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Pollution is harmful to human physical health and wellbeing. What is less well established is the relationship between adolescent mental health - a growing public health concern - and pollution. In response, we systematically reviewed studies documenting associations between pollution and mental health in adolescents. We searched Africa Wide, Medline, PsycArticles, PsycInfo, PubMed, CINAHL, ERIC, SciELO, Scopus, and Web of Science Core Collection for studies published up to 10 April 2020 that investigated exposure to any pollutant and symptoms of anxiety; depression; disruptive, impulse-control, and conduct disorders; neurodevelopmental disorders; psychosis; or substance abuse in 10-24-year-olds (i.e., adolescents as per expanded and more inclusive definition of adolescence). This identified 2291 records and we assessed 128 papers for inclusion. We used a narrative synthesis to coalesce the studies' findings. This review is registered on PROSPERO, CRD42020176664. Seventeen studies from Asia, Europe, the Middle East, and North America were included. Air and water pollution exposure was associated with elevated symptoms of depression, generalised anxiety, psychosis, and/or disruptive, impulse control and conduct disorder. Exposure to lead and solvents was associated with neurodevelopmental impairments. Most studies neglected factors that could have supported the mental health resilience of adolescents exposed to pollution. Notwithstanding the limited quality of most reviewed studies, results suggest that pollution exposure is a risk to adolescent mental health. High-quality research is urgently required, including the factors and processes that protect the mental health of pollution-exposed adolescents. Studies with adolescents living in low- and lower middle-income countries and the southern hemisphere must be prioritized.

Additional file 2. APA PsycArticles search strategy, modified as needed for other electronic databases

Additional file 1. PRISMA-P 2015 Checklist. This checklist has been adapted for use with protocol submissions to Systematic Reviews from Table 3 in Moher D et al: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Systematic Reviews 2015 4:1

Vitamin D has been shown in a wide variety of species to play critical roles in neurodevelopment. Vitamin D deficiency disrupts development of the brain and can cause lasting behavioral dysfunction. Zebrafish have become an important model for the study of development in general and neurodevelopment in particular. Zebrafish were used in the current study to characterize the effects of developmental vitamin D deficiency on behavioral function. Adult zebrafish that had been chronically fed a vitamin D deficient or replete diets were bred and the offspring were continued on those diets. The offspring were behaviorally tested as adults. In the novel tank diving test the vitamin D deficient diet significantly lowered the vertical position of fish indicative of more anxiety-like behavior. In the novel tank diving test swimming activity was also significantly decreased by vitamin D deficiency. Startle response was increased by developmental vitamin D deficiency during the early part of the test. No significant effects of vitamin D deficiency were seen with social affiliation and predatory stimulus avoidance tests. These results indicate a phenotype of vitamin D deficiency characterized by more anxiety-like behavior. This result was relatively specific inasmuch as few or no behavioral effects were seen in other behavioral tests.

Background Whilst there is little uncertainty about the deleterious impact of pollution on human and planetary health, pollution’s impact on child and adolescent mental health is less well understood. This is particularly true for young people in underdeveloped and developing world contexts, about whom research is generally lacking. Furthermore, although child and adolescent resilience continues to be a research priority, little attention has been paid to child and adolescent pathways of resilience in the face or aftermath of pollution exposure. Methods We will conduct a systematic review that adheres to PRISMA guidelines. Its purpose is to document what is known about how pollution affects child and adolescent mental health, along with what protects children and adolescents against these effects and associations across diverse contexts. Discussion Systematic insights are likely to support psychologists and other mental health practitioners to better support child and adolescent wellbeing globally regardless of exposure to pollution. Systematic review registration We have registered the review protocol in PROSPERO (registration: CRD42020176664).

Atlantic killifish inhabiting polluted sites along the east coast of the U.S. have evolved resistance to toxic effects of contaminants. One such contaminated site is the Acushnet River estuary, near New Bedford Harbor (NBH), Massachusetts, which is characterized by very high PCB concentrations in the sediments and in the tissues of resident killifish. Though killifish at this site appear to be thriving, the metabolic costs of survival in a highly contaminated environment are not well understood. In this study we compared the hepatic metabolite profiles of resistant (NBH) and sensitive populations (Scorton Creek (SC), Sandwich, MA) using a targeted metabolomics approach in which polar metabolites were extracted from adult fish livers and quantified. Our results revealed differences in the levels of several metabolites between fish from the two sites. The majority of these metabolites are associated with one-carbon metabolism, an important pathway that supports multiple physiological processes including DNA and protein methylation, nucleic acid biosynthesis and amino acid metabolism. We measured the gene expression of DNA methylation (DNA methyltransferase 1, dnmt1) and demethylation genes (Ten-Eleven Translocation (TET) genes) in the two populations, and observed lower levels of dnmt1 and higher levels of TET gene expression in the NBH livers, suggesting possible differences in DNA methylation profiles. Consistent with this, the two populations differed significantly in the levels of 5-methylcytosine and 5-hydroxy methylcytosine nucleotides. Overall, our results suggest that the unique hepatic metabolite signatures observed in NBH and SC reflect the adaptive mechanisms for survival in their respective habitats.

