By Kat Mowle, UNF MS Student
If you grew up watching Jaws, there’s a good chance you fear sharks when you go swimming in the ocean. In reality, the vast majority of sharks are completely harmless, and you are more likely to be struck by lightning than die from a fatal shark attack. If you think about it, sharks actually have more of a reason to fear us than we have to fear them, because many shark populations are in decline due to factors like overfishing, pollution, and loss of habitat. Shark and ray species are more vulnerable to overexploitation than bony fish are because sharks tend to mature at a later age, produce fewer offspring, and grow slower. Additionally, mating events for some shark and ray species may occur only every two to three years. All of these factors combined mean that sharks and rays are particularly vulnerable to overexploitation, and it is absolutely critical that we work to understand the biology and life history of these organisms so we can better manage their populations (Hoenig and Gruber, 1990; Stevens et al., 2000).
Traditionally, understanding the reproductive cycles of sharks has required lethal sampling in order to examine changes in their reproductive tracts throughout the year. This approach is unsustainable for threatened species, and cannot be used for endangered species (Hammerschlag and Sulikowski, 2011). In more recent years, scientists have moved to developing non-lethal methods for assessing reproduction of sharks and rays, which is what my advisor, Dr. Jim Gelsleichter, works on at the University of North Florida. Ultrasonography can be used to determine pregnancy, just like with humans. Scientists are also able to measure the concentrations of reproductive hormones in the blood of sharks to determine when various reproductive events occur. For male sharks, high plasma concentrations of testosterone occur during the peak time of sperm production, indicating when a male is capable of mating. For females, high concentrations of estrogen (E2) correlate with the development of eggs in their ova, indicating when a female is ready for fertilization and pregnancy (Awruch et al., 2008; Awruch, 2013; Sulikowski et al., 2007).
While these methods have proven to be useful for many species, analyzing only E2 does not always provide researchers with the full picture of a species’ reproduction. As mentioned earlier, some shark species only reproduce every two or three years. How often an individual reproduces is referred to as reproductive periodicity, with females that reproduce every year termed annual, every two years biennial, and every three years triennial. For females of species with more unusual patterns of reproductive periodicity, examining levels of E2 in their plasma doesn’t always give the full picture of their reproductive cycle. This is where my research comes in. My master’s research focuses on measuring vitellogenin (Vtg) in the plasma of elasmobranchs, specifically focusing on the bonnethead shark, Sphyrna tiburo (Figure 1).
Figure 1. Bonnethead shark (Sphyrna tiburo) specimen
Vtg is a protein that is produced by the liver during the time of follicular development, when female sharks have new ova developing in their ovaries. Thus, determining when Vtg is produced is a good indicator of when follicular development occurs. If researchers are able to couple measurements of plasma Vtg with a nonlethal determination of pregnancy (such as ultrasonography), then a new nonlethal method for determining reproductive periodicity can be developed. For example, if a female has Vtg present in her plasma during pregnancy, that indicates the female will have eggs ready to be fertilized after she gives birth, and thus she will likely give birth again the next year. So this female would be an annual reproducer. On the other hand, if a female does not have Vtg present during pregnancy, she will likely need to take time off to grow eggs before she is ready to mate again; such a female would likely be a biennial or triennial reproducer. These measurements do depend on the length of a female’s gestation period, as the bonnethead shark is an annual reproducer, but does not produce Vtg during pregnancy. However, this species has a fairly short gestation period of only 4 to 5 months, giving females time to develop new eggs and still be able to mate and give birth every year.
My research focuses specifically on measuring Vtg in the plasma of female bonnethead sharks and characterizing the process in this species. In order to do this, I collect blood samples from female bonnethead sharks out in the field (Figure 2). I then centrifuge the blood to separate the plasma and analyze the plasma for the presence of vitellogenin using a process called Western blotting or immunoblotting. This process doesn’t give us quantitative results, but is a good method for determining whether or not Vtg is present in the plasma of a female bonnethead shark.
Figure 2. Obtaining a blood sample from a mature female bonnethead shark.
We ultimately were able to detect both Vtg itself and a likely component protein (lipovitellin, or Lv) in the plasma of female S. tiburo. Since it is known that Vtg breaks down fairly quickly if plasma is not stored immediately or not stored with a protease inhibitor, we chose to classify females with only the Lv component protein in their plasma as also likely producing Vtg (Figure 3). It was determined that the proteins being detected were likely Vtg (~200 kD) and Lv (~70 kD) based on what had been observed for another elasmobranch species (Perez and Callard, 1992).
Figure 3. Immunoblots showing the detection of Vtg and Lv within the plasma of female S. tiburo. a) Detection of Vtg within plasma of females sampled in March (Lanes 2-8) and early May (lane 12). b) Detection of Lv within the plasma of mature female S. tiburo. The Lv protein is outlined in the boxes.
Looking at both the detection of Vtg and Lv as evidence of Vtg production occurring, it was determined that the highest numbers of females were producing Vtg in March and April, which matches previous studies on this species (Parsons, 1993). The protein was also produced in May for one individual. What was interesting was we found evidence of the protein’s production beginning as early as August for some individuals, with the protein continuing to be found in the plasma from August to December (Figure 4).
Figure 4. Proportion of mature S.tiburo females that were determined to have either vitellogenin or the putative lipovitellin protein present within their plasma during each month.
This finding suggests that female bonnethead sharks begin developing new eggs within their ovaries immediately after they give birth; production is not limited to the spring time period, which was suggested by previous studies that focused on when the highest number of large yolky eggs were observable in the ovaries. The variations we observed in when Vtg was produced are likely due to variations between populations. It has been observed, for example, that females in populations in South Florida give birth earlier than those in North Florida, which would explain why Vtg production is observed in August for females in South Florida but not until October for females captured in South Carolina (Lombardi-Carlson et al., 2003).
As I have been conducting my master’s thesis research, we have also been testing whether we can measure Vtg in the plasma of other elasmobranch species. We focused on the bonnethead because our antibody against Vtg was developed specifically for this species. But, as noted, measurement of Vtg would be particularly useful for clarifying the reproductive periodicity of other species, such as the blacknose shark (Carcharhinus acronotus), which seems to be capable of both annual and biennial reproduction in the Atlantic (Driggers et al., 2004). Measuring the concentrations of E2 in the plasma of this species does not effectively answer questions about the reproductive status of females of this species; researchers are unable to determine if a female is resting or reproductively active.
So far we have confirmed that we are able to measure Vtg (or at least component proteins of the larger Vtg protein) in the plasma of eight other elasmobranch species. These results indicate that the methods I have developed in my study will be useful for studying reproduction of other species. Particularly, these methods will be a good nonlethal method for characterizing reproductive periodicity. Having a good understanding of exactly how often a species reproduces is critical to management of a population, as the population growth depends on how many females are actually contributing to the next generation in a given year. Thus, the methods developed specifically throughout my master’s thesis will hopefully continue to be used and will act as an ideal new nonlethal method for determining reproductive periodicity, providing crucial information about a species’ reproduction to managers.
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