Molecular Pathways Leading to Malpigmentation in Flounder: Part Three of Three

Stock enhancement – supplementing natural populations by releasing large numbers of hatchery-produced fish – is one of the tools that fishery managers use to maintain healthy fisheries. Texas Parks and Wildlife Department (TPWD) has been at it for decades, and they recently celebrated the release of their one billionth fish. The effectiveness of stock enhancement depends on releasing a lot of fish and on their survival after release. In response to a widespread decline in Southern Flounder populations throughout the Gulf of Mexico and southeastern United States, several states have begun stock enhancement programs for Southern Flounder. However, hatchery production methods for flounder are not as well established as they are for other species, such as red drum. Because of this, just producing large numbers of flounder for release is challenging. But, another problem is that a large portion of hatchery-produced fish lack almost all coloration on their eyed side. These malpigmented fish, called pseudo-albino, are almost entirely white. Since the eyed-side pigmentation allows flounders to camouflage, it is essential for their survival.

The first two parts of this series of articles explained how researchers at the University of Texas Marine Science Institute’s Fisheries and Mariculture Laboratory (FAML) conducted experiments on Southern Flounder and California Halibut and applied the tools of molecular biology to gain a better understanding of malpigmentation in flatfishes. The results of that research showed that the reason pseudo-albino flounder are mostly white is not because the pigment cells on the eyed side of the fish lack pigment, but rather, the pigment cells are not there. The researchers found differences in the activity of three genes in Southern Flounder that were associated with malpigmentation rate (the percentage of fish in a spawn that become pseudo-albino). One gene, in particular, had the same relationship with malpigmentation rate in California Halibut. That gene is known to play a key role in creating pigment cells during development. Importantly, that relationship between gene activity and malpigmentation rate occurs when flounder and halibut are 5 to 6 weeks old, weeks before malpigmentation can be observed. This gene could potentially be used to screen batches of flounder to determine whether the malpigmentation rate for a particular spawn will be so high that it might not be worth rearing the fish any further.

Although having an early detection system for malpigmentation could be useful, it would be better to know how to reduce or prevent malpigmentation altogether. With that in mind, the researchers noticed that the incidence of malpigmentation was consistently higher in fish reared in the TPWD hatchery compared to the FAML laboratory. On average 30% of hatchery fish were pseudo-albinos, while the laboratory averaged 5%. Dr. Lee Fuiman and Cynthia Faulk sat down with TPWD’s Dr. Christopher Mace and Ashley Fincannon and put together a list of the differences in how the two facilities reared flounder, thinking that one or more of these differences might be responsible for the discrepancy in malpigmentation rates. Two things piqued their interest: differences in food and differences in lighting. They agreed to start by exploring the effect of the food.

Almost all marine fish are very small when they start feeding and they will not eat dry food. They will only eat live animals that are small enough to consume and easy enough to catch. And, they need to be fed a lot because young fish have a very high metabolism. So, hatcheries must produce millions of live, microscopic animals every day. The first food marine fish larvae receive is rotifers – tiny, soft-bodied animals. Rotifers are not very nutritious, but they can be immersed in an enrichment product, which the rotifers consume, and that fortifies them with fatty acids, amino acids, and minerals before being fed to the fish. When flounder are about 3 weeks old, they can eat slightly larger animals, and brine shrimp are the convenient choice. Brine shrimp can be purchased as dry cysts (eggs) and hatched out on demand by placing them in seawater for a day. Newly hatched brine shrimp have a yolk sac that provides nutrition for the brine shrimp and for a fish that eats it. But, the nutritional value of the yolk varies and the brine shrimp use up the yolk quickly, so the nutritional value of brine shrimp to the fish diminishes over time. In some hatchery protocols, brine shrimp are enriched in the same way as rotifers, but the process of enriching brine shrimp adds significantly to an already expensive and labor intensive fish production operation. The FAML protocol for rearing flounder uses enriched brine shrimp, but the protocol used by the TPWD hatchery feeds newly hatched brine shrimp to flounder. Could this difference in larval diet explain the large difference in malpigmentation rates between FAML and TPWD? The researchers conducted an experiment in the CCA Texas Laboratory for Marine Larviculture at FAML to determine whether the differences in malpigmentation rates between the two facilities were due to the enrichment of the brine shrimp.

The research team strip-spawned five female flounder from the broodstock at FAML. They took 10,000 eggs from a single spawn and put half the eggs into one rearing tank and the other half into a different rearing tank. They repeated this for the other four spawns. The eggs were incubated and all 10 tanks of fish were reared identically, with one exception. Starting about three weeks after hatching, one tank of flounder from each spawn was fed newly hatched brine shrimp (the hatchery diet) and the other tank from each spawn was fed enriched brine shrimp (the laboratory diet). Eight weeks after hatching, 100 fish were removed from each tank and the normal and malpigmented fish were counted to determine the malpigmentation rate.

The results showed a clear improvement in malpigmentation rate when fish were fed enriched brine shrimp. For example, one spawn had 42% pseudo-albino fish when they were fed newly hatched brine shrimp but only 6% pseudo-albino fish when they were fed the enriched food. For all five spawns, the average malpigmentation rate decreased from 18% to 4% due to the enriched diet.

“The research has provided compelling evidence for our stocking centers to try using enriched foods. While it adds time and resources, if this step can reduce malpigmentation, it’ll drastically improve the success of the released fingerlings,” said Ashley Fincannon, CCA/CPL Marine Development Center Hatchery Manager. “TPWD is committed to ensuring the success and sustainment of the Southern Flounder population in Texas and ensuring there’s plenty of fish for future generations.”

The next step in this research collaboration is to determine whether the same degree of improvement in flounder pigmentation that was observed in the lab happens when the enriched diet is applied in hatchery-scale production. The CCA/CPL Marine Development Center in Corpus Christi is planning to feed enriched brine shrimp to larval flounder this fall, and FAML staff will determine the malpigmentation rates in those production systems to see how well the laboratory results translate to the real world. Partnerships like that of FAML and TPWD provide a great collaboration to help sustain fish populations long into the future.

The research described here was made possible by funding from the Texas Legislature allocated to the Texas Gulf Coast Research Center at the University of Texas Marine Science Institute.

– Dr. Lee A. Fuiman is a Professor at The University of Texas Marine Science Institute in Port Aransas and Director of the Fisheries and Mariculture Laboratory.