Pre-adult developmental dietary
status influences adult negative
O status dietético durante o desenvolvimento influência o comportamento
de geotaxia negativa em diferentes genótipos de Drosophila melanogaster
Estado de la dieta durante la influencia del desarrollo del comportamiento
geotaxia negativa en diferentes genotipos de Drosophila melanogaster
*Pós Graduação em Diversidade Cultural e Inclusão Social
Universidade Feevale, Novo Hamburgo, RS
**Departamento de Genética, Instituto de Biociências
Universidade Federal do Rio Grande do Sul (UFRGS)
***Departamento de Estatística, Instituto de Matemática
Universidade Federal do Rio Grande do Sul
Geraldine Alves dos Santos* | Sidia Maria Callegari-Jacques** ***
Vera Lúcia Valente** | Gabriela Fernandes Vera**
Anelise Batista** | Alexandre Martins**
Julhana Pohlmann** | Thays Soliman**
Bruna Vieira** | Cinthia Mesquita**
Tássia Lazzari** | Gilson Luis da Cunha*
Negative geotaxis is an innate escape response in which flies climb the walls of a container after being tapped to its bottom. This climbing speed is known to decrease with age in a genotype-dependent fashion in Drosophila. In the present paper we describe, for the first time, the influence of developmental dietary status on this behavior. We observed significantly different performances in negative geotaxis among adult individuals of Drosophila melanogaster developed in standard and fat-rich media. Our results suggest that fat-rich nutrition during development can modify negative geotaxis in adult Drosophila individuals. However, some genotypes seem to be benefited or, at least, not affected by the dietary status adopted.
Keywords: Aging. Development. Dietary fat. Locomotor senescence. Negative geotaxis. Drosophila.
A geotaxia negative é uma resposta inata de fuga na qual moscas escalam as paredes de um recipiente após serem lançadas ao fundo do mesmo. Sabe-se que a velocidade de escalada decresce com o avanço da idade, de modo dependente do genótipo. No presente artigo, nós descrevemos, pela primeira vez, a influência da dieta durante o desenvolvimento sobre esse comportamento. Nós observamos desempenhos diferentes entre indivíduos de Drosophila melanogaster que se desenvolveram em meio de cultura padrão e meio rico em gordura. Os resultados sugerem que uma nutrição rica em gordura durante o desenvolvimento pode modificar a geotaxia negativa em indivíduos adultos de Drosophila. Entretanto, alguns genótipos parecem se beneficiar, ou, ao menos, não serem afetados pelo tipo de dieta adotado.
Unitermos: Envelhecimento. Dieta de gorduras. Senescência locomotora. Geotaxia Negativa. Drosophila.
Recepção: 23/08/2015 - Aceitação: 30/11/2015
|EFDeportes.com, Revista Digital. Buenos Aires, Año 20, Nº 211, Diciembre de 2015. http://www.efdeportes.com/||
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Aging is a mosaic of processes that lead to the decline in physiological performance and in stress resistance. However, these processes do not always occur uniformly among different species, strains, individuals or even tissues of one same individual.
Growing evidence has shown that different individuals of the same species can achieve the same longevity through different physiological mechanisms (Vettraino, Buck and Arking, 2001). Environmental factors like diets, temperature, drugs and physical activity are also involved in the modulation of aging (Avanesian, Khodayari, Felgner and Jafari, 2010).
