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Parental whole life cycle exposure to dietary methylmercury in zebrafish (Danio rerio) affects the behavior of offspring Francisco Xavier Mora-Zamorano, Rebekah Klingler, Cheryl Murphy, Niladri Basu, Jessica Head, PhD Head, and Michael John Carvan III Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00223 • Publication Date (Web): 29 Mar 2016 Downloaded from http://pubs.acs.org on March 30, 2016

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Environmental Science & Technology

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Parental whole life cycle exposure to dietary

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methylmercury in zebrafish (Danio rerio) affects the

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behavior of offspring

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Francisco X. Mora-Zamorano†, Rebekah Klingler†, Cheryl Murphy††, Niladri Basu‡‡, Jessica

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Head‡‡, Michael J. Carvan III*†

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†School of Freshwater Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin,

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USA

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†† Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan, USA

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‡‡School of Dietetics and Human Nutrition, McGill University, Ste-Anne-de-Bellevue, Quebec,

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Canada

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Keywords: Fish, aquatic toxicology, mercury, animal behavior

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ACS Paragon Plus Environment

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ABSTRACT

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Methylmercury (MeHg) is an established neurotoxicant of concern to fish-eating organisms.

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While most studies have focused on the fish consumers, much less is known about the effects of

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MeHg on the fish themselves, especially following exposures to chronic and environmentally

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relevant scenarios. Here we evaluated the behavioral effects of developmental MeHg insult by

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exposing parental generations of zebrafish to environmentally realistic MeHg diets (0, 1, 3 and

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10 ppm) throughout their whole life span. Upon reaching adulthood, their offspring were

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analyzed through a series of behavioral tests, including the visual-motor response (VMR) assay,

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analysis of spontaneous swimming and evaluation of foraging efficiency. The VMR assay

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identified decreased locomotor output in the 6 day post-fertilization (dpf) offspring of fish

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exposed to 3 and 10 ppm MeHg. However, in a second test 7 dpf fish revealed an increase in

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locomotor activity in all MeHg exposures tested. Increases in locomotion continued to be

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observed until 16 dpf, which coincided with increased foraging efficiency. These results suggest

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an association between MeHg and hyperactivity, and imply that fish chronically exposed to

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MeHg in the wild may be vulnerable to predation.

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INTRODUCTION

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Mercury (Hg) is a neurotoxic heavy metal that is released into the environment by

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anthropogenic and natural sources, with coal combustion and artisanal gold mining being the

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primary anthropogenic sources1, 2. Worldwide atmospheric deposition causes inorganic Hg to be

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incorporated into aquatic ecosystems3, after which bacteria metabolize this form of Hg into

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organic methylmercury (MeHg)4.

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MeHg uptake by fish occurs primarily through dietary exposure5 which subsequently leads to

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bioaccumulation and biomagnification6. In most cases, more than 90% of total Hg (THg) in fish

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muscle tissue is in the form of MeHg7, 8 and maternal burdens of this pollutant can be transferred

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to the eggs during oogenesis9. Maternal transfer of MeHg is particularly threatening to the

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offspring, due to the high susceptibility of developing embryos to environmental contaminants10.

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Fish are especially relevant models for behavioral screening of aquatic toxicants and have been

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used for a long period11. They are particularly relevant due to their direct relationship with the

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aquatic ecosystem in which the exposure occurs. Among the various aquatic model organisms,

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zebrafish larvae are particularly well suited for large-scale behavioral toxicology due to their

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small size, fast development and the capacity of a single adult zebrafish breeding pair to produce

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200-300 embryos12.

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To interact with its environment and survive, zebrafish larvae exhibit a complex behavioral

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repertoire. Spontaneous swimming is the most fundamental behavioral output in zebrafish larvae,

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however, they also exhibit more complex behaviors like a variety of startle responses and prey

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tracking13, 14 all of which can be potentially compromised by exposure to a neurotoxicant.

