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On the origin of animals and placental mammals: a critique of literalist readings of the fossil record.

The fossil record is incomplete, as evidenced by the pervasive presence of ghost lineages throughout the Tree of Life. For example, across placental mammals, at least 720 Myr of basal lineages are ghost lineages, that is, lineages that have left no fossil evidence of their past history. In contrast, some studies have suggested that the fossil record is a faithful temporal archive of evolutionary history and thus the times of diversification of clades must be close to the ages of their oldest fossils. Such literalist interpretations have been contradicted by analysis of molecular datasets which, in many cases, indicate that groups including placental mammals and animals may have originated at times substantially older than their fossil records. Some of those studies have further argued that, in the case of animals and placental mammals, molecular clocks are uninformative, suffer from characteristic pathologies, and thus cannot distinguish between recent and ancient hypotheses of diversification. Here, we reexamine these two cases and show, using Bayesian model selection theory, that the explosive diversification models previously proposed for animals and placental mammals have a posterior probability of ∼0. We show the characteristic pathologies purportedly discovered do not exist, highlight errors in previous analyses, and provide advice on best practice for molecular-clock dating analysis.

Animals

Viral zoonosis and human cancer: a perspective.

Zoonotic viruses, which are pathogens naturally transmitted from animals to humans, pose a significant and evolving challenge to public health. Although most known zoonotic viruses do not exhibit the persistence typically necessary for viral oncogenesis, the potential cancer-causing effects of these infections remain unclear. Persistent infection, latency, or abortive replication within susceptible but non-permissive human cells may allow some animal-origin viruses to evade immune clearance, disrupt host cell signaling, and induce genomic instability-key features of cancer development. Evidence from both in vitro and in vivo studies indicates that certain animal viruses can enter human cells, integrate their genetic material, or express oncogenic proteins, even without completing full replication. These mechanisms resemble those of established human oncoviruses and suggest that, under specific host and environmental conditions, zoonotic viruses could contribute to neoplastic transformation. Given the increasing frequency of human-animal interactions through companionship, agriculture, wildlife trade, and food production, multidisciplinary research combining virology, oncology, and epidemiology is essential. Such efforts should focus on sensitive molecular detection, mechanistic studies, and population-based investigations to better understand the long-term cancer risks associated with zoonotic viral infections and to guide effective prevention strategies.

Cancer

Improved quality of life and prolonged survival with add-on homeopathic treatment in patients with non-small cell lung cancer: a prospective, randomized, placebo-controlled, double-blind, three-arm, multicenter study.

