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

It's no longer unexpected to hear about the decline of the Southern Flounder populations, as the imposition of bag limits and size restrictions conspicuously illustrates the severity of the fisheries’ downturn.

The University of Texas Marine Science Institute’s Fisheries and Mariculture Laboratory (FAML) has been at the forefront of Southern Flounder research since the first-ever captive Southern Flounder spawn in 1978. Protocols introduced by FAML and other research institutions for mass rearing of larvae of this species have been implemented by hatchery facilities in various states. The Stock Enhancement Program led by the Texas Parks and Wildlife Department (TPWD) is one of them, working to recover wild Southern Flounder populations across the Texas coast. Stock enhancement programs support the natural supply of flounder to rebuild the declining flounder populations. TPWD fish hatcheries are actively involved in helping jumpstart the recovery process by supplementing hatchery fish with wild populations. Despite the program’s diligent endeavors, the success of a stock enhancement program is dependent on not only the number of fish reared and released, but also the survival of the stocked fish.

TPWD is continually looking to improve their methods to provide the stocked fish with the best chance for survival. One of the problems that have plagued flatfish produced by hatcheries is malpigmentation. Flatfish species, from flounder to halibut, sole, and tongue sole possess pigmentation that matches their surroundings to avoid predators and increase their survival. Flounder produced in hatcheries can have a notably high rate of malpigmentation, the most common form is pseudo-albinism, where the upper or eyed side of the flounder lacks most pigmentation so that it is white. Pseudo-albinism does not show up until around metamorphosis, which can be a month after hatching and after a substantial amount of resources are used for producing the fish. TPWD is activity working with the University of Texas Marine Science Institute to not only figure out the causes of pseudo-albinism, but also a mechanism for earlier detection. This collaboration should prove valuable because TPWD has seen malpigmentation rates up to 30% in hatchery-reared Southern Flounder. Intriguingly, only about 10% of FAML-raised flounder have pseudo-albino characteristics. In the wild, instances of malpigmentation remain undocumented, largely because predators quickly pick off prey that visibly stand out. Hatchery-reared flounder with malpigmentation experience lower survival rates compared to those with normal pigmentation. For this reason, corrective actions to reduce the incidence of malpigmentation within stock enhancement programs are imperative for the fate of Southern Flounder.

New preliminary data suggested that broodstock diet may play an important role in determining adult pigmentation. In collaboration with TPWD, Dr. Lee Fuiman, the Director of FAML, conducted a study through a grant provided by the Texas Parks and Wildlife Department and the U.S. Fish and Wildlife Service to determine if the high malpigmentation rates are caused by maternal diet. To test this, researchers divided Southern Flounder broodstock into two groups, with each group being fed a maturation diet consisting of either sardines or shrimp. A combination of both diets is a common practice for marine fish culture; however, researchers tested the two diet extremes to observe if malpigmentation is influenced by broodstock diet.

Researchers selected six key genes in pigment cell development to study. This was undertaken to identify the physiological pathway that might be affected and to pinpoint precisely when during development malpigmentation genes are expressed. Understanding which genes are involved in pigmentation and when should help researchers develop an earlier detection method and possibly develop corrective measures. Broodstock spawns were collected and measured at three developmental stages: pre-metamorphosis (27 days posthatching [dph]), mid-metamorphosis (34 dph), and post-metamorphosis (55 dph). At these stages, the little fish are between one-quarter and one-half an inch long. The expression of the genes was compared to the malpigmentation rates for the batches of fish produced.

As it turns out, the experiments produced low malpigmentation rates, between 1 and 15%, in the juvenile fish for both broodstock diets. While the diet did not provide a silver bullet answer, the genetic testing did provide valuable insight. Results from the pre-metamorphosis stage provided little information about which genes were involved. However, upon testing the mid-metamorphosis stage, three of the six indicator genes were lower when the malpigmentation rate was higher. Two of these genes showed the same differences between normally pigmented and pseudo-albino post-metamorphosis fish, confirming the results seen in the earlier mid-metamorphosis stage.

Even though the malpigmentation rates were low, the results suggest that the two genes may serve as useful indicators of malpigmentation in Southern Flounder, and that those indicators can be applied as early as 34 dph. To confirm the utility of these genes as true indicators, the researchers at FAML need to look to other hatcheries that produce fish with high malpigmentation rates. Collaborating with Hubbs-SeaWorld Research Institute (HSWRI) provided just the partnership needed. HSWRI rears California Halibut, a species very closely related to the Southern Flounder. At HSWRI, hatchery-produced halibut exhibit rates of 40% to almost 100% malpigmentation! With such high rates, researchers aim to cross-reference the expression levels with the same genes in the flounder to determine if the genes are useful early indicators of malpigmentation.

With the spotlight now on genetic screening as a possible tool in flounder conservation, the research opens new avenues for mitigating malpigmentation in commercial and stock enhancement culture of flatfish.