Influence of activity in Drosophila melanogaster longevity. Role of temperature.

Jorge Monje Martn. Diego Fernndez Garayzbal.

*Corresponding author. E-mail addresses:

jorge_vk_10@hotmail.com diego.fernandez01@estudiante.uam.es

Introduction
Organisms longevity has always fascinated humans since the beginning of science. Aristotle already wrote down on his On Longevity and Shortness of Life (350 B.C.E.) The reasons for some animals being long-lived and others short-lived, and, in a word, causes of the length and brevity of life call for investigation. Indeed, these longevity differences are shown clearly in nature. One of the first evident differences we can find when comparing organisms is that larger ones usually live longer than smaller ones (60 years Elephant versus 2 years common mouse). Generally small animals suffer greater heat loss per unit area, thus they need high metabolic rate to reduce their losses. On 1928 Pearl published his experiments and his rate living theory where longevity was defined as a factor inversely dependent to metabolic rate. (Live fast die young). Hence small animals due to their higher metabolic rates tend to live shorter and viceversa. Such theory gained molecular respect when Harman in 1958 developed his aging theory dependent on the discharge of free oxygen radicals during metabolism which destabilize cellular structure. (Harman, 1956; Harman, 1981) Considering the context after rate of living theory born, new research lines emerged seeking to reduce metabolic rate of model animals to see how it affect to longevity. Numerous studies restricted the amount of nutrients that animals ingest, it was found for many of them that longevity was increased with this caloric restriction presumably by a decline on cellular damage caused by high metabolism. (McCay, McCrowell and Maynard 1935; Walford, 1988) However, it is evident that some species remain unresponsive to the size and metabolic rate rule. Some birds like eagles have similar weigh and metabolic rate to some medium mammals like dogs, but they are much more long-lived. In 2004 Illoyd Demetrius highlighted the importance of considering evolutionary history to know the metabolic equilibrium of one given species. By this way it is possible to know how it will react to metabolic rate changes. Some species have obtained traits around evolution that make them able to be more long lived (top-predators are not predated, antioxidants). (Demetrius, 2004). On our experiment we will have to consider this evolutionary perspective of our animal model. We have worked with fruit flies Drosophila melanogaster lines subjected to 24 fluctuatingtemperature cycles. We will measure its Activity in terms of locomotor activity (LA) which is the capacity of move at expense of metabolic expense. In Drosophila LA gets influenced by traits like population density, temperature, luminosity, humidity (Sewel 1979; Medioni 1966). Drosophila melanogaster activity had been used for more than 40 years to study the circadian rhythms. The first endogenous factor (gene period) that responds to one exogenous factor was found by Benzer and Konpka on 1971 Mutants showed shorter or longer rhythms, or arrhythmic behavior. (Konopka and Benzer, 1971).

Since then, lots of genes have been founded and which are the external factors they respond to. (Dunlap, 1999) Our experiment studies the role of activity on longevity, depending on temperature, which is one environmental variable which generate cyclic behaviors. We start out from two assumptions: -1 Considering that Drosophila melanogaster is an exothermic insect, so its activity, and therefore its metabolic rate and cellular damage increase with temperature. -2 Considering, as Demetrius proposed, the natural history of the specie. D.melanogaster is a specie with short life-cycle that occurs when situations are favorable. Longevity has not been developed on his evolution. Its life strategy is a very fast succession of generations with great offspring. Hence, its longevity will be significantly affected by environmental changes. Our main hypothesis to demonstrate is: Fluctuating temperatures generate different circadian activity peaks in D.melanogaster which suppose one accumulation of stress that can decrease longevity significantly.

Materials and Methods.

We developed two different experiments both under the same conditions. The variable factor is the Temperature. 1 Longevity experiment: The main objective is to see if the longevity gets influenced by the different temperature conditions. 2 Activity experiment: The goal is to show the global activity of different flies lines each experiment day and the activity per hour. Both experiments are closely related and its results will be related and used to give a global conclusion. For our statistical analysis we used JMP 10.lnk. Flies: Two different Drosophila melanogaster lines: A y G. Three replicates inside each line: A1, A2, A3 / G1, G2, G3 The origin of these flies is on Coffs Harbour, Australia. In May 2009 a single breed was set mixing 150 isofemale lines (10males&10females). 5 generations were succeed (12L, 12D) from this single breed at 1C 25C with standar Drosophila medium (yeast and oatmeal agar) Then, two different lines (A and G) were split and were subjected under two different temperature conditions and 12Light, 12Dark during 65 and 75 generations respectively. In each condition three replicate lines (A1, A2, A3 / G1, G2, G3) were conserved in each generation to give more statistical power to our analysis.