The effects of prenatal exposure to cigarette smoke remain a subject of major interest, especially as it relates to neural development and adverse behavioral outcomes. Several studies have investigated the developmental toxicity of cigarette smoke components in a zebrafish model, showing that developmental exposure to total particulate matter (TPM; particulate phase of cigarette smoke) leads to adverse physiological aberrations and locomotor hyperactivity. Thus, the current study examines whether developmental TPM exposure of zebrafish embryos/larvae (F0) leads to physiological and behavioral alterations, and whether adverse effects are observed in adult fish and the next generation (F1; i.e. F0 offspring). We also examine whether behavioral effects are associated with changes in neural development, stress response, neurotransmitters, and bioenergetics. We demonstrate that TPM exposure during F0 development increased the incidence of deformities in F0 larvae, but F1 larvae did not exhibit any deformities. TPM exposure also resulted in swimming hyperactivity in F0 larvae and several behavioral changes were noted in F0 fish when they grew into adulthood. These behavioral changes were generally not associated with changes in markers of neural development in larvae, stress response in F0 adults, and concentration of neurotransmitters (acetylcholine, dopamine, and serotonin) in F0 adult brain. There were also no changes in F0 or F1 embryonic oxygen consumption rate (OCR; marker of bioenergetics and mitochondrial health); however, the OCR in the brain of F0 males was reduced with TPM. We conclude that developmental exposure to TPM affects larval physiology and induces hyperactive swimming behavior, but these effects do not persist in F1 larvae. Moreover, developmental TPM exposure leads to long-lasting sex-specific behavioral outcomes in the F0 adult fish.

As the older class of brominated flame retardants (BFRs) are phased out of commercial use because of findings of neurotoxicity with developmental exposure, a newer class of flame retardants have been introduced, the organophosphate flame retardants (OPFRs). Presently, little is known about the potential for developmental neurotoxicity or the behavioral consequences of OPFR exposure. Our aim was to characterize the life-long neurobehavioral effects of 4 widely used OPFRs using the zebrafish model. Zebrafish embryos were exposed to 0.1% DMSO (vehicle control); or one of the following treatments; isopropylated phenyl phosphate (IPP) (0.01, 0.03, 0.1, 0.3 µM); butylphenyl diphenyl phosphate (BPDP) (0.003, 0.03, 0.3, 3 µM); 2-ethylhexyl diphenyl phosphate (EHDP) (0.03, 0.3, 1 µM); isodecyl diphenyl phosphate (IDDP) (0.1, 0.3, 1, 10 µM) from 0- to 5-days postfertilization. On Day 6, the larvae were tested for motility under alternating dark and light conditions. Finally, at 5–7 months of age the exposed fish and controls were tested on a battery of behavioral tests to assess emotional function, sensorimotor response, social interaction and predator evasion. These tests showed chemical-specific short-term effects of altered motility in larvae in all of the tested compounds, and long-term impairment of anxiety-related behavior in adults following IPP, BPDP, or EHDP exposures. Our results show that OPFRs may not be a safe alternative to the phased-out BFRs and may cause behavioral impacts throughout the lifespan. Further research should evaluate the risk to mammalian experimental models and humans.

Background Polybrominated diphenyl ethers (PBDEs) were widely used as flame retardants until the early 2000s, mainly in home furnishings and electronics. The persistence of PBDEs in the environment leads to continued ubiquitous exposure to low levels, with infants and children experiencing higher exposures than adults. Accumulating evidence suggest that low-level exposures during early life stages can affect brain development and lead to long-term behavioral impairments. We investigated the effects of zebrafish exposure to low doses of the two prominent PBDEs; 2,2′,4,4′,5,-Pentabromodiphenyl ether (BDE-99) and 2,2′,4,4′,-Tetrabromodiphenyl ether (BDE-47), during embryo-development on short- and long-term behavioral endpoints. We included the organophosphate pesticide chlorpyrifos (CPF) due to its well documented neurotoxicity across species from zebrafish to humans. Methods Zebrafish embryos were exposed to the following individual treatments; 0.1% DMSO (vehicle control); 0.3 μM CPF; 0.01, 0.03, 0.1, 0.3 μM BDE-47; 0.003, 0.03, 0.3, 1, 3, 10, 20 μM BDE-99 from 5 until 120 h post fertilization (hpf). Low exposure levels were determined as those not causing immediate overt toxicity, and behavior assays were conducted in the low-level range. At 144 hpf the larvae were tested for locomotor activity. At approximately 6 months of age adult zebrafish were tested in a behavioral battery including assays for anxiety-related behavior, sensorimotor response and habituation, social interaction, and predator avoidance. Results In the short-term, larval locomotor activity was reduced in larvae treated with 0.3 μM CPF and 0.1 μM BDE-47. BDE-99 treatment caused non-monotonic dose effects, with 0.3 μM causing hyperactivity and 1 μM or higher causing hypoactivity. In the long-term, adult anxiety-related behavior was reduced in all treatments as measured in both the novel tank dive test and tap test. Discussion We show that exposure of zebrafish embryos to low concentrations of the brominated flame retardants BDE-47 and BDE-99, and the organophosphate pesticide CPF, caused both short- and long-term behavioral impairments. Interestingly, we also found that at very low exposure concentrations, where there were no visible effects on larval activity, adult behavior was still strongly affected.

