Int. J. Adv. Sci. Eng. Vol. 2 No.3 153-157 (2016) 153 ISSN 2349 5359 Heterologous induction of allo-tetraploidy and early embryonic development in Buenos Aires , Hyphessobrycon Anisitsi (Eigenmann, 1902) C. Justin David1,2* 1Biotechnology & Bioinformatics, School of Ocean Science & Technology, Kerala University of Fisheries & Ocean Studies, Kochi- 682506, Kerala 2Department of Behavior and Physiology, School of Biological Sciences, Madurai Kamaraj University, Madurai – 625 021, Tamilnadu India

ABSTRACT: Selective breeding, hybridization, triploidy and tetraploidy by heterospecific insemination are genetic methods for generating male or female population capable of producing diploid (2n) or triploid (3n) gametes. The generated allotetraploids (4n) will thus serve as gamete providers for generating auto and allo- triploids, tetraploids and androgenetic clones. In the present study, we generated an allo-tetraploid population by crossing wild type black widow tetra (WT), Gymnocorymbus ternetzi (Boulenger) females with either wild type gray or recessive golden androgenetic (BT), Hyphessobrycon anisitsi (Eigenmann), males followed by arresting the Ist mitotic division of hybrid embryos with thermal shock to study their early embryonic development and survival. The 22-min post fertilization hybrid embryos were subjected to heat shock at 41 C for 2 min or cold shock at 4 C for 2 min; maximum hatching success of tetraploids ranged between 2 - 5%. Survival of tetraploid tetras was lowered by embryonic mortality especially after gastrulation stages. Karyotyping and erythrocyte measurements confirmed successful induction of tetraploidy. The tetraploid red blood cell (RBC) nuclear volume increased 2.2 times than its diploid counterpart and the chromosome numbers doubled (94) in tetraploids. Causes for such high incidence of mortality at hatching and reasons for survival of interspecific allotetraploids are discussed. KEYWORDS: heterospecific insemination, allotetraploidy, triploidy, androgenetic tetra, double shock © 2016 mahendrapublications.com, All rights reserved 1.0 . INTRODUCTION Naturally occurring tetraploid race were reported in characid tetra, Buenos Aires tetra (BT) cyprinids Carassius auratus (Kobayashi et al. 1976), the caudovittatus using heterologous eggs of black WT, African Barbus (Agnese et al. 1990), Misgurnus Gymnocorymbus ternetzi, (ii) optimize protocols for shocking anguillicaudatus (Arai et al. 1991a; 1991b), Cobitid, Cobitis temperature and duration for arresting the first mitotic biwae (Kusunoki, et al. 1994) and the silurids Clarias cleavage and (iii) study early embryonic development and batrachus (Pandey and Lakra, 1997) and Heteropneustes survival of allotetraploid larval population. fossilis (Pandian and Koteeswaran 1999). Although there is a long list of unsuccessful attempts to generate tetraploids 2.0 MATERIAL AND METHODS (Pandian and Koteeswaran 1998), viable tetraploids capable 2.1. Fish of breeding have been generated in about half a dozen To induce hybrid tetraploidy, Buenos Aires Tetra, (BT) belonging to the cyprinids (e.g. M. anguillicaudatus, Hemigrammus caudovittatus (Ahl, 1923) and widow tetra Arai et al. 1993; M. mizolepis, Nam et al. 2001), the salmonids (WT), Gymnocorymbus ternetzi (Boulenger, 1895) was Oncorhynchus mykiss (Thorgaard et al.1981; Blanc et al. chosen. Short generation time (4 months), oviparity and 1987; Chourrout and Nakayama 1987). Androgenotes amenability for stripping eggs and sperm, fortnightly generated using diploid sperm from tetraploid rainbow breeding and availability of strains with contrasting colours trout showed improved survival (Thorgaard et al.1990). (Table 1) make these tetras ideally suited for induction of Interestingly, production of tetraploids may also pave way tetraploidy. The following crosses were made: Experimental 1 2 for the production of triploids (Chourrout et al. 1986) and series (I) wild type gray BT (X Y ) ♂ x wild type black WT 1 2 1 2 penta and hexaploids (Thorgaard et al. 1990; Arai et al. (X X ) ♀ and series (II) wild type gray BT (X Y ) ♂ x wild type 1 2 1993), as many tetraploid males are known to mature and black WT (X X ) ♀, involving double shocks (heat shock produce unreduced sperm ranging from haploid to triploid followed by cold one) to suppress the first mitotic cleavage. (Arai et al. 1991b, 1993). Briefly, suppression of the first In all the crosses wild type (X1X2) black widow tetra (WT), G. mitotic cleavage is the common step used to induce ternetzi female, served as egg donor. For each experiment, 5- tetraploidy. Of many methods, that involving hybridization 10 individuals were used for collection of gametes. not only induced tetraploidy but also improved the yield Estimations reported in Tables 2 to 5 are based on Mean (Cherfas et al. 1994). Hence in the present study, an attempt SD of 5-15 replicates. was made to (i) induce hybrid/allotetraploidy in the

