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Ecotoxicology and Human Environmental Health

Lifelong exposure to dioxin-like PCBs alters paternal offspring care behavior and reduces male fish reproductive success David P. Coulter, Kara E. Huff Hartz, Maria S Sepulveda, Amy Godfrey, James E. Garvey, and Michael J. Lydy Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b03460 • Publication Date (Web): 01 Aug 2019 Downloaded from pubs.acs.org on August 2, 2019

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Environmental Science & Technology

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Lifelong exposure to dioxin-like PCBs alters

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paternal offspring care behavior and reduces male

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fish reproductive success

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David P. Coultera*, Kara E. Huff Hartza, Maria S. Sepúlvedab,c, Amy Godfreyb, James E.

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Garveya, Michael J. Lydya

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aCenter

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Illinois University, Life Science II Room 251, 1125 Lincoln Drive, Carbondale, IL USA 62901

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bDepartment

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Lafayette, IN USA 47907

for Fisheries, Aquaculture, and Aquatic Sciences and Department of Zoology, Southern

of Forestry and Natural Resources, Purdue University, 195 Marsteller St., West

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cLyles

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USA 47907

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KEYWORDS: dioxin-like PCBs; paternal offspring care; Pimephales promelas; endocrine

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disruption; anti-androgenic; fathead minnow

School of Civil Engineering, Purdue University, 195 Marsteller St., West Lafayette, IN

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ACS Paragon Plus Environment

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ABSTRACT

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Offspring survival, cohort performance, and ultimately population dynamics are strongly

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influenced by maternal characteristics (e.g., fecundity), whereas paternal contribution is often

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considered limited to genetic-driven fitness of males through sexual selection. However, male

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contribution to reproductive success can be particularly influential in species exhibiting paternal

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offspring care. Polychlorinated biphenyls (PCBs) are widespread, persistent contaminants that can

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disrupt maternal reproductive processes and negatively affect offspring. In contrast, how PCBs

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affect paternal reproductive success is largely unknown, but could ultimately affect population

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dynamics. We examined the effects of lifelong PCB exposure on the reproductive processes of

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male fathead minnows (Pimephales promelas), a species exhibiting sole paternal offspring care,

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by examining endocrine-associated gene expression, testes histology, secondary sexual

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characteristics, courtship ability, offspring care, and offspring survival. PCBs minimized male

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secondary sexual characteristics, but did not affect gonadal endpoints or inhibit ability to court

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females. Fathers exposed to high concentrations of dioxin-like PCBs had changes in gene

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expression, reduced offspring care behavior, and higher embryo mortality, possibly due to fathers

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spending less time within nests and less frequently tending to embryos. Through complex

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interactions among gene expression, physical characteristics, and behavior, PCBs inhibit paternal

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reproductive success and have the potential to suppress population size.


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INTRODUCTION

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Offspring survival and performance, and subsequent population demographics, are

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strongly influenced by maternal characteristics and offspring investment.1,2 For example, female

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size, age, and condition may affect the number, size, survival, and condition of offspring.1,3,4 In

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comparison, less research has examined the role of paternal contribution to offspring and

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subsequent population dynamics. When it is examined, male reproductive contribution is

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oftentimes determined by the quantity and quality of sperm,5 which can be positively correlated

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with male phenotype, including coloration6 and physical ornamentation.5 These characteristics

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might improve offspring survival and could ultimately influence population dynamics and

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abundance.

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Male reproductive contributions may become particularly influential in species exhibiting

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paternal offspring care. Male behaviors, including obtaining a nesting location7, defending

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offspring from competitors and predators8, and tending to nests and offspring9, have been shown

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to affect offspring fate. While a gradient of paternal care strategies exists, males have a stronger

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effect on population dynamics in species where they care solely for offspring after birth.10

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Parental reproductive effects are ultimately determined by a combination of parental

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characteristics and environmental conditions.3,11 Extreme fluctuations in temperature or

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precipitation, for example, can affect maternal nutritional and energetic provisioning from

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somatic tissue to gonads and, therefore, influence biochemical composition and viability of

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offspring.3,12 Natural variability in environmental conditions likely has relatively minor long-

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term negative effects on many populations because such fluctuations oftentimes last for a

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relatively short duration and affect only a few reproductive events (e.g., short-term drought13).


