Using Proales similis (Rotifera) for toxicity assessment in marine waters.

There is a need to develop more animal species for assessing toxicity in marine environments. Cyst-based toxicity tests using invertebrates are especially fast, technically simple, cost-effective, and sensitive to a variety of toxicants. Over the past 30 years, a variety of toxicity endpoints have been measured using the marine rotifer Brachionus plicatilis hatched from cysts, including mortality, reproduction, ingestion, swimming, enzyme activity, and gene expression. A consensus has developed that the most ecologically relevant toxicity measurements should be made using more than one species. Furthermore, it has been noted that the rotifer species toxicant sensitivity distribution is much broader than which endpoint is measured. This implies that toxicity should be measured with the simplest, fastest, least expensive test available on as many species as feasible. If a battery of test species is to be used to estimate toxicity, diapause egg-based toxicity tests that do not require culturing of test animals will be key. In this paper, we describe how diapause eggs of a new marine rotifer, Proales similis, can be produced, stored and hatched under controlled conditions to produce animals for toxicity tests. Methods are described for quantifying the toxicity of copper, mercury and cadmium based on mortality, ingestion, reproduction, and diapause egg hatching endpoints. We found that reproduction and ingestion endpoints were generally more sensitive to the metals than mortality or diapause egg hatching. When the copper sensitivity of P. similis was compared to Brachionus manjavacas and B. plicatilis using an ingestion test, similar EC50s were observed. In contrast, the B. rotundiformis ingestion EC50 for copper was about 4× more sensitive. Although diapause egg hatching was not the most sensitive endpoint, it is the most ecologically relevant for assessing sediment toxicity. Our discovery of diapausing eggs in the P. similis life cycle has created a conundrum. We have not observed males or sex in P. similis populations, which is a direct contradiction to the orthodox view of the monogonont rotifer life cycle. Work is needed to clarify how diapause egg production is accomplished by P. similis and whether sexual reproduction is involved.

Repurposed FDA-approved drugs targeting genes influencing aging can extend lifespan and healthspan in rotifers.

Pharmaceutical interventions can slow aging in animals, and have advantages because their dose can be tightly regulated and the timing of the intervention can be closely controlled. They also may complement environmental interventions like caloric restriction by acting additively. A fertile source for therapies slowing aging is FDA approved drugs whose safety has been investigated. Because drugs bind to several protein targets, they cause multiple effects, many of which have not been characterized. It is possible that some of the side effects of drugs prescribed for one therapy may have benefits in retarding aging. We used computationally guided drug screening for prioritizing drug targets to produce a short list of candidate compounds for in vivo testing. We applied the virtual ligand screening approach FINDSITEcomb for screening potential anti-aging protein targets against FDA approved drugs listed in DrugBank. A short list of 31 promising compounds was screened using a multi-tiered approach with rotifers as an animal model of aging. Primary and secondary survival screens and cohort life table experiments identified four drugs capable of extending rotifer lifespan by 8-42%. Exposures to 1 µM erythromycin, 5 µM carglumic acid, 3 µM capecitabine, and 1 µM ivermectin, extended rotifer lifespan without significant effect on reproduction. Some drugs also extended healthspan, as estimated by mitochondria activity and mobility (swimming speed). Our most promising result is that rotifer lifespan was extended by 7-8.9% even when treatment was started in middle age.

Freshwater toxicity testing using rehydrated Philodina sp. (Rotifera) as test animals.

Rotifers have become widely used in aquatic toxicology as a rapid screening test for toxicity. The commercial availability of diapausing embryos (cysts) have facilitated their popularity because test animals can be obtained without having to master the details of culturing. Other rotifer species have life stages capable of surviving desiccation and also could be used in non-culture systems for toxicity assessment. In this article, we describe a system for toxicity testing in freshwater based on rehydrating desiccated bdelloid rotifers in the genus Philodina. These animals can remain in this anhydrobiotic state for more than one year and then rehydrate within hours to provide animals for toxicity tests. We describe three endpoints: a 1.5 h ingestion test, a 24 h mortality test, and a five day reproductive test. The latter test requires feeding and a method using a dried commercial product is explained. Using desiccated rotifers and dried food in toxicity tests make this system especially attractive because of its flexibility and low threshold of biological expertise required to execute the tests. The use of the Philodina toxicity test is illustrated with four metals: copper, lead, mercury and cadmium. Reproduction generally was the most sensitive endpoint, with EC50s of 0.33, 0.44, 0.60, and 0.12 mg/L, respectively. Ingestion was a close second with EC50s of 0.13, 1.64, 0.64, and 6.26 mg/L, respectively.