*Corresponding Author: [email protected] Received: 28.12.2015 Accepted: 02.02.2016 Published on: 25.02.2016

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Int. J. Adv. Sci. Eng. Vol. 2 No.3 153-157 (2016) 154 ISSN 2349 5359

Table 1. Phenotypic markers in Hemigrammus caudovittatus 2.4.3. Erythrocyte measurements: Ploidy was also (BT) ♂ and Gymnocorymbus ternetzi (WT) ♀ chosen for ascertained by measuring the erythrocytes. Prior to induction of hybrid tetraploid immersing the selected fry in acetic acid for karyotyping, it was streaked on a glass slide for preparation of blood Gymnocory Hemigrammus smears; the smear was fixed in methanol and stained in 5% mbus caudovittatus Giemsa. The longer diameter of 20 erythrocytes and nuclei Marker ternetzi was measured using an ocular micrometer calibrated with a WT ♀ BT ♂ stage micrometer following David (2004). Strain Black wild Gray Golden 3.0. STATISTICAL ANALYSIS type wild recessive and The calculated values were expressed as Mean SD. ANOVA type rogenote was performed to estimate the level of significance among shape Fusiform Slender Slender the corresponding data at a level of P<0.05. All data were nalyzed using Sigma STAT (Ver 9.0). Body vertical Present Absent Absent stripes 4.0 RESULTS AND DISCUSSION colour Black Gray Gold 4.1.1. Tetraploidization: An experiment was made to know Colour of fins Black Red Golden the compatibility between BT male and WT female and to yellow estimate the hybrid survival. Table 2 shows that the selected Tail Deeply Normal Normal cross was compatible and resulted in the production of 78 th morphology forked 2.13% hybrid hatchlings; hybrids survival on the 30 day Eye colour Black Black Red was 57 2.17%, a level at which survival was sustained until 350 days. It is known from earlier observations that the age of 22-min old embryos of WT and 25-min old embryos of BT 2.2 Gametes and fertilization is the optimal for successful suppression of the first mitotic For each series, about 100 eggs from wild type black WT cleavage in WT (David and Pandian 2006a) and BT (David females were stripped into a thin plastic petri-dish (1mm and Pandian 2006b), respectively. For this, the 22-min and thickness) containing Ringer’s solution. Fertilization was 25-min old diploid hybrid embryos, resulting from the cross achieved by mixing 1 ml of fresh diluted milt from the between BT male and normal black WT female, were selected male with WT eggs in a petri-dish and subsequent exposed to 41oC for 2 min; 19 and 2 % of progenies were addition of 3 ml of fresh tap water and gentle swirling of the tetraploids, when shock was given to the hybrid embryos at dish for 45 sec ensured activation of embryonic the age of 22 and 25 min, respectively (Table 3). Hence, the development. The developing embryos were maintained at 22-min old hybrid embryos were exposed to 41oC for 2 min 26 2oC. in all the series and an additional cold shock at 4oC for 2 min 2.3Tetraploidization in series II (Table 4). 2.3.1 Thermal shocking: In all the experimental series, tetraploidization was achieved by arresting the first mitotic cleavage. The embryos were heat shocked by suspending them in plastic containers (7 cm diameter and 3 cm depth with a nylon mesh 0.5mm at the bottom) submerged in water bath, in which the desired temperature was maintained, exposed to optimal shocking temperature for the required duration. After the heat or cold shock treatment, the embryos were immersed in sterilized tap water (26 1oC) to return back to pre-shock temperature. From earlier observations, it is known that an exposure of

22-min old WT embryos successfully suppressed the first mitotic cleavage (David and Pandian 2006a). Hence, the 22- Figure 1. Representative samples of (Ai) golden BT (2n), (Aii) min old hybrid embryos were shocked at 41oC for 2 min. black WT (2n), (Bi) tetraploid hybrid – 3 day old and (Bii) 30-day However, an additional cold shock at 4oC for 2 min was also old 4n hybid tetraploid. Note the transformation of body shape to given to the embryos of II series. fusiform in the 30-day old fry .