Methylmercury (MeHg) is a ubiquitous pollutant shown to cause developmental neurotoxicity, even at low levels. However, there is still a large gap in our understanding of the mechanisms linking early-life exposure to life-long behavioural impairments. Our aim was to characterise the short- and long-term effects of developmental exposure to low doses of MeHg on anxiety-related behaviours in zebrafish, and to test the involvement of neurological pathways related to stress-response. Zebrafish embryos were exposed to sub-acute doses of MeHg (0, 5, 10, 15, 30 nM) throughout embryo-development, and tested for anxiety-related behaviours and locomotor activity at larval (light/dark locomotor activity) and adult (novel tank and tap assays) life-stages. Exposure to all doses of MeHg caused increased anxiety-related responses, heightened response to the transition from light to dark in larvae, and a stronger dive response in adults. In addition, impairment in locomotor activity was observed in the higher doses in both larvae and adults. Finally, the expressions of several neural stress-response genes from the HPI-axis and dopaminergic system were found to be disrupted in both life-stages. Our results provide important insights into dose-dependent differences in exposure outcomes, the development of delayed effects over the life-time of exposed individuals and the potential mechanisms underlying these effects.

Early life exposure to environmental chemicals can have long-term consequences that are not always apparent until later in life. We recently demonstrated that developmental exposure of zebrafish to low, nonembryotoxic levels of 3,3’,4,4’,5-pentachlorobiphenyl (PCB126) did not affect larval behavior, but caused changes in adult behavior. The objective of this study was to investigate the underlying molecular basis for adult behavioral phenotypes resulting from early life exposure to PCB126. We exposed zebrafish embryos to PCB126 during early development and measured transcriptional profiles in whole embryos, larvae and adult male brains using RNA-sequencing. Early life exposure to 0.3 nM PCB126 induced cyp1a transcript levels in 2-dpf embryos, but not in 5-dpf larvae, suggesting transient activation of aryl hydrocarbon receptor with this treatment. No significant induction of cyp1a was observed in the brains of adults exposed as embryos to PCB126. However, a total of 2209 and 1628 genes were differentially expressed in 0.3 and 1.2 nM PCB126-exposed groups, respectively. KEGG pathway analyses of upregulated genes in the brain suggest enrichment of calcium signaling, MAPK and notch signaling, and lysine degradation pathways. Calcium is an important signaling molecule in the brain and altered calcium homeostasis could affect neurobehavior. The downregulated genes in the brain were enriched with oxidative phosphorylation and various metabolic pathways, suggesting that the metabolic capacity of the brain is impaired. Overall, our results suggest that PCB exposure during sensitive periods of early development alters normal development of the brain by reprogramming gene expression patterns, which may result in alterations in adult behavior.

Vitamin D receptor (VDR) signaling is important for optimal neurobehavioral development. Disruption of VDR signaling by environmental toxicants during early development might contribute to the etiology of behavioral dysfunction. In the current set of studies, we examined ten compounds known to affect VDR function in vitro for neurobehavioral effects in vivo in zebrafish. Zebrafish embryos were exposed to concentrations of the compounds in their water during the first 5 days post-fertilization. On day 5, the embryos were tested in an alternating light-dark locomotor assay using a computerized video tracking system. We found that most of the compounds produced significant changes in locomotor behavior in exposed zebrafish larvae, although the direction of the effect (i.e., hypo- or hyperactivity) and the sensitivity of the effect to changes in illumination condition varied across the compounds. The nature of the behavioral effects generally corresponded to the effects these compounds have been shown to exert on VDR. These studies lay a foundation for further investigation to determine whether behavioral dysfunction persists into adulthood and if so which behavioral functions are affected. Zebrafish can be useful for screening compounds identified in high throughput in vitro assays to provide an initial test for how those compounds would affect construction and behavioral function of a complex nervous system, helping to bridge the gap between in vitro neurotoxicity assays and mammalian models for risk assessment in humans.

Farmers are often chronically exposed to insecticides, which may present health risks including increased risk of neurobehavioral impairment during adulthood and across aging. Experimental animal studies complement epidemiological studies to help determine the cause-and-effect relationship between chronic adult insecticide exposure and behavioral dysfunction. With the zebrafish model, we examined short and long-term neurobehavioral effects of exposure to either an organochlorine insecticide, dichlorodiphenyltrichloroethane (DDT) or an organophosphate insecticide chlorpyrifos (CPF). Adult fish were exposed continuously for either two or 5 weeks (10–30 nM DDT, 0.3–3 μM CPF), with short- and long-term effects assessed at 1-week post-exposure and at 14 months of age respectively. The behavioral test battery included tests of locomotor activity, tap startle, social behavior, anxiety, predator avoidance and learning. Long-term effects on neurochemical indices of cholinergic function were also assessed. Two weeks of DDT exposure had only slight effects on locomotor activity, while a longer five-week exposure led to hypoactivity and increased anxiety-like diving responses and predator avoidance at 1-week post-exposure. When tested at 14 months of age, these fish showed hypoactivity and increased startle responses. Cholinergic function was not found to be significantly altered by DDT. The two-week CPF exposure led to reductions in anxiety-like diving and increases in shoaling responses at the 1-week time point, but these effects did not persist through 14 months of age. Nevertheless, there were persistent decrements in cholinergic presynaptic activity. A five-week CPF exposure led to long-term effects including locomotor hyperactivity and impaired predator avoidance at 14 months of age, although no effects were apparent at the 1-week time point. These studies documented neurobehavioral effects of adult exposure to chronic doses of either organochlorine or organophosphate pesticides that can be characterized in zebrafish. Zebrafish provide a low-cost model that has a variety of advantages for mechanistic studies and may be used to expand our understanding of neurobehavioral toxicity in adulthood, including the potential for such toxicity to influence behavior and development during aging.

Previous studies on pre-molt gastroliths have shown a typical onion-like morphology of layers of amorphous mineral (mostly calcium carbonate) and chitin, resulting from the continuous deposition and densification of amorphous mineral spheres on a chitin-matrix during time. To investigate the consequences of this layered growth on the local structure and composition of the gastrolith, we performed spatially-resolved Raman, X-ray and SEM-EDS analysis on complete pre-molt gastrolith cross-sections. Results show that especially the abundance of inorganic phosphate, phosphoenolpyruvate (PEP)/citrate and proteins is not uniform throughout the organ but changes from layer to layer. Based on these results we can conclude that ACC stabilization in the gastrolith takes place by more than one compound and not by only one of these additives.

The use of electronic cigarettes (e-cigarettes) is increasing despite insufficient information concerning their long-term effects, including the effects of maternal e-cigarette use on pre- and postnatal development. Our previous study demonstrated that developmental exposure to 1,2-propanediol (a principal component of e-cigarette liquid) affected early development of zebrafish, causing reduced growth, deformities, and hyperactive swimming behavior in larvae. The current study extends assessment of the developmental toxicity of 1,2-propanediol by examining additional long-term behavioral effects. We demonstrate that embryonic/larval exposure of zebrafish to 1,2-propanediol (0.625% or 1.25%) not only affected behavioral parameters in the larvae, but also caused persisting behavioral effects in adults after early developmental exposure. Additional parameters, including neural and vascular development in larvae, stress response in adults, and concentration of neurotransmitters dopamine and serotonin in adult brain were examined, in order to explain the behavioral differences. These additional assessments did not find 1,2-propanediol exposure to significantly affect Tg(Neurog1:GFP) or the transcript abundance of neural genes (Neurog1, Ascl1a, Elavl3, and Lef1). Vascular development was not found to be affected by 1,2-propanediol exposure, as inferred from experiments with Tg(Flk1:eGFP) zebrafish; however, transcript abundance of vascular genes (Flk1, Vegf, Tie-2, and Angpt1) was decreased. No statistically significant changes were noted for plasma cortisol or brain neurotransmitters in adult fish. Lastly, analysis of gene transcripts involved with 1,2-propanediol metabolism (Adh5, Aldh2.1, and Ldha) showed an increase in Adh5 transcript. This is the first study to demonstrate that developmental exposure to 1,2-propanediol has long-term neurobehavioral consequences in adult zebrafish, showing that e-cigarettes contain substances potentially harmful to neurodevelopment.