Repurposing FDA-approved drugs for anti-aging therapies.

There is great interest in drugs that are capable of modulating multiple aging pathways, thereby delaying the onset and progression of aging. Effective strategies for drug development include the repurposing of existing drugs already approved by the FDA for human therapy. FDA approved drugs have known mechanisms of action and have been thoroughly screened for safety. Although there has been extensive scientific activity in repurposing drugs for disease therapy, there has been little testing of these drugs for their effects on aging. The pool of FDA approved drugs therefore represents a large reservoir of drug candidates with substantial potential for anti-aging therapy. In this paper we employ FINDSITEcomb, a powerful ligand homology modeling program, to identify binding partners for proteins produced by temperature sensing genes that have been implicated in aging. This list of drugs with potential to modulate aging rates was then tested experimentally for lifespan and healthspan extension using a small invertebrate model. Three protein targets of the rotifer Brachionus manjavacas corresponding to products of the transient receptor potential gene 7, ribosomal protein S6 polypeptide 2 gene, or forkhead box C gene, were screened against a compound library consisting of DrugBank drugs including 1347 FDA approved, non-nutraceutical molecules. Twenty nine drugs ranked in the top 1 % for binding to each target were subsequently included in our experimental analysis. Continuous exposure of rotifers to 1 µM naproxen significantly extended rotifer mean lifespan by 14 %. We used three endpoints to estimate rotifer health: swimming speed (mobility proxy), reproduction (overall vitality), and mitochondria activity (cellular senescence proxy). The natural decline in swimming speed with aging was more gradual when rotifers were exposed to three drugs, so that on day 6, mean swimming speed of females was 1.19 mm/s for naproxen (P = 0.038), 1.20 for fludarabine (P = 0.040), 1.35 for hydralazine (P = 0.038), as compared to 0.88 mm/s in the control. The average reproduction of control females in the second half of their reproductive lifespan was 1.08 per day. In contrast, females treated with 1 µM naproxen produced 1.4 offspring per day (P = 0.027) and females treated with 10 µM fludarabine or 1 µM hydralazine produced 1.72 (P = <0.001) and 1.66 (P = 0.001) offspring per day, respectively. Mitochondrial activity naturally declines with rotifer aging, but B. manjavacas treated with 1 µM hydralazine or 10 µM fludarabine retained 49 % (P = 0.038) and 89 % (P = 0.002) greater mitochondria activity, respectively, than untreated controls. Our results demonstrate that coupling computation to experimentation can quickly identify new drug candidates with anti-aging potential. Screening drugs for anti-aging effects using a rotifer bioassay is a powerful first step in identifying compounds worthy of follow-up in vertebrate models. Even if lifespan extension is not observed, certain drugs could improve healthspan, slowing age-dependent losses in mobility and vitality.

Rotifers as experimental tools for investigating aging.

Comparative biogerontology has much to contribute to the study of aging. A broad range of aging rates has evolved to meet environmental challenges, and understanding these adaptations can produce valuable insights into aging. The supra Phylum Lophotrochozoa is particularly understudied and has several groups that have intriguing patterns of aging. Members of the lophotrochozoan phylum Rotifera are particularly useful for aging studies because cohort life tables can be conducted with them easily, and biochemical and genomic tools are available for examining aging mechanisms. This paper reviews a variety of caloric restriction regimens, small molecule inhibitors, and dietary supplements that extend rotifer lifespan, as well as important interactions between caloric restriction and genotype, antioxidant supplements, and TOR and JNK pathways, and the use of RNAi to identify key genes involved in modulating the aging response. Examples of how rapamycin and JNK inhibitor exposure keeps mortality rates low during the reproductive phase of the life cycle are presented, and the ease of conducting life table experiments to screen natural products from red algae for life extending effects is illustrated. Finally, experimental evolution to produce longer-lived rotifer individuals is demonstrated, and future directions to determine the genetic basis of aging are discussed.

