Developmental & Comparative Immunology

Turbot (Scophthalmus maximus) is an important aquaculture species, but the genomic organization and expressed diversity of its antibody repertoire remain incompletely characterized. In this study, we annotated the immunoglobulin heavy chain (IGH) locus using the haplotype resolved fScoMax1.1 genome assembly, and we used this as a reference to profile the expressed turbot IgM, IgD and IgT repertoires in skin and spleen. The primary IGH locus was located on chromosome 19, spanned approximately 72 kb, and contained 25 IGHV genes, including 24 functional genes and one pseudogene, together with three IGHD, seven IGHJ and three IGHC genes corresponding to IgT, IgM and IgD. Comparison with the alternate fScoMax1.1 haplotype and a second turbot genome assembly showed conserved IGHD, IGHJ and IGHC content, whereas IGHV gene number differed among assemblies. High throughput 5RACE repertoire sequencing revealed isotype and tissue associated differences in expressed IGH diversity. IgM represented the dominant productive repertoire in both skin and spleen and showed the highest clonotypic diversity, particularly in spleen. IgD displayed an intermediate profile, whereas IgT was more enriched in skin and exhibited the strongest clonal restriction. IGHV subgroup usage was dominated by IGHV3 in IgM and IgD, whereas IgT showed a distinct profile characterized by preferential use of IGHV4, especially in skin. Gene level analysis further showed broad IGHV-IGHJ pairing in IgM and IgD, with preferential use IGHJ3 segment, while IgT sequences paired exclusively with IGHJT. Clonotype sharing between skin and spleen was isotype dependent, being strongest for IgT, intermediate for IgM, and negligible for IgD, suggesting that clonal expansion did not necessarily predict inter tissue trafficking. Together, these results provide a curated genomic and expressed repertoire framework for turbot IGH genes and reveal isotype specific organization of antibody diversity, with IgT displaying a particular repertoire pattern.

The migratory endoparasitic pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, causing significant economic and ecological losses in conifer forest ecosystems in Europe and Asia. Understanding the molecular mechanisms regulating PWN parasitism-related genes may lead to new sustainable solutions for control. Based on previous PWN transcriptomic datasets from the pre-parasitic and parasitic stages and from the pharyngeal gland cells (GC), an in silico analysis was performed to identify transcription factors (TF) highly expressed in the GC. Seven candidates TF genes were selected, and their spatial expression validated by in situ hybridisation. From those, two GC-expressed TFs, BXY_079 and BXY_022, each encoding zinc finger domains, were successfully knocked down by RNA interference. Transcriptomic data from silenced BXY_079 and BXY_022 TFs, analysed with existing life cycle specific transcriptomic data, showed that both TFs control genes expressed at similar times, by repressing male-related genes while activating genes expressed during the J3 and D3 stages, yet each represents the extreme of the others minor function. In addition to these common roles, BXY_079 also activates parasitism-related genes in the J2 stage. These BXY_079-activated parasitism-related genes predominantly encode proteins with lytic functions, including secreted peptidases and glycoside hydrolases. Consistent with their proposed role in parasitism, these genes are highly expressed during the parasitic juvenile stages and are likely involved in nematode feeding, tissue penetration, and migration within the host. In contrast, BXY_022 also represses the expression of several genes related to the reproduction system, such as major sperm proteins and cytosolic motility proteins, particularly in the adult male stage. Taken together, both dual-functional TFs work together, non-redundantly, to regulate gene expression across the life cycle, while each is additionally specialised to regulate diverse and distinct gene sets: ranging from genes implicated in lytic parasitic functions to sexual dimorphism.

