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.

Parental fish rearing and breeding

We initiated our experiment in February 2021 with 600 freshly laid (<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.

Thermal treatments during ‘Early’ and ‘Late’ ontogeny

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.

Size and spawning success in fish exposed to thermal treatments during ‘Early’ and ‘Late’ ontogeny

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.

Female reproductive traits: Egg counts and measurements

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.

Male reproductive traits: Sperm concentration

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

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.