Rotenone alters behavior and reproductive functions of freshwater catfish, Mystus cavasius, through deficits of dopaminergic neurons in the brain

Highlights

• Rotenone lowered dopamine (DA) in the brain of Mystus cavasius.

• TH-positive dopaminergic neurons declined in middle diencephalon exposed to rotenone.

• Disruption of DA neurons disrupts spontaneous movement and feeding behavior.

• Dopamine neuronal degeneration stimulated ovarian development.

Abstract

Rotenone, commonly used as a pesticide in agriculture and as a piscicide in aquaculture, is a toxic compound that causes dopaminergic neuronal cell loss in the substantia nigra pars compacta of the brain. At the neuroendocrine level, dopamine (DA) drives behavioral (locomotion, emotion, feeding, and social interactions, etc.) and reproductive functions of fish. In the current investigation, we examined effects of rotenone toxicity on neurobehavioral and reproductive functions in whole brain and in selected brain regions in an Indian freshwater catfish, locally known as gulsha (Mystus cavasius). After fish were exposed to water containing rotenone at 0, 2.5, 25, and 250 μg/L for 2 days, significant reductions of DA, 3,4-dihydroxyphenylacetic acid (DOPAC; a DA metabolite), and their ratio (DOPAC/DA) were observed in whole brain at 250 μg/L ambient concentrations of rotenone. When fish were treated with rotenone at 250 μg/L concentration for 2 days, there was a significant reduction of DA, DOPAC and DOPAC/DA in diencephalon, DA and DOPAC in pituitary, and only DA in the telencephalon, compared with control fish. In parallel, numbers of tyrosine hydroxylase-positive (TH⁺) neurons declined significantly in the diencephalon and pituitary after rotenone treatment. Slowed, spontaneous movement and reduced feeding behavior were observed in rotenone-treated fish. Rotenone treatment resulted in a significantly higher gonadosomatic index with many mature vitellogenic oocytes in ovaries and lowered dopaminergic activity in these fish. These results indicate that rotenone influences neurobehavioral and reproductive functions through dopaminergic neuronal cell loss in gulsha brain.

Introduction

Environmental crises are among the most serious challenges we face, exacerbated in recent decades by rapid population growth, food shortages, land scarcity, and intensified agricultural practices. Due to a lack of awareness, farmers often use excess pesticides, or use them inappropriately, hoping to enhance crop production (Uddin et al., 2016). Use of pesticides on agricultural crops may lead to contamination of aquatic environments through spray drift, leaching, and runoff (Van den Brink, 2013; Rahman et al., 2020). Fish exposed to pesticides are often more susceptible to diseases, and they display stunted growth, and increased mortality (Shahjahan et al., 2017, 2019; Islam et al., 2019).

Rotenone is an odorless, colorless, crystalline isoflavone found in the seeds, stems, and roots of many plants belonging to the family Leguminosae. It is commercially used as a pesticide in agriculture and as a piscicide in aquaculture, and it is hazardous to aquatic life. It can easily traverse the blood-brain-barrier, causing neurobehavioral impairment through inhibition of mitochondrial complex-I of the electron transport chain, and it specifically damages dopaminergic neurons in the substantia nigra pars compacta in mammals (Xiong et al., 2012; Nandipati and Litvan, 2016). Similar effects of rotenone toxicity have been documented in some teleost species (Walsh-Monteiro et al., 2014; Melo et al., 2015; Lv et al., 2019), and also in some invertebrates (pond snails, Lymnaea stagnalis, Drosophila, and Platyhelminthes) (Vehovszky et al., 2007). Rotenone treatment reportedly reduces locomotion due to decreased levels of dopamine in brain of zebrafish (Wang et al., 2017), and rainbow trout (Cheng and Farrell, 2007). Immunolocalization reveals that tyrosine hydroxylase, the limiting enzyme of dopamine synthesis, declined in brain of zebrafish after rotenone treatment (Fontaine et al., 2015). These studies support the hypothesis that rotenone destroys central dopaminergic neurons in different regions of the brain, which may alter behavior and reproductive activities in fish.

