DOI: 10.5061/dryad.qz612jmm4
title: Multiple stressors lead to complex responses in reproductive behaviors in an African cichlid

description: Exposure to multiple environmental stressors is a common occurrence that
 can affect organisms in predictable or unpredictable ways. Hypoxia and
 turbidity in aquatic environments are two stressors that can affect
 reproductive behaviors by altering energy availability and the visual
 environment, respectively. Here we examine the relative effects of
 population and the rearing environment (oxygen concentration and
 turbidity) on reproductive behaviors. We reared cichlid fish (the Egyptian
 mouthbrooder, Pseudocrenilabrus multicolor) from two populations (a swamp
 and river) until sexual maturity, in a full factorial design
 (hypoxic/normoxic x clear/turbid) and then quantified male competitive and
 courtship behaviors and female preference under their respective rearing
 conditions. Overall, we found that the rearing environment was more
 important than population for determining behavior, indicating there were
 few heritable differences in reproductive behavior between the two
 populations. Unexpectedly, males in the hypoxic rearing treatment
 performed more competitive and courtship behaviors. Under turbid
 conditions males performed fewer competitive and courtship behaviors. We
 predicted that females would prefer males from their own population.
 However, under the hypoxic and turbid combination females from both
 populations preferred males from the other population. Our results suggest
 that reproductive behaviors are affected by interactions between male
 traits, female preference, and environmental conditions.
description: <strong>Rearing Experiment</strong>
 Fish were collected from one swamp site (Lwamunda) and one river
 site (Ndyabusole) in the Lake Nabugabo region of Uganda during May 2018.
 The swamp site is characterized by hypoxic (0.79 ± 0.1 mg/L, mean ± SE
 point in time measurements) and clear (2.02 ± 0.3 NTU) conditions, while
 the river site is normoxic (6.84 ± 0.2 mg/L) and moderately turbid (18.81
 ± 1.1 NTU; mean ± SE); measurements were taken between June and August in
 2015, 2016, and 2017 from Oldham (2018). The wild-caught fish were
 transported to the laboratory for a full-sibling split-brood rearing
 experiment. The two populations were held separately and allowed to breed
 until they produced ten independent broods (10 different male-female
 pairs) from each population. To ensure that each brood was produced by
 different parents, one male and three females from a single population
 were isolated until the first brood was observed.
 <em>Pseudocrenilabrus multicolor </em>are maternal
 mouthbrooders. After a female was determined to be holding a brood
 (distended jaw, refusal of food, and anti-social behavior) she was
 isolated to continue brooding. When the female released her brood
 (approximately 2-3 weeks), the young were housed in separate aquaria for
 one week then the brood was split randomly in a full-factorial design with
 two factors (oxygen treatment and turbidity treatment) with each having
 two levels (hypoxic or normoxic and clear or turbid, respectively). Broods
 from each treatment (oxygen and turbidity) and population (swamp or river)
 were spread evenly across 80, 19-liter aquaria to account for minor
 differences in temperature and light availability (10 tanks per
 population/treatment). Weekly water changes were conducted in each tank to
 maintain water quality. In the hypoxic tanks, oxygen
 was gradually reduced over a period of one week by bubbling nitrogen gas
 into the water and placing bubble wrap over the surface of the water. To
 maintain normoxic (mean ± SE: 7.52 ± 0.001 mg/L
 O<sub>2</sub>), and hypoxic conditions (mean ± SE: 2.27 ± 0.01
 mg/L O<sub>2</sub>), DO concentrations were measured once a
 week in normoxic tanks and three to seven times a week in hypoxic tanks
 using a YSI Pro2030 multimeter probe<strong> </strong>(see
 Supplementary 1 for full summary of rearing conditions). DO was adjusted
 as needed by bubbling ambient air or nitrogen gas into the tanks. In the
 turbid tanks, we gradually increased turbidity over a period of one week
 by adding ~1.5 mL of bentonite clay solution (70 g in 700 mL water) each
 day. To maintain clear (mean ± SE: 1.31 ± 0.03 NTU) and turbid (mean ± SE:
 16.6 ± 0.14 NTU) conditions, turbidity was measured once a week in clear
 tanks and one to two times a week in turbid conditions using a Hach 2100Q
 portable turbidimeter<strong> </strong>(Supplementary
 1).<strong> </strong>Turbidity was then adjusted as needed
 during weekly water changes, or for turbid tanks, by adding bentonite clay
 solution. Temperature was consistent across all tanks (mean ± SE: 24.9 ±
