I. Introduction

Finfish, molluscs and crustaceans exhibit a wide diversity of modes of reproduction. The common mode is dioecy, in which male and female reproductive organs are in separate individuals and each individual is of one sex. Most species currently used in aquaculture are dioecious.

Non-dioecious modes of reproduction are known to exist in fish, molluscan and crustacean species -- including in some important aquacultural species. While the modes of reproduction are quite variable, and often very complex, two general categories exist: hermaphroditism and parthenogenesis. Hermaphroditic organisms possess both male and female reproductive organs. Depending on the particular characteristics of their reproductive development and behavior, hermaphrodites may reproduce through cross- or self- fertilization. Parthenogenesis includes all modes of reproduction where one or more genomes are inherited clonally (Moore 1984). Reproduction in true parthenogens requires neither the presence of sperm to induce embryogenesis nor incorporation of DNA from a male for reproduction of viable progeny.

Considerable variation exists in the specific characteristics of non-dioecious reproduction in fish and shellfish. In order to assess the risks of releases of genetically modified non-dioecious GMOs, knowledge of the specific characteristics of their reproduction is essential. This Supporting Text provides:

1) background on non-dioecious modes of reproduction; and
2) risk management guidance for non-dioecious GMOs of high concern.

II. Background on Non-Dioecious Modes of Reproduction

Fishes
All known parthenogenetic fishes require at least physical stimulus of sperm to induce embryogenesis (Dawley 1989). Depending on the parthenogenetic species, males from the same or different species are involved in the reproduction. Consult Dawley (1989) for an explanation of two different modes of male involvement - gynogenesis and hybridogenesis. Families with species which are parthenogenic include Poeciliidae, Atherinidae, Cyprinidae, Cobitidae (Dawley 1989). Researchers working with fish in the family Poeciliidae may begin their examination of the potential for parthenogenesis in their research organisms by consulting the following references: for Poecilia formosa, Dawley (1989), Monaco et al. (1984), and Schartl et al. (1995); for Poeciliopsis spp., Dawley (1989) and Vrijenhoek (1984).

Currently, only a single species of self-fertilizing hermaphroditic fish is known: Rivulus marmoratus (Soto et al. 1992, Turner et al. 1992). Simultaneous and sequential hermaphroditic fish are known to exist within 14 families of teleost fishes (Shapiro 1984), some of aquaculture interest, such as the sea breams (family Sparidae, Buxton and Garrett 1990) and the groupers (family Serranidae, Fischer and Peterson 1987). It is critically important to determine whether or not a particular GMO within such families is hermaphroditic. Sequentially hermaphroditic fish may be protoandrous or protogynous (Sadovy and Shapiro 1987, Debas et al. 1990). In some sequential hermaphrodites, sex change results from social factors (Fricke and Fricke 1977, Shapiro 1984, Dawley 1989, Sunobe and Nakozono 1993).

Molluscs and Crustaceans
Among the 5600 known mollusc genera, 40% are either simultaneous or sequential hermaphrodites, including 9% of bivalves (Heller 1993). In bivalves, many types of hermaphroditism have been found (Peralta 1988). Hermaphroditism in bivalves has been thought to be limited to small, brooding species. In addition, those hermaphroditic species which spawn eggs for external fertilization are thought to self-fertilize indavertently, or with less fit progeny resulting. However, selfed larvae of an aquacultural species, the bay scallop Argopecten irradians, have been found to grow as well as outcrossed larval cultures (Wilbur and Gaffney 1991). Increased incidence of hermaphroditism in triploid oysters has been documented through histological studies, though it is uncertain if such hermaphrodites yield functional gametes (Allen and Downing 1990).

Parthenogenesis is known to occur in strains of aquacultural crustacean species, such as Artemia (brine shrimp) (Triantaphyllidis et al. 1993) and Daphnia spp. (Hebert et al. 1993). These species are cultured and marketed to provide live foods for some aquacultural fish species. Results of a study of the freshwater crustacean, Candonocypris novozelandiae suggest that a sexual strain was displaced by a parthenogenetic strain in a disrupted habitat (Chaplin 1993). Two hermaphroditic individuals of the marine shrimp species (Penaeus vannamei) were found among broodstock on a shrimp farm and were suggested to be hermaphroditic as a result of environmental conditions encountered in captivity. (Perez-Farfante and Robertson 1992).

III. Risk Management Guidance: Non-Dioecious GMOs of High Concern

Compared to cross-fertilizing GMOs, organisms which reproduce by selfing can be particularly useful research organisms because of reduced variation in the genotypes of their progeny. However, accidental escapes of such organisms pose particularly high risks. Accidental escape of a single GMO individual with either reproductive mode could result in the establishment of an entire population of GMO descendants. Following the precautionary principle, this high potential for population establishment demands that research with these types of GMOs employ the most stringent level of confinement possible.

Researchers using GMOs which are self-fertilizing hermaphrodites or true parthenogens are directed to Risk Management to manage for no/negligible accidental escapees. If one of the biological barriers used is induced sterility, permanence of sterility is essential.

IV. Clarification of Terms for Other Non-Dioecious GMOs
Researchers using other non-dioecious GMOs may proceed through the Flowcharts using the following clarifications as a guide to applying the questions to their research. The appropriate outcome will be based on responses to the questions, as modified with the clarifications below.

Interbreeding and Hybridization.
In the case of parthenogenic GMOs, questions about "interbreeding" or "hybridization" refer to the presence of species in the accessible ecosystem with which the parthenogenic GMO can interact and reproduce. Specifically, it refers to the presence of species in which males have the capability to initiate embryogenesis in the parthenogen, regardless of whether or not the males actually contribute DNA to progeny. In the case of hermaphroditic GMOs, "interbreeding" and "hybridization" refer to the potential for cross-fertilization of these GMOs with other hermaphroditic individuals, or with any other species.

Immediate potential for introgression.
For parthenogens, the immediate potential for introgression and consequent effects are generally not a concern. Instead, the primary concern is the potential establishment of a viable GMO population and any direct or indirect adverse effects on other organisms or ecosystem structure and processes. Hermaphroditic GMOs with potential for cross-fertilization do pose concern of immediate potential for introgression.

Interspecific hybrid involving Parthenogenic GMOs and Introgressive Hybridization.
Two cases of parthenogenic "hybridization" are presently known to exist. In one case, the male contributes DNA and sperm (hybridogenesis in Poeciliopsis). In the second case, the sperm usually only triggers embryogenesis (gynogenesis in Poecilia); Schartl et al. (1995) documented an exception where males of another species contributed microchromosomes to offspring of matings with the all- female P. formosa. In either case, the progeny should be considered interspecifc hybrids for the purpose of these Flowcharts. In either case, if the males are a protected species, reproductive competition whereby too many males mate with parthenogenic GMO females of another species rather than with females of their own species is a greater risk than is introgressive hybridization.

Reproductively mature.
Identification of reproductively mature individuals among sequential hermaphrodites should be considered carefully. Consult experts for advice on age at which individuals should be examined and proper methods to determine maturity.

Abiotic factors of accessible ecosystem.
For non-dioecious GMOs, in addition to considering whether or not abiotic factors might preclude reproduction, the possibility that abiotic factors might induce reproduction should also be examined.

In the case of GMOs which are thought to be dioecious, the potential for abiotic factors to trigger non-dioecious reproduction should also be considered, with consultation from experts. In one study, environmental factors in captive rearing conditions of an outdoor shrimp farm was suspected to have triggered a low incidence of hermaphroditism in the farmed population (Perez-Farfante and Roberston 1992).