2.4. Confirmation of tetraploidy While 13 to 14 % of the karyotyped fry were tetraploids in 2.4.1. Embryonic development: Phenotypic characters and the I series, it was as high as 22 % for the II series produced developmental stages of BT and WT embryos were studied after double shocks (Table 5). Clearly, the double shock in detail (David & Pandian 2006a, 2006b), by monitoring treatment generated higher percentage of tetraploids and them under strereozoom microscope (Nikon, Japan). ensured higher survival at hatching. In both series, survival 2.4.2. Karyotyping: For Karyotyping, freshly hatched fry of tetraploids at hatching was 11 % and 21 % for the hybrid were kept in 0.01% colchicine solution for 6 hr. Karyotyping tetraploid progenies, whose respective embryos suffered a was made following Kligerman and Bloom (1977). Ploidy single (I series) or double (II series) shock(s), respectively. level of each randomly selected individual was determined However, survival on the 30th day was the highest (1.25%) from 20 5 metaphase spreads for the normal hybrid tetraploids (I series I: Table 5). Remarkably, the chromosomes of diploid and tetraploid

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Int. J. Adv. Sci. Eng. Vol. 2 No.3 153-157 (2016) 155 ISSN 2349 5359 larvae showed difference in numbers and exhibited Since more than 78% of BT-WT hybrids survived for longer differences in both phenotypic features and genetic makeup than 350 days, karyotypic incompatibility is not likely to be (allo-tetraploidy; Figure 1). the major reason (David, 2004). High homozygosity and aneuploidy appears to be more important for the embryonic 4.1.2. Confirmation of tetraploidy: mortality. Secondly, thermal induction especially at high Most remarkably, the body shape of all 30-day-old temperature is known to include high frequency of tetraploids generated was fusiform, exactly resembling that mosaicism; hence among the hatched fry ~80% suffered of female WT, irrespective of the difference in paternal aneuploidy. However, from flow cytometric and Coulter genomic contribution from the slender (wild type) gray counting analyses, Yamazaki and Goodier (1993) provided normal (X1Y2) male. Randomly selected progenies were evidences for the mosaicism in the presumptive 3n and 4n karyotyped. Figure 3A shows that they were tetraploids. Ctenopharyngodon idella as the reason for its inability to Besides, measurements of the longest diameter of survive longer Hence, it is likely that aneuploidy and erythrocyte cell (14.23 0.03 m) and its nucleus (9.43 mosaicism may be the major reasons responsible for the 0.04 m) also confirmed that the randomly selected embryonic mortality. Among the 11 – 21% hatched 4n fry, progenies in these series were indeed tetraploids [Fig. 3(B)]. more than 80% suffered mortality within 30 days of Since most of the presumptive tetraploids succumbed prior hatching. In the tetraploids, as the cell size is known to to hatching, embryonic events were more precisely observed enlarge, the number of cells is decreased (Pandian and for delayed, distorted and arrested development. Figure 3 Koteeswaran 1998); consequently, the tetraploid cells are represents the estimates of embryonic mortality, mostly due not exactly double the size of diploids. For instance, Cassani to the arresting and the consequent bursting of the embryo, et al. (1990) estimated the decreases in cell number of the suffered at the selected stages by the presumptive tetraploid tetraploid grass carp C. idella to be 31% and 54% for a 30- embryos generated in the 2 experimental series. min and 5-hr old fry. Consequent to the larger cell size, one or more of physiological activities are slowed down or hindered. In BT-WT hybrid tetraploids too, the surface area of erythrocyte nucleus increased from 9.46 0.05 m2 in diploids to 33.49 0.09 m2 in tetraploids which may also negatively affect the nucleo-cytoplasmic ratio in cells. Hence it is likely that the reduced cell surface area may also be responsible for the post-embryonic mortality provided that nuclear genome increased corresponding to ploidy.

Figure 3. Survival (%) at selected developmental stages of control 2n fry and hybrid tetraploid tetra. Among the experimental series, the II series involving heat and cold shocks not only generated more tetraploids but also Figure 2. Karyotypes (vertical column A) and erythrocytes ensured higher survival at hatching. This observation (vertical column B) of the tetra hybrid tetraploids. First confirms the earlier report by Nam et al. (1999), who horizontal column shows representative figures for normal obtained a 3% yield of 4n mud loach, Misgurnus mizolepis diploid BT, The photographs in II, III and IV rows show when he attempted auto tetraploidy using thermal shocks. representative figures for hybrid tetraploids from the first So far, those who have successfully induced tetraploidy second and third series of experiments. Scales represent 5 m (A) showed Karyotype, erythrocyte measurement and flow cytometric analyses (e.g. Misgurnus mizolepis, Nam et al. and 10 m (B) 1999, Nam et al. 2001 as evidences for achieving tetraploidy. Hence, in the present study karyoptype and nuclear 5.0 DISCUSSION measurements served as the This attempt has successfully generated hybrid tetraploids ACKNOWLEDGEMENTS of the chosen tetras namely BT and WT. However, about 80 The author expresses his gratitude to Prof. T.J. Pandian, % embryos suffered heavy mortality before attaining tail Madurai Kamaraj University for guidance and support. bud stage (Fig. 3). The causes for the observed embryonic Financial support by the Department of Science & mortality may be (i) mosaicism, (ii) aneuploidy, (iii) reduced Technology, New Delhi and Kerala University of Fisheries & cell surface, (iv) wrong cytological events and or (v) Ocean Studies for infrastructure is gratefully acknowledged enhanced homozygosity (Pandian and Koteeswaran, 1998).

