Game of Hybridization leads to Accidental Birth

Avanti Basu

16th April, 2021

Abstract

Russian sturgeon(Acipenser gueldenstaedtii; functional tetraploid) from the family Acipenseridae and American paddlefish(Polyodon spathula; functional diploid) from the family Polyodontidae were hybridized. The process of hybridization was repeated using eggs from three sturgeon and sperm from four paddlefish individuals. Survival in all hybrid family groups ranged from 62% to 74% 30 days after hatching, which was the first successful hybridization between these two species and between members of their families. The two ploidy levels in hybrid were revealed by flow cytometry, chromosome analysis, and microsatellite analyses. The chromosome numbers of the hybrids ranged between 156-184 in “functional” triploids and 300-310 in “ “functional” pentaploids. In hybrids, the size and number of dorsal and ventral scutes (external bony plate) correlated with the ploidy levels as well as with the calculated ratio of the maternal and paternal chromosome sets. Irrespective of the ploidy level, an extra haploid cell lineage was found in three hybrid individuals suggesting polyspermy.

1. Introduction

Polyploidy is frequent in interspecific hybrids of fishes. Sturgeons represent ancestral ray-finned fishes in the class Actinopterygii, which originated during the Jurassic era, 200 million years ago. They diverged before the teleost (a large group comprising all ray-finned fishes) specific genome duplication from the other ray-finned fishes. In the family Polyodontidae, there is no further genome duplication and they are considered “functional diploids”(2n) with around 120 chromosomes. American paddlefish(P.spathula) and Chinese paddlefish(Psephurus gladius) are the only two species in the family. In Acipenseridae, at least one additional genome duplication happened during the evolution of the Atlantic clade of the family, and more during the shortnose sturgeon (Acipenser bevirostrum) and the pacific clade evolution. Most of the species in the Atlantic clade, including the Russian sturgeon, are considered “functional tetraploid” (4n) with approx. 250 chromosomes. Besides the large phylogenetic distance, representatives of Polyodontidae and Acipenseridae differ in their gross morphology as well as feeding behavior, preferred habitat, etc. In hybrids of Acipenseridae, odd ploidy plasticity (the capacity of organisms to alter the phenotype in response to changes in their environment) may occur, which highly depends on the ploidy level of the parental species. During an experiment to produce gynogenic (the nucleus of sperm is unable to form syngamy with the ovum nucleus), Russian sturgeon progeny, a negative control was initiated using non-irradiated American paddlefish sperms and eggs from the Russian sturgeon. Unexpectedly, the control cross resulted in viable hybrids. This experiment addressed whether simultaneous genome duplication occurs and how it affects the ploidy level of hybrid progenies. Finally, it described the relationship between the external morphology and ploidy level in the hybrids of species with such great phylogenetic distance.

2. Materials and Methods

2.1. Reproduction and Nursing 
Irradiated paddlefish sperm had been used for gynogenesis. As a control of the success for gynogenesis, parallel fertilization is done using untreated American paddlefish sperm. Three females from Russian sturgeon and four males from American paddlefish were crossed for the control and produced five parent hybrid combinations. To induce ovulation and spermiation, the breeders were injected with LHRH(luteinizing hormone-releasing hormone) analog Des-gly^10(D-Ala^6) LHRH ethylamide(40micro g/kg for females and 20micro g/kg for males). Eggs were stripped 48-52 hours post-injection and milt (the semen of male fish) was stripped 48 hours post-injection. According to the common aquaculture practice, immediately before fertilization, milt was diluted with water at a ratio of 1:200, and 2mL of this diluted solution was added to egg samples weighing 300 g. A 2min 36 degree Celsius heat shock was applied 18 min post-fertilization for retention of the second polar body. Fertilization rates at 48 hours post-fertilization and survival at hatching were determined by counting 3 x 100 eggs in each parent combination. Two thousand newly hatched fry from each of the treatment groups was stocked into 400 L volume tanks with through-flow water for nursing. On the eighth day after hatching, fry received frozen brine shrimp nauplii and mosquito larvae followed dry feed feeding from the twentieth day. After reaching the size of 3 cm, the fishes were transferred into large tanks.