Crustaceans, like most mineralized invertebrates, adopted calcium carbonate mineralization for bulk skeleton reinforcement. Here, we show that a major part of the crustacean class Malacostraca (which includes lobsters, crayfishes, prawns and shrimps) shifted toward the formation of calcium phosphate as the main mineral at specified locations of the mandibular teeth. In these structures, calcium phosphate is not merely co-precipitated with the bulk calcium carbonate but rather creates specialized structures in which a layer of calcium phosphate, frequently in the form of crystalline fluorapatite, is mounted over a calcareous “jaw”. From a functional perspective, the co-existence of carbonate and phosphate mineralization demonstrates a biomineralization system that provides a versatile route to control the physico-chemical properties of skeletal elements. This system enables the deposition of amorphous calcium carbonate, amorphous calcium phosphate, calcite and apatite at various skeletal locations, as well as combinations of these minerals, to form graded composites materials. This study demonstrates the widespread occurrence of the dual mineralization strategy in the Malacostraca, suggesting that in terms of evolution, this feature of phosphatic teeth did not evolve independently in the different groups but rather represents an early common trait.

It has been suggested that gastroliths are a good model for the study of biomineralization and extracellular organic matrix assembly, since they can serve as simplified cases of more complex mineralized biological structures, such as cuticles, bone, and teeth. Proteins are known to be key players in biomineralized chitinous matrices in general and in gastrolith assembly in particular. Here, we present a structural model that is based on the integration into the gastrolith assembly of the gastrolith proteins identified thus far. The model allows the assignment of these proteins to the different aspects of gastrolith construction, based on their characteristics. The model also predicts the existence of several additional proteins yet to be identified.

Bioavailable calcium is maintained by some crustaceans, in particular freshwater crayfish, by stabilizing amorphous calcium carbonate (ACC) within reservoir organs—gastroliths, readily providing the Ca 2+ needed to build a new exoskeleton. Despite the key scientific and biomedical importance of the in situ molecular-level picture of biogenic ACC and its stabilization in a bioavailable form, its description has eluded efforts to date. Herein, using multinuclear NMR, we accomplish in situ molecular-level characterization of ACC within intact gastroliths of the crayfish Cherax quadricarinatus . In addition to the known CaCO 3 , chitin scaffold and inorganic phosphate (Pi), we identify within the gastrolith two primary metabolites, citrate and phosphoenolpyruvate (PEP) and quantify their abundance by applying solution NMR techniques to the gastrolith “soluble matrix.” The long-standing question on the physico-chemical state of ACC stabilizing, P-bearing moieties within the gastrolith is answered directly by the application of solid state rotational-echo double-resonance (REDOR) and transferred-echo double-resonance (TEDOR) NMR to the intact gastroliths: Pi and PEP are found molecularly dispersed throughout the ACC as a solid solution. Citrate carboxylates are found Å from a phosphate (intermolecular C⋯P distance), an interaction that must be mediated by Ca 2+ . The high abundance and extensive interactions of these molecules with the ACC matrix identify them as the central constituents stabilizing the bioavailable form of calcium. This study further emphasizes that it is imperative to characterize the intact biogenic CaCO 3 . Solid state NMR spectroscopy is shown to be a robust and accessible means of determining composition, internal structure, and molecular functionality in situ.

Stable amorphous calcium carbonate (ACC) is a unique material produced naturally exclusively as a biomineral. It was demonstrated that proteins extracted from biogenic stable ACC induce and stabilize synthetic ACC in vitro. Polyphosphate molecules were similarly shown to induce amorphous calcium carbonate formation in vitro. Accordingly, we tested the hypothesis that biogenic ACC induction and stabilization is mediated by the phosphorylated residues of phosphoproteins. We show that extracellular organic matrix extracted from gastroliths of the red claw crayfish Cherax quadricarinatus induce stable ACC formation in vitro. The proteinaceous fraction of this organic matrix is highly phosphorylated and is incorporated into the ACC mineral phase during precipitation. We have identified the major phosphoproteins of the organic matrix and showed that they have high calcium binding capacity. Based on the above, in vitro precipitation experiments with single phosphoamino acids were performed, indicating that phosphoserine or phosphothreonine alone can induce the formation of highly stable ACC. The results indicate that phosphoproteins may play a major role in the control of ACC formation and stabilization and that their phosphoamino acid moieties are key components in this process.

