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Captive zebrafish (Danio rerio) exhibit a limited repertoire of mating behaviors, likely due to the somewhat unnatural environment of aquaria. Observations in their natural habitat led us to believe that a depth gradient within the mating setup would positively affect fish mating. By tilting the tank to produce a depth gradient, we observed novel behaviors along with a preference for oviposition in the shallow area. Although we did not see an increase in the likelihood of a pair of fish to mate, we did see an increase in the embryo output in both adults and juveniles. In the adults, tilting led to a significant increase in embryo production (436±35 tilted vs. 362±34 untilted; p<0.05). A similar effect was seen in juvenile fish as they progressed through sexual maturity. These results suggest that tilting of mating cages in the laboratory setting will lead to demonstrable improvements in embryo production for zebrafish researchers, and highlights the possibility of other manipulations to increase fecundity.
Zebrafish are found in the area circumnavigated by Pakistan, Myanmar, Nepal, and Kamatara India. Though it is thought that they retreat to the nearby streams during the dry season (Engeszer et al., 2006), they are typically seen in clear, shallow floodplains such as lakes and rice cultivation areas (Spence et al., 2006). It is here that they have been observed to spawn on outskirts of flooded paddies, where the water depth is shallower (Rowena Spence, personal observation). Reasons behind this behavior have not been well explored but may include predator avoidance, sunlight exposure, or food availability.
Spawning behaviors are typically observed starting between 6 and 12 weeks postfertilization, corresponding to the appearance of sexual dimorphism (Darrow and Harris, 2004). Mating in the wild is timed with the monsoon season, although females have been found with mature ovum year round, suggesting food availability may also play a role (Spence et al., 2006). Daily photoperiods have been shown to be a key factor in the induction of mating in both domesticated and wild-caught fish, with the first hour of sunlight being the busiest for spawning events (Darrow and Harris, 2004; Spence et al., 2007). It has also been noted that the addition of cold water in the aquaria, simulating a heavy rain, can induce additional spawning (Breder and Rosen, 1966; Rowena Spence, personal observation). In addition to the aforementioned season and food availability, successful matings are dependent upon a number of factors within the tank. Pheromones released by females can suppress other females' fecundity; additionally, mating chambers with less than 200mL affect the clutch size negatively (Goolish et al., 1998; Gerlach, 2006).
Previous studies of zebrafish in a lab setting described basic mating behaviors, including elliptical movements made by the male around the female and the subsequent oviposition of 5–20 unfertilized eggs, which are then fertilized by released sperm (Guthrie, 1993; Darrow and Harris, 2004; KemajouNjiwa et al., 2004). Observation of the zebrafish in their natural environments suggests that they prefer to mate along the shallow shorelines (Rowena Spence, personal observation). However, in the laboratory setting, the fish are typically exposed to a uniform depth. This led us to hypothesize that recapitulating the natural preference for shallow mating areas in the laboratory would lead to the augmentation of fecundity and a more diverse set of mating behaviors. Given the popularity of the zebrafish for both behavioral and genetic studies, interventions that increase embryo output and behavioral expression are likely to directly enhance the utility of this model organism.
Two different zebrafish strains were used in this study. Research was carried out on either Tuebingen (Tu) or AB (wildtype) stock. These were housed at the zebrafish facility at Children's Hospital Boston or Dana Farber Cancer Institute. At Children's Hospital Boston, the Tu stocks are kept on 14:10 light–dark cycle at 28°C. All fish were raised in a density of 4 fish/L and kept in solid Schwartz Tank Systems (Maschmühlenweg 40–42–37081, Göttingen, Germany). Unless otherwise noted, these fish were fed three times a day with a mixture of Artemia and Tetramin Flake (Herrenteich 78 49324, Melle, Germany). At Dana Farber Cancer Institute, the AB stock was raised and housed at 2 fish/L in varying sized AHAB Tanks (Aquatic Habitats, Apopka, FL). Their light cycle is 14:10 light–dark and are kept at 29°C. Feeding consists of two daily feeds of Artemia and one feed with a mixture of Tetramin flakes and Larval Diet AP100 (Zeigler Brothers, Gardners, PA).
All matings were set up in 3 L Schwartz Tank (11×21.5cm) with a screen-bottom insert of the same dimensions placed inside (Fig. 1A, B). The mating pair is placed inside the insert, while the embryos are able to fall through the holes of the screen (2×2mm), protecting them from possible adult predation.
The night before observation, fish at the age of 7 months were put into several mating pairs with dividers in the setup to keep the male and female separated. Each tank received a fake plant that was put in the same location in each tank. In the morning, tanks were either left flat creating a uniform depth (4cm) (Fig. 1A) or tilted, producing a depth gradient (from 0 to 4cm) (Fig. 1B). The dividers were pulled one at a time and videotaped from a side and bird's eye view, although only representative videos are shown. Observations made from videos were not analyzed quantitatively but used merely to show alteration in behavior patterns.
