DOI: 10.5061/dryad.08kprr575
title: Alteration of reproductive behaviors by aromatase inhibition is population-dependent in an African cichlid fish

description: Although hormones are vital to an organism’s ability to respond to
 environmental stressors, they can be directly altered by the environment
 and impact reproductive behavior. For example, in some fishes, aquatic
 hypoxia (low dissolved oxygen) inhibits the aromatase enzyme that converts
 testosterone to estradiol. Here, we examined the effects of short-term
 aromatase inhibition on reproductive behavior in male Pseudocrenilabrus
 multicolor, a widespread African cichlid, from one normoxic river
 population and one hypoxic swamp population. We further tested the
 response of females to treated and untreated males. We predicted that
 aromatase inhibition would decrease courtship and competitive behaviors,
 but the swamp population would be less affected given generational
 exposure to hypoxia. Specifically, we compared competition and courtship
 behavior of males treated with a short-term exposure to an aromatase
 inhibitor with control fish from the two populations. We found that both
 courtship and competitive behaviors were affected by the interaction
 between treatment and population. River fish performed fewer courtship and
 competitive behaviors under the aromatase inhibition treatment while the
 behavior of swamp males was unaffected. Additionally, we found that
 females from the swamp population preferred males from the aromatase
 inhibition treatment and river females preferred control males. While we
 found behavioral effects of short-term aromatase inhibition, we did not
 find any effects on male nuptial coloration. Overall, these results
 indicate that the effects of short-term aromatase inhibition on behavior
 could depend on local adaptation in response to hypoxia.
description: Male-Male Competition Experiment
 To compare differences in aggression between males treated with
 an aromatase inhibitor and control males and the two populations, we
 measured aggression during male-male competition trials (n = 19 trials; 9
 trials river population, 10 trials swamp population). Specifically, we
 measured aggression of males when competing with an individual from the
 same population but exposed to the opposite treatment (e.g., aromatase
 males vs control male). From the 30-minute male-male competition trial
 videos, we quantified the number of lateral displays (male is
 perpendicular to the other fish and spreads all fins), charges (rapid
 movement towards the opposing male) as well as the time active (i.e.,
 moving in the water column vs maintaining a stationary position) of each
 male in the trial. Male Courtship &
 Female Preference Experiment After the male
 competition trials, we compared female preference and male courtship
 behaviors between males in the control and aromatase inhibited group and
 the two populations. Specifically, after the male-competition trials were
 completed, we conducted a 50% water change in both male tanks (renewing
 the fadrozole concentration in the aromatase inhibited group). After the
 water change was completed, a third tank containing a single female was
 placed between the two tanks containing the males, with black plastic
 visually isolating all tanks (Supplementary Figure 1). We placed a piece
 of PVC pipe as a shelter in the center tank for the female to hide in. The
 fish were allowed to acclimate to their tanks overnight (~24 hours). After
 acclimation, the fish were filmed for 30 min to record all movement and
 interactions (n = 20/trials; 10/population). Trials where females did not
 leave the hiding spot were excluded in the analysis of male courtship and
 female preference (n =14 trials included). In each trial, we recorded the
 amount of time a female spent within 7 cm (~1.5 body lengths) of each
 male. For males, we quantified the number of and quivers (full body
 shaking) as well as time spent active. Lateral displays were also recorded
 during male courtship trials as these displays may occur during aggressive
 encounters with other males and during courtship of females (Alphen et
 al., 2004; Baerends and Roon, 1950; Gray et al., 2012). However, because
 males could see the other male during trials, we cannot distinguish
 between lateral displays directed at the other male and the focal female.
 All videos were analyzed using BORIS (Friard and Gamba, 2016) and the
 observer was blind to the population and treatment of the fish.
 Aromatase Inhibition To
 quantify the effect of the aromatase inhibitor on circulating hormone
 levels, we measured excreted estradiol and testosterone in holding water
 using ELISA assays according to manufacturer instructions (estradiol
 501890, testosterone 582701, Cayman Chemical). The correlation between the
 concentrations of testosterone and estradiol in plasma and the
 concentration excreted by the gills has been established in male
 <em>P. multicolor</em> (Friesen et al., 2012a; Friesen et al.,
 2012b) as well as other species of fish (Friesen et al., 2012a; Kidd et
 al., 2010). Therefore, we used a holding water method that is ideal for
 small fish to collect hormone samples after the completion of the last
 behavioral trial. We placed individual males in glass containers
 containing 200 mL of clean treated water for one hour. After collection,
 the hormones from the holding water were extracted onto Sep-Pak Plus C18
 SPE cartridges using a vacuum manifold and then frozen at -20 ℃ until
 further processing. We extracted hormones from the cartridges using 4 mL
 of ethyl acetate and then dried the sample under a stream of nitrogen gas.