In the crustacean Cherax quadricarinatus, alterations of multi-transcript expression patterns between intermolt and late premolt stages were identified in the hypodermis and in the gastrolith disk via a cDNA microarray. The gastrolith disk is a specialized epithelium forming the gastroliths at premolt. The gastroliths are deposits of calcium carbonate derived from the digested cuticle contributing the mineral to the newly formed exoskeleton at postmolt. The late premolt stage was characterized by a dramatic general up-regulation of genes in the gastrolith disk. This phenomenon is explained by the gastrolith disk function rapid formation of the relatively large gastrolith during a short period of time. Besides genes of general importance for this dramatic change, three genes related to the chitin-protein-mineral structure were identified. The cDNA and the deduced protein of the novel one of them, the chitin deacetylase 1 (Cq-CDA1) was fully characterized and its resemblance to already characterized structural proteins of the gastrolith matrix was described. Cq-CDA1 characteristics strongly indicate its participation in the gastrolith construction, although its protein product was not identified yet in the gastrolith. In addition, many differentially expressed genes with unknown function were elucidated. An unexpected milder down-regulation was observed in the hypodermis.

Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endocrine manipulation, causing them to hypertrophy. Transcripts from these glands were used in the construction of an AG cDNA subtractive library. Screening of the library revealed an AG-specific gene, termed the M. rosenbergii insulin-like AG (Mr-IAG) gene. The cDNA of this gene was then cloned and fully sequenced. The cysteine backbone of the predicted mature Mr-IAG peptide (B and A chains) showed high similarity to that of other crustacean AG-specific insulin-like peptides. In vivo silencing of the gene, by injecting the prawns with Mr-IAG double-stranded RNA, temporarily prevented the regeneration of male secondary sexual characteristics, accompanied by a lag in molt and a reduction in growth parameters, which are typically higher in males of the species. In terms of reproductive parameters, silencing of Mr-IAG led to the arrest of testicular spermatogenesis and of spermatophore development in the terminal ampullae of the sperm duct, accompanied by hypertrophy and hyperplasia of the AGs. This study constitutes the first report of the silencing of a gene expressed specifically in the AG, which caused a transient adverse effect on male phenotypical gender differences and spermatogenesis. Temporal silencing of a newly identified insulin-like gene from prawn androgenic gland inhibits primary spermatogenesis, male secondary sex characteristics, and growth.

Despite the proclamation of Lowenstam and Weiner that crustaceans are the “champions of mineral mobilization and deposition of the animal kingdom,” relatively few proteins from the two main calcification sites in these animals, i.e., the exoskeleton and the transient calcium storage organs, have been identified, sequenced, and their roles elucidated. Here, a 65-kDa protein (GAP 65) from the gastrolith of the crayfish, Cherax quadricarinatus , is fully characterized and its function in the mineralization of amorphous calcium carbonate (ACC) of the extracellular matrix is demonstrated. GAP 65 is a negatively charged glycoprotein that possesses three predicted domains: a chitin-binding domain 2, a low-density lipoprotein receptor class A domain, and a polysaccharide deacetylase domain. Expression of GAP 65 was localized to columnar epithelial cells of the gastrolith disk during premolt. In vivo administration of GAP 65 dsRNA resulted in a significant reduction of GAP 65 transcript levels in the gastrolith disk. Such gene silencing also caused dramatic structural and morphological deformities in the chitinous-ACC extracellular matrix structure. ACC deposited in these gastroliths appeared to be sparsely packed with large elongated cavities compared with the normal gastrolith, where ACC is densely compacted. ACC spherules deposited in these gastroliths are significantly larger than normal. GAP 65, moreover, inhibited calcium carbonate crystallization in vitro and stabilized synthetic ACC. Thus, GAP 65 is the first protein shown to have dual function, involved both in extracellular matrix formation and in mineral deposition during biomineralization.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants. The most toxic PCBs are the non-ortho-substituted (“dioxin-like”) congeners that act through the aryl hydrocarbon receptor (AHR) pathway. In humans, perinatal exposure to dioxin-like PCBs is associated with neurodevelopmental toxicity in children. Yet, the full potential for later-life neurobehavioral effects that result from early-life low level exposure to dioxin-like PCBs is not well understood. The objective of this study was to determine the effects of developmental exposure to low levels of dioxin-like PCBs on early-and later-life behavioral phenotypes using zebrafish as a model system. We exposed zebrafish embryos to either vehicle (DMSO) or low concentrations of PCB126 (0.3, 0.6, 1.2 nM) for 20 hours (4–24 hours post fertilization), and then reared them to adulthood in clean water. Locomotor activity was tested at two larval stages (7 and 14 days post fertilization). Adult fish were tested for anxiety-related behavior using the novel tank and shoaling assays. Adult behavioral assays were repeated several times on the same group of fish and effects on intra-and inter-trial habituation were determined. While there was no effect of PCB126 on larval locomotor activity in response to changes in light conditions, developmental exposure to PCB126 resulted in impaired short-and long-term habituation to a novel environment in adult zebrafish. Cyp1a induction was measured as an indicator for AHR activation. Despite high induction at early stages, cyp1a expression was not induced in the brains of developmentally exposed adult fish that showed altered behavior, suggesting that AHR was not activated at this stage. Our results demonstrate the effectiveness of the zebrafish model in detecting subtle and delayed behavioral effects resulting from developmental exposure to an environmental contaminant.

Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network that constitutes the scaffold upon which cuticles form. The chitin fibers that form this network are closely associated with specific structural proteins, while the cuticular matrix contains many additional structural, enzymatic and other proteins. We study the crayfish gastrolith as a simple model for the assembly of calcified cuticular structures, with particular focus on the proteins involved in this process. The present study integrates a gastrolith-forming epithelium transcriptomic library with data from mass spectrometry analysis of proteins extracted from the gastrolith matrix to obtain a near-complete picture of gastrolith protein content. Using native protein separation we identified 24 matrix proteins, of which 14 are novel. Further analysis led to discovery of three putative protein complexes, all containing GAP 65 the most abundant gastrolith structural protein. Using immunological methods we further studied the role of GAP 65 in the gastrolith matrix and forming epithelium, as well as in the newly identified protein complexes. We propose that gastrolith matrix construction is a sequential process in which protein complexes are dynamically assembled and disassembled around GAP 65, thus changing their functional properties to perform each step in the construction process. Biological significance The scientific interest on which this study is based arises from three main features of gastroliths: (1) Gastroliths possess partial analogy to cuticles both in structural and molecular properties, and may be regarded, with the appropriate reservations (see Introduction), as simple models for cuticle assembly. At the same time, gastroliths are terminally assembled during a well-defined period, which can be controlled in the laboratory, making them significantly easier to study than cuticles. (2) Gastroliths, like the crayfish exoskeleton, contain stable amorphous calcium carbonate (ACC) rather than crystalline calcite. The biological mechanism for the stabilization of a naturally unstable, but at the same time biologically highly available, calcium carbonate polymorph is of great interest from the pharmaceutical point of view. (3) The gastrolith organic matrix is based on a highly structured chitin network that interacts with a variety of substances. This biologically manipulated, biodegradable structure is in itself of biotechnological and pharmaceutical potential. A growing body of evidence indicates that proteins play central roles in all above aspects of gastrolith construction. This study offers the first comprehensive screening of gastrolith proteins, and we believe that the analysis presented in this work can not only help reveal basic biological questions regarding assembly of mineralized and non-mineralized cuticular structures, but may also serve as basis for applied research in the fields of agriculture (e.g. cuticle-based pest management), health (e.g. bioavailable calcium supplements and biodegradable drug carriers) and materials science (e.g. non-toxic scaffolds for water purification).

In crustaceans, like all arthropods, growth is accompanied by a molting cycle. This cycle comprises major physiological events in which mineralized chitinous structures are built and degraded. These events are in turn governed by genes whose patterns of expression are presumably linked to the molting cycle. To study these genes we performed next generation sequencing and constructed a molt-related transcriptomic library from two exoskeletal-forming tissues of the crayfish Cherax quadricarinatus, namely the gastrolith and the mandible cuticle-forming epithelium. To simplify the study of such a complex process as molting, a novel approach, binary patterning of gene expression, was employed. This approach revealed that key genes involved in the synthesis and breakdown of chitin exhibit a molt-related pattern in the gastrolith-forming epithelium. On the other hand, the same genes in the mandible cuticle-forming epithelium showed a molt-independent pattern of expression. Genes related to the metabolism of glucosamine-6-phosphate, a chitin precursor synthesized from simple sugars, showed a molt-related pattern of expression in both tissues. The binary patterning approach unfolds typical patterns of gene expression during the molt cycle of a crustacean. The use of such a simplifying integrative tool for assessing gene patterning seems appropriate for the study of complex biological processes.

Summary Gastroliths are transient extracellular calcium deposits formed by the crayfish Cherax quadricarinatus von Martens on both sides of the stomach wall during pre-molt. Gastroliths are made of a rigid chitinous organic matrix, constructed as sclerotized chitin-protein microfibrils within which calcium carbonate is deposited. Although gastroliths share many characteristics with the exoskeleton, they are simpler in structure and relatively homogenous in composition, making them an excellent cuticle-like model for the study of cuticular proteins. In searching for molt-related proteins involved in gastrolith formation, two integrated approaches were employed, namely the isolation and mass spectrometric analysis of proteins from the gastrolith matrix, and 454-sequencing of mRNAs from both the gastrolith-forming and sub-cuticular epithelia. SDS-PAGE separation of gastrolith proteins revealed a set of bands at apparent molecular weights of 75-85 kDa, of which peptide sequencing following mass spectrometry matched the deduced amino acid sequences of seven hemocyanin transcripts. This assignment was then examined by immunoblot analysis using anti-hemocyanin antibodies, also used to determine the spatial distribution of the proteins in situ. Apart from contributing to oxygen transport, crustacean hemocyanins were previously suggested as being involved in several aspects of the molt cycle, including hardening of the new post-molt exoskeleton via phenoloxidation. The phenoloxidase activity of gastrolith hemocyanins was demonstrated. It was also noted that hemocyanin transcript expression during pre-molt was specific to the hepatopancreas. Our results thus reflect a set of functionally versatile proteins, expressed in a remote metabolic tissue and dispersed via the hemolymph to perform different roles in various organs and structures.