BACKGROUND: Alongside conventional anticancer treatment, add-on homeopathy might help to alleviate adverse effects of conventional therapy. AIM: The aim of this study was to replicate previous studies on the effect of adjunctive homeopathy on quality of life (QoL) and survival in non-small cell lung cancer (NSCLC) patients. METHOD: In this prospective, randomized, placebo-controlled, double-blind, three-arm multicenter phase III study with quadruple-checked data analysis, we investigated the potential effects of an add-on homeopathic treatment compared to placebo in patients with stage IV NSCLC in terms of QoL. Ninety-eight received either individualized homeopathic medicinal products (HMPs; n&#x2009;=&#x2009;51) or placebo (n&#x2009;=&#x2009;47) in a double-blinded fashion. Fifty-two control patients without homeopathic treatment were only observed in terms of their survival rate. The ingredients of the various HMPs were mainly prepared of plant, mineral, or animal origin. The data entry and statistical analysis were subject to an exceptional quadruple-checked data analysis process. The analysis presented in this article was inspired by our earlier report of this trial published in The Oncologist in 2020, which was retracted by that journal in November 2025 after two corrections; a majority of the co-authors disagreed with this decision. The present article is based on the same trial dataset but was deliberately designed to highlight the unique research methodology: design and preparation by a lead statistician, data entry, data clearing and independent statistical evaluation were performed in four mutually independent steps, reporting follows the CONSORT statement, and the interpretation of the findings has been reframed conservatively. RESULTS: Global health status (QoL) was higher in the homeopathy group than in the placebo group after 9&#xa0;weeks and after 18&#xa0;weeks (p&#x2009;<&#x2009;0.001). With the exception of cognitive functioning at 9&#xa0;weeks and of pain, diarrhea and financial difficulties at 9&#xa0;weeks, all functional and symptom scales of the EORTC QLQ-C30 favored the homeopathy group (p&#x2009;<&#x2009;0.001 for the multivariate comparisons), with between-group differences exceeding the threshold of 10 points that is generally regarded as clinically meaningful. Median survival time over the 730-day observation period was 435&#xa0;days in the homeopathy group, 257&#xa0;days in the placebo group (p&#x2009;=&#x2009;0.010), and 228&#xa0;days in the non-randomized control group (p&#x2009;<&#x2009;0.001); the corresponding 2-year survival rates were 45.1%, 23.4%, and 13.5% (homeopathy vs. placebo p&#x2009;=&#x2009;0.020; homeopathy vs. control p&#x2009;<&#x2009;0.001). The difference between the placebo group and the non-randomized control group was not statistically significant (p&#x2009;=&#x2009;0.154). CONCLUSION: In this trial, add-on homeopathy was associated with better quality of life across most functional and symptom domains, with clinically meaningful effect sizes congruently to a previous open study. Survival time was significantly longer in the homeopathy group compared to both the placebo and control groups. Independent replication, ideally within contemporary immuno-oncological treatment regimens is required. TRIALS REGISTRATION: ClinicalTrials.gov; No.: NCT01509612; January 7, 2012.

Humans

Cross-Kingdom Genomic Conservation of Putative Human Sleep-Related Genes: Phylogenomic Evidence From Chlamydomonas reinhardtii.

Sleep is a widespread and evolutionarily conserved process observed in diverse organisms, from jellyfish to mammals, hinting at its origin as a life-supporting mechanism over 500 million years ago. Although its fundamental purpose and mechanisms remain unclear, sleep's evolution and adaptive significance continue to be debated. This study explores the evolutionary origins of sleep using Chlamydomonas reinhardtii as a model organism, identifying 112 putative sleep-related genes across species and highlighting the evolutionary conservation of sleep-regulatory pathways. Additionally, discovering uncharacterized proteins with high sequence similarity and significant e-values suggests unexplored roles in sleep regulation, underscoring the potential of C. reinhardtii to reveal new insights into the molecular basis of sleep. This study provides a foundation for identifying previously unknown sleep-associated proteins, particularly within single-celled organisms, which may offer novel perspectives on the biological role of sleep. The study demonstrates that phylogenomic analysis of diverse model organisms can expand our understanding of the evolutionary trajectory of sleep and its fundamental function, paving the way for further research in sleep biology and its health implications. Overall, the fundamental functions of sleep observed in higher animal phyla originated from its primordial activities, demonstrating an evolutionary continuum wherein more specialized tasks were integrated with sleep's essential restorative properties.

Chlamydomonas reinhardtii

Patterns of antimicrobial resistance genes in pathogens across One Health sectors in Ireland: an in silico approach.

As part of a rapid risk assessment, an in silico approach was used to detect antimicrobial resistance (AMR) in pathogenic isolates from humans, animals, and the environment. A total of 11,670 genomic data sets were retrieved from the NCBI Pathogen Detection system for Ireland, which represented 47 pathogenic species, including Salmonella enterica, Escherichia coli/Shigella spp., Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus faecium. Identifying the most critical pathogenic strains over time is essential, as these organisms significantly contribute to mortality, morbidity, and hospitalization. The analysis identified 799 antimicrobial resistance genes (ARGs), including their allelic diversity, 117 plasmid replicons, and 274 virulence factors. Several critical ARGs, particularly those conferring resistance to beta-lactams, aminoglycosides, quinolones, and colistin, were common across isolates originating from human, animal, and environmental sources, suggesting shared resistance profiles across One Health sectors. Klebsiella pneumoniae, E. coli/Shigella spp., S. enterica, and S. aureus were the dominant hosts of these ARGs and associated mobile genetic elements. Increasing resistance across major antibiotic classes aligned with trends reported across other European countries. This study provides a national-scale in silico comparison of AMR across pathogens and One Health sectors using publicly available genomic data. The findings help reinforce Ireland's AMR surveillance by showing which resistance genes are present and how they spread across critical pathogens in humans, animals, and the environment. These findings highlight the urgent need for improved antibiotic stewardship and integrated One Health surveillance to limit the emergence and spread of AMR.IMPORTANCEAntimicrobial resistance (AMR) is a growing threat to human, animal, and environmental health. This study used publicly available genomic data to identify antimicrobial resistance genes (ARGs) in key bacterial pathogens circulating in Ireland. By analyzing over 11,000 genomes from humans, animals, and the environment, we found that several dangerous resistance genes, including those against last-resort antibiotics, were widespread across different sources. The study highlights which bacteria and resistance genes are most critical and how they may spread between humans, animals, and the environment. These insights provide a national snapshot of AMR, supporting more effective monitoring and prevention strategies. By revealing patterns of resistance and modes of transmission, our findings underscore the importance of coordinated antibiotic stewardship and One Health approaches to slow the emergence and spread of resistant infections, protecting public health and ensuring antibiotics remain effective.

Humans

Transferable IncHI2-Associated blaLAP-2 and blaCTX-M-55 Resistance Platforms in Foodborne Salmonella.

Extended-spectrum &#x3b2;-lactamase genes in foodborne Salmonella enterica can disseminate through mobile multidrug-resistance platforms. IncHI2 plasmids are important resistance vehicles capable of carrying complex resistance regions and facilitating their horizontal transfer across diverse bacterial backgrounds, but the transfer and genomic organization of IncHI2 elements co-carrying blaLAP-2 and blaCTX-M-55 remain insufficiently characterized. This study investigated two multidrug-resistant foodborne isolates recovered in Shanghai in 2022: Salmonella Agona ST13 isolate Sal22C150 and Salmonella Havana ST1527 isolate Sal22P208. Antimicrobial susceptibility testing, whole-genome sequencing, conjugation, plasmid-retention analysis, comparative genomics, as well as strain- and plasmid-level phylogenetic analyses were performed. Both isolates exhibited broad antimicrobial resistance, including resistance to extended-spectrum cephalosporins. In both isolates, blaLAP-2 and blaCTX-M-55 co-transferred with the IncHI2 replicon to Escherichia coli J53 at frequencies of (4.95 &#xb1; 0.41) &#xd7; 10-5 and (4.46 &#xb1; 0.42) &#xd7; 10-6 transconjugants per donor cell, respectively. All tested plasmid markers remained detectable through 20 passages without antimicrobial selection. Complete assembly of Sal22P208 confirmed the location of the three &#x3b2;-lactamase genes on the 275,096 bp IncHI2 plasmid pSal22P208. The plasmid contained a conserved conjugative backbone and mosaic accessory regions carrying 15 antimicrobial-resistance determinants together with mercury- and tellurium-resistance loci. SNP-based analysis placed pSal22P208 within a closely related cluster containing six reference IncHI2 plasmids differing by fewer than 30 SNPs and recovered from Salmonella and E. coli of animal, food, and human origin, suggesting a broad distribution of this plasmid lineage across diverse bacterial and ecological backgrounds. Sal22P208 additionally contained a Tn3-associated chromosomal multidrug-resistance region between rpmJ and rpmE that shared extensive structural similarity with a region in Citrobacter braakii LBA3. These findings highlight the role of transferable IncHI2 resistance platforms in the horizontal dissemination and short-term post-transfer maintenance of linked resistance determinants, while chromosomally integrated resistance regions may provide an additional route for the accumulation and inheritance of multidrug resistance in foodborne Salmonella.

IncHI2 plasmid

The genomic origin of the unique chaetognath body plan.

