Developmental & Comparative Immunology

Cetacean pathology is a cornerstone for population and marine ecosystem health monitoring, allowing clear differentiation among natural and anthropogenic threats. Previous studies in the Canary Islands reported natural causes of death in 59.4% (1999-2005) and 81% (2006-2012) of stranded cetaceans, versus anthropogenic causes in 33.3% and 19%, respectively. This study aimed to determine the causes of death (CD), pathologic findings, and epidemiological patterns of 316 cetaceans stranded in the Canary Islands between 2013 and 2018. The CDs were classified in pathologic entities (PEs) emphasizing natural versus anthropic origins. Of 316 animals, 224 (70.9%) from 18 species were suitable for pathological investigations. Among natural PEE, natural pathology associated with good nutritional status (NP-GNS) and natural pathology associated with significant loss of nutritional status (NP-LNS) represented 43/224 (19.2%) and 36/224 (16%) cases, respectively. Natural pathology with undetermined nutritional status (NP-UNS) occurred in 19/224 (8.5%) animals. Intra- and interspecific traumatic interactions (ITI) represented 30/224 (13.4%) cases, followed by neonatal/perinatal pathology (NPN) 19/224 (8.5%) and live-stranding stress and/or capture myopathy (LS-CM) 18/224 (8%). Infectious and parasitic diseases predominated in natural PEs. Anthropogenic PEs included interaction with fishing activities (IFA) in 17/224 (7.6%) cases, vessel collisions (VC) in 9/22 (4%) cases, and foreign body-associated pathology (FBAP) in 3/224 (1.3%) animals. Overall, anthropogenic causes accounted for 12.9% of deaths, natural causes for 73.6%, and the CD could not be established in 30/194 (13.4%) cases. This study reaffirms the trends concerning recognized PEs (NP-GNS, NP-LNS, NP-UNS, ITI, NPN, LS-CM, IFA, VC, and FBAP), expands the body of knowledge on cetacean pathology in the Canary Islands, and reports novel findings including mixed infections, clostridiosis in uncommon species, uremic syndrome secondary to urethral nematodiasis, gas embolism in unusual species, epibiont stomatitis, congenital musculo-skeletal malformations, or neoplastic processes. These findings advance understanding of cetacean mortality patterns and support conservation and management strategies.

The early evolutionary history of modern domestic horses (Equus caballus/E. ferus caballus), known as the DOM2 lineage, is well documented due to numerous archaeological and ancient DNA (aDNA) studies. Although many uncertainties remain in the domestication timeline, current evidence suggests that the domestication of modern horses began in the Pontic-Caspian steppe at least [~]2700 BCE (before common era), or even earlier. However, it is not known how long remnant wild horse populations survived or when domestic horses were introduced into Northern Europe. In this study, we review the current knowledge of horse domestication, focusing on Northern Europe. We analysed prehistoric horses from western Russia to assess the body sizes of wild horses from the Ivanovskaya site (5900-3800 BCE) in the Pontic-Caspian steppe, and the body weight of one Lithuanian wild horse (4000-3800 BCE). Additionally, we analysed body sizes of Late Bronze Age-Early Roman Age horses (1100 BCE-300 CE; common era) and re-analysed body sizes and estimated rider weights of historic domestic horses from Lithuania (100-1400 CE). We searched for pathological changes and signs of bit wear indicative of bridling. Furthermore, we investigated maternal genetic diversity by sequencing ancient mitochondrial DNA. We found that wild horses from Ivanovskaya were intermediate in body size between earlier and more recent horses of the Eurasian Steppe, and that the Lithuanian wild horse weighed only [~]270 kg and Late Bronze Age-Early Roman Age horses 200-300 kg. Lithuanian domestic horses were pony-sized (< 130 cm on average). Bit wear was confirmed on one tooth, the oldest domestic horse in Lithuania (799-570 cal BCE). Another tooth showed signs of the Equine Odontoclastic Tooth Resorption and Hypercementosis (EOTRH) condition. Mitochondrial DNA was successfully amplified from one Ivanovskaya wild horse along with 25 other ancient samples, including Lithuanias oldest domestic horse. mtDNA diversity was high, revealing several maternal lineages.

Toll-like receptors (TLRs) are vital components of the innate immune system, recognizing both exogenous pathogens signals (PAMPs) and internal stress signals (DAMPs). TLR2 is unique among the human (Homo sapiens) TLR family members, as it contains a large cavity for binding hydrophobic ligands, such as lipoteichoic acid (LTA) and di/triacyl lipopeptides (Pam2/3CSK4). This study analyzed the structural phylogeny of cavity presence in the TLR2 lineage in vertebrates (vTLR) enabled by AI protein structure predictions and explored the potential convergent evolution of similar features in invertebrates (iTLRs). Analysis of AI models of TLR2s shows that this cavity is consistently present in TRL2 orthologs across jawed vertebrates (Gnathostomata). In jawless vertebrates (Cyclostomatha), these cavities were found in lamprey (Petromyzon marinus) TLR2 model, but only in some extant hagfish (Myxini), suggesting an ancestral origin in basal vertebrates followed by lineage-specific losses. TLR2 paralogs were found in several species, with a similar central cavity but potentially different ligand specificities. In silico ligand docking showed Pam2CSK4 binds to this cavity in all TLRs and paralogs consistently, demonstrating the conserved function of the ligand-binding pocket in gram-positive bacteria recognition across TLR2 branches. Changes in the TLR2 cavity size and shape in some vertebrate groups show the evolution of this DAMP recognition mechanism adapted to its respective pathogens. iTLRs form a separate phylogenetic branch with distinct structural features, but in literature some are considered to be TLR2 orthologs. Indeed, TLRs from some species of Helobdella and Ciona, contain a cavity with some similarity to that in the vTLR2 lineage. However, detailed structural comparisons of their location in the LRR domain and the structural details of the models suggest that their cavities have developed independently from that in TLR2s. Smaller cavities are present in other branches of the LRR family, but show different locations, shapes, and features, indicating that the binding of small ligands in the internal cavities within the LRR domains evolved multiple times in the LRR domain family history.

Animals sense and integrate complex external cues to make developmental decisions that help them better survive and adapt to their natural habitats. Under environmental adversity, nematodes can enter an alternative developmental pathway to form a diapautic and stress-resistant stage, termed the dauer larvae. While dauer formation has been well characterized in Caenorhabditis elegans, how environmental factors influence analogous stages in other nematode species remains largely unexplored. This study examines how symbiotic bacteria, temperature, and pheromones affect the formation of the infective juvenile (IJ), a dauer-like stage, of the insect-parasitic nematode Steinernema hermaphroditum. In contrast to C. elegans, where dauer entry is promoted by heat, IJ development in S. hermaphroditum development is enhanced by reduced temperature. Moreover, the presence and absence of live symbiotic bacterium Xenorhabdus griffiniae functions as an ON-and-OFF switch that regulates the host IJ formation. Crude pheromone extracts from S. hermaphroditum liquid culture do not robustly induce IJ formation in a dose-responsive manner, unlike the potent pheromone-driven dauer entry observed in C. elegans. Nutrient-rich liver-kidney media that mimics host insect environment showed IJ entry induction in a pheromone-dependent manner. These data suggest that external cues, such as temperature, microbial diet, and pheromone, are perceived differently by S. hermaphroditum in comparison to that of C. elegans, reflecting species-specific adaptations to distinct ecological niches and life history strategies.