Fish at the age of 9 months were put into 20 groups of five fish with the female-to-male ratio of 3:2. They were assigned and kept in a numbered tank for the duration of the study, which allowed for the same group of fish to be set up each week. Once a week for 2 weeks, they were put into the mating setup to allow collection of embryos.
Half of the tanks were tilted, and the tilting status was switched the following week to allow a crossover observation to be conducted. Setups were done the night before at 1500h, taken down at 1200h the next day, and always left overnight in the same location. This consistent schedule allowed for the control of exposure time and surrounding environment, two factors thought to influence mating success. After taking the fish out of the setup, the tanks containing embryos were carefully lifted, so not to alter embryo location, and a grid was placed beneath the tank (Fig. 1C). Rows on the grid designate a particular end of the tank. On those tanks that were tilted, row A is consistently the shallow area, which gradually deepens into row P. Grid alignment was the same for the untilted tanks to avoid variance due to the environmental differences such as ambient light and noise. The embryo numbers for each grid block were counted and recorded. A replicate study was carried out at the Dana Farber fish facility using the AB strain of fish. Studies were done concurrently to avoid possible anomalies in mating success due to seasonal changes, weather, and the like. Data were manually entered into GenePattern (Broad Institute, Cambridge, MA) to create heat maps based on the number of embryos in each quadrant.
At the age of 7 months, 20 groups of five fish each with the female-to-male ratio of 3:2 were put into numbered tanks similar to the site preference methods. The time and location was also kept constant, while the tilting condition switched each week for a 6-week period. After fish were removed, the total number of embryos was counted and recorded.
Fish were put into groups of six unsexed fish at the age of 6 weeks and fed two meals per day. Again each group was assigned a numbered tank and setup each week at the same time and same location. For the juveniles, the tilting condition was kept constant from week to week for the 6-week study.
As in the site preference methods, fish were put into 20 groups, numbered, and kept separate. To look at the likelihood that a pair of fish would mate, we used individual pairs of Tu with the female-to-male ratio of 1:1. These fish were set up for 3 weeks in a row in a crossover design. Fish were rated with either a “yes,” they mated, or “no,” they did not. A clutch that was dead or contained less than 10 embryos was considered a “no.”
For comparisons of tilting effects in the adults, total embryo production for each tank (n=10 in each condition per week) was compared using ANOVA with repeated measures. Because there was no significant effect of time in the adults, p-values represent the results of an unpaired t-test for totals in each condition.
For juvenile comparisons, embryo production was ascertained in the same manner as for the adults. In this case, the nonparametric Wilcoxon rank sum test was utilized in lieu of the Student's t-test due to unequal variances in the groups.
Fish were recorded and observed expressing mating behaviors in both the tilted and untilted condition. General descriptions of mating behaviors are described in Table 1. In the untilted condition (Supplemental Video S1, available online at www.liebertpub.com), which produces a uniform depth, we see the male chasing the female throughout the entire tank space and also making vertical undulations using the entirety of the water column. Just before oviposition, the male may quiver and then follow by wrapping himself around the female. In the tilted condition (Supplemental Video S2, available online at www.liebertpub.com), a depth gradient is present, forming a shallow end and a deep end. The female waited in the shallow end for the male to arrive or was escorted there by the male. On an odd occasion, we observed the male waiting in the shallow area for the female to join him. No such waiting behavior was seen in the untitled condition. Just before oviposition, they circled each other in a head-to-tail fashion, followed by the male adjusting his position to swim alongside her, typically pinning her. While staying in the shallow end of the tank, the male began the quiver followed by the wrap around behavior. The complete wrap around behavior is shown clearly in Supplemental Video S3 (available online at www.liebertpub.com).
Next we tested oviposition site preference in the presence and absence of a depth gradient. Visual heat maps were used to demonstrate the effects of tilting on mating site preference (n= 10 tanks per group). In Figure 2A, the effect of moving from an untilted to a tilted configuration is shown. In the untilted position, the majority of the embryos (red indicates greater number) were found in the corners and ends of the tank, with no preference for either end. In contrast, tilting resulted in the majority of the embryos to be found in the shallow end of the tank. The converse is seen in Figure 2B. Here we show the effect of moving from a tilted to an untilted configuration on oviposition, demonstrating a site preference for the shallow area that is lost when the condition is changed to a uniform depth. These data were independently verified at the Dana Farber facility and show similar results (Supplemental Fig. S1, available online at www.liebertpub.com). Summed heat maps were created to remove variation caused by facility and fish strain. The results confirm the oviposition site preference for the shallow area (Supplemental Fig. S2, available online at www.liebertpub.com).
