DOI: 10.5061/dryad.k0p2ngfdg
title: Multiple stressors disrupt sex hormone and fitness outcomes: Effects of turbidity and hypoxia on an African cichlid fish

description: Freshwater organisms are faced with a complex array of environmental
 stressors that can negatively affect endocrine function and subsequent
 fitness outcomes. Hypoxia and turbidity are examples of two environmental
 stressors that are increasing due to human activities that could lead to
 endocrine disruption and reduced reproductive output. Our research
 addresses how hypoxia and elevated turbidity affect traits related to
 reproductive success, specifically sex hormone concentrations, investment
 in reproductive tissues, and body size. We used wild fish from two
 populations (a river and a swamp) of an African cichlid, Pseudocrenilabrus
 multicolor, to produce offspring that were reared in a full factorial
 split brood rearing experiment  (hypoxic/normoxic x
 clear/turbid). River and swamp populations represent divergent habitat
 types with respect to the stressors of interest, being well-oxygenated but
 turbid or hypoxic and clear, respectively. Overall, we found evidence for
 plastic responses to both stressors. Specifically, we found that there was
 an interactive effect of oxygen and turbidity on testosterone in males
 from both populations. Additionally, males of both populations reared
 under hypoxic conditions were significantly smaller in both mass and
 standard length than those raised under normoxic conditions, and males
 reared under hypoxic conditions invested less in reproductive tissues
 (quantified as gonadosomatic index). Hypoxia and turbidity are experienced
 naturally by this species and these environmental stressors did not affect
 the number of eggs laid by females when experienced in the absence of
 another stressor (i.e., normoxic/turbid or hypoxic/clear). However, there
 was an interactive effect of hypoxia and turbidity as females reared and
 maintained in this treatment combination laid fewer eggs. This research
 underscores the importance of considering the possibility of stressor
 interactions when determining how anthropogenic stressors affect fitness
 outcomes.
description: <em>Rearing Experiment</em>
 Research was conducted under approval from The Ohio State
 University Institutional Animal Care and Use Committee (2014A00000055-R1).
 To assess the effects of hypoxia and turbidity on hormone levels and
 fitness metrics, we measured testosterone and estradiol in F1 <em>P.
 multicolor</em> males derived from wild-caught fish reared in a
 split brood rearing experiment (full factorial, hypoxic/normoxic x
 clear/turbid treatments). Fish used as parents in this rearing study were
 collected from one swamp site (Lwamunda) and one river site (Ndyabusole)
 in the Lake Nabugabo region of Uganda in May 2018. The swamp site is
 typically hypoxic (0.79 ± 0.1 mg/L, mean ± SE point in time measurements)
 and clear, albeit tannin-stained (2.02 ± 0.3 Nephelometric turbidity
 units; NTU) while the river site is typically normoxic (6.84 ± 0.2 mg/L)
 and moderately turbid (18.81 ± 1.1 NTU); measurements were taken between
 June and August in 2015, 2016, and 2017 from Oldham (2018). Ten broods
 from each of two populations (one river and one swamp population) were
 reared under each combination of rearing conditions. Once a female
 released her brood, the young were housed for one week under normoxic,
 clear conditions before being split randomly into the four different
 treatment groups. In the hypoxic treatment group, the oxygen was reduced
 gradually over a period of one week by placing bubble wrap over the
 surface of the water. In the turbid tanks, turbidity was gradually
 increased over a one week period by adding approximately 1.5 mL of
 bentonite clay solution (100g clay/ L water) Dissolved oxygen
 concentrations were measured throughout the rearing experiment (DO:
 hypoxic mean ± SE = 2.27 ± 0.01 mg/L O<sub>2</sub> or normoxic
 mean = 7.52 ± 0.001 mg/L O<sub>2</sub>) using a YSI Pro2030
 multimeter probe 3-7 days per week and was adjusted as needed by adjusting
 bubble wrap, and bubbling water with ambient air or nitrogen gas.
