DOI: 10.5061/dryad.7h44j0zx1
title: Differential reproductive plasticity under thermal variability in a freshwater fish (Danio rerio)

description: Human-driven increases in global mean temperatures are associated with
 concomitant increases in thermal variability. Yet, few studies have
 explored the impacts of thermal variability on fitness-related traits,
 limiting our ability to predict how organisms will respond to dynamic
 thermal changes. Among the myriad organismal responses to thermal
 variability, one of the most proximate to fitness – and, thus, a
 population’s ability to persist - is reproduction. Here, we examine how a
 model freshwater fish (Danio rerio) responds to diel thermal fluctuations
 that span the species’ viable developmental range of temperatures. We
 specifically investigate reproductive performance metrics including
 spawning success, fecundity, egg provisioning, and sperm concentration.
 Notably, we apply thermal variability treatments during two ontogenetic
 timepoints to disentangle the relative effects of developmental plasticity
 and reversible acclimation. We found evidence of direct, negative effects
 of thermal variability during later ontogenetic stages on reproductive
 performance metrics. We also found complex interactive effects of early
 and late-life exposure to thermal variability, with evidence of beneficial
 acclimation of spawning success and modification of the relationship
 between fecundity and egg provisioning. Our findings illuminate the
 plastic life-history modifications that fish may undergo as their thermal
 environments become increasingly variable.
description: <strong>Parental fish rearing and
 breeding</strong> We initiated our experiment in
 February 2021 with 600 freshly laid (&lt;4 h post-fertilization) F0
 zebrafish eggs from an ancestral stock of three wild-type AB lineages
 acquired from the Dalhousie University Zebrafish Core Facility (ZCF) that
 were previously reared under standard conditions.
 <strong>Thermal treatments during ‘Early’ and ‘Late’
 ontogeny</strong> We manipulated thermal
 variability during ‘Early’ (embryonic and larval stages; 0 – 29 days
 post-fertilization) and ‘Late’ (juvenile and adult stages; 30+ days post –
 fertilization) ontogeny of F0 ­­fish, employing a full-factorial,
 split-clutch design. The Early period ultimately represented the
 developmental plasticity treatment, whereas the Late period represented
 the acclimation treatment. We randomly and evenly split
 clutches of freshly-laid eggs from three lineages (pairings) of F0
 zebrafish into Constant or Fluctuating treatments. Fish remained in these
 treatments until the onset of the Late ontogenetic period, we once again
 split groups into Constant and Fluctuating treatments, representing the
 acclimation treatment. This factorial design resulted in four groups that
 experienced a combination of Early and Late thermal treatments:
 Constant-Constant (CC), Constant-Fluctuating (CF), Fluctuating-Constant
 (FC), and Fluctuating-Fluctuating (FF). We selected 27
 °C as our Constant temperature treatment, and a diel fluctuation from 22 –
 32 °C as our Fluctuating treatment. Warm temperatures were set to peak at
 12:00 pm, and cool temperatures were set to peak at 12:00 am. These
 thermal treatments were designed to have different magnitudes of
 variability while maintaining an equal thermal mean. Whereas temperatures
 ranging from 26 - 28 °C are often considered constant ‘optimal’
 temperatures for laboratory zebrafish (e.g., promoting growth, fecundity,
 and immune responses), 22 – 32 °C represents the maximum range of
 temperatures under which zebrafish develop normally, representing the
 extreme developmental thermal boundary beyond which high levels of
 mortality, deformation, and thermal stress occur. Yet, during reproductive
 season in natural habitats, temperatures tend to vary from ~23 – 31 °C. As
 such, the Fluctuating regime represents a physiologically challenging but
 ecologically realistic range of temperatures.
