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Issue: February 2007

Aquarium Science Male or Female? Gender Determination in Fish

Author: Donna M. Recktenwalt

RECK 0207
Photographer: MP. & C. Piednoir
A scientific explanation of why fish are born the sex they are, as well as a look at how gender can be manipulated by outside factors.

For fishkeepers, one of the great pleasures of the hobby comes from having their fish spawn successfully and (for some species) care for their eggs and fry. Such success is affirmation that the fishkeeper has done a number of things right. They have gotten a compatible breeding pair or group; provided acceptable water, temperature, and environmental conditions; and fed the breeder fish well enough to foster development of healthy eggs and sperm, thus (usually) resulting in healthy fry.

If you’re one of the many who have spawned their fish—whether one or more of the relatively easy species, or some of the more difficult ones—congratulations! You’ve achieved something that most casual aquarists have never done.

But the task of breeding fish doesn’t end with a successful spawning. Now comes the process of growing out the fry. Sufficient room, clean water with regular water changes, and frequent feedings of nutritious food usually do the job. After this most of us would probably expect that, when the juvenile fish sex out, there would be roughly the same number of males as females. Instead, many times aquarists find that they have nearly all males, or nearly all females. What happened?

There are many possible causes, from loss due to disease (the weaker fry), to severe predation by faster-growing siblings, to aggressive males attacking their younger brothers. There are many theories for skewed sex ratios, and there has been much discussion among hobbyists on the topic, but all lead to a single key question: Just when and how is gender determined in developing fish?

For the casual aquarium owner, the topic of gender determination in fish is probably of little interest. If you simply “keep a few pretty fish,” you don’t need to know if the fish are male or female, only that they are attractive, interesting, and will live in your aquarium. Since there is little visual difference between males and females in many aquarium species, gender doesn’t matter to the casual aquarium owner. However, for hobbyist breeders and commercial fish breeders that supply the aquarium and seafood trades, gender is a far more important matter.

First, in order to produce large numbers of saleable fish, you must begin with healthy adult fish of both sexes. With only a few exceptions, the only way to get viable spawn and fry that can be grown out for market is to fertilize a female’s eggs with a male’s sperm.

Under ideal conditions, and ignoring such outside factors as disease and predation, the expected ratio of male to female should be about 50/50, or a ratio of 1:1. At least, that’s what most of us would like to believe. It doesn’t always work out that way.

When it comes to gender determination, nature has been far from narrow-minded. In fish, gender can be determined in two basic ways: “genotypic,” where the gender is determined by chromosomes, and “environmental,” where gender in the developing fry is determined or heavily influenced by variables of chemistry and temperature.

Genotypic Sex Determination

Most of us think of gender determination in mammalian terms, where the sex of the offspring is determined at the time of conception, when fertilization of the egg mates up copies of the chromosomes from each parent. Since female mammals can provide only the genetic code for female (X) and the male can provide a code for either female (X) or male (Y), random chance would suggest that each offspring has a 50/50 chance of being female (XX) or male (XY). This genetic sexual determination is subsequently further reinforced and refined by the changing cascade of hormone and enzyme production occurring during embryonic gestation.

The system is not entirely flawless. The chemical coding inherited by the offspring can go awry, through mutation or incomplete transfer of information. The sequence of chemical triggers during early growth may err at any point, leading to variations in development, but on the whole the system works quite well.

A variation of this pattern occurs in some communal species, such as ants, termites, and honeybees.

Honeybees (Apis sp.) live in a communal, matriarchal, hive society, dominated by an egg-laying fertile queen. All members of the hive are her direct offspring. The majority of these are female worker bees, hatched from the queen’s fertilized eggs. Male bees, whose only purpose is to mate with the queen, are produced from her unfertilized eggs. Should the colony outgrow the hive, or the queen grow too old, the worker bees produce a few larger than usual cells in the comb, and feed a special diet to the female larvae contained therein. These resulting queens mate, then either fight for supremacy of the hive or establish new ones.

