Multicellular Research Articles

Landscape, demographic, and dispersal parameters influence the spread and establishment of an aquatic biological invasion

AbstractHuman‐mediated species introductions are contributing to the biotic homogenization of global flora and fauna. Despite extensive research, we lack simple methods of predicting how and where an introduced species will spread and establish, particularly in species with complex life histories in aquatic ecosystems. We predict that spread can be modeled simply using the characteristics of the invading population, specifically species growth rate and dispersal capacity. In addition, we predict that the establishment of introduced species should be explained by the characteristics of the receiving ecosystem. Using the brown trout (Salmo trutta) invasion on the Island of Newfoundland as a case study, we fit and test a reaction–diffusion model with brown trout population data collected from the literature. Next, we use statistical models to assess the influence of a suite of abiotic (conductivity, pH, turbidity, calcium), biotic (Atlantic salmon occurrence), and landscape (watershed relief, watershed area, distance to original introduction) variables on brown trout establishment (i.e., presence–absence) patterns. We find that observed coastal spread in Newfoundland is slow (~4.4 km/year), and that it lies on the lower end of the range of predictions made by the reaction–diffusion model parameterized based on the estimates of growth rate and movement from the literature (predicted spread range 1.4 to 92 km/year). Also, we did not find evidence for a relationship between abiotic or biotic variables and brown trout establishment. However, we did observe that landscape variables of the distance to trout introduction point and estuary area may explain establishment patterns along the south coast of Newfoundland. Our results suggest the importance of using population‐specific parameterization and the need to integrate regional landscape factors that are generally applicable across biological invasions (e.g., distance to introduction), and those that are more specific to the ecology of the invader (e.g., estuary area). Our study contextualizes the mechanisms that contribute to a slow invasion by an aquatic species with a complex life history and reveals that future studies need to integrate a variety of methods to elucidate the processes governing invasions.

Treatments and the Perspectives of Developing a Vaccine for Chagas Disease.

Chagas disease (CD) treatment and vaccine development are critical due to the significant health burden caused by the disease, especially in Latin America. Current treatments include benznidazole and nifurtimox, which are most effective in the acute phase of the disease but less so in the chronic phase, often with significant side effects. Here, using the available literature, we summarize the progress in vaccine development and new treatments that promise to reduce CD incidence and improve the quality of life for those at risk, particularly in endemic regions. New treatment options, such as posaconazole and fexinidazole, are being explored to improve efficacy and reduce adverse effects. Vaccine development for CD remains a high priority. The complex life stages and genetic diversity of Trypanosoma cruzi present challenges, but several promising vaccine candidates are under investigation. These efforts focus on stimulating a protective immune response through various innovative approaches.

The immunity gap in primates.

Vertebrates exhibit sexual dimorphism in response to infectious diseases and in morbidity and mortality rates to various pathogens. Females are generally more immunocompetent than males, despite their increased reproductive burden and the immunosuppressive effects of gestation. In addition, females generally have lower incidences of cancer compared to males; however, they have higher rates of autoimmune disorders. These sex differences may be a result of life history differences, sexual selection, genetics, and/or the physiological effects of hormones. As highly social mammals with complex life histories, primates offer a unique opportunity to investigate the evolution of enhanced female immunocompetence. This review aims to examine the evidence of this immunity gap, understand current hypotheses for its evolution, and explore the potential role of X chromosome specific genes and heterozygosity within this framework.

Heat tolerance, oxidative stress response tuning and robust gene activation in early-stage Drosophila melanogaster embryos.

In organisms with complex life cycles, life stages that are most susceptible to environmental stress may determine species persistence in the face of climate change. Early embryos of Drosophila melanogaster are particularly sensitive to acute heat stress, yet tropical embryos have higher heat tolerance than temperate embryos, suggesting adaptive variation in embryonic heat tolerance. We compared transcriptomic responses to heat stress among tropical and temperate embryos to elucidate the gene regulatory basis of divergence in embryonic heat tolerance. The transcriptomes of tropical and temperate embryos differed in both constitutive and heat-stress-induced responses of the expression of relatively few genes, including genes involved in oxidative stress. Most of the transcriptomic response to heat stress was shared among all embryos. Embryos shifted the expression of thousands of genes, including increases in the expression of heat shock genes, suggesting robust zygotic gene activation and demonstrating that, contrary to previous reports, early embryos are not transcriptionally silent. The involvement of oxidative stress genes corroborates recent reports on the critical role of redox homeostasis in coordinating developmental transitions. By characterizing adaptive variation in the transcriptomic basis of embryonic heat tolerance, this study is a novel contribution to the literature on developmental physiology and developmental genetics.

A realist review of the causes of, and current interventions to address 'missingness' in health care.

This protocol describes a realist review exploring the problem of "missingness" in healthcare, defined as the repeated tendency not to take up offers of care that has a negative impact on the person and their life chances. More specifically, the review looks at the phenomenon of patients missing multiple appointments in primary care in the UK - at the causal factors that influence how patients come to be "missing" in this way, and what interventions might support uptake and "presence" in healthcare. Background research informing this project suggests that a high rate of missed appointments predicted high premature death rates, and patients were more likely to have multiple long-term health conditions and experience significant socioeconomic disadvantage. Most research in this field focuses on population- or service-level characteristics of patients who miss appointments, often making no distinction between causes of single missed appointments and of multiple missed appointments. There have therefore been no interventions for 'missingness', accounting for the complex life circumstances or common mechanisms that cause people to repeatedly miss appointments. We use a realist review approach to explore what causes missingness - and what might prevent or address it - for whom, and in what circumstances. The review uses an iterative approach of database searching, citation-tracking and sourcing grey literature, with selected articles providing insight into the causal dynamics underpinning missed appointments and the interventions designed to address them. The findings of this review will be combined with the findings of a qualitative empirical study and the contributions of a Stakeholder Advisory Group (STAG) to inform the development of a programme theory that seeks to explain how missingness occurs, whom it affects and under what circumstances. This will be used to develop a complex intervention to address multiple missed appointments in primary care. CRD42022346006.

Mouse Models for the Study of Borrelia burgdorferi Infection.

Lyme disease, a tickborne illness caused by Borrelia burgdorferi, is an emerging, significant public health concern. B. burgdorferi infections are challenging to study because of their complex life cycle that requires adaptation to both ticks and mammalian hosts for long-term survival and transmission. Bacterial adaptation is accomplished through extensive gene expression alterations in response to environmental cues that remain to be more fully explored. Mouse models of infection serve as valuable tools for studying B. burgdorferi adaptation to the mammalian host and the spirochete's ability to cause persistent infections and thus to interact with and evade the immune system. This article details three mouse models that differ in their primary methods of infection: infestation with B. burgdorferi infected ticks, intradermal inoculation of culture-grown spirochetes, and infection via subcutaneous transplantation of infected tissue. Each method offers unique advantages and limitations. Tick infestation is the route of natural transmission but presents logistical challenges. Syringe inoculation is easy and provides precise control over the infectious dose, but infection is with culture-adapted bacteria. Transplantation of infected tissue introduces mammalian-host-adapted B. burgdorferi in precise anatomical locations, but misses the transfer of tick factors affecting immunity. Detailed protocols are provided for each of the three infection routes, and pros and cons of each method are outlined to help researchers identify the best approach for a research question to be addressed. A protocol is also provided for the treatment of mice with antibiotics that reliably eliminates detectable spirochetes from the animals. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Syringe inoculation of mice with cultured B. burgdorferi and collection of necropsy tissues Basic Protocol 2: Infection of mice with B. burgdorferi via tick infestation Basic Protocol 3: Infection of mice with host-adapted B. burgdorferi via tissue transplant Support Protocol: Clearance of B. burgdorferi by antibiotic treatment.

Navigating challenges and opportunities: perspectives on digital service development in substance use disorder treatment

BackgroundSome people with substance use disorders (SUD) can experience multiple co-occurring social problems. Digital solutions have been developed to support effective and cost-effective social welfare and healthcare in addictions treatment. Given the varying severity of problems from alcohol and other drug use, digital service tools can save money and provide tailored care.ObjectiveIn this study we aimed to understand the perspectives of those who develop digital service tools on people with SUD and treatment encounters. As a case, we interviewed those who have been involved in the development of a digital client segmentation tool The Navigator.MethodsTen (N = 10) semi-structured interviews were conducted with professionals involved in digital client segmentation tool development and were analysed with inductive content analysis. Participants were asked about the development of the Navigator from the perspectives of their own role as developers, the clients, the effectiveness of the services, and decision-making processes.FindingsSome people with SUD may face several obstacles when using digital services. Digital divide, feared or experienced stigma and biased attitudes, complex life situations, and difficulties in committing to treatment were identified as challenges. Nevertheless, digital solutions can offer the clients alternative ways of using the services that can better meet their individual needs. The anonymity and facelessness of digital solutions can reduce the fear of immediate judgement. Implementing digital solutions in substance use work poses challenges due to chronic staff shortages. Digitalisation often results in the creation of multiple simultaneously managed channels, potentially reducing time-consumption but increasing the perceived workload. There is a call for multi-professionalism, acknowledging inequalities between various disciplines within the field.

Comparisons of isotopic niche between females and male alternative reproductive tactics of Chinook salmon (Oncorhynchus tshawytscha) from Lake Ontario

Chinook salmon (Oncorhynchus tshawytscha) have complex life histories with two distinct male alternative reproductive tactics (ARTs). The larger and older “hooknose” males battle for position to spawn with nesting females whereas the precociously maturing “jack” males use a sneaking tactic to gain access to females. Although dietary niche is known to differ between ARTs of other fish taxa, no research to date has examined these differences in a salmonid. Here, we used δ15N and δ13C stable isotope analyses to compare the dietary niches of female, hooknose male, and jack male Chinook salmon sampled from a Lake Ontario tributary. We found that all three life histories shared similar δ15N values, whereas jack males had greater δ13C values compared to hooknose males, with females having intermediate values. Jack males also had notably little overlap in their isotopic niche space with hooknose males and females, indicating that jack males occupy a distinct isotopic niche in Lake Ontario. We discuss habitat partitioning and differences in dietary niche as explanatory factors. Altogether, these data unveil novel distinctions in isotopic niche within the complex life history strategies of Chinook salmon in Lake Ontario that can inform management decisions and provide avenues for future research.

History of Earth evolution

The paper delves deeply into crucial subjects spanning the birth of our solar system to humanitys ascendancy on Earth. It aims to enable humankind to gain a deeper understanding of our own origins and the Earths genesis and evolution. Given that Earth is our sole habitat, we must gain insights into the conditions necessary for it to sustain life or identify potential terrestrial planets suitable for human habitation. Throughout its history, Earth has weathered countless encounters with meteorites, comets, and asteroids, dating back to the formation of the solar system. These events, though rare, played a pivotal role in shaping our planets atmosphere, magnetic field, and unique environmental characteristics. Perhaps the most extraordinary aspect of Earths history is the emergence of life itselfa true marvel. Starting with single-celled protists, life evolved into complex multicellular organisms. Yet, a pivotal moment in Earths history came with a momentous global extinction event triggered by the rise of oxygen. This event reshaped the planets natural ecosystem, leading to the extinction of many species and the emergence of new ones. Humanitys own journey, navigating the depths of history, eventually led to our dominance over the Earth.

Progress on the Regulation of the Host Immune Response by Parasite-Derived Exosomes.

Exosomes are membrane-bound structures released by cells into the external environment that carry a significant amount of important cargo, such as proteins, DNA, RNA, and lipids. They play a crucial role in intercellular communication. Parasites have complex life cycles and can release exosomes at different stages. Exosomes released by parasitic pathogens or infected cells contain parasitic nucleic acids, antigenic molecules, virulence factors, drug-resistant proteins, proteases, lipids, etc. These components can regulate host gene expression across species or modulate signaling pathways, thereby dampening or activating host immune responses, causing pathological damage, and participating in disease progression. This review focuses on the means by which parasitic exosomes modulate host immune responses, elaborates on the pathogenic mechanisms of parasites, clarifies the interactions between parasites and hosts, and provides a theoretical basis and research directions for the prevention and treatment of parasitic diseases.

The Orphan G Protein-Coupled Receptor GPR52 is a Novel Regulator of Breast Cancer Multicellular Organization.

G protein-coupled receptors (GPCRs) are the largest class of membrane-bound receptors and transmit critical signals from the extracellular to the intracellular spaces. Transcriptomic data of resected breast tumors shows that low mRNA expression of the orphan GPCR GPR52 correlates with reduced overall survival in breast cancer patients, leading to the hypothesis that loss of GPR52 supports breast cancer progression. CRISPR-Cas9 was used to knockout GPR52 in human triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, and in the non-cancerous breast epithelial cell line, MCF10A. Loss of GPR52 was found to be associated with increased cell-cell interaction in 2D cultures, altered 3D spheroid morphology, and increased propensity to organize and invade collectively in Matrigel. Furthermore, GPR52 loss was associated with features of EMT in MDA-MB-468 cells. To determine the in vivo impact of GPR52 loss, MDA-MB-468 cells were injected into zebrafish and loss of GPR52 was associated with a greater total cancer area compared to control cells. RNA-sequencing and proteomic analyses of GPR52-null breast cancer cells reveal an increased cAMP signaling signature. Consistently, we found that treatment of wild-type (WT) cells with forskolin, which stimulates production of cAMP, induces some phenotypic changes associated with GPR52 loss, and inhibition of cAMP production rescued some of the GPR52 KO phenotypes. Overall, our results reveal GPR52 loss as a potential mechanism by which breast cancer progression may occur and support the investigation of GPR52 agonism as a therapeutic option in breast cancer.

Peculiarities of the existence of a focus cyatocephalosis among wild and cultivated salmon fishes in the reservoirs of the Izhora upland of the Leningrad region

One of the conditions for maintaining fish health in aquaculture is to control and prevent the presence of disease agents in the water supply sources of fish farms. The main advantages of industrial aquaculture to prevent the spread of parasites can be considered the use of groundwater and pellet feed. The use of an underground water source completely eliminates the possibility of the spread of parasites. In the case of using river, lakes or pounds water the consumption of pellets feed by fish should exclude their eating of invertebrates — intermediate hosts of parasites with a complex life cycle. However, we have found singles Cyathocephalus truncatus (Pallas, 1781) cestodes in juvenile rainbow trout in industrial conditions. This served as the basis for research on the spread of this parasite both in natural reservoirs and fish farms of various types located within the Izhora upland. Cyathocephalus truncatus cestode is a dangerous parasite with a complex life cycle that is widespread mainly in salmon fish. The paper presents experimental data highlighting the impact of fish farming factors and nutritional selectivity on the spread of the parasite. Special attention was paid to the infestation of Salvelinus lepecheni grown in the FSGCR in order to replenish the natural population. As a result of our research conducted over three years, fish of this species have not been invaded by the parasite in both industrial and experimental conditions. This indicates the safety of juveniles reproduced in the FSGCR for settlement in natural reservoirs. In order to determine the boundaries of the natural focus of cyatocephalosis, 106 specimens of salmon fish from 8 reservoirs were tested for infection. Methods of preventing disease in flowing pools are proposed. It has been suggested that the preservation of the focus is ensured by the progenis of the cestode procercoids, excluding the need for a definitive host (fish) to maintain the helminth population in isolated hydraulic water sources.

Temporal and spatial variation of morphological traits and genetic structure in Phengaris teleius myrmecophilous butterflies following habitat and climate changes three decades after reintroduction

A successful reintroduction of Phengaris teleius performed in the Netherlands by translocating 86 individuals from a Polish metapopulation in 1990 represents a unique opportunity to study changes in butterflies from a source and reintroduced metapopulation after such a common conservation practice. Using multilevel comparisons, we tested morphological and genetic changes that occurred after 30 generations since the reintroduction. We also assessed the climatic and connectivity changes that occurred over time in both metapopulation networks. Unexpectedly, we found more significant morphological changes in the current individuals from the source metapopulation, where both sexes had bigger hindwings with different shapes in comparison to the individuals from the original metapopulation in the year of the reintroduction and the ones from the current reintroduced metapopulation. The butterflies from the Dutch metapopulation also had smaller thorax width compared to the ones from the current source metapopulation. The observed morphological changes can be shaped by various factors like changes in climatic conditions and habitat connectivity. Additionally, the genetic analysis revealed a differentiation between the source and reintroduced metapopulation. We found a loss of half of the allelic richness and a bottleneck effect in the reintroduced metapopulation compared to the current Polish one. Our results show that Phengaris butterflies have the potential to adapt to new habitats and respond to climatic changes despite their complex life cycle. A proper long-term habitat management in reintroduced butterfly metapopulations and habitat restoration are key factors influencing the success of reintroduction.

Transcriptomic analyses of differentially expressed human genes, micro RNAs and long-non-coding RNAs in severe, symptomatic and asymptomatic malaria infection

Malaria transmission and endemicity in Africa remains hugely disproportionate compared to the rest of the world. The complex life cycle of P. falciparum (Pf) between the vertebrate human host and the anopheline vector results in differential expression of genes within and between hosts. An in-depth understanding of Pf interaction with various human genes through regulatory elements will pave way for identification of newer tools in the arsenal for malaria control. Therefore, the regulatory elements (REs) involved in the over- or under-expression of various host immune genes hold the key to elucidating alternative control measures that can be applied for disease surveillance, prompt diagnosis and treatment. We carried out an RNAseq analysis to identify differentially expressed genes and network elucidation of non-coding RNAs and target genes associated with immune response in individuals with different clinical outcomes. Raw RNAseq datasets, retrieved for analyses include individuals with severe (Gambia—20), symptomatic (Burkina Faso—15), asymptomatic (Mali—16) malaria as well as uninfected controls (Tanzania—20; Mali—36). Of the total 107 datasets retrieved, we identified 5534 differentially expressed genes (DEGs) among disease and control groups. A peculiar pattern of DEGs was observed, with individuals presenting with severe/symptomatic malaria having the highest and most diverse upregulated genes, while a reverse phenomenon was recorded among asymptomatic and uninfected individuals. In addition, we identified 141 differentially expressed micro RNA (miRNA), of which 78 and 63 were upregulated and downregulated respectively. Interactome analysis revealed a moderate interaction between DEGs and miRNAs. Of all identified miRNA, five were unique (hsa-mir-32, hsa-mir-25, hsa-mir-221, hsa-mir-29 and hsa-mir-148) because of their connectivity to several genes, including hsa-mir-221 connected to 16 genes. Six-hundred and eight differentially expressed long non coding RNA (lncRNA) were also identified, including SLC7A11, LINC01524 among the upregulated ones. Our study provides important insight into host immune genes undergoing differential expression under different malaria conditions. It also identified unique miRNAs and lncRNAs that modify and/or regulate the expression of various immune genes. These regulatory elements we surmise, have the potential to serve a diagnostic purpose in discriminating between individuals with severe/symptomatic malaria and those with asymptomatic infection or uninfected, following further clinical validation from field isolates.

Toward a Natural History of Microbial Life

For most of Earth's history life was microbial, with archaeal and bacterial cells mediating biogeochemical cycles through their metabolisms and ecologies. This diversity was sufficient to maintain a habitable planet across dramatic environmental transitions during the Archean and Proterozoic Eons. However, our knowledge of the first 3 billion years of the biosphere pales in comparison to the rich narrative of complex life documented through the Phanerozoic geological record. In this review, we attempt to lay out a microbial natural history framework that highlights recent and ongoing research unifying microbiology, geochemistry, and traditional organismal evolutionary biology, and we propose six broadly applicable principles to aid in these endeavors. In this way, the evolutionary history of microbial life—once considered only a prelude to the much more storied history of complex metazoan life in the Phanerozoic—is finally coming into its own. ▪The outlines of microbial natural history are now starting to appear through the integration of genomic and geological records.▪Microorganisms drive Earth's biogeochemical cycles, and their natural history reflects a coevolution with the planet.▪Past environmental changes have induced microbial biotic transitions, marked by extinction, taxonomic shifts, and new metabolisms and ecologies.▪Microbial evolution can benefit from a historical perspective of processes and successions as established by macropaleontology.

Predicting fatigue life with the IMWCW under complex loading

The Integration Modified Wöhler Curve Method (IMWCW), a novel approach for predicting fatigue life under complex strain-based non-proportional loading paths is introduced in this research. This method integrates concepts from the Modified Wöhler Curve Method (MWCW) with the principle of damage incremental integration to provide a more nuanced prediction of material fatigue. Besides two established E-N curves (strain-number of cycles) for uniaxial tension and torsion, a new fundamental E-N curve that characterizes the strain-life relationship under complete non-proportional loading paths is introduced. A novel strain path decomposition technique is presented, which dissects an infinite number of instantaneous micro-elements along any loading path into components parallel and perpendicular to the proportional loading direction. It is assumed that each instant will cause fatigue damage to the material. A concurrent model for the incremental integration of damage is formulated, allowing for the calculation of cumulative damage. The model is further expanded to provide analytical expressions for proportional loading and non-proportional loading, linking them to three fundamental E-N curve, so as to calibrate the parameters required for the integration model. The empirical validity of the model is confirmed through experimental testing on 7 different metallic materials under 20 loading conditions, with 391 data points analyzed. The results demonstrate that 91% of the model's predictions fall within a scatter band of 2, confirming its robustness and reliability in forecasting fatigue life in complex, real-world scenarios.

Effective mechanical potential of cell-cell interaction in tissues harboring cavity and in cell sheet toward morphogenesis.

Measuring mechanical forces of cell-cell interactions is important for studying morphogenesis in multicellular organisms. We previously reported an image-based statistical method for inferring effective mechanical potentials of pairwise cell-cell interactions by fitting cell tracking data with a theoretical model. However, whether this method is applicable to tissues with non-cellular components such as cavities remains elusive. Here we evaluated the applicability of the method to cavity-harboring tissues. Using synthetic data generated by simulations, we found that the effect of expanding cavities was added to the pregiven potentials used in the simulations, resulting in the inferred effective potentials having an additional repulsive component derived from the expanding cavities. Interestingly, simulations by using the effective potentials reproduced the cavity-harboring structures. Then, we applied our method to the mouse blastocysts, and found that the inferred effective potentials can reproduce the cavity-harboring structures. Pairwise potentials with additional repulsive components were also detected in two-dimensional cell sheets, by which curved sheets including tubes and cups were simulated. We conclude that our inference method is applicable to tissues harboring cavities and cell sheets, and the resultant effective potentials are useful to simulate the morphologies.

Genome-wide analysis of transcription-coupled repair reveals novel transcription events in Caenorhabditis elegans.

Bulky DNA adducts such as those induced by ultraviolet light are removed from the genomes of multicellular organisms by nucleotide excision repair, which occurs through two distinct mechanisms, global repair, requiring the DNA damage recognition-factor XPC (xeroderma pigmentosum complementation group C), and transcription-coupled repair (TCR), which does not. TCR is initiated when elongating RNA polymerase II encounters DNA damage, and thus analysis of genome-wide excision repair in XPC-mutants only repairing by TCR provides a unique opportunity to map transcription events missed by methods dependent on capturing RNA transcription products and thus limited by their stability and/or modifications (5'-capping or 3'-polyadenylation). Here, we have performed eXcision Repair-sequencing (XR-seq) in the model organism Caenorhabditis elegans to generate genome-wide repair maps in a wild-type strain with normal excision repair, a strain lacking TCR (csb-1), and a strain that only repairs by TCR (xpc-1). Analysis of the intersections between the xpc-1 XR-seq repair maps with RNA-mapping datasets (RNA-seq, long- and short-capped RNA-seq) reveal previously unrecognized sites of transcription and further enhance our understanding of the genome of this important model organism.

Defining context-dependent m6A RNA methylomes in Arabidopsis

N6-Methyladenosine (m6A) prevalently occurs on cellular RNA across almost all kingdoms of life. It governs RNA fate and is essential for development and stress responses. However, the dynamic, context-dependent m6A methylomes across tissues and in response to various stimuli remain largely unknown in multicellular organisms. Here, we generate a comprehensive census that identifies m6A methylomes in 100 samples during development or following exposure to various external conditions in Arabidopsis thaliana. We demonstrate that m6A is a suitable biomarker to reflect the developmental lineage, and that various stimuli rapidly affect m6A methylomes that constitute the regulatory network required for an effective response to the stimuli. Integrative analyses of the census and its correlation with m6A regulators identify multiple layers of regulation on highly context-dependent m6A modification in response to diverse developmental and environmental stimuli, providing insights into m6A modification dynamics in the myriad contexts of multicellular organisms.

Exploring the unknown: how can we improve single-cell RNAseq cell type annotations in non-model organisms?

Single-cell RNA sequencing (scRNAseq) is a powerful tool to describe cell types in multicellular organisms across the animal kingdom. In standard scRNAseq analysis pipelines, clusters of cells with similar transcriptional signatures are given cell type labels based on marker genes, which infer specialized known characteristics. Since these analyses are designed for model organisms, such as humans and mice, problems arise when attempting to label cell types of distantly related, non-model species that have unique or divergent cell types. Consequently, this leads to limited discovery of novel species-specific cell types and potential mis-annotation of cell types in non-model species when using scRNAseq. To address this problem, we discuss recently published approaches that help annotate scRNAseq clusters for any non-model organism. We first suggest that annotating with an evolutionary context of cell lineages will aid in the discovery of novel cell types and provide a marker-free approach to compare cell types across distantly related species. Second, machine learning has greatly improved bioinformatic analyses, so we highlight some open-source programs that use reference-free approaches to annotate cell clusters. Lastly, we propose the use of unannotated genes as potential cell markers for non-model organisms, as many do not have fully annotated genomes and this data is often disregarded. Improving single-cell annotations will aid the discovery of novel cell types and enhance our understanding of non-model organisms at a cellular level. By unifying approaches to annotate cell types in non-model organisms, we can increase the confidence of cell annotation label transfer and the flexibility to discover novel cell types.