Alternative Model Organism Research Articles

Temporal cueing enhances neuronal and behavioral discrimination performance in rat whisker system

Since sensory systems operate with a finite quantity of processing resources, an animal would benefit from prioritizing processing of sensory stimuli within a time window that is expected to provide key information. This behavioral manifestation of such prioritization is known as attention. Here, we investigate attention with temporal cueing and its neuronal correlates in the rat primary vibrissal somatosensory (vS1) cortex. Rats were trained in a simple whisker vibration detection task. A vibration was presented at one of two spatial locations (left or right), sometimes after an unknown time interval and sometimes after receiving an auditory cue. The auditory cue provided temporal but not spatial information about the vibration. We found that for all rats ( n = 6), the auditory cue consistently enhanced detection of the vibration stimulus. Neuronal activity in vS1 cortex reflected the observed behavioral enhancement from temporal cueing with single units responded differentially to the whisker vibration stimulus when it was temporally predicted by the auditory cue, exhibiting an enhanced signal-to-noise ratio. Our findings indicate that rats are capable of prioritizing processing within a specified time window and provide evidence that the primary sensory cortex may participate in the temporal allocation of resources. NEW & NOTEWORTHY We demonstrate a novel paradigm of temporal cueing in rats. In a two-alternative whisker detection task, an auditory cue provided information about the timing of the stimulus but not the correct choice. In the presence of cue, detection was faster and more accurate, and neuronal activity from the primary somatosensory cortex revealed enhanced representation of vibrations. These results thus establish the rat as an alternative model organism to primates for studying temporal attention.

Microbiome Alteration in Type 2 Diabetes Mellitus Model of Zebrafish

Understanding the gut microbiota in metabolic disorders, including type 2 diabetes mellitus (T2DM), is now gaining importance due to its potential role in disease risk and progression. We previously established a zebrafish model of T2DM, which shows glucose intolerance with insulin resistance and responds to anti-diabetic drugs. In this study, we analysed the gut microbiota of T2DM zebrafish by deep sequencing the 16S rRNA V3-V4 hypervariable regions, and imputed a functional profile using predictive metagenomic tools. While control and T2DM zebrafish were fed with the same kind of feed, the gut microbiota in T2DM group was less diverse than that of the control. Predictive metagenomics profiling using PICRUSt revealed functional alternation of the KEGG pathways in T2DM zebrafish. Several amino acid metabolism pathways (arginine, proline, and phenylalanine) were downregulated in the T2DM group, similar to what has been previously reported in humans. In summary, we profiled the gut microbiome in T2DM zebrafish, which revealed functional similarities in gut bacterial environments between these zebrafish and T2DM affected humans. T2DM zebrafish can become an alternative model organism to study host-bacterial interactions in human obesity and related diseases.

Divergent mechanisms for regulating growth and development after imaginal disc damage in the tobacco hornworm, Manduca sexta.

Holometabolous insects have been able to radiate to vast ecological niches as adults through the evolution of adult-specific structures such as wings, antennae and eyes. These structures arise from imaginal discs that show regenerative capacity when damaged. During imaginal disc regeneration, development has been shown to be delayed in the fruit fly Drosophila melanogaster, but how conserved the delay-inducing mechanisms are across holometabolous insects has not been assessed. The goal of this research was to develop the hornworm Manduca sexta as an alternative model organism to study such damage-induced mechanisms, with the advantage of a larger hemolymph volume enabling access to the hormonal responses to imaginal disc damage. Upon whole-body X-ray exposure, we noted that the imaginal discs were selectively damaged, as assessed by TUNEL and Acridine Orange stains. Moreover, development was delayed, predominantly at the pupal-to-adult transition, with a concomitant delay in the prepupal ecdysteroid peak. The delays to eclosion were dose dependent, with some ability for repair of damaged tissues. We noted a shift in critical weight, as assessed by the point at which starvation no longer impacted developmental timing, without a change in growth rate, which was uncoupled from juvenile hormone clearance in the body. The developmental profile was different from that of D. melanogaster, which suggests species differences may exist in the mechanisms delaying development.