 0.02°C). The number of fish in each brood ranged from
 27-105 (mean ± SE: 42.25 ± 4.65), so the initial number of fish in each
 tank ranged from 6-25 fish. Fish from each brood were randomly divided
 between treatments. Note that at one week old we could not determine sex,
 so sex ratios varied across tanks. To decrease and homogenize density, the
 number of fish in each tank was reduced to ten at six weeks and further
 reduced to six fish at 20 weeks post-release. The fish were reared under
 their respective treatment conditions until they were ~1.5 years old when
 they were used for the behavioral experiments outlined below. At the time
 of the behavioral experiments, all fish were sexually mature, e.g., males
 had red spots on anal fin, blue lips, and yellow coloration on ventral
 surface. <strong>Male-Male
 Competition</strong> To test the effects of
 oxygen, turbidity, and population on aggression, we conducted male-male
 competition trials. Male competition trials were conducted in the same
 environmental conditions that the fish were raised under (Summary of trial
 rearing conditions; Supplementary 2). Hypoxic conditions were achieved by
 bubbling nitrogen gas into the trial tanks and placing bubble wrap over
 each compartment to help maintain low oxygen conditions, while normoxic
 tanks had air stones to maintain their respective treatment conditions.
 Bentonite clay solution was added to turbid trial tanks to increase
 turbidity. Males were size-matched (less than 8% difference by standard
 length; distance from snout to caudal peduncle, range 4.4-6.3 cm; mean ±
 SD: 5.13 ± 0.46 cm) within trials and were from different broods of the
 same population so that siblings were not used in a trial together. DO,
 turbidity, and temperature measurements were taken from the center of the
 tank at the start of acclimation and immediately after the trial (for
 conceptual diagram see Figure 1A; for snapshot from trials see
 Supplementary Figure 1). An opaque barrier was placed between the two
 males (barrier likely did not block olfactory cues) during an overnight
 acclimation (18-22 hours). Note that because sex ratios varied between
 tanks, our pre-experimentation housing was not standardized. Accordingly,
 social status of males and prior experience of males and females likely
 differed across trials. To initiate the trial the opaque barrier was
 replaced with a clear one so that the males could interact without direct
 contact to prevent injury, and trials were filmed from the side for 30
 min. Full water changes were performed between trials. Trials were only
 included in the analysis if the males approached each other (n = 33 trials
 analyzed out of 60; 2-5 trials per treatment, sample sizes in
 Supplementary Table 2). Note, that while not quantified formally, there
 was generally higher mortality in the hypoxia turbid combination,
 particularly in the river population. Therefore, sample sizes in this
 treatment combination (river/hypoxic/turbid) in the male-male competition
 trial were low (two trials) due to a lack of size-matched males that could
 be tested. We quantified the number of lateral displays (where the male
 places himself perpendicular to the opposing male), charges (rapid
 movement towards the opposing male with mouth closed), and bites (rapid
 movement towards the opposing male with mouth open), as well as the time
 active (i.e., moving in the water column). All videos for mate choice and
 male competition were analyzed by one observer using BORIS (Friard and
 Gamba 2016). The observer was blind to the population of fish in the
 trials. <strong>Mate
 Choice</strong> To test the effects of oxygen,
 turbidity, and population on reproductive behaviors in males and females,
 we compared female preference and male courtship behaviors between fish
 reared under the four different treatment combinations and from the swamp
 and river populations. Specifically, we conducted mate choice trials on
 fish under the treatment conditions that the fish was reared under (e.g.,
 a female raised in hypoxic/clear conditions was placed in a mate choice
 tank with the choice of two males, one from each population – swamp and
 river, that were also raised under hypoxic/clear conditions). The male
 from the same population as the female is referred to as the native male
 while a male from the different population is referred to as non-native
 male. All fish within a trial were from different broods so that siblings
 were not used in a trial together. Immediately before the overnight
 acclimation (18-22 hours), the fish were photographed to quantify color
 (see below) and males were size-matched (less than 8% difference by
 standard length; range 3.9-6.1 cm; mean ± SD: 4.98 ± 0.49 cm).