The emergence of animal phyla, each with their unique body plan, was a rapid event in the history of animal life, yet its genomic underpinnings are still poorly understood1. Here we investigate at the genomic, regulatory and cellular levels, the origin of one of the most distinctive animal phyla, the chaetognaths, whose organismal characteristics have historically complicated their phylogenetic placement2,3. We show that these characteristics are reflected at the cell-type level by the expression of genes that originated in the chaetognath lineage, contributing to adaptation to planktonic life at the sensory and structural levels4. Similarly to other members of gnathiferans (which also include rotifers and several other microscopic phyla)5,6, chaetognaths have undergone accelerated genomic evolution with gene loss and chromosomal fusions7,8. Furthermore, they secondarily duplicated thousands of genes9,10, without evidence for a whole-genome duplication, yielding, for instance, tandemly expanded Hox genes, as well as many phylum-specific genes. We also detected repeat-rich highly methylated neocentromeres and a simplified DNA methylation toolkit that is involved in mobile element repression rather than transcriptional control. Consistent with fossil evidence11,12, our observations suggest that chaetognaths emerged after a phase of morphological simplification through a reinvention of organ systems paralleled by massive genomic reorganization, explaining the uniqueness of their body plan.

Animals

Cellular imbalance in proximal and distal lung of CFTR-/- sheep in utero and at birth.

BACKGROUND: The Lung is the major focus of therapeutic approaches for the inherited disorder cystic fibrosis (CF) as without treatment lung disease is life-limiting. However, the initiating events that predispose the CF lung to cycles of infection, inflammation and resultant tissue damage are still unclear. Inflammation may occur in the CF lung prior to birth in human and several large animal models suggesting an in utero origin for the disease and encouraging further studies prior to birth. METHODS: Here we used the sheep model of CF (CFTR-/-) and age-matched wild-type (WT) sheep of the same breed to investigate the single cell transcriptomes of proximal and distal lung tissue at 80&#xa0;days and 120&#xa0;days of gestation and at term (147&#xa0;days). Single cell RNA-seq was performed on tissues from 4 to 7 animals of each genotype (WT and CFTR-/-) at each time point. RESULTS: At term, FOXJ1-expressing ciliated cells are overrepresented in both lung regions from CFTR-/- lambs, while secretory epithelial and basal cells are underrepresented in proximal lung, as are T cells and monocytes in distal lung. The imbalance in ciliated and basal cells was confirmed by immunohistochemistry. At 120&#xa0;days of gestation, lymphoid cells are slightly more abundant in proximal and distal lung from CFTR-/- animals compared to WT, consistent with the transient CF-associated inflammatory response in utero. At 80&#xa0;days of gestation, T and B cells are underrepresented in both lung regions. CONCLUSIONS: The differences in epithelial cell abundance observed in the CFTR-/- lambs at term may reflect sequelae from the loss of CFTR on lung development and differentiation in utero. These findings provide novel insights into the cellular mechanisms of pathology and may be relevant to the design of new therapeutic approaches for CF lung disease.

Animals

Multiple introductions of equine influenza virus into the United Kingdom resulted in widespread outbreaks and lineage replacement.

Influenza A viruses (IAVs) are prime examples of emerging viruses in humans and animals. IAV circulation in domestic animals poses a pandemic risk as it provides new opportunities for zoonotic infections. The recent emergence of H5N1 IAV in cows and subsequent spread over multiple states within the USA, together with reports of spillover infections in humans, cats and mice highlight this issue. The horse is a domestic animal in which an avian-origin IAV lineage has been circulating for >60 years. In 2018/19, a Florida Clade 1 (FC1) virus triggered one of the largest epizootics recorded in the UK, which led to the replacement of the Equine Influenza Virus (EIV) Florida Clade 2 (FC2) lineage that had been circulating in the country since 2003. We integrated geographical, epidemiological, and virus genetic data to determine the virological and ecological factors leading to this epizootic. By combining newly-sequenced EIV complete genomes derived from UK outbreaks with existing genomic and epidemiological information, we reconstructed the nationwide viral spread and analysed the global evolution of EIV. We show that there was a single EIV FC1 introduction from the USA into Europe, and multiple independent virus introductions from Europe to the UK. At the UK level, three English regions (East, West Midlands, and North-West) were the main sources of virus during the epizootic, and the number of affected premises together with the number of horses in the local area were found as key predictors of viral spread within the country. At the global level, phylogeographic analysis evidenced a source-sink model for intercontinental EIV migration, with a source population evolving in the USA and directly or indirectly seeding viral lineages into sink populations in other continents. Our results provide insight on the underlying factors that influence IAV spread in domestic animals.

