Beyond population size: whole-genome data reveal bottleneck legacies in the peninsular Italian wolf.

Preserving genetic diversity and adaptive potential while avoiding inbreeding depression is crucial for the long-term conservation of natural populations. Despite demographic increases, traces of past bottleneck events at the genomic level should be carefully considered for population management. From this perspective, the peninsular Italian wolf is a paradigmatic case. After being on the brink of extinction in the late 1960s, peninsular Italian wolves rebounded and recolonized most of the peninsula aided by conservation measures, including habitat and legal protection. Notwithstanding their demographic recovery, a comprehensive understanding of the genomic consequences of the historical bottleneck in Italian wolves is still lacking. To fill this gap, we sequenced whole genomes of thirteen individuals sampled in the core historical range of the species in Central Italy to conduct population genomic analyses, including a comparison with wolves from two highly-inbred wolf populations (i.e., Scandinavia and Isle Royale). We found that peninsular Italian wolves, despite their recent recovery, still exhibit relatively low genetic diversity, a small effective population size, signatures of inbreeding, and a non-negligible genetic load. Our findings indicate that the peninsular Italian wolf population is still susceptible to bottleneck legacies, which could lead to local inbreeding depression in case of population reduction or fragmentations. This study emphasizes the importance of considering key genetic parameters to design appropriate long-term conservation management plans.

Moyamoya disease presenting with tubular dysfunction in a child: pitfalls in diagnosing an atypical hyponatremic-hypertensive syndrome.

BACKGROUND: Moyamoya disease, a cause of pediatric stroke, has been shown to affect furthermore extra-cranial districts, mostly the kidney arterial site, resulting in steno-occlusive changes. Unilateral renal artery stenosis accounts for 8%-10% out of cases of renovascular hypertension in childhood, however it rarely underlies a hyponatremic-hypertensive syndrome (HHS). CASE PRESENTATION: We describe an 18-month-old boy with a recent history of polyuria and polydipsia, who presented an acute febrile gastroenteritis with neurological impairment, severe dehydration, hyponatremia, hypokalemia, kidney tubular dysfunction, and elevated aldosterone and renin even with a normal blood pressure. Fluid and electrolytes correction was performed, with complete recovery. An abdominal ultrasound displayed a smaller right kidney. A brain magnetic resonance and an electroencephalogram did not show any relevant abnormalities. Five months later, the child experienced a left-side hemiparesis after a traumatic concussion, and a severe hypertension. A brain tomography documented a cerebral ischemia. Brain and kidney angiographic studies displayed puff of smoke findings of internal right carotid artery branches and a steno-occlusive pattern of right renal artery, respectively. Hence, moyamoya disease with HHS secondary to unilateral renal artery stenosis was diagnosed. After an unsuccessful antiplatelet and antihypertensive pharmacological treatment, the boy underwent a renal angioplasty and a cerebral STA-MCA bypass (direct superficial temporal artery-to-middle cerebral artery bypass), resulting in a significant improvement of both neurological and kidney disease. CONCLUSIONS: Although the association between unilateral renal artery stenosis and HHS has been previously shown, this is the first report of atypical HHS, with hypertension preceded by tubular dysfunction, recognized in the framework of moyamoya disease.

Comparing effects and action mechanisms of BPA and BPS on HTR-8/SVneo placental cells†.

Bisphenol A (BPA) is one of the most investigated compound as a suspected endocrine disrupting chemical. It has been found at nM concentrations in the maternal serum, cord serum, and amniotic fluid and also permeates placental tissues. Attempts are being made to replace BPA with the analog Bisphenol S (BPS). Also BPS was found in maternal and umbilical cord serum, and urine samples from a large population of pregnant women. A few studies investigated BPA impact on the placentation process, and even less are available for BPS. This work aimed to elucidate and compare the effects of BPA and BPS on physiological functions of HTR-8/SVneo cells, derived from extravillous trophoblast of first-trimester pregnancy. Proliferation and migration ability of trophoblast cells were assessed in vitro after exposure to BPA or BPS (10-13-10-3 M). Further, induction of the inflammatory response by the bisphenols was studied. To provide insight into the molecular pathways implicated in the responses, experiments were carried out in the presence or absence of tamoxifen as estrogen receptors (ERs) blocker, and U0126 as ERK1/2 phosphorylation inhibitor. Data indicate that BPA significantly affects both proliferation and migration of HTR-8/SVneo cells, through ER and ERK1/2 mediated processes. Differently, BPS only acts on proliferation, again through ER and ERK1/2 mediated processes. BPS, but not BPA, induces secretion of interleukins 6 and 8. Such effect is inhibited by blocking ERK1/2 phosphorylation. To the best of our knowledge, these are the first data showing that BPS affects trophoblast functions through ER/MAPK modulation.