 In each trial, the trial tank was divided into three compartments
 (Figure 1B). Sand was included in the trial tanks so that males could
 build territorial pits overnight. We randomly assigned two males from the
 same rearing treatment but different populations (swamp population and
 river population) on either end of the trial tank. Males were isolated
 from each other and not able to physically interact. The female was placed
 in the center compartment, also physically isolated from both males. Only
 females that were not mouthbrooding were selected for use in trials.
 However, we did not record whether females were gravid at the time of the
 trial, so female receptivity to male courtship likely varied. A piece of
 PVC pipe in the middle of the tank was provided as a shelter so the female
 could choose not to interact with either male. The fish were allowed to
 acclimate to the tank overnight during which time opaque barriers
 prevented the fish from seeing each other (barriers likely did not prevent
 olfactory cues). After acclimation, the opaque barriers were removed
 (leaving clear barriers), and the fish were filmed from the side for 30
 min to record all movement and interactions. Some males were reused, but
 they were given a least one week in between testing, and males never
 encountered another male more than twice.  Only trials where females left
 the hiding spot were included in analysis (n = 81 trials analyzed out of
 105; 10-11 females per treatment/population Supplementary Table
 2). The treatment conditions were created and
 maintained as in competition trials (described above). Videos were
 recorded from the side and analyzed using BORIS by one observer that was
 blind to the population. In each videotaped 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 lateral displays and quivers (shaking
 movement of the whole body), as well as time spent active.
 <strong>Male Color</strong> Male
 color was calculated using a previously established protocol (Maan et al.
 2004). Briefly, before overnight acclimation, males were placed in a
 small, clear box (i.e., photo cuvette) filled with water and gently
 pressed against the front of the box using a grey board to be photographed
 (Canon PowerShot SX740 set to automatic). Using Adobe Photoshop, the
 photos were white balance corrected using a white standard placed on the
 front of the cuvette. The area of the fish (excluding fins and eyes)
 containing red (hue = 0-26 and 232-255, saturation = 40-97%) and yellow
 (hue = 27-45, saturation = 40-97%) pixels relative to the total number of
 pixels of the fish body was quantified. This value was used to calculate
 the overall % red + % yellow color to evaluate overall differences in red
 and yellow coloration as these colors typically resulting from carotenoid
 pigments may be correlated with fitness proxies and female preference in
 cichlids (Maan et al. 2006; Sefc et al. 2014).
 <strong>Statistics</strong> All
 analyses were conducted in R, version 4.3.0 (R Core Team 2019), and we
 used the lme4 package, version 1.10-35.1 (Bates et al. 2015) for
 generalized linear mixed models (GLMM) and linear mixed models (LMM). To
 determine the potential independent and interacting influences of oxygen
 and turbidity on behavior, we modeled these treatments as separate fixed
 factors and included an interaction term between oxygen and turbidity in
 all models. We also included population and standard length in all models.
 Brood was included as a random effect in all GLMM’s and LMM’s to account
 for relatedness between siblings. All data and analyses are available on
 Dryad. For the male competition trials, we modeled the
 number of competitive behaviors (# of lateral displays + charges + bites)
 and activity (time spent moving through the water column). All models
 included oxygen, turbidity, the interaction between oxygen and turbidity,
 and population as fixed factors, standard length as a covariate and trial
 and brood as random effects. Due to overdispersion, a GLMM with a negative
 binomial distribution was used to model the # of competitive behaviors
 while activity (s) was modeled using an LMM. Color data
 (% red + % yellow coloration) for males used in the mate choice trials was
 analyzed using a LMM which included oxygen, turbidity, the interaction
 between oxygen and turbidity, and population as fixed factors, standard
 length as a covariate, and brood as random effect. Because some fish were
 used in more than one trial (90 unique males were used in mate choice
 trials), we only included the color measurement for the male’s first trial
 in this analysis. For males in the mate choice trials,
 we also tested for an effect of oxygen, turbidity, population, and
 standard length on the number of male courtship behaviors (# of lateral
 displays + # of quivers) and activity (time spent moving through the water
 column). Because some males were used in more than one mate choice trial
 (90 unique males were used in mate choice trials; 8-13 males per
 population/treatment combination, Supplementary 3), we only analyzed male
 behavior from each male’s first mate choice trial. All models included
 oxygen, turbidity, the interaction between oxygen and turbidity, and
 population as fixed factors and standard length as a covariate. A GLMM
 with a Poisson distribution and a log link was used to model the # of
 courtship behaviors. Activity was modeled using an LMM.