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These effects are also mitigated by species evolving under natural variability in environmental

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conditions and adjusting reproductive strategies accordingly.4 Anthropogenic alterations to

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ecosystems, however, have the potential to cause long-term population effects. Chemical

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contaminants (e.g., polychlorinated biphenyls; PCBs) are particularly problematic as they are

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globally distributed oftentimes at high concentrations14, target numerous pathways including

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reproductive endpoints15, bioaccumulate16, and remain in the environment for decades17, thereby

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negatively affecting organisms throughout their lives and influencing numerous successive

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reproductive events.

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How PCBs affect male reproductive contribution is largely unknown. Similar to other

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contaminants, some PCBs act as endocrine-disrupting compounds (e.g., dioxin-like or anti-

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androgenic congeners) where they reduce testosterone levels and can produce intersex gonadal

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tissue15,18,19. Observational studies suggest that PCBs may adversely affect aspects of paternal

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offspring care. Field studies with birds revealed that males with increased PCB concentrations

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spent less time defending nests containing eggs20 and were more frequently absent from nests for

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longer periods21. Long-term effects on offspring survival have also occurred, where elevated

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PCB concentrations in male fish spawned with uncontaminated females produced fewer

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offspring.22 These studies support the assertion that PCB contamination reduces male

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reproduction but provide sporadic information about mechanisms. A comprehensive

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understanding of how lifelong PCB exposure affects the suite of male reproductive endpoints

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would allow for more accurate predictions of how contaminants affect populations. Specifically,

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population models are generally parameterized with data on maternal reproductive rates23, but

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accuracy of model predictions would be improved if data on paternal and maternal reproductive

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contributions were incorporated, particularly in species exhibiting paternal offspring care.


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We conducted a laboratory experiment designed to identify the mechanisms by which

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PCB contamination affects male reproductive contribution from individuals to populations. We

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dosed male fathead minnows (Pimephales promelas), a widely distributed cavity-nesting species

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exhibiting male secondary sexual characteristics and sole paternal offspring care, with one of two

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PCB mixtures throughout their lives. Once mature, we bred males with uncontaminated females

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and evaluated effects on 1) expression of genes from brain tissue involved in reproductive

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endocrine pathways, 2) testicular cellular development, 3) secondary sexual characteristics, 4)

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courtship and reproductive behavior, 5) offspring survival, and 6) long-term offspring

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performance. We then used a simplified population model to illustrate the extent to which the

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PCB effects on reproductive endpoints observed in our experiments could have on population

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dynamics and overall abundance.

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MATERIALS AND METHODS

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Experimental design

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The experiment consisted of three treatments: a control and two PCB treatments with

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different congener mixtures and concentrations. Each treatment consisted of a recirculating

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freshwater system of ten 20-L aquaria. Twenty-five, thirty-day-old fathead minnows produced

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from a breeding colony (provided by US Environmental Protection Agency, Duluth, Minnesota,

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USA) were placed into each aquarium and reared for six months to maturity. Throughout this

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time, fish were dosed to treatment-specific PCB levels by feeding daily on freshwater

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oligochaete worms, Lumbriculus variegatus. Control L. variegatus cultures were reared in

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uncontaminated sediment, cultures for one PCB treatment (hereafter PCB-1) were reared in


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control sediment mixed 30 d prior with Aroclor 1254, and cultures for the second PCB treatment

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(hereafter PCB-2) were reared in sediment collected from a known, contaminated field site in a

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local lake (see Supporting Information).24 Each day, 50 L. variegatus were removed from each

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treatment culture and provided in equal amounts to fathead minnows (~28 mg L. variegatus·tank-

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1·day-1).

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similar across treatments (χ2 = 1.8, df = 2, P = 0.41). See the Supporting Information (SI) for

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complete details on experimental design.

