Modification of Morphology and Function of Integument ...

Modification of Morphology and Function of Integument Mitochondria‐Rich Cells in Tilapia Larvae (Oreochromis mossambicus) Acclimated to Ambient Chloride Levels Author(s): Li‐Yih Lin and Pung‐Pung Hwang Reviewed work(s): Source: Physiological and Biochemical Zoology, Vol. 74, No. 4 (July/August 2001), pp. 469-476 Published by: The University of Chicago Press Stable URL: http://www.jstor.org/stable/10.1086/322159 . Accessed: 03/09/2012 02:53 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp

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Modification of Morphology and Function of Integument Mitochondria-Rich Cells in Tilapia Larvae (Oreochromis mossambicus) Acclimated to Ambient Chloride Levels Li-Yih Lin1 Pung-Pung Hwang 2,* 1 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China; 2Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China Accepted 1/8/01

ABSTRACT Similar to those of the gills of adults, three types of mitochondria-rich (MR) cells with different morphologies of apical surfaces (wavy convex, shallow basin, and deep hole) were identified on the integument of freshwater-acclimated tilapia larvae (Oreochromis mossambicus). The object of this study is to test the hypothesis that these subtype cells may represent MR cells equipped with variable efficiencies in Cl⫺ uptake. Larvae acclimated to low-Cl⫺ ([Cl⫺] p 0.001–0.007 mM) water developed higher densities of MR cells than those acclimated to high-Cl⫺ ([Cl⫺] p 7.3–7.9 mM) water. The percentage of wavyconvex-type cells in total MR cells was higher in low-Cl⫺acclimated larvae than in high-Cl⫺-acclimated larvae, which displayed only deep-hole type. In addition, Cl⫺ influx rates of whole larva measured with 36Cl⫺ showed a coincident correlation with MR cell densities, that is, low-Cl⫺ larvae displayed higher Cl⫺ influx rates than did high-Cl⫺ larva, suggesting that tilapia larvae develop a higher density of MR cells with larger apical surfaces (wavy-convex type) to boost Cl⫺ uptake in Cl⫺deficient water. The distinct types of apical surfaces may represent different phases of MR cells that possess different efficiencies of Cl⫺ uptake. Increased apical membrane surface areas of MR cells may provide larvae with rapid regulation of Cl⫺ before new MR cells differentiate.

* Corresponding author; e-mail: [email protected]. Physiological and Biochemical Zoology 74(4):469–476. 2001. 䉷 2001 by The University of Chicago. All rights reserved. 1522-2152/2001/7404-00111$03.00

Introduction Gills are the most important organs responsible for ion regulation in adult teleosts. Mitochondria-rich (MR) cells are the major sites for active transport of ions in branchial epithelium; these cells secrete ions in seawater-adapted fish and absorb ions in freshwater-adapted fish. The role of MR cells in freshwater teleost gills in transepithelial Ca2⫹ and Cl⫺ uptake has been clearly established, yet its role in Na⫹ uptake is currently being debated (reviewed by Perry 1997). In embryos and larvae of several teleost species, MR cells have been identified in the epithelia covering the yolk and body, and these extrabranchial MR cells are considered to be the site of ionic regulation during the early developmental stages when the functional gills are not yet well developed (Guggino 1980; Hwang and Hirano 1985; Hwang 1989; Ayson et al. 1994; Hwang et al. 1994, 1999; Shiraishi et al. 1997; Hiroi et al. 1999). Morphological changes of MR cells in hormone-treated, softfreshwater, or acid/alkaline-induced fish were reported in several freshwater teleosts (Perry and Laurent 1989; Laurent and Perry 1990; Perry et al. 1992; Laurent et al. 1994; Perry and Goss 1994). These morphological alterations of MR cells were suggested to be a crucial mechanism for ionic regulation. In our previous studies, three types of apical surfaces of MR cells were identified in gills of freshwater-acclimated (FW) tilapia (Lee et al. 1996). The relative abundance of these three subtypes of MR cells—wavy convex, shallow basin, and deep hole—varied with external ionic compositions of hypotonic media, suggesting that these subtypes might be functionally distinct (Chang et al. 2001). In the same species, Van der Heijden et al. (1997) also reported different apical surfaces of MR cells. However, the structure-function relation of these MR cell subtypes is still unclear. The most significant difference between the previously defined three MR cell subtypes is their configuration on the apical surface. Wavy-convex cells are equipped with the largest apical surface, while those of shallow basin and deep hole are the intermediate and the smallest, respectively. In a recent prevailing model of Cl⫺ uptake of MR cells, Cl⫺ is believed to be transported actively across the apical membrane (reviewed by Perry 1997), and the larger apical membrane is supposed to provide higher capability in active transport. Therefore, we reasonably hypothesized that the cell subtypes may represent MR cells equipped with variable efficiencies of