Genomic evidence for the Old divergence of Southern European wolf populations.

The grey wolf (Canis lupus) is one of the most widely distributed mammals in which a variety of distinct populations have been described. However, given their currently fragmented distribution and recent history of human-induced population decline, little is known about the events that led to their differentiation. Based on the analysis of whole canid genomes, we examined the divergence times between Southern European wolf populations and their ancient demographic history. We found that all present-day Eurasian wolves share a common ancestor ca 36 000 years ago, supporting the hypothesis that all extant wolves derive from a single population that subsequently expanded after the Last Glacial Maximum. We also estimated that the currently isolated European populations of the Iberian Peninsula, Italy and the Dinarics-Balkans diverged very closely in time, ca 10 500 years ago, and maintained negligible gene flow ever since. This indicates that the current genetic and morphological distinctiveness of Iberian and Italian wolves can be attributed to their isolation dating back to the end of the Pleistocene, predating the recent human-induced extinction of wolves in Central Europe by several millennia.

The Sicilian Wolf: Genetic Identity of a Recently Extinct Insular Population.

Historically, many local grey wolf (Canis lupus) populations have undergone substantial reductions in size or become extinct. Among these, the wolf population once living in Sicily, the largest island in the Mediterranean Sea, was completely eradicated by human activity in the early decades of the 20th century. To gain a better understanding of the genetic identity of the Sicilian wolf, we used techniques for the study of ancient DNA to analyze the mitochondrial (mt) variability of six specimens stored in Italian museums. We were able to amplify a diagnostic mtDNA fragment of the control region (CR) in four of the samples. Two of the samples shared the same haplotype, differing by two substitutions from the currently most diffused Italian wolf haplotype (W14) and one substitution from the only other Italian haplotype (W16). The third sample showed a previously unreported wolf-like haplotype, and the fourth a haplotype commonly found in dogs. All of the wolf haplotypes analyzed in this study belonged to the mitochondrial haplogroup that includes haplotypes detected in all the known European Pleistocene wolves and in several modern southern European populations. Unfortunately, this endemic island population, which exhibited unique mtDNA variability, was definitively lost before it was possible to understand its taxonomic uniqueness and conservational value.

Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog.

BACKGROUND: Genomic methods can provide extraordinary tools to explore the genetic background of wild species and domestic breeds, optimize breeding practices, monitor and limit the spread of recessive diseases, and discourage illegal crossings. In this study we analysed a panel of 170k Single Nucleotide Polymorphisms with a combination of multivariate, Bayesian and outlier gene approaches to examine the genome-wide diversity and inbreeding levels in a recent wolf x dog cross-breed, the Czechoslovakian Wolfdog, which is becoming increasingly popular across Europe. RESULTS: Pairwise FST values, multivariate and assignment procedures indicated that the Czechoslovakian Wolfdog was significantly differentiated from all the other analysed breeds and also well-distinguished from both parental populations (Carpathian wolves and German Shepherds). Coherently with the low number of founders involved in the breed selection, the individual inbreeding levels calculated from homozygosity regions were relatively high and comparable with those derived from the pedigree data. In contrast, the coefficient of relatedness between individuals estimated from the pedigrees often underestimated the identity-by-descent scores determined using genetic profiles. The timing of the admixture and the effective population size trends estimated from the LD patterns reflected the documented history of the breed. Ancestry reconstruction methods identified more than 300 genes with excess of wolf ancestry compared to random expectations, mainly related to key morphological features, and more than 2000 genes with excess of dog ancestry, playing important roles in lipid metabolism, in the regulation of circadian rhythms, in learning and memory processes, and in sociability, such as the COMT gene, which has been described as a candidate gene for the latter trait in dogs. CONCLUSIONS: In this study we successfully applied genome-wide procedures to reconstruct the history of the Czechoslovakian Wolfdog, assess individual wolf ancestry proportions and, thanks to the availability of a well-annotated reference genome, identify possible candidate genes for wolf-like and dog-like phenotypic traits typical of this breed, including commonly inherited disorders. Moreover, through the identification of ancestry-informative markers, these genomic approaches could provide tools for forensic applications to unmask illegal crossings with wolves and uncontrolled trades of recent and undeclared wolfdog hybrids.