Animals

The evolution of cnidarian stinging cells supports a Precambrian radiation of animal predators.

Cnidarians-the phylum including sea anemones, corals, jellyfish, and hydroids-are one of the oldest groups of predatory animals. Nearly all cnidarians are carnivores that use stinging cells called cnidocytes to ensnare and/or envenom their prey. However, there is considerable diversity in cnidocyte form and function. Tracing the evolutionary history of cnidocytes may therefore provide a proxy for early animal feeding strategies. In this study, we generated a time-calibrated molecular clock of cnidarians and performed ancestral state reconstruction on 12 cnidocyte types to test the hypothesis that the original cnidocyte was involved in prey capture. We conclude that the first cnidarians had only the simplest and least specialized cnidocyte type (the isorhiza) which was just as likely to be used for adhesion and/or defense as the capture of prey. A rapid diversification of specialized cnidocytes occurred through the Ediacaran (~654-574 million years ago), with major subgroups developing unique sets of cnidocytes to match their distinct feeding styles. These results are robust to changes in the molecular clock model, and are consistent with growing evidence for an Ediacaran diversification of animals. Our work also provides insight into the evolution of this complex cell type, suggesting that convergence of forms is rare, with the mastigophore being an interesting counterexample.

Animals

Limitations of serial cloning in mammals: unresolved donor-cell genomic integrity challenges broad claims of cloning limits.

Wakayama et al. describe an extraordinary 20-year serial cloning study in mice, concluding that serial cloning in mammals is ultimately limited by the accumulation of genetic anomalies. However, their whole-genome sequencing (WGS) analysis characterized selected cloned animals but did not include matched genomic profiling of the corresponding cumulus cell (CC)-donor mice, the source CC populations, or developmental stages. Because each reconstructed embryo originated from a single CC nucleus and re-cloned animals were used to advance the lineage, pre-existing somatic variation could have entered the lineage and subsequently been propagated. Consequently, variants detected in later generations cannot be assigned definitively to pre-existing donor-cell mosaicism, donor-cell handling, somatic cell nuclear transfer manipulation, or early embryogenesis. Thus, the observed decline cannot be attributed exclusively to genetic lesions arising during repeated cloning, but the unresolved genomic status of the lineage-founding donor cells remains a plausible but unproven contributor. The study therefore demonstrates the transmission and propagation of genetic lesions through serial cloning more directly than it establishes that all initiating lesions arose because of repeated cloning. Paired genomic profiling of donor-cell populations, embryos, and offspring would help resolve the origins of accumulated genetic lesions and determine whether donor-cell screening could extend serial cloning.

Animals

Accounting for voluntary waiting period changes in US dairy herds: Adjusting daughter pregnancy rate and introducing first-service to conception.