470 L.-Y. Lin and P.-P. Hwang Table 1: Ion compositions of four different hypotonic media Ion ⫺

Cl (mM) Na⫹ (mM) Ca2⫹ (mM) Mg2⫹ (mM) K⫹ (mM) pH Total alkalinity (mg/L)

Normal

N-Na-L-Cl

H-Na-L-Cl

H-Na-H-Cl

.480–.520 .420–.487 .142–.158 .160–.172 .151–.178 6.70–6.87 118–137

.001–.003 .535–.559 .176–.188 .167–.187 .157–.167 6.70–6.88 89–102

.005–.007 10.771–10.360 .167–.175 .186–.208 .153–.176 6.74–6.86 85–95

7.314–7.880 9.267–10.035 .186–.190 .148–.168 .148–.168 6.70–6.78 85–100

Cl⫺ uptake. In this study, tilapia yolk sac larvae, which also display MR cell subtypes in their integument, were used to test this hypothesis. The morphological and density changes of MR cells and whole-body Cl⫺ influx in larvae acclimated to the hypotonic media with different compositions of Na⫹ and Cl⫺ were examined. Material and Methods Animals and Various Hypotonic Media Mature adult tilapia from the Tainan branch of the Taiwan Fisheries Research Institute were reared in circulating freshwater at 26⬚–28⬚C under a photoperiod of 12–14 h of lighting (Hwang et al. 1994). Tilapia eggs and larvae are available year

round from mature adults that are reared in controlled conditions as described above. Fertilized eggs were retrieved from the mouths of females that had started mouth breeding, as described in Hwang et al. (1994). Four kinds of artificial freshwater, high-Na⫹-low-Cl⫺ (H-NaL-Cl), high-NaCl (H-Na-H-Cl), normal-Na⫹-low-Cl⫺ (N-NaL-Cl), and Normal, were prepared by adding appropriate amounts of NaCl, Na2SO4, MgSO4, K2HPO4, KH2PO4, and CaSO4 to deionized water (Table 1). Ion concentrations of the Normal medium were near the ranges of local freshwater. Temperature of the media was kept between 26⬚ and 28⬚C. During the experiments, the larvae were not fed, and the media were changed daily to maintain water quality. Acclimation Experiments Experiment 1. To test our hypothesis, we examined the correlation between the density and ratio of MR cell apical surfaces on integument and whole Cl⫺ influx in larvae acclimated to the four media, H-Na-L-Cl, H-Na-H-Cl, N-Na-L-Cl, and Nor-

Figure 1. Three distinct types of mitochondria-rich cell apical openings observed on the body surface of tilapia larva with SEM. They were identified as wavy convex (thick arrow), shallow basin (arrowhead), and deep hole (thin arrow).

Figure 2. Changes of mitochondria-rich cell densities on tilapia larvae after transfer to four different media. Cell density gradually increased with time in larvae transferred to Cl⫺-deficient media (H-Na-L-Cl and N-Na-L-Cl), in contrast to declining gradually in larvae transferred to high-Cl⫺ medium. The curve connects the mean values. Comparisons by ANOVA were made among the four groups at the same time point. Different letters indicate significant differences.