In crustaceans, molting is known to be under the control of neuropeptide hormones synthesized and secreted from the eyestalk ganglia. While the role of molt-inhibiting hormone (MIH) in regulating molting has been described in several species using classical methods, an in vivo specific MIH targeted manipulation has not been described yet. In the present study, an MIH cDNA was isolated and sequenced from the eyestalk ganglia of the Australian freshwater red claw crayfish Cherax quadricarinatus (Cq) by 5' and 3' RACE. We analyzed the putative Cq-MIH based on sequence homology, a three dimensional structure model and transcript's tissue specificity. We further examined the involvement of Cq-MIH in the control of molt in the crayfish through RNAi by in vivo injections of Cq-MIH double-stranded RNA, which resulted in, similarly to eyestalk ablation, acceleration of molt cycles. This acceleration was reflected by a significant reduction (up to 32%) in molt interval and an increased rate in molt mineralization index (MMI), which correlated with the induction of ecdysteroid hormones compared to control. Altogether, this study provides a proof of function for the involvement of the Cq-MIH gene in molt regulation in the crayfish.

Bioavailable calcium is maintained by some crustaceans, in particular freshwater crayfish, by stabilizing amorphous calcium carbonate (ACC) within reservoir organs--gastroliths, readily providing the Ca(2+) needed to build a new exoskeleton. Despite the key scientific and biomedical importance of the in situ molecular-level picture of biogenic ACC and its stabilization in a bioavailable form, its description has eluded efforts to date. Herein, using multinuclear NMR, we accomplish in situ molecular-level characterization of ACC within intact gastroliths of the crayfish Cherax quadricarinatus. In addition to the known CaCO(3), chitin scaffold and inorganic phosphate (Pi), we identify within the gastrolith two primary metabolites, citrate and phosphoenolpyruvate (PEP) and quantify their abundance by applying solution NMR techniques to the gastrolith "soluble matrix." The long-standing question on the physico-chemical state of ACC stabilizing, P-bearing moieties within the gastrolith is answered directly by the application of solid state rotational-echo double-resonance (REDOR) and transferred-echo double-resonance (TEDOR) NMR to the intact gastroliths: Pi and PEP are found molecularly dispersed throughout the ACC as a solid solution. Citrate carboxylates are found5 Å from a phosphate (intermolecular CP distance), an interaction that must be mediated by Ca(2+). The high abundance and extensive interactions of these molecules with the ACC matrix identify them as the central constituents stabilizing the bioavailable form of calcium. This study further emphasizes that it is imperative to characterize the intact biogenic CaCO(3). Solid state NMR spectroscopy is shown to be a robust and accessible means of determining composition, internal structure, and molecular functionality in situ.

Members of the insulin family of hormones are generally not regarded as gender-specific, although there is sporadic evidence for the possible involvement of insulin pathways in sexual differentiation. In crustaceans, sexual differentiation is controlled by the androgenic gland (AG), an organ unique to males. To date, attempts to identify active AG factors in decapods through either classical purification methods or sequence similarity with isopod AG hormones have proven unsuccessful. In the present study, the first subtractive cDNA library from a decapod AG was constructed from the red-claw crayfish Cherax quadricarinatus. During library screening, an AG-specific gene, expressed exclusively in males even at early stages of maturation and termed Cq-IAG (C. quadricarinatus insulin-like AG factor), was discovered. In situ hybridization of Cq-IAG confirmed the exclusive localization of its expression to the AG. Following cloning and complete sequencing of the gene, its cDNA was found to contain 1445 nucleotides encoding a deduced translation product of 176 amino acids. The proposed protein sequence encompasses Cys residue and putative cleaved peptide patterns whose linear and 3D organization are similar to those of members of the insulin/insulin-like growth factor/relaxin family and their receptor recognition surface. The identification of Cq-IAG is the first report of a pro-insulin-like gene expressed in a decapod crustacean in a gender-specific manner. Its expression in a male-specific endocrine gland controlling sex differentiation supports the notion that insulin may have evolved in the context of regulating sexual differentiation.