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A number of methods have been proposed to assess neurotoxicity in zebrafish, and they

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include, among others, the analysis of the response to abrupt light changes referred to as the


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visualmotor response (VMR)15, 16, as well as the analysis of free swimming with computer vision

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algorithms11. Foraging efficiency, is a lesser studied behavioral endpoint and assays in zebrafish

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larvae have mainly focused on larvae preying on paramecia13,

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previous efforts have been made to analyze the effects of a neurotoxicant on the ontology of

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foraging in zebrafish larvae, however quantifying this endpoint is crucial to truly understand

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some of the broader ecological implications of MeHg exposure. Foraging is a life-sustaining

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activity that integrates a number of skills that a fish must accurately display in order to hunt,

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perceive and capture prey. For this reason, the data gathered from foraging assays is especially

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suitable to be translated into an ecological context.

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. To our knowledge, no

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The objective of this study was to elucidate the behavioral effects of MeHg on the offspring of

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zebrafish that had been exposed to environmentally relevant doses of dietary MeHg (0, 1, 3 and

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10 ppm) throughout their whole life cycle. Such an approach integrates four important concepts

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that realistically mimic MeHg exposure in aquatic ecosystems: 1) exposure to MeHg occurs

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primarily though the diet; 2) fish that inhabit MeHg contaminated ecosystems are likely exposed

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to this contaminant throughout their whole life; 3) MeHg accumulates in the ovaries of female

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fish throughout their life span; 4) once female fish reach sexual maturity, MeHg can be

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transferred from the ovary to the developing embryos. Here, we hypothesized that the

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fundamental behavioral paradigms tested in our study (i.e. VMR, spontaneous swimming and

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foraging efficiency) would be altered by MeHg in a dose-dependent manner, and that such

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behavioral alterations would give insight into the degree at which MeHg affects swimming,

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foraging and predator evasion in zebrafish larvae.

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MATERIALS AND METHODS

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Fish husbandry. All of the animal protocols described hereafter were approved by the

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Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin -

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Milwaukee. Wild type zebrafish (Danio rerio) larvae used in this study were from the EK strain,

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which was originally obtained from EkkWill Waterlife Resources (Ruskin, Florida, USA;

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maintained in the laboratory for well over 15 generations). These fish were raised in the NIEHS

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Children’s Environmental Health Core Center at the University of Wisconsin – Milwaukee

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(Milwaukee WI, USA). All zebrafish embryos were reared in E2 embryo medium (15mM NaCl,

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0.5mM KCl, 1mM MgSO4, 150µM KH2PO4, 50µM Na2HPO4, 1mM CaCl2, 0.7mM NaHCO3),

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and were incubated at 28°C on a 14h:10h light:dark cycle.

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MeHg supplemented diet preparation. An initial 3mM stock solution (in ethanol) of MeHg

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chloride (Sigma-Aldrich Co., St. Louis MO, USA) was used to make all of the required dilutions

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to obtain the desired MeHg concentrations in the diets (specifications of diets and MeHg

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concentrations will be discussed in the section below). Adult zebrafish diets were treated with

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MeHg in batches of 500g of food. After weighing the food, a calculated amount of MeHg stock

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solution was mixed into 950mL of ethanol, subsequently this solution was mixed into the food.

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Vehicle control diets (“0 ppm MeHg” diets) were prepared by mixing 950mL of ethanol into

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500g of food. The preparations were stirred three times daily under a fume hood for 4 days until

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all the ethanol had evaporated completely.

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Larval micropellet diets were prepared in batches of 50g. A volume of 250mL of ethanol was

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used to mix in the MeHg into the food; larval vehicle control diets were prepared by mixing


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250mL of ethanol into 50g of food. As with the adult flakes, the larval food was stirred three times daily under a fume hood until the ethanol evaporated completely.

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Dietary MeHg exposures. Dietary exposures began in 8 month old females (G0) which were

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fed with a prepared diet (Biodiet starter, Bio-Oregon,Westbrook, ME, USA; 4% body weight per

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day) containing nominal MeHg at nominal concentrations of 0, 0.5, 5 and 50 ppm for 9 weeks

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(supporting information, Table S1), as described by Liu and collaborators19. These dietary MeHg

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concentrations were known to render embryonic MeHg burdens that would be suitable for our

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study. The females were housed in 3L flow-though tanks (10 fish per tank); 3 tanks were kept for

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each of the assigned MeHg supplemented diets. Additionally, untreated males were kept in