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CO2 in a critical-point drier (Hitachi HCP-2, Tokyo, Japan) and sputter coated for 3 min with a gold-palladium complex in a vacuum evaporator (Eiko 1B-2, Tokyo, Japan). The coated specimens were examined in an SEM (Hitachi S-2500, Tokyo, Japan) at an accelerating voltage of 15 kV. MR cell density of tilapia larvae was counted with the number (cell per unit surface area) of apical openings on the larval body surface. MR cells with apical openings are considered to be functional MR cells. The criteria of cell type classification of MR cells followed Lee et al. (1996). Five areas (60 # 105 mm2 p 6,300 mm2 each) on the body surface of one larva were chosen for counting at #1,000 magnification to measure the densities of different types of MR cells. The locations of the counting areas were as close as possible in different individuals. Five individuals per group were analyzed. Figure 3. Percentage changes of wavy-convex type in total mitochondria-rich cells on tilapia larvae after transfer to four different media. Ratio of wavy-convex type significantly increased in larvae transferred to Cl⫺-deficient media (H-Na-L-Cl and N-Na-L-Cl). In contrast, no wavy-convex type was observed on larvae transferred to H-Na-H-Cl medium. The curve connects the mean values. Comparisons by ANOVA were made among the four groups at the same time point. Different letters indicate significant differences.

mal. Freshwater larvae from the same brood were transferred to the four hypotonic media at the second day after hatching. Fifty individuals were maintained in 1,000 mL of artificial medium, which was aerated and renewed daily. Then the larvae were sampled at 12, 24, 36, and 48 h after transfer for scanning electron microscope (SEM) observation and quantification of MR cells (five individuals for each test). In addition, at 24 h, larvae that were acclimated to various media were also sampled for measurement of whole-larva Cl⫺ influx (10 individuals for each test). Experiment 2. On the basis of the results of experiment 1, we further monitored short-term changes in density and morphology of apical openings of MR cells in larvae subjected to environmental Cl⫺ fluctuation. Two-day-old tilapia larvae were acclimated to H-Na-H-Cl medium. After 24 h of acclimation, the larvae were then transferred acutely to H-Na-L-Cl medium. At 0 (in H-Na-H-Cl), 4, and 10 h after transfer, larvae were sampled for SEM observation and quantification of MR cells (five individuals for each test). SEM Observations and Quantifications Sampled larvae were anesthetized and then fixed at 4⬚C in phosphate-buffered 5% glutaraldehyde/4% paraformaldehyde overnight. After rinsing with 0.1 M PB, the specimens were postfixed with 1% osmium tetraoxide in 0.2 M PB for another 1 h. After rinsing with phosphate buffer (PB) and dehydration with ethanol, the specimens were critical-point dried with liquid

Measurement of Cl⫺ Influxes Measurement of whole-larva Cl⫺ influxes with radioactive Na36Cl followed the previous report by Guggino (1980) and the modification by Hwang et al. (1994, 1996). Larvae from the four different acclimation media were transferred to the tracer medium, which was prepared with Normal medium (Table 1) but with an appropriate amount of Na36Cl added (980 mCi mL⫺1; Amersham, Piscataway, N.J.). After rinsing briefly with deionized water, larvae from different acclimation media were transferred to the tracer media for 1 h. In the preliminary experiments, the relation between exposure time and accumulated radioactivities of the tracer was determined to be linear within 6 h. Thus, 1 h of exposure to the tracer was chosen for the calculation of Cl⫺ influx rates. After exposure, the larvae were rinsed in an isotope-free water bath three times for a total of 5 min. After the 5-min rinse, the radioactivity of the subsequent rinse water was checked; it did not significantly differ from the background. Larvae were anesthetized and then digested with tissue solubilizer (Soluene-350, Packard, Meriden, Conn.) in a counting vial at 50⬚C for 4 h. After solubilization, counting solution (Hionic-Fluor, Packard, Meriden, Conn.) was added into each vial. The radio activities of both the incubation medium and the solubilized larvae were measured by liquid scintillation counter (LS 6500, Beckman, Fullerton, Calif.). The Cl⫺ influx rate was calculated by the formula Jin p Q larva X ⫺1T ⫺1W ⫺1, where Jin is the influx rate (nmol mg⫺1 h⫺1), Qlarva is the radioactivity of the larva (counts min⫺1 individual⫺1 at the end of the incubation), X is the specific activity of the incubation medium (counts min⫺1 nmol⫺1), T is incubation time (h), and W is wet body mass (mg).

472 L.-Y. Lin and P.-P. Hwang

Figure 4. Comparisons of Cl⫺ influx rates of larvae acclimated to the four media for 24 h. Larvae acclimated to Cl⫺-deficient media (H-NaL-Cl and N-Na-L-Cl) displayed a higher rate of Cl⫺ uptake than did normal larvae. In contrast, the Cl⫺ influx rate of larvae acclimated to HNa-H-Cl was lower than that of normal larvae. Comparisons by ANOVA were made among the four groups. Different letters indicate significant differences.