Conditions The conditions are: Fluctuating, line A: Cabinet with 24h changing temperature regimen (Mean 1C 27C) Constant, line G: Cabinet provided always with the same temperature ( 1C 27C). Three different light intensity types in both cabinets during one day (Morning light, noon light, afternoon light). Temperature controlled and registered with data logger. 1 Longevity experiment 180 flies (90 males and 90 females) extracted randomly from the global mass bred in each subline (LA1, LA2, LA3/LG1, LG2, LG3).30 flies per vial. The density of each vial was controlled. We registered the number of dead flies each two days during all their life. 2 Activity experiment 6 experiment days each 5 days during one month for each line. (Day 5, Day 10, Day 15, Day 20, Day 25, Day 30). To measure LA we used a Locomotor Activity Monitor (TriKinetics). It has 4 racks with 30 holes each one that can be filled by small glass vials (5 mm in diameter). A single specimen can be placed inside one of these vials. The machine counts, with an infrared cell in the middle, the number of times that the fly passes through the photocell, each 5 seconds during the whole experiment day.

Results Figures and tables.

*Note: All figures show a 95% confidence interval

LINE A G

SEX M F M F

5 22,88 30,55 34,64 34,91

10 20,82 37,18 32,48 23,13

AGE 15 20 9,75 22,22 25,92 29,01 17,58 20,47 19,17 28,37

25 23,07 22,74 25,12 24,90

30 17,19 22,39 22,41 14,03

F 22,66 28,82 58,80 51,35

P <,0001*** <,0001*** <,0001*** <,0001***

Table 1: Activity index value (AIV) of flies during their first 30 days of life.

AGE 5 10 15 20 25 30 F M 22,39 17,16 20,45

SEX F M F M F M F M F M

SQ MEAN 30,55 22,88 37,18 20,82 25,92 9,75 29,01 22,22 22,74 23,07

F 14,02 119,44 141,86 21,74 0,0718

P 0,00002*** <,0001*** <,0001*** <,0001*** 0,7888

<,0001***

Table 2: Activity (AIV) of flies under fluctuating conditions

Figure 1: Mean of locomotor activity vs. Age under fluctuating conditions

AGE 5 10 15 20 25 30

SEX F M F M F M F M F M F M

SQ MEAN 34,91 34,64 23,13 32,48 19,17 17,58 28,36 20,47 24,90 25,12 14,03 22,41

F 0,0208 42,19 3,1043 25,3865 0,0238 36,74

P 0,8853 <,0001*** 0,0782 <,0001*** 0,8775 <,0001***

Table 3: Activity (AIV) of flies living under constant conditions

Figure 2: Mean locomotor activity vs. Age under constant conditions

AGE 5 10 15 20 25 30

LINE A G A G A G A G A G A G

SQ MEAN 22,88 34,64 20,82 32,48 9,75 17,58 22,22 20,47 23,07 25,12 17,16 22,41

F 37,1459 53,3383 38,0089 2,7108 1,7156 16,61

P <,0001*** <,0001*** <,0001*** 0,0998 0,1906 <,0001***

Table 4: Activity (AIV) of males in both conditions

Figure 3: Mean locomotor activity vs. Age in males under both conditions

AGE 5 10 15 20 25 30

LINE A G A G A G A G A G A G

SQ MEAN 30,55 34,91 37,18 23,13 25,92 19,17 29,01 28,37 22,74 28,38 22,39 14,03

F 5,1737 111,54 29,3463 0,1152 8,7265 43,7850

P 0,0232* <,0001*** <,0001*** 0,7344 0,0032** <,0001***

Table 5: Activity (AIV) of females in both conditions

Figure 4: Mean locomotor activity vs. Age in both conditions. We can see the Forever young phenomena at 20 day on G lines.

Figure 5: Mean longevity in fluctuating F= 43,07 P<0,0001***

Figure 6: Mean Longevity vs. Sex in constant F= 73,84 P<0,0001***

Figure 7: Mean Longevity vs. Line (males)F= 17,11 P<0,0001***

Figure 8: Mean Longevity vs. Lines (females) F= 33,47 P<0,0001***

Figure 9: Female activity mean hour per hour. We can see significant differences in hours 7, 15, 16, 17 and 20, with P<0,05. Mean A: 26,67. Mean G:22,88

Figure 10: Male activity mean hour per hour. Mean A:19,73. Mean G:24,91

Main results: Our analysis finds differences in locomotor activity through age. There is a decline in locomotor activity when flies become older (see Table 1). We also have find significant differences between sexes and lines (fluctuant A and constant G). Females are usually more active than males under fluctuant conditions (Table 2 & Figure 1) but those differences are not so clear between the males and females in the constant cabinet, being even higher the mean of the males (Table 3 & Figure2). We found a non-expected result. We can see how total activity does not decrease in every experiment day (Table 1 & Figure 1 Figure 2 Figure 3 Figure 4) or inclusive, it increases in some degree. This happens at 15th-20th day, depending on the line and in few replicates the increase of activity becomes significant. We called this non expected result Forever young phenomena. About the effect of the constant and fluctuant temperatures, there is a clear pattern in males. Constant males show more activity than fluctuating males (Table 4 & Figure 3).

