Hayatta kalma Süre deneyinde<em> Brachionus</em> Rotiferler: Maternal Koşullarının Senkronizasyon
Summary
Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.
Abstract
Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.
Introduction
Rotiferler filumunu Rotifera 1 oluşturduğu mikroskopik kozmopolit zooplankton (<1 mm) 'dir. Bu yaklaşık 1000 somatik hücreler gibi karakteristik bir tekerlek gibi siliyer cihazından oluşan basit bir vücut düzeni hareket kabiliyeti ve besleme için kullanılan korona, adı vardır. Çoğu rotifer yaklaşık 1.600 ve 500 tür, sırasıyla 2 ihtiva sınıflar Monogononta veya bdelloidea aittir. Bdelloid rotifer zorunlu parthenogenesis 3 ile yeniden oluşturma sırasında Monogonont rotiferler genellikle hem eşeyli ve eşeysiz üreme evreleri (döngüsel parthenogenesis) var. Deneylerde, yüksek tekrarlanabilirliği sağlanır genetik olarak özdeş rotifer bireyler elde etmek mümkündür. Buna ek olarak, bu kadar kısa bir ömrü, kültür kolaylığı, genomik ve transkriptomik dizi verilerine 4-7 kullanılabilirliği ve uzak benzersiz bir filogenetik pozisyon gibi model organizmalar gibi birçok başka avantajları varrthropods ve nematodların 8. Rotifer Bu nedenle ekolojik, toksikolojik omurgasız modelleri vaat ve çalışmalar 9-12 yaşlanma vardır.
Çevresel strese ya da kimyasallara maruz altında hayatta kalma süresi bu araştırma alanlarında 13-19 bir çok ölçülen parametredir. Bu annelerinin çevre koşullarına maruz olduğu rotiferler hayatta kalma süresini ölçme, ancak, dikkatli gereklidir. Yani, monogonont rotiferi Brachionus manjavacas olarak, yaşlı annelerden gelen kadın yavrular genç annelerden daha kısa bir ömrü var; Ancak, anne kalori kısıtlaması (CR) kısmen ileri anne yaşı 20 zararlı etkilerini dengeler. B. plicatilis, anne CR yavru uzun ömürlü, açlık altında uzun yaşam süresi ve antioksidan enzimler 21,22 gelişmiş ifadesi ile ilişkili yüksek oksidatif stres direnci sağlar. anne yaşı etkisiAyrıca bdelloid rotiferler 23 gözlenmiştir. Bu nedenle, deneysel rotifer koşulları dikkatlice sağkalım süresi ölçümleri önce birkaç nesil boyunca senkronize edilmelidir.
Burada birkaç nesil boyunca kültür koşulları senkronizasyon aşağıdaki Brachionus rotifer sağkalım süresinin ölçümü için bir protokol sağlar. Aralıklı açlık (IF), rotiferler periyodik beslenir CR bir varyasyon nedeniyle uzun ömürlü 22,24 tarihinde IF tanınmış etkilerine senkronizasyon etkisini ortaya çıkarmak için uygulanmıştır.
Protocol
Representative Results
Discussion
Mevcut protokolü Brachionus rotiferler sağkalım süresini ölçmek için bir yöntem tarif eder. Kritik adım birkaç nesil boyunca rotifer koşulları eşitleme olduğunu. Deneysel rotifer iyi senkronize edildiğinde birkaç önceki çalışmalarda 18,24,37,38 belirtildiği gibi, tipik bir tip I sağkalım eğrisi çok az erken mortalite ile görülmektedir. onların hayatta kalma süresi standart sapmaları dolayısıyla yüksek istatistiksel güç sonuçlanan kötü senkronize rotifer göre dah…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Biz filme katkılarından dolayı George Jarvis, Martha Bock ve Bette Hecox-Lea, Deniz Biyoloji Laboratuvarı müteşekkiriz.