Environmental (Temperature) Sexual Determination

In the reptile world, members of the crocodile and turtle families, and a few lizards, lay eggs that incubate in “nests.” These structures can be as simple as small depressions scratched in the ground, or more complex creations dug underground or created from piles of soil and rotting vegetation. Gender of the young is then determined by the temperature of the nest interior.

Among turtles and lizards, incubation is left entirely to chance. Alligators, crocodiles, and caiman supply varying degrees of parental care for their eggs. The female usually guards her nest and cares for it, often removing or adding material to help maintain the proper internal temperature. At the optimum temperature for a given species, an equal number of male and female offspring are produced. Incubation at either end of the acceptable temperature range normally produces offspring of all one gender. The closer the temperature to the ideal for the species, the closer the offspring are to a gender ratio of 1:1.

Temperature not only affects gender determination in cold-blooded animals, it also influences the length of the incubation period, growth and activity rates, nesting intervals, and species distribution, among other factors.

Influencing Gender in Developing Fish

Ichthyologists have long known that among some species of fish “gender” is a slippery concept. In some fish, gender can be altered by surgical and/or social changes during adulthood. This is possible since research has revealed that the gene cells of fish retain their bipotentiality to differentiate into male or female until full sexual maturation and beyond. A number of reef fish, and some freshwater species as well, may actually change sex several times during their lifetime. Labroides dimidiatus, the saltwater cleaner fish, lives in harems of females attended by a single male. When the male dies, a female from the group actually changes sex and takes over the male role, not only behaving like a male, but actually fertilizing the eggs of the other females.

Research also seems to indicate that the sex of developing eggs and fry may be directly affected by a variety of factors. Depending on the species, these may include the age of the spawning adults; the temperature, pH, and DH of the water; light levels; and the subtle mix of chemicals, hormones, and hormone-like substances in the water. This may help to explain why some aquarists have problems with severely skewed sex ratios in spawns from a certain species, while other aquarists working with the same fish have no problems at all, or find that the sex ratios are skewed the opposite way. This may occur because no two aquarists have exactly identical water conditions.

Gender Manipulation

Hobbyist breeders may encounter spawns that are heavily skewed toward one sex or the other. Commercial producers of food fish often prefer the predominance of one sex.

Commercial fish breeders, who spawn and raise fish in vast numbers for human consumption, have learned how to manipulate some of these developmental variables to their benefit, reducing aggression and markedly improving growth rates. Such gender manipulation may be achieved by chromosomal manipulation during fertilization and the embryonic stages, control of hormone levels during and just after hatching, and by temperature during the juvenile phases. However, research with a number of species has shown that outside (human) manipulation of gender in developing eggs and fry is only possible during very specific, limited time periods.

Chromosomal manipulation is limited to the few seconds and minutes during and immediately after fertilization of the egg and during its very early cleavages. Manipulation at this point can produce a hatch of all females, all males, or completely sterile individuals.

Hormonal manipulation is restricted to a few minutes just before or after hatching (using the immersion tech­nique) or to a few days after hatching for ornamental fish. In food fishes (such as carp and salmon) this may be ex­tended to a few months, when using dietary administration of hor­mones.

Thermal manipulation may be the simplest method for gender control in fish. Although only limited research has been conducted in this area, results seem to show that the technique works best with juvenile fish, and that lower tem­per­a­tures result in females and higher tem­pera­tures result in males. Actual sex determination is triggered by thermal con­trol of certain enzymes.

In large-scale aquaculture, manipulation for gender has been practiced for some time, using hormone and/or temperature treatments to produce all-one-sex spawns that grow better and produce more high-quality harvestable biomass at lower costs. Among tilapia, males grow more quickly than females, so single sex populations are highly desirable. These can be produced by manually sorting the fry for sex, by administering hormones to all the fry (male and female) during key growth periods, or by crossing different species.

Hormone treatment has also been used in the aquarium trade to produce all male spawns, and pressure treatments have been effectively used to produce sterile populations of desirable decorative species.

