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Toxicity of Malathion during Senegalese sole, Solea senegalensis larval development and metamorphosis: Effects on AChE and CYP1A enzymatic gene expression and protein systems, thyroid ontogeny, eye migration and skeletogenesis J.B. Ortiz-Delgado1*, E. Scala2, G. Albendín2, V. Funes3, C. Sarasquete1, M. I. Arufe2, M. Manchado3, J.M. Arellano2 1 Dto. de Biología Marina y Acuicultura, Instituto de Ciencias Marinas de Andalucía/ICMAN-CSIC, Puerto Real, Cádiz, Spain 2 Dto. de Toxicología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Cádiz, Spain 3 Dto. de Biología Molecular, IFAPA Centro el Toruño, Cádiz, Spain *Presenting author contact: [email protected] Abstract In last few decades, the use of pesticides has increased dramatically worldwide, especially in developing countries in order to protect stored grains, standing crop and prevent diseases spread by insect vectors (malaria, sleeping sickness, etc.). Pesticides include a wide variety of insecticides, herbicides, fungicides and other biocides. Organophophates (OP) insecticides like malathion elicit toxicity primarily through irreversible inhibition of acetyl cholinesterase (AChE) at cholinergic synapses. Inhibition of AChE leads to accumulation of acetylcholine in the synapses, which in turns leads to prolonged activation of cholinergic receptors and overt signs of cholinergic toxicity. As a result, the inhibition of cholinesterase activity has been well accepted as a specific biomarker of exposure to pesticides. Inhibition of AChE enzyme levels can be correlated to induction levels of AChE mRNA due to an increase of acetylcholine in the synapses, as well as by a potential over-gene expression mediated by the excess of the neurotransmitter. One of the serious consequences of increased use of pesticides is the chemical contamination of freshwater and estuarine ecosystems, which are the ultimate storehouse of pesticide residues, and thus adversely affect the biota. Pesticides, even at a very low concentration are reported to have hazardous impacts on fish biology like mortality and/or retardation in growth, development and reproduction. Pesticides can also affect levels of somatic and metabolic hormones implicated in regulation of metabolic activities and body growth like the thyroid hormones, thyroxine (T4) and triiodothyronine (T3). Flatfish, Senegalese sole, Solea senegalensis, used in this study as experimental model, is an interesting commercial pleuronectiform species that suffer an asymmetric skull development and eye migration during metamorphosis process, which is mediated entirely by thyroid, between other hormones. Because Malathion can induce decline in the thyroid hormone levels by arresting thyroxine secretion, exposure of early life stages of Solea senegalensis to this contaminant can alter the normal metamorphosis process (failure of eye migration and adaptation to benthic life). The aim of this work was to study chronic effects of environmental relevant concentrations of Malathion (ranged between 50 and 200 µg/L) on Senegalese sole larval development and metamorphosis during the first month of larval life. Effects on thyroid gland development were detected by means of histological and immnunohistochemical approaches by using commercial antibodies and differences between control and exposed fish were detected in terms of timing of apparition and number of thyroid follicles and their functionality (presence of thyroid hormones within the colloid) at different larval ages. In parallel, differences in skull development, failure of eye migration and malformations in vertebrae and caudal complex are analyzed during metamorphic phase (from 13 to 20 days post hatching, dph). AChE enzymatic activity was also measured in Sole specimens exposed to malathion and changes of AChE gene expression during experimental period are studied by means of Real Time PCR/q-PCR and in situ hybridization. AChE was also studied at tissue protein level by means of immunohistochemistry. Interestingly, because malathion is metabolized by hepatic enzymes, being one of the enzyme systems the hepatic microsomal cytochrome P450-dependent monooxygenase (CYP1A subfamily), changes in the expression of CYP1A are compared in terms of gene expression patterns and protein accumulation in response to Malathion exposure. Finally, histopathological disorders are studied in target organs (i.e. liver, gills,). This work was funded by MICIIN (project AGL2010-15951). V. Funes is a recipient of a Postdoctoral contract (DOC-INIA Subprogram). E. Scala is a recipient of an ERASMUS grant (UCA student/ ERASMUS programme).