Aquaculture Magazine

October / November 2014

Triploidy – Background and Rationle

By C. Greg Lutz

A key feature in the formation of eggs and sperm in virtually all the aquatic species one might wish to culture involves a halving of the number of chromosomes (and therefor the genetic material) through a process called meiosis.

By Greg Lutz

This halving is necessary so that when eggs (1N) and sperm (1N) combine to form new individuals, the offspring possess the same total number of chromosomes (2N) as their parents. As a general rule, the process involves two cell divisions, which result in the formation of four 1N sperm cells in males, or one egg and two (or in some cases three) “polar bodies” in females. During egg maturation or activation, the first polar body (typically 2N) may be expelled directly from the egg, as is the case in many fishes, but in some aquatic species it may divide at the time of the first meiotic division. Subsequently, the second polar body (1N) is lost, leaving a 1N set of chromosomes in the egg to pair up with those from the sperm (Fig. 1).

Over the years, many researchers have searched for ways to disrupt this process by creating organisms, referred to as “triploids,” that have three chromosome sets (3N). A 3N complement of chromosomes cannot be divided equally, and this results in sterility. The ultimate goal of this type of research is to develop organisms that are biologically viable in every respect except gamete formation.

The gametes of many fish and shellfish can be manipulated to produce triploid offspring. Sometimes the goal may be faster growth, with energy normally required for maturation and spawning becoming available for weight gain. Sometimes enhanced survival is possible due to reduction in the physiological stress normally association with the spawning season. In other cases, it may be desirable for fish to be sterile in order to protect intellectual property or to preclude the establishment of populations by animals which may inadvertently escape from production facilities. A well-known example involves the use of triploid grass carp for vegetation control.

From a production standpoint, the bio-economics of triploidy depend greatly on the relationship between an organism’s natural life history and the production cycle in question. If the species being cultured is typically harvested prior to the onset of sexual maturity, then triploidy may result in few if any benefits (and occasionally a number of deficiencies). However, if market size is not reached until after one or two spawning seasons, the improved growth efficiency realized from sterility may be significant.

 

Examples of Negative Results

Liu et al. found no differences in growth (as measured by shell length and/or body weight) between diploid and triploid blacklip abalone over a 50-day grow-out period. Although food intake was significantly higher in triploids, their conversion efficiency was significantly lower. Diploid abalone converted 1 g of dry food into 0.58 g of body weight, as compared to only 0.44 g for triploids. Ultimately, triploids would have a higher cost of production for a similar sized animal.

Mori et al. evaluated the performance of triploid barfin flounder (Verasper moseri) and found that both males and females appeared to be functionally sterile. However, triploid males grew more slowly than male diploids, and triploid females exhibited similar or slower growth than female diploids. Similarly, over an experimental period of 76 days, Segato et al. found that juvenile shi drum (Umbrina cirrosa) triploids performed poorly when compared to diploid controls. Triploids exhibited reduced protein retention, with significantly lower specific growth rates and final body weights. Compared to diploids, triploids had larger amounts of coelomatic fat, higher liver lipid content and lower crude protein content.

 

Examples of Positive Results

Xiang et al. evaluated the physiology and performance of triploid Chinese shrimp, Penaeus chinensis, produced by heat shock. They found that although these triploids did not exhibit improved growth during early life history, at the onset of maturation they began to grow faster than their diploid counterparts. Triploids appeared to be sterile based on the status of their reproductive organs. Similarly, Cal et al. found that growth of triploid turbot was similar to that of diploids for the first year of life, but thereafter triploids out-grew diploids, with weights that were 10 to 12 percent higher from 24 to 48 months of age. During this same period, survival of diploids was 92 percent, as compared to 100 percent for triploids. The authors surmised this difference was attributable to a lack of spawning-associated stress and mortality. While diploids exhibited a sex ratio of 1 male to 0.6 females at 47 months, there were 3.3 female triploids for every male. This combined with a significant dress-out advantage for triploid females (14.3 percent over their diploid counterparts) indicated that commercial production of large turbot could benefit substantially from the use of triploids.

 

Disease Resistance

Triploid organisms generally have larger, but fewer, cells than their diploid counterparts – throughout all types of body tissues. The immune function of triploids is often influenced in different ways as a result. In the Xiang et al. study cited above, triploid shrimp had fewer, but larger haemocytes. Budino et al. produced triploid turbot to study their immune systems. In this case as well, triploid individuals had larger immune cells, but the numbers of erythrocytes, leucocytes and thrombocytes were lower than in diploids. Since the larger size of these blood components in triploids was offset by reduced numbers, total respiratory burst and phagocytosis activities were similar in diploids and triploids.

