Developmental & Comparative Immunology

The honey bee colony (Apis mellifera) acts as a superorganism, with a dual immune system that operates at the individual and social level. However, the linkages between immune mechanisms across the two levels remain poorly understood, despite the relevance for developing effective breeding strategies to improve honey bee disease resistance. Hygienic behavior involving the removal of unhealthy brood is a key component of honey bee social immunity and is highly effective at limiting parasites and pathogens in the colony. While this form of hygienic behavior can reduce brood diseases, parasites infecting adult bees primarily, such as Nosema ceranae, are not directly impacted by the behavior. However, when using the Unhealthy Brood Odor (UBeeO) assay to quantify hygienic behavior performance, hygienic colonies have been shown to maintain lower Nosema spp. loads over time and overall compared to non-hygienic colonies. To investigate the mechanisms driving reduced Nosema spp. in hygienic colonies, we conducted a series of field and lab experiments to test the innate immune performance of individual bees. We evaluated several factors across hygienic and non-hygienic bees including (1) differences in N. ceranae infection levels, (2) survival probability, (3) Vitellogenin and Hymenoptaecin gene expression, and (4) amount of N. ceranae inoculant consumed. We found that hygienic bees consumed less of the inoculant, exhibited upregulated Vitellogenin gene expression at peak N. ceranae infection, showed a positive relationship between Hymenoptaecin gene expression and N. ceranae infection levels, and had greater survivability when infected with N. ceranae, compared to non-hygienic bees. Here, we present new findings that link colony hygienic behavior performance to individual-level resistance and tolerance mechanisms in response to N. ceranae, suggesting broader implications for the success of selective breeding programs targeting hygienic traits.

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.

Waterborne horizontal transmission of viral diseases in fish relies on the release of infectious virus particles (termed shedding) into the aquatic environment. Both the rate and duration of shedding are critical for efficient viral spread, making interventions that reduce shedding valuable for disease control. While vaccines primarily aim to protect individuals from infection and severe disease, they should ideally also limit pathogen transmission by reducing shedding. In this study, we evaluated the capacity of two commercial vaccines - Clynav (DNA vaccine) and AlphaJect Micro 1-PD (inactivated whole-virus vaccine) - to reduce Salmonid alphavirus subtype 3 (SAV3) shedding following experimental infection of Atlantic salmon post-smolts. In individually housed fish, the AlphaJect Micro 1-PD vaccine significantly reduced the proportion of SAV3-shedding fish, the duration of shedding, and the cumulative shedding. The Clynav vaccine significantly reduced the shedding duration and also reduced the proportion of shedding fish. In cohort tanks with concurrent Tenacibaculum dicentrarchi co-infection, the AlphaJect Micro 1-PD vaccine significantly reduced the cumulative shedding but increased the number of shedding days. These results demonstrate the potential of vaccines to limit SAV3 transmission, while also highlighting how co-infections likely influence vaccine efficacy.

Glutathione has important roles in diverse infections, yet its involvement in the interaction between the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd) and its amphibian hosts is still unclear. Using in vitro assays and a cell infection model, we examined how glutathione influences Bd virulence traits and cellular host disease resistance. For Bd, inhibition of glutathione reductase rapidly killed zoospores, indicating that glutathione is essential for this pathogen. In addition, exposure to exogenous glutathione promoted the potential for virulence through accelerated and increased zoospore release. In host amphibian cells, Bd infection decreased intracellular glutathione content and increased reactive oxygen species, suggesting that chytridiomycosis pathogenesis may involve oxidative stress. Depletion of host glutathione before exposure to Bd increased infection severity and Bd growth, whereas amphibian cells with slightly elevated glutathione levels were partially protected against Bd. However, manipulation of host glutathione levels after the establishment of Bd infection did not impact its intracellular growth, implying that the host glutathione-mediated resistance only occurs during the initial Bd invasion process. Importantly, this effect of glutathione on host resistance is not a general response to pathogens, as it was not observed in cells exposed to viral pathogen FV3. As glutathione increased both infectious zoospore production and host resistance to zoospore infection, our study suggests that this antioxidant may play an important role in the host/pathogen interaction during chytridiomycosis. Thus, environmental conditions and therapeutic approaches that affect glutathione systems in the host and/or pathogen have the potential to alter chytridiomycosis dynamics and should be further explored.

Shrimp and other arthropods are capable of specific, adaptive immune responses to viruses based on viral copy DNA (vcDNA) fragments in the host genome called endogenous viral elements (EVE). These may produce negative sense RNA transcripts leading to an RNA interference (RNAi) defense response against cognate viruses. We first reported high-frequency-read sequences (HFRS) of white spot syndrome virus EVE (named WSSV-EVE 4,6,8) in a WSSV-free breeding stock of whiteleg shrimp (Penaeus vannamei). Here we describe screening for the same HFRS-EVE in a captured giant tiger shrimp (Penaeus monodon) breeding stock, also free of WSSV. WSSV-EVE 4,6,8 was detected in some of the P. monodon stock individuals with positive or negative RNA expression. Eight broodstock individuals were selected for mating in 4 crosses. The offspring from these crosses were grown sufficiently to allow tagging and pleopod sampling for DNA and RNA analysis prior to challenge with WSSV. This allowed for Mendelian analysis of EVE inheritance and for its expression or not in the offspring, together with analysis of their relationships to survival and WSSV infection level after challenge. The results revealed that EVE inheritance was Mendelian, but that their RNA expression or not was independently controlled. In Crosses 1 and 2, all the offspring died and none of them carried 2 or more of the expressed EVE in their parental shrimp. In contrast, 100% of 10 arbitrarily selected surviving shrimp from Cross 3 and 90% from Cross 4 carried and expressed 2 or more of the 3 expressed EVE transmitted from the parental shrimp. These results reveal a potential protocol for development of viral tolerant shrimp stocks.

Rift Valley fever (RVF) is a zoonotic arboviral disease that causes adverse pregnancy outcomes and high mortality in domestic and wild ruminants. The disease is caused by the RVF virus (RVFV), which is transmitted by mosquitoes from several genera, mainly Aedes and Culex. However, whether ruminants can become infected by horizontal virus transmission remains unclear. In addition, how the route of RVFV inoculation may influence RVF pathogenesis and the host immune response in animals is still largely unknown. With this aim, we conducted a comparative experimental study in which young sheep were either inoculated subcutaneously (SC) or intranasally (IN) with the virulent RVFV 56/74 strain. We then evaluated disease dynamics, viremia, virus excretion, tissue damage, and the humoral immune response. We also aimed to determine whether RVFV can be transmitted from infected to in-contact animals, and to assess whether the inoculation route may influence virus excretion and the likelihood of subsequent horizontal transmission. The results showed that SC inoculated sheep had a shorter incubation period, an earlier onset of viremia, and an earlier seroconversion. In contrast, IN inoculated animals developed higher rectal temperatures, reached higher peak viremia, and developed a more robust neutralizing antibody response. They also exhibited increased concentrations of analytes indicative of moderate but more severe hepatic injury compared with the subcutaneous group, along with more pronounced histopathological damage in the central nervous system. These results demonstrate the influence of the route of inoculation on RVF pathogenesis and the host immune response. Our results also confirmed the horizontal transmission of RVFV between SC inoculated sheep and in-contact animals housed in the same room, a phenomenon not observed in the IN inoculated group. This finding underscores the influence of the inoculation route on virus transmission and the potentially significant role of horizontal transmission in RVF epidemiology and disease control. Author summaryAccording to the World Health Organization (WHO), RVFV is considered a priority pathogen due to its ability to strain animal and public health systems, especially in developing countries. RVF outbreaks have occurred across most of Africa and, since 2000, in the Arabian Peninsula. Evidence of RVFV circulation in North Africa further highlights the threat to Europe, where competent mosquito vectors are present. How the inoculation route shapes disease dynamics and hosts immunity is still largely unknown. Similarly, whether the virus can spread between infected and non-infected animals without competent vectors remains unclear. A comparative infection in which young sheep were inoculated SC or IN with the RVFV 56/74 strain showed that SC inoculated sheep had a shorter incubation period, an earlier onset of viremia, and earlier seroconversion. However, rectal temperature and peak viremia were higher in IN inoculated sheep, which also showed evidence of moderate but more severe hepatic damage, accompanied by greater central nervous system damage. Only the in-contact animals housed in the subcutaneous group became infected, demonstrating horizontal transmission. Our results show that the route of inoculation influences disease progression and that RVFV can be transmitted among sheep in the absence of mosquitoes.

This report describes lethal Nannizziopsis-associated dermatomycosis in a breeding colony of the family Diplodactylidae (Correlophus ciliatus and Rhacodactylus auriculatus). After introducing one gecko from overseas, three with indirect contact history died due to severe skin lesions. Extensive lesions were observed on the toe pads and ventral surface, along with necrotic dermatitis and cellulitis associated with fungi forming hyphae. Subsequently, four geckos developed diarrhea, melena, emaciation, and fungal dermatitis of the toe pads and died. Histopathologically, the fungal morphologies observed in the skin lesions of the seven geckos were consistent, and Nannizziopsis arthrosporioides was isolated and identified in two of them. To our knowledge, this is the first report of a fatal outbreak of N. arthrosporioides in geckos.

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.

Plumage colour in domestic geese is an important economic trait and a selection target since the early days of domestication. In European domestic geese of greylag goose (Anser anser) origin, white plumage colour is known to be caused by two independent loci, one causing white spotting and one causing sex-linked dilution, together producing white plumage. Strong candidate mutations have been identified upstream of the EDNRB2/LOC106047519 gene (endothelin receptor B-like) and within the sex-linked MLANA gene (melan-A). To confirm these candidate mutations, we genotyped differently coloured European domestic goose breeds, wild greylag geese, Chinese domestic geese (derived from swan goose A. cygnoid) and European and Chinese domestic geese crossbreeds. One base pair deletion in the MLANA gene (NW_013185876.1: g.950,868 C > -) was confirmed to cause sex-linked dilution, and thus autosexing (almost white gander and goose diluted grey). However, mutation upstream of EDNRB2/LOC106047519 (NW_013185915.1: g. 775,151 G > T) was not the causative mutation for saddleback pattern but strongly linked to it in European domestic geese. We sequenced the EDNRB2 gene and coding sequence of a neighbouring VAMP7 gene (vesicle-associated membrane protein 7) but found no genetic variaion linked to colour. Additionally, we sequenced the coding sequence of TYRP1 (tyrosinase related protein 1), a candidate gene for buff colouration, but no variation linked to colour was found. Further, we genotyped a 14-bp insertion in exon 3 of the EDNRB2 gene, known to be causative of the white phenotype in the Chinese domestic goose, and identified it in one European domestic goose individual.

In chickens and humans, classical class I molecules of the major histocompatibility complex (MHC) can have a hierarchy of correlated properties, including cell surface expression and peptide repertoire. Chicken BF2 alleles that are less well-expressed on the cell surface and bind a very wide range of peptides are expressed by MHC haplotypes that confer protection from a variety of economically-important infectious diseases, while certain human HLA-B alleles that are well-expressed and bind a narrow range of peptides lead to slow progression from HIV infection to AIDS. Understanding the impact of these promiscuous generalists and fastidious specialists is of considerable interest. The promiscuous BF2 molecule from the chicken B21 haplotype, BF2*21:01, binds a wide range of peptides by remodelling the peptide-binding site, allowing co-variation of the anchor residues at peptide positions P2 and Pc-2, and binding of an anchor residue at Pc. By using in vitro refolding assays with peptides and peptide libraries, determining thermostability and crystal structures, and analysing a chicken B21 cell line by immunopeptidomics, we found that BF2*21:01 will accommodate many possible combinations at P2 and Pc-2, as well as several hydrophobic amino acids at Pc. However, marked preferences for particular peptide lengths, particular amino acids at the three anchor residues, combinations of amino acids at P2 and Pc-2, and amino acids at P3 and Pc-3 affecting stability lead to high frequencies of major peptides while still allowing the possibility of presenting a wide peptide repertoire. These simplifying principles may eventually allow predictions of pathogen peptides with stable binding for this iconic promiscuous class I molecule, as well as providing the data for more sophisticated peptide prediction methods.

The North American deermouse Peromyscus leucopus is reservoir for several zoonotic agents, including bacterial, protozoan, and viral. It is remarkable for indiscernible or limited fitness consequences of these infections, a trait known as infection tolerance. But experimental infections have largely been of pathogens that P. leucopus naturally harbors. We asked whether infection tolerance extended to an agent, like SARS-CoV-2 virus, it had presumably not encountered before. Following protocols for experiments with mice and hamsters, we infected 8 female and 8 male P. leucopus of heterogeneous stock and compared responses of these animals on days 3 or 6 to those of 14 controls inoculated with virus-free medium. Serologic and virologic confirmation of infection was obtained for all exposed deermice. Moderate inflammation in lungs was histologically evident in infected animals, but no histological changes were noted in brains, even when viral RNA was present. Fourteen (88%) animals displayed no or only mild sickness; two had more severe illness. Genome-wide RNA-seq revealed an interferon-stimulated response on day 3 superceded mainly by a cell-mediated response by day 6. In brains transcription of the interferon-stimulated genes Isg15 and Mx2 positively correlated with viral RNA levels. The findings confirmed susceptibility of this species of Peromyscus to SARS-CoV-2 virus. For most infected outbred animals the immune response was swift and effective in controlling the pathogen and without evidence of excessive inflammation. Whatever is the basis for P. leucopus trait of infection tolerance, it extended to at least one pathogen that for it would be novel. ImportancePeromyscus leucopus is North American rodent that is reservoir for several agents of human disease, while exhibiting minimal illness, a phenotype termed infection tolerance. Whether this trait is pathogen-specific or represents a broader strategy has remained uncertain. By experimentally infecting P. leucopus with SARS-CoV-2 virus, which it is unlikely to have encountered, we investigated whether infection tolerance extends to a novel virus. Despite disseminated infection and lung pathology, most animals showed only mild or no disease. Expression analyses revealed early interferon-stimulated responses followed by cell-mediated responses with only limited production of inflammatory mediators interferon-gamma and nitric oxide synthase 2. Compared with results with a mouse model of infection, deermice displayed higher baseline expression of antiviral genes and quicker resolution of interferon responses. These findings suggest that infection tolerance is a strategy that limits immunopathology generally while resisting microbes, which has implications for understanding reservoir competence and host resilience.

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.

Immunity against pathogens is costly and can adversely affect fitness through resource allocation or physiological trade-offs. Infection by multiple pathogens may further worsen the effects if hosts require the activation of multiple immune responses, which in turn can elevate the overall immunological costs. Poor body condition and stressful environments can also exacerbate these trade-offs. In this study, we experimentally tested these possibilities using Tribolium castaneum populations that were evolving against either a single or a set of coinfecting bacterial pathogens. Contrary to our expectations, none of these evolved beetles showed any measurable trade-offs between pathogen resistance vs major fitness traits, such as reproduction or lifespan. Instead, they appeared to increase reproductive success and resistance to starvation, suggesting improved body condition that could mask underlying fitness costs. However, evolved beetles showed reduced quinone production, an externally secreted antimicrobial defence, indicating trade-offs between internal vs external immunity. Finally, we identified reproductive costs only under limited resource availability, but not under suboptimal resource quality, suggesting that trade-offs can be highly condition-dependent. Overall, this study provides a unique comparison across pathogens and infection types, highlighting the importance of analysing variation in life-history traits within relevant ecological contexts to understand fitness costs of evolving immunity.

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.

Equine endometrosis is a major cause of subfertility in mares characterized by fibrotic remodeling of the endometrium. Although transforming growth factor beta 1 (TGF-{beta}1) is implicated in fibrogenesis, the relationship between endometrosis severity and transcripts associated with tissue maintenance and proliferation remains incompletely defined. Present study evaluated endometrial mRNA expression of IGF1, MKI67, TGFB1, and ACTA2 in relation to endometrosis severity and defined histopathological features. Forty-seven endometrial samples were graded according to the modified Kenney and Doig (KD) categories. Relative mRNA expression was quantified by RT-qPCR and histopathology was extended using a standardized feature-based assessment. TGFB1 mRNA expression was higher in category I+ than in categories I and III (p = 0.041) and in samples with glandular basal lamina disruption (p = 0.020). MKI67 mRNA expression was lower in samples with luminal epithelial erosion (p = 0.049). IGF1 mRNA expression correlated negatively with KD category ({rho} = -0.401, p = 0.015), glandular degeneration ({rho} = -0.340, p = 0.043), overall inflammatory infiltration ({rho} = -0.387, p = 0.020), lymphocytic infiltration ({rho} = -0.426, p = 0.010), and neutrophilic infiltration ({rho} = -0.448, p = 0.006). MKI67 correlated positively with ESR1 ({rho} = 0.887, p < 0.001). These findings indicate that early endometrosis-compatible lesions are associated with increased TGFB1 transcription and that epithelial damage is accompanied by reduced MKI67 expression. The inverse associations between IGF1 expression and both lesion severity and inflammatory infiltration support a link between progressive histopathological changes and reduced expression of a growth factor involved in tissue maintenance.

Edhazardia aedis is an obligate microsporidian parasite of the arthropod vector Aedes aegypti, which is responsible for the spread of several vertebrate pathogens of global health importance. E. aedis can be highly virulent to Ae. aegypti and infection has severely detrimental effects on multiple life history traits that are relevant to the vectoral capacity of Ae. aegypti, including longevity, body size, propensity to host-seek and blood-feed, and reproductive capacity. Because E. aedis is also highly specific to Ae. aegypti and is incapable of completing its full life cycle in any other mosquito species, E. aedis merits investigation as a novel tool for biological vector control. In the present study, we queried the effect of E. aedis infection on the bacterial microbiota of adult female Ae. aegypti using high-throughput amplicon sequencing of the 16S rRNA gene. Analysis of sequencing data revealed that the bacterial microbiota community is strikingly robust to E. aedis infection, as we observed no significant effect on alpha or beta diversity, differential abundance of any taxa, predicted metabolic function profile, or overall bacterial load. The data show that E. aedis, despite dramatically impacting the health and fitness of the adult female mosquito, does not affect the microbiota. These results provide unique insight into tripartite relationships (or lack thereof) between hosts, pathogens, and the microbiota.

It has been well established that females have a more active immune system. Females respond better to vaccines, are more resistant to somatic cell cancers, display better pathogen responses, present antigens better, and, conversely, are more prone to autoimmune diseases compared to male counterparts. Though these trends have been observed across normal and pathogenic states, the mechanisms underlying these sex differences have not been fully explained. Some hormonal effects on immune cell populations have been reported, but much less is known about effects contributed by genes on the sex chromosomes, for example those that are more highly expressed in females due to X inactivation escape, or Y-linked genes those unique to males. Here we use the Four Core Genotypes (FCG) mouse model and wildtype XY male mice to disentangle the effects of sex hormones, sex chromosome complement, and their interactions on baseline B and T cell populations in the periphery and T cells in the thymus. We test the effects of a previously described X-Y chromosomal translocation and those of the Sry transgene insertion on chromosome 3. We observe that mice harboring the Sry transgene show significant depletion of peripheral CD8+ T cell subpopulations. In the thymus, the XY XY,but not the XX males, show significant decrease to both CD8+ and CD4+ single positive T cells and an increase to CD4/CD8 double positive T cells. We also show that Y chromosome-bearing mice exhibit depletion in splenic marginal zone B cells. Our data suggests that the gonadal sex is the strongest contributor to this phenotype. Our studies define a critical framework for the use of this model and provide valuable data to assess the use of the FCGs model, especially for diseases involving the immune response.

We present here a normal table for post-embryonic development in the California newt (Taricha torosa), part of a genus of newts frequently studied for their toxicity and role within a predator-prey relationship. We generated the table by observing larvae collected as eggs in the wild and hatched and reared in the lab through metamorphosis. Building upon an established table consisting of 40 embryonic stages of development, our table consists of 13 stages based on discrete anatomical changes, primarily in limb development, and concludes at Stages 12-13 when the larvae undergo metamorphosis. We also describe more gradual phenotypic changes and their correlation to discrete stages in the developmental timeline. Finally, we illustrate the variability of the timing for reaching these stages in a controlled lab environment, demonstrating that time from hatching is not a reliable metric for standardizing results for diverse studies involving developing larvae. This staging table and accompanying observations will facilitate cross-study integration of research with larval T. torosa.

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.