 Turbidity (clear: 1.31 ± 0.03 NTU or turbid: 16.6 ± 0.14 NTU) was measured
 1-2 days per week using a Hach2100Q portable turbidimeter and was adjusted
 as needed during weekly water changes or through the addition of the
 bentonite clay solution. Fish were fed Tetramin Tropical crisps once a
 day, <em>ad libitum </em>for five minutes. The treatments and
 populations were dispersed randomly across 80 aquaria to minimize small
 differences in light and temperature (mean ± SE: 24.9 ± 0.02 ℃ )(Williams,
 2023). We maintained fish in a 12L:12D photoperiod. All fish were mature
 at the time of sampling. To distinguish between fish within a tank we used
 white visible implant elastomer tags. We tagged 348 fish (156 males and
 192 females) when they were between 345 to 386 days old.
 The results reported here are part of a larger study where male
 and female reproductive behaviors (Williams<em> et al.</em>,
 2024) were examined using the fish described here. For additional details
 of the rearing experiment components see (Williams, 2023,
 Williams<em>, et al.</em>, 2024) and<strong>
 </strong>(Tiarks, 2024). We used subsets for measures of hormones (n
 = 77; when males were between 466 to 514 days old) and egg count (n = 37
 when females were between 615 to 794 days old). After hormone and
 fecundity and reproductive behavior (Williams<em>, et
 al.</em>, 2024) data were collected, we sampled fish for
 morphological metrics (n = 255) and GSI (n = 234). The age of fish ranged
 from 571 to 827 at the time of final sampling due to restrictions to
 laboratory access during the COVID-19 pandemic.
 <em>Hormone Collection</em>
 Hormone samples were collected from a subset of adult males
 between June 24<sup>th</sup>, 2020, and July
 5<sup>th</sup>, 2020, when fish (n = 77) were between 466 to
 514 days old (fish were maintained at treatment conditions from 7 days
 post-release).<strong> </strong>Hormones were collected
 between 09:30 to 13:30 to minimize diel variation in hormone
 concentrations (Cowan<em> et al.</em>, 2017). We collected
 hormone samples using a non-invasive method that has been previously
 validated in <em>P. multicolor</em> and other species of fish
 (Friesen<em>, et al.</em>, 2012, Kidd<em> et
 al.</em>, 2010). Hormone samples were collected by placing
 individual fish in glass containers containing 200 mL of clean treated
 water for one hour, during which time hormones are released from the gills
 at a rate that is highly correlated with the concentration in plasma
 (Friesen<em>, et al.</em>, 2012). The oxygen and turbidity of
 the sampling water matched the rearing treatment the fish was raised in.
 Hormone samples were extracted onto Sep-Pak Plus C18 SPE cartridges and
 frozen at -20 ℃ until further processing. Hormones were extracted from the
 cartridges using ethyl acetate and dried under a stream of nitrogen gas.
 Samples were then reconstituted in assay kit buffer (582701 and 582251,
 Cayman Chemical) according to manufacturer instructions. To quantify
 background levels of hormones in the water, four water samples not
 containing fish were also collected, and the concentrations of
 testosterone and estradiol were measured to establish a baseline level.
 The average baseline measurement of hormones (mean ± SE = 85.3 ± 12.7
 pg/ml testosterone and 119.0 ± 17.0 pg/ml estradiol),<strong>
 </strong>was subtracted from the hormone measurements. Sample
 measurements lower than the baseline water measurements were replaced with
 zeroes for analysis (5 testosterone, 38 estradiol measurements). Because
 estradiol measurements were below the detection threshold for many
 individuals, we did not statistically analyze estradiol by itself, only
 the ratio of testosterone/estradiol. Estradiol averages are presented in
 Supplementary Figure 1. <em>Morphological
 Measurements</em> Standard length (cm; length
 from tip of snout to end of caudal peduncle) and mass (g) were collected
 before euthanizing (in 1:10 Eugenol: Ethanol solution) males (n = 118) and
 females (n = 137; see Supplementary Table 1 for treatment sample sizes) at
 the end of the rearing experiment. <em>Fitness
 Metrics</em> To understand the effects of the
 rearing environment and population on investment in reproduction
 (Brewer<em> et al.</em>, 2008), we analyzed GSI in a subset of
 males (n = 107) and females (n = 127). The gonads were removed, and we