Vetesntk et al. found similar patterns in the erythtocyte profiles of diploid and triploid crucian carp. Erythrocyte counts were lower in triploids, but mean corpuscular volume and haemoglobin content increased in these animals. As a result, overall haematocrit values and corpuscular haemoglobin did not differ significantly between triploids and diploids. Beyea et al. reported similar observations in triploid shortnose sturgeon.

 

Genetic Effects

There are several approaches to inducing triploidy in aquatic organisms. In general, more techniques are available for mollusks than for finfish. Induction of “meiotic” triploidy, a typical approach for finfish, involves applying thermal, pressure or chemical ‘shocks’ to newly-fertilized eggs, with the resultant disruption of the mechanisms that would otherwise force the second polar body out of the egg. The sperm contributes 1N to the soon-to-be-developing zygote, as do both the egg pro-nucleus and the second polar body. In this way, 3 sets of chromosomes (one paternal, two maternal) combine within the nucleus of the fertilized egg, and all 3 sets replicate with each cell division as the zygote begins its development (Fig. 1).

The fact that the triploid offspring produced in this way have unequal inheritance, with two maternal sets of chromosomes as opposed to one paternal set, has led to some interesting observations in recent years. Park et al. examined diploid and triploid reciprocal hybrids between the Mud Loach and the Cyprinid Loach and found that although diploid hybrids were intermediate to the parental species in terms of body weight, growth of triploid hybrids was similar to that of their maternal parents. Similar relationships were apparent in terms of body proportions, with diploid hybrids being somewhat intermediate in form and triploid hybrids resembling their maternal species. Triploid hybrids were sterile, while diploids were not.

Johnson et al. reported on differences in heritability and maternal effects in diploid and triploid Chinook salmon resulting from “dosage effects.” They found that triploidy resulted in significantly higher levels of phenotypic variance for growth and survival-related traits. Triploidy also appeared to alter the variation patterns for these traits, but the opposite was true for lysozyme activity. This increased fitness-related trait might be accounted for by an increased level of heterozygosity. Duchemin et al. reported a similar reduction in environmental sensitivity in triploid Pacific oysters when compared to diploid counterparts.

 

Interploid Triploids

In numerous studies, performance of triploid aquatic organisms has been inferior to that of diploid controls, at least during early life stages. Many studies suggest that the shocks applied to newly-fertilized eggs in order to induce triploidy may be partially to blame. However, “interploid” triploids can sometimes be produced without the use of physiological shocks, by crossing tetraploid (4N) individuals with normal diploids. Tetraploids, which typically produce 2N gametes, have been produced in a variety of aquatic organisms. In the production of tetraploids, normal (1N) eggs and sperm combine to form a normal, viable 2N diploid zygote. The 2N chromosomes then replicate in preparation for the first cell division, or “first cleavage”, but temperature, pressure or chemical shocks are applied at the precise moment to prevent this division, leaving a 4N chromosomal complement in the cell (Fig. 1). From this point on, chromosomal replication and cell division proceed normally, but each cell will now contain a 4N complement of chromosomes: 2N of paternal origin and 2N of maternal origin.

Li et al. reported on the production of interploid triploids in the blunt snout bream (Megalobrama amblycephala). Two types of interploids were produced: 2N female by 4N male, and the reciprocal 4N female by 2N male. The authors referred to these as “negative” and “positive” interploids, respectively. Negative interploids exhibited similar fertilization, embryonic development, hatching rates and post-hatching growth and survival as diploid controls, while the positive interploids (from 4N females) were inferior in all these characteristics.

 

Take Home Message

The number of species evaluated for triploidy continues to increase, as does our understanding of methods and techniques for inducing triploidy. Nonetheless, this approach to genetic manipulation often results in reduced performance, at least until a size and age at which sexual maturation would normally take place.

 

 

C. Greg Lutz, has a PhD in Wildlife and Fisheries Science from the Louisiana State University. His interests include recirculating system technology and population dynamics, quantitative genetics and multivariate analyses and the use of web based technology for result-demonstration methods.

editorinchief@dpinternationalinc.com

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