Large Differences in Aging Phenotype between Strains of the Short-Lived Annual Fish Nothobranchius furzeri methods
Aim. Evidence-backed execution summary for Large Differences in Aging Phenotype between Strains of the Short-Lived Annual Fish Nothobranchius furzeri methods from Large Differences in Aging Phenotype between Strains of the Short-Lived Annual Fish Nothobranchius furzeri.
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This experiment, in seven questions
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Shopping and prep list
What do I need before I start?
fish
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Captive lifespan of F1 and F2 generations
reagent used in the protocol.
- Use
- Maximum likelihood estimates (MLE) of the demographic parameters for three F2 isolates and the inbred laboratory stain GRZ were obtained using WinModest software. These are presented in. Differences in lifespan are accounted for by differences in the demographic rate of aging (the b parameter of the model). This pa...
Fish culture
reagent used in the protocol.
- Use
- Eggs were maintained on wet peat moss at room temperature in sealed Petri dishes. When embryos had developed, eggs were hatched by flushing the peat with tap water at 16-18°C. Embryos were scooped up and transferred to a clean vessel. Fry were fed with newly hatched Artemia nauplii for the first 2 weeks a...
Survival assay
reagent used in the protocol.
- Use
- Surviving fish were counted every week starting from the forth week. Mortality in fishes younger than 4 weeks usually occurred in the first days after hatching. Dead fish were not counted because they decay fast in water and may be eaten by their tankmates before they are noticed. To compute differences among treatm...
Survival assay
reagent used in the protocol.
- Use
- Demographic analysis was performed using WinModest software ( http://www.hcoa.org/scott/softw-winmodest.asp ).
Histology and histochemistry
reagent used in the protocol.
- Use
- Fishes were euthanized with MS-222 and cooled on crushed ice for 5 min before dissection. Target tissues were dissected and fixed by immersion in 4% paraformaldehyde/0.1 M phosphate buffer (pH 7.4). Fishes analyzed in Pisa were infiltrated with 30% sucrose to ensure cryoprotection, embedded in Tissuetek (Reichart-Ju...
Open-field-like assay
reagent used in the protocol.
- Use
- Single fish were scored for locomotor activity in a 20-l test-tank at the same temperature as the home tank. Video recordings were made using a digital video camera from above and the water level was kept very low to minimize displacement on the z -axis, which would not be picked up by the camera while recording fro...
Molecular phylogeny
reagent used in the protocol.
- Use
- Amplicons were cloned in a pGEMTeasy vector and sequenced in both directions. Sequences were aligned using the program CLUSTAL W followed by manual inspection and modification. Sequences have been deposited in GenBank under accession numbers EF464684-EF464713. For analysis, the primer sequences were trimmed t...
Captive lifespan of F1 and F2 generations
Maximum likelihood estimates (MLE) of the demographic parameters for three F2 isolates and the inbred laboratory stain GRZ were obtained using WinModest software. These are presented in. Differences in lifespan are accounted for by differences in the demographic rate of aging (the b parameter of the model). This pa...
- Use
- Maximum likelihood estimates (MLE) of the demographic parameters for three F2 isolates and the inbred laboratory stain GRZ were obtained using WinModest software. These are presented in. Differences in lifespan are accounted for by differences in the demographic rate of aging (the b parameter of the model). This pa...
Histology and histochemistry
Fluorescence analysis of images for both lipofuscin and Fluoro-Jade B staining was performed using Metamorph software.
- Use
- Fluorescence analysis of images for both lipofuscin and Fluoro-Jade B staining was performed using Metamorph software.
Methods
A form similar to N. furzeri was discovered in 1999 on an amateur collection trip in the lower Limpopo River drainage system in southern Mozambique. This habitat is located approximately 300 km south of the original collection point in the GRZ National Park. The form is very similar in shape to N. furzeri but has a...
- Use
- A form similar to N. furzeri was discovered in 1999 on an amateur collection trip in the lower Limpopo River drainage system in southern Mozambique. This habitat is located approximately 300 km south of the original collection point in the GRZ National Park. The form is very similar in shape to N. furzeri but has a...
Survival assay
Demographic analysis was performed using WinModest software ( http://www.hcoa.org/scott/softw-winmodest.asp ).
- Use
- Demographic analysis was performed using WinModest software ( http://www.hcoa.org/scott/softw-winmodest.asp ).