 calculated GSI using the following formula: GSI = (gonad mass/mass)
 *100. To quantify the effects of the rearing treatments
 on fitness more directly, we placed a subset of sexually mature females
 from the rearing experiment in aquaria with non-sibling males of the same
 rearing treatment and population group. The males and females were not
 used as parents more than one time, so each brood has a distinct
 combination of parents. Rearing treatment conditions were maintained in
 these aquaria. Females were observed daily, and when they were determined
 to be carrying a brood, we removed the brood from her mouth, counted the
 number of eggs, and weighed the brood (ME104E, Mettler Toledo analytical
 balance) to determine brood mass (g; n = 37 broods; 2-6 broods per
 treatment/population combination). We then calculated the average egg mass
 by dividing the brood mass by the number of eggs. Average egg mass and
 number of eggs in the brood were used as proxies for reproductive
 success. <em>Statistics</em>
 All analyses were conducted in R, version 4.3.0 (R Core Team,
 2024). Linear mixed models (LMM) were performed using
 <em>Lme4</em>, version 1.10-35.1 (Bates<em> et
 al.</em>, 2015). To understand the interacting and independent
 effects of oxygen, turbidity, and population on sex hormones and fitness
 metrics, all models contained oxygen, turbidity, the interaction between
 oxygen and turbidity, and population as fixed factors. We assessed
 homogeneity of variance by examining residual plots and the assumption of
 normality by examining Q-Q plots. If data did not meet the model
 assumptions, they were transformed. Because fish were genetically related
 (siblings), we also included brood as a random effect in all models of
 hormones, GSI, and size. Averages are presented as mean ± SE. Results were
 considered significant at α &lt; 0.05 (Thiese<em> et
 al.</em>, 2016).
 <em>Hormones</em> In males,
 testosterone and the ratio of testosterone to estradiol (an indicator of
 aromatase enzyme activity<strong> </strong>(Friesen<em>,
 et al.</em>, 2012, Shang<em> et al.</em>, 2006))  were
 log transformed to improve model assumptions. We analyzed both
 testosterone and the ratio of testosterone to estradiol using LMM’s with
 oxygen, turbidity, the interaction between oxygen and turbidity, and
 population as fixed factors, log-transformed standard length as a
 covariate, and brood as a random effect. The time a sample was collected
 was initially included as a covariate in all hormone models, but it was
 not significant and subsequently removed from all models.
 <em>Body Size</em> We analyzed,
 log-transformed standard length and log-transformed mass in males and
 females separately using LMM’s with oxygen, turbidity, and the interaction
 between oxygen and turbidity as fixed effects and brood as a random
 effect. Because sampling for size data was completed over approximately
 eight months, age at the time of sampling was also included as a covariate
 in these models. <em>GSI</em>
 To improve model assumptions, GSI was log transformed. We
 analyzed GSI separately for males and females using LMM’s with brood as a
 random effect and oxygen, turbidity, the interaction between oxygen and
 turbidity, and population as fixed factors and log-transformed SL as a
 covariate. For females, an additional fixed factor, Brooding (Yes/No), was
 included to account for females that were mouth brooding at the time of
 sampling, as their GSI was generally much lower (0.825 ± 0.124) than
 non-brooding females (3.516 ± 0.321). <em>Fitness
 Metrics</em> Finally, we analyzed the effects of
 rearing treatment on egg number using a Poisson model (GLM) with oxygen,
 turbidity, interaction between oxygen and turbidity as fixed factors, and
 the mass of the mother as a covariate. One brood was determined to be
 influential (Cook’s distance &gt; 1) and was removed from all models.
 Additionally, the sample size of the hypoxic/turbid treatment combination
 produced by river females was low (n = 2), so the river and swamp
 populations were pooled for the analyses of egg number, batch weight, and
 egg mass. We also analyzed the batch weight of the broods (g) and egg mass
 (g; batch weight/ # of eggs) using linear models with oxygen, turbidity,
 the interaction between oxygen and turbidity as fixed factors, and the
 mass of the mother as a covariate.
description: <strong>Excel </strong>is required to open the excel
 files Rearingexperimentdissectiondata_morph, Rearing_Experiment_Hormones,
 Broodinfo, Brood_release_dates, F2Ftinesstracking, and
 Reartankinfo <strong>R </strong>is required
 to run the scripts F2rscript_final.R, and
 Hormones_morph_analysis.R
description: # Multiple Stressors Disrupt Sex Hormones and Fitness Outcomes --- This
 dataset includes morphological and fitness data of the cichlid
 *Pseudocrenilabrus multicolor* in response to rearing under hypoxic and
 turbid conditions. This dataset includes seven excel files and two R
 scripts, F2rscript_final.R and Hormones_morph_analysis.R. Both scripts
 were analyzed using R version 4.3.0.  F2rscript_final.R uses the input
 files F2Fitnesstracking.xlsx, Broodinfo.xlsx, Brood_release_dates.xlsx,
 and Reartankinfo.xlsx to analyze egg mass, brood mass, and egg number. The
 script Hormones_morph_analysis.R uses the input
 files Rearingexperimentdissectiondata_morph.xlsx, Rearing_Experiment_Hormones.xlsx, Broodinfo.xlsx, and Brood_release_dates.xlsx to analyze sex hormones, gonadosomatic index, standard length, and mass. ## Description of the data and file structure ### **2020\_VIE\_.xlsx** This excel file contains information on when fish were tagged. Note: Empty cells are samples where that data was not collected for that fish. *Fish:* Fish id number *Side:* The side the fish was tagged on * L= left * R=right *Number:* the number of VIE tags used *Tank:* the rearing tank the fish came from *Do*: Oxygen rearing treatment (Normoxic or Hypoxic) *Turb:* Turbidity rearing treatment (Clear or turbid) *RT:* Rearing treatment * HT= Hypoxic, turbid * NC= Normoxic, clear * NT= Normoxic, turbid * HC= Hypoxic, Clear *Pop:* Population * NYA= Nydabusole, river population * LWA= Lwamunda, swamp population *Brood:* Brood number *Sex:* Sex (male or female) *TagDate:* Date fish was tagged ### **Brood\_release**\_**dates** This dataset includes the days that each brood were born (i.e., the day we removed the brood from the mothers) *Brood*: the brood and population # *Release_Date:* the day the brood was born ### **Broodinfo** This excel file includes the brood that was in each tank. *Reartank*: The tank #  *Brood*: The brood # ### **F2Fitnesstracking** This dataset includes the fitness data for each population. *BroodNo:* The brood number *Mother*: The id of the mother *Father:* The id of the father *Pop*: Population (LWA swamp population or NYA river population) *Treatment*: Rearing treatment * HT= Hypoxic, turbid * NC= Normoxic, clear * NT= Normoxic, turbid * HC= Hypoxic, Clear *Datestarted:* The date the mother/father pair was created *Datefound:* The date a brood was found *Number:* The number of eggs in the brood *Mass:* The total mass (g) of the brood *Pics:* Picture number of the brood *Mothersl:* The standard length of the mother (cm) *Mothermass*: The mass of the mother (g) ### **Rearing\_Experiment\_Hormones\_final** This dataset includes information on sex hormones collected in males. *Fish:* The fish id *Collection_Time*: The time the hormone sample was collected *Testosterone (pg/ml):* The testosterone concentration *Testosterone*: the testosterone excretion rate (pg/ml/hr) *Estradiol (pg/ml)*: The estradiol concentration *Estradiol:* The estradiol excretion rate (pg/ml/hr) *SL: *Standard Length (cm) *Mass*: the mass in g *Hormones_**collected_**Date*: The date the hormones were collected *Pop*: Population (LWA swamp population or NYA river population) *RT*: Rearing treatment * HT= Hypoxic, turbid * NC= Normoxic, clear * NT= Normoxic, turbid * HC= Hypoxic, Clear *Brood*: The brood # *Reartank*: The tank the fish were reared in ### **Rearingexperimentdissectiondata\_morph** This dataset contains the size, mass, sex and gonad mass of the fish at the time of dissection Note: Empty cells are those where we did not collect that data point for that particular fish (i.e., we did not dissect gonads for all fish) *Fish:* The fish id *Date*: Date of Dissection *Reartank*: The tank the fish were reared in *Pop*: Population (LWA swamp population or NYA river population) *RT*: Rearing treatment * HT= Hypoxic, turbid * NC= Normoxic, clear * NT= Normoxic, turbid * HC= Hypoxic, Clear *TL:* Total Length (cm) *SL:* Standard Length (cm) *Mass:* Mass (g) *Gonad_Mass:* Gonad mass (mg) *Sex:* F= Female, M=Male *Brooding*: Was the fish brooding at time of dissection  * Y= Yes * N= No * NA= Not applicable (for males) ### **Reartankinfo.xlsx** This dataset tells which fish were in each rearing tank *Fish*: The fish id *Reartank*: The tank the fish was reared in