 <strong>Size and spawning success in fish exposed to
 thermal treatments during ‘Early’ and ‘Late’
 ontogeny</strong> In May 2021, when fish were
 sexually mature (120 d old), we began breeding experiments. Zebrafish are
 seasonal batch-spawners, having the ability to spawn continuously after
 reaching sexual maturity, though reproductive effort is largely expended
 during the monsoon season in their natural habitats. Further, in the wild,
 they generally exhibit an annual life cycle, typically experiencing only
 one reproductive season. We conducted breedings once per week over the
 course of five weeks for each lineage to attain an estimate of female
 fecundity. A one-week rest period between spawnings has been shown to be
 sufficient to allow zebrafish to recuperate their reproductive
 investment. To measure spawning success, which we
 define as the production of any eggs by a breeding pair, we randomly
 selected pairs of sibling males and females from the same treatment tank.
 Sibling pairs, rather than between-family crosses, were selected so that
 we could later delineate family-level effects from treatment effects. We
 placed breeding pairs in a zebrafish breeding box connected to the
 flow-through system the afternoon before breeding. We separated males and
 females using a clear plastic divider and placed identical sterilized
 plastic plants in each female’s compartment to stimulate egg production.
 The next morning, immediately after the onset of the light period at 08:00
 h, we disconnected the flow-through system from breeding tanks and removed
 dividers. We elevated one end of each tank by 5 cm to create a gradient of
 water depth to stimulate breeding and allowed pairs to breed for 3 h.
 After this period, fish were sedated via inhalation of buffered MS-222
 [80mg/L], weighed, and measured for standard length (SL) as our metric of
 body size, before being returned to their tank of origin.
 <strong>Female reproductive traits: Egg counts and
 measurements</strong> Female zebrafish will
 occasionally produce necrotic, ‘non-viable’ eggs, the result of resorption
 of mature ova; these non-viable eggs are identified by an opaque and
 asymmetric appearance. We collected and sorted F1 eggs from each spawning
 event, separating out non-viable eggs. We placed viable eggs in petri
 dishes filled with E3 embryo medium, and photographed them under a
 dissecting microscope, using a 0.001 cm micrometer for size calibration.
 We took the production of any eggs (viable or non-viable) to indicate that
 spawning took place (i.e., breeding conditions stimulated the female to
 produce eggs), but only included counts of viable eggs in our estimates of
 fecundity. We present fecundity data as the raw count of viable
 eggs. To estimate egg provisioning, we measured
 equatorial yolk diameter for a random subsample of up to 10 viable eggs
 per spawning using ImageJ (National Institutes of Health, Bethesda, MD).
 Yolk volume is a common and suitable proxy for maternal provisioning of
 eggs, and is relevant given its correlation with offspring fitness in
 oviparous ectotherms. We present yolk size data as the average diameter of
 ~10 eggs per clutch (mm). We converted these average diameters to a volume
 using the equation Volume = 4/3*pi*(Diameter/2)^3.
 <strong>Male reproductive traits: Sperm
 concentration</strong> Sperm concentration and
 volume are both significantly correlated with sperm quality in zebrafish,
 leading to higher rates of fertilization and lower rates of offspring
 deformity. In September 2021, two weeks after the last breeding, we
 measured sperm concentration and volume from 8-12 randomly selected F0
 males per treatment to use as a proxy for male reproductive quality.  We
 collected and measured sperm from anesthetized males taken directly from
 home tanks, using 10 µl glass microcapillary tubes. A visual assessment of
 the collected sperm was then made to minimize urine or faeces
 contamination in samples; poor quality samples indicated by low opacity or
 fecal content were removed from analyses. Known volumes of sperm from each
 male were then diluted with 4 microlitres of E400 medium. The absorbance
 of the resulting sample was measured at 400 nm using a Nanodrop
 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and sperm
 concentration (sperm cells/mL) was estimated by using a
 hemocytometer-calibrated standard curve. The sperm concentration data are
 presented after conversion from spectrophotometer values to concentration
 values using the hemocytometer-calibrated standard curve available at
 ZIRC: https://zebrafish.org/wiki/_media/protocols/cryo/zirc_rmmb_freezing_protocol.pdf
description: Data files are presented in Excel format, but they can be opened
 and viewed using the R statistical computing environment. We also include
 the R code for analyses and visualizations.