Many fishes possess specialized epidermal cells that are ruptured by the teeth of predators, thus reliably indicating the presence of an actively foraging predator. Understanding the evolution of these cells has intrigued evolutionary ecologists because the release of these alarm chemicals is not voluntary. Here, we show that predation pressure does not influence alarm cell production in fishes. Alarm cell production is stimulated by exposure to skin-penetrating pathogens (water moulds: Saprolegnia ferax and Saprolegnia parasitica), skin-penetrating parasites (larval trematodes: Teleorchis sp. and Uvulifer sp.) and correlated with exposure to UV radiation. Suppression of the immune system with environmentally relevant levels of Cd inhibits alarm cell production of fishes challenged with Saprolegnia. These data are the first evidence that alarm substance cells have an immune function against ubiquitous environmental challenges to epidermal integrity. Our results indicate that these specialized cells arose and are maintained by natural selection owing to selfish benefits unrelated to predator-prey interactions. Cell contents released when these cells are damaged in predator attacks have secondarily acquired an ecological role as alarm cues because selection favours receivers to detect and respond adaptively to public information about predation.

Mercury, one of the most toxic elements, exists in various chemical forms each with different toxicities and health implications. Some methylated mercury forms, one of which exists in fish and other seafood products, pose a potential threat, especially during embryonic and early postnatal development. Despite global concerns, little is known about the mechanisms underlying transport and toxicity of different mercury species. To investigate the impact of different mercury chemical forms on vertebrate development, we have successfully combined the zebrafish, a well-established developmental biology model system, with synchrotron-based X-ray fluorescence imaging. Our work revealed substantial differences in tissue-specific accumulation patterns of mercury in zebrafish larvae exposed to four different mercury formulations in water. Methylmercury species not only resulted in overall higher mercury burdens but also targeted different cells and tissues than their inorganic counterparts, thus revealing a significant role of speciation in cellular and molecular targeting and mercury sequestration. For methylmercury species, the highest mercury concentrations were in the eye lens epithelial cells, independent of the formulation ligand (chloride versusl-cysteine). For inorganic mercury species, in absence of l-cysteine, the olfactory epithelium and kidney accumulated the greatest amounts of mercury. However, with l-cysteine present in the treatment solution, mercuric bis-l-cysteineate species dominated the treatment, significantly decreasing uptake. Our results clearly demonstrate that the common differentiation between organic and inorganic mercury is not sufficient to determine the toxicity of various mercury species.

Using synchrotron x-ray fluorescence mapping, we have examined the uptake and localization of organic mercury in zebrafish larvae. Strikingly, the greatest accumulation of methyl and ethyl mercury compounds was highly localized in the rapidly dividing lens epithelium, with lower levels going to brain, optic nerve, and various other organs. The data suggest that the reported impairment of visual processes by mercury may arise not only from previously reported neurological effects, but also from direct effects on the ocular tissue. This novel approach is a powerful tool for directly investigating the molecular toxicology of heavy metals, and should be equally applicable to the study of a wide range of elements in developing embryos.methylmercury ͉ thimerosal ͉ x-ray fluorescence mapping ͉ eye lens T oxic organic mercury compounds worry many communities worldwide, yet the detailed mechanisms underlying their transport and toxicity remain uncertain (1). These neurotoxic compounds are particularly insidious due to the latency in onset of toxic symptoms and have caused several devastating masspoisonings of humans. Adults are affected, but exposure in utero has resulted in severe consequences such as microcephaly, cerebropalsy, seizures, and mental retardation. Methylmercury (MeHg) compounds are actively transported across cell membranes (2) although not, as originally thought, by molecular mimicry of methionine (3). Beyond this, however, our knowledge of the mechanisms underlying organic mercury (Hg) toxicity is fragmentary. MeHg-induced changes in cellular Ca 2ϩ have been shown to be important (4-7), as has oxidative stress (4), and glutamate metabolism (8). In both yeast and human cells, overexpression of the ubiquitin-targeting enzyme Cdc34 confers protection against the cytotoxic effects of MeHg, which leads to the suggestion that an unknown protein containing a signal for ubiquitination by Cdc34 is involved in the development of the cytotoxic effects of MeHg (9-11).Zebrafish (Danio rerio) are common model organisms for the study of embryonic development, and have found increasing use in vertebrate toxicology (12). While embryonic and larval zebrafish previously have been used to study the toxic effects of MeHg exposure at the whole body (13,14) and at the molecular level (15), there are no available data on MeHg uptake and accumulation with respect to different tissues and organs. Such information is critical to properly integrate information on tissue and cell specific impacts of Hg with uptake and accumulation of the metal at the whole animal level. We present herein a novel approach to the investigation of heavy-metal toxicity using synchrotron x-ray fluorescence imaging (16) to directly image metal localization within zebrafish larvae and to observe remarkable differential accumulation of organic Hg using this technique. Strikingly, we find that both methyl and ethyl Hg derivatives are concentrated to the greatest degree in lens epithelium with lower levels present in the brain, optic nerve, and other organs....

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The toxic effects of cadmium and other heavy metals have been well established, and many of these and other environmental pollutants are known to be embryotoxic or teratogenic. However, it has proven difficult to identify individual cells that respond to toxicants among the wide range of cell populations in an intact animal, particularly during early development when cells are continually changing their molecular and physiologic characteristics as they differentiate. Here we report the establishment of an in vivo system that uses hsp70 gene activation as a measure of cadmium toxicity in living early larvae of transgenic zebrafish carrying a stably integrated hsp70-enhanced green fluorescent protein (eGFP) reporter gene. We demonstrate that eGFP expression in this strain of fish acts as an accurate and reproducible indicator of cell-specific induction of hsp70 gene expression. Furthermore, the transgene responds in a dose-dependent manner at concentrations similar to those observed for morphologic indicators of early-life-stage toxicity and is sensitive enough to detect cadmium at doses below the median combined adverse effect concentration and the median lethal concentration. The stable nature of this transgenic line should allow for extremely rapid and reproducible toxicologic profiling of embryos and larvae throughout development.