Understanding behavioral differences between non-native and closely related endangered species could be important to aid conservation management. In volume 169 of Zoology, Bels et al. (2025) reported on their comparison of display-action-patterns (DAP) between native Iguana delicatissima and non-native iguanas present on islands of the Guadeloupe Archipelago in the Caribbean Lesser Antilles. Here, we address conceptual and methodological concerns about their work and reanalyze their data given our proposed corrections, primarily a literature-informed adjustment of their "species" category. We additionally utilize online videos from South American mainland I. iguana populations, from where the non-native iguanas in the Guadeloupe Archipelago originate, to better understand the different DAPs between native and non-native iguanas in the Guadeloupe Archipelago. Significant differences in DAP characteristics among "species" categories (native I. delicatissima, non-native iguanas, and hybrids) show that Bels et al. (2025) oversimplified their data analyses by merging all non-native populations into one group. This result indicates the presence of behavioral variation among subpopulations within widely hybridizing iguanid populations, which has been poorly studied. Additionally, videos from mainland populations across two major mitochondrial clades of Iguana iguana show that non-native iguanas on Guadeloupe retained DAP characteristics of those populations from which they originate. We discuss these findings in light of the proposed hypotheses put forward by Bels et al. (2025), of which two can be excluded. Overall, our reanalysis shows that studies focusing on characteristics within settings of complex hybridization in diverse species should acknowledge this complexity.

Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development.

Helminths are parasitic worms that secrete a variety of immune-regulating molecules to modulate the hosts inflammatory responses, enabling them to persist within the host over a long period of time, even decades. Their capacity to control host responses has prompted research into helminth-derived molecules as potential therapies for controlling excessive immune and inflammatory activity across a range of diseases. This systematic review with cross-study quantitative analysis aims to synthesize the published data on helminth-derived immunomodulatory peptides/polypeptides/proteins (HDIPs) with a focus on determining the extent of their disease-modifying and anti-inflammatory potential in in vivo animal models of inflammatory disease. In accordance with PRISMA 2020 guidelines, a predefined systematic search of the PubMed, Web of Science and Medline databases identified relevant studies published up to February 2026, and 65 articles were included after screening. We found that, although the HDIPs were assessed in multiple different disease models, most published studies assessed their potential in mouse models of colitis, asthma, arthritis and sepsis. Twenty species from which >65 isolated HDIPs were derived were tested in these models, with the trematode, Fasciola hepatica, and the nematode, Acanthocheilonema viteae, the most explored species. A common property of the HDIPs was the ability to significantly reduce disease severity across the in vivo animal models of inflammatory disease, underpinned by a decrease in pro-inflammatory cytokine levels and an increase in anti-inflammatory cytokine levels. Overall, this systematic review with cross-study quantitative analysis not only synthesizes the existing literature in this field but also highlights the disease-modifying and anti-inflammatory potential of HDIPs for a range of diseases in which immunoregulatory therapeutics may improve disease outcomes. It also encourages accelerated advancement of these helminth-derived molecules into first-in-human clinical trials.

Introducing cognitive enrichment from an early age has the potential to enhance an animals capacity to learn both simple and complex tasks, promote neural plasticity, and support cognitive development. This is applicable for young cattle who are at a critical stage in their development and could benefit from the influence cognitive enrichment has on their behavioral expression. This study aims to explore the effects cognitive enrichment has on weaned dairy calves through analyzing behavioral measures of voluntary participation and short-term behavioral reactions to enrichment exposure. Our study involved a total of five pairs of weaned calves (n=8 treatment; n=2 control). The treatment groups were presented with three variations of a puzzle box, each equipped with unique challenges that offer different solutions (push, slide, pull). These boxes were provided to the calves twice daily over the span of nine days in an isolated corridor located behind their pen. We hypothesized that motivated calves would consistently engage with cognitive enrichment voluntarily over time and express directed natural behaviors, reflecting sustained participation across repeated trials. Results demonstrated that calves consistently visited the cognitive enrichment area across trials, with an average latency of 75.7 {+/-} 47.0s from the pen to the enrichment. Secondly, the calves spent a significant proportion of trial time within the enrichment area at 65% (870.1 {+/-} 21s). Lastly, all calves expressed a broad range of behaviors in line with their natural exploration within the enrichment area, while the puzzle box treatment groups expressed higher durations of behavioral expressions when compared to the control (F=11.7, p<0.0001). Combined, these results indicate the calves motivations to voluntarily participate in a cognitive challenge. While these are promising findings for cognitive enrichment and its applicability to dairy calves, further work is needed to understand broader parameters. Specifically, how can social dynamics influence enrichment interaction in groups, how can this type of enrichment be implemented on farms, and what are the long-term effects to providing cognitive enrichment in the early stages of development.