Hydra as an alternative model organism for toxicity testing: Study using the endocrine disrupting chemical Bisphenol A

Abstract In the recent times use of vertebrate animal models in the toxicity testing of chemical entities has come under criticism and surveillance in view of lack of relevance as surrogates to humans and also ethical considerations. Though in vitro and in silico tools can obviate animal models in toxicity testing, use of some simple organisms, the sentience level of which is not a big issue, as animal model is also considered pertinent. Hydra, a simple freshwater cnidarian is already a model organism for studies in developmental biology, and is also thought to be suitable for environmental toxicity assessments. Our group has conducted studies along this direction and found it appropriate for testing a few bulk as well as nano-materials. In the present study, we have tested Bisphenol A (BPA), an environmental chemical which is also an endocrine disruptor, in Hydra towards furtherance of biotechnological application of this simple organism. We worked out the LC50 of BPA adopting the morphological assay. Effective sub-lethal doses were fixed and tested for toxicity adopting regeneration and feeding assays. Since the anatomy of Hydra is such that all the cells in its two body layers can potentially be continuously exposed to the toxicant present in the water, the present study justifies another of its application viz., environmental risk assessment.

Silkworm, Bombyx mori, as an alternative model organism in toxicological research

Silkworms, Bombyx mori, are a promising model animal in health safety and environmental pollution assessment due to their sensitivity to chemical compounds like pesticides, drugs, and heavy metals, in addition to other features like their low cost and body characteristics and their full genome sequencing. In this review, we summarize the silkworm advantages as a model organism in toxicological research. Graphical abstract ᅟ.

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish.

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

Comparative genomic analysis of the human and nematode Caenorhabditis elegans uncovers potential reproductive genes and disease associations in humans.

Reproduction is an important biological process. However, studies of human reproduction at the molecular level are limited due to the difficulty of performing in vivo studies. Hence, a mechanistic understanding of human reproduction remains still poor. Thus, it is important to use an alternative model organism for mechanistic studies of human reproduction. In this study, we used the nematode Caenorhabditis elegans as a model for studying human reproduction and identified 61 human and 535 worm reproductive genes through a combination of comparative genomic and Gene Ontology (GO) analyses. Interestingly, in terms of sex specificity, the number of male-specific genes was greater than the number of female-specific genes. Gene enrichment analysis identified biologically significant processes such as protein localization to cajal bodies/telomeres/nuclear bodies/chromosomes, helicase activity, pyrimidine biosynthesis, and determination of adult lifespan. Regarding the analysis of human reproductive diseases among the identified genes, 10 and 12 genes were identified in the human- and C. elegans-based analyses, respectively. In addition, RNA interference knockdown of a newly identified F52H2.6/DHCR24 gene increased brood size and ovulation/egg-laying rate in C. elegans. Therefore, gene identification, disease associations, and a proof-of-concept experiment using C. elegans will not only provide insights into mechanistic study of human reproduction, but also demonstrate the utility in studying human reproduction.

Genetic linkage between altered tooth and eye development in lens-ablated Astyanax mexicanus

The phenotype of lens-ablated Mexican tetra (Astyanax mexicanus) compared to wild-type surface fish has been described and includes, among other effects, eye degeneration, changes in tooth number and cranial bone changes. Here, we investigate the spatiotemporal expression patterns of several key genes involved in the development of these structures. Specifically, we show that the expression of pitx2, bmp4 and shh is altered in the eye, oral jaw, nasal pit and forebrain in these lens-ablated fish. Furthermore, for the first time, we show altered pitx2 expression in the cavefish, which also has altered eye and tooth phenotypes. We thus provide evidence for a genetic linkage between the eye and tooth modules in this fish species. Furthermore, the altered pitx2 expression pattern, together with the described morphological features of the lens-ablated fish suggests that Astyanax mexicanus could be considered as an alternative teleost model organism in which to study Axenfeld–Rieger syndrome (ARS), a rare autosomal dominant developmental disorder that is associated with PITX2 and which has both ocular and non-ocular abnormalities.

Changes in Hepatic Expression of Hyaluronan Synthases (HAS2 and HAS3) After Treatment with Steptozotocin in Channel Catfish