The genetic evaluation of female fertility traits in dairy cattle in the United States has progressed over the past 2 decades, with 4 additional traits integrated into the national evaluation system since the introduction of daughter pregnancy rate (DPR) in 2004. However, concerns have arisen in the dairy sector, with reports of producers extending the voluntary waiting period (VWP), which is the time of initial breeding after calving, due to more persistent lactation yields. Since its inception, DPR calculations in the US evaluation have assumed a fixed 50-d VWP, which may not reflect modern reproductive strategies. Furthermore, producers may determine that some of their cows should have their VWP extended while others may follow the standard time. A re-evaluation of the current female fertility evaluation is necessary to ensure traits align with current management practices. Therefore, this study explores the addition of a potential new trait, First Service to Conception (FSC), along with a revised DPR formula that permits more flexibility with VWP. About 32 million records from the National Cooperator Database, covering 5 major dairy breeds (2003-2023), were used for this study. Data for cows calving before 2003 were unavailable because insemination records were not recorded prior. The findings suggest FSC enhances female fertility evaluations, providing a more comprehensive reproductive assessment independent of VWP. Another approach was an adjustment to the DPR calculation (DPRadj) to account for herd VWP on a herd-year and lactation group basis. The mean DPR value was 45.06%, while DPRadj increased the mean value to 52.58%. The mean FSC was 62.64 d. In an analysis using first lactation cows for cow traits and heifers for heifer traits, genetic correlations of FSC with other fertility traits were 0.97 with Cow Conception Rate (CCR) and DPR, 0.98 with DPRadj, 0.40 with Heifer Conception Rate (HCR), and 0.32 with Early First Calving (EFC). Predicted transmitting abilities (PTA) were calculated to assess the implications of adding FSC to, or replacing DPR with DPRadj within the CDCB multi-trait evaluation index, Net Merit $ (NM$). The highest correlation among the top 10% of bulls occurred when replacing DPR with DPRadj (0.99). Adding FSC to the original model reduced the latter correlation to 0.88, which indicates slight re-ranking of top bulls. Among animals with PTA reliability above 50%, the lowest correlation was between the original index and the index replacing DPR with FSC (0.95). This work advances genetic evaluation in dairy cattle, improving reproductive efficiency and productivity.

daughter pregnancy rate

Investigating the zoonotic origins of ESBL-producing E. coli in community-acquired urinary tract infections in Ecuador.

Extended-spectrum &#x3b2;-lactamase-producing Escherichia coli (ESBL-producing E. coli) pose a growing global health threat. Although Latin America has been identified as a global hotspot of antimicrobial resistance, the zoonotic contribution to drug-resistant infections in the region remains poorly defined. We analyzed 137 clinical ESBL-producing E. coli isolates from urinary tract infections (UTIs) in Quito, Ecuador, applying a Bayesian latent class model informed by host-associated mobile genetic elements to estimate the fraction of infections attributable to food-animal sources. We estimated that 25.5% (35/137) of UTI isolates were putative zoonotic cases. This proportion rose to 42.5% after excluding ST131-H30, a human-associated pandemic lineage. Putative zoonotic isolates were enriched for animal-associated &#x3b2;-lactamase genes (e.g., blaTEM-1B, blaCTX-M-65), lacked human-associated markers such as blaOXA-1, and exhibited diverse antimicrobial resistance gene profiles resembling those observed among food-animal isolates. These isolates were also enriched for ColV-associated virulence genes typically linked to avian pathogenic E. coli. Putative zoonotic strains contributed substantially to third-generation cephalosporin-resistant UTIs in Quito, Ecuador, challenging assumptions derived from high-income settings that such infections are driven predominantly by human-to-human transmission. These findings highlight the importance of integrated One Health surveillance and mitigation, particularly in low- and middle-income countries where gaps in water, sanitation, and hygiene (WASH) may interact with antimicrobial use in food production to amplify antimicrobial resistance transmission.IMPORTANCEESBL-producing E. coli have rapidly emerged as a major global antimicrobial resistance threat. In Latin America, cephalosporins are commonly used in food-animal production, fueling the emergence of ESBL-producing E. coli. In low- and middle-income countries, excessive antimicrobial use driven by poorly regulated over-the-counter sales, combined with inadequate water, sanitation, and hygiene (WASH) infrastructure, can facilitate antimicrobial-resistant pathogen transmission from food animals to humans. Using a novel statistical-genomic approach, we found that over one in four cephalosporin-resistant UTIs in Quito, Ecuador, may be caused by E. coli strains originating from food animals. Our findings highlight the public health risks associated with antimicrobial use in food-animal production and the role of environmental and infrastructure-related vulnerabilities. As global demand for animal protein continues rising in middle-income countries, controlling zoonotic antimicrobial resistance transmission becomes increasingly urgent for protecting human health through integrated One Health strategies.

