Liña de auga e fangos 3.9.1.1 Conduccións e accesorios

A final method is to select directly on longevity, that is, to measure the longevity of individual families and then breed only from the longest lived. The biggest problem with this approach is that it is difficult to predict the longevity of an organism before it has died, and fecundity declines with age. Due to the scrutiny required during these experiments, it has also been necessary to keep the population sizes small, leading to inbreeding, low initial variation and a high risk of bottlenecks and fixation.

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Despite these drawbacks, direct selection remains the only way to unambiguously select for longer life, without introducing bias into the evolutionary path taken, i.e. selecting on stress resistance may extend lifespan as a result of changes in stress resistance but selecting on lifespan makes it more likely that a novel or unexpected mechanism is responsible for any observed lifespan extension (Zwaan, Bijlsma and Hoekstra, 1995).

The first attempt to select directly on lifespan yielded mixed results (Lints et al., 1979). Breeding pairs were established and bred approximately 15 days before 75% population mortality was expected. Once 75% mortality was reached, the progeny of the longest living pairs were then used to set up the next generation. Lifespan was not increased in the selected lines relative to the controls although lifespan of both the selected and control lines increased markedly compared to the base population. It was reasoned that these unusual results were likely due to uncontrolled environmental factors rather than a hereditary element to ageing. This conclusion was challenged however, and a later interpretation by Baret et al. suggested that a small lifespan extension was observed to a small extent in the original experiment (Fukui, Pletcher and Curtsinger, 1995).

More recently, Zwaan et al. (1995) used an updated familial selection method in order to avoid some of the pitfalls experienced previously. The method exploits the plasticity of Drosophila lifespan, which as a poikilotherm, can be modulated effectively by changing the environmental temperature. In short, breeding pairs produced offspring, some offspring were reared at 15°C, slowing ageing, while the others had their lifespan measured at 29°C, speeding ageing. By the time enough data was gathered from the lifespan assays to determine the longest-lived families, the siblings reared at 15°C were reaching a biological age of about young-adulthood and so the siblings of the longest-lived flies reared at 29°C could be selected to form the breeding pairs of the next generation. This method was successful in extending lifespan by about 28% in females and 10% in males after 4 generations and did not cause the same alteration of the reproductive schedule as seen in previous experiments, although there was a significant reduction in total lifetime progeny in the selected flies. The response to selection was more extreme when tested at 29°C, suggesting at least a minor role of temperature in the lifespan extension.

6

Conclusion

Selection experiments continue to be a useful method for the study of ageing. However, it is important to consider the selection methodology and how appropriate it is to answer your

questions. Although selection experiments are time-consuming, with careful planning they can be a powerful method to develop a suitable model organism for an experiment, without the need to resort to mutagenic, transgenic or chemical interventions.

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We have designed and carried out a selection experiment to select directly on lifespan, using the familial method of Zwaan et al. We used wild-caught flies to maximise initial variation and lowered the temperature of the lifespan assays to 27°C to reduce the potential bias towards heat-shock genes in the selection. The purpose of this selection was to investigate the transcription profile of long-lived organisms, and so the transcriptome of the selected flies was later characterised using RNA-Seq, and a range of phenotypes were tested to guide interpretation of the transcriptome data.

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Materials and Methods

7

Collection and Identification of Drosophila melanogaster

7.1

Collection of Flies

Drosophila were collected by aspirator from Williamson’s Park Butterfly House and a domestic glasshouse in Lancaster, UK, October 2013. Around 20 individuals were collected at each location and transported to the lab in bottles containing stock medium (Table 16). Flies from separate locations were kept apart at this stage.

Females were isolated and transferred individually to vials of cornmeal medium (Table 16) and allowed to lay. After 2 days, females were tipped to fresh vials and the larvae reared under standard conditions. On eclosion, flies were examined to determine their species.

7.2

Species Identification

Collected flies were identified as D. melanogaster morphologically and based on mating

compatibility. Morphology of males was examined under a dissecting microscope using the criteria outlined in (Ashburner, 1989).

The most reliable characteristic to identify D. melanogaster is the male genital structure located on the epandrium (Figure 2A). The phallus has flexible lateral expansions and a further expansion aligned with the claspers. The anal plates lack a ventral process and teeth (Figure 2Error! Reference

source not found.B) and the genital arch has expansions wider than they are long, with a further

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Figure 3. Sketches of the heads of (A) D.

simulans, and (B) D. melanogaster from the side, showing the wider cheeks of D. melanogaster

(Burla, 1951).

To further differentiate with D. simulans, the most similar species to D. melanogaster in terms of morphology, cheek width and maxillary palp bristle number were examined. The cheek of D. simulans is narrower than that of D. melanogaster (Figure 3) and the maxillary palps, located at the mouth, have fewer bristles (Figure 4).

Figure 2. Morphology of male D. melanogaster. (A) Shows the whole body morphology, and location of the genitalia at the epandrium (Weigmann et al., 2003). (B) Shows the male genitalia in a wild-type

D. melanogaster noting the Ap, anal plate; Cl, claspers; GA, genital arch; Lp, lateral plate (Gorfinkiel, Sánchez and Guerrero, 1999). (C) Shows a close up of the genital arch in D. melanogaster (left) and D. simulans (right), both genital arch images are at the same magnification and the scale line measures 100 micrometres (Coyne, 1983).

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Finally, five virgin females from each isofemale line were outcrossed to males from the lab strain w- Dahomey and the resulting progeny were mated and allowed to lay. The presence of viable larvae produced by this F2 generation confirm the original parents as D. melanogaster and rule out the possibility of D. melanogaster/D. simulans hybrids contaminating the stock.

In total, 18 lines were identified as being D. melanogaster and were used to set up the base population for selection.