Immune memory responses are rapid and qualitatively distinct from primary responses. They typically develop in the presence of antigen-experienced memory T and B cells and pre-existing antibodies. Although the contribution of T and B cells to recall responses is well defined, the contribution of antibody "memory" and the mechanisms by which pre-existing antibodies modulate the development of germinal center and plasma cell responses is not precisely understood. Here we report on mechanisms that mediate antibody enhancement of germinal center (GC) and plasmablast (PB) compartments, and the parallel process by which they change the affinity threshold for B cell recruitment into immune responses. The data indicate that antibody-mediated enhancement of GC and PB responses is Fc gamma receptor (Fc{gamma}R) dependent and largely complement receptor 1 and 2 (CR1/2) independent. In contrast, the reduction in the affinity threshold for GC entry is independent of both Fc{gamma}Rs and CR1/2. SummaryCipolla et al. show that antibody can modulate immune responses via both Fc gamma receptor dependent and independent mechanisms. These mechanisms influence both the magnitude and composition of the germinal center response.

As global reptile populations continue to decline, improving reproductive success in managed populations of listed species, such as Phrynosoma cornutum (the Texas horned lizard) has become increasingly critical for species survival. One understudied area of reproductive research in reptile species is gamete collection and storage, a crucial component for maintaining genetic diversity. In Texas, semen was collected from wild P. cornutum (n = 20) in June 2025. Semen collection was performed via electroejaculation (EEJ) under alfaxalone anesthesia. Prior to semen collection, snout-vent-lengths (SVL) and weights were recorded and testes measurements were taken using a portable ultrasound. Average sperm motility and concentration across all lizards was 83.7% and 85.7 x 106 sperm/mL, respectively. While lizards with longer SVLs had higher sperm motility, weight and testis size did not affect sperm parameters. Samples were extended in INRA96 and divided for use in cold-storage longevity or cryopreservation trials. Samples under cold-storage conditions were assessed for motility daily for 10 days. Motility was not significantly reduced until 48 hours post-collection and maintained 19% motility at day 10. For cryopreservation, samples were diluted 1:1 in INRAFreeze cryopreservation media and frozen in liquid nitrogen, then immediately thawed. Average post-thaw sperm motility was 13.9%, with the highest post-thaw motility recorded at 38.2%. This is the first report of semen storage and cryopreservation in Phrynosoma and provides valuable insight into semen storage potential in reptile species.

In mammals, most of the iron is found in the heme of red blood cells (RBCs), which must be recycled to support erythropoiesis in the bone marrow. Splenic red pulp macrophages (RPMs) play a crucial role in this process by phagocytosing senescent RBCs, metabolizing the heme and releasing iron back into the blood. Free cytoplasmic iron generates toxic reactive oxygen species, yet iron-specific adaptations of RPMs are not well documented. We previously reported that autophagy prevents ferroptosis in Langerhans cells, a cutaneous phagocyte subset. Thus, we hypothesized that autophagy may be important for the regulation of RPM metabolism and their maintenance of systemic iron homeostasis. To study this, we used Atg5flox/flox and Cd169cre mouse models to delete ATG5 in CD169+ macrophages, including RPMs. Atg5-deficient RPMs were decreased in number, and the remaining ones showed increased generation of toxic lipid peroxides. Spleens of Atg5{Delta}Cd169 mice were enlarged and contained more RBCs. Finally, autophagy impairment in RPMs exacerbated RBC loss in a model of phenylhydrazine-induced anemia. Our findings exemplify how dysregulation of macrophage metabolism alters their function and can disrupt tissue homeostasis upon challenge.

