Fertilization and cytogenetic examination of interspecific reciprocal hybridization between the scallops, Chlamys farreri and Mimachlamys nobilis

Abstract

Crossbreeding is a powerful tool for improving productivity and profitability in aquaculture. We conducted a pilot study of an artificial cross between two important cultivated scallops in China, Chlamys farreri and Mimachlamys nobilis, to test the feasibility of interspecific hybridization. Reciprocal hybridization experiments were performed using a single-pair mating strategy (M. nobilis ♀ × C. farreri ♂ and C. farreri ♀ × M. nobilis ♂). The fertilization of each pair was tracked using fluorescence staining of the gametes, and the chromosomes of the F1 hybrid larvae were examined via conventional karyotyping and genomic in situ hybridization (GISH). We observed moderate fertilization success in both interspecific crosses, although the overall fertilization was generally less rapid than that of intraspecific crosses. Conventional karyotyping showed that 70.4% of the viable F1 larvae in M. nobilis ♀ × C. farreri ♂ and 55.4% in C. farreri ♀ × M. nobilis ♂ comprised hybrid karyotypes (2n = 35 = 6m+5sm+11st+13t), and the results were further confirmed by GISH. Interestingly, we detected a few F1 from the M. nobilis ♀ × C. farreri ♂ cross that appeared to have developed gynogenetically. In addition, chromosome fragmentations, aneuploids and allopolyploids were observed in some F1 individuals. Our study presents evidence that the artificial cross between M. nobilis and C. farreri is experimentally possible. Further investigations of the potential heterosis of the viable F1 offspring at various developmental stages should be conducted to obtain a comprehensive evaluation of the feasibility of crossbreeding between these two scallop species.

Figure 1. Cytological observation of the fertilization in M. nobilis ♀ × C. farreri ♂ crosses by fluorescent microscope.

(1) Sperm bound to the egg. (2) Sperm penetrating the egg. (3) Meiotic anaphase I of the egg. (4) Release of the first polar body. (5) Meiotic anaphase II of the egg. (6) Release of the second polar body; female and male pronuclei are formed. (7, 8) Fusion of female and male pronuclei. (9) Formation of polar lobe and the first mitotic division. SP: Sperm. CH: Chromosome. SN: Sperm nucleus. PB1: First polar body. PB2: Second polar body. MP: Male pronucleus. FP: Female pronucleus. PL: Polar lobe. Bar = 20 µm.

Figure 2. Chromosomes and karyotypes of M. nobilis (A), C. farreri (B), and the F1 hybrids (C). Bar  =  5 µm.

Figure 3. Representative metaphase chromosomes and karyotypes of the F1 hybrids of M. nobilis ♀ × C. farreri ♂ (A, B) and C. farreri ♀× M. nobilis ♂ (C, D) examined by GISH.

CF: chromosomes from C. farreri. MN: chromosomes from M. nobilis. Chromosomes are painted by FITC (green) and counterstained by PI (red). In (A, C), the chromosomes originated from M. nobilis are identified in green using the labeled genome DNA probes from M. nobilis. In (B, D), the chromosomes from C. farreri are identified in green using the labeled genome DNA probes from C. farreri. Bars = 5 µm.

Figure 4. Observations of gynogenetic eggs derived from the interspecific cross of M. nobilis ♀ × C. farreri ♂.

(1) The egg has released the first polar body with a sperm binding to it. (2) The egg proceeds to meiotic anaphase II without incorporating a sperm nucleus. (3, 4) The egg releases the second polar body without incorporating a sperm nucleus. SP: Sperm. PB1: First polar body. PB2: Second polar body. FP: Female pronucleus. Bar = 20 µm.

Figure 5. Chromosomes of a gynogen-like hybrid examined by GISH.

(A) All of the chromosomes are painted green using the M. nobilis genomic DNA probes. (B) No C. farreri chromosomes or chromosome segments were found when using the C. farreri genomic DNA as probes. Bars = 5 µm.

Figure 6. Examples of chromosome elimination (A) and an allotetraploid (B, C) in the F1 hybrids.

(A) Chromosomes are identified by GISH using the C. farreri genomic DNA probes. The green chromosomes are from C. farreri, and the red chromosomes are from M. nobilis. Some chromosomes from M. nobilis were eliminated in the metaphase spread. A chromosome fragment originating from M. nobilis is marked with a single arrow. (B, C) Chromosomes are identified by GISH using the M. nobilis genomic DNA probes. The 16 (n) chromosomes originating from M. nobilis are shown with FITC fluorescence (green), and the 57 (3n) chromosomes originating from C. farreri are counterstained with PI (red). Bars = 5 µm.