After growing fish in experimental aquaria to seven months old, final fish density was

107 108 109

Spawning trials At seven months old, one adult male fathead minnow from each replicate aquarium was

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randomly selected to remain in the aquarium for spawning trials, with extra fish removed from

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the study (see Supporting Information). Two uncontaminated females (seven months old from a

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separately held colony) were placed into each aquarium at this time along with a nesting tile (102

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mm long x 102 mm wide x 51 mm deep). After 24 h, breeding behavior was recorded (Go-Pro

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Hero 3, GoPro, Inc., San Mateo, CA, USA) for 15 min, with the camera placed outside of

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aquaria facing nest openings and camera lights covered. Recordings were used to quantify

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breeding behavior (first five min discarded as acclimation) and included: duration each male

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stayed inside the nest; duration a female stayed inside the nest; frequency a male contacted the

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underside of the nest (i.e., nest maintenance); frequency a male circled inside the nest (i.e., nest

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guarding); and, frequency a female entered the nest.25

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Fish were allowed to breed for seven days. Nests were inspected daily to determine the number of viable embryos present. All adult fish were removed five days after first laying


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embryos to prevent adults from eating hatched larvae. Larvae were fed uncontaminated, 24 h old

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brine shrimp (Artemia) twice daily for 30 d post-hatch to assess possible paternal treatment

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effects on offspring foraging ability and development. After 30 d post-hatch, larvae were

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euthanized and measured for total body length and mass.

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Male fathead minnows used in spawning trials were further examined for possible effects

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of life-long PCB exposure. Males were visually inspected while spawning for the presence of a

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dark colored body and dark vertical bands, which are both secondary sexual characteristics.

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Euthanized males were photographed on their left side for geometric morphometric analysis to

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quantify body shape. Body length and mass were measured and the brain and pituitary were

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removed, flash-frozen in liquid nitrogen, and stored at -80°C for gene expression analyses.

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Additional secondary sexual characteristics assessed included the fatpad (fleshy dorsal pad

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anterior to the dorsal fin), which males use for cleaning nests, and nuptial tubercles (epidermal

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protuberances on the face), used to stimulate females during breeding or in territoriality. Fatpads

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were categorically scored based on the visual appearance on the body and were also assessed as

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relative size by removing from the body and weighing to quantify fatpad index (fatpad mass /

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body mass * 100). Nuptial tubercles were scored into one of three categories representing

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increasing tubercle size, where higher scores reflect larger tubercle size. See the Supporting

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Information for information on fatpad and tubercle scoring. Nuptial tubercles were also

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quantified as total number. Testes were also weighed to determine gonadosomatic index (GSI;

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gonad mass / body mass * 100) and preserved in 10% neutral-buffered formalin for histological

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analyses. Fulton’s condition factor (K), an index of body condition, was calculated for offspring

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as (M / L3)(100000), where M is total body mass (g) and L is total length (mm).

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PCB quantification

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Due to the cost of PCB analysis, three randomly selected males per treatment were

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analyzed for PCB concentration following spawning trials. Concentration was quantified as total

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PCBs (119 congeners), dioxin-like PCBs (12 congeners), and anti-androgenic PCBs (18

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congeners). See the Supporting Information for complete details. Dioxin-like congeners were

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examined because they have been found to alter maternal offspring care behavior in other

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organisms26-28 and to be estrogenic.29 Anti-androgenic congeners were examined due to their

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endocrine-disrupting potential in males.30,31

153 154 155

Gene expression Expression of genes involved in reproductive function and known to be influenced by

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PCBs was quantified in males using real-time polymerase chain reaction (see Supporting

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Information). Genes examined from pituitary include luteinizing hormone β (lhb), follicle

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stimulating hormone β (fshb), and thyroid stimulating hormone (tshb), and genes examined from

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brain tissue included aromatase (cyp19b) and gonadotropin releasing hormone receptor (gnrhr).

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Although brain and pituitary were initially collected on 30 total males, gene expression was only

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quantified on samples where personnel could ensure that the pituitary was present in the tissue

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sample after being removed from -80°C in order to guarantee comparable data across samples

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(17 total males).

164 165

Histological analysis of testes


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Testes were examined because PCBs have been found to affect gonadal tissue

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development across taxa.18,19 One testis was collected from each male and fixed in 10% buffered

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formalin, although five testes were ultimately not retained within tissue cassettes after

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preservation and were not analyzed. Hematoxylin and eosin (H & E) longitudinal sections were

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prepared following standard procedures and sections examined under a light microscope (10-

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40X) for staging and evaluation of any abnormalities.32 The thickness of germinal epithelium and

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the percent area occupied by different cell types (primary spermatocyte, secondary spermatocyte,

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spermatid, spermatozoa or spermatogonia) were measured from each testes on five seminiferous

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tubules per section, with three total sections (100 μm spacing between sections) examined per

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male.