Disentangling Timing of Admixture, Patterns of Introgression, and Phenotypic Indicators in a Hybridizing Wolf Population.

Hybridization is a natural or anthropogenic process that can deeply affect the genetic make-up of populations, possibly decreasing individual fitness but sometimes favoring local adaptations. The population of Italian wolves (Canis lupus), after protracted demographic declines and isolation, is currently expanding in anthropic areas, with documented cases of hybridization with stray domestic dogs. However, identifying admixture patterns in deeply introgressed populations is far from trivial. In this study, we used a panel of 170,000 SNPs analyzed with multivariate, Bayesian and local ancestry reconstruction methods to identify hybrids, estimate their ancestry proportions and timing since admixture. Moreover, we carried out preliminary genotype-phenotype association analyses to identify the genetic bases of three phenotypic traits (black coat, white claws, and spur on the hind legs) putative indicators of hybridization. Results showed no sharp subdivisions between nonadmixed wolves and hybrids, indicating that recurrent hybridization and deep introgression might have started mostly at the beginning of the population reexpansion. In hybrids, we identified a number of genomic regions with excess of ancestry in one of the parental populations, and regions with excess or resistance to introgression compared with neutral expectations. The three morphological traits showed significant genotype-phenotype associations, with a single genomic region for black coats and white claws, and with multiple genomic regions for the spur. In all cases the associated haplotypes were likely derived from dogs. In conclusion, we show that the use of multiple genome-wide ancestry reconstructions allows clarifying the admixture dynamics even in highly introgressed populations, and supports their conservation management.

Correction to: Parma consensus statement on metabolic disruptors.

CORRECTION: After publication of the article [1], it has been brought to our attention that the thirteenth author of this article has had their name spelt incorrectly. In the original article the spelling "Laura Rizzir" was used. In fact the correct spelling should be "Laura Rizzi".

Assessing the environmental hazard of individual and combined pharmaceuticals: acute and chronic toxicity of fluoxetine and propranolol in the crustacean Daphnia magna.

Pharmaceuticals are widespread emerging contaminants and, like all pollutants, are present in combination with others in the ecosystems. The aim of the present work was to evaluate the toxic response of the crustacean Daphnia magna exposed to individual and combined pharmaceuticals. Fluoxetine, a selective serotonin re-uptake inhibitor widely prescribed as antidepressant, and propranolol, a non-selective ß-adrenergic receptor-blocking agent used to treat hypertension, were tested. Several experimental trials of an acute immobilization test and a chronic reproduction test were performed. Single chemicals were first tested separately. Toxicity of binary mixtures was then assessed using a fixed ratio experimental design. Five concentrations and 5 percentages of each substance in the mixture (0, 25, 50, 75, and 100%) were tested. The MIXTOX model was applied to analyze the experimental results. This tool is a stepwise statistical procedure that evaluates if and how observed data deviate from a reference model, either concentration addition (CA) or independent action (IA), and provides significance testing for synergism, antagonism, or more complex interactions. Acute EC50 values ranged from 6.4 to 7.8 mg/L for propranolol and from 6.4 to 9.1 mg/L for fluoxetine. Chronic EC50 values ranged from 0.59 to 1.00 mg/L for propranolol and from 0.23 to 0.24 mg/L for fluoxetine. Results showed a significant antagonism between chemicals in both the acute and the chronic mixture tests when CA was adopted as the reference model, while absence of interactive effects when IA was used.

Insights into the regulation of the MXR response in haemocytes of the Mediterranean mussel (Mytilus galloprovincialis).

This study investigated functional and transcriptional modulation of the Multixenobiotic resistance (MXR) system as a cytoprotective mechanism contributing to the physiological chemoresistance of haemocytes in the Mediterranean mussel. Basal transport activity was assessed using the model substrate rhodamine 123 and specific inhibitors for the MXR-related transporters P-glycoprotein (ABCB mRNA) and Multidrug resistance-related protein (ABCC mRNA). Results showed that MXR activity in mussel haemocytes was mainly supported by the Mrp-mediated efflux. In agreement, ABCC was expressed at higher levels than ABCB. Activation of the cyclic-AMP (cAMP) dependent protein kinase A (PKA) resulted in increased rhodamine efflux, which was counteracted by the selective PKA inhibitor H89. Although serotonin, a physiological modulator of cAMP/PKA signaling and ABCB transcription in haemocytes, did not affect basal MXR transport, the environmental pharmaceuticals fluoxetine, propranolol, and carbamazepine, which interact in different ways with the adrenergic and serotoninergic pathways, were showed to act as modulators and substrates of MXR-related transporters and to affect cell viability. While the increased MXR activity may have lowered the cytotoxic effects of propranolol and carbamazepine, the lack of MXR efflux induction by fluoxetine may play a role in the observed cytotoxicity of the compound.

Choosy Wolves? Heterozygote Advantage But No Evidence of MHC-Based Disassortative Mating.

A variety of nonrandom mate choice strategies, including disassortative mating, are used by vertebrate species to avoid inbreeding, maintain heterozygosity and increase fitness. Disassortative mating may be mediated by the major histocompatibility complex (MHC), an important gene cluster controlling immune responses to pathogens. We investigated the patterns of mate choice in 26 wild-living breeding pairs of gray wolf (Canis lupus) that were identified through noninvasive genetic methods and genotyped at 3 MHC class II and 12 autosomal microsatellite (STR) loci. We tested for deviations from random mating and evaluated the covariance of genetic variables at functional and STR markers with fitness proxies deduced from pedigree reconstructions. Results did not show evidences of MHC-based disassortative mating. Rather we found a higher peptide similarity between mates at MHC loci as compared with random expectations. Fitness values were positively correlated with heterozygosity of the breeders at both MHC and STR loci, whereas they decreased with relatedness at STRs. These findings may indicate fitness advantages for breeders that, while avoiding highly related mates, are more similar at the MHC and have high levels of heterozygosity overall. Such a pattern of MHC-assortative mating may reflect local coadaptation of the breeders, while a reduction in genetic diversity may be balanced by heterozygote advantages.

Identification of TLR4 as the receptor that recognizes Shiga toxins in human neutrophils.

Hemolytic uremic syndrome (HUS) caused by intestinal Shiga toxin-producing Escherichia coli infections is a worldwide health problem, as dramatically exemplified by the German outbreak occurred in summer 2011 and by a constant burden of cases in children. Shiga toxins (Stx) play a pivotal role in HUS by triggering endothelial damage in kidney and brain through globotriaosylceramide (Gb3Cer) receptor targeting. Moreover, Stx interact with human neutrophils, as experimentally demonstrated in vitro and as observed in patients with HUS. A neutrophil-protective role on endothelial damage (sequestration of circulating toxins) and a causative role in toxin delivery from the gut to the kidney (piggyback transport) have been suggested in different studies. However, the receptor that recognizes Stx in human neutrophils, which do not express Gb3Cer, has not been identified. In this study, by competition and functional experiments with appropriate agonists and antagonists (LPS, anti-TLR4 Abs, respectively), we have identified TLR4 as the receptor that specifically recognizes Stx1 and Stx2 in human neutrophils. Accordingly, these treatments displaced both toxin variants from neutrophils and, upon challenge with Stx1 or Stx2, neutrophils displayed the same pattern of cytokine expression as in response to LPS (assessed by quantitative RT-PCR, ELISA, or multiplexed Luminex-based immunoassays). Moreover, data were supported by adequate controls excluding any potential interference of contaminating LPS in Stx-binding and activation of neutrophils. The identification of the Stx-receptor on neutrophils provides additional elements to foster the understanding of the pathophysiology of HUS and could have an important effect on the development of therapeutic strategies.

Parma consensus statement on metabolic disruptors.

A multidisciplinary group of experts gathered in Parma Italy for a workshop hosted by the University of Parma, May 16-18, 2014 to address concerns about the potential relationship between environmental metabolic disrupting chemicals, obesity and related metabolic disorders. The objectives of the workshop were to: 1. Review findings related to the role of environmental chemicals, referred to as "metabolic disruptors", in obesity and metabolic syndrome with special attention to recent discoveries from animal model and epidemiology studies; 2. Identify conclusions that could be drawn with confidence from existing animal and human data; 3. Develop predictions based on current data; and 4. Identify critical knowledge gaps and areas of uncertainty. The consensus statements are intended to aid in expanding understanding of the role of metabolic disruptors in the obesity and metabolic disease epidemics, to move the field forward by assessing the current state of the science and to identify research needs on the role of environmental chemical exposures in these diseases. We propose broadening the definition of obesogens to that of metabolic disruptors, to encompass chemicals that play a role in altered susceptibility to obesity, diabetes and related metabolic disorders including metabolic syndrome.

Neuroendocrine functions of monoamines in invertebrates: Focus on bivalve molluscs.

Monoamines (MA) such as serotonin, catecholamines (dopamine, norepinephrine, epinephrine), and trace amines (octopamine, tyramine), are neurotransmitters and neuroendocrine modulators in vertebrates, that contribute to adaptation to the environment. Although MA are conserved in evolution, information is still fragmentary in invertebrates, given the diversity of phyla and species. However, MA are crucial in homeostatic processes in these organisms, where the absence of canonical endocrine glands in many groups implies that the modulation of physiological functions is essentially neuroendocrine. In this review, we summarize available information on MA systems in invertebrates, with focus on bivalve molluscs, that are widespread in different aquatic environments, where they are subjected to a variety of environmental stimuli. Available data are reviewed on the presence of the different MA in bivalve tissues, their metabolism, target cells, signaling pathways, and the physiological functions modulated in larval and adult stages. Research gaps and perspectives are highlighted, in order to enrich the framework of knowledge on MA neuroendocrine functions, and on their role in adaptation to ongoing and future environmental changes.

Identification of the core competencies of pediatric hematology-oncology nurses: A Delphi study.

PURPOSE: To identify the core competencies of the pediatric hematology-oncology nurse. METHODS: A Delphi study was conducted. After identifying the competencies of the pediatric hematology-oncology nurse through a literature review, a questionnaire was created to be administered to a panel of experts to obtain consensus. The panel of experts consisted of 19 nurses. For each competence identified, it was necessary to evaluate the competence in terms of "understanding" and "relevance", assigning a score from 0 (not at all) to 4 (completely), according to a Likert scale. Then, the experts identified what could be the necessary "level of work experience" within which a nurse should become autonomous in every competency choosing among the levels: beginner, intermediate, expert. Consensus among the experts was reached in two rounds. RESULTS: After submitting the document to two rounds, a final document was obtained with a total of 126 competencies, divided into nine areas. Fifty-eight competencies fall into the "beginner" level, 46 competencies into the "intermediate" level and 22 competencies into the "expert" level. CONCLUSIONS: The document produced by this study could be a starting point for organizing specific training courses for nurses in the pediatric hematology-oncology department. A clear description of specific competencies for pediatric hematology-oncology nurses would help ensure the best care for the child.

Single exposure of food-derived polyethylene and polystyrene microplastics profoundly affects gut microbiome in an in vitro colon model.

Microplastics (MPs) are widespread contaminants highly persistent in the environment and present in matrices to which humans are extensively exposed, including food and beverages. MP ingestion occurs in adults and children and is becoming an emerging public health issue. The gastrointestinal system is the most exposed to MP contamination, which can alter its physiology starting from changes in the microbiome. This study investigates by an omic approach the impact of a single intake of a mixture of polyethylene (PE) and polystyrene (PS) MPs on the ecology and metabolic activity of the colon microbiota of healthy volunteers, in an in vitro intestinal model. PE and PS MPs were pooled together in a homogeneous mix, digested with the INFOGEST system, and fermented with MICODE (multi-unit in vitro colon model) at loads that by literature correspond to the possible intake of food-derived MPs of a single meal. Results demonstrated that MPs induced an opportunistic bacteria overgrowth (Enterobacteriaceae, Desulfovibrio spp., Clostridium group I and Atopobium - Collinsella group) and a contextual reduction on abundances of all the beneficial taxa analyzed, with the sole exception of Lactobacillales. This microbiota shift was consistent with the changes recorded in the bacterial metabolic activity.

Estimating distribution and abundance of wide-ranging species with integrated spatial models: Opportunities revealed by the first wolf assessment in south-central Italy.

Estimating demographic parameters for wide-ranging and elusive species living at low density is challenging, especially at the scale of an entire country. To produce wolf distribution and abundance estimates for the whole south-central portion of the Italian wolf population, we developed an integrated spatial model, based on the data collected during a 7-month sampling campaign in 2020-2021. Data collection comprised an extensive survey of wolf presence signs, and an intensive survey in 13 sampling areas, aimed at collecting non-invasive genetic samples (NGS). The model comprised (i) a single-season, multiple data-source, multi-event occupancy model and (ii) a spatially explicit capture-recapture model. The information about species' absence was used to inform local density estimates. We also performed a simulation-based assessment, to estimate the best conditions for optimizing sub-sampling and population modelling in the future. The integrated spatial model estimated that 74.2% of the study area in south-central Italy (95% CIs = 70.5% to 77.9%) was occupied by wolves, for a total extent of the wolf distribution of 108,534 km2 (95% CIs = 103,200 to 114,000). The estimate of total population size for the Apennine wolf population was of 2557 individuals (SD = 171.5; 95% CIs = 2127 to 2844). Simulations suggested that the integrated spatial model was associated with an average tendency to slightly underestimate population size. Also, the main contribution of the integrated approach was to increase precision in the abundance estimates, whereas it did not affect accuracy significantly. In the future, the area subject to NGS should be increased to at least 30%, while at least a similar proportion should be sampled for presence-absence data, to further improve the accuracy of population size estimates and avoid the risk of underestimation. This approach could be applied to other wide-ranging species and in other geographical areas, but specific a priori evaluations of model requirements and expected performance should be made.

MHC variability in an isolated wolf population in Italy.

Small, isolated populations may experience increased extinction risk due to reduced genetic variability at important functional genes, thus decreasing the population's adaptive potential. The major histocompatibility complex (MHC), a key immunological gene cluster, usually shows high variability maintained by positive or balancing selection in response to challenges by pathogens. Here we investigated for the first time, the variability of 3 MHC class II genes (DRB1, DQA1, and DQB1) in 94 samples collected from Italian wolves. The Italian wolf population has been long isolated south of the Alps and is presently recovering from a recent bottleneck that decreased the population to less than 100 individuals. Despite the bottleneck, Italian wolves show remarkable MHC variability with 6-9 alleles per locus, including 2 recently described alleles at DRB1. MHC sequences show signatures of historical selective pressures (high d N/d S ratio, ω > 1.74) but no evidence of ongoing selection. Variation at the MHC genes and 12 background microsatellite loci were not apparently affected by the recent bottleneck. Although MHC alleles of domestic dog origin were detected in 8 genetically admixed individuals, these alleles were rare or absent in nonadmixed wolves. Thus, despite known hybridization events between domestic dogs and Italian wolves, the Italian wolf population does not appear affected by deep introgression of domestic dog MHC alleles.

Bioaccumulation of algal toxins and changes in physiological parameters in Mediterranean mussels from the North Adriatic Sea (Italy).

The Northwestern Adriatic Sea is a commercially important area in aquaculture, accounting for about 90% of the Italian mussel production, and it was subjected to recurring cases of mussel farm closures due to toxic algae poisoning. A spatial and temporal survey of four sites along the North Adriatic Sea coasts of Emilia Romagna (Italy) was undertaken to study the possible impairments of physiological parameters in Mytilus galloprovincialis naturally exposed to algal toxins. The sites were selected as part of the monitoring network for the assessment of algal toxins bioaccumulation by the competent Authority. Samples positive to paralytic shellfish toxins and to lipophilic toxins were detected through the mouse bioassay. Lipophilic toxins were assessed by HPLC. Decreasing yessotoxins (YTX) levels were observed in mussels from June to December, while homo-YTX contents increased concomitantly. Lysosome membrane stability (LMS), glutathione S-transferase and catalase activities, and multixenobiotic resistance (MXR)-related gene expressions were assessed as parameters related to the mussel health status and widely utilized in environmental biomonitoring. Levels of cAMP were also measured, as possibly involved in the algal toxin mechanisms of action. Low LMS values were observed in hemocytes from mussels positive to the mouse bioassay. MXR-related gene expressions were greatly inhibited in mussels positive to the mouse bioassay. Clear correlations were established between increasing homo-YTX contents (and decreasing YTX) and increasing cAMP levels in the tissues. Similarly, significant correlations were established between the increase of homo-YTX and cAMP levels, and the expressions of three MXR-related genes at submaximal toxin concentrations. In conclusion, YTXs may affect mussel physiological parameters, including hemocyte functionality, gene expression and cell signaling.