Statistics

Experiment 1

Values were compared using a one-way ANOVA (Tukey’s pairwise method). Values are presented as the mean Ⳳ SD. Results Morphology of MR Cell Apical Openings Figure 1 shows the three types of apical openings of MR cells displayed on the body surface of tilapia larvae acclimated to the N-Na-L-Cl medium. According to previous definitions by Lee et al. (1996), they are identified as wavy-convex-, shallowbasin-, and deep-hole-type openings. In terms of the diameter of these three opening types measured under SEM observation, 86% of the deep-hole-type openings were in the range of 0.5–1.5 mm, 80% of shallow basin were between 2 and 4 mm, and 84% of wavy convex were between 6 and 8 mm.

Figure 2 shows the density change of MR cells (with apical openings) in larvae acclimated to four different media. H-NaL-Cl-acclimated larvae (H-Na-L-Cl larvae) and N-Na-L-Cl-acclimated larvae (N-Na-L-Cl larvae) displayed an increase in cell density with time, in contrast to H-Na-H-Cl larvae, whose cell density decreased with time. At 36 h after transfer, cell densities of the H-Na-L-Cl and N-Na-L-Cl groups were 40% higher than that of the Normal group; however, that of the H-Na-H-Cl group decreased to 30% of the Normal group value. These data indicate that MR cell density is negatively correlated with Cl⫺ level in the water. In terms of cell types, H-Na-L-Cl and N-Na-L-Cl larvae displayed higher ratios of wavy-convex-type cells in total MR cells than did the Normal group. In contrast, H-Na-H-Cl larvae displayed a unique type with a smaller apical opening (deep-

Table 2: Comparison of mitochondria-rich cell density, ratio of wavy-convex cell, and Cl⫺ influx rate in larvae acclimated to four different media for 24 h ⫹

[Na ] (mM) [Cl⫺] (mM) Cell density (102 cells mm⫺2) Ratio of wavy convex (%) Cl⫺ influx rate (nmol h⫺1 mg⫺1)

Normal

N-Na-L-Cl

H-Na-H-Cl

H-Na-L-Cl

.5 .5 4.4 Ⳳ 1.4ab 10.6 Ⳳ 9.0a 1.8 Ⳳ .4a

.5 .005 6.6 Ⳳ 3.0a 37.6 Ⳳ 7.5b 8.8 Ⳳ 1.6b

10 10 2.8 Ⳳ 1.6b 0 Ⳳ 0a .3 Ⳳ .1c

10 .005 6.2 Ⳳ 1.3a 50.8 Ⳳ 6.5c 10.6 Ⳳ 1.8d

Note. Mean Ⳳ SD (N p 5 for the cell density and ratio, N p 10 for the influx) is indicated. Comparisons by ANOVA were made among the four groups in each parameter. Different letters indicate significant differences.

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hole type) after 24-h acclimation (Fig. 3). These data indicate that larvae developed MR cells with larger apical surfaces (wavyconvex type) in low-Cl⫺ medium, which is in contrast to the type with smaller apical surfaces (deep-hole type) in high-Cl⫺ medium. Interestingly, significant differences were found between the two low-Cl groups (H-Na-L-Cl and N-Na-L-Cl) in terms of cell density and ratio of the cell type (Figs. 2, 3). The H-Na-L-Cl group displayed a higher density than did the NNa-L-Cl group at 48 h after transfer but displayed a higher ratio of wavy-convex cell type during the acclimation. Figure 4 compares the Cl⫺ influx rates of larvae acclimated with different media for 24 h. Results show that larvae acclimated to low-Cl⫺ media (H-Na-L-Cl and N-Na-L-Cl groups) developed higher Cl⫺ influx rates than those of larvae acclimated to Normal medium. In contrast, larvae in high-NaCl medium displayed the lowest Cl⫺ influx rate. Coincidentally, the difference in displayed cell type between the two L-Cl groups was also reflected on Cl⫺ uptake capability. We summarized and compared the above results in Table 2, which clearly elucidates the correlation between environmental Cl⫺ and the three parameters, MR cell density, ratio of wavyconvex cells, and whole-larval Cl⫺ influxes. Comparing the groups that were constant in Na⫹ concentration but different in Cl⫺ level (Normal vs. N-Na-L-Cl and H-Na-H-Cl vs. H-NaL-Cl), we concluded that a Cl⫺ deficit could induce a higher density of MR cells with larger apical surface areas and, consequently, a higher Cl⫺ uptake ability. However, the correlation between Na⫹ and MR cell densities or cell-type ratios does not seem to support the model of Na⫹ absorption in MR cells when we compared the N-Na-L-Cl and H-Na-L-Cl groups.