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Population modelling The potential for long-term, population-level consequences to emerge from results

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observed during spawning trials were assessed using a deterministic life-stage model (stagePop

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package in R33). Embryo, larval, juvenile, and adult life stages were used in the model and, based

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on spawning trial results, three simulations were performed. One simulated population growth

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under baseline conditions where the probability of embryos surviving to larvae was the mean

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embryo survival (98%) from the control treatment, whereas the other simulations used mean

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embryo survival from the PCB-1 (86%) and PCB-2 (97%) treatments. In order to illustrate

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effects of reduced embryo survival on a growing population, simulations started with 100

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individuals, had a carrying capacity of 10,000 individuals, and were simulated for 100 years.

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Data analysis

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See the Supporting Information for complete details of statistical analyses.

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RESULTS

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Treatment PCB Concentrations

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Male fathead minnows differed in total PCB concentration (χ2 = 86, df = 2, P < 0.001),

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where control males had lower total PCB concentration than both PCB treatments, which were

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not statistically different from each other (Figure 1). Mean (SE) estimated oral dose of all PCB

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congeners was 4.9·10-5 (1.7·10-6) mg PCBs·kg fish-1·day-1 for control males; 0.006 (0.0003)

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mg·kg-1·day-1 for PCB-1 males; and 0.011 (0.0004) mg·kg-1·day-1 for PCB-2 males. Total

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concentration of dioxin-like congeners in males also differed across all treatments (χ2 = 637, df =

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2, P < 0.001) and was highest for the PCB-1 treatment and intermediate for the PCB-2 treatment

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(Figure 1; SI Table S6). Total concentration of anti-androgenic congeners was lower for the

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control treatment compared to the two PCB treatments, which did not differ (χ2 = 107, df = 2, P

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< 0.001; Figure 1; SI Table S6). Concentrations of total PCBs and dioxin-like PCBs in L.

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variegatus fed to male fathead minnows remained stable throughout the study (See SI Table S5).

204 205 206

Gene expression and histology


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Expression of lhb was upregulated in males exposed to the PCB-1 treatment compared to

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control males but was similar to males in the PCB-2 treatment (χ2 = 6.8, df = 2, P = 0.03; Figure

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2). Expression of fshb also differed among treatments (χ2 = 7.8, df = 2, P = 0.02), where males in

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the PCB-1 treatment had increased fshb expression compared to males in the PCB-2 treatment,

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and expression did not differ between the control and both PCB treatments (Figure 2).

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Expression of gnrhr (χ2 = 0.9, df = 2, P = 0.63), cyp19b (χ2 = 0.7, df = 2, P = 0.78), and tshb (χ2

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= 2.9, df = 2, P = 0.23) did not differ among treatments (Figure 2).

214 215

Histological sections of testes revealed no difference in thickness of germinal epithelium

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among treatments (F2,22 = 1.3, P = 0.31). Composition of sperm cell type was marginally

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significant across all treatments (F2,22 = 2.2, P = 0.06), but corrected pairwise comparisons

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identified no significant differences across treatments.

219 220

Physical characteristics

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Geometric morphometric analysis identified differences in male fathead minnow

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morphology across treatments (F2,27 = 3.2, P < 0.001), where morphology of control males was

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different from males in both PCB treatments (all F2,27 ≥ 3.7, P ≤ 0.004), and male morphology

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was similar between PCB treatments (F2,27 = 1.4, P = 0.17). Control males had deeper bodies

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than males in the two PCB treatments, particularly having a larger head and anterior half of the

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body (Figure 3A). All control males had dark colored bodies which was statistically more than

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the 64% (PCB-1) and 55% (PCB-2) of males with dark coloration in both PCB treatments


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(Fisher’s exact test: P = 0.002; Figure 3B). In contrast, the number of males with vertical

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banding on their bodies was similar for all treatments (overall mean = 25% of males; Fisher’s

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exact test: P = 0.71). Fatpad index (relative fatpad mass) was similar among treatments (F2,27 =

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0.3, P = 0.74), but fatpad score (categorical scoring of fatpad appearance on the body) was higher

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for control males than males in both PCB treatments (χ2 = 10.6, df = 2, P = 0.005; Figure 3C).