Based on the above data, we then asked whether tilting affected overall embryo production in both adults and juveniles. For the adult studies, 20 individual tanks of five fish, with the female-to-male ratio of 3:2, were set up 6 consecutive weeks, alternating the tilted and untilted conditions (n=60 tanks for each cohort). The total number of embryos was significantly greater in the tilted (436±35) versus untilted (362±34) conditions (p=0.0262, paired t-test) (Fig. 3A). This study confirmed that similar to oviposition site preference, the preference for a tilt was not retained and rather was environmentally induced. Therefore, to simplify our approach, we did not follow the crossover design in the juvenile studies.
In the juvenile groups, tilting over a 6-week period (n=60 tanks for each cohort) led to a statistically significant increase in embryo production when compared to the untilted group (262±42 vs. 165±12) (Fig. 3B). This effect was primarily seen in the latter 3 weeks of testing, suggesting that tilting has a more pronounced effect as the juveniles approach sexual maturity (p=0.036, Wilcoxon exact rank sum test).
In trying to understand the effects of tilting on a given pair's likelihood of mating, we looked at clutch production with respect to the tilting condition. Twenty couples (n=10 for each condition) with the female-to-male ratio of 1:1 were set up for 3 consecutive weeks with alternating tilting condition. Over the 3-week period the mean number of tilted tanks that successfully mated was 5.7±1.15, while the average number of untilted tanks was 5.3±1.5. These results give no significant effect of tilting on the likelihood that a given pair of robust fish would mate.
The zebrafish is a useful model organism for studies that include early development, behavior, ecology, cancer, and stem cells. In the laboratory, a setting that severely disrupts the natural environment in which the fish normally reside, the success of mating is a critical determinant of these types of research. Because the zebrafish appear to naturally prefer shallow mating areas, our study addressed whether we could increase embryo production in the captive setting by providing a depth gradient for mating pairs.
Fish that were set up in the typical fashion experience a uniform depth of water of 4cm and express a particular set of mating behaviors. They include use of the entire tank space and water column with no strong preference for one particular end although edges and corners seem to be preferred over the middle area of the tank. However, when the insert is tilted to create a depth gradient of 4–0cm (theoretical shore), the fish demonstrate a broader range of behaviors than in the untilted condition. For example, actions that are unique to the tilted condition such as waiting and circling may be intrinsic to the species, but not expressed previously in a lab environment due to the unnatural characteristics of the mating tank. Carrying out similar studies with wild-caught fish with more natural substrate and conditions would get us closer to the reasons behind the results observed.
Tilting increased absolute embryo production in both juvenile and adult matings, suggesting that this is a modifiable factor in laboratory settings. When using a cutoff of at least 10 embryos per mating, tilting did not increase the likelihood that a particular pair of fish would mate, only that the clutch size would be increased. Whether the chance of mating success in a given pair is influenced by the robustness of that particular strain is unknown, but future studies in obstinate maters will help define the general utility of this method.
The affinity for mating locations may be due to facilitating the male in his induction of mating. By pinning the female against the tank wall, the male can ease his work of pushing against the female either with his nose or during quiver. While in the shallow area, the friction caused by the female's belly on the bottom may also be of aid to the process and hence lead to the tendency to mate there.
An alternative explanation may be that the feeling of the insert bottom on belly of the female is similar to that of a preferred gravel substrate (Spence et al., 2007). This particular type of surface combined with a shallower depth is thought to increase water exchange and oxygen availability, offers protection from cannibalism, and exposes them to warmer temperatures (Martin and Swiderski, 2001; Spence et al., 2007).
Some of the environmental factors known to affect fecundity in fish that are typically controlled in the lab are pH, food availability, and temperature (Winn, 1960; Reisman and Cade, 1967; Lee and Gerkin, 1979, 1980; Wootton, 1999). However, the aspect of topography and environmental stimulation is ignored in the lab mating setup. By overlooking factors such as substrate and presence of natural plant matter, both of which fish show a preference for (Stacey et al., 1979; Spence et al., 2007), we are limiting their natural behavior expression, and in doing so, we may be negatively affecting their lay rates.
Natural habitat items such as plants, substrate, and topography have all been shown to be preferred strata for fish mating. By simply altering the mating environment in lab zebrafish to include a more natural environment, we uncovered behaviors not previously described. This approach to exposing natural behavior opens doors to studying other inherent animal behaviors, why they've evolved, and how they are inherited. The results also have immediate implications for captive fish, which not only may facilitate research, but may also improve husbandry standards. By taking steps toward maximizing the sexual stimulation of the fish, we have not only shortened generation time and saved valuable tank space, but also increased embryo production, which is vital for the success of genetic screens, microinjections, and chemical screening. When combined with other variables such as food availability and vegetation, our aim is to further augment the integration of natural life history into the lab setting.
The authors thank Donna Neuberg for her help with statistical analyses.
No competing financial interests exist.