Techniques for Rapidly Sampling Six Crucial Organs in Adult Xenopus.

Xenopus has been a powerful model organism for understanding vertebrate development and disease for over a hundred years. While experimental analysis and dissection techniques of the embryo have been well documented, descriptions of adult Xenopus structures and organs, together with techniques for working with adults, have not been updated to take into consideration the requirements of such modern approaches as quantitative proteomics and single-cell transcriptomics. The cell-type and gene-centric perspectives require contrasting observations in embryonic stages to those in adult tissues. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location all along the larval to adult transition, most notably during massive metamorphosis remodeling. Establishing robust standards for organ identification and dissection is crucial to ensure datasets resulting from studies performed at different laboratories can be consistent. The present protocol identifies six of the organs in the adult Xenopus, demonstrating methods for dissection and sampling of the heart ventricle, liver, fat body, pancreas, paired kidney, and skin of the adult Xenopus. Depending on the preservation methods, the dissected organs can be used for quantitative proteomics, single cell/nuclei transcriptomics, in situ hybridization, immunohistochemistry, histology, etc. This protocol aims to standardize tissue sampling and facilitate multi-lab investigations of the adult organ systems.

Effective Rapid Blood Perfusion in Xenopus.

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol in Xenopus, aimed at a consistent and drastic reduction of blood within all tissues, is defined. Perfusion is carried out by inserting a needle directly into the ventricle of the heart and pumping heparinized phosphate-buffered saline (PBS) through the vascular system. The procedure can be completed in approximately 10 min per animal. The blood is dominated by a few highly abundant proteins and cell types, creating numerous issues as these proteins mask most other molecules and cell types of interest. The reproducible characterization of adult Xenopus tissues with quantitative proteomics and single-cell transcriptomics will benefit from applying this protocol prior to organ sampling. The protocols for tissue sampling are defined in companion papers. These procedures are aimed at the standardization of practices across Xenopus of different sex, age, and health status, specifically X. laevis and X. tropicalis.

Effective Rapid Blood Perfusion in Xenopus.

Xenopus has been a powerful model organism for understanding vertebrate development and disease for over a hundred years. Here we define a rapid blood perfusion protocol in Xenopus aimed at a consistent and drastic reduction of blood across tissues. Perfusion is done by inserting a needle directly into the ventricle and pumping heparin in PBS through the vascular system. The whole procedure should take about 10 minutes per frog. Blood is dominated by a few highly abundant proteins and cell types which create numerous issues by masking most other molecules and cell types of interest. Reproducible characterization of adult Xenopus tissues with quantitative proteomics and single cell transcriptomics will gain from applying this protocol prior to organ dissections defined in companion papers. The procedure is aimed at standardization of practice across the animals of different gender, age and Xenopus species, specifically X.laevis and X.tropicalis . SUMMARY: An effective rapid blood perfusion protocol to prepare tissue samples for transcriptomics and proteomics studies.

Inverse Lansing Effect: Maternal Age and Provisioning Affecting Daughters' Longevity and Male Offspring Production.

AbstractMaternal age effects on offspring life history are known in a variety of organisms, with offspring of older mothers typically having lower life expectancy (the Lansing effect). However, there is no consensus on the generality and mechanisms of this pattern. We tested predictions of the Lansing effect in several Daphnia magna clones and observed clone-specific magnitude and direction of the maternal age effect on offspring longevity. We also report ambidirectional, genotype-specific effects of maternal age on the propensity of daughters to produce male offspring. Focusing on two clones with contrasting life histories, we demonstrate that maternal age effects can be explained by lipid provisioning of embryos by mothers of different ages. Individuals from a single-generation maternal age reversal treatment showed intermediate life span and intermediate lipid content at birth. In the clone characterized by the "inverse Lansing effect," neonates produced by older mothers showed higher mitochondrial membrane potential in neural tissues than their counterparts born to younger mothers. We conclude that an inverse Lansing effect is possible and hypothesize that it may be caused by age-specific maternal lipid provisioning creating a calorically restricted environment during embryonic development, which in turn reduces fecundity and increases life span in offspring.

Intelligent high-throughput intervention testing platform in Daphnia.

We present a novel platform for testing the effects of interventions on the life- and healthspan of a short-lived freshwater organism with complex behavior and physiology-the planktonic crustacean Daphnia magna. Within this platform, dozens of complex behavioral features of both routine motion and response to stimuli are continuously quantified over large synchronized cohorts via an automated phenotyping pipeline. We build predictive machine-learning models calibrated using chronological age and extrapolate onto phenotypic age. We further apply the model to estimate the phenotypic age under pharmacological perturbation. Our platform provides a scalable framework for drug screening and characterization in both life-long and instant assays as illustrated using a long-term dose-response profile of metformin and a short-term assay of well-studied substances such as caffeine and alcohol.

Lack of age-related respiratory changes in Daphnia.

Aging is a multifaceted process of accumulation of damage and waste in cells and tissues; age-related changes in mitochondria and in respiratory metabolism have the focus of aging research for decades. Studies of aging in nematodes, flies and mammals all revealed age-related decline in respiratory functions, with somewhat controversial causative role. Here we investigated age-related changes in respiration rates, lactate/pyruvate ratio, a commonly used proxy for NADH/NAD+ balance, and mitochondrial membrane potential in 4 genotypes of an emerging model organism for aging research, a cyclic parthenogen Daphnia magna. We show that total body weight-adjusted respiration rate decreased with age, although this decrease was small in magnitude and could be fully accounted for by the decrease in locomotion and feeding activity. Neither total respiration normalized by protein content, nor basal respiration rate measured in anaesthetized animals decreased with age. Lactate/pyruvate ratio and mitochondrial membrane potential (∆Ψmt) showed no age-related changes, with possible exceptions of ∆Ψmt in epipodites (excretory and gas exchange organs) in which ∆Ψmt decreased with age and in the optical lobe of the brain, in which ∆Ψmt showed a maximum at middle age. We conclude that actuarial senescence in Daphnia is not caused by a decline in respiratory metabolism and discuss possible mechanisms of maintaining mitochondrial healthspan throughout the lifespan.