We present the first radiographic ageing framework for common dolphins (Delphinus delphis), based on ossification and epiphyseal fusion patterns in the pectoral flipper, demonstrating higher reliability for chronological age estimation than currently available epigenetic approaches for this species. Using individuals of known dental age, we calibrated two modelling approaches to predict dental age from radiographic bone scores: 1) a univariate polynomial regression using a total bone score (sum of 16 scores across all assessed flipper bones), and 2) a multivariate canonical analysis of principal coordinates (CAP) incorporating 16 individual bone-score variables. Both approaches successfully predicted dental age from skeletal ossification patterns. For an age range of 0 to 24 years, polynomial regression demonstrated high predictive accuracy with median absolute errors (MAEs) of 1.25 years in females (Spearmans {rho} = 0.93, R{superscript 2} = 0.90) and 1.08 years in males ({rho} = 0.95, R{superscript 2} = 0.86). The CAP model yielded MAEs of 1.35 years in females ({rho} = 0.90, R{superscript 2} = 0.85) and 1.80 years in males ({rho} = 0.94, R{superscript 2} = 0.84). Notably, both radiographic bone ageing models achieved equal or lower median absolute errors and higher coefficients of determination than a recently developed epigenetic clock for common dolphins derived from the same population (MAE = 1.80, Pearsons correlation (r) = 0.91, R{superscript 2} = 0.82). When applying the bone ageing models to individuals of unknown dental age, both models produced age estimates consistent with expected life-history stages (foetus, neonate, juvenile, subadult, adult), although accuracy declined in dolphins above 20 years, likely as a consequence of subtle age-related variation in skeletal changes in this species. Radiographic ageing provides an accurate non-invasive tool for demographic assessment to support conservation management of common dolphins.

The outcome of a Plasmodium infection depends on the timely regulation of the robust pro-inflammatory response required to eliminate the parasite, but this response can cause tissue damage if not properly controlled. IL-10 is an important regulatory cytokine that prevents immunopathology during many Plasmodium infections; however, this protection comes at the expense of less effective parasite control. This is illustrated by infection with P. yoelii, in which mice exhibit a lower parasite load in the absence of IL-10. However, the immune components that limit parasite burden in the absence of IL-10 remain poorly understood. Abolishing IL-10 led to a predicted increase in TH1 polarization and higher production of IL-12 and IFN-{gamma}. However, the enhanced production of these cytokines did not explain the improved parasite control seen in Il10-/- mice. Loss of IL-10 signaling reduced the accumulation of germinal center B cells and plasmablasts in the spleen, indicating a role for IL-10 in supporting the humoral response. However, although B cells are essential for survival, they do not play a critical role in early parasite control in IL-10-deficient mice. Moreover, Il10-/-mice lacking IFN-{gamma} and B cells can limit early parasite expansion, suggesting that IL-10 suppresses host-protective pathways beyond the functions of B cells and IFN-{gamma} in parasite control.

Expression of the Csf1r gene in cells of the mononuclear phagocyte lineage is regulated by a conserved enhancer, the fms-intronic regulatory element (FIRE). In mice with a germ-line deletion of FIRE (Fireko) CSF1R expression is undetectable in bone marrow progenitors and classical monocytes. Fireko mice lack subpopulations of macrophages in the brain and periphery but develop normally. Here we show that loss of CSF1R expression in Fireko mice is partly overcome by CSF2 in vitro and inflammatory recruitment in vitro. Analysis of heterozygous mutant mice and deletion of the conserved AP1 motif in FIRE provide evidence that continuous receptor synthesis determines CSF1 responsiveness. The absence of macrophages in kidney and heart of Fireko mice was not associated with detectable loss of physiological function. In a model of renal injury macrophage recruitment and histopathology were similar in WT and Fireko mice. Tissue resident macrophages that were depleted in Fireko mice, including microglia, were replaced by donor-derived cells following intraperitoneal adoptive transfer of wild-type bone marrow at weaning. The Fireko mouse provides a novel platform to dissect the functions of tissue resident macrophages in development, homeostasis and pathology. Summary StatementThis study describes a unique model of selective tissue resident macrophage deficiency arising from dysregulated expression of the mouse Csf1r gene.

