Part 2 Chromosome Manipulations Polyploidy - Case study 1 Chromosome manipulation technology Gynogenesis and androgenesis – Case study 2 Cryopreservation of gametes MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Oyster Culture                                                                In 1996 (FAO) over 1,200,000 tons of the Pacific Oyster (Crassostrea gigas) were produced in the world. Although it is a Japanese species it has been introduced in Australia, North America, France and New Zealand, always voluntarily; Why? Because it was the only way to enhance the production of oyster in these countries. Are there alternatives? MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Oyster Paper MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

YES! Polyploidization, Hybridization between closely related species, Genetic selection MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Attractiveness of Polyploidy Sterility: Reduced environmental impact of escapees; No diversion of energies towards maturation; Faster growth Disease resistance Simple technique MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Polyploidization in Oysters What is it and how does it work? Various ways to do it: Suppress polar body I or II formation during meiosis using cytochalasin B; Pressure shocks; Heat shocks; 6-DMAP treatment of eggs; Mate tetraploids with diploids. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Mitosis MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Meiosis MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Triploid Oysters MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Tetraploid Oysters MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Protocols for the induction of triploidy Allow fertilization to occur; Shortly after (e.g. 10 min in rainbow trout) treat eggs in order to inhibit extrusion of 2nd polar body: Add cytochalasin B or heat shock or cold shock or pressure shock the eggs. Thermal shocks are easier to implement although pressure shocks have produced better and more robust results (more expensive equipment required). MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

The standard method is to induce triploidy by treatment of newly fertilized eggs with CB to prevent extrusion of PB2 (Allen et al., 1989). The alternative method, possible because of our development of tetraploid oysters, is by mating tetraploid and diploids (Guo et al., 1996). MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Growth of Triploid vs. Diploid Oysters MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Problems with Triploids For many species triploids are not allowed by law (e.g. sea bass in Europe); Although sterile many triploids differentiate and develop gonads to some extent (mosaics) , so growth advantage is not always there; MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

PAPER Growth Trials with Triploid Bass MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Triploid Hybrids Triploids where one diploid set of chromosomes comes from one species and one haploid set comes from another. e.g. grass carp x common carp or rainbow trout x brook trout; Often show increased survival; Sterility is more sure; Can often reproduce the growth advantage of triploids without the mortality or deformity rates sometimes seen in triploids (e.g. coho x chinook salmon triploids) MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Gynogenesis Diploid individuals with both set of chromosomes from their mothers; In species with homogametic females it will produce all-female lines; Two main applications: Sex control for the production of all-female lines in species where females mature larger than the commercial size; Rapid inbreeding for the generation of inbred lines (mainly useful for research purposes, but potentially also useful for the production of hybrids between inbred lines with resulting heterosis) MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Protocols for Gynogenesis Sterilize sperm using radiation or chemical treatments; Allow fertilization to occur; Shortly after (e.g. 10 min in rainbow trout) treat eggs in order to inhibit extrusion of 2nd polar body: Add cytochalasin B or heat shock or cold shock or pressure shock the eggs. Thermal shocks are easier to implement although pressure shocks have produced better and more robust results (more expensive equipment required). Treatment applied in the first division will produce partially homozygous diploids; A late treatment results in totally homozygous diploids. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Problems of Gynogenetic Lines Highly Inbred, with low survival rates, poor growth, high deformities, etc.; This can be reversed if a gynogenetic, inbred population is hormonally sex-reversed and then mated with normal females. The offspring will be all females and outbred. As a means of controlling reproduction there is a risk that introduced males will lead to establishment of an unwanted population; MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Gynogenetic Seabass Lines MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Cryopreservation of Gametes MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

The benefits of cryopreservation in aquaculture species include the following: Cryopreservation can be used to improve hatchery operations by providing sperm on demand and simplifying the timing of induced spawning. Frozen sperm can enhance efficient use of facilities and create new opportunities in the hatchery by eliminating the need to maintain live males. Valuable genetic lineages, such as endangered species, research models or improved farmed strains, can be protected by storing frozen sperm. This could be critical for marine species such as shellfish, where valuable broodstocks must be stored in natural waters. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

The benefits of cryopreservation in aquaculture species include the following (cont.): Sperm can be used in breeding programs to create new, improved lines and shape the genetic resources available for aquaculture operations. A dramatic example of this is in the dairy industry, which relies almost entirely upon cryo-preserved sperm to produce improvements in milk yields. Cryopreserved sperm of aquatic species will likely become an entirely new industry within the coming decade. Large, highly valuable global markets for cryopreserved sperm of aquatic species are on the horizon. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection