Aquaculture

Pseudomonas aeruginosa is a clinically significant bacterial pathogen that poses a serious threat to aquaculture. However, there are limited information on Massilia isolates against pathogenic P. aeruginosa in aquaculture. In the present study, a facultative predator, M. varians isolate P2-4, was isolated from aquaculture sediment using Chinese mitten crab Eriocheir sinensis-pathogenic P. aeruginosa as the prey bacterium, and its genomic feature, bacteriolysis-related genes, safety, bacteriolytic spectrum, and in vitro and in vivo antibacterial effects against pathogenic P. aeruginosa in E. sinensis were further characterized. Isolate P2-4 consisted of one chromosome and one plasmid (with a total of 75 tRNAs, 7 5S rRNAs, 7 16S rRNAs, 7 23S rRNAs, 34 sRNAs, 5,238 coding genes, 20 genomic islands, 1 prophage, 23 insertion sequences, and 102 repeat sequences), and harbored 19 bacteriolysis-related genes (pilA, pilB, pilC, pilD, pilF, pilG, pilH, pilM, pilO, pilP, pilQ, pilS, pilR, pilT, mltA, mltB, mltC, mltD, and dacB) associated with cellular motility and cell wall lysis. In addition, the isolate carried no virulence genes, was unable to produce haemolysin, hydrogen sulfide, nitrite and ammonia, and avirulent in E. sinensis with a 7-day acute intraperitoneal LD50 value of above 5.0 x 108 CFU/mL. Furthermore, the isolate possessed a wide bacteriolytic spectrum against pathogenic Shewanella algae, Aeromonas caviae, A. hydrophila, and Photobacterium damselae besides P. aeruginosa, exhibited bacteriolysis rates of 99.35% to 99.99% towards the pathogenic P. aeruginosa at 1.0x103 to 1.0x10{square} CFU/mL, and displayed relative percentage survivals of 42.31% to 73.08% against P. aeruginosa infection in E. sinensis at doses of 6.0 x 103 to 6.0 x 105 CFU/g diet. To our knowledge, this study for the first time demonstrates a M. varians strain as a potential biocontrol agent against pathogenic P. aeruginosa in aquaculture.

Telomere length (TL) is increasingly used in ecology as a biomarker of individual quality and environmental stress, yet research on non-model species with complex life histories remains limited. Because TL varies among tissues and across ages in a species-specific manner, identifying non-lethal tissues that reliably reflect whole-organism telomere dynamics is essential for longitudinal telomere studies in the field. This study aimed to evaluate tissue-specific TL in Japanese eel (Anguilla japonica), an endangered catadromous fish. We first mapped the chromosomal distribution of telomeric sequences using fluorescent in situ hybridization (FISH), the first application of this method in this species. We then tested whether muscle and caudal fin, which can be sampled easily and non-lethally, can serve as suitable proxy tissues for TL measurements in wild individuals. Relative telomere length (RTL) was quantified by qPCR in blood, brain, caudal fin, gonads, heart, liver, and muscle. FISH analysis confirmed telomeric repeats at all chromosomal ends, with only weak interstitial signals on three chromosomal pairs unlikely to affect qPCR-based estimates. A generalized additive mixed model and Wilcoxons signed-rank tests revealed significant inter-tissue differences: RTL was shortest in the brain and muscle and longest in liver, blood and caudal fin. Muscle and caudal fin RTL were significantly correlated with RTL in many other tissues, supporting their use as proxy tissues for longitudinal TL monitoring, including responses to environmental variation. Both total length and age were tested as explanatory variables for RTL, and the model including total length showed a better fit than the age-based model. Non-linear relationships between RTL and total length observed in several tissues suggest physiological shifts associated with growth and sexual differentiation. Overall, these findings advance understanding of telomere dynamics in eels and establish muscle and caudal fin as suitable tissues for repeated, non-lethal TL assessment in ecological and conservation contexts.