Histology and histochemistry
Intracellular accumulation of lipofuscin during aging was detected in brain and liver tissues from young and old fishes as light blue autofluorescent granules under UV excitation. For quantification, images were acquired using a Leica confocal microscope (in Pisa) or a Zeiss LSM (in Jena) at an excitation wavelength...
- Use
- Intracellular accumulation of lipofuscin during aging was detected in brain and liver tissues from young and old fishes as light blue autofluorescent granules under UV excitation. For quantification, images were acquired using a Leica confocal microscope (in Pisa) or a Zeiss LSM (in Jena) at an excitation wavelength...
Open-field-like assay
Single fish were scored for locomotor activity in a 20-l test-tank at the same temperature as the home tank. Video recordings were made using a digital video camera from above and the water level was kept very low to minimize displacement on the z -axis, which would not be picked up by the camera while recording fro...
- Use
- Single fish were scored for locomotor activity in a 20-l test-tank at the same temperature as the home tank. Video recordings were made using a digital video camera from above and the water level was kept very low to minimize displacement on the z -axis, which would not be picked up by the camera while recording fro...
Captive lifespan of F1 and F2 generations
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Maximum likelihood estimates (MLE) of the demographic parameters for three F2 isolates and the inbred laboratory stain GRZ were obtained using WinModest software. These are presented in. Differences in lifespan are accounted for by differences in the demographic rate of aging (the b parameter of the model). This pa...
Survival assay
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Surviving fish were counted every week starting from the forth week. Mortality in fishes younger than 4 weeks usually occurred in the first days after hatching. Dead fish were not counted because they decay fast in water and may be eaten by their tankmates before they are noticed. To compute differences among treatm...
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Methodology/Principal Findings
We characterized captive survival and age-related traits in F1 and F2 offspring of wild-caught N. furzeri. Wild-derived N. furzeri lines showed expression of lipofuscin and neurodegeneration at age 21 weeks. Median lifespan in the laboratory varied from to 20 to 23 weeks and maximum lifespan from 25 to 32 weeks. These data demonstrate that rapid age-dependent decline and short lifespan are natural characteristics of this species. The N. furzeri distribution range overlaps with gradients in altitude and aridity. Fish from more arid habitats are expected to experience a shorter survival window in the wild. We tested whether captive lines stemming from semi-arid and sub-humid habitats differ in longevity and expression of age-related traits. We detected a clear difference in age-dependent cognitive decline and a slight difference in lifespan (16% for median, 15% for maximum lifespan) be...
Captive lifespan of F1 and F2 generations
Analysis of lifespan in the F2 generation focussed on MZM-04/10 and MZM-04/03 ( ) as representatives of northern and southern populations. Inspection of eggs after 2 months of incubation revealed the presence of developed embryos and these were then hatched. The F2 generation of MZM-04/10 P, MZM-04/10 G and MZM-04/10 T isolates showed similar survivorship characteristics and were pooled. The median lifespan of this pooled group was 20 weeks, with 10% survivorship at 23 weeks. The median lifespan of the MZM-04/03 isolate was 23 weeks, with 10% survivorship at 29 weeks. These values were within the ranges recorded for the F1 generation of the same populations. Differences in longevity between F2 MZM-04/03 and pooled MZM-04/10 are statistically significant (log rank test, P <0.05).
Captive lifespan of F1 and F2 generations
Unexpectedly, a strong effect of incubation time on post-hatch lifespan was detected in the F2 generation of at least one isolate. Inspection of F2 eggs of the MZM-04/10 P isolate revealed the presence of many embryos still vital after 12 months of incubation. These were hatched in four different "cohorts" and they all showed an extremely short lifespan similar to the GRZ laboratory strain. The pooled data are reported in. This isolate was named MZM-04/10 Plate. By contrast, attempts to hatch F2 eggs of the MZM-04/03 isolate after 12-month incubation resulted in only seven vital embryos. These developed into longer-lived individuals than the late-hatched MZM-04/10 Plate (age at death 10-15 weeks). At this stage, the study was interrupted because the junior authors (A.C., E.T., D.V.) had to relocate for lack of funding and the fish colony was moved from SNS Pisa to...
Captive lifespan of F1 and F2 generations
Maximum likelihood estimates (MLE) of the demographic parameters for three F2 isolates and the inbred laboratory stain GRZ were obtained using WinModest software. These are presented in. Differences in lifespan are accounted for by differences in the demographic rate of aging (the b parameter of the model). This parameter is almost six-fold higher for the GRZ strain compared to the MZM-04/03 isolate. MLE of baseline mortality (the a parameter of the model) showed large confidence intervals, and differences among isolates were not statistically significant.