Exposure to copper, one of the prevalent contaminants in aquatic environments, has wide-ranging adverse immunological effects. However, it is unclear if copper induced immune changes are due to alterations in lymphoid tissues or the result of direct immune cell toxicity. Therefore, to understand the mechanistic action of copper immunomodulation, we utilized an emerging immunotoxicologic model, threespine stickleback fish (Gasterosteus aculeatus). We exposed stickleback fish to dietary copper for a period of 14 days and examined its effect on the spleen, including histopathologic changes in splenic architecture and resident melanomacrophage centers (MMC) populations. We found that dietary copper exposure decreases splenic MMC coverage, suggesting copper is suppressing this phagocyte population. We found no histopathological differences between control and copper groups. Quantification of splenic compartments demonstrated that there is no significant difference in red or white pulp between the control and copper groups, suggesting that reduced MMC coverage is not due to the expansion of other splenic regions. Overall, results from this study suggest that copper toxicity leads to melanomacrophage suppression without damaging or altering the splenic secondary immune tissue structure. Future studies should examine the effect of copper on melanomacrophage viability, development, and function to better understand the mechanism behind MMC reduction. Impact statementThis study demonstrates that dietary copper reduces splenic MMCs but does not cause histopathological damage or alter other compartments of spleen, including the red and white pulp, suggesting that copper-induced immune modulation is the result of direct melanomacrophage toxicity and not due to secondary immune tissue damage. MMC assays could be a useful tool for monitoring heavy metal and other contaminant exposures in aquatic organisms.

Black-footed ferrets (Mustela nigripes) are highly susceptible to sylvatic plague caused by Yersinia pestis, but the genetic basis of this vulnerability remains poorly understood. Here, comparative immunogenomic analyses across Carnivora species identified a conserved class of immunoglobulin lambda variable (IGLV) genes with unusually long antigen-binding sites (CDRL1) that are common among Caniformia species but absent in Feliformia species. First discovered in the domestic ferret (Mustela putorius furo), these genes encode tyrosine-rich and anionic motifs resembling the chemokine receptor CCR5 and contain experimentally validated sulfotyrosines previously associated with pathogen-interacting interfaces. Evolutionary analyses revealed distinct selective pressures across Caniformia lineages and showed strong purifying selection acting on long-CDRL1 IGLV genes in mustelids and bears. Antibody repertoire sequencing demonstrated that these genes are actively utilized in expressed repertoires and that their usage correlates with evolutionary conservation. Functional analyses of monoclonal antibodies derived from the long-CDRL1 IGLV gene identified an antibody that significantly reduced intracellular Y. pestis survival in macrophages and revealed a positive correlation between anti-plague activity and sulfotyrosine signal. Notably, all analyzed black-footed ferrets carried a frameshifting deletion in the long-CDRL1 IGLV gene resulting in loss of its expression in antibody repertoires. Together, these findings uncover a germline-encoded immunoglobulin feature conserved across dog-like carnivores and suggest a potential link between antibody germline variation and immune responses to plague.

The recent shift in Bangladesh from tradition backyard rearing system to modern commercial layer farming has made the birds immune to infectious diseases. Pediculosis, however, continues to pose a challenge in modern production system due to its invasive nature, often going unnoticed and neglected as it is typically non-lethal, yet capable of causing significant production losses. Lice infestation is a persistent threat in poultry production; however, its implications in battery-caged commercial layer hens in Bangladesh remain insufficiently characterized. The study aimed to identify the causative louse species and evaluate its associations with clinical pathology, hematological alteration and productive performance in 30 white-feathered (15 infested + 15 non-infested) and 30 brown-feathered (15 infested + 15 non-infested) laying birds from two commercial farms in Tangail. Morphological characterization confirmed the parasite as Menacanthus stamineus, distinguished by a dorsoventrally flattened body, parabolicallly rounded head wider than long, concealed club-shaped antennae, an oblong-oval abdomen with fine setae and three pairs of short legs each bearing paired claws. Infested birds exhibited consistent clinical pathology, including pale combs, petechial hemorrhages around the vent, severe feather damage with alopecic and exudative areas and incidence of irregular and broken-shelled eggs. Production performance analysis revealed significant reduction in hen-day egg production, egg weight, and feed intake, accompanied by significantly increased feed conversion ratios. Hematological evaluation demonstrated significantly reduced hemoglobin concentration, hematocrit and erythrocyte counts in infested hens, indicating mild anemia and compromised oxygen-carrying capacity. Collectively, pediculosis was strongly associated with lice-induced self-inflicted injury and cannibalism, systemic physiological stress, impaired erythropoiesis, reduced production efficiency and compromised welfare in caged laying hens. To best of our knowledge, it was the first integrative reports from Bangladesh documenting M. stramineus infestation in battery-caged commercial layer system with concurrent evidence of hematological disruption and measurable productivity losses, underscoring its epidemiological and economic significance and urgent need for targeted, evidence-based ectoparasite control strategies.