Antioxidants can extend lifespan of Brachionus manjavacas (Rotifera), but only in a few combinations.

Animal cells are protected from oxidative damage by an antioxidant network operating as a coordinated system, with strong synergistic interactions. Lifespan studies with whole animals are expensive and laborious, so there has been little investigation of which antioxidant interactions might be useful for life extension. Animals in the phylum Rotifera are particularly promising models for aging studies because they are small (0.1-1 mm), have short, two-week lifespan, display typical patterns of animal aging, and have well characterized, easy to measure phenotypes of aging and senescence. One class of interventions that has consistently produced significant rotifer life extension is antioxidants. Although the mechanism of antioxidant effects on animal aging remains controversial, the ability of some antioxidant supplements to extend rotifer lifespan was unequivocal. We found that exposing rotifers to certain combinations of antioxidant supplements can produce up to about 20% longer lifespan, but that most antioxidants have no effect. We performed life table tests with 20 single antioxidants and none yielded significant rotifer life extension. We tested 60 two-way combinations of selected antioxidants and only seven (12%) produced significant rotifer life extension. None of the 20 three- and four-way antioxidant combinations tested yielded significant rotifer life extension. These observations suggest that dietary exposure of antioxidants can extend rotifer lifespan, but most antioxidants do not. We observed significant rotifer life extension only when antioxidants were paired with trolox, N-acetyl cysteine, L: -carnosine, or EUK-8. This illustrates that antioxidant treatments capable of rotifer life extension are patchily distributed in the parameter space, so large regions must be searched to find them. It furthermore underscores the value of the rotifer model to conduct rapid, facile life table experiments with many treatments, which makes such a search feasible. Although some antioxidants extended rotifer lifespan, they likely did so by another mechanism than direct antioxidation.

Moderately lower temperatures greatly extend the lifespan of Brachionus manjavacas (Rotifera): Thermodynamics or gene regulation?

Environmental temperature greatly affects lifespan in a wide variety of animals, but the exact mechanisms underlying this effect are still largely unknown. A moderate temperature decrease from 22°C to 16°C extends the lifespan of the monogonont rotifer Brachionus manjavacas by up to 163%. Thermodynamic effects on metabolism contribute to this increase in longevity, but are not the only cause. When rotifers are exposed to 16°C for four days and then transfered to 22°C, they survive until day 13 at nearly identical rates as rotifers maintained at 16°C continuously. This persistence of the higher survival for nine days after transfer to 22°C suggests that low temperature exposure alters the expression of genes that affect the rate of aging. The relative persistence of the gene regulation effect suggests that it may play an even larger role in slowing aging than the thermodynamic effects. The life extending effects of these short-term low temperature treatments are largest when the exposure happens early in the life cycle, demonstrating the importance of early development. There is no advantage to lowering the temperature below 16°C to 11° or 5°C. Rotifers exposed to 16°C also displayed increased resistance to heat, starvation, oxidative and osmotic stress. Reproductive rates at 16°C were lower than those at 22°C, but because they reproduce longer, there is no significant change in the lifetime fecundity of females. To investigate which genes contribute to these effects, the expression of specific temperature sensing genes was knocked down using RNAi. Of 12 genes tested, RNAi knockdown of four eliminated the survival enhancing effects of the four-day cold treatment: TRP7, forkhead box C, Y-box factor, and ribosomal protein S6. This demonstrates that active gene regulation is an important factor in temperature mediated life extension, and that these particular genes play an integral role in these pathways. As a thermoresponsive sensor, TRP7 may be responsible for triggering the signaling cascade contributing to temperature mediated life extension. The TRP genes may also provide especially promising candidates for targeted gene manipulations or pharmacological interventions capable of mimicking the effects of low temperature exposure. These results support recent theories of aging that claim rate of aging is determined by an actively regulated genetic mechanism rather than an accumulation of molecular damage.