Materials
| Sodium chloride | Wako | 190-13921 | |
| Magnesium chloride | Wako | 136-03995 | |
| Magnesium sulfate | Wako | 131-00427 | |
| Potassium chloride | Wako | 168-22111 | |
| Calcium chloride | Wako | 035-00455 | |
| Sodium bicarbonate | Wako | 199-05985 | |
| Sodium bromide | Wako | 190-01515 | |
| Membrane filter (0.45 µm pore size) | Millipore | HAWP04700 | |
| Culture plate, 6-well, non-treated | Thomas Scientific | 6902D01 | Flat bottom |
| Culture plate, 48-well, non-treated | Thomas Scientific | 6902D07 | Flat bottom |
| Tetraselmis, Living | Carolina Biological Supply Company | 152610 | |
| PRISM 6 | GraphPad Software | Version 6.0d |
Riferimenti
- Wallace, R. L., Snell, T. W., Ricci, C., Nogrady, T. . Rotifera Vol.1: Biology, ecology and systematics. , (2006).
- Segers, H. . Annotated checklist of the rotifers (Phylum Rotifera), with notes on nomenclature, taxonomy and distribution. , (2007).
- Mark Welch, D. B., Meselson, M. Evidence for the evolution of bdelloid rotifers without sexual reproduction or genetic exchange. Science. 288 (5469), 1211-1215 (2000).
- Suga, K., Mark Welch, D., Tanaka, Y., Sakakura, Y., Hagiwara, A. Analysis of expressed sequence tags of the cyclically parthenogenetic rotifer Brachionus plicatilis. PLoS ONE. 2, e671 (2007).
- Denekamp, N. Y., et al. Discovering genes associated with dormancy in the monogonont rotifer Brachionus plicatilis. BMC Genomics. 10, 108 (2009).
- Lee, J. -. S., et al. Sequence analysis of genomic DNA (680 Mb) by GS-FLX-Titanium sequencer in the monogonont rotifer, Brachionus ibericus. Hydrobiologia. 662 (1), 65-75 (2010).
- Flot, J. -. F., et al. Genomic evidence for ameiotic evolution in the bdelloid rotifer Adineta vaga. Nature. 500 (7463), 453-457 (2013).
- Dunn, C. W., et al. Broad phylogenomic sampling improves resolution of the animal tree of life. Nature. 452 (7188), 745-749 (2008).
- Yoshinaga, T., Kaneko, G., Kinoshita, S., Tsukamoto, K., Watabe, S. The molecular mechanisms of life history alterations in a rotifer: a novel approach in population dynamics. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 136 (4), 715-722 (2003).
- Dahms, H. -. U., Hagiwara, A., Lee, J. -. S. Ecotoxicology, ecophysiology, and mechanistic studies with rotifers. Aquat. Toxicol. 101 (1), 1-12 (2011).
- Snell, T. W. Rotifers as models for the biology of aging. Int. Rev. Hydrobiol. 99 (1-2), 84-95 (2014).
- Snell, T. W., Johnston, R. K., Gribble, K. E., Mark Welch, D. B. Rotifers as experimental tools for investigating aging. Invertebr. Reprod. Dev. 59, 5-10 (2015).
- Kaneko, G., et al. Molecular characterization of Mn-superoxide dismutase and gene expression studies in dietary restricted Brachionus plicatilis rotifers. Hydrobiologia. 546, 117-123 (2005).
- Yoshinaga, T., et al. Insulin-like growth factor signaling pathway involved in regulating longevity of rotifers. Hydrobiologia. 546, 347-352 (2005).
- Ozaki, Y., Kaneko, G., Yanagawa, Y., Watabe, S. Calorie restriction in the rotifer Brachionus plicatilis enhances hypoxia tolerance in association with the increased mRNA levels of glycolytic enzymes. Hydrobiologia. 649 (1), 267-277 (2010).
- Kailasam, M., et al. Effects of calorie restriction on the expression of manganese superoxide dismutase and catalase under oxidative stress conditions in the rotifer Brachionus plicatilis. Fish. Sci. 77 (3), 403-409 (2011).
- Garcìa-Garcìa, G., Sarma, S., Núñez-Orti, A. R., Nandini, S. Effects of the mixture of two endocrine disruptors (ethinylestradiol and levonorgestrel) on selected ecological endpoints of Anuraeopsis fissa and Brachionus calyciflorus (Rotifera). Int. Rev. Hydrobiol. 99 (1-2), 166-172 (2014).
- Yang, J., Mu, Y., Dong, S., Jiang, Q., Yang, J. Changes in the expression of four heat shock proteins during the aging process in Brachionus calyciflorus (rotifera). Cell Stress Chaperones. 19 (1), 33-52 (2014).
- Han, J., et al. Sublethal gamma irradiation affects reproductive impairment and elevates antioxidant enzyme and DNA repair activities in the monogonont rotifer Brachionus koreanus. Aquat. Toxicol. 155, 101-109 (2014).