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Male body length (F2,27 = 0.1, P = 0.88), body mass (F2,27 = 0.2, P = 0.78), gonadosomatic index

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(i.e., relative gonad mass; F2,27 = 0.7, P = 0.49), total number of nuptial tubercles (used to

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stimulate females and in territoriality; χ2 = 6.1, df = 2, P = 0.05; no differences after pair-wise

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comparisons), and nuptial tubercle score (F2,27 = 1.2, P = 0.31) were similar across treatments

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(Table 1).

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Table 1. Mean (SE) response variables of male fathead minnow physical characteristics after six

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month exposure to PCB treatments that were not statistically different among treatments (n = 10

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per treatment).

242 243 244 245

Response

Control

PCB-1

PCB-2

% with vertical bands

33

18

23

Body length (mm)

58 (2)

58 (2)

59 (1)

Body mass (g)

2.5 (0.5)

2.4 (0.7)

2.5 (0.5)

Fatpad index (%)a

4.6 (0.4)

4.2 (0.9)

4.0 (0.3)

Type 1 tuberclesb

9 (1)

10 (1)

13 (2)

Type 2 tuberclesc

8 (1)

5 (2)

7 (1)

Type 3 tuberclesd

1 (0.3)

1 (0.3)

1 (0.3)

GSIe

1.4 (0.1)

1.6 (0.2)

1.6 (0.2)

aFatpad

index: relative fatpad mass. bType 1 tubercles: small nuptial tubercles present. cType 2 tubercles: nuptial tubercles with a radial base, the presence of furrows, and a jagged vertical appearance. dType 3 tubercles: pronounced tubercles with a large base and deep furrows. eGSI: gonadosomatic index.

246 247 248

Spawning behavior and success Male fathead minnows in the PCB-1 treatment spent, on average, approximately 50% less

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time inside spawning nests (χ2 = 6.5, df = 2, P = 0.04) than males in the other treatments (Figure

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4A), and males in the PCB-1 treatment circled 69% less often within the spawning nest, on

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average, than control males (χ2 = 6.4, df = 2, P = 0.04; Figure 4B). Males contacted the underside

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of the nest (i.e., nest maintenance) equally across treatments (χ2 = 2.3, df = 2, P = 0.10; Table 2),

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and the number of times females entered nests (χ2 = 0.1, df = 2, P = 0.71) and the duration

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females stayed inside nests (χ2 = 3.1, df = 2, P = 0.21) was similar among treatments (Table 2).


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The number of embryos laid on nesting tiles was variable and similar across treatments

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(χ2 = 0.1, df = 2, P = 0.95), although a higher percentage of embryos in the PCB-1 treatment

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became nonviable before hatch compared to the control (χ2 = 6.9, df = 2, P = 0.03; Figure 4C).

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There were no differences in body length (χ2 = 2.5, df = 2, P = 0.29), body mass (χ2 = 1.1, df = 2,

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P = 0.57), or body condition (Fulton’s K; χ2 = 3.7, df = 2, P = 0.47; Table 2) of larvae surviving

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from hatch to 30 d post-hatch. However, the density of 30 d post-hatch larvae in each tank was a

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significant covariate in analyses (P < 0.001).

262 263

Table 2. Mean (SE) response variables of male fathead minnow spawning behavior, spawning

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success, and characteristics of 30 d post-hatch (dph) offspring across PCB treatments. No response

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variables were statistically significant across treatments (n = 10 per treatment; 30 dph larvae:

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control n = 18; PCB-1 n = 24; PCB-2 n = 49).

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Response

Control

PCB-1

PCB-2

P-value

Frequency male touched nest

198 (48)

77 (39)

167 (41)

0.10

Frequency female entered nest

6 (2)

6 (1)

5 (2)

0.71

Seconds female in nest

226 (70)

222 (64)

105 (49)

0.21

Eggs laid

168 (39)

178 (48)

191 (53)

0.95

Larval survival to 30 dph (%)a

14 (12)

2 (1)

3 (1)

0.38

30 dph larval body length (mm)

17 (2)

17 (1)

16 (1)

0.29

30 dph larval body mass (g)

0.0858 (0.0328)

0.0537 (0.0156)

0.0442 (0.0044)

0.57

30 dph larval condition

1.15 (0.14)

0.86 (0.03)

0.87 (0.02)

0.47

aLarval

survival to 30 dph: percentage of hatched larvae surviving to 30 dph.