Caloric restriction, the most successful experimental intervention in aging, is known to extend longevity and to improve functional parameters in old age, for different vertebrate and invertebrate species (You, Sonntag, Leng and Carter, 2007). Caloric restriction may play a role in slowing physiological impairments in locomotion, sensory functions and cognition (Witte, Fobker, Gellner, Knecht and Flöel, 2009) as well as in retarding the development of age-related pathologies (Colman, Beasley, Allison and Weindruch, 2008). With opposing effects, traits like obesity, as well as fat-rich diets, are often cited as factors causing longevity shortening and faster onset of age-related pathologies in a large variety of species (Sun, Seeberger, Alberico, Wang, Wheeler, Schauss et al., 2010; Bruce-Keller, White, Gupta, Knight, Pistell, Ingram et al., 2010) . More than a risk factor for age-related pathologies, fat-rich diets are pointed as a source of perturbation to normal gene expression, being implicated in common diseases with late-onset phenotypes through interactions between the epigenome, the genome and the environment (Feinberg, 2007). In the fruit fly Drosophila, the effects of fat rich diets on aging are poorly studied, although all available papers show a clear life shortening effect (Driver, Wallis, Cosopodiotis and Ettershank, 1986; Vermeulen, Van De Zande, Bijlsma, 2006). However, there are no current studies on age-related functional impairments influenced by dietary fat in Drosophila. Tests of age-related functional decline in motor activity of model organisms were used as tools to clarify the contributions of genetic and environmental influences acting in the lowering of movement speed (Rhodenizer, Martin, Bhandari, Pletcher and Grotewiel, 2008).The fruit fly Drosophila melanogaster is one of the most important experimental models in aging research. This insect has been used in order to locate the main genes controlling longevity and stress resistance, the regulation of their expression and the developmental effects of pre-adult environment in adult lifespan (Arking, 2001). Larval starvation induced by overcrowded culture media during pre-adult development was studied in extreme caloric restriction and has been observed to extend adult lifespan and stress resistance in different strains (Da Cunha and Oliveira, 1996; Rion and Kawecki, 2007; Driver, Wallis, Cosopodiotis and Ettershank, 1986; Vermeulen, Van De Zande, Bijlsma, 2006). This result suggests that one of the effects of larval starvation is the activation of developmentally regulated genes with influence on lifespan.
Among the several phenotypes observed during aging, negative geotaxis is especially useful to evaluate functional senescence in Drosophila. Negative geotaxis is an innate escape response in which flies climb the walls of a container after being tapped to its bottom (Gargano, Martin, Bhandari, and Grotewiel, 2005). This escape response is known to decline with age in Drosophila (Rhodenizer, Martin, Bhandari, Pletcher, Grotewiel, 2008). Environmental and genetic factors acting on the aging process can affect motor activity of the flies as well (Avanesian, Khodayari, Felgner and Jafari, 2010). Recently, it was observed that lamotrigine extends Drosophila longevity, although causing impairments to locomotor activity (Avanesian, Khodayari, Felgner and Jafari, 2010; Burger, Buechel and Kawecki, 2010). Caloric restriction was equally efficient in order to extend drosophila lifespan, however, it showed no effects on cognitive aging in this insect or neuronal dysfunction in Drosophila models of Alzheimer's disease (Kerr, Augustin, Piper, Gandy, Allen, Lovestone et al., 2009). These data indicate the increasing need of studies addressing different aspects of functional aging in Drosophila.
Due to the lack of data on functional impairments induced by fat rich diets on aging in Drosophila, and particularly, on fat consumption during pre-adult development, the present study aimed to test one wild type and four mutant strains of Drosophila melanogaster developed under standard and fat-rich culture media, in order to assess if these different genotypes can be influenced by pre-adult nutrition conditions in their adult negative geotaxis responses.
2. Material and methods
2.1. Drosophila strains
The following Drosophila strains were used:
Wild type Oregon-R controls (Da Cunha and Oliveira, 1996).
ADP60 mutants, showing an obesity phenotype. After 1 week of ad libitum feeding, the fat body cells of ADP60 flies contain greatly enlarged lipid droplets compared to controls. In addition, the mutant animals show a twofold higher accumulation of triglycerides, and a variable, slight reduction of glycogen levels to 70-80% of normal levels. ADP60adults show increased resistance to starvation compared to control flies (Häder, Müller, Aguilera, Eulenberg, Steuernage, Ciossek et al, 2003) IDH ngb2, mutants deficient in enzyme activity for NADP-dependent isocitric dehydrogenase. This enzyme displays the same electrophoretic mobility as IdhS. The mutants show only 5% residual enzyme activity (Burkhart, Montgomery, Langley, 1984).