The corticotropin-releasing factor (CRF) system bidirectionally interacts with cytokines and other immune-related components in mammals. However, the nature of these interactions remains poorly characterized in other vertebrates, including teleost fishes. To gain insight into the relationship between immune responses and the CRF system in teleosts, we explored how CRF system components were transcriptionally regulated in immune organs of rainbow trout (Oncorhynchus mykiss). We first characterized the CRF system in the spleen and head kidney--two primary immune organs in teleosts--and found that many CRF system components were present in both tissues, but splenic expression was consistently greater. Changes in the abundance of splenic CRF system components following vaccination (which transiently stimulated inflammatory responses and cytokine production) indicated contrasting and time-dependent regulation of CRF receptor 1 (CRFR1; suppression) and CRFR2 (stimulation) activities in response to an inflammatory challenge. Using spleen explant cultures, we then evaluated whether these effects were mediated by either of nuclear factor kappa B (NF-{kappa}B; a pro-inflammatory transcription factor) or cortisol (an anti-inflammatory hormone). At baseline, cultured spleens increased cytokine production and exhibited transcriptional changes in CRF system components comparable to those observed following vaccination. Cortisol treatment and NF-{kappa}B inhibition both attenuated the rise in cytokine transcription; however, cortisol treatment generally affected transcripts influencing CRFR1 activity, while NF-{kappa}B inhibition reduced CRFR2 activity. Overall, our data provide novel insight into CRF system regulation in the spleen and suggest that cortisol and inflammatory cytokines differentially regulate CRFR1 and CRFR2 activity within this organ.

The corticotropin-releasing factor (CRF) system is a major neural regulator of stress responses in vertebrates. However, stress-related roles for the CRF system in other tissues--and whether these roles vary between stressor types--remain unclear. To address this gap, we first characterized the CRF system in the gills and intestine of rainbow trout (Oncorhynchus mykiss) and then evaluated how it is transcriptionally regulated following either an immune (vaccination) or osmotic (seawater transfer) stressor. Additionally, since the CRF system is involved in food intake regulation, we also evaluated whether feeding state affects the intestinal CRF system. Vaccination against Vibrio anguillarum reduced CRF system activity in the intestine--as indicated by elevations in CRF binding protein transcripts paired with reductions in ligand (crfa2) and receptor (crfr1b) transcripts--but did not affect the gill CRF system. In contrast, seawater transfer caused the abundance of most CRF system transcripts to increase in the middle (but not posterior) portion of the intestine, while transcript levels of CRF binding proteins and receptors in the gills declined. Finally, levels of CRF system transcripts in the intestine varied with feeding state in a region-specific manner. In the middle intestine, transcript levels of most components declined with fasting and increased when feeding was resumed, whereas the opposite pattern occurred in the posterior intestine. Overall, our results implicate the peripheral CRF system as a stressor- and epithelial tissue-specific modulator of immune and osmoregulatory functions in teleosts.

Human norovirus (hNoV) is the leading global cause of acute gastroenteritis, imposing a substantial health and economic burden worldwide. Progress in understanding hNoV pathogenesis has been hindered by the lack of tractable small-animal models that recapitulate symptomatic infection. Although zebrafish larvae support hNoV replication, infection remains asymptomatic, limiting their utility for studying disease mechanisms and host-pathogen interactions. In this study, we report that the zebrafish embryo infection model, in which microinjection of hNoV at the early cell stage, resulted in robust systemic viral replication accompanied by overt pathological manifestations, including pericardial and renal edema, yolk and cranial opacity, and mortality by 3 days post-infection. Disease severity displayed marked individual variability and correlated closely with viral burden. Integrated multi-omics analyses, including bulk transcriptomics, untargeted metabolomics, and single-cell RNA sequencing, demonstrated that embryonic infection elicits a stronger and more coordinated antiviral response than larval-stage infection, while enabling widespread viral dissemination across diverse cell lineages. Approximately two-thirds of infected cells were derived from the nervous system or neural crest lineages, providing a potential mechanistic basis for the neurological complications occasionally reported in hNoV-infected patients. Furthermore, we identified a developmental stage-dependent role for extracellular vesicle (EV)-associated hNoV transmission: free virions mediated more efficient infection and higher symptomatic incidence in immunologically immature embryos, whereas EV-associated virions exhibited enhanced infectivity in more immunocompetent larvae. Together, these findings establish the zebrafish embryo as a versatile and accessible in vivo platform for studying symptomatic hNoV infection, reveal host maturity-dependent viral transmission strategies, and provide new opportunities for mechanistic studies and high-throughput evaluation of antiviral and vaccine candidates. Author summaryHuman norovirus is the leading cause of stomach flu worldwide but studying it has been difficult because the lack of a simple, small-animal model that actually gets sick from the virus. While older zebrafish larvae can harbor the virus, they do not show symptoms. In this study, we successfully created a new model by injecting human norovirus into zebrafish embryos at their early cell stage. Unlike the older larvae, these embryos developed clear symptoms, including fluid buildup around the heart and kidneys, tissue cloudiness, and death within three days. Using advanced genetic and metabolic tracking, we discovered that the virus spreads widely throughout the embryos body, particularly targeting cells in the nervous system. This link might explain why human patients occasionally suffer from neurological symptoms. Additionally, our study revealed that the virus changes its transmission strategy based on the animals age: it travels freely to infect vulnerable embryos but hides inside lipid vesicles to infect older, more immune-developed larvae. Ultimately, these findings provide a practical, efficient animal model to better understand norovirus sickness and rapidly test new vaccines and treatments.

Naturally occurring variation in the bovine mannose receptor C-type 1 gene (MRC1) may shape macrophage responses to Mycobacterium (M.) bovis, a key driver of bovine tuberculosis (bTB). We identified four coding region SNPs in MRC1 across Bos taurus (Holstein Friesian, Brown Swiss) and Bos indicus (Boran, Sahiwal) cattle breeds, including a non-synonymous variant, rs380943118 (c.2963G>A; Ser988Asn) in C-type lectin-like domain (CTLD) 6, most prevalent in Sahiwal cattle. Structural modelling suggested that the S988N substitution, which is spatially separated from the monosaccharide binding site of CTLD4, might indirectly affect glycan binding, perhaps through a conformational change in the receptor. Monocyte-derived macrophages upregulated MR expression during differentiation, with heterozygous (G/A) animals showing higher MR expression and increased uptake of GFP-M. bovis BCG, although differences were not statistically significant. Anti-CD206 blockade did not inhibit BCG internalization, either indicating that this specific antibody did not bind to a CTLD involved in ligand binding or that MR is not the sole entry receptor. These results highlight naturally occurring MRC1 polymorphisms that may influence MR structure and macrophage function, providing a foundation for future studies to assess their role in bTB susceptibility.

Influenza C (ICV) and influenza D (IDV) viruses belong to the Orthomyxoviridae family and are classified in the genera Gammainfluenzavirus and Deltainfluenzavirus, respectively. Although the main reservoir of ICV is humans, IDV is mainly found in cattle. To date, the zoonotic potential of IDV has not been fully elucidated. ICV and IDV share about 50% homology at the genetic level, and both express hemagglutinin esterase fusion (HEF) glycoproteins on the surface for the dual purpose of binding the receptor and releasing new virions. Using pseudotyped viruses (PVs) in a pseudotyped virus-based microneutralisation assay (pMN), some bovine serum samples showed strong neutralisation of both ICV and IDV. In silico analyses were performed to explore the molecular basis of this phenomenon. HEF structures were recovered from the Protein Data Bank, epitopes were predicted using BepiPred, and sialic acid receptor docking was evaluated with HDOCK. Five potential epitopes were selected, and mutual substitutions of amino acid residues were introduced to generate mutant ICV and IDV HEFs and corresponding PVs. Although only mutant IDV PVs were successfully produced, a reference ICV antiserum showed high neutralising activity against one construct, indicating the exposure of an ICV-like antigenic site within the IDV framework. Herein we provide evidence consistent with the existence of antigenic sites shared between ICV and IDV, which could be exploited for cross-protective vaccine design, through integrated computational and experimental investigations.

Photoreceptor outer segments are sensory cilia whose maintenance depends on a balance between basal disc renewal and tip shedding, controlled by intraflagellar transport and axonemal microtubule organization. Microtubule plus-end proteins regulate microtubule dynamics and are strong candidates for roles in this process. In this study, mCherry-tagged EB1, EB3, and DCX were overexpressed in zebrafish (Danio rerio) cone photoreceptors under a cone-specific promoter. Eyes were examined at 5 and 10 dpf, and eyecup depth, diameter, and cone photoreceptor area were quantified relative to uninjected controls. At 5 dpf, all three constructs produced eyes indistinguishable from those of controls. By 10 dpf, all three constructs significantly increased eye cup depth and cone photoreceptor area. EB1 and DCX also significantly increased eye cup diameter. EB1 and, more severely, EB3 also caused retinal holes, mainly in the retinal pigment epithelium and at the outer nuclear/outer plexiform layer, along with misshapen cells near the inner plexiform layer. DXC did not cause retinal holes, but, like EB1 and EB3, produced enlarged, bulbous cone outer segments. The results show that overexpression of any of the three +TIPs results in a similar eye and photoreceptor overgrowth phenotype, while also producing construct-specific defects: EB1 and EB3 disrupt the broader retinal architecture, whereas DCX produces enlarged eyes. The shared outer segment hypertrophy suggests an imbalance between cargo delivery at the basal end and shedding of the distal tips. The organomegaly may reflect altered progenitor signaling in the ciliary marginal zone.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus with epidemic potential. Despite the threat NiV poses, no therapeutics are licensed to treat infection. Studies have shown that monoclonal antibodies (mAb) can protect animals against NiV and the related Hendra virus (HeV). The best studied mAb, m102.4, has been used to treat infected patients on a compassionate basis, and has entered clinical trials. However, there is a need to define additional mAbs with therapeutic potential, which could be combined with m102.4 to improve neutralising potency and breadth. Here, we isolated five high affinity mAbs from an mRNA immunised pig, which bound the G glycoprotein derived from NiV Malaysia strain (NiV-M), and one of which (mAb A2) also bound HeV G. Aligned with this, all mAbs neutralised NiV-M pseudovirus but only mAb A2 neutralised pseudovirus representing the NiV Bangladesh (NiV-B) strain. mAb A2 and the most potent NiV-M neutralising mAb, C1, showed minimal competition with each other and m102.4, suggesting recognition of non-overlapping epitopes. Single-particle cryogenic electron microscopy of the NiV-M G receptor binding domain complexed to A1 and C2 Fab fragments revealed distinct epitopes that did not overlap with the receptor-binding site, targeted by m102.4, suggesting action through steric impedance of receptor binding or interference downstream of receptor engagement. Inoculation of mAb A2 to hamsters did not provide complete protection against NiV-B challenge (60% survival), however, a split dose of mAb A2 and m102.4 provided the same protection as m102.4 alone (100% survival). Collectively, these data demonstrate the potential of the porcine model for isolation of therapeutic candidate mAbs, which contribute both to our understanding of the NiV G antigenic landscape, and the development of mAb combinations, that exert complementary mechanisms of neutralisation, for therapeutic intervention.

Viruses make long (>40 bp) double stranded RNA (LdsRNA) during replication, which stimulates the innate antiviral immune response. In vertebrates, LdsRNA can induce the type I interferon response (IFN) or the antiviral RNA interference response (dsRNAi) to limit viral replication. Extracellular vesicles (EVs) have previously been shown to carry a variety of nucleic acids for intercellular signaling, and insects and plants have been shown to package LdsRNA in EVs as part of their antiviral immune response. We hypothesized that a similar phenomenon occurs in vertebrates in which EVs traffic LdsRNA between cells during viral infection to induce an antiviral response in naive cells. In this study we showed that both vesicular stomatitus virus (VSV)-derived and in vitro transcribed (ivt)-LdsRNA can be packaged into EVs. LdsRNA was detectable by immunoblots in EVs extracted by both differential ultracentrifugation from ivt-LdsRNA treated U937 and ExoQuick-TCTM precipitation from VSV-infected U937 cells (dsRNA-EVs) but not uninfected controls (control EVs). Isolated EVs were roughly 100 nm in diameter and were able to protect LdsRNA from degradation by RNase III. LdsRNA delivery by dsRNA-EVs was visualized in HEL-299 cells via immunocytochemistry (ICC). The LdsRNA-EVs protected against infection from HCoV-229E, while control EVs did not. Together these results indicate EVs can package and deliver long dsRNA to provide antiviral protection in naive vertebrate cells. Author SummaryWhen viruses infect cells, they produce double-stranded RNA, a molecule that alerts the body to the presence of infection and triggers antiviral defenses. Previous studies have shown that cells can release small membrane-bound packages called extracellular vesicles, which carry biological messages to other cells. However, it was not known whether antiviral double-stranded RNA could be transported in these vesicles and shared with neighboring cells. In this study, we investigated whether human cells package double-stranded RNA into extracellular vesicles and whether this cargo helps protect other cells from viral infection. We found that both synthetic and virus-derived double-stranded RNA were incorporated into extracellular vesicles and shielded from degradation. These vesicles successfully delivered double-stranded RNA to untreated cells, substantially protecting these cells from infection with a human coronavirus. Our findings suggest that cells can communicate antiviral warnings to neighboring cells by packaging double-stranded RNA into extracellular vesicles. This work reveals a previously unrecognized way that antiviral protection may spread through tissues during infection. By extending antiviral signals beyond directly infected cells, extracellular vesicles may help coordinate a broader host defense response. Understanding this natural communication system could also inform the development of new RNA-based antiviral therapies.

Background: Viral myocarditis presents a significant burden of disease, particularly among children and young adults. However, clinical guidelines and treatment strategies for pediatric patients are derived from those for adult patients due to a lack of pediatric data. Current animal models of viral myocarditis use adult mice, so conclusions from these models cannot necessarily be extrapolated to the pediatric population. We sought to develop a juvenile mouse model of myocarditis to examine differences between these two distinct clinical populations. Methods: Male and female BALB/c 3-4-week-old 'juvenile' and 8-week-old 'adult' mice were infected intraperitoneally with 103 PFU of heart-passaged coxsackievirus B3. Sera was used to evaluate testosterone and estradiol levels. Cardiac histological evaluations included overall inflammation, fibrosis, and specific cell-type infiltration. RNA was extracted from cardiac tissue and evaluated for changes in gene expression of cell-type markers, complement components, and NLRP3 inflammasome components. Results: Juvenile mice exhibited more severe inflammation than adult mice but no sex differences in overall inflammation. Juvenile mice demonstrated increased infiltration of CD11b+ cells, F4/80+ cells, and CD3+ T-cells vs. adults. Inflammasome genes NLRP3 and caspase-1 were significantly increased in juvenile compared with adult myocarditis. Conclusions: This paper is the first to describe a juvenile mouse model of coxsackievirus B3 myocarditis and provides a direct comparison to a translational adult mouse model. Juvenile mice had greater cardiac inflammation than adults. This model replicates clinical populations and provides a valuable tool to study age as a factor in the pathogenesis of myocarditis.

Tonate virus (TONV) is a neglected mosquito-borne alphavirus of the Venezuelan equine encephalitis complex associated with febrile illness, encephalitis, and fetal central nervous system abnormalities. Yet, host pathways that sense TONV infection and restrict its replication remain poorly defined. Here, we investigated the interaction between TONV and the type I interferon (IFN-I) system in human cells. We show that TONV infection led to the accumulation of cytosolic double-stranded RNA and a robust IFN response. RIG-I and MDA5 depletion as well as that of MAVS and IRF3 strongly reduced TONV-induced IFN response. Disruption of RIG-I, MDA5, MAVS, or IRF3 resulted in an increase of dsRNA-positive cells and a higher viral RNA accumulation. Finally, we found that treatment with exogenous IFN-I strongly inhibits TONV replication reducing both viral RNA loads and infectious particle production. Thus, together, our results identify the RIG-I/MDA5-MAVS-IRF3 axis as a major pathway sensing TONV infection and establishing an IFN-I-dependent antiviral state, providing the first molecular characterization of TONV innate immune sensing in human cells.

