Internal embryonic development in a non-copulatory, egg-laying teleost, the three-spined stickleback, Gasterosteus aculeatus

* Article.
* Open Access.
* Published: 20 February 2019.
* Laura L. Dean,
* Shaun Robertson,
* Muayad Mahmud &
* Andrew D. C. MacColl.

## **Abstract:**

The switch from egg-laying to retaining and giving birth to live young is a major transition in the history of life.

Despite its repeated evolution across the fishes, records of intermediate phenotypes are vanishingly rare, with only two known cases in existence of normally egg-laying fish species retaining embryos within the ovaries.

We report the discovery of a third occurrence, in which well-developed embryos were found in the ovaries of a three-spined stickleback (***Gasterosteus aculeatus***), a non-copulatory, normally oviparous species.

Extracted from the parent fish, these embryos hatched and grew to adulthood.

Genetic and physiological examination of the parent fish and offspring ruled out development by parthenogenesis and hermaphroditism, therefore implicating internal fertilisation by a male stickleback.

This extremely rare phenomenon may have been facilitated in this population by an unusual tendency for females to become egg-bound, and suggests that some major transitions may arise almost spontaneously.

## **Introduction:**

Major transitions, involving multifaceted and synergistic changes in multiple traits, pose an evolutionary conundrum because incomplete intermediate forms are unlikely to be functional1.

Consistent with this, partially developed major transitions are extremely rare2,3, and rightly celebrated when discovered4,5,6.

The transition from oviparity to retaining developing embryos internally and giving birth to live young involves multiple interrelated changes in physiology and behaviour and as such, has a good claim to being a major transition in the history of life7,8,9.

In most animal species, females reproduce oviparously, laying eggs that develop and hatch in the external environment7.

In many lineages, including teleost fish, some degree of embryo retention has evolved, ranging from ovoviviparity (*egg retention*) to matrotrophy (*placental nourishment and live birth*).

However, intraspecific variation in such traits is rare (but see Surget-Groba, *et al*.10), suggesting that they are under strong selection.

Despite the >35 independent evolutionary origins of ovoviviparity across fish lineages7, the huge number of extant egg-laying species (*~90% of bony and 43% of cartilaginous fish*), and the colossal number of these that must have been examined by fish biologists, records of normally oviparous fish retaining developing embryos are incredibly rare. There are only two previous records of this phenomenon.

Multiple specimens of ***Hemilepidotus gilberti*,** a marine sculpin, were discovered with eyed embryos in the ovaries of spent females, but all embryos died early in development or were deformed and would not have survived post-hatching11.

A single three-spined stickleback (***Gasterosteus aculeatus***, *Linnaeus, 1758, hereafter ‘stickleback’*) was also found with well-developed embryos retained in the ovaries12, but embryos were not assessed for deformities or signs of life. We document a second occurrence of this phenomenon in stickleback, in which embryos, once removed from the mother, hatched and grew to adulthood without physical abnormalities.

Stickleback are a non-copulatory, oviparous, sexually reproducing species, with a simple XY sex-determining system13.

Female stickleback are batch spawners14 and can become egg-bound (*with eggs overripening and hardening in the reproductive tract)* if they fail to either spawn or spontaneously release their eggs15,16.

Retained eggs are lost for reproduction and obstruct the release of further clutches, ultimately killing the female17,18.

Despite this, egg-bound females are common in some stickleback populations17,19,20.

The prolonged retention of overripe ovules is considered a non-adaptive ‘accident’16,18, but ovule retention is necessary for internal fertilisation, which is an essential step in the transition from oviparity to live-bearing8, and thus could be considered a preadaptation to this transition.

Preadaptations that arise as non-adaptive by-products of other adaptations, or initially evolve to serve a different function, may facilitate major transitions by subsequently becoming useful in a different role21,22,23,24.

We discovered an egg-bound marine stickleback in a saline coastal lagoon on North Uist, Scottish Western Isles with ovaries containing well-developed, eyed embryos.

This phenomenon has at least three possible explanations, which make distinct, testable predictions: (i) the ova developed by parthenogenesis i.e. by artificial stimulation by heat, chemical or physical shock, in the absence of sperm. Parthenogenesis is common in some animal species25, and gynogenesis (development in which the embryo contains only maternal chromosomes due to activation of an egg by a sperm that degenerates without fusing with the egg nucleus) occurs in some fish26. Gynogenesis has been stimulated artificially in stickleback27, but neither gynogenesis, nor true parthenogenesis have been recorded in natural stickleback populations. This hypothesis requires the parent fish to be genetically and physiologically female and the offspring to be genetically identical to the parent. (ii) Embryos were produced hermaphroditically. Synchronous hermaphroditism, whereby mature individuals possess both functional ovaries and tests, is common in certain teleost species, although all but a single species are outcrossing28. *Kryptolebias marmoratus*, a mangrove killifish, is the only known hermaphroditic teleost species that predominantly self-fertilises, and is capable of releasing both fertilised and unfertilised eggs29,30. Synchronous hermaphroditism can be chemically induced in stickleback31, and an apparently hermaphroditic stickleback population exists in Alaska, from which the single other individual carrying developing embryos was collected12, making hermaphroditism an apparently strong possibility. This requires that the parent fish possesses both male and female sex organs, and the offspring carry only alleles present in the parent. (iii) Finally, eggs could have been internally fertilised by the sperm of another stickleback entering the reproductive tract from the external environment, which was the case in *H. gilberti*11. This requires the parent fish to be genetically and physiologically female, and the offspring to carry alleles from the father that were absent in the mother. We examined the gonads of the parent fish and performed genetic sex testing, alongside analysis of the allelic composition of parent and offspring to test these hypotheses. To examine the possibility that the presence of the embryos is related to a tendency by fish in the same population to retain eggs, we compared the prevalence of egg-bound gravid females across stickleback populations on North Uist.

## Results

### Analysis of gonads

The parent fish possessed ovaries containing two clutches of eggs, the older (proximal to the cloaca) of which contained many developing embryos (Fig. 1A,B). Embryos were distributed throughout the older clutch and had reached stage 21 of 24 stages of embryonic development in stickleback32, Fig. 1C,D. Embryo viability was confirmed by the observation of a heartbeat, and no physical abnormalities, or deformities were observed. Three independent researchers agreed that the gonads of the parent fish contained no testicular tissue (Supplementary Fig. S1), and none was evident elsewhