Sex diversity in the 21st century: Concepts, frameworks, and approaches for the future of neuroendocrinology.

Sex is ubiquitous and variable throughout the animal kingdom. Historically, scientists have used reductionist methodologies that rely on a priori sex categorizations, in which two discrete sexes are inextricably linked with gamete type. However, this binarized operationalization does not adequately reflect the diversity of sex observed in nature. This is due, in part, to the fact that sex exists across many levels of biological analysis, including genetic, molecular, cellular, morphological, behavioral, and population levels. Furthermore, the biological mechanisms governing sex are embedded in complex networks that dynamically interact with other systems. To produce the most accurate and scientifically rigorous work examining sex in neuroendocrinology and to capture the full range of sex variability and diversity present in animal systems, we must critically assess the frameworks, experimental designs, and analytical methods used in our research. In this perspective piece, we first propose a new conceptual framework to guide the integrative study of sex. Then, we provide practical guidance on research approaches for studying sex-associated variables, including factors to consider in study design, selection of model organisms, experimental methodologies, and statistical analyses. We invite fellow scientists to conscientiously apply these modernized approaches to advance our biological understanding of sex and to encourage academically and socially responsible outcomes of our work. By expanding our conceptual frameworks and methodological approaches to the study of sex, we will gain insight into the unique ways that sex exists across levels of biological organization to produce the vast array of variability and diversity observed in nature.

A phylogenetics-based nomenclature system for steroid receptors in teleost fishes.

Teleost fishes have emerged as tractable models for studying the neuroendocrine regulation of social behavior via molecular genetic techniques, such as CRISPR/Cas9 gene editing. Moreover, teleosts provide an opportunity to investigate the evolution of steroid receptors and their functions, as species within this lineage possess novel steroid receptor paralogs that resulted from a teleost-specific whole genome duplication. Although teleost fishes have grown in popularity as models for behavioral neuroendocrinology, there is not a consistent nomenclature system for steroid receptors and their genes, which may impede a clear understanding of steroid receptor paralogs and their functions. Here, we used a phylogenetic approach to assess the relatedness of protein sequences encoding steroid receptor paralogs in 18 species from 12 different orders of the Infraclass Teleostei. While most similarly named sequences grouped based on the established phylogeny of the teleost lineage, our analysis revealed several inconsistencies in the nomenclature of steroid receptor paralogs, particularly for sequences encoding estrogen receptor beta (ERß). Based on our results, we propose a nomenclature system for teleosts in which Greek symbols refer to proteins and numbers refer to genes encoding different subtypes of steroid receptors within the five major groups of this nuclear receptor subfamily. Collectively, our results bridge a critical gap by providing a cohesive naming system for steroid receptors in teleost fishes, which will serve to improve communication, promote collaboration, and enhance our understanding of the evolution and function of steroid receptors across vertebrates.

A seasonal switch hypothesis for the neuroendocrine control of aggression.

Aggression is a well-studied social behavior that is universally exhibited by animals across a wide range of contexts. Prevailing knowledge suggests gonadal steroids primarily mediate aggression; however, this is based mainly on studies of male-male aggression in laboratory rodents. When males and females of other species, including humans, are examined, a positive relationship between gonadal steroids and aggression is less substantiated. For instance, hamsters housed in short 'winter-like' days show increased aggression compared with long-day housed hamsters, despite relatively low circulating gonadal steroids. These results suggest alternative, non-gonadal mechanisms controlling aggression. Here, we propose the seasonal switch hypothesis, which employs a multidisciplinary approach to describe how seasonal variation in extra-gonadal steroids, orchestrated by melatonin, drives context-specific changes in aggression.

Sex and seasonal differences in neural steroid sensitivity predict territorial aggression in Siberian hamsters.

Many animals display marked changes in physiology and behavior on a seasonal timescale, including non-reproductive social behaviors (e.g., aggression). Previous studies from our lab suggest that the pineal hormone melatonin acts via steroid hormones to regulate seasonal aggression in Siberian hamsters (Phodopus sungorus), a species in which both males and females display increased non-breeding aggression. The neural actions of melatonin on steroids and aggressive behavior, however, are relatively unexplored. Here, we housed male and female hamsters in long-day photoperiods (LDs, characteristic of breeding season) or short-day photoperiods (SDs, characteristic of non-breeding season) and administered timed melatonin (M) or control injections. Following 10 weeks of treatment, we quantified aggressive behavior and neural steroid sensitivity by measuring the relative mRNA expression of two steroidogenic enzymes (aromatase and 5α-reductase 3) and estrogen receptor 1 in brain regions associated with aggression or reproduction [medial preoptic area (MPOA), anterior hypothalamus (AH), arcuate nucleus (ARC), and periaqueductal gray (PAG)] via quantitative PCR. Although LD-M and SD males and females displayed increased aggression and similar changes in gene expression in the ARC, there were sex-specific effects of treatment with melatonin and SDs on gene expression in the MPOA, AH, and PAG. Furthermore, males and females exhibited different relationships between neural gene expression and aggression in response to melatonin and SDs. Collectively, these findings support a role for melatonin in regulating seasonal variation in neural steroid sensitivity and aggression and reveal how distinct neuroendocrine responses may modulate a similar behavioral phenotype in male and female hamsters.

Sex-specific endocrine regulation of seasonal aggression in Siberian hamsters.

Coordinating physiological and behavioural processes across the annual cycle is essential in enabling individuals to maximize fitness. While the mechanisms underlying seasonal reproduction and its associated behaviours are well characterized, fewer studies have examined the hormonal basis of non-reproductive social behaviours (e.g. aggression) on a seasonal time scale. Our previous work suggests that the pineal hormone melatonin facilitates a 'seasonal switch' in neuroendocrine regulation of aggression in male and female Siberian hamsters (Phodopus sungorus), specifically by acting on the adrenal glands to increase the production of the androgen dehydroepiandrosterone (DHEA) during the short-day (SD) photoperiods of the non-breeding season. Here, we provide evidence that the activity of 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3ß-HSD), a key enzyme within the steroidogenic pathway that mediates DHEA synthesis and metabolism, varies in a sex-specific and melatonin-dependent manner. Although both male and female hamsters displayed increased aggression in response to SDs and SD-like melatonin, only males showed an increase in adrenal 3ß-HSD activity. Conversely, SD and melatonin-treated females exhibited reductions in both adrenal and neural 3ß-HSD activity. Collectively, these results suggest a potential role for 3ß-HSD in modulating non-breeding aggression and, more broadly, demonstrate how distinct neuroendocrine mechanisms may underlie the same behavioural phenotype in males and females.

Winter madness: Melatonin as a neuroendocrine regulator of seasonal aggression.

Individuals of virtually all vertebrate species are exposed to annual fluctuations in the deterioration and renewal of their environments. As such, organisms have evolved to restrict energetically expensive processes and activities to a specific time of the year. Thus, the precise timing of physiology and behavior is critical for individual reproductive success and subsequent fitness. Although the majority of research on seasonality has focused on seasonal reproduction, pronounced fluctuations in other non-reproductive social behaviors, including agonistic behaviors (e.g., aggression), also occur. To date, most studies that have investigated the neuroendocrine mechanisms underlying seasonal aggression have focused on the role of photoperiod (i.e., day length); prior findings have demonstrated that some seasonally breeding species housed in short "winter-like" photoperiods display increased aggression compared with those housed in long "summer-like" photoperiods, despite inhibited reproduction and low gonadal steroid levels. While fewer studies have examined how the hormonal correlates of environmental cues regulate seasonal aggression, our previous work suggests that the pineal hormone melatonin acts to increase non-breeding aggression in Siberian hamsters (Phodopus sungorus) by altering steroid hormone secretion. This review addresses the physiological and cellular mechanisms underlying seasonal plasticity in aggressive and non-aggressive social behaviors, including a key role for melatonin in facilitating a "neuroendocrine switch" to alternative physiological mechanisms of aggression across the annual cycle. Collectively, these studies highlight novel and important mechanisms by which melatonin regulates aggressive behavior in vertebrates and provide a more comprehensive understanding of the neuroendocrine bases of seasonal social behaviors broadly.

Uncovering the seasonal brain: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a biochemical approach for studying seasonal social behaviors.

Many animals show pronounced changes in physiology and behavior across the annual cycle, and these adaptations enable individuals to prioritize investing in the neuroendocrine mechanisms underlying reproduction and/or survival based on the time of year. While prior research has offered valuable insight into how seasonal variation in neuroendocrine processes regulates social behavior, the majority of these studies have investigated how a single hormone influences a single behavioral phenotype. Given that hormones are synthesized and metabolized via complex biochemical pathways and often act in concert to control social behavior, these approaches provide a limited view of how hormones regulate seasonal changes in behavior. In this review, we discuss how seasonal influences on hormones, the brain, and social behavior can be studied using liquid chromatography-tandem mass spectrometry (LC-MS/MS), an analytical chemistry technique that enables researchers to simultaneously quantify the concentrations of multiple hormones and the activities of their synthetic enzymes. First, we examine studies that have investigated seasonal plasticity in brain-behavior interactions, specifically by focusing on how two groups of hormones, sex steroids and nonapeptides, regulate sexual and aggressive behavior. Then, we explain the operations of LC-MS/MS, highlight studies that have used LC-MS/MS to study the neuroendocrine mechanisms underlying social behavior, both within and outside of a seasonal context, and discuss potential applications for LC-MS/MS in the field of behavioral neuroendocrinology. We propose that this cutting-edge technology will provide a more comprehensive understanding of how the multitude of hormones that comprise complex neuroendocrine networks affect seasonal variation in the brain and behavior.

Adrenal MT1 melatonin receptor expression is linked with seasonal variation in social behavior in male Siberian hamsters.

Many animals exhibit pronounced changes in physiology and behavior on a seasonal basis, and these adaptations have evolved to promote survival and reproductive success. While the neuroendocrine pathways mediating seasonal reproduction are well-studied, far less is known about the mechanisms underlying seasonal changes in social behavior, particularly outside of the context of the breeding season. Our previous work suggests that seasonal changes in melatonin secretion are important in regulating aggression in Siberian hamsters (Phodopus sungorus); it is unclear, however, how melatonin acts via its receptors to modulate seasonal variation in social behavior. In this study, we infused a MT1 melatonin receptor-expressing (MT1) or control (CON) lentivirus into the adrenal glands of male Siberian hamsters. We then housed hamsters in long-day (LD) or short-day (SD) photoperiods, administered timed melatonin or control injections, and quantified aggressive and non-aggressive social behaviors (e.g., investigation, self-grooming) following 10 weeks of treatment. LD hamsters infused with the MT1 lentivirus had significantly higher adrenal mt1 expression than LD CON hamsters, as determined via quantitative PCR. While melatonin administration was necessary to induce SD-like reductions in body and relative reproductive mass, only LD hamsters infused with the MT1 lentivirus displayed SD-like changes in social behavior, including increased aggression and decreased investigation and grooming. In addition, SD CON and LD hamsters infused with the MT1 lentivirus exhibited similar relationships between adrenal mt1 expression and aggressive behavior. Together, our findings suggest a role for adrenal MT1 receptor signaling in regulating behavior, but not energetics or reproduction in seasonally breeding species.

Increased polyamine levels and maintenance of γ-aminobutyric acid (Gaba) homeostasis in the gills is indicative of osmotic plasticity in killifish.

The Fundulus genus of killifish includes species that inhabit marshes along the U.S. Atlantic coast and the Gulf of Mexico, but differ in their ability to adjust rapidly to fluctuations in salinity. Previous work suggests that euryhaline killifish stimulate polyamine biosynthesis and accumulate putrescine in the gills during acute hypoosmotic challenge. Despite evidence that polyamines have an osmoregulatory role in euryhaline killifish species, their function in marine species is unknown. Furthermore, the consequences of hypoosmotic-induced changes in polyamine synthesis on downstream pathways, such as Æ´-aminobutyric acid (Gaba) production, have yet to be explored. Here, we examined the effects of acute hypoosmotic exposure on polyamine, glutamate, and Gaba levels in the gills of a marine (F. majalis) and two euryhaline killifish species (F. heteroclitus and F. grandis). Fish acclimated to 32 ppt or 12 ppt water were transferred to fresh water, and concentrations of glutamate (Glu), Gaba, and the polyamines putrescine (Put), spermidine (Spd), and spermine (Spm) were measured in the gills using high-performance liquid chromatography. F. heteroclitus and F. grandis exhibited an increase in gill Put concentration, but showed no change in Glu or Gaba levels following freshwater transfer. F. heteroclitus also accumulated Spd in the gills, whereas F. grandis showed transient increases in Spd and Spm levels. In contrast, gill Put, Spm, Glu, and Gaba levels decreased in F. majalis following freshwater transfer. Together, these findings suggest that increasing polyamine levels and maintaining Glu and Gaba levels in the gills may enable euryhaline teleosts to acclimate to shifts in environmental salinity.

Melatonin-dependent changes in neurosteroids are associated with increased aggression in a seasonally breeding rodent.

