations suitable for affecting protein synthesis are accompanied by toxic side effects of the drug. Even smaller doses of the drug led to chromatolysis, necrosis and/or electrical abnormality (Nakajima, 1969). Hence, toxic effects could cause avoidance learning observed in the current study.

Aversive conditioning

The avoidance reaction observed at 1 d post treatment could also be caused by a taste aversive reaction (Garcia and Kimeldorf, 1957). Application of the drug in the 1 h or 2 h following treatment with the HP and the odour could have caused an aversive form of STM which was not affected by the drug’s disruption of protein synthesis. This could have been induced by classical forward conditioning, as the CS (the odour) was preceding the drug. Studies in rodents or snails (Garcia et al., 1955; Sugai et al., 2007) have shown that taste aversion learning can usually be induced by only one conditioning trial and does not necessarily require a short spacing between CS and US, leading to a long-lasting avoidance.

Taste avoidance STM would, however, not explain the lack of avoidance response at 1 h testing, as so far, a delayed response in aversive taste conditioning has not been shown yet. However, the exposure of 1 h might have not been sufficient to cause an aversion, contrary to an exposure of 2 h. In this case, it could be suggested, that 2 h post exposure to ACD induced aversive STM lasting 1 d, as only long-term taste aversion memory depends on protein synthesis (Rosenblum et al., 1993; Gal-Ben-Ari and Rosenblum, 2011) and consequently the degradation of proteins (Rodriguez-Ortiz et al., 2011), but not STM (Houpt and Berlin, 1999). Tully (Tully et al., 1994) demonstrated in his study about memory formation in Drosophila that feeding 35 mM of the proteinsynthesis inhibitor Cycloheximide (CHX) 12-15 h before training and until 24 h after training did not lead to aversive behaviour or any deleterious effects of the drug. Deleterious effects of CHX such as death of the animal, increased after 24 h from 19% dead animals to 100% dead animals 96 h later, contrary to feeding with sucrose, which resulted in 3% and 12% dead animals, respectively (Tully et al., 1994).

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In summary, the avoidance response observed in the ACD group at 1 d following conditioning could be due to an aversive STM formation. However, considering the toxicity of the drug, it is likely to be due to toxic effect occurring at 1 d, which has also been shown in other studies (Squire and Barondes, 1970).