Streptozotocin (STZ) is commonly used experimentally to induce hyperglycemia in rodents. As an alternative model organism for investigating human obesity, channel catfish show a genetic predisposition toward an accelerated growth rate resulting in a phenotype similar to obese humans. In our previous studies, STZ treatment resulted in hypoglycemia and abnormal liver morphology in channel catfish, indicating that STZ treatment possibly induces liver damage. The mechanisms involved in the STZ‐induced hypoglycemic and tissue damage have never been examined. Another noted marker for liver damage stems from hyaluronan synthases (HAS), which have been associated with tissue damage in both humans and rodents. We hypothesized the expression of HAS mRNA would change in response to liver damage induced by STZ in channel catfish. Juvenile channel catfish were treated with 0, 3.6, 36, 180, or 360 mg STZ/kg body weight (n=4 fish/treatment) on day 0. Liver samples were collected on day 7. Hepatic expression of HAS2 and HAS3 mRNA was examined, using real‐time PCR. Expression of HAS2 and HAS3 mRNA decreased in response to an increased dose of STZ (p < 0.05 and p < 0.10, respectively). These results suggest STZ induces severe liver damage that down regulates the expression of HAS2 and HAS3 mRNA. However, the effects of STZ on gene expression appear to be global, and the tissue damage induced by STZ is not reversible by changes in the amount or activity of HAS.Support or Funding InformationThis research was funded by the APS and a grant from the National Science Foundation Integrative Organismal Systems (IOS) (Grant #IOS‐1238831). It was also supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Teratogenic potential of nanoencapsulated vitamin A evaluated on an alternative model organism, the tunicate Ciona intestinalis

Nano-encapsulation is a technology used to pack substances in order to enhance their stability and bioavailability, but this packing may interact with living systems, causing unexpected toxicity. Vitamin A (vit A) is a substance that has received attention, because in developed countries, the increasing availability of supplements is leading to its excessive intake. This study aims to compare teratogenic effects caused by exposure to the traditional formulation of vit A versus nano-encapsulated vit A. We used ascidian embryos as an alternative model. Ascidians are marine organisms closely related to vertebrates that share with them a body plan and developmental programme, including the morphogenetic role of retinoic acid (RA). Our data showed that the adverse effects of exposure to the same concentration of the two formulations were different, suggesting that the nano-encapsulation increased the bioavailability of the molecule, which could be better absorbed and metabolised to RA, the effective teratogenic substance.

Breeding Zebrafish: A Review of Different Methods and a Discussion on Standardization

In recent years, a rapidly increasing number of scientific papers have been published that utilize zebrafish (Danio rerio) as an alternative model organism in the study of a wide range of biological phenomena from cancer to behavior. This is, in large part, due to the prolific nature, relative ease of maintenance, and sufficiently high genetic homology of zebrafish to humans. With the surge of zebrafish use in animal research, the variations in methodologies of breeding and husbandry of this species have also increased. Investigators usually focus on the development and implementation of rigorous laboratory control that is specific to their studies. We suggest that the same scrutiny and attention may be required for the methods of breeding and housing of zebrafish. This article reviews a variety of zebrafish husbandry and breeding techniques and conditions employed around the world. It discusses factors ranging from numerous aspects of rearing/housing conditions through the sex ratio of the breeding group to the composition of the diet of zebrafish that may vary across laboratories. It provides some feedback on the potential pros and cons of the different methods. It argues that there is a substantial need for systematic analysis of these methods, that is, the effects of environmental factors on zebrafish health and breeding. It also discusses the question as to whether some degree of standardization of these methods is needed to enhance cross-laboratory comparability of results.

A global community effort to decipher the unique biology of annual killifish.

Over the past 50 years, annual killifishes arose as alternative model organisms for studies of vertebrate biology. The annual fish offers exceptional advantages for studies of genetics, genomics, developmental biology, population dynamics, ecology, biogeography, and evolution. They inhabit extremely variable freshwater environments in Africa and South America, have a short lifespan and a set of unique and fascinating developmental characteristics. Embryos survive within the dry substrate during the dry season, whereas the adult population dies. Thus, the survival of the populations is entirely dependent on the buried embryos that hatch the next rainy season. Although Old and New World species share similarities in their life cycle, they also have different adaptive responses associated with climate-related selective pressures. Therefore, contrasting different species from these areas is essential to understand unique adaptations to heterogeneous environment. A network of laboratories (United States, Czech Republic, Italy, Brazil, Chile, and Uruguay) is working and collaborating on many aspects of the biology of annual fishes. Participating researchers share projects and cross-training undergraduate and graduate students. These efforts resulted in two International Symposia (2010 and 2015) that took place in Montevideo and an international book. Herein, we summarize the progress made by this global community of scientists. Developmental Dynamics 246:807-811, 2017. © 2017 Wiley Periodicals, Inc.

Caenorhabditis elegans as a powerful alternative model organism to promote research in genetic toxicology and biomedicine.