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separate 2L tanks (5 fish per tank, 1 male tank for each female tank in this study). Upon initiating

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dietary exposures, the females were paired with the untreated males (ratio of 10 females to 5

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males) for mating every 2-3 weeks to collect embryos for THg accumulation assessment. The

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purpose of this first exposure was to supply MeHg spiked diets to the females, in order to quickly

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obtain embryos with a range of different maternally transferred MeHg burdens. Therefore, once

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the embryos had reached a range of statistically different THg concentrations ranging from 0.005

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to 1 ppm, they were assigned to rearing tanks to be raised to adulthood (9 2L polycarbonate tanks

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per MeHg exposure concentration, 60 embryos per tank). This new generation of fish (G1) was

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exposed to dietary MeHg throughout its whole life span, and its offspring (G2) was later analyzed

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for behavioral impairments (supporting information, Figure S1). Dietary MeHg exposure of the

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G1 fish began at 7 dpf. The G1 larvae were fed ad libitum with a micropellet diet (Golden Pearls,

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Brine Shrimp Direct, Ogden, UT, USA) supplemented with nominal MeHg concentrations of 0,

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1, 3, and 10 ppm. The sizes of the food pellets were adjusted throughout the development of the

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fish from 50-100µm sized pellets (7-14 dpf), to a mixture of 50-100µm and 100-200µm sized


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pellets (15-30 dpf); to exclusively 100-200µm sized pellets (31-120 dpf). From 4 months of age

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onward, the fish were fed with a crushed flake diet (Pentair Aquatic Eco-Systems Aquatox food,

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Apopka FL, USA), also containing 0, 1, 3, and 10 ppm of MeHg (supporting information, Table

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S2). Upon the development of sexual characteristics, the fish were sorted by sex. Male and

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female fish were raised separately (3 breeding tanks of males and 3 of females per MeHg dietary

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exposure), however, both male and female fish continued to receive their designated

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experimental diets throughout their whole life span. Females were kept in 3L flow-through tanks

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(12 per tank) and males were kept in 2L flow-through tanks (6 per tank). The fish were then bred

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at 8 months of age (ratio of 12 females to 6 males) and a clutch of embryos was obtained from

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each of the three replicate male and female breeding tanks, for each of the MeHg exposure

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groups. Since this study was concerned with the behavioral effects of embryonic MeHg burdens

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as a consequence of parental dietary MeHg exposure, the newly spawned embryos (G2) were no

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longer raised on MeHg supplemented diets.

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Assessment of embryo mortality and early life stage toxicity (ELS-Tox). In order to

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evaluate embryo mortality due to MeHg exposure, all embryos were collected and counted

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immediately after spawning; the next morning, at 24 hpf, all unfertilized eggs were counted and

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discarded. Additionally, ELS-Tox scoring was performed to screen for observable teratogenic

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effects of MeHg exposure, this was performed by assigning the embryos a score of 0-4

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depending on the severity of morphological abnormalities, (0 = normal, 1 = one morphological

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anomaly, 2 = two morphological anomalies, 3 = more than two morphological anomalies, and 4

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= dead) as described by Heiden and collaborators20. ELS-Tox scoring was performed in 24, 72

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and 144 hpf embryos (pools of 10 embryos per each MeHg exposure dose tested, in triplicate).


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Analysis of Hg contents in diets and tissues. Total mercury (THg) content in tissues and in

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all MeHg supplemented diets were directly analyzed using a Direct Mercury Analyzer 80

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(DMA-80, Milestone Inc, Shelton, CT, USA) as described by Basu and collaborators21. The

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maternally transferred Hg burdens in both G1 and G2 embryos were analyzed from triplicate

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pools of two hundred embryos (4 hpf) for each exposure group. The morning after spawning, the

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ovaries were excised from three G1 adult females per dose, in triplicate, to assess THg in whole

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ovary tissue.

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VMR assay. The VMR assay has been suggested as a screening method to be used as an

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integral part of behavioral assessment in fish16. The experiment consists of quantifying the

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response of multiple zebrafish larvae reacting to sudden changes in light intensity. Here we

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performed a modified version of this assay, originally published by Emran and collaborators22.