ESBL-producing E. coli

Genome-wide SNP-based genomic diversity and population structure analysis in alpaca populations from Europe and Peru.

This study aimed to analyze the genetic diversity and population structure of alpacas in Germany, Switzerland, and Austria (German-speaking regions, GSR) and to compare with that of the country of origin of the species (Peru). A total of 179 animals from GSR and 151 from Peru were genotyped with a species-specific 76k SNP array. The observed and expected heterozygosity was 0.305 and 0.311 for GSR and 0.310 and 0.312 for Peru. The mean FROH values were 0.029 for GSR and 0.023 for Peru. In general, results show that breeders in both analyzed regions efficiently maintain genetic diversity. Principal component analysis identified the GSR and Peru populations as separate from each other, but the relative proximity of both clusters indicates the shared genetic heritage. FST and XPEHH methods identified genomic regions under selection for traits such as coat color and adaptation. Genome-wide association studies comparing black and brown with white or gray alpacas identified associated genome regions containing the ASIP and KIT genes, respectively. The association of a recently identified keratin locus on chromosome 16 with differences in fleece type in alpacas was confirmed, while the putative causality of a TRPV3 variant was rejected.

Animals

Developmental and genomic insight into the origin of the tardigrade body plan.

Tardigrada is an ancient lineage of miniaturized animals. As an outgroup of the well-studied Arthropoda and Onychophora, studies of tardigrades hold the potential to reveal important insights into body plan evolution in Panarthropoda. Previous studies have revealed interesting facets of tardigrade development and genomics that suggest that a highly compact body plan is a derived condition of this lineage, rather than it representing an ancestral state of Panarthropoda. This conclusion was based on studies of several species from Eutardigrada. We review these studies and expand on them by analyzing the publicly available genome and transcriptome assemblies of Echiniscus testudo, a representative of Heterotardigrada. These new analyses allow us to phylogenetically reconstruct important features of genome evolution in Tardigrada. We use available data from tardigrades to interrogate several recent models of body plan evolution in Panarthropoda. Although anterior segments of panarthropods are highly diverse in terms of anatomy and development, both within individuals and between species, we conclude that a simple one-to-one alignment of anterior segments across Panarthropoda is the best available model of segmental homology. In addition to providing important insight into body plan diversification within Panarthropoda, we speculate that studies of tardigrades may reveal generalizable pathways to miniaturization.

Animals

Ex situ reared black-footed ferrets exhibit altered sperm DNA methylation.

Many endangered species rely on ex situ management for survival when external threats exist on the landscape. Yet, ex situ settings pose challenges through space limitation, altered environment, and diet. This can lead to environmentally determined inbreeding depression, where ex situ animals exhibit reduced reproductive fitness compared with their in situ counterparts, despite originating from the same genetic stock. We investigated epigenetic differences as a potential mechanism underlying environmentally determined inbreeding depression in black-footed ferrets (Mustela nigripes), a North American endemic species reliant on ex situ conservation. More specifically, we explored how environmental context may influence sperm DNA methylation in samples collected from 12 ex situ and 5 in situ males. Average sperm DNA methylation was significantly higher in ex situ individuals. We additionally identified more than&#x2009;500 differentially methylated regions between ex situ and in situ sperm samples that were enriched for gene ontology terms pertaining to reproduction and development. Putative genes of interest included NPR2, WEE2, SLC15A1, PDE10A, PIP5K1B, CACNA1E, and CACNA1A, all of which have previously been linked to spermatogenesis, sperm motility, or fertilization in mammals. Results suggest that environmental conditions may alter sperm DNA methylation in black-footed ferrets, with possible links to decreased reproductive success in ex situ settings. These findings provide valuable insights into the molecular mechanisms underlying environmentally determined inbreeding depression in black-footed ferrets and other conservation-reliant species, and can serve as a foundation for future research on improving reproductive health in endangered wildlife.