Development and use of assisted reproductive technologies (ARTs) in non-domestic species provides novel tools for species conservation. As a first step towards in vitro embryo production, we developed an OPU technique for two antelope species, scimitar horned oryx (Oryx dammah) and roan antelope (Hippotragus equinus) utilizing a custom-made needle guide and existing OPU equipment utilized by livestock and human practitioners. Females were anesthetized and placed in sternal recumbency for transvaginal OPUs. Prior to OPUs (36 - 45 hours), SHO and roan were either hormonally stimulated with follicle stimulating hormone (FSH, 140 or 250IU) as a single injection or not. A total of 32 and 26 OPUs were completed in SHO (n=10) and roan (n=7), respectively, representing one to four OPUs per animal at monthly intervals. A total of 141 oocytes were recovered from 215 follicles in SHO and 31 oocytes from 58 follicles in roan. FSH dose (250IU) increased (P<0.05) the number of follicles aspirated and the number of oocytes recovered in SHO. No effects of FSH were observed in roan (P>0.05). Good quality oocytes were recovered from all females and procedures were conducted in four consecutive months with no evidence of scar tissue buildup or reduced capacity to recover quality oocytes. These ARTs can be used to develop in vitro embryo production tools for population management and the preservation of female genetics; bolstering genetic diversity and guarding against extinction.

Conventional semen cryopreservation involves equilibration at 4{degrees}C and optimum freezing rates. We hypothesized that a cholesterol-based semen extender obviates the need for equilibration, minimizing total processing time for semen cryopreservation. Experiments were conducted to determine the effects of semen extender (egg yolk- or cholesterol-based) and freezing method (routine or fast) on post-thaw sperm characteristics and fertility of beef and bison semen. In Experiment 1, beef semen diluted in tris-egg yolk-glycerol (TEYG) or cholesterol-cyclodextrin tris-glycerol (CCTG) extender underwent routine or fast freezing method. Cholesterol from animal and plant origins were compared. The routine method included 90-min equilibration at 4{degrees}C and routine freezing (RE-RF, total time 97 min) whereas the fast method included no equilibration and fast freezing (NE-FF, total time 14 min). Post-thaw sperm quality was assessed by CASA, and in vitro fertilization. Post-thaw sperm motility was not affected by the origin of cholesterol (animal or plant), but was lowest in the TEYG NE-FF group (24% vs 43-51%, P < 0.05). In vitro cleavage and blastocyst development rates did not differ between RE-RF and NE-FF groups. In Experiment 2, bison semen was diluted in TEYG or plant-CCTG extender and frozen as in Experiment 1. Post-thaw sperm motility was lowest in the TEYG NE-FF group (10% vs 39-51%, P < 0.05). In Experiment 3, beef semen diluted in TEYG or plant-CCTG extender underwent either a routine (RE-RF) or modified freezing (NE-RF, total time 25 min) method. Post-thaw sperm characteristics did not differ between extenders but were greater using routine freezing (RE-RF) compared to the modified method of freezing (NE-RF). Pregnancy rates were similar between extenders (TEYG vs plant-CCTG) using the modified freezing method without equilibration and insemination at 72 h after progesterone device removal. In conclusion, beef and bison semen diluted in cholesterol-based extender may be cryopreserved without equilibration.

The genus Trichiurus is the most economically valuable fish in the family Trichiuridae, currently recognized to include 10 valid species. However, historically numerous morphologically similar congeners have been erroneously assigned as synonyms or subspecies of T. lepturus. In this study, we examined 16 hairtail specimens collected from the southern waters of Java Island, Indonesia. Integrated morphological and mitochondrial phylogenetic analyses (COX1 and 16S rRNA), compared against global Trichiurus sequences, revealed that these specimens form an independent lineage that diverged early from other congeners. Consequently, we describe this lineage as a previously undescribed cryptic species. Diagnostic characters include: first anal-fin spine below 36th-37th dorsal-fin rays; anus below 35th-36th dorsal-fin rays; anteriormost tip of supraoccipital well posterior to posterior distal margin of eye; anterior margin of the pectoral-fin spine non-serrated; fangs on both jaws with barb-like processes; upper jaw long, mean 16.6% (15.5-17.6%) of preanal length; snout short, 12.0% (10.9-13.1%) of preanal length; eye small, diameter 5.3% (4.3-5.7%) of preanal length; and absence of hyperostosis on dorsal cranium. We herein propose the name Trichiurus javaensis sp. nov., and provide a formal morphological description and diagnostic characterization of this species.

Intraspecific morphological variability presents a complex challenge for biological systematics and biomonitoring, particularly for organisms with high phenotypic plasticity, such as zooplankton. Morphological differences between individuals of the water flea species Bosmina longirostris (Crustacea: Cladocera) are difficult to distinguish visually, parthenogenetic females look morphologically uniform within the species; nevertheless, they demonstrate differences attributable to their geographic origin and developmental stage. A reference dataset of microscopic images was created for the study, including populations from two geographically separated regions (seven ones from European Russia and seven ones from Sakhalin Island in the Pacific Ocean (Far East of Russia) and two age groups, demonstrating the ability of a neural network classify to successfully the intraspecific morphological variation. This study demonstrates that deep learning methods are prospective for the detection and understanding of fine morphological intraspecific differences in the cladocerans.

Regulation of water permeability in the collecting duct is important for osmoregulatory acclimation in teleost fish. In hyperosmotic environments such as seawater (SW), the teleost kidney functions as a site of divalent ion excretion. The collecting ducts reabsorb Na+, Cl-, and water, thereby reducing urine volume and producing small amounts of isotonic urine with high concentrations of divalent ions. In hypoosmotic environments such as freshwater (FW) or low-salinity brackish water (BW), the kidney produces large volumes of hypotonic urine and serves as a site of water excretion; under these conditions, the collecting ducts reabsorb Na+ and Cl- but not water. To identify aquaporins (Aqps) involved in regulating water permeability in the collecting ducts of teleosts, we analyzed renal Aqp expression in a euryhaline marine fish, the Japanese pufferfish (Takifugu rubripes), which possesses 16 Aqp genes in its genome, seven of which (Aqp1aa, 1ab, 3a, 4a, 7, 8bb, and 11a) are expressed in the kidney. Quantitative RT-PCR analysis showed that Aqp1aa and Aqp4a were highly expressed in collecting duct tissues, and that Aqp1aa expression was markedly reduced in fish acclimated to BW. Immunohistochemistry revealed apical localization of Aqp1aa and basolateral localization of Aqp4 in collecting duct cells, with apical Aqp1aa downregulated in BW. These results suggest that Aqp1aa and Aqp4 mediate water reabsorption in SW and that downregulation of Aqp1aa contributes to hypotonic urine production in BW. NEW & NOTEWORTHYRegulation of water permeability in the collecting duct is important for osmoregulation in teleost fish. Expression analyses of aquaporins (Aqps) in the marine pufferfish Takifugu rubripes showed that Aqp1aa and Aqp4a are highly expressed in the collecting duct and localized to the apical and basolateral membranes, respectively. Renal Aqp1aa expression was markedly reduced in fish acclimated to hypoosmotic brackish water. These results indicate that collecting duct water permeability is regulated by Aqp1aa expression.

In quantitative genetics, candidate SNPs are identified through genotype-phenotype associations inferred with genome-wide association studies (GWAS). In this study, we explore an alternative approach to detect genetic variants with non-neutral effects by tracking temporal trends in allele frequency in a winter wheat (Triticum aestivum L.) breeding population over an eight-year period, from which signals of selection may be inferred. Selection signatures were inferred with a generalized linear model, where we modeled trends in allele frequency as a function of time (crossing year). These signatures of selection were used to prioritize variants. Associations between phenotypic performance and individual load of prioritized variants were then investigated. Furthermore, we assessed whether incorporating selection information into a genomic best linear unbiased prediction (GBLUP) model improves model performance in terms of quality of fit and prediction ability. Our findings indicate that the inferred signals of selection are effective in identifying non-neutral variants. Variants under strong negative selection were associated with a decrease in protein content adjusted for grain yield (p-value < 0.01), while genetic variants that had been under moderate to high levels of positive selection were associated with increased grain yield (p-value < 0.01). However, incorporating selection information did not improve prediction accuracy. In conclusion, temporal trends in allele frequency can be used to detect non-neutral variants. The proposed approach may hence complement traditional quantitative genetic methods for detecting non-neutral genetic variation. This approach may allow breeders to detect non-neutral variants earlier in the breeding cycle, without resorting to phenotypic data.