 We quantified female interest in male courtship as the time a
 female spent within 7 cm (approximately 1.5 body lengths) of either male
 using a LM with oxygen, turbidity, the interaction between oxygen and
 turbidity, and population as fixed factors. We also modeled the preference
 of a female for males from her own population using a LM. Preference was
 quantified by subtracting the time spent with the non-native male from the
 time spent with the male from her own population. A positive value
 indicated that a female spent more time with male from her own population.
 We calculated a PCA loading factor that combined standard length and the
 overall % of red + yellow color, as color and size are positively
 correlated in males of this species. We also calculated relative courtship
 behavior (# of courtship behaviors by male from native population- # of
 courtship behaviors by non-native male). A positive value for relative
 courtship indicated that the male from the local population engaged in
 more courtship behaviors. The relative differences of the PCA loading
 factor and time spent in courtship were included in the model of female
 preference. We used a SEM to understand the direct and
 indirect effects of oxygen, turbidity, and population on female
 preference. We used the results from our GLMM and LMM’s on male traits as
 well as previous studies to develop our <em>a priori</em>
 model (Figure 2; (Atkinson and Gray 2022; Gray et al. 2012; McNeil et al.
 2016)). Our SEM was modeled using lavaan (Rosseel 2012). To test the
 assumption of multivariate normality, we examined the χ<sup>2
 </sup>Q-Q plot from the MVN package (Korkmaz et al. 2014). To
 improve assumptions of multivariate normality, female preference (time
 spent with native or non-native male) and male courtship behavior (# of
 courtship behaviors) were square root transformed. Transformed data met
 assumptions of multivariate normality. For variables that were expected to
 be related, but without an obvious causal relationship, we included the
 covariance between residuals (male courtship, male size, and the time a
 female spent with either male). Whether our a priori model adequately fit
 the data was determined by examining the Model χ<sup>2 
 </sup>(<em>P</em> &gt; 0.05), and approximate fit
 indices (Comparative Fit Index &gt; 0.95, Standardized root mean
 square residual &lt; 0.10) (Grace 2020). We did not remove
 nonsignificant paths from our final model.
description: <strong>Excel </strong>is required to open the files
 dfcomp, dfcourtbyfish, and dfcourtbytrial.
 <strong>R </strong>is required to run the scripts
 Competition_script.R, Courtship_script.R and
 Rearing_env_data_script.R
description: # Multiple Stressors Lead to Complex Responses in Reproductive Behaviors
 This dataset includes competitive and courtship behaviors of the cichlid
 *Pseudocrenilabrus multicolor* in response to rearing under hypoxic and
 turbid conditions. Behaviors were quantified during mate choice trials and
 male-male competition trials. Behaviors quantified include time a female
 spent within 7 cm of a male, activity, lateral displays, quivers, and
 charges. This dataset includes four excel files. ## Description of the
 data and file structure ### rearing\_env\_data.xlsx The
 rearing_env_data.xlsx file contains the weekly environmental measurements
 during rearing for all fish and was analyzed in the R script
 Rearing_env_data_script. *Tank*: Tank number that the fish was reared in
 (1-80). *Date:* Date measurement was taken. *Population:* Population of
 fish in tank corresponding to LWA (Lwamunda swamp) or NYA (Nydabusole
 river) *Brood:* Brood designation of fish in tank *RT:* Rearing treatment
 * HC= Hypoxic, Clear * HT= Hypoxic, Turbid * NC= Normoxic, Clear * NT=
 Normoxic, Turbid *Turb:* Turbidity measured in Nephelomtric turbidity
 units, days when measurements were not taken have been left blank *DO:*
 Dissolved oxygen measured in mg/L, days when measurements were not taken
 have been left blank *Temp:* Temperature measured in degrees Celsius, days
 when measurements were not taken have been left blank *Cond:* Conductivity
 measured in microSeimens/ cm, days when measurements were not taken have