 Samples were then reconstituted in assay kit buffer (582701, and 582251,
 Cayman Chemical) according to manufacturer instructions. Pharmaceuticals,
 including estradiol, have been detected in water samples at water
 treatment plants in Ohio even after water treatment (Dutta et al., 2024),
 so we measured the average baseline level of hormones in water that did
 not contain fish. The baseline measurement (mean ± SE; 85.3 ± 12.7 pg/ml
 testosterone and 119.0 ± 17.0 pg/ml estradiol),<strong>
 </strong>was subtracted from the sample hormone measurements. Three
 males had estradiol measurements that were indistinguishable from baseline
 (2 control males and one aromatase-inhibited male). Pooled samples were
 measured at the start and end of each plate to determine inter-assay
 variability (testosterone 27.8% CV,  n = 3 assays; estradiol 24.9% CV; n =
 2 assays) and intra- assay variability (testosterone 7.9% CV; estradiol
 16.4% CV). Note that the concentrations of hormones in the pooled sample
 were low which likely contributed to the moderately high CV, but samples
 were distributed equally across plates to minimize the effects of intra-
 and inter-assay variability. Male
 Color Because color can influence female
 preference, we also compared male color between the control and aromatase
 inhibition treatments by taking spectrophotometric measurements. Following
 euthanasia in eugenol, we took spectrophotometric measurements using a JAZ
 spectrophotometer (Jazz, Ocean Optics, Dunedin, FL, USA) connected to a
 bifurcated fiber optic cable (QR400-7-UV-VIS<strong>,
 </strong>Ocean Optics)<strong> </strong>following Gray
 et al. (2011). We measured spectral characteristics of color at three
 positions along the abdomen (Supplementary Figure 2). These three
 measurements were then averaged. We applied a LOESS
 smoothing function to the spectrophotometric data to reduce noise (span =
 0.2). To address negative values, we added the absolute value of the most
 negative number to the entire curve. We used Pavo<strong>
 </strong>(Maia et al., 2019) to calculate the λ<sub>max
 </sub>(hue or wavelength of maximum reflectance; H1 in Pavo, but
 restricted between 400 nm to 700 nm) and R<sub>max
 </sub>(brightness or maximum reflectance; B3 in Pavo but restricted
 between 400 nm to 700 nm) for each male.
 Statistics All analyses
 were conducted in R version 4.4.1 (R Core Team, 2024). For linear mixed
 models (LMM) and generalized linear mixed models (GLMM) we used the lme4
 package (Bates et al., 2015; R Core Team, 2024). Results were considered
 significant at α &lt; 0.05. Effect sizes were calculated as partial
 eta squared (η<sup>2</sup>) using the effectsize package
 (Ben-Shacar et al., 2020), except for GLMM’s where we estimated the
 overall variance explained by the model (conditional
 R<sup>2</sup>) and variance explained by the fixed effects
 (marginal R<sup>2</sup>) using the MuMIn package (Bartoń,
 2023). All behavior models included the aromatase inhibition treatment
 (control or fadrozole), population (swamp or river), the interaction
 between aromatase inhibition treatment and population.
 We measured the effects of fadrozole on the testosterone
 excretion rate, estradiol excretion rate, and the ratio of testosterone:
 estradiol between males in the control and aromatase inhibition treatment
 using LM’s with aromatase inhibition treatment and populations as fixed
 factors and standard length as a covariate. The testosterone excretion
 rate, estradiol excretion rate, and the ratio of testosterone to estradiol
 were log10 transformed to meet model fit assumptions.
 To test whether the number of competitive behaviors (# of lateral
 displays + # of charges) and activity (time spent moving through the water
 column) were affected by the aromatase inhibition treatment or population
 during male competition trials, all models included treatment, population,
 and the interaction between treatment and population as fixed factors.
 Because males from the swamp population were on average slightly larger
 than males from the river population, standard length was included as a
 covariate in all behavior models (including courtship models outlined
 below). For all male competition models, trial was included as a random
 effect to account for lack of independence between male behaviors within a
 trial (i.e., the two males interacted with each other). A Poisson model
 with a log link was used to model the # of competitive behaviors. Activity
 was also modeled using an LMM. We also tested whether
 the number of male courtship behaviors (# of lateral displays + # of
 quivers) and activity (time spent moving through the water column) were
 affected by the aromatase inhibition treatment or population during mate
 choice trials. A Poisson model with a log link was used to model the # of
 courtship behaviors. Activity was modeled using an LMM. Trial was included
 as a random effect to account for lack of independence of male behaviors
 within a trial. We quantified female preference during
 the mate choice trails as the relative amount of time a female spent with
 the control male (time spent with control male – time spent with aromatase
 inhibited males). We excluded trials where the females did not leave the
 center of the tank and one influential point (Cooke’s distance &gt;
 1), for a total of 13 trials included in the analysis. Population,
 relative time spent in courtship behaviors (time control male spent in
 courtship behaviors – time aromatase inhibited male spent in courtship
 behaviors, the relative ventral λ <sub>max </sub>(λ
 <sub>max </sub>control male – λ <sub>max
 </sub>aromatase inhibited male)<strong><sub>
 </sub></strong>and relative ventral R<sub>max
 </sub>(R<sub>max </sub>control male – R<sub>max
 </sub>aromatase inhibited male), were also included in the female
 preference model. We tested for differences in spectral
 characteristics of male color (λ <sub>max</sub> and R
 <sub>max</sub>) due to aromatase inhibition treatment or
 population on the ventral regions of the fish where males are typically
 brighter. Aromatase inhibition treatment, population, standard length, and
 the interaction between aromatase inhibition treatment and population were
 included as fixed factors in all models. For males, the ventral λ
 <sub>max </sub>was square root transformed.