Glycerol extends lifespan of Brachionus manjavacas (Rotifera) and protects against stressors.

Diet has profound effects on animal longevity and manipulation of nutrient sensing pathways is one of the primary interventions capable of lifespan extension. This often is done through caloric restriction (CR) and a variety of CR mimics have been identified that produce life extending effects without adhering to the rigorous CR dietary regimen. Glycerol is a dietary supplement capable mimicking CR by shifting metabolism away from glycolysis and towards oxidative phosphorylation. Glycerol supplementation has a number of beneficial effects, including lifespan extension, improved stress resistance, and enhanced locomotory and mitochondria activity in older age classes. Using rotifers as a model, we show that supplements of 150-300mM glycerol produced 40-50% extension of mean lifespan. This effect was produced by raising glycerol concentration only three times higher than its baseline concentration in rotifer tissues. Glycerol supplementation decreased rotifer reliance on glycolysis and reduced the pro-aging effects of glucose. Glycerol also acted as a chemical chaperone, mitigating damage by protein aggregation. Glycerol treatment improved rotifer swimming performance in older age classes and maintained more mitochondrial activity. Glycerol treatment provided increased resistance to starvation, heat, oxidation, and osmotic stress, but not UV stress. When glycerol was co-administered with the hexokinase inhibitor 2-deoxyglucose, the lifespan extending effect of glycerol was enhanced. Co-administration of glycerol with inhibitors like 2-deoxyglucose can lower their efficacious doses, thereby reducing their toxic side effects.

Joint inhibition of TOR and JNK pathways interacts to extend the lifespan of Brachionus manjavacas (Rotifera).

The TOR kinase pathway is central in modulating aging in a variety of animal models. The target of rapamycin (TOR) integrates a complex network of signals from growth conditions, nutrient availability, energy status, and physiological stresses and matches an organism's growth rate to the resource environment. Important remaining problems are the identification of the pathways that interact with TOR and their characterization as additive or synergistic. One of the most versatile stress sensors in metazoans is the Jun-N-terminal kinase (JNK) signaling pathway. JNK is an evolutionarily conserved stress-activated protein kinase that is induced by a range of stressors, including UV irradiation, reactive oxygen species, DNA damage, heat, and bacterial antigens. JNK is thought to interact with the TOR pathway, but its effects on TOR are poorly understood. We used the rotifer Brachionus manjavacas as a model animal to probe the regulation of TOR and JNK pathways and explore their interaction. The effect of various chemical inhibitors was examined in life table and stressor challenge experiments. A survey of 12 inhibitors revealed two, rapamycin and JNK inhibitor, that significantly extended lifespan of B. manjavacas. At 1 µM concentration, exposure to rapamycin or JNK inhibitor extended mean rotifer lifespan by 35% and maximum lifespan by 37%. Exposure to both rapamycin and JNK inhibitor simultaneously extended mean rotifer lifespan by 65% more than either alone. Exposure to a combination of rapamycin and JNK inhibitors conveyed greater protection to starvation, UV and osmotic stress than either inhibitor alone. RNAi knockdown of TOR and JNK gene expression was investigated for its ability to extend rotifer lifespan. RNAi knockdown of the TOR gene resulted in 29% extension of the mean lifespan compared to control and knockdown of the JNK gene resulted in 51% mean lifespan extension. In addition to the lifespan, we quantified mitochondria activity using the fluorescent marker MitoTracker and lysosome activity using LysoTracker. Treatment of rotifers with JNK inhibitor enhanced mitochondria activity nearly 3-fold, whereas rapamycin treatment had no significant effect. Treatment of rotifers with rapamycin or JNK inhibitor reduced lysosome activity in 1, 3 and 8 day old animals, but treatment with both inhibitors did not produce any additive effect. We conclude that inhibition of TOR and JNK pathways significantly extends the lifespan of B. manjavacas. These pathways interact so that inhibition of both simultaneously acts additively to extend rotifer lifespan more than the inhibition of either alone.