 been left blank *Ph:* measured pH, days when measurements were not taken
 have been left blank ### dfcourtbyfish.xlsx dfcourtbyfish.xlsx holds data
 from the mate choice trials arranged by individual fish and was analyzed
 using the script Courtship_script.R. *pca1:* PCA combining standard length
 and % yellow and red *Brood:* Brood a fish came from *Trial:* Trial id
 number *Fish:* Fish id number *Reartank:* Tank a fish was reared in
 *trial_date:* Date of trial *Population:* Population of male relative to
 female, cells were left blank if the row pertains to a female * Same=
 Male's population was the same as the female's population. *
 Different= Male's population was not the same as the female's
 population. *Pop:* Population of origin * LWA= Lwamunda swamp * NYA=
 Nydabusole river *Quivercount:* number of quivers, cells were left blank
 if the row pertains to a female *Chargecount*: number of charges, cells
 were left blank if the row pertains to a female *Displaycount:* number of
 lateral displays, cells were left blank if the row pertains to a female
 *Quiver*: time spent in quivers (s), cells were left blank if the row
 pertains to a female *Display*: time spent in lateral displays (s), cells
 were left blank if the row pertains to a female *Timespent:* time a female
 spent within 7 cm of a male (s) *Turb:* Turbidity measured in Nephelomtric
 turbidity units *DO:* Dissolved oxygen measured in mg/L *Temp:*
 Temperature measured in degrees Celsius. *RT:* Rearing treatment * HC=
 Hypoxic, Clear * HT= Hypoxic, Turbid * NC= Normoxic, Clear * NT= Normoxic,
 Turbid *SL*: Standard length measured in cm *Mass:* mass measured in g
 *Sex:* Sex of fish (male or female) *Side:* Trial tank compartment (Left,
 Middle, Right) *Oxygen:* Oxygen treatment (Hypoxia or Normoxia)
 *Turbidity*: Turbidity treatment (Clear or turbid) *Redandyellow:* % red
 and % yellow pixels *Activity:* time spent active (s), cells were left
 blank if the row pertains to a female *Reproductivetime:* time spent in
 reproductive activities (s), cells were left blank if the row pertains to
 a female *allcourt*: total # of courtship behaviors (lateral displays and
 quivers), cells were left blank if the row pertains to a female
 *sqrtcourtprop:* the square root of the number of courtship
 behaviors/minute active, cells were left blank if the row pertains to a
 female ### dfcourtbytrial.xlsx dfcourtbytrial.xlsx holds data from the
 mate choice trials arranged by trial and was analyzed using the script
 Courtship_script.R. *Trial:* Trial id number *Brood:* Brood id *Fish:*
 Fish id number *Reartank:* Tank a fish was reared in *trial_date:* Date of
 trial *Pop:* Population of origin * LWA= Lwamunda swamp * NYA= Nydabusole
 river *Turb:* Turbidity measured in Nephelomtric turbidity units *DO:*
 Dissolved oxygen measured in mg/L *Temp:* Temperature measured in degrees
 Celsius. *RT:* Rearing treatment * HC= Hypoxic, Clear * HT= Hypoxic,
 Turbid * NC= Normoxic, Clear * NT= Normoxic, Turbid *Oxygen:* Oxygen
 treatment (Hypoxia or Normoxia) *Turbidity*: Turbidity treatment (Clear or
 turbid) *pca1same:* Pca combining SL and red+yellow color for males in the
 trial of the same population as the female. *Broodsame:* Brood id of the
 male from the same population as the female *Fishsame:* Fish id of the
 male from the same population as the female *Reartanksame:* Rearing tank
 of the male from the same population as the female *Popsame*: population
 of the male from the same population as the female *Timespentsame*: time
 (s) a female spent near the male from the same population
 *SLsame*: Standard length (cm) of the male from the same population as the
 female *Sidesame*: trial tank compartment of the male (Left or Right) from
 the same population as the female *Redandyellowsame*: % red and % yellow
 of the male from the same population as the female *Activitysame*: time
 spent active (s) by the male from the same population as the female
 *Reproductivetimesame*: time spent (s) in reproductive activities by male
 from the same population as the female *reproductivepropsame*: proportion
 of time active spent in reproductive activities by male from the same
 population as the female *allcourtsame*: # of courtship behaviors
 (display+quivers) by male from the same population as the female