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After 10 minutes of acclimation in the dark, the larvae underwent two cycles of alternating 10

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minute light and dark periods (for a total of 50 min). The locomotor activity of each fish was

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monitored with a DanioVision system (Noldus Information Technology, Leesburg, VA, USA).

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The light intensity during all light periods of the VMR assay was 221.75 lux (Fisher Scientific

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Traceable Dual-Range Light Meter, Pittsburgh, PA, USA). All VMR experiments were

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conducted from 12:00pm to 6:00pm to limit the effects of circadian rhythms16. The total distance

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traveled of each fish was analyzed using Ethovision software version 8.0 (Noldus Information

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Technology); individual fish (6 dpf) were observed in 24-well plates and tracked at a frame rate

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of 25 frames per second. A total of 126 larvae per exposure group were analyzed.

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Analysis of 7 dpf larval zebrafish swimming behavior. A custom made behavior

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observation chamber was designed for the purpose of this experiment. This chamber (10.25”

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deep × 18” wide × 14.24” tall) was constructed out of black polyethylene to block extraneous


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light. Within it, a manifold was placed to hold up to four Logitech C920 webcams facing

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downwards. Underneath the chamber, a flat 22” (Acer P221W) computer screen was used as a

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light source to enhance the contrast of the video recordings. The locomotor activity of the larvae

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was analyzed using a free and open source machine vision algorithm (python-ctrax

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www.ctrax.sourceforge.net) and tracking errors were corrected using the “fixerrors” MATLAB

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toolbox provided by the ctrax developers. All raw trajectory data was imported into a custom

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Microsoft Excel macro (Microsoft, Redmond, WA, USA) to calculate the rate of travel

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(mm/5min), swimming speed (mm/s), percentage of time active (“% activity”), minimum speed

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(mm/s) net-to-gross displacement ratio (NGDR) and maximum speed (mm/s). Additionally,

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since zebrafish larvae swim in a characteristic scoot-and-glide pattern, the frequency of these

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“scoots” (Hz) was also analyzed. A total of 180 fish tracks were analyzed for each of the four

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exposure groups.

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;

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Free swimming and foraging efficiency at 8, 12 and 16 dpf. The swimming performance of

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the larval zebrafish was further monitored at 8, 12 and 16 dpf. Immediately after each assay, the

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foraging efficiency of the larvae was also monitored. At 9:00am, on the day of the analysis, 25

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larvae were transferred to 10cm diameter glass Petri dishes containing 50mL of 28°C E2 embryo

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medium. The dish was then transferred to the recording chamber and the fish were allowed to

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acclimate for 5 minutes, after which they were recorded for 10 minutes. All recordings were

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carried out from 12:00pm to 6:00pm. A 30 second fragment was randomly selected from the 10

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minute clips to analyze the spontaneous swimming of the larvae. The behavioral parameters

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analyzed included % activity and NGDR. Immediately after the recording of free swimming,

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foraging efficiency was measured by introducing 6 Artemia nauplii per fish (i.e. 25 fish per dish

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foraging on 150 nauplii) into the Petri dish. The larvae were allowed to feed for 10 minutes, after


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which the remaining nauplii were counted. At the end of each experiment, the fish were returned

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to 2L tanks to be housed until the next experimental time point; the same fish were observed at 8,

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12 and 16 dpf. A total of 150 fish tracks were analyzed per exposure group.

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Data processing and statistical analysis. Statistical analyses were conducted with SigmaPlot

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software version 11.0 (Systat Software, San Jose CA, USA). All data was tested for normality

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with the Shapiro-Wilk test. Normal data were analyzed via ANOVA with subsequent assessment

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of multiple pair-wise comparisons via the Holm-Sidak method. Non-normal data were analyzed

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using Kruskal-Wallis ANOVA on ranks and multiple pair-wise comparisons were evaluated with

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Dunn’s method. Measured concentrations of THg in the embryos were log transformed prior to

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statistical analysis with one-way ANOVA due to the 3-to-12-fold differences between exposure

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groups. The data from the VMR, free swimming and foraging efficiency assays were analyzed

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with repeated measures two-way ANOVA. For scoot frequency analysis data, Gaussian curves

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were fitted using the dynamic fitting function in SigmaPlot. A P-value

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