Dopamine (DA) is a hormone and a monoamine neurotransmitter synthesized by the tyrosine hydroxylase and aromatic l-amino acid decarboxylase. It is essential in the central nervous systems of vertebrates, including teleosts (Badruzzaman et al., 2013). However, the distribution of dopaminergic neurons and behavioral phenotypes are not well documented in teleosts. Dopaminergic neurons are widely distributed in different regions of fish brain, implying that DA mediates neuroendocrine control of various behavioral and physiological activities in fish. It has been reported that telencephalic DA modulates locomotor activity, while hypothalamic DA controls release of gonadotropins from the pituitary (Vidal et al., 2004; Thompson et al., 2008; Ikegami et al., 2015). However, in addition to the gonadotropin releasing hormone (GnRH) stimulatory system, DA has been identified as an inhibitor of reproduction via the brain-pituitary-gonad (BPG) axis in goldfish, Carassius auratus (Peter et al., 1978; Chang and Peter, 1983; Kah et al., 1987), African catfish, Clarias gariepinus (De Leeuw et al., 1988), common carp, Cyprinus crpio (Peng et al., 1994), rainbow trout, Oncorhynchus mykiss (Vacher et al., 2002), gray mullet, Mugil cephalus (Aizen et al., 2005), zebrafish, Danio rerio (Levavi-Sivan et al., 2010), Nile tilapia, Oreochromis niloticus (Levavi-Sivan et al., 2004), European eel, Anguilla Anguilla (Dufur et al., 2010) and damselfish, Chrysiptera cyanea (Badruzzaman et al., 2013). DA regulates gonadotropic secretion as an inhibitor of luteinizing hormone (LH) synthesis and release from the pituitary. For example, in pituitary fragments cultured in vitro, the DA D2-type agonist, apomorphine, significantly reduced GnRH synthesis in goldfish brain (Levavi-Sivan et al., 2004). In contrast, the inhibitory effect of DA on GnRH-stimulated LH secretion was eliminated by treatment with a DA D2-receptor antagonist that increased plasma LH concentrations in tench, Tinca tinca (Podhorec et al., 2012). These findings suggest that DA interacts with the reproductive endocrine axis in fish.

Dopamine receptors are prominent G-protein-coupled receptors in the central nervous systems of vertebrates. These are classified into two types (D1 and D2) and are linked to adenylyl cyclase activation (D1-like) or inhibition (D2-like). In zebrafish pituitary, three DA D2-receptor subtypes are expressed in LH-producing cells indicating that DA binds to D2-receptors on pituitary gonadotropic cells and affect the reproductive endocrine axis in fish (Fontaine et al., 2013, 2015). Additionally, two DA D1-receptor subtypes (D_1A and D₂) have been identified in the forebrain and part of the mesencephalon in an African cichlid fish, Astatotilapia burtoni, modulating their behavior (O’Connell et al., 2011).

The gulsha, Mystus cavasius, is a species of freshwater catfish belonging to the family Bagridae and it is commonly found in rivers, lakes, and canals in and around the Indian subcontinent. The reproductive season of the gulsha ranges from April to September, peaking in July (Badruzzaman et al., 2017). Since the brain governs physiological responses of fish to their environments, the gulsha is a suitable model to understand physiopharamcological effects of rotenone on behavioral and reproductive activity at the endocrine level. As rotenone has long been used extensively in aquacultural ponds to eradicate undesirable fish, it is of prime importance to better understand its impact upon fish. Chronic exposure to environmental rotenone selectively damages dopaminergic neurons in the brain, which in turn causes behavioral impairment in vertebrates, including teleosts (Wang et al., 2017). In the present study, we examined the effects of rotenone on dopaminergic activity in the brain of Mystus cavasius. Locomotor activity, feeding behavior, and ovarian development of the fish in the rotenone-treated group were compared to those in the untreated control group.