 

2.2.Microsatellite Marker Analysis 
From the female Russian sturgeons and male American paddlefish parents and 19 hybrid offspring, fin clips were collected. Genomic DNA was extracted using the standard protocol of the E.Z.N.A Tissue Kit (Omega Bio-Tek, USA). Four pairs of microsatellite primers were used and three microsatellite loci were developed for American paddlefish and the fourth locus was developed for shovelnose sturgeon. For capillary electrophoretic analysis of microsatellite alleles, the universal fluorescent labeling method was used. To each reaction mixture, one tail-specific oligo was added. Amplified, labeled products were subjected to capillary electrophoresis on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, USA). GeneMapper 4.0 software was used to determine the fragment size of the detected alleles.

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A “sturddlefish”, hybrid of Russian sturgeon and American
paddlefish accidentally bred in a nursery in Hungary.

2.3.Flow Cytometry and DNA Content Analysis 
DNA content of RBCs was measured by flow cytometry using propidium iodide staining. Blood cells were centrifuged at 500x g for 5 min and resuspended in 500 micro L PBS to set the cell concentration to 10^7mL. Then, 4500 micro L 70% ethanol was added to the samples and the cells were fixed and permeabilized for 30 min at room temperature. The samples were washed twice in PBS. The ethanol-fixed cells were stained with 40 micro g/mL PI for 30 min. 
Flow cytometry measurements were carried out on 98 hybrid fishes, two Russian sturgeons, and four male American paddlefish. Upon DNA content analysis, the mean PI fluorescence intensity of each progeny was normalized to the mean PI fluorescence intensity measured in the case of the three male Americans.

2.4. Chromosome Analysis 
It was carried out on tissue culture cells from eight ten-month-old hybrid individuals. The fibroblast culture was set up from the pectoral fin and the culture medium was changed twice a week. When the fibroblast monolayer was almost confluent in the flasks and 40 micro L of KaryoMAX^TM Colcemid^TM Solution. The cells were trypsinized after two hours and after 7 min centrifugation was resuspended in 0.56% KCL aa hypotonic treatment. The cell suspension was spread on slides, dried at room temperature, and then stained with 5% fresh Giemsa solution for 7-8 min. From each individual, five slides were prepared and at least 30 metaphase spreads per individual were examined to determine the chromosome number. 

2.5.Morphometry
21 morphometric and four meristic characters on 218 American paddlefish x Russian sturgeon hybrids, 49 American paddlefish, and 50 Russian sturgeon individuals were measured with a digital caliper after clove oil narcotization. The meristic characters covered the number of dorsal, lateral, ventral scutes and the number of barbels. The age of the examined fish varied between 228 and 258 days. 
Based on their origin and genome size, the fishes were divided into five groups:
American paddlefish, Russian sturgeon, small genome size hybrids, large genome size hybrids, and hybrids without genome size data. For each morphometric and meristic character to reveal differences between groups, a series of non-parametric univariate tests and pairwise comparisons were done. Principal Component Analysis was performed on the entire standardized dataset to evaluate the morphometric variation. Since scute size was absent in all paddlefishes and hybrids, it was excluded from the multivariate tests.

3.Results

3.1. Fertilization, Survival and Growth of hybrids 
The fertilization rate varied from 86-93% and the hatching rate from 78-85% in the five parent combinations. Survival after hatching at 30days ranged from 62-74% and at 180 days from 49-68%.In individual weights, substantial differences were observed within the groups.

3.2. Microsatellite Analysis 
No common allele was found between the two species. The Psp-28, Psp-32, and Spl-101 were disomic while the Psp-29 was tetrasomic on the male American paddlefishes. All of the detected alleles of the four loci in the case of American paddlefish were different from the Russian sturgeon. The Psp-29 was disomic, while Psp-28, Psp-32, and Spl-101 were tetrasomic on female Russian sturgeon. All the hybrid individuals inherited one paternal allele from the disomic and two alleles from the tetrasomic alleles loci. The small genome hybrid(SH)individuals inherited one maternal allele from Psp-28 or Psp-29 and two alleles from the tetrasomic loci while the LH hybrids inherited two, three, or four alleles in the case of tetrasomic markers, but at least one of the two maternal tetrasomic loci revealed the presence of two maternal genomes. The presence of polyspermy was not suggested by any of the genotypes.