Animals

Comparative Embryology and Transcriptomics of Asellus infernus, an Isopod Crustacean From Sulfidic Groundwater.

Sulfidic caves are harsh and extreme environments characterized by limited oxygen, low pH, and the presence of hydrogen sulfide. Amazingly, animals can live in sulfidic caves, one such animal being Asellus infernus, a representative of the Asellus aquaticus species complex, originating from Movile Cave and from old wells that represent windows of access to a sulfidic groundwater ecosystem located in southeast Romania. Little previous work has been done on lab-reared populations of A. infernus as they have been historically difficult to raise in the lab. Here, we develop resources for A. infernus, examining questions of timing of morphological differences in cave versus surface individuals, whether the environment (lab-bred vs. wild-caught) influenced size characteristics, and the genes and pathways showing differential expression between cave and surface samples. We found that A. infernus did not develop pigmentation embryonically, and juveniles had increased body length and longer antenna II as compared to surface individuals. Furthermore, we found that some of these measures differed between wild-caught and lab-reared juveniles for a given population, indicating that environmental differences can also influence these size characteristics. In addition, differential expression between cave and surface samples and allele-specific expression studies within F1 hybrids identified multiple genes, including those involved in sulfide metabolism and phototransduction. Strikingly, molecular convergence of genes involved in sulfide detoxification was observed between A. infernus and previous work on a fish that lives in both cave and sulfidic environments, Poecilia mexicana. In sum, we were able to develop embryonic and genomic tools for A. infernus, a model for understanding cave adaptation and adaptation to sulfidic environments.

Animals

Novel bacterial hosts and mobile genetic structure of tet(X) variants in tetracycline-contaminated aquatic environment uncovered by culture and long-read metagenomics.

Clinically important tigecycline (3rd-generation tetracycline) resistance tet(X) variants were inferred to have evolutionarily originated from environmental bacteria, and have been recognized among environment, human and animals. However, genetic basis for environmental proliferation and dissemination of tet(X) variants remains ambiguous. This study profiled tet(X) variants at gene, contig, isolate, and community levels in environmental community subjected to long-term stepwise increasing oxytetracycline (1st-generation tetracycline) or tigecycline pressure using long-term microcosm experiments, quantitative PCR, bacterial isolation, whole-genome sequencing, and Nanopore-based long-read metagenomics. We confirmed that both oxytetracycline and tigecycline enriched the abundance of tetracycline resistance genes especially oxytetracycline-enriched tet(X3). Unexpectedly diverse bacterial hosts and genetic structure of tet(X)-positive mobile elements in the environment microbiome were identified using bacterial isolation and long-read Nanopore metagenomics. Pseudomonas defluvii was first reported to carry tet(X3) in the chromosome, forming IS26-tet(X3)-res-ISCR2 circular intermediate to transfer between different DNA molecules. Database mining revealed similar mobile segments have prevailed among animal-derived Acinetobacter species. Unlike the widely reported ISCR2-mediated transfer of tet(X6), we identified a novel mobile multidrug transposon TnAs3 where tet(X6) and class 1 integron co-transferred as its passenger region. Mobile tet(X2)-ere(D)-aadS-erm(F)-blaOXA-347 segment was annotated in Runella, and co-occurrences of tet(X2) and ere(D), aadS, blaOXA-347 were also found in Flavobacterium, Arsenicibacter, Chryseobacterium and Pedobacter. Overall, tetracycline-contaminated aquatic microbiome harboured diverse mobile tet(X)-positive segments which have not yet been acquired by clinical pathogens, and thus served as the genetic pool of tet(X) variants together with indigenous bacterial hosts, especially the newly reported Pseudomonas defluvii. Reducing pollution of older-generation tetracyclines would be a proactive way to mitigate environmental evolution and possible clinical effects of tet(X) variants.

Metagenomics