Although sex manipulation in developing eggs and fry has been widely prac­ticed in many commercially important species for some time, there is evidence that over the long run it may result in stunted growth, sterility, and other undesirable secondary effects. It should also be noted that no one species has been thoroughly stu­died us­ing all the potential methods of gender manip­ulation.

Sex Ratio and Environment

Experiments with the Atlantic silverside Menidia menidia proved that the sex ratio could be influenced by environment, and that the sex determination of the fry was controlled by both the genotype and temperature during a specific stage of larval development. Surprisingly, spawn from different females varied in their responsiveness to changing temperatures. In general, the combination of lower temperatures and higher pH seemed to produce high numbers of females.

Keeping fry of various Apistogramma species at higher temperatures, especially during the first months, has resulted in the production of more males, and higher pH values produced more females. Best results, at least in one study, indicated that the most even sex ratio distributions for the study species occurred when water temperatures were maintained at about 78°F (26°C), with pH levels about 6.8 for the first month.

Among killifish, the Cynolebias species may react in a similar manner, although to fish from narrower temperature ranges, such as the rainforest Aphyosemion, pH may matter more. Aquarists working with killifish have observed direct links between water parameters and sex ratios. Among some of the Aphyosemion species, soft water (pH 6.5, 30 ppm hardness) at 72° to 76°F (22 to 24°C) resulted in 90 percent females. Harder water (pH 7.4, DH 140) at the same temperature produced mostly males. Cooler, moderately hard water resulted in roughly even sex distribution. Other aquarists have noted that reducing water hardness by adding reverse osmosis or rainwater produced more females.

Considerable anecdotal evidence also indicates that a number of variables may have an effect on sex differentiation. One noted breeder observed that if he raised a large group of Nothobranchius fry under crowded conditions, he invariably got larger numbers of males. Smaller batches of fry raised under less-cramped conditions usually (but not always) produced more even numbers of males and females.

Breeders in other countries have also observed the phenomena of skewed sex ratios. In Soderjanie I razvedenie aquariumnih rib (1991), A. S. Polonskii wrote: “The temperature and chemical composition of the water can affect sex ratio in the fry of Cyprinodontidae. For example, at 22° to 25°C (72° to 77°F) most of the fry will be females; if the temperature is not constant, most of the fry will be males. At the same pH 6.0, Epiplatys dageti will have more female offspring (>90%) in the soft water (dH about 5 degrees); in the hard water (dH 24) about 90 percent of the fry will be males. Aphyosemion gabunense in the acidic water (pH 5.0) will give more females, at higher pH (of) 6.5 more males.”

Another factor that has seen recent study is the presence of chemical residues from manmade pesticides and herbicides. These may mimic sex hormones, delaying sexual maturity in males and disrupting breeding habits. Such chemicals have been shown to cause sex ratio skewing in salmonids and may even be responsible for decreased sperm counts in humans. Male fish that develop in the presence of such hormones tend to be increasingly female-like, and some may even produce proteins associated with egg production. The suspect chemicals may come from a variety of sources and may have effects at very low concentrations.

Sometimes, though, there is no apparent cause for what we see in our tanks. Two tanks of the same species, located side by side and maintained under identical conditions, may produce totally different mixtures of male vs. female. Certain species have long been known to produce fry of predominantly one sex. Some of the Rivulus species are noted for this problem. Other species are notorious for cannibalism among the fry, with the faster-growing fry preying on their slower-growing siblings. This can result in a heavy bias toward one sex or the other.

More to Be Learned

The effects of so many variables on the sex of developing spawn and fry may prove highly frustrating to hobbyists who breed their fish, but they can cause serious problems and have major economic effects on the commercial aquaculture industry. This has lead to research with results that seem to indicate that in most freshwater fish, temperature and pH have the most direct effect on the sex of developing fry.

But even the experts freely admit that there is much about fish embryology and development that they still do not know. Sex determination can happen at a number of points during the egg and larval stages of development, with factors including population density and water temperature, pH and DH, among others, having direct effects. Most experts do agree that gender determination is probably not due to a single factor, such as pH or temperature, but to a combination of influences working together.