About females the results are no so clear. Constant females are more active in 5 th and 25th days, but less active in 10th, 15th and 30th days. Even, the P value is more significant when fluctuating females are the most active flies (Table 5 & Figure 4). About longevity, females live longer than males under both conditions (P<0,0001***) and flies living under fluctuating temperatures live shorter than same-sex flies living under constant temperatures (P<0,0001***) (Figures 5 to 8). Seeing the 24 hours tables (Figure 9 and Figure 10) we can see a significant increase on fluctuating flies at the noon light (11h,12h,13h,14h,15h) in both fluctuating males and fluctuating females. This increase coincides with higher temperatures on fluctuating conditions. The highest activity peak is on the evening light (from 18h to 20h). In males we can see this increase with not significant differences between G and A. But in females we can see a highest peak for the fluctuant ones.

Discussion.
As we predict in our main hypothesis, fluctuating temperatures are a stress factor and, as a consequence, they reduce the longevity of the flies. But the effect on flies activity varies depending on the sex. The activity of A males is lower than G males, but these differences are not so clear in females, because sometimes A females are even more active. This different pattern between males and females could occur due to the higher necessity for males of coping with desiccation, so males will use more energy to deal with desiccation and less energy to move. This effect is not seen in females due to their bigger body size, a characteristic that made females more able to cope with desiccation. Because of this, females have more energy available to move, and we can see that this stressing condition makes females to move more by mean. If we look at Figure 9 we can see how the evening activity peak is significantly higher for the fluctuating flies. It can have led to an increase of its stress (Due to oxygen radicals because of the higher metabolic rate). We can consider this fact as the stress factor (thermal stress) that have led a global decrease on fluctuating flies longevity. If we look at males on Figure 10 there is not significant differences between A and G maybe because they have to cope with desiccation

Linked with the longevity experiment, the higher activity of fluctuant females is in concordance with their lower longevity. But the opposite occur with the males. The less active males (fluctuant) are those who live shorter. The reason of their shorter live may lie in their stress, which is not shown as activity (this happen with females), but in other physiological activities not analyzed here as the maintenance of their water balance. This results are on concordance with the second point that we underline in our introduction about considering, as Demetrius proposed, the natural history of the animal. As they have not

developed its longevity during their evolution, little changes on some environmental conditions can affect to longevity significantly. About the forever young phenomena first we have to say is that it could be random phenomena, and it has not a great statistical power. However, it is garish to see how they have had an activity-behavioral rejuvenescence. N.N. Moghadam had a result on her recent investigation that could be related with our forever young phenomena. Her flies showed at the 20th day a lipid membrane composition typical from younger flies. (Moghadam et.al. 2011). So it could be interesting to get more research on this aspect.

Acknowledgments
We are very grateful with every people on this course (Teachers, students, student coordinators) because it gave us the opportunity of work as a real researchers for the first time. Special thanks to Neda Nasiri for her patience, help and advice since the beginning. Thanks also to Tommaso Manenti for investing his time helping us with data analysis.

Bibliography
Demetrius L. Caloric restriction, metabolic rate and entropy. Journal Gerontology: Biological Sciences 59:902-915, September, 2004. Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol. (3):298300. July 1956 Harman D The aging process. Proc Natl Acad Sci U S A; 78(11): 71247128 November 1981. McCay, C. M.; Crowell, Mary F.; Crowell "Prolonging the Life Span". The Scientific Monthly 39 (5): 405414. November, 1934 Medioni, J. Effet inhibiteur de la lumirre blanche sur l'activit6 locomotrice "'spontanre'" de la drosophile (Drosophila melanogaster Meigen). C. R. Sdances Soc. Biol. 160, 667-671, 1966. Moghadham, N.N; Holmstrup, M; Pertoldi, C; Loeschke, V. Age-induced perturbation in cell membrane phospholipid fatty acid profile of Drosophila melanogaster longevity selected and corresponding control lines not published yet. 2012 Pearl, R. The Rate of Living, Being an Account of Some Experimental Studies on the Biology of Life Duration. New York: Alfred A. Knop. 1928 Konopka, R; Benzer, S; Clock mutants of Drosophila melanogaster proc. Nat. Acad. Sci. USA Vol. 68, no. 9, pp. 21122116, September 1971 Sewell, D.F. Effect of temperature and density variation on locomotor activity in Drosophila melanogaster: a comparison of behavioural measures. Anim. Behav. 27, 312-313, 1979. Jay C. Dunlap Molecular Bases for Circadian Clocks Cell, Vol. 96, 271290, January 22, 1999.