Men nnc1 flies (deficient for NADP dependent malic enzyme). Enzyme extracted from MennNC1 homozygotes is kinetically indistinguishable from wild-type enzyme. It was proposed that MennNC1 is a cis-acting regulatory mutant. The mutant enzyme has only 3-5% of the normal activity (Burkhart, Montgomery, Langley, 1984).
CAT n1 individuals (deficient in enzyme activity for catalase). The Catn1 mutation determines a nonfunctioning protein. Decreased viability and longevity of these acatalasemic flies can be restored by transformation with the wild-type catalase gene. It suggests that the lethality of these genotypes is due solely to the lack of catalase (Griswold, Matthews, Bewley, Mahaffey, 1993).
ADP60 mutants were kindly provided by Dr. Ronald Khünlein. Oregon-R strain is regularly maintained at the Drosophila Lab in the genetics Department at UFRGS. All the remaining strains were provided by the Bloomington stock center.
2.2. Culture media and fly handling
The standard culture medium was prepared with 160 g of pre-boiled corn meal, 44 g of molasses, 20 g of soy flour, 36 g of dehydrated yeast, 16 g of agar, 3 g of nipagin, 1.3 ml of propionic acid, 10 ml of ethanol and 1000 ml of distilled water.
The fat-rich medium consisted of the standard medium supplemented with the addition of 200 g of commercially available culinary vegetal hydrogenated fat (Gordura vegetal Primor, Bunge Alimentos, Brazil).
The flies were maintained at 25 °C and 65% relative humidity, under permanent light.
Each strain was divided in two groups, one that laid eggs in fat-rich medium, and one that laid eggs in standard medium. Egg laying time was 3 hours for all strains. Following eclosion, adults grown in fat-rich and in standard media were then separately placed in new tubes with standard medium. The fat-rich medium group was used only for the negative geotaxis experiments and disposed of thereafter in order to avoid selection for fat tolerance in the strains employed in the experiments.
2.3. Negative geotaxis
Negative Geotaxis Assay was performed as follows. Adult males were collected at the age of 6 days by cold anesthesia (exposure at 0 °C for 3 minutes) and allowed to recover for at least 18 hours. They were then placed into separate vials, one tube per strain, 25 individuals in each tube. The individuals were assayed at the ages of 1 and 3 weeks. Individuals older than 3 weeks were not available among the group developed in fat rich medium, since fat consumption reduced maximum lifespan to 23 days (data not shown). The reduced survival in ages above 3 weeks prevented comparisons to older individuals cultured under standard conditions. This age limit imposed by fat rich diets has been observed in other recent studies (Sun, Seeberger, Alberico, Wang, Wheeler, Schauss, et al., 2010). The individuals to be assayed were placed in the negative geotaxis vials 30 minutes before the beginning of the test. All tests were conducted at 25 °C and 65% relative humidity, under permanent light.
A rack containing the negative geotaxis vials was sharply hit 3 times against the workbench. At the last blow, a photograph was taken using a digital camera (Canon power shot SD630, 6.0 megapixels, Japan) previously positioned 30 cm away from the apparatus, with a 4-second delay. The individuals able to climb to a distance equal or higher than 8 cm in this time interval were counted in the photos. Each strain was tested 5 times. The flies were given 5 minutes of rest between each assay.
2.4. Statistical analysis
The average numbers of climbs of each strain were analyzed by the Repeated Measures ANOVA test using the SPSS software and Duncan’s multiple pairwise comparisons between means. Differences between group means were considered statistically significant if p ≤0.05.
Table 1 presents the observed mean number of climbings in individuals 1 and 3 week old, for the five different samples.