- Gribble, K. E., Jarvis, G., Bock, M., Mark Welch, D. B. Maternal caloric restriction partially rescues the deleterious effects of advanced maternal age on offspring. Aging Cell. 13 (4), 623-630 (2014).
- Yoshinaga, T., Hagiwara, A., Tsukamoto, K. Effect of periodical starvation on the survival of offspring in the rotifer Brachionus plicatilis. Fish. Sci. 67 (2), 373-374 (2001).
- Kaneko, G., et al. Calorie restriction-induced maternal longevity is transmitted to their daughters in a rotifer. Funct. Ecol. 25 (1), 209-216 (2011).
- Lansing, A. I. A transmissible, cumulative, and reversible factor in aging. J. Gerontol. 2 (3), 228-239 (1947).
- Yoshinaga, T., Hagiwara, A., Tsukamoto, K. Effect of periodical starvation on the life history of Brachionus plicatilis O. F. Müller (Rotifera): a possible strategy for population stability. J. Exp. Mar. Biol. Ecol. 253 (2), 253-260 (2000).
- Gribble, K. E., Kaido, O., Jarvis, G., Mark Welch, D. B. Patterns of intraspecific variability in the response to caloric restriction. Exp. Gerontol. 51, 28-37 (2014).
- Snell, T. W., Johnston, R. K. Glycerol extends lifespan of Brachionus manjavacas (Rotifera) and protects against stressors. Exp. Gerontol. 57, 47-56 (2014).
- Kim, H. -. J., Hagiwara, A. Effect of female aging on the morphology and hatchability of resting eggs in the rotifer Brachionus plicatilis Müller. Hydrobiologia. 662 (1), 107-111 (2011).
- Kim, H. -. J., et al. Light-dependent transcriptional events during resting egg hatching of the rotifer Brachionus manjavacas. Mar. Genomics. 20, 25-31 (2015).
- Gribble, K. E., Welch, D. B. M. Life-span extension by caloric restriction is determined by type and level of food reduction and by reproductive mode in Brachionus manjavacas (Rotifera). J. Gerontol. A Biol. Sci. Med. Sci. 68 (4), 349-358 (2013).
- Kaneko, G., Kinoshita, S., Yoshinaga, T., Tsukamoto, K., Watabe, S. Changes in expression patterns of stress protein genes during population growth of the rotifer Brachionus plicatilis. Fish. Sci. 68 (6), 1317-1323 (2002).
- Kim, H. J., Sawada, C., Hagiwara, A. Behavior and reproduction of the rotifer Brachionus plicatilis species complex under different light wavelengths and intensities. Int. Rev. Hydrobiol. 99 (1-2), 151-156 (2014).
- Yoshinaga, T., Hagiwara, A., Tsukamoto, K. Effect of conditioned media on the asexual reproduction of the monogonont rotifer Brachionus plicatilis O. F. Müller. Hydrobiologia. 412, 103-110 (1999).
- Ohmori, F., Kaneko, G., Saito, T., Watabe, S. A novel growth-promoting protein in the conditioned media from the rotifer Brachionus plicatilis at an early exponential growth phase. Hydrobiologia. 667 (1), 101-117 (2011).
- Collet, D. . Modelling Survival Data in Medical Research. , 151-193 (1993).
- Bouliotis, G., Billingham, L. Crossing survival curves: alternatives to the log-rank test. Trials. 12, A137 (2011).
- Yang, J., et al. Changes in expression of manganese superoxide dismutase, copper and zinc superoxide dismutase and catalase in Brachionus calyciflorus during the aging process. PloS ONE. 8 (2), e57186 (2013).
- Snell, T. W., Johnston, R. K., Rabeneck, B., Zipperer, C., Teat, S. Joint inhibition of TOR and JNK pathways interacts to extend the lifespan of Brachionus manjavacas (Rotifera). Exp. Gerontol. 52, 55-69 (2014).
- Klass, M. R. Aging in nematode Caenorhabditis-elegans – major biological and environmental-factors influencing life-span. Mech. Ageing Dev. 6 (6), 413-429 (1977).
- Priest, N. K., Mackowiak, B., Promislow, D. E. L. The role of parental age effects on the evolution of aging. Evolution. 56 (5), 927-935 (2002).