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Male whole-body total PCB concentration and concentration of anti-androgenic

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congeners were not related to the duration males stayed within nests (Figure 5A; anti-androgenic

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congeners: R2 < 0.001, P = 0.99), frequency males circled inside of nests (Figure 5B; anti-

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androgenic congeners: R2 < 0.001, P = 0.99), or the proportion of clutches becoming nonviable

272

(total PCBs: R2 = 0.49, P = 0.11; anti-androgenic congeners: R2 = 0.48, P = 0.11). In contrast,

273

male whole-body concentration of dioxin-like congeners was negatively related to duration in

274

nests (Figure 5C) and number of times males circled in nests (Figure 5D), but was not related to

275

proportion of clutches becoming nonviable (R2 = 0.03, P = 0.64).

276 277 278

Population model Model simulations indicated that reduced embryo survival representing that observed in

279

the spawning trials (98% control; 86% PCB-1; 97% PCB-1) can have a disproportionate effect

280

on population size and alter population trajectories (Figure 6). The simulated PCB-1 population

281

grew more slowly than the control and PCB-2 populations throughout the time simulated, and

282

had 39% lower adult abundance than the control at the largest difference between populations.

283

After 100 years, the PCB-1 population had 10% lower adult abundance than the control

284

population.

285 286 287 288

DISCUSSION Our results demonstrated that sublethal, lifelong PCB exposure affects a suite of male reproductive endpoints, including gene expression in the brain, male secondary sexual


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characteristics, and offspring care behavior. However, embryo survival was reduced only in

290

males exposed to high concentrations of dioxin-like PCB congeners, where male offspring care

291

behaviors were reduced. Past studies have determined that PCBs can affect individual male

292

reproductive responses (e.g., gonadal effects18) or paternal behavior21, whereas we have

293

experimentally identified mechanisms by which male reproductive success is influenced when

294

exposed to contaminants throughout life. Our work also identified that paternal exposure to PCB

295

mixtures with greater dioxin-like PCBs led to reductions in embryo survival. In a broader

296

ecological context, we also demonstrated the role that males exhibiting paternal offspring care

297

can provide to female reproductive success22 and to subsequent population dynamics.

298

The reduced male reproductive success (elevated embryo mortality) observed in one PCB

299

treatment (PCB-1) may have been due to altered male offspring care behavior, with males

300

spending less time inside nests and less frequently tending to nests. These paternal behaviors are

301

essential to prevent embryo mortality due to fungal infection by providing embryos with

302

sufficient dissolved oxygen (tail fanning) and reducing fungal spread by removing embryos that

303

develop fungus.25 While we observed effects on other male reproductive endpoints, it is unclear

304

the degree to which these contributed to reduced male reproductive success. Contaminated males

305

had diminished secondary sexual characteristics compared to control males, but this did not alter

306

male ability to court females or affect the number of eggs laid in nests. Similarly, gonadal

307

endpoints examined suggest that fertility may not have been affected since these endpoints (GSI,

308

sperm cell development, germinal epithelial thickness) were similar to control males. It is

309

possible, though, that reduced male fertility occurred in the PCB-1 treatment, since we did not

310

directly quantify sperm production or quality. Along with reduced paternal offspring care

311

behavior, this may have contributed to decreased male reproductive success. While we attempted


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312

to assess effects of paternal contaminant exposure on long-term (30 d post-hatch) offspring

313

performance, these results were inconclusive. Density of 30 d post-hatch offspring was a

314

significant covariate when comparing larval final length and mass across PCB treatments,

315

indicating larvae were inadvertently food-limited during the grow-out period which likely

316

masked treatment effects, including survival to 30 d post-hatch. However, the long-term effects

317

of paternal PCB exposure on offspring survival has previously been reported22, where negative

318

relationships were found between paternal PCB concentration and abundance of offspring alive

319

at the end of their first year of life.