As hobbyist breeders, what does all this mean to us? Quite simply, that there is no simple answer to the question of when a fish becomes male or female. What can you do if faced with a skewed sex ratio in a batch of fry? Unfortunately, by the time you can identify the problem, there’s nothing you can do—at least for that batch of fry. But when your fish produce the next batch of fry, try a little experimenting.

Separate the spawn into two or more tanks, or if you have the room, place two fry each in a series of small containers. Often this method will result in a number of pairs (one male, one female).

Change the pH of the water.

Raise or lower the temperature.

Change water hardness by adding more soft or hard water.

Perform more frequent water changes.

Simply give the fry more room.

There’s no guarantee that any of these techniques will work for you, but they might improve your chances of achieving a more even gender balance in the developing fish. Isn’t a little hard work and inconvenience well worth the possibility of improving the sex ratio of your next batch of fry?

 

References

Chapman, Frank A. “Culture of Hybrid Tilapia: A Reference Profile.” University of Florida Cooperative Extension Service. http://edis.ifas.ufl.edu/BODY_FA012. (Accessed July 2004.)

D’Cotta, H., A. Fostier, Y. Guiguen, M. Govoroun, and J. F. Baroiller. “Search for genes involved in the

temperature-induced gonadal sex differentiation in the tilapia, Oreochromis niloticus.” 2001. The Journal of

Experimental Zoology 2990 (6):574585. Referenced in Genetic Computation Limited, http://www.genecomp.com/January_2002.htm. (Accessed July 2004.)

Duchin, Moon. “Biological Sex and Sex Differentiation.” University of Chicago. http://www.math.uchicago.edu/~mduchin/gstu/biology.html. (Accessed July 2004.)

Haqq, Christopher M., and Patricia K. Donahoe. “Regulation of Sexual Dimorphism in Mammals.” Physiological Reviews, Vol. 78, No. 1, January 1998, pp. 133. (Accessed July 2004.)

Johnson, Pete. “Sex Ratios.” http://www.thekrib.com. (Accessed July 2004.)

Koning, Ross E. “The Biology of the Honeybee, Apis Mellifera.” Plant Physiology Website, 1994. http://plantphys.info/plants_human/bees/bees.html. (Accessed July 2004.)

Madge, David, DSc “Temperature and Sex Determination in Reptiles with Reference to Chelonians.” Testudo, Volume 2, Number 3, 1985. http://www.deancloseprep.gloucs.sch.uk/chelonia/testudo/articles/v2n3sex.html. (Accessed July 2004.)

Nakamura, Masaru, PhD “Estrogen regulation of sexual plasticity in fish.” Nakamura Laboratory: Sesoko Station, http://www.geocities.com/bhandr/nakamura_lab.html. (Accessed July 2004.)

Pandian, T. J. and R. Koteeswaran. “Lability and sex differentiation in fish.” http://tejas.serc.iisc.ernet.in/~currsci/feb25/articles26.htm. (Accessed July 2004.)

Pieau, C., M. Girondot, G. Desvages, M. Darizzi, N. Richard-Mercier, P. Zaborski. 1995. “Temperature variation and sex determination in reptilia.” Experimental Medicine, 13:516523 [in Japanese, translation from the original English by Dr. S. Ohno.] http://www.ese.u.psud.fr/epc/cosnervation/Publi/texte/BD_EM95.html. (Accessed July 2004.)

Raloff, J. “Common pollutants undermine masculinity.” Science News Online, Vol. 155, No. 14 (April 3, 1999). htp://www.sciencenews.org/pages/sn_arc99/4_3_99/fob3.htm. (Accessed July 2004.)

Robinson, Jim. “A Controlled Experiment Concerning Skewed Sex Ratios in Simpsonichthys.” http://fins.actwin.com/killietalk/month.200011/msg00123.html. (Accessed July 2004.)

Romer, U., and W. Beisenherz. “Environmental determination of sex in Apistogramma (Cichlidae) and two other freshwater fishes (Teleostei).” Journal of Fish Biology. V. 48, p. 714725.

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