Table 1. Negative geotaxis expressed as climbing means in individuals
one and three weeks old developed in standard and fat rich media
We observed a significant triple interaction among strain, diet and week (F = 2.661; df= 4, 40 ; p = 0.046) in the within-subjects tests of the repeated measures ANOVA, meaning that induced diet effects on negative geotaxis were strain dependent and influenced by age as well. The between-subjects analyses also revealed interaction effect between strain and diet (F = 5,876; df = 4, 40; p = 0.003). These results prompted us to compare diets separately by strain and age of individuals. The results of the pairwise comparisons are presented in Figure 1 (A and B).
Figure 1. Effect of development under fat-rich media on negative geotaxis of adults 1 and 3 weeks old (A and B respectively): average climbings and standard errors
Stars indicated statistically significant differences between
diets within strain, using Duncan’s test at the 0.05 level
Among all 1-week-old individuals cultured in standard medium, CATn1 had the highest negative geotaxis values, followed respectively by Mennnc1, IDHngb2, ADP60 and Oregon-R. It is interesting to remark that mutants deficient in catalase, malic enzyme and NADP dependent iscocytric dehydrogenase showed better performance in negative geotaxis than the Wild type Oregon-R. The same pattern, however, was not observed in 3-weeks old individuals.
The lowest values in the NG test among all strains, ages and treatments were those obtained by Mennnc1, 3-weeks old individuals cultured in fat rich media. This strain’s score in negative geotaxis seemed to be strongly affected both by age and diet.
In the fat-rich medium, among the 1-week-old individuals of all strains, the
order of successful climbing was IDHngb2> CATn1 > Men nnc1 > ADP60>
, Development in fat-rich medium produced lower
climbing rates both in younger and older CATn1, Men nnc1 and Oregon individuals.
No statistically significant differences between the climbing rates were
observed for ADP60 flies.
Curiously, displaying a radically opposed behavior, IDH ngb2 individuals 1 week old, grown in fat-rich media, had higher negative geotaxis performances than their counterparts from standard media and showed no significant differences at 3 weeks old.
According to Rhodenizer et al. (2008) the decline in negative geotaxis observed in Drosophila is age and strain-dependent. In the present paper we report, for the first time, the effects of development under fat-rich nutrition conditions in adult performance of five different Drosophila melanogaster genotypes in the Negative geotaxis test., For three of the five strains studied, the performances of individuals developed in fat-rich culture medium were poorer as compared to the performance of their counterparts grown in standard medium. Drosophila physiology is not identical to mammal physiology. However, at cellular level the mechanisms induced by dietary manipulation could trigger similar responses in gene expression (Haigis and Guarente, 2006). There is a reasonable set of evidences for Drosophila showing that dietary conditions in pre-adult development can modulate lifespan of adult flies through changes in gene expression regulation (Arking, Burde, Graves, Hari, Feldman, Zeevi et al., 2000). In the past decade, different mechanisms were proposed in order to explain the effects of caloric restriction or high caloric intake on Drosophila aging. Two of these mechanisms, the dSIR2 mediated histone modifications and the DNA methylation are among the most studied in Drosophila and in mammals. Both mechanisms consider caloric restriction as a factor of gene silencing or, at least, as a gene expression attenuator. A high fat intake is expected to promote opposing effects through different pathways: a) reducing the amount of NAD available for the sirtuins could slow down its histone deacetylase activity (Guarente 2005) and b) a fat-rich diet could disrupt the normal methylation patterns observed in both development and aging (Burdge, Lillycrop, Phillips, Slater-Jefferies, Jackson and Hanson, 2009). So far, in Drosophila, the most plausible candidate of these two mechanisms seems to be the sirtuins-dependent one, since it is the best documented and the most experimentally tested. However, the hypothesis of methylation disruption has been widely investigated in mammals and cannot be entirely ruled out in Drosophila, because recent evidences showed that intactness of dDnmt2, the only DNA methyl transferase responsible for DNA methylation in fruit flies, is required for the maintenance of normal life span. Overexpression of the same gene confers increased resistance to paraquat in Drosophila melanogaster (Lin, Tang, Reddy, Shen,2005) Paraquat resistance is often correlated to extended lifespan phenotypes in Drosophila melanogaster (Arking, 2001).