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Table 2. Survival of hybrids of the cross between gray WT, Gymnocorymbus ternetzi female and golden androgenetic BT, Hemigrammus caudovittatus male. Each value is the mean ( SD) of 15 replicates. * as percentage of eggs used.

Hatching success Survival Survival Egg Series on 3rd day* on 30th day* (no) (no) (%) (no) (%) (no) (%) Control 1060 ± 996 ± 964 ± 946 ± 94 ± 3.12 91 ± 2.12 89 ± 1.13 WT 30 7.23 1.23 1.43 Control 1040 ± 998 ± 971 ± 953 ± 96 ± 2.23 93 ± 1.53 91 ± 2.46 BT 13 6.71 2.16 2.02 1030 ± 803 ± 731 ± 587 ± Hybrid 78 ± 1.71 78 ± 2.13 57 ± 2.17 23 9.04 2.42 1.46 Table 3. Optimization of age of the hybrid embryo chosen for heat shocking at 41 C for 2 min to arrest the first mitotic cleavage for induction of hybrid tetraploidy using eggs of black WT, Gymnocorymbus ternetzi and sperm of gray BT, Hemigrammus caudovittatus. Karyotyping was made on randomly selected 2-3 day old fry. Values are given for 10 estimates (mean SD). * as percentage of karyotyped fry.

Karyotyped Observed ploidy Incidence of Hatching success Age of shocked WT fry (no) 4n embryo egg (min) (no) (no) (%) (no) 2n -4n 4n (%)*

526 ± 94 ± Control 560 ± 20 36 36 - - - 5.23 2.13 258 ± 41 ± 22 630 ± 16 67 42 12 13 19 6.13 1.33 97± 18 ± 25 540 ± 23 43 29 13 1 2 3.13 1.72 Table 4. Optimization of cold shock (4 C) duration chosen for induction of hybrid tetraploidy following heat shock at 41 C for 2 min to 22 min old hybrid embryos. to arrest the first mitotic cleavage. Eggs of black WT, Gymnocorymbus ternetzi were fertilized with sperm of gray BT, Hemigrammus caudovittatus. Karyotyping was made on randomly selected 2-3 day old fry. Values are given for 15 estimates (mean SD). * as percentage of karyotyped fry Hatching success Karyotyped Observed ploidy Incidence Duration of cold WT Fry (no) of 4n shock egg (no) (min) (no) (no) (%) 2n -4n 4n (%)* 0 210 ± 12 88 ± 4.23 42 ± 2.31 26 26 - - - 1 330 ± 21 142 ± 7.26 36 ± 1.43 38 32 5 1 3 2 320 ± 16 115 ± 5.14 34 ± 3.12 46 32 4 10 22 3 340 ± 29 114 ± 3.74 27 ± 2.46 36 27 7 2 6 Table 5. Survival of hybrid tetraploid produced in series I: black WT ♀and gray BT♂, whose 22 min old hybrid embryos were subjected to a single shock at 41 C for 2 min. series II: black WT ♀and gray BT♂, whose 22 min old hybrid embryos were subjected to double shocks first at 41 C for 2 min and later at 4 C for 2 min. Randomly selected 2-3 day old fry were karyotyped. * as percentage of hatching. –4n represents hypotetraploidy Hatching success Karyotyped Observed ploidy Incidence Duration of cold WT Fry (no) of 4n shock egg (no) (min) (no) (no) (%) 2n -4n 4n (%)* 0 210 ± 12 88 ± 4.23 42 ± 2.31 26 26 - - - 1 330 ± 21 142 ± 7.26 36 ± 1.43 38 32 5 1 3 2 320 ± 16 115 ± 5.14 34 ± 3.12 46 32 4 10 22 3 340 ± 29 114 ± 3.74 27 ± 2.46 36 27 7 2 6

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