In view of increased life expectancy the risk for disturbed integrity of genetic information increases. This inevitably holds the implication for higher incidence of age-related diseases leading to considerable cost increase in health care systems. To develop preventive strategies it is crucial to evaluate external and internal noxae as possible threats to our DNA. Especially the interplay of DNA damage response (DDR) and DNA repair (DR) mechanisms needs further deciphering. Moreover, there is a distinct need for alternative in vivo test systems for basic research and also risk assessment in toxicology. Especially the evaluation of combinational toxicity of environmentally present genotoxins and adverse effects of clinically used DNA damaging anticancer drugs is a major challenge for modern toxicology. This review focuses on the applicability of Caenorhabditis elegans as a model organism to unravel and tackle scientific questions related to the biological consequences of genotoxin exposure and highlights methods for studying DDR and DR. In this regard large-scale in vivo screens of mixtures of chemicals and extensive parallel sequencing are highlighted as unique advantages of C. elegans. In addition, concise information regarding evolutionary conserved molecular mechanisms of the DDR and DR as well as currently available data obtained from the use of prototypical genotoxins and preferential read-outs of genotoxin testing are discussed. The use of established protocols, which are already available in the community, is encouraged to facilitate and further improve the implementation of C. elegans as a powerful genetic model system in genetic toxicology and biomedicine.

Gene Disruption of Zebrafish Sbds Phenocopies Human Shwachman-Diamond Syndrome but Suggests More Global and Lineage Defects

Introduction: Shwachman-Diamond Syndrome (SDS) is a complex disorder resulting in short stature, neutropenia, bony dysplasia, and pancreatic malabsorption. Individuals with SDS are also at increased risk for developing myelodysplastic syndrome and/or acute myeloid leukemia (AML). Its estimated incidence of 1 in 77,000 births makes SDS difficult to study and develop newer therapies. SDS results from mutations in the SBDS gene. Genetic ablation of Sbds results in early embryonic lethality (ED 7.5) in mice. Zebrafish provide an attractive, alternative model organism to study human diseases. Genome analysis of zebrafish (Danio rerio) revealed the presence of a single sbds gene, encoding a protein 90% identical to the human orthologue.Methods: We used CRISPR/Cas9 genome editing technique to generate indel mutations in sbds andestablished multiple zebrafish lines. We used zebrafish sbds mutants to analyze the function of this gene during vertebrate development and in the pathophysiology of SDS.Results: Western blotting showed progressive decrease of Sbds expression with no detectable protein at 8 days post fertilization (dpf). These results suggest that the presence of Sbds during early stages of the development is due to maternal deposition, which was confirmed by qPCR and immunoblot demonstration of sbds transcript and proteins in in all stages of the development including the 1 cell-stage. Unlike Sbds-/- mice, homozygous mutant sbdsnu132live up to 6 weeks. They display marked growth retardation. We also observed a significantly low number of neutrophils at 5 dpf and 15 dpf. No differences were found in macrophages and hemoglobinization. Interestingly, multiple organs, especially the pancreas, liver, digestive tract and eye showed histologic evidence of atrophy at 21 dpf. Differences were also observed in fat deposition and bone formation. These defects were not seen at 15 dpf, suggesting a critical stage in development dependent on functional sbds.Conclusions: To our knowledge, we established the first zebrafish model of SDS, making it a highly relevant model to investigate its pathophysiology and develop new small molecule therapies. Comparison of null states between mice and zebrafish suggest that loss of Sbds results in two phenotypes: an early embryonic stage required for viability and development and a post-embryonic (larval) stage critical for granulocyte production and normal size. Data will be presented on bone formation, neutrophil chemotaxis, metabolomics, and RNAseq in the zebrafish model.This work was supported by grants from the NIH R21CA15920, Shwachman-Diamond Syndrome Foundation and the Alex Turnquist Memorial Research fund of Shwachman-Diamond America. DisclosuresNo relevant conflicts of interest to declare.

Steroid Androgen Exposure during Development Has No Effect on Reproductive Physiology of Biomphalaria glabrata.