Age-related markers: histology
We repeated the analysis of brain Fluoro-Jade B and liver lipofuscin in FLI in Jena after relocation on GRZ and F6 generation of MZM-04/03 using specimens bred in Jena. In addition, we analyzed expression of brain lipofuscin. Three brain areas were analyzed separately: telencephalon, optic tectum and hindbrain. Expression of liver lipofuscin was higher in the GRZ strain at 11 weeks compared to age-matched MZM-04/03 fishes, but was significantly lower than in 21-week-old MZM-04/03 ( ). Lipofuscin accumulation was also accelerated in the brain of GRZ strain ( ), a pattern paralleled by faster expression of Fluoro-Jade B positivity ( ). This acceleration affected the three brain areas equally. Quantification of both age markers in the brain revealed that 11-week-old GRZ individuals exhibited a level of expression comparable to that in 21-week-old MZM-04/03 individuals ( ).
Age-related markers: histology
Data for the telencephalon, optic tectum and hindbrain are presented separately. Error bars represent the standard error of the mean. Significance is reported only for comparison between GRZ 11 weeks and MZM-04/03 11 weeks. Student's t -test, * P <0.05, ** P <0.01.
Age-related markers: behavior
Aging in the inbred GRZ strain of N. furzeri is associated with reduced open-field exploration. Open-field exploration was quantified at 5 weeks and 9 weeks of age in GRZ and in F2 progeny of MZM-04/10 G and MZM-04/3 strains. Less open-field exploration was observed only in the short-lived GRZ strain. On the other hand, the MZM-04/03 line showed an increase in exploratory activity in this time window. ( )
Age-related markers: behavior
Age-dependent impairment of learning and memory is a hallmark of aging in complex organisms and is observed in many model systems, including Drosophila. Age-dependent cognitive decline was also recently described in zebrafish. A form of age-dependent learning decline is observed in the GRZ strain between 5 and 9 weeks of age and can be detected using a protocol of active avoidance in a modified version of the shuttle box test. We quantified age-dependent learning impairment in F2 progeny of MZM-04/10 Plate, MZM-04/10 G, MZM-04/3 and MZM-04/02 isolates. MZM-04/10 Plate and MZM-04/10 G showed a marked decrease in learning performance between 5 and 9 weeks of age that is reminiscent of the decline described in the GRZ strain. The MZM-04/02 and MZM-04/03 lines showed lower performance at 5 weeks of age compared to MZM-04/10 Plate and MZM-04/10 G. However, their learning performance d...
Measurement outputs
What raw and processed outputs should exist?
We characterized captive survival and age-related traits in F1 and F2 offspring of wild-caught N. furzeri. Wild-derived N. furzeri lines showed expression of lipofuscin and neu...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The analysis revealed that N. furzeri forms a well-supported clade that also contains its sympatric species N. orthonotus and the parapatric species N. kunthae. This clade is s...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The survival of the F1 captive generation was recorded for MZM-04/03, MZM-04/06 and MZM-04/10 P. The number of individuals followed over their lifespan is limited because F1 in...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis of lifespan in the F2 generation focussed on MZM-04/10 and MZM-04/03 ( ) as representatives of northern and southern populations. Inspection of eggs after 2 months of i...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Populations of N.
from paperScoring or quantification
Quantify the primary readouts for this experiment: We characterized captive survival and age-related traits in F1 and F2 offspring of wild-caught N. furzeri. Wild-derived N. furzeri lines showed expression of lipofuscin and neu...; The analysis revealed that N. furzeri forms a well-supported clade that also contains its sympatric species N. orthonotus and the parapatric species N. kunthae. This clade is s...; The survival of the F1 captive generation was recorded for MZM-04/03, MZM-04/06 and MZM-04/10 P. The number of individuals followed over their lifespan is limited because F1 in...; Analysis of lifespan in the F2 generation focussed on MZM-04/10 and MZM-04/03 ( ) as representatives of northern and southern populations. Inspection of eggs after 2 months of i....
from paperStatistical comparison
Populations of N. furzeri showed a significant level of genetic differentiation depending on their geographic origin. Three samples collected in the lower Limpopo basin clustere...; A partial sequence of the mitochondrial locus cox1 was used to define the relationship between N. furzeri and its sympatric/parapatric species, as well as to test for significan...; Analysis of lifespan in the F2 generation focussed on MZM-04/10 and MZM-04/03 ( ) as representatives of northern and southern populations. Inspection of eggs after 2 months of i...; Maximum likelihood estimates (MLE) of the demographic parameters for three F2 isolates and the inbred laboratory stain GRZ were obtained using WinModest software. These are pres...