In Toxoplasma gondii, microneme proteins (MICs) are secreted components of the apical complex that play central roles in motility, host cell attachment, and invasion. Because proteins at the host-parasite interface are often predicted to evolve rapidly, MICs have been suggested as candidates for adaptive diversification. We tested this expectation using comparative analyses of three relatively understudied microneme proteins, MIC13, MIC12, and MIC16. Coding sequences were assembled from GenBank and ToxoDB, aligned by translation, and analyzed using maximum-likelihood phylogenetics, codon-based tests of selection, and predicted protein structure. MIC13 was represented by 51 sequences, MIC12 by 30, and MIC16 by 34, spanning multiple T. gondii haplogroups and including Hammondia hammondi and Neospora caninum as outgroups. All three genes were highly conserved among T. gondii strains, but their phylogenetic trees were topologically incongruent, indicating that individual MICs do not recover a single shared strain history. Contrary to expectation, no positively selected codons were detected in any gene. Instead, purifying selection was detected at one site in MIC13 and 15 sites in MIC12, while no significant codon-specific selection was detected in MIC16. Several constrained MIC12 sites overlapped annotated EGF and calcium-binding EGF-like domains, consistent with structural conservation of extracellular adhesion modules. AlphaFold prediction of MIC13 supported two sialic acid-binding micronemal adhesive repeat regions, but the single constrained MIC13 site did not overlap these motifs. Together, these results indicate that MIC13, MIC12, and MIC16 are shaped more by sequence conservation and heterogeneous gene histories than by strong recurrent positive selection. These findings refine expectations for microneme evolution in T. gondii and highlight conserved domains that may be important for parasite invasion and future functional study.

Parasitism is one of the key, structural, interspecific interactions in ecology. One remarkable parasitic strategy that has been documented in multiple systems is the behavioral manipulation of hosts to increase parasite fitness. While not yet documented in humans, we propose that a ubiquitous zoonotic parasite - Toxoplasma gondii - may change human behavior to favor the parasite by increasing the fitness of the parasites definitive host - cats. Specifically, we assess the possibility that human behavioral changes resulting from chronic, latent T. gondii infection lead to measurable changes in attitudes, actions and dopaminergic responses towards cats that function to increase domestic cat fitness. We assessed the potential role of humans in the T. gondii lifecycle by identifying and testing behavioral changes in humans that benefit the parasite; specifically, human affection for cats. We assessed T. gondii infection status in 68 participants using T. gondii serum antibody testing, and assessed their attitudes towards cats in three ways: i) surveys, ii) participant behavior in the presence of domestic cats, and iii) participant oxytocin levels before and after interactions with cats to assess dopaminergic changes. Only 2 of 68 participants were positive for T. gondii antibodies, limiting statistical power. However, our results indicated that T. gondii-positive participants both reported a greater affection for cats in surveys, and spent more time engaged with cats during behavioral trials than T. gondii-negative participants (87% of study time engaging with cats vs 75%). Oxytocin results were inconclusive.

Iron is an essential nutrient for all types of organisms, including insects and the microbes that infect them. We predicted that insects fed an iron-supplemented diet would accumulate more iron in their hemolymph, and, because infectious microbes acquire iron from their hosts, that this extra iron would increase the severity of bacterial infections. To test this hypothesis, we studied the effects of dietary iron supplementation on infection outcomes in Manduca sexta (tobacco hornworm). Larvae were fed an artificial diet, with or without antibiotics, or the same diets supplemented with 10 mM iron. Control and iron-treated larvae were inoculated with non-pathogenic Escherichia coli or the entomopathogenic Enterococcus faecalis, and bacterial load and larval survival were measured. We found that dietary iron supplementation increased the iron content of hemolymph by approximately 20 fold; however, contrary to our prediction, this increase in iron did not result in an increase in the bacterial load of either E. coli or E. faecalis. The effect of iron supplementation on survival was more complicated. As expected, for larvae inoculated with nonpathogenic E. coli, iron supplementation had no effect. For larvae inoculated with E. faecalis, the effect of iron supplementation depended on whether antibiotics were present in the diet. Without antibiotics, iron supplementation prolonged larval survival; with antibiotics, iron supplementation decreased larval survival. The results of this study do not support the hypothesis that dietary iron supplementation increases infection severity in M. sexta. Instead, the results support the viewpoint that the relationship between dietary iron and infection outcome is complex.