Experiment 2 According to the above experiment, we further asked whether the different types of apical openings were transformed from each other. Therefore, we acclimated larvae to H-Na-H-Cl medium to induce only deep-hole-type openings (Fig. 5A) and then withdrew Cl⫺ from the medium to follow their morphological changes. Results showed that the shallow-basin type of MR cells were observed on larval integument at 4 h after transfer (Fig. 5B), when the wavy-convex type was barely observed (!3%; Fig. 6). At 10 h after transfer, the percentage of wavyconvex type had increased to about 35% of total MR cells (Fig. 6). In addition, the cell density increased dramatically within

Figure 5. SEM pictures showing the morphological changes of mitochondria-rich cells (arrowheads) on larvae preacclimated to H-Na-HCl medium for 24 h and then acutely transferred to H-Na-L-Cl medium. After preacclimation to H-Na-H-Cl for 24 h, the larvae displayed unique deep-hole cells (a). However, after transfer to H-Na-L-Cl medium for 4 h, shallow-basin cells were observed on the larvae (b). Wavy-convex cells showed up at 10 h after transfer (c).

474 L.-Y. Lin and P.-P. Hwang

Figure 6. Percentage changes of wavy-convex type of total mitochondria-rich cells in tilapia larvae preacclimated to H-Na-H-Cl medium for 24 h and then acutely transferred to H-Na-L-Cl medium. No significant increase in wavy-convex cells was observed on larvae at 4 h after transfer. However, wavy-convex cells dramatically increased to about 35% of total MR cells by 10 h after transfer. The curve connects the mean values. Comparisons achieved by ANOVA were made among different time points. Different letters indicate significant differences.

4 h after transfer (Fig. 7). Taking all the data together, we concluded that wavy-convex-type cells were transformed from the deep-hole type through the intermediate shallow-basin type.

Discussion It is well known that the apical surfaces of MR cells recessed to form apical crypts in seawater-acclimated fishes but were flush or slightly raised above the adjacent pavement cells in most FW fishes (reviewed by Perry and Laurent 1993). In the special case of tilapia, Oreochromis mossambicus, recessed apical surface of MR cells were also found in FW individuals (Wendelaar Bonga and van der Meij 1989; Lee et al. 1996; van der Heijden et al. 1997). In our previous study on freshwater tilapia, we categorized these MR cells with different apical surface as wavy-convex, shallow-basin, and deep-hole subtypes (Lee et al. 1996). Larger than 6 mm in maximum dimension, apical membranes of the wavy-convex MR cells had arrays of microvilli, which gave a rough surface appearance to these cells. The ovoid apertures of shallow-basin cells, which measured 2–4 mm in maximum dimension, occasionally ornamented with short microvilli. The deep-hole cells, which measured about 0.5–1.5 mm in maximum dimension, were narrow, deep, and round to oval pores in which little or no internal structure was visible. However, the structure-function relation of these MR cell subtypes is still unclear. In absorptive epithelial cells, such as enterocytes of intestines and intercalated cells of renal collecting ducts, a microvilli-rich apical membrane is a critical, common characteristic and is thought to provide large surface areas for ef-

fective transport of ions or molecules. In addition, the enlargement of the apical surface of freshwater MR cells and increase in cell density were reported in salmonid fishes acclimated to low-NaCl water (Laurent et al. 1985; Perry and Laurent 1989; Greco et al. 1996). Indeed, these results showed that both mature MR cell density and the surface area of apical membranes increased (increase of wavy-convex type represented the increase of apical surface area) in tilapia larvae subjected to environmental Cl⫺ deficits. In this study, we clearly elucidated the correlation between environmental Cl⫺ level and morphological changes of MR cells. We especially focused on short-term changes in morphology and density of MR cells. Using the strategy of preacclimating larvae to H-Na-H-Cl medium to induce single deep-hole-type cells and then withdrawing Cl⫺ in the medium, the morphological change of MR cells could be more easily elucidated. Intermediate-sized shallow-basin openings appeared at 4 h after Cl⫺ withdrawal when only a few wavyconvex type had appeared. Six hours later, the ratio of wavyconvex type had dramatically increased to 35%. According to Tsai and Hwang (1998), the generation time of gill epithelial cells is about 4 d, and it takes a few days for the epithelial cells to differentiate into MR cells. The increase of the wavy-convex type after exposure to Cl⫺-deficient water for 10 h is comparatively rapid, and hence, these wavy-convex MR cells are quite unlikely to be derived from undifferentiated epithelial cells. More likely, the wavy-convex type is transformed from MR cells of other types. Moreover, we suggest that the distinct types of apical surfaces in tilapia larvae represent different phases of MR cells possessing different efficiencies of Cl⫺ transport. Through modulating the apical membrane of MR cells and putative transporters on it, larvae are able to quickly and effectively regulate Cl⫺ transport.

Figure 7. Changes in mitochondria-rich cell density in tilapia larvae preacclimated to H-Na-H-Cl medium for 24 h and then acutely transferred to H-Na-L-Cl medium. Cell density dramatically more than doubled within 4 h. Comparisons by ANOVA were made among different time points. Different letters indicate significant differences.

Mitochondria-Rich Cells in Tilapia Larvae The role of MR cells in freshwater teleost gills in transepithelial Ca2⫹ and Cl⫺ uptake has been clearly established, yet their role in Na⫹ uptake is still controversial (reviewed by Perry 1997). In these results, the density and apical surface size of MR cells were positively correlated with a deficient level of Cl⫺ in the water, implying that MR cells are responsible for Cl⫺ uptake (Figs. 2, 3; Table 2). However, the correlation between Na⫹ and MR cell densities or cell-type ratios did not support the idea that MR cells are also responsible for Na⫹ uptake when we compared the N-Na-L-Cl with H-Na-L-Cl groups (Figs. 2, 3; Table 2). H-Na-L-Cl larvae displayed more wavy-convex cells than did N-Na-L-Cl larvae, implying that pavement cells may be responsible for Na⫹ uptake and may, consequently, compete for larval surface area with MR cells that expanded their apical surface for Cl⫺ absorption in Cl⫺-deficient water. As Na⫹ source was sufficient in the H-Na-L-Cl group, MR cells were able to expand their apical surfaces without competition from pavement cells. In the prevailing model of transcellular Cl⫺ absorption, Cl⫺ is thought to be transported actively through the apical membrane of MR cells by Cl⫺/HCO⫺3 exchangers (reviewed by Perry 1997). Thus, the increase of apical membrane area may allow more Cl⫺/HCO⫺3 exchangers to be inserted, which, consequently, produce a more powerful active transport of Cl⫺. In contrast, when the Cl⫺ level in the water is raised, the apical membrane and the exchangers on it could be internalized to reserve energy and maintain Cl⫺ balance. Therefore, the reconstruction of apical surface seems to be critical for modulating Cl⫺ uptake activities. The modulation of apical surface structure might be achieved through membrane turn over, cytoskeleton reorganization, and other intracellular modifications. Perry and Laurent (1993) proposed a model to describe the adjustment of MR cell apical surface. In this model, ion transport activity of MR cells was regulated passively by the covering of surrounding pavement cells. The activity was reduced when the covering of pavement cells inactivated the transport proteins on apical membrane of MR cells. Our ongoing experiment is to test these models and figure out the regulatory machinery underlying the morphological changes of MR cells. Acknowledgments This study was supported by grants to P.-P.H. from the National Science Council (NSC 89-2311-B001-066) and Academia Sinica (Major Group-Research Project), Taiwan, Republic of China. Literature Cited Ayson F.G., T. Kaneko, S. Hasegawa, and T. Hirano. 1994. Development of mitochondria-rich cells in the yolk-sac membrane of embryos and larvae of tilapia, Oreochromis

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