The heterotrimeric G-protein transducin (Gt) is among the key proteins mediating phototransduction in rod and cone cells of the vertebrate retina. Even though this protein has been extensively characterized in mammals, little is known about its expression patterns in migratory songbirds. Here we characterised Gt expression in the European robin, a night-migratory songbird known for its light-dependent magnetoreception. The mechanism underlying magnetoreception is not fully understood, but one well-supported hypothesis involves a radical-pair formation in the blue light receptor cryptochrome type 4a. The - and {gamma}-subunits of cone specific transducin have been identified as possible interaction partners of cryptochrome 4a. Therefore, we analysed the expression patterns of various G-protein subunits in bird photoreceptors. Specifically, we combined single cell RNA sequencing and immunohistochemistry, and tested for protein interaction by pulldown, co-immunoprecipitation, and NanoBiT luminescence assays. We show that genes for G-protein subunits GNB1 and GNB3 (coding for Gt{beta}1 and Gt{beta}3, respectively) are predominantly expressed in rods and cones. Among {gamma}-subunits, GNGT2 (coding for Gt{gamma}T2) was the principal isoform in cones, whereas GNG11 (coding for Gt{gamma}11) was associated with rods. In contrast, we did not detect GNG10 (coding for Gt{gamma}10) expression in either photoreceptor type. Interaction assays demonstrated that all three {beta}{gamma} combinations; {beta}{gamma}T2, {beta}{gamma}10, and {beta}{gamma}11, can associate in vitro. These findings indicate that {beta}{gamma} dimer formation in vivo is likely constrained by the photoreceptor-specific expression of the respective subunits. Furthermore, the absence of GNG10 expression in rods and cones does not support a role of this {gamma}-subunit in photoreceptor-based magnetoreception.

The foundation of coral reef ecosystems centered around the nutritional relationship between corals and intracellular algal symbionts. Although these symbioses are highly obligate for coral hosts, many partnerships are re-established anew with each coral generation. Furthermore, climate change destabilizes the symbiosis, and the cellular mechanisms underlying successful symbiont colonization of hosts and host development, and how they are affected by thermal stress are poorly understood. Here, we explored the effect of algal species and thermal treatments on symbiont and host cell proliferation by offering Acropora tenuis larvae one of four algal species pre-exposed to elevated or ambient temperature. In addition, we characterized the cell-surface glycome of each species-temperature combination to understand its role in symbiont recognition and proliferation. We found that thermal pre-treatment negatively affected algal photosynthetic efficiency and initial symbiont density in hosts, but did not affect symbiont colonization rate or cell proliferation. In contrast, host cell proliferation was affected in a species-specific manner. Thermal pre-treatment of B. minutum and D. trenchii negatively affected host cell proliferation compared to control symbionts, whereas thermal treatment of S. microadriaticum did not affect developmental outcomes. Further, uptake of thermally pre-treated D. trenchii decreased host cell proliferation below that of larvae not offered any symbionts, indicating that this relationship is costly to host development despite the high thermal tolerance of this species. Algal surface glycan composition varied across species but not by thermal pre-treatment, suggesting reductions in density of thermally pre-treated algae may be due to changes in physiology rather than altered surface chemistry. Further, variation in glycan abundance across species did not track differences in colonization rate or symbiont density, hinting towards a smaller role of glycans in host-symbiont specificity.

Vertebrate sex determination is highly diverse, with distinct mechanisms that have evolved repeatedly and independently across phylogeny. The sea lamprey (Petromyzon marinus) is an extant jawless vertebrate that remains sexually labile during its larval stage that lasts around 3-20 years. Their gonad contains bipotential, female, and male germ cells before metamorphosis. To examine the genetic programs in germ cells at different developmental stages, we profiled transcriptomes of dissociated gonadal cells from larvae and recently metamorphosed juvenile (transformers, sex-determined) using fluorescence-activated cell sorting (BD FACS Aria IIu) and PIPseqTM (Fluent BioSciences/Illumina). 66,676 cells were sequenced and classified into 19 cell clusters according to their gene expression profiles. Primordial germ cells differentially expressed genes encoding various heat-shock proteins, hormones, growth factors, and their receptors, and proteins sensitive to iron, nitrogen starvation, sugar and lipid metabolisms, and oxidative stress, providing possible links between environmental factors, cellular functions, and cell fate determinants. Germ cells in larvae had enriched expression of genes involved in DNA replication, transcription, translation, apoptosis, autophagy, cell cycle, cell differentiation, cell adhesion and migration, cell survival, chromatin remodeling, and proliferation. Specifically, female germ cells differentially expressed various transcript isoforms of AEP1, CUZD1, S100A1, and ZP genes, whereas male germ cells differentially expressed BOLL and PIWIL1. Pseudotime trajectory and Gene Ontology (GO) analysis revealed that genes with changing expressions along the female differentiation pathway were involved in binding of sperm to zona pellucida, egg coat formation, prevention of polyspermy and positive regulation of acrosome reaction. On the other hand, genes with changing expressions along the male differentiation pathway were involved in ribosome assembly and translation. Several WNT5 transcripts were differentially expressed in germ cells or somatic cells. Our results suggest that sea lamprey sex determination and sexual differentiation likely involve the coordinated action of numerous genes that interact with various environmental factors and regulate germ cell specification and development.