320

One mechanism by which PCBs affect paternal reproductive ability is via endocrine

321

disruption. Several studies have reported that PCBs can interact with nuclear receptors, including

322

the estrogen (ERα/β), androgen (AR), and thyroid (TR) receptors, leading to estrogenic, anti-

323

estrogenic, and anti-androgenic effects, as well as thyroid disruption.34,35 Male fathead minnows

324

exposed to PCB mixtures in our study were altered via morphological changes and decreased

325

paternal offspring care behavioral. We also observed an upregulation in the expression of lhb in

326

the PCB-1 treatment, and while the average fsh expression for the PCB-1 group was nearly twice

327

that of the control, differences were not significant due to the overall high amount of variability

328

in the expression of this gene. An up-regulation of lhb and fsh is suggestive of an anti-androgenic

329

compensatory response by the pituitary driven by a decrease in androgen circulating levels,

330

which although not quantified in our study, has been reported as a common response after PCB

331

exposure in several male animal models, including humans.29,36,37 No changes in the expression

332

of gnrhr, cyp19b, and tshb were detected.

333

Differences in offspring care between PCB treatments illustrate how the specific

334

chemical mixture to which males are exposed affects male reproductive behavior and success.


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335

Poorer paternal offspring care in males from the PCB-1 treatment was likely due to their elevated

336

concentration of dioxin-like congeners. Laboratory studies with female rats have shown altered

337

maternal care caused by dioxin-like congeners, although effects are complex and sometimes in

338

contrast to our findings with male fish. Female rats fed a dioxin-like PCB congener spent more

339

time building28 and staying in nests26-28, more time grooming28 and nursing offspring26,28, and

340

had upregulation of oxytocin receptor (OXTR) gene expression, which is positively associated

341

with maternal offspring care.28 Data from a variety of taxa demonstrate that dioxin-like PCB

342

congeners can affect parental behavior, and future work designed to quantify dose-response

343

relationships might be able to better identify and predict these trends. However, it is unclear why

344

we did not observe diminished paternal offspring care and embryo survival in our PCB-2

345

treatment, which had males with intermediate dioxin-like PCB concentrations. Specific dioxin-

346

like congeners may play a large role in affecting male reproductive behavior, since

347

concentrations of specific dioxin-like congeners varied between our PCB treatments.

348

Additionally, a threshold concentration of one or more specific congeners may be needed to

349

elicit a behavioral response.38

350

Anti-androgenic PCB congeners potentially contributed to diminished paternal

351

reproductive success. PCB-126 is considered one of the most potent anti-androgenic PCBs;

352

however, we did not detect this congener in our fish. Instead, we detected several other known

353

anti-androgenic PCBs at concentrations > 500 ng·g-1 in both mixtures, including PCBs 101, 138,

354

and 153. A major anti-androgenic PCB (PCB-118) was detected in PCB-1 at 1,225 ng·g-1, but

355

only at a concentration of 217 ng·g-1 in PCB-2. Additional anti-androgenic PCBs (PCBs- 47,

356

170, 180) were only detected in PCB-2. Follow-up studies are required to determine the role of

357

these congeners on diminished paternal reproductive success. However, regardless of the


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358

mechanism, our results demonstrate how species exhibiting paternal offspring care can respond

359

quite differently to mixtures depending on the specific chemical composition.

360

Model simulations illustrated how slight reductions in embryo survival at levels observed

361

in our experiment can affect species at the population level. These model simulations were

362

simplistic demonstrations of potential effects of reduced embryo survival and do not incorporate

363

complex interactions that would affect wild populations, although recent work using a large-

364

scale model also demonstrated strong population-level consequences due to PCB exposure in

365

whales.23 Some of the interactions not incorporated into our model could act to magnify

366

contaminant effects on population dynamics. For example, males leaving nests unattended for

367

long periods of time could not only have increased offspring mortality from lack of offspring

368

care (e.g., fungal infections), but could also have additional mortality of offspring from nest

369

predation, which alone can be a substantial source of offspring mortality.9 Also, environmental

370

stressors including altered temperature39 or additional contaminants40 could further reduce the

371

number of offspring reaching sexual maturity. In contrast, wild population abundances may be

372

minimally affected by inhibited male reproductive success due to intraspecific competition

373

among breeding males. Contaminant loads vary among individuals within a system41, so highly

374

contaminated males could be less competitive than less contaminated males for nest sites or

375

females25 and, therefore, have fewer breeding attempts. The ability of contaminated males to

376

compete for nesting sites and females is unknown, though, and needs further investigation.

377

Finally, quantifying the combined effects of PCBs on maternal and paternal reproductive success

378

will improve population models, since both sexes ultimately become dosed in the wild.