from paperReporting output
Report representative outputs alongside summary comparisons for We characterized captive survival and age-related traits in F1 and F2 offspring of wild-caught N. furzeri. Wild-derived N. furzeri lines showed expression of lipofuscin and neu..., The analysis revealed that N. furzeri forms a well-supported clade that also contains its sympatric species N. orthonotus and the parapatric species N. kunthae. This clade is s..., The survival of the F1 captive generation was recorded for MZM-04/03, MZM-04/06 and MZM-04/10 P. The number of individuals followed over their lifespan is limited because F1 in..., Analysis of lifespan in the F2 generation focussed on MZM-04/10 and MZM-04/03 ( ) as representatives of northern and southern populations. Inspection of eggs after 2 months of i....
inferred from protocolStructured statistical methods
Populations of N. furzeri showed a significant level of genetic differentiation depending on their geographic origin. Three samples collected in the lower Limpopo basin clustere...; A partial sequence of the mitochondrial locus cox1 was used to define the relationship between N. furzeri and its sympatric/parapatric species, as well as to test for significan...; Analysis of lifespan in the F2 generation focussed on MZM-04/10 and MZM-04/03 ( ) as representatives of northern and southern populations. Inspection of eggs after 2 months of i...; Maximum likelihood estimates (MLE) of the demographic parameters for three F2 isolates and the inbred laboratory stain GRZ were obtained using WinModest software. These are pres...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
We characterized captive survival and age-related traits in F1 and F2 offspring of wild-caught N. furzeri. Wild-derived N. furzeri lines showed expression of lipofuscin and neurodegeneration at age 21 weeks. Median lifespan in the laboratory varied from to 20 to 23 weeks and maximum lifespan from 25 to 32 weeks. These data demonstrate that rapid age-dependent decline and short lifespan are natural characteristics of this species. The N. furzeri distribution range overlaps with gradients in altitude and aridity. Fish from more arid habitats are expected to experience a shorter survival window in the wild. We tested whether captive lines stemming from semi-arid and sub-humid habitats differ in longevity and expression of age-related traits. We detected a clear difference in age-dependent cognitive decline and a slight difference in lifespan (16% for median, 15% for maximum lifespan) between these lines. Finally, we observed shorter lifespan and accelerated expression of age-related markers in the inbred laboratory strain compared to these wild-derived lines.
Analysis of lifespan in the F2 generation focussed on MZM-04/10 and MZM-04/03 ( ) as representatives of northern and southern populations. Inspection of eggs after 2 months of incubation revealed the presence of developed embryos and these were then hatched. The F2 generation of MZM-04/10 P, MZM-04/10 G and MZM-04/10 T isolates showed similar survivorship characteristics and were pooled. The median lifespan of this pooled group was 20 weeks, with 10% survivorship at 23 weeks. The median lifespan of the MZM-04/03 isolate was 23 weeks, with 10% survivorship at 29 weeks. These values were within the ranges recorded for the F1 generation of the same populations. Differences in longevity between F2 MZM-04/03 and pooled MZM-04/10 are statistically significant (log rank test, P <0.05).
Unexpectedly, a strong effect of incubation time on post-hatch lifespan was detected in the F2 generation of at least one isolate. Inspection of F2 eggs of the MZM-04/10 P isolate revealed the presence of many embryos still vital after 12 months of incubation. These were hatched in four different "cohorts" and they all showed an extremely short lifespan similar to the GRZ laboratory strain. The pooled data are reported in. This isolate was named MZM-04/10 Plate. By contrast, attempts to hatch F2 eggs of the MZM-04/03 isolate after 12-month incubation resulted in only seven vital embryos. These developed into longer-lived individuals than the late-hatched MZM-04/10 Plate (age at death 10-15 weeks). At this stage, the study was interrupted because the junior authors (A.C., E.T., D.V.) had to relocate for lack of funding and the fish colony was moved from SNS Pisa to FLI in Jena. Analysis of subsequent generations performed in Jena revealed that the extremely short-lived phenotype observed in the F2 generation of MZM-04/10 Plate is not genetically fixed (Supplementary ).
Maximum likelihood estimates (MLE) of the demographic parameters for three F2 isolates and the inbred laboratory stain GRZ were obtained using WinModest software. These are presented in. Differences in lifespan are accounted for by differences in the demographic rate of aging (the b parameter of the model). This parameter is almost six-fold higher for the GRZ strain compared to the MZM-04/03 isolate. MLE of baseline mortality (the a parameter of the model) showed large confidence intervals, and differences among isolates were not statistically significant.
We repeated the analysis of brain Fluoro-Jade B and liver lipofuscin in FLI in Jena after relocation on GRZ and F6 generation of MZM-04/03 using specimens bred in Jena. In addition, we analyzed expression of brain lipofuscin. Three brain areas were analyzed separately: telencephalon, optic tectum and hindbrain. Expression of liver lipofuscin was higher in the GRZ strain at 11 weeks compared to age-matched MZM-04/03 fishes, but was significantly lower than in 21-week-old MZM-04/03 ( ). Lipofuscin accumulation was also accelerated in the brain of GRZ strain ( ), a pattern paralleled by faster expression of Fluoro-Jade B positivity ( ). This acceleration affected the three brain areas equally. Quantification of both age markers in the brain revealed that 11-week-old GRZ individuals exhibited a level of expression comparable to that in 21-week-old MZM-04/03 individuals ( ).
Data for the telencephalon, optic tectum and hindbrain are presented separately. Error bars represent the standard error of the mean. Significance is reported only for comparison between GRZ 11 weeks and MZM-04/03 11 weeks. Student's t -test, * P <0.05, ** P <0.01.
Aging in the inbred GRZ strain of N. furzeri is associated with reduced open-field exploration. Open-field exploration was quantified at 5 weeks and 9 weeks of age in GRZ and in F2 progeny of MZM-04/10 G and MZM-04/3 strains. Less open-field exploration was observed only in the short-lived GRZ strain. On the other hand, the MZM-04/03 line showed an increase in exploratory activity in this time window. ( )
Age-dependent impairment of learning and memory is a hallmark of aging in complex organisms and is observed in many model systems, including Drosophila. Age-dependent cognitive decline was also recently described in zebrafish. A form of age-dependent learning decline is observed in the GRZ strain between 5 and 9 weeks of age and can be detected using a protocol of active avoidance in a modified version of the shuttle box test. We quantified age-dependent learning impairment in F2 progeny of MZM-04/10 Plate, MZM-04/10 G, MZM-04/3 and MZM-04/02 isolates. MZM-04/10 Plate and MZM-04/10 G showed a marked decrease in learning performance between 5 and 9 weeks of age that is reminiscent of the decline described in the GRZ strain. The MZM-04/02 and MZM-04/03 lines showed lower performance at 5 weeks of age compared to MZM-04/10 Plate and MZM-04/10 G. However, their learning performance did not decrease further at 9 weeks of age ( ). Therefore, the age-dependent decline in conditioning did not correlate with captive longevity, but with the geographic origin of the animals tested.
Machine-readable layer
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"text": "We characterized captive survival and age-related traits in F1 and F2 offspring of wild-caught N. furzeri. Wild-derived N. furzeri lines showed expression of lipofuscin and neurodegeneration at age 21 weeks. Median lifespan in the laboratory varied from to 20 to 23 weeks and maximum lifespan from 25 to 32 weeks. These data demonstrate that rapid age-dependent decline and short lifespan are natural characteristics of this species. The N. furzeri distribution range overlaps with gradients in altitude and aridity. Fish from more arid habitats are expected to experience a shorter survival window in the wild. We tested whether captive lines stemming from semi-arid and sub-humid habitats differ in longevity and expression of age-related traits. We detected a clear difference in age-dependent cognitive decline and a slight difference in lifespan (16% for median, 15% for maximum lifespan) be..."
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"text": "Age-dependent impairment of learning and memory is a hallmark of aging in complex organisms and is observed in many model systems, including Drosophila. Age-dependent cognitive decline was also recently described in zebrafish. A form of age-dependent learning decline is observed in the GRZ strain between 5 and 9 weeks of age and can be detected using a protocol of active avoidance in a modified version of the shuttle box test. We quantified age-dependent learning impairment in F2 progeny of MZM-04/10 Plate, MZM-04/10 G, MZM-04/3 and MZM-04/02 isolates. MZM-04/10 Plate and MZM-04/10 G showed a marked decrease in learning performance between 5 and 9 weeks of age that is reminiscent of the decline described in the GRZ strain. The MZM-04/02 and MZM-04/03 lines showed lower performance at 5 weeks of age compared to MZM-04/10 Plate and MZM-04/10 G. However, their learning performance d..."
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