In the zebrafish visual system, accurate retinotopic mapping occurs throughout life as new neurons are generated and integrated into existing circuitry in both the retina and optic tectum (OT). To explore how OT development changes relative to innervation from the retina, we examined cell death and proliferation in genetic and surgical models that disrupt retinal innervation of the OT. Specifically, we compared lakritz (lak) mutants, which have no optic nerves due to a lesion in the atoh7 gene, with either wild-type or one-eyed fish generated through surgical eye removal. We observed elevated cell death, fewer proliferating progenitors, and fewer sox2+ OT neuroepithelial stem cells in lak mutant and denervated OT lobes. To examine whether light-mediated vision contributes to proliferation and survival in the optic tectum, we reared fish in constant darkness and then compared survival and proliferation of OT cells in innervated and non-innervated tecta. We found that OT cells were still more likely to survive and proliferate in the presence of optic nerve innervation even when fish were reared in the dark. To identify molecular pathways that could regulate OT growth, we examined the expression of known mitogens in the zebrafish optic tectum and found evidence that Wnt/{beta}-catenin pathway activity could promote innervation-dependent proliferation in lak mutant tecta. Expression of both wnt3a and the Wnt/{beta}-catenin target gene axin2, as detected by in situ hybridization and RT-qPCR, is decreased in non-innervated tectal lobes. Further supporting an innervation-dependent role for Wnt/{beta}-catenin pathway activation in the zebrafish OT, we found that lak mutants treated with a Wnt-pathway agonist, BIO, exhibited levels of OT cell proliferation that were indistinguishable from wild-type. Together these findings suggest that progenitor cells in the optic tectum produce Wnt3a in response to innervation by the optic nerve, providing new insight into how a vertebrate visual system coordinates growth across its sensory and recipient tissues.

Advances in immunophenotyping of tumour-infiltrating lymphocytes (TILs) have improved our understanding of prognostic biomarkers and immune targets in human melanoma. However, whether the tumour-immune landscape in canine oral malignant melanoma (COMM) is concordant with that of human melanoma has not been properly defined. To address this gap, we developed a 16-colour spectral flow cytometry panel to characterise TIL phenotypic and functional profiles in COMM. Validation using mitogen-stimulated peripheral blood mononuclear cells from healthy dogs (n = 5) demonstrated robust identification of major T cell lineages, including regulatory T cells (Tregs) and memory subsets, and reliable evaluation of their activation and exhaustion status. COMM patients (n = 8) displayed a distinct protumour microenvironment, characterised by an increased proportion of Tregs, enrichment of tumour-specific exhausted-like T cells co-expressing programmed cell death protein 1 (PD-1) and tumour necrosis factor receptor 2 (TNFR2), and a reduction in cytotoxic CD8 and natural killer T (NKT) cell populations compared with tissue-resident (n = 4) and circulating (n = 8) lymphocytes. Analysis of additional solid tumours, including a mast cell tumour, nerve sheath tumour and adrenal cortical carcinoma, further supported the capability of the panel to identify similar patterns of immune dysregulation across diverse canine tumour landscapes. Collectively, this work describes the first detailed evaluation of canine TIL immunophenotyping using spectral flow cytometry and provides insights into the immunosuppressive mechanisms shaping the tumour microenvironment in COMM. These findings not only increase our understanding of canine tumour immunology but also identify potential immune targets and support ongoing comparative immuno-oncology efforts.