Giant pangolin (Smutsia gigantea) is one of the least studied pangolin species worldwide, with no published hematological and biochemical data available. We report the first blood parameters from a rehabilitated adult male from Campo Maan National Park (southern Cameroon). Hematological and biochemical findings are described and discussed in relation to available data from other pangolin species. These preliminary results provide the first reference framework for this species and highlight their relevance for clinical assessment, health monitoring, and conservation management.

Soil-transmitted helminth (STH) infections are a major public health burden, and there are programmes of mass drug administration that attempt to ameliorate the harm that they cause. There has been increasing use of genomics to study STH infections and other parasitic nematodes, with particular interest in whole genome sequencing (WGS). For such studies, samples are commonly stored frozen, but in settings where these infections are endemic this can be difficult, and so there would be advantages to having ambient temperature storage methods. We investigated two ambient temperature storage methods - FTA cards and DESS buffer - for infective larvae of the rat parasites Nippostrongylus brasiliensis and Strongyloides ratti, prior to DNA extraction and then WGS. Our results showed that for individual larvae stored on FTA cards or in DESS buffer, this resulted in a lower proportion of sequence reads that mapped to the reference genomes, compared to the frozen control samples. Generally, for individual larvae, DESS-storage resulted in better sequencing results than FTA-storage. However, for pools of 10 or 50 larvae, then these ambient temperature storage methods generally resulted in comparable sequence read mapping to the frozen control samples.

BackgroundThe global rise of multidrug-resistant (MDR) bacteria represents a critical public health threat, and Romania ranks amongst the most affected countries in Europe. As conventional therapy increasingly fails, bacteriophage therapy has re-emerged as a promising alternative to antibiotics. Urban rivers, contaminated with resistant bacterial strains, represent an underexplored and accessible reservoir for the isolation of lytic phages with therapeutic potential. MethodsTwo bacteriophages, 17M_Ec17_D and 22C_Ec22_D, were isolated from the Dambovita River, Bucharest, Romania, using MDR E. coli as host bacteria. Phage characterization included plaque morphology, transmission electron microscopy, and host range assessment by spot assay against 30 MDR E. coli isolates. Whole genome sequencing was performed on Illumina MiSeq and Oxford Nanopore Technologies MinION platforms, followed by bioinformatic analysis including taxonomic classification, lifestyle prediction, and functional annotation. ResultsBoth phages formed clear plaques and were classified as Kayfunavirus (17M_Ec17_D, Podoviridae-like) and Kagunavirus (22C_Ec22_D, Siphoviridae-like) with nucleotide similarities of 89.2% and 71.4% to their closest relatives, respectively, suggesting both are candidates for novel species. Host range analysis revealed lytic activity against 13% and 10% of tested MDR isolates, with complementary infection profiles. Genomic analysis confirmed a strictly lytic lifestyle for both phages, supported by the presence of holin and spanin genes and the absence of lysogenic modules, antibiotic resistance genes, and virulence factors. ConclusionsTo the best of our knowledge, this is the first study conducted in Romania to isolate and genomically characterize lytic bacteriophages targeting MDR E. coli. The characterized phages represent safe therapeutic candidates whose complementary host ranges suggest potential application as part of phage cocktail to broaden antimicrobial coverage against MDR infections.