Despite the unclear nature of the mechanisms acting during the development of the strains used in the present study, ADP60 mutants — in all ages and media — exhibited an unusually stable behavior. This gene was identified as an evolutionarily conserved WD40/tetratricopeptide-repeat-domain protein (Häder, Müller, Aguilera, Eulenberg, Steuernagel, Ciossek et al., 2003), and was shown to be a conserved dosage-sensitive anti-obesity gene (Suh, Zeve, McKay, Seo, Salo, Li et al., 2003). Loss of ADP activity promotes increased fat storage, which extends the lifespan of mutant flies under starvation conditions. Curiously, in ADP60 individuals, there were no differences in NG performance in individuals grown in either the standard or the fat-rich medium, suggesting that this genotype, in the conditions of the present study, is a factor of greater influence as compared to the environment (i.e. diets) over the responses to the NG test.
Since oxidative stress is one of the best known causal factors in motor activity decline and neurodegenerative diseases, it was expected that CAT n1 individuals should present the lowest performances, at every experimental condition defined. Furthermore, considering Malic enzyme and IDH-NADP as important sources of reducing power, their deficient genotypes (Men nnc1 and IDH ngb2) would be expected to present lower performances than those shown by the Oregon-R strain. Remarkably, the highest performance observed for the IDH ngb2 strain was seen in individuals developed in fat-rich medium. Taking this observation into account, this set of results suggests that, despite the generally harmful effects of fat-rich diets on development, the range of responses to these conditions can vary widely among different genotypes. Additional studies, combining molecular, biochemical and behavioral assays, are needed in order to identify the mechanisms underlying the decline of motor activity in different genetic backgrounds.
The authors thank Dr. Ronald Khünlein and the Bloomington Drosophila Stock Center for providing the mutant strains and to CNPq by grants to Vera Lúcia da Silva Valente.
Arking, R., Burde, V., Graves, K., Hari, R., Feldman, E., Zeevi, A., Soliman, S., Saraiya, A., Buck, S., Vettraino, J. & Sathrasala, K. (2000). Identical longevity phenotypes are characterized by different patterns of gene expression and oxidative damage. Exp Gerontol. 35, 353-73.
Arking, R. (2001). Gene expression and regulation in the extended longevity phenotypes of Drosophila. Ann N Y Acad Sci., 928,157-67.
Avanesian, A., Khodayari, B., Felgner, J. S. & Jafari, M. (2010). Lamotrigine extends lifespan but compromises health span in Drosophila melanogaster. Biogerontology, 11 (1), 45-52.
Bruce-Keller, A. J., White, C. L., Gupta, S., Knight, A. G., Pistell, P. J., Ingram, D. K., Morrison, C. D. & Keller, J. N. (2010). NOX activity in brain aging: Exacerbation by high fat diet. Free Radic Biol Med., 25. Epub ahead of print.
Burdge, G. C., Lillycrop, K. A., Phillips, E. S., Slater-Jefferies, J. L., Jackson, A. A. & Hanson, M. A. (2009). Folic acid supplementation during the juvenile-pubertal period in rats modifies the phenotype and epigenotype induced by prenatal nutrition. J Nutr., 139, 1054-60.
Burger, J. M., Buechel, S. D. & Kawecki, T. J. (2010). Dietary restriction affects lifespan but not cognitive aging in Drosophila melanogaster. Aging Cell, 12. Epub ahead of print.
Burkhart, B. D., Montgomery, E., Langley, C. H. & Voelker, R. A. (1984). Characterization of Allozyme Null and Low Activity Alleles from Two Natural Populations of Drosophila melanogaster. Genetics, 107(2), 295-306.
Colman, R. J., Beasley, T. M., Allison, D. B. & Weindruch, R. (2008). Attenuation of sarcopenia by dietary restriction in rhesus monkeys. J Gerontol A Biol Sci Med Sci., 63, 556-9.