 

3.3. Flow Cytometry and DNA Content Analysis 
Based on this, two groups were made: A small genome size group(SH) and a large genome size group(LH). Based on their parents, the hybrids were divided into five subgroups. The majority of the tested progenies belonged to the small genome size group in each parent combination case. There was no statistically significant difference in mean DNA content among subgroups of the SH group but some outlier individuals with statistically different DNA content were identified.

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Some of the hybrid offspring have more scutes, or bony scales like their sturgeon mothers and others have longer snouts, like their paddlefish fathers.

3.4.Chromosome Analysis and Ploidy Level in Different Cytometry Class Groups
The triploid hybrids were formed by one maternal gamete(2n) from a functional tetraploid female Russian sturgeon and one paternal gamete(n)from a functional diploid paddlefish. 
Detailed karyotype analysis was performed on one randomly chosen SH and LH individual. The karyotype of triploid individuals consisted of 70 meta and submetacentric, 12 acrocentrics, and 94 microchromosomes. The analysis in the case of pentaploid individuals showed the occurrence of 118 metacentric and submetacentric, 18 acrocentrics, and 174 microchromosomes. In both cases, two large acrocentric chromosomes were found.

 

3.5. Morphometry
The difference between American paddlefish and Russian sturgeon occurred in morphometric characters related to rostrum length which were found higher in American paddlefish. The rostrum length-related characters of hybrids differed from the parent species’ and further differences occurred between SH and LH groups. The upper caudal lobe and pectoral fin lengths showed similar patterns but the LHs differed marginally from the Russian sturgeons. The size and number of scutes were higher in the Russian sturgeon, lower in the SH group, and intermediate in the LH group. The hybrids were closer to the Russian sturgeon. The hybrids could not be divided into distinct subgroups but the LH individuals projected closer to the Russian sturgeon.

4.Conclusion

The experiment marked the very first profitable hybridization between these two species-Russian sturgeon and American paddlefish. Both these fish are referred to as “fossil fish” because of their ancient lineage and slow evolution. The researchers had tried to breed the sturgeon and paddlefish in the lab since they are both endangered species. To exploit the advantages of heterosis(increase in yield of hybrid organism over those of its parents) the sturgeon hybrids are used in aquaculture. It is estimated that hybrids account for approximately 35% and 20% of global sturgeon meat and caviar production respectively. The new hybrid can play an important role in adapting pond aquaculture to the challenges of climate change if the planktivorous(planktonic food)feeding habit of parent species of paddlefish is inherited to a certain extent.

Reference 

  1. [1] C. Sallan, L. (2014). Major issues in the origin of ray-finned fish. Retrieved from https://doi.org/10.1111/brv.12086
    [2] Zuogang(2006). Age and Biogeography of major clades in Sturgeons and Paddlefishes(Pisces:Acipenseriformes)
    [3] Leggatt, R. (2003). Occurrence of polyploidy in the fish
    [4] Rajkov, J. (2014). Evolution of Polyploidy and Functional Diploidization in Sturgeons:Microsatellite Analysis in 10  Sturgeon species 
    [5] Havelka, M.,  & Arai, K. (2018). Hybridization and Polyploidization in Sturgeon 
    [6] Stech, L., & Linhart, O. (1999). Minimally Invasive Surgical Removal of Ovulated Eggs from Paddlefish 
    [7] Fontana, F., & Lanfrendi, M., (2009). Karyotypic Characterization of Acipenser  gueldenstaedti with C-, AgNo3 and fluorescence banding techniques 
    [8] D. Mims, S., & Shelton, W. L.(2015). Paddlefish Aquaculture 
    [9] Earley, E. M.(1975). Chromosome preparations from monolayer cell cultures

About the Author

IMG_20200301_093623 - Avanti Basu.jpg

Avanti is from Assembly Of Christ School in Class XII. Interested in learning more about human genetics. She is incomplete without Music.