Seaweed cultivation has recently gained increased attention in North-West Europe as a sustainable source of biomass for biobased products. However, yields need to increase to make the seaweed sector economically viable. To achieve this, higher yielding varieties can be bred but this requires variation for yield and yield-related traits among genotypes. To reliably select high-yielding genotypes, an understanding is required of how both within-farm and between-farm environmental differences affect phenotypes and how to identify simple and reliable proxies for yield. In this study we evaluated growth of nine Saccharina latissima genotypes on two farms, 12 km apart, within the same season. We observed a threefold difference in yield among genotypes, demonstrating the potential for improvement through selection and breeding. Blade thickness and blade size-related traits were strongly correlated with yield, highlighting their potential to serve as rapid and non-destructive proxies for yield, thereby accelerating selection. Furthermore, we demonstrated the importance of adequate replication in farm trials to improve genotype performance estimation by correcting for within-farm spatial variation. Moreover, phenotypic variation was most explained by the genotype and environment, highlighting the importance of both genotype and site selection. Although genotype by environment interactions (GxE) were significant, its contributions were small, indicating stable genotype ranking across farms. Overall, these results are promising for breeding improved S. latissima as it indicates that genotype performance is consistent across close by locations and that local S. latissima populations harbour substantial phenotypic variation that can be used to breed for increased yield. Highlights- Local genetic resources harbour substantial variation in yield and morphology for breeding. - Minor GxE allows for breeding across farms. - Blade thickness and blade size related traits are good predictors of yield. - Correction for on-farm spatial variation improves genotype performance estimation.

Edible seaweed has the potential to become a valuable marine resource for food applications due to its potential health benefits and ecological sustainability. The brown seaweed Alaria esculenta is rich in essential minerals, vitamins, and dietary fibers, making it a nutritious food source. Fermentation, as a traditional preservation method, can enhance seaweed shelf-life and be useful for the development of new foods/ beverages. In this study, the effects of fermentation of A. esculenta, by the lactic acid bacterium (LAB) Lactiplantibacillus plantarum, on the nutritional profile, and the content of potentially toxic elements, was investigated. L. plantarum was successfully cultivated on A. esculenta using two modes of operation, submerged (SmF) and solid-state fermentation (SSF), resulting in production of cells and lactic acid, and reduction of the pH to below 4.3 within 3 days, which was not achieved in parallel spontaneous fermentations using indigenous seaweed microbiota. A. esculenta s macro-nutritional profile was altered, reducing mannitol but increasing fucose and glucose content (after acid hydrolysis) while also concentrating the protein content. LAB fermentation significantly increased the concentration of antioxidant phenolic compounds, such as phloroglucinol, syringic acid, and epicatechin, compared to untreated samples. However, lipophilic compounds like carotenoids decreased after both spontaneous and LAB-fermentation. A reduction in total mineral content was observed after LAB fermentation and water soaking, and SmF with L. plantarum effectively reduced arsenic and iodine levels. Overall, fermentation using L. plantarum showed potential as a bio-preservation method for the edible brown seaweed, A. esculenta, improving its nutritional profile and enhancing food safety.

For over four decades, the bivalve Anomalocardia flexuosa has been recorded in Hong Kong coastal waters. However, the known native distribution of this heavily exploited commercial species is restricted to the Atlantic coast of South America, raising questions about the biogeographical validity of the Hong Kong populations. By employing an integrative taxonomic approach combining morphological re-evaluations and molecular phylogenetic analysis of the COI gene, we confirm that the species in Shui Hau, Hong Kong, China, has been historically misidentified. The population belongs to Cryptonema producta (syn. Anomalocardia producta).

The high efficiency of genome editing presents a challenge when modifying genes associated with viability, welfare, or fertility issues, as implementation of the technology frequently results in mosaic animals with bi-allelic mutations. Combining deactivated Cas9 (dCas9) with Cas9 has been proposed as a strategy to protect one of the two target alleles from editing. We piloted this strategy with 11 genes that are reported as homozygous lethal or associated with welfare issues. We showed that the viability of founders was significantly increased when using 80:20 or 90:10 dCas9:Cas9 ratios, whereas the 70:30 ratio did not yield an equivalent protective effect. The associated overall production rate of mutated founder per manipulated embryo was significantly higher for the 80:20 ratio. Concomitantly, an increased proportion of dCas9 was associated with a significant increase in retention of unedited target alleles but, importantly, did not hinder germline transmission. In addition, editing genes in a paralog cluster with a combination of dCas9 and Cas9 reduced unwanted off-target editing, illustrating a further potential applicability of this approach. This study defines the optimal ratio between dCas9 and Cas9 for strategies aimed at achieving mono-allelic mutations within mosaic founders and proposes a means to reduce the incidence of off-target effects in experiments with limited gRNA options.

Colibacillosis, caused by Avian Pathogenic Escherichia coli (APEC), result in substantial economic losses in global poultry production. The emergence of multidrug-resistant (MDR) APEC poses zoonotic risks through horizontal transfer of antimicrobial resistance (AMR) genes. Bacteriophage therapy emerges as a safe alternative to antibiotherapy; however, comprehensive characterization of phages targeting MDR-APEC from diverse geographical regions remains limited. We isolated five lytic bacteriophages from poultry fecal samples collected from five Indian states and characterized them through morphological analysis, physiological stability testing, whole-genome sequencing, and in vivo efficacy assessment. Host range was determined against APEC isolates, and therapeutic potential was validated in Galleria mellonella infection model. All five phages showed Myovirus-like morphology and stability across physiologically relevant temperatures (up to 55-70{degrees}C) and pH conditions (3-11). Their genome size ranges from 170 to 356 kb, belonging to three distinct genera; Dhakavirus, Gaprivervirus, and Asteriusvirus. Genomic analysis confirmed absence of antimicrobial resistance, virulence, toxin, or lysogeny genes. 51 APEC strains were isolated, of which 23 (45.1%) were MDR. Individual phages lysed 37-51% of tested APEC and 17-39% of MDR strains. Three Escherichia phages (fBSZT1, fUAMT1, fPKPT2) significantly improved larval survival to 60-80% at MOI 10 in G. mellonella infection models compared to untreated controls. This study establishes a well-characterized phage bank targeting MDR-APEC strains, providing foundation for developing phage-based interventions to reduce antibiotic dependency and mitigate AMR transmission risks under One Health framework.

The relationship between Campylobacter levels in broiler caeca and on carcass skin is central to quantitative microbial risk assessment along the poultry production chain, underpinning modelling of intervention impacts, including EFSA assessments of the public health impact of control measures. However, this relationship is typically inferred from monitoring data generated under sampling designs that do not preserve pairing between specimens and may involve pooling. In this study, we used a simulation framework to evaluate whether commonly used sampling strategies allow reliable recovery of the caecal-skin relationship. A simulated broiler population was generated, assigning caecal and skin loads to individual birds based on a specified linear relationship. Sampling was conducted under paired and unpaired designs, with and without pooling, reflecting approaches used in surveillance programmes and in policy-oriented models. Regression models were fitted to sampled data across 1,000 simulations for a range of assumed slopes. Under paired sampling, estimated slopes closely matched the true relationship across most scenarios. In contrast, unpaired sampling consistently failed to recover the association, with estimated slopes centred around zero regardless of the true slope. These findings were robust to variation in within-flock prevalence, residual error, and intercept. The results show that sampling design fundamentally affects identifiability of relationships between stages of the production chain. This has implications for interpretation of parameters derived from monitoring data and used in quantitative Campylobacter risk assessments informing policy. Parameters derived from unpaired and pooled monitoring data should therefore be interpreted with caution when used to support risk assessment and decision-making. Campylobacter; broiler chickens; sampling strategy; unpaired sampling; carcass contamination; quantitative microbial risk assessment; simulation.