Aggression is a complex social behaviour that allows individuals to compete for access to limited resources (eg, mates, food and territories). Excessive or inappropriate aggression, however, has become problematic in modern societies, and current treatments are largely ineffective. Although previous work in mammals suggests that aggressive behaviour varies seasonally, seasonality is largely overlooked when developing clinical treatments for inappropriate aggression. Here, we investigated how the hormone melatonin regulates seasonal changes in neurosteroid levels and aggressive behaviour in Siberian hamsters, a rodent model of seasonal aggression. Specifically, we housed males in long-day (LD) or short-day (SD) photoperiods, administered timed s.c. melatonin injections (which mimic a SD-like signal) or control injections, and measured aggression using a resident-intruder paradigm after 9 weeks of treatment. Moreover, we quantified five steroid hormones in circulation and in brain regions associated with aggressive behaviour (lateral septum, anterior hypothalamus, medial amygdala and periaqueductal gray) using liquid chromatography-tandem mass spectrometry. SD hamsters and LD hamsters administered timed melatonin injections (LD-M) displayed increased aggression and exhibited region-specific decreases in neural dehydroepiandrosterone, testosterone and oestradiol, but showed no changes in progesterone or cortisol. Male hamsters also showed distinct associations between neurosteroids and aggressive behaviour, in which neural progesterone and dehydroepiandrosterone were positively correlated with aggression in all treatment groups, whereas neural testosterone, oestradiol and cortisol were negatively correlated with aggression only in LD-M and SD hamsters. Collectively, these results provide insight into a novel neuroendocrine mechanism of mammalian aggression, in which melatonin reduces neurosteroid levels and elevates aggressive behaviour.

Seasonal patterns of melatonin alter aggressive phenotypes of female Siberian hamsters.

Many animal species exhibit year-round aggression, a behaviour that allows individuals to compete for limited resources in their environment (eg, food and mates). Interestingly, this high degree of territoriality persists during the non-breeding season, despite low levels of circulating gonadal steroids (ie, testosterone [T] and oestradiol [E2 ]). Our previous work suggests that the pineal hormone melatonin mediates a 'seasonal switch' from gonadal to adrenal regulation of aggression in Siberian hamsters (Phodopus sungorus); solitary, seasonally breeding mammals that display increased aggression during the short, 'winter-like' days (SDs) of the non-breeding season. To test the hypothesis that melatonin elevates non-breeding aggression by increasing circulating and neural steroid metabolism, we housed female hamsters in long days (LDs) or SDs, administered them timed or mis-timed melatonin injections (mimic or do not mimic a SD-like signal, respectively), and measured aggression, circulating hormone profiles and aromatase (ARO) immunoreactivity in brain regions associated with aggressive or reproductive behaviours (paraventricular hypothalamic nucleus [PVN], periaqueductal gray [PAG] and ventral tegmental area [VTA]). Females that were responsive to SD photoperiods (SD-R) and LD females given timed melatonin injections (Mel-T) exhibited gonadal regression and reduced circulating E2 , but increased aggression and circulating dehydroepiandrosterone (DHEA). Furthermore, aggressive challenges differentially altered circulating hormone profiles across seasonal phenotypes; reproductively inactive females (ie, SD-R and Mel-T females) reduced circulating DHEA and T, but increased E2 after an aggressive interaction, whereas reproductively active females (ie, LD females, SD non-responder females and LD females given mis-timed melatonin injections) solely increased circulating E2 . Although no differences in neural ARO abundance were observed, LD and SD-R females showed distinct associations between ARO cell density and aggressive behaviour in the PVN, PAG and VTA. Taken together, these results suggest that melatonin increases non-breeding aggression by elevating circulating steroid metabolism after an aggressive encounter and by regulating behaviourally relevant neural circuits in a region-specific manner.

Melatonin mediates seasonal transitions in aggressive behavior and circulating androgen profiles in male Siberian hamsters.

Some seasonally-breeding animals are more aggressive during the short, "winter-like" days (SD) of the non-breeding season, despite gonadal regression and reduced circulating androgen levels. While the mechanisms underlying SD increases in aggression are not well understood, previous work from our lab suggests that pineal melatonin (MEL) and the adrenal androgen dehydroepiandrosterone (DHEA) are important in facilitating non-breeding aggression in Siberian hamsters (Phodopus sungorus). To characterize the role of MEL in modulating seasonal transitions in aggressive behavior, we housed male hamsters in long days (LD) or SD, treated them with timed MEL (M) or saline injections, and measured aggression after 3, 6, and 9 weeks. Furthermore, to assess whether MEL mediates seasonal shifts in gonadal and adrenal androgen synthesis, serum testosterone (T) and DHEA concentrations were quantified 36 h before and immediately following an aggressive encounter. LD-M and SD males exhibited similar physiological and behavioral responses to treatment. Specifically, both LD-M and SD males displayed higher levels of aggression than LD males and reduced circulating DHEA and T in response to an aggressive encounter, whereas LD males elevated circulating androgens. Interestingly, LD and SD males exhibited distinct relationships between circulating androgens and aggressive behavior, in which changes in serum T following an aggressive interaction (∆T) were negatively correlated with aggression in LD males, while ∆DHEA was positively correlated with aggression in SD males. Collectively, these findings suggest that SD males transition from synthesis to metabolism of circulating androgens following an aggressive encounter, a mechanism that is modulated by MEL.

Photoperiod modulates the gut microbiome and aggressive behavior in Siberian hamsters.

Seasonally breeding animals undergo shifts in physiology and behavior in response to changes in photoperiod (day length). Interestingly, some species, such as Siberian hamsters (Phodopus sungorus), are more aggressive during the short-day photoperiods of the non-breeding season, despite gonadal regression. While our previous data suggest that Siberian hamsters employ a 'seasonal switch' from gonadal to adrenal regulation of aggression during short-day photoperiods, there is emerging evidence that the gut microbiome, an environment of symbiotic bacteria within the gastrointestinal tract, may also change seasonally and modulate social behaviors. The goal of this study was to compare seasonal shifts in the gut microbiome, circulating levels of adrenal dehydroepiandrosterone (DHEA) and aggression in male and female Siberian hamsters. Hamsters were housed in either long-day (LD) or short-day (SD) photoperiods for 9 weeks. Fecal samples were collected and behaviors were recorded following 3, 6 and 9 weeks of housing, and circulating DHEA was measured at week 9. SD females that were responsive to changes in photoperiod (SD-R), but not SD-R males, displayed increased aggression following 9 weeks of treatment. SD-R males and females also exhibited distinct changes in the relative abundance of gut bacterial phyla and families, yet showed no change in circulating DHEA. The relative abundance of some bacterial families (e.g. Anaeroplasmataceae in females) was associated with aggression in SD-R but not LD or SD non-responder (SD-NR) hamsters after 9 weeks of treatment. Collectively, this study provides insight into the complex role of the microbiome in regulating social behavior in seasonally breeding species.

Neural Androgen Synthesis and Aggression: Insights From a Seasonally Breeding Rodent.

Aggression is an essential social behavior that promotes survival and reproductive fitness across animal systems. While research on the neuroendocrine mechanisms underlying this complex behavior has traditionally focused on the classic neuroendocrine model, in which circulating gonadal steroids are transported to the brain and directly mediate neural circuits relevant to aggression, recent studies have suggested that this paradigm is oversimplified. Work on seasonal mammals that exhibit territorial aggression outside of the breeding season, such as Siberian hamsters (Phodopus sungorus), has been particularly useful in elucidating alternate mechanisms. These animals display elevated levels of aggression during the non-breeding season, in spite of gonadal regression and reduced levels of circulating androgens. Our laboratory has provided considerable evidence that the adrenal hormone precursor dehydroepiandrosterone (DHEA) is important in maintaining aggression in both male and female Siberian hamsters during the non-breeding season, a mechanism that appears to be evolutionarily-conserved in some seasonal rodent and avian species. This review will discuss research on the neuroendocrine mechanisms of aggression in Siberian hamsters, a species that displays robust neural, physiological, and behavioral changes on a seasonal basis. Furthermore, we will address how these findings support a novel neuroendocrine pathway for territorial aggression in seasonal animals, in which adrenal DHEA likely serves as an essential precursor for neural androgen synthesis during the non-breeding season.

Changes to Intestinal Transport Physiology and Carbonate Production at Various CO2 Levels in a Marine Teleost, the Gulf Toadfish (Opsanus beta).

Most marine teleosts defend blood pH during high CO2 exposure by sustaining elevated levels of HCO3(-) in body fluids. In contrast to the gill, where measures are taken to achieve net base retention, elevated CO2 leads to base loss in the intestine of marine teleosts studied to date. This loss is thought to occur through transport pathways previously demonstrated to be involved with routine osmoregulation in marine teleosts. The main objective of this study was to characterize the intestinal transport physiology of the gulf toadfish (Opsanus beta) when exposed to varied levels of CO2: control, 5,000, 10,000, and 20,000 µatm CO2 (0.04, 0.5, 1, and 2 kPa, respectively). Results of this study suggest that intestinal apical anion exchange is highly responsive to hypercarbia, evidenced by a dose-dependent increase in intestinal luminal HCO3(-) (mmol L(-1)) that was mirrored by a reduction in Cl(-) (mmol L(-1)). Despite activation of HCO3(-) transport pathways typically used during osmoregulation, fractional fluid absorption was only significantly lower at the highest level of CO2. Although increased HCO3(-) excretion could provide more substrate for intestinally produced carbonates, carbonate production was not significantly increased during hypercarbia at the levels tested. This study is among the first to thoroughly characterize how compensation for elevated CO2 affects transport physiology and carbonate production in the marine fish intestine. This deeper understanding may be particularly relevant when considering the impacts of future predicted ocean acidification, where prolonged base loss may alter the energetic cost of acid-base balance or osmoregulation in marine fish.

Growth inhibition in early life-stage tests predicts full life-cycle toxicity effects of lead in the freshwater pulmonate snail, Lymnaea stagnalis.

The freshwater pulmonate snail, Lymnaea stagnalis, is the most sensitive freshwater organism tested to date for several metals (Co, Cu, Pb, Ni) based on 28 d early life-stage (ELS) tests in which growth was the most sensitive endpoint. The United States Environmental Protection Agency (USEPA) has expressed concern that growth in 28 d ELS tests with mollusks may overpredict toxicity because of the potential for recovery in a full life-cycle (LC) test. Consequently, the USEPA only accepts the survival endpoint for these tests in establishing water quality criteria (WQC). To address this concern, the current study aimed to test the sensitivity of L. stagnalis to Pb in a 56 d full LC test evaluating survival, growth, reproductive and embryonic growth endpoints and compare the estimated effect levels to those established using the 28 d ELS test design. The most sensitive endpoints in this study were 28 d growth and 56 d egg mass production, both with a NOEC of <1.0 µg L(-1) and a LOEC of 1.0 µg L(-1), showing that the ELS growth endpoint is predictive of the 56 d reproduction endpoint. Snails exposed to 1.0 and 2.7 µg L(-1) Pb showed full and partial recovery from growth inhibition between 28 and 56 d. While this recovery supports the USEPA's concern about the 28 d growth endpoint; considering the reproductive lifespan of L. stagnalis and the recovery dose-response, we conclude that the 28 d growth endpoint will be within a factor of 3 of full LC endpoints. This is consistent with the level of precision previously determined for fish ELS tests, which the USEPA accepts for WQC derivation, and suggests that tests using 28 d ELS growth endpoint for L. stagnalis may be acceptable for inclusion in WQC derivation.

Investigations into the mechanism of lead toxicity to the freshwater pulmonate snail, Lymnaea stagnalis.

The freshwater pulmonate snail, Lymnaea stagnalis, is the most sensitive aquatic organism tested to date for Pb with an estimated EC20 for juvenile snail growth of 3 µg l⁻¹. A previous study supported the hypothesis that this hypersensitivity to Pb was due to an extremely high Ca²âº uptake rate needed to support shell formation. The current study sought to build upon this working hypothesis and develop a mechanistic predictive model for inhibition of snail growth as a function of Pb exposure. Initial experiments confirmed previous predictions that juvenile snails have net Ca²âº uptake rates of 7000-8000 nmol g⁻¹ h⁻¹, approximately 100-fold higher than observed in a typical freshwater fish. However, an initial time course study revealed that the onset of growth inhibition occurs at least 4d prior to inhibition of net Ca²âº flux in Pb-exposed snails indicating the latter is not the primary mechanism of action. Qualitative observations during this experiment indicated snail feeding was inhibited in a dose-dependent manner. A subsequent experiment demonstrated that when food is withheld from snails for even 24 h, net Ca²âº uptake is significantly (∼50%) reduced. A second time course study demonstrated quantitatively that snail feeding is inhibited by Pb exposure by up to 98% at relatively high Pb concentrations (57 µg l⁻¹) but no inhibition was observed at ≤ 10 µg l⁻¹ Pb indicating feeding inhibition is not causing observed growth effects at concentrations approximating the EC20 of 3 µg l⁻¹ Pb. A final experiment testing whether Pb-induced growth effects are related to inhibition of carbonic anhydrase activity in the snail mantle also failed to demonstrate an effect. We conclude that while both feeding and net Ca²âº uptake in snails are affected by Pb exposure, they appear to be secondary effects. The primary mechanism of action explaining L. stagnalis hypersensitivity to Pb remains to be identified.