 *reproductivecount30same*: # of courtship behaviors (display+quivers)/
 minute trial by male from the same population as the female
 *pca1different:* Pca combining SL and red+yellow color for males in the
 trial not of the same population as the female. *Brooddifferent:* Brood id
 of the male not from the same population as the female *Fishdifferent:*
 Fish id of the male not from the same population as the female
 *Reartankdifferent:* Rearing tank of the male not from the same population
 as the female *Popdifferent*: population of the male not from the same
 population as the female *Timespentdifferent*: time (s) a female spent
 near the male not from the same population *SLdifferent*: Standard length
 (cm) of the male not from the same population as the female
 *Sidedifferent*: trial tank compartment of the male (Left or Right) not
 from the same population as the female *Redandyellowdifferent*: % red and
 % yellow of the male not from the same population as the female
 *Activitydifferent*: time spent active (s) by the male not from the same
 population as the female *Reproductivetimedifferent*: time spent (s) in
 reproductive activities by male not from the same population as the female
 *reproductivepropdifferent*: proportion of time active spent in
 reproductive activities by male not from the same population as the female
 *allcourtdifferent*: # of courtship behaviors (display+quivers) by male
 not from the same population as the female
 *reproductivecount30different*: # of courtship behaviors
 (display+quivers)/ minute trial by male not from the same population as
 the female *sqrtTimespentdifferent*: square root of time (s) female spent
 with male not from the same population *Timespentdifference*: time (s)
 spent with native male - time spent with non-native male
 *Redandyellowdifference*: % red and % yellow of native male - time spent
 with non-native male *SLdifference:* standard length (cm) of native male -
 standard length of non-native male *Timespenttotal*: time a female spent
 within 7 cm of either male *sqrtreproductivecount30different*: sqrt of #
 of courtship behaviors (display+quivers)/ minute trial by male not from
 the same population as the female *sqrtreproductivecount30same*: sqrt of #
 of courtship behaviors (display+quivers)/ minute trial by male from the
 same population as the female *sqrttimespentsame:* sqrt of time spent with
 male from the same population *allcourtdifference:* # of courtship
 behaviors performed by native male- # of courtship behaviors performed by
 non-native male *pca1difference:* PCA1 of native male - PCA1 of non-native
 male ### dfcomp.xlsx The dfcomp.xlsx file includes data from the
 competition trials and was analyzed using the script Competition_script.R.
 *Brood:* Brood a fish came from *Reartank:* Tank a fish was reared in
 *Fish:* Fish id number *Trial:* Trial id number *trial_date:* Date of
 trial *Active:* time spent active (s) *Charge*: # of charges *Retreat:* #
 of retreats *Displaycount:* number of lateral displays *Display*: time
 spent in lateral displays (s) *ASR:* time spent in aquatic surface
 respiration (s) *Bite*: number of bites *Quivercount:* number of quivers
 *Quiver*: time spent in quivers (s) *DO:* Dissolved oxygen measured in
 mg/L *Temp:* Temperature measured in degrees Celsius. *Turb:* Turbidity
 measured in Nephelomtric turbidity units *Pop:* Population of origin *
 LWA= Lwamunda swamp * NYA= Nydabusole river *RT:* Rearing treatment * HC=
 Hypoxic, Clear * HT= Hypoxic, Turbid * NC= Normoxic, Clear * NT= Normoxic,
 Turbid *SL*: Standard length measured in cm *Mass:* mass measured in g
 *Side:* Trial tank compartment (Left or Right) *Pit*: Did fish build a
 territorial pit (Yes or No), if the presence of a pit was not recorded the
 cell was left blank *Oxygen:* Oxygen treatment (Hypoxia or Normoxia)
 *Turbidity*: Turbidity treatment (Clear or Turbid) *Release_date*: Date of
 release from mother (akin to birthday) *diff_in_days:* age at time of
 trial *aggtotal*: total number of competitive behaviors (# of
 charges+bites+displays) *aggrate*: # of competitive behaviors/minute
 active *logaggrate:* log of # of competitive behaviors/minute active
 *Redandyellow:* % red and % yellow pixels of males