Many pathogens, both those with human spillover potential as well as avian-specific viruses, are maintained in wild bird populations. While routine surveillance for influenza A viruses (IAVs) is performed annually, surveillance for other pathogens is limited. Sampling of wild birds is time-consuming, labour-intensive, often limited in sample size, and involves handling of wild and potentially infected birds, posing an increased risk of direct exposure for personnel. Additional methods for surveillance are needed given these significant challenges. Longitudinal fecal and sediment sampling was performed at various sites in southern Manitoba, Canada, particularly focused in Winnipeg from May to October 2025. Sites were chosen based on the suitability of the area for waterfowl habitat, the presence of waterfowl in the area, as well as proximity to reported outbreaks of H5N1 influenza virus. Fecal and sediment samples were collected and screened for the presence of influenza A virus (IAV), Newcastle disease virus (NDV), avian reovirus (ARV), and avian poxvirus (APXV). In total, 782 combined fecal and sediment samples were collected. Of the 714 fecal samples, 34 tested positive for IAV RNA (4.8% prevalence). None of the IAV-positive fecal samples tested positive for H5 RNA. Of the 68 sediments, 15 were positive for IAV RNA (22.1% prevalence), four of which were positive for H5 RNA. NDV RNA positivity was low, with only four positive fecal samples (0.6% prevalence) that were all collected on the same day. ARV RNA positivity was also low, with five positive sediment samples (7.4% prevalence in sediment samples). None of the samples tested positive for APXV DNA. This study builds on previous work showing the utility of environmental sampling for a variety of avian and zoonotic pathogens using a One Health approach that is low-risk, efficient, and high-throughput.

The marbled teal (Marmaronetta angustirostris) is a widely distributed but declining waterfowl species, classified as Near Threatened globally and Critically Endangered in Spain. Despite ongoing conservation actions, including ex situ management and population reinforcement programmes, the genomic consequences of long-term captivity, inbreeding, and patterns of functional genetic variation remain unknown due to the absence of a species-specific reference genome. Here, we present the first chromosome-level genome assembly for this species. The genome was generated using PacBio HiFi long reads and Omni-C data, yielding a 1.15Gb assembly with a scaffold N50 of 76.95Mb. A total of 97.16% of the assembly was anchored into 36 chromosome-scale scaffolds, including the Z and W sex chromosomes. BUSCO analysis recovered 99.2% of conserved avian genes. Gene prediction was performed using both ab initio and homology-based strategies, resulting in 16,048 protein-coding genes. This resource provides a foundation for genomewide analyses of inbreeding, demographic history, and adaptive variation, and will support evidencebased in situ and ex situ conservation strategies for this threatened species.

Gastropods are a highly diverse and often overlooked taxonomic group of significant ecological and economic importance. Some terrestrial gastropods are critical pests of commercial agriculture and home gardens worldwide. Malacopathogenic nematodes offer an effective biological control method of managing pest slugs and snails as a natural enemy. Pellioditis (syn. Phasmarhabditis) hermaphrodita and Pellioditis (syn. Phasmarhabditis) californica are two species of biocontrol nematodes that have been commercialized, sold as Nemaslug(R) and Nemaslug(R) 2.0 respectively on three continents. Although there is interest in bringing Nemaslug(R) products to the US, they are currently not permitted due to limited knowledge on their North American distribution and effects on non-target and native species. In this study, we investigated the impact of P. hermaphrodita and P. californica on Ariolimax columbianus across two slug-host life stages, in laboratory infectivity assays. The objectives were to 1. determine whether P. hermaphrodita and P. californica nematodes impact survival of A. columbianus, and 2. evaluate whether there are differential effects on survival in juvenile and adult life stages of A. columbianus, in laboratory infectivity trials. We found that P. hermaphrodita caused significant mortality in A. columbianus with 100% mortality observed in both juvenile and adult slug hosts. The P. californica treatment had significant effects on the juvenile A. columbianus group only, with 80% mortality. By contrast, only 16% of unexposed control juveniles and 4% of control adult slugs died during the experiment. These results indicate that P. hermaphrodita and P. californica are lethal to the native, non-target Pacific banana slug (A. columbianus) under laboratory conditions, with mortality differing between juvenile and adult host life stages. Given the ecological importance of A. columbianus, these findings raise concerns for potential non-target effects of P. californica and P. hermaphrodita on terrestrial gastropod communities and emphasize the need for testing biocontrol agents against multiple life stages.