description: These data were analyzed in Boris (video analysis software), and
 analyzed using R.
description: # Population Variation in the Effects of Aromatase Inhibition on
 Reproductive Behaviors in male African Cichlid fish, Pseudocrenilabrus
 multicolor --- This data includes color and behavioral measurements of
 male cichlid* Pseudocrenilabrus multicolor that were in either control or
 aromatase inhibited treatments. This dataset includes two R scripts
 (Hormone Manipulationfinal.R and spec_fad.R), three datasheets and one zip
 folder containing spectrophometric data. The script Hormone
 Manipulationfinal.R uses FadTrials.csv, Fad_hormone_data_R.xlsx, and
 FADbymale.xlsx as input files to analyze behavioral data. The script
 spec_fad.R uses the files in the Fad_renamed zip folder as input to
 analyze spectral color data. ## Description of the data and file structure
 ### FadTrials.csv This dataset contains information from all behavioral
 trials for males and females. Data cells are empty if data was not
 collected for that individual. *Trial:* The trial id number. *Trial_type*:
 Trial type (either mate choice or male competition) *Fish*: Fish id number
 or the vat collection number *Pop*: Population * BWA= Bwera swamp * BUN=
 Bunoga river *Quivercount:* number of quivers *Chargecount*: number of
 charges *Displaycount:* the number of lateral displays *Quiver:* Time
 spent quivering (s) *Display*: Time spent in lateral display (s)
 *Timespent*: the time a female spent near that male (s) *Activity:*
 overall activity (s) DO: dissolved oxygen (mg/L) Temp: temperature (C)
 Treatment: * Control * FAD=Fadrozole SL: Standard length (cm) Sex: female
 or male Side: the side of the experimental tank a fish was in (Left, right
 or middle) Fish_Hid: N= No, Y= Yes, whether male approached other male or
 if female stayed in hiding spot FemaleHid: N=No, Y= Yes, whether female
 stayed in hiding spot ### Fad\_hormone\_data\_R.xlsx This dataset contains
 information on fish hormone data. Data cells are empty if data was not
 collected for that individual. Fish: Fish id number Testosterone (pg/ml):
 Raw testosterone data Testosterone: testosterone excretion rate (pg/ml/hr)
 Estradiol: (pg/ml): raw estradiol data Estradiol: Estradiol excretion rate
 (pg/ml/hr) GSI: Gonadosomatic Index ### FADbymale.xlsx This
 dataset contains output from spectral analyses for each male used to
 analyze the potential effects of fadrozole on male color. Datacells are
 empty if data was not collected for that individual. The different values
 represent different ways to analyze color as defined in the R package
 Pavo. including B3 (the maximum reflectance) and H1 (the wavelength at the
 maximum reflectance). See the Pavo package handbook for more information
 on these color
 descriptors: [https://book.colrverse.com/spectral-shape-descriptors.html](https://book.colrverse.com/spectral-shape-descriptors.html)  Fish: Fish id number B3Belly123: average B3 (the maximum reflectance (%); a measure of brightness) of three spots along belly; H1Belly123: average H1 (the wavelength at the maximum reflectance, measure of hue) of three spots value along belly B3Dorsal123: average B3 (the maximum reflectance (%);a measure of brightness) of three spots value along dorsal region H1Dorsal123: average H1 (measure of hue) value of three spots along dorsal region ### Fad\_renamed This zip folder contains raw jaz files that are analyzed in the spec_fad R script. Jaz files can be viewed and were analyzed in R, but they can also be opened in a text editor or in excel. Each jaz file represents the spectral measurements for a single spot on an individual fish. Jaz files were used to derive the B3 and H1 values provided in the FADbymale.xlsx file. Each file name contains information on the treatment (CON= control, FAD= fadrozole), population (BWA=bwera swamp, BUN= bunoga river), Fish ID, position name (Belly 1-3, Peduncle4, Blank, Dorsal, Lip), and position sequence (1-9). For example, file CON_BUN_FAD007_Belly1_5 contains the spectral data of the belly 1 position (the fifth measurement in the measurement sequence) for the fish FAD007 which was a river fish in control conditions. ## Code/Software This data was analyzed in R. Excel is required to open excel files. The package Pavo in R was used to analyze spectrophotometric color measurements.