Da Cunha, G. L. & De Oliveira, A. K. (1996). Citric acid cycle: a mainstream metabolic pathway influencing life span in Drosophila melanogaster? Exp Gerontol., 31, 705-15.
Driver, C. J., Wallis, R., Cosopodiotis, G. & Ettershank. G. (1986). Is a fat metabolite the major diet dependent accelerator of aging? Exp Gerontology, 21(6), 497-50.
Feinberg, A. P. (2007). Phenotypic plasticity and the epigenetics of human disease. Nature, 447(7143), 433-40.
Gargano, J. W., Martin, I., Bhandari, P. & Grotewiel, M. S. (2005). Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila. Exp Gerontol, 40 (5), 386-95.
Griswold, C. M., Matthews, A. L., Bewley, K. E. & Mahaffey, J. W. (1993). Molecular characterization and rescue of acatalasemic mutants of Drosophila melanogaster. Genetics, 134(3), 781-8a.
Guarente, L. (2005). Calorie restriction and SIR2 genes--towards a mechanism. Mech Ageing Dev., 126, 923-8.
Häder, T., Müller, S., Aguilera, M., Eulenberg, K. G., Steuernagel, A. & Ciossek, T., et al. (2003). Control of triglyceride storage by a WD40/TPR-domain protein. EMBO Rep., 4, 511-6.
Haigis, M. C. & Guarente, L. P. (2006). Mammalian sirtuins-emerging roles in physiology, aging, and calorie restriction. Genes Dev., 20, 2913-21.
Kerr, F., Augustin, H., Piper, M. D., Gandy, C., Allen, M. J., Lovestone, S. & Partridge L. (2009). Dietary restriction delays aging, but not neuronal dysfunction, in Drosophila models of Alzheimer's disease. Neurobiol Aging, 5. Epub ahead of print.
Lin, M. J., Tang, L. Y., Reddy, M. N. & Shen, C. K. (2005). DNA Methyltransferase Gene dDnmt2 and longevity of Drosophila. J Biol Chem., 280, 861-4.
Rhodenizer, D., Martin, I., Bhandari, P., Pletcher, S. D. & Grotewiel, M. (2008). Genetic and environmental factors impact age-related impairment of negative geotaxis in Drosophila by altering age-dependent climbing speed. Exp Geront., 43, 739-48.
Rion, S. & Kawecki, T. J. (2007). Evolutionary biology of starvation resistance: what we have learned from Drosophila. J. Evol Biol., 20, 1655-64.
Suh, J. M., Zeve, D., McKay, R., Seo, J., Salo, Z., Li, R., et al. (2007). Adipose is a conserved Dosage-Sensitive Antiobesity Gene. Cell Metab., 6(3), 195–207.
Sun, X., Seeberger, J., Alberico, T., Wang, C., Wheeler, C. T., Schauss, A. G. & Zou S. (2010). Açai palm fruit (Euterpe oleracea Mart.) pulp improves survival of flies on a high fat diet. Exp Gerontol., 45(3), 243-51.
Vermeulen, C. J., Van De Zande, L. & Bijlsma, R. (2006). Developmental and age-specific effects of selection on divergent virgin life span on fat content and starvation resistance in Drosophila melanogaster. J Insect Physiol., 52, 910-9.
Vettraino, J., Buck, S. & Arking, R. (2001). Direct selection for paraquat resistance in Drosophila results in a different extended longevity phenotype. J Gerontol A Biol Sci Med Sci., 56, B415-25.
Witte, A. V., Fobker, M., Gellner, R., Knecht, S. & Flöel, A. (2009). Caloric restriction improves memory in elderly humans. Proc Natl Acad Sci., 106, 1255-60.
You, T., Sonntag, W. E., Leng, X. & Carter, C. S. (2007). Lifelong caloric restriction and interleukin-6 secretion from adipose tissue: effects on physical performance decline in aged rats. J Gerontol A Biol Sci Med Sci., 62, 1082-7.
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