379 380

The population-level consequences of inhibited male reproductive contribution resulting from PCB exposure to populations are obviously complex. For example, PCBs can not only


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381

affect reproductive endpoints, but can influence many other physiological pathways (e.g.,

382

metabolic42) that have the potential to also cause population-level effects. Nevertheless, evidence

383

from our study, similar work finding long-term paternal effects on offspring survival22, and

384

whole ecosystem studies43 demonstrate the potential for contaminant mixtures to alter population

385

dynamics. Future research should focus on bridging information gaps among individual-level

386

consequences of contaminants on reproductive success, species population dynamics, and

387

ecosystem interactions to understand the broader ecological consequences of specific

388

contaminant mixtures.

389 390

ACKNOWLEDGEMENTS

391

Amy Turlington, Federico Sinche, Andy Coursey, L. Zoe Almeida, and Tim Sesterhenn provided

392

laboratory and data analysis assistance. Funding was provided by the Center for Fisheries,

393

Aquaculture, and Aquatic Sciences at Southern Illinois University-Carbondale. The authors

394

declare no competing financial interests.

395 396

ASSOCIATED CONTENT

397

SUPPORTING INFORMATION

398

The Supporting Information is available free of charge on the ACS Publications website. Further

399

details about experimental design; materials and methods; sample processing for PCB

400

concentrations, gene expression, and histology; data and statistical analyses; results (PDF)


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REFERENCES

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Ecology 2010, 91, 2862−2873.

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size in a freshwater fish. Ecosphere 2016, 7, e01304.

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Figure captions

517

Figure 1. Mean (SE) whole-body concentration of all PCB congeners (total), anti-androgenic

518

congeners, and dioxin-like congeners from male fathead minnows after six-month exposure to

519

PCBs (n = 3 males per treatment). Different letters denote differences across treatments within

520

congener categories.

521

Figure 2. Mean (SE) gene expression from brain (gnrhr, cyp19b) and pituitary (lhb, fshb, tshb)

522

samples of male fathead minnows exposed to PCB treatments for six months during development

523

(control n = 5; PCB-1 n=6; PCB-2 n = 6). Different letters indicate differences in expression across

524

treatments for each gene. Reference gene was ribosomal protein L8 (rpl8) and expression was

525

normalized to the control.

526

Figure 3. Differences in secondary sexual characteristics of male fathead minnows exposed to one

527

of three PCB treatments for six months during development. (A) Warped outline drawing (scale

528

factor: 3) illustrating mean morphology of males from the control and males from both PCB

529

treatments. (B) Percent of mature males from each treatment having dark body coloration and (C)

530

mean (SE) fatpad score. Different letters indicate statistical differences among treatments (n = 10

531

per treatment).

532

Figure 4. Differences in spawning behavior and success in male fathead minnows exposed to one

533

of three PCB treatments for six months during development. Mean (SE) (A) duration males

534

remained in the spawning nest, (B) frequency males circled inside of nests (indicator of nest

535

guarding), and (C) percent clutch mortality. Different letters indicate differences across treatments

536

(n = 10 per treatment).


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537

Figure 5. Relationships between male fathead minnow whole-body PCB concentration and

538

spawning behavior (points represent individual males; n = 3 per treatment). Total PCB

539

concentration was not related to (A) duration males spent inside spawning nests or (B) frequency

540

males circled inside nests (nest guarding). However, concentration of dioxin-like congeners was

541

negatively related to (C) time in nests and (D) nest guarding. Several points represent multiple

542

individuals that had similar PCB concentrations or spawning behavioral responses. PCB

543

concentrations below reporting limit were set to 0 ng·g-1. Similar analyses with anti-androgenic

544

PCBs were not significant.

545

Figure 6. Modeled fathead minnow population growth under baseline embryo survival rates

546

(Control: mean 98% embryo survival), similar embryo survival rates from the PCB-2 treatment

547

(mean 97% survival), and reduced embryo survival rates from the PCB-1 treatment (mean 86%

548

survival). The arrow indicates the percent difference in adult abundance at the point of maximum

549

difference in population trajectories.

550


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AUTHOR INFORMATION

552

Corresponding Author

553

*E-mail: [email protected]. Phone: (618) 453-2608

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TOC ART

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