Gastropod mollusks have been proposed as alternative models for male reproductive toxicity testing, due to similarities in their reproductive anatomy compared to mammals, together with evidence that endocrine disrupting chemicals can cause effects in some mollusks analogous to those seen in mammals. To test this hypothesis, we used the freshwater pulmonate snail, Biomphalaria glabrata, for which various genetic tools and a draft genome have recently become available, to investigate the effects of two steroid androgens on the development of mollusk secondary sexual organs. Here we present the results of exposures to two potent androgens, the vertebrate steroid; 5α-dihydrotestosterone (DHT) and the pharmaceutical anabolic steroid; 17α-methyltestosterone (MT), under continuous flow-through conditions throughout embryonic development and up to sexual maturity. Secondary sexual gland morphology, histopathology and differential gene expression analysis were used to determine whether steroid androgens stimulated or inhibited organ development. No significant differences between tissues from control and exposed snails were identified, suggesting that these androgens elicited no biologically detectable response normally associated with exposure to androgens in vertebrate model systems. Identifying no effect of androgens in this mollusk is significant, not only in the context of the suitability of mollusks as alternative model organisms for testing vertebrate androgen receptor agonists but also, if applicable to other similar mollusks, in terms of the likely impacts of androgens and anti-androgenic pollutants present in the aquatic environment.

Sensory Prioritization in Rats: Behavioral Performance and Neuronal Correlates.

Detection of low-amplitude events is critical to survival; for example, to warn prey of predators. To formulate a response, decision-making systems must extract minute neuronal signals from the sensory modality that provides key information. Here, we identify the behavioral and neuronal correlates of sensory prioritization in rats. Rats were trained to detect whisker vibrations or visual flickers. Stimuli were embedded in two contexts in which either visual or whisker modality was more likely to occur. When a stimulus was presented in the high-likelihood context, detection was faster and more reliable. Neuronal recording from the vibrissal cortex revealed enhanced representation of vibrations in the prioritized context. These results establish the rat as an alternative model organism to primates for studying attention.

Expression of miRNA-122 Induced by Liver Toxicants in Zebrafish.

MicroRNA-122 (miRNA-122), also known as liver-specific miRNA, has recently been shown to be a potent biomarker in response to liver injury in mammals. The objective of this study was to examine its expression in response to toxicant treatment and acute liver damage, using the zebrafish system as an alternative model organism. For the hepatotoxicity assay, larval zebrafish were arrayed in 24-well plates. Adult zebrafish were also tested and arrayed in 200 mL cages. Animals were exposed to liver toxicants (tamoxifen or acetaminophen) at various doses, and miRNA-122 expression levels were analyzed using qRT-PCR in dissected liver, brain, heart, and intestine, separately. Our results showed no significant changes in miRNA-122 expression level in tamoxifen-treated larvae; however, miRNA-122 expression was highly induced in tamoxifen-treated adults in a tissue-specific manner. In addition, we observed a histological change in adult liver (0.5 μM) and cell death in larval liver (5 μM) at different doses of tamoxifen. These results indicated that miRNA-122 may be utilized as a liver-specific biomarker for acute liver toxicity in zebrafish.

Ultrastructural Mapping of the Zebrafish Gastrointestinal System as a Basis for Experimental Drug Studies.

Research in the field of gastroenterology is increasingly focused on the use of alternative nonrodent model organisms to provide new experimental tools to study chronic diseases. The zebrafish is a particularly valuable experimental platform to explore organ and cell structure-function relationships under relevant biological and pathobiological settings. This is due to its optical transparency and its close-to-human genetic makeup. To-date, the structure-function properties of the GIS of the zebrafish are relatively unexplored and limited to histology and fluorescent microscopy. Occasionally those studies include EM of a given subcellular process but lack the required full histological picture. In this work, we employed a novel combined biomolecular imaging approach in order to cross-correlate 3D ultrastructure over different length scales (optical-, X-ray micro-CT, and high-resolution EM). Our correlated imaging studies and subsequent data modelling provide to our knowledge the first detailed 3D picture of the zebrafish larvae GIS. Our results provide unequivocally a limit of confidence for studying various digestive disorders and drug delivery pathways in the zebrafish.

Using zebrafish in systems toxicology for developmental toxicity testing.

With the high cost and the long-term assessment of developmental toxicity testing in mammals, the vertebrate zebrafish has become a useful alternative model organism for high-throughput developmental toxicity testing. Zebrafish is also very favorable for the 3R perspective in toxicology; however, the methodologies used by research groups vary greatly, posing considerable challenges to integrative analysis. In this review, we discuss zebrafish developmental toxicity testing, focusing on the methods of chemical exposure, the assessment of morphological abnormalities, housing conditions and their effects on the production of healthy embryos, and future directions. Zebrafish as a systems toxicology model has the potential to elucidate developmental toxicity pathways, and to provide a sound basis for human health risk assessments.