Autophagy is a catabolic process used for the degradation of organelles and proteins. Macroautophagy involves the formation of autophagosomes and subsequent fusion with lysosomes to mediate cargo degradation. It also functions as a cellular defence mechanism, known as xenophagy, during infection. Previous studies show that different viruses manipulate the autophagy pathway of the host cell to assure successful replication and/or virion assembly. Vaccinia virus (VACV), the prototypic poxvirus, replicates exclusively in the cytoplasm of host cells. It is known that VACV infection causes LC3 lipidation and prevents autophagosome formation, yet the double membrane vesicles formed during autophagy do not serve as the source of the mature VACV membrane. To date the viral protein(s) causing increased LC3 lipidation have not been identified. Here we developed an image-based screening approach based on LC3 granularity to identify candidate VACV genes affecting its lipidation. We identify several candidate viral membrane proteins as effectors of LC3 lipidation, suggesting that the interplay between VACV and autophagy is more directed than previously thought.

Unbiased classification of equine dendritic cells (DC) is necessary to address various research questions such as the role of DC subsets in immune-mediated diseases of horses. We applied single-cell RNA sequencing (scRNA-seq) on DC enriched from the blood of two horses. All main DC subsets were detected by key gene expression, including conventional DC type 1 (cDC1; XCR1) and type 2 (cDC2; FCER1A, CD1E) as well as plasmacytoid DC (pDC; TCF4). In addition, we detected a small cluster of hematopoietic progenitors, as well as transitional DC (tDC; FCER1A, TCF4) and putative DC type 3 (DC3; FLT3, CD163). Our data confirms the previously reported phenotype of equine pDC (Flt3+MHC-IIlowCADM1lowCD172aint), cDC1 (Flt3+MHC-IIhighCADM1highCD172alow-int) and cDC2 (Flt3+MHC-IIhighCADM1intCD172ahigh), while also highlighting considerable CD14 expression for cDC2. Two subclusters of equine cDC2 were found to be enriched in FCER1A or CX3CR1 transcripts (cDC2.1 and cDC2.2, respectively), with suggested enhanced extravasation and T-cell stimulatory capacities of the latter. Conservation of DC subsets across species (horse, pig, human, mouse) was illustrated by enrichment analyses with subset-specific gene signatures and by cross-species data integration with publicly available scRNA-seq datasets. Our atlas of equine blood DC is a valuable resource for comparative analyses, and it forms the foundation for understanding the involvement of distinct DC subsets in infections and immune-mediated pathologies.

In wildlife forensic practice, species identification and estimation of the Minimum Number of Individuals (MNI) for highly processed specimens have long relied on weight-based conversion methods, which may result in underestimation of the number of individuals involved in a case. Focusing on confiscated casque products of the helmeted hornbill (Rhinoplax vigil), this study combines macroscopic morphological examination with mitochondrial DNA barcoding (16S rRNA, COI, and Cytb) to explore a more robust approach for individual quantification. The results demonstrate that the conventional "weight-based" approach overlooks critical biological information contained in anatomical structures and cannot accurately reflect the actual number of individuals involved. Based on this, we propose an anatomy-based criterion centered on the principle of structural uniqueness: specimens retaining biologically unique beak or casque structures should be directly assigned to a single individual, whereas weight-based estimation should only be applied when original anatomical features are entirely absent. In addition, considering material loss during processing, we propose approximately 85 g as a reference threshold for estimating the number of individuals in heavily processed solid casque products. This approach improves the scientific rigor and accuracy of forensic identification and provides reliable technical support for the conviction, sentencing, and law enforcement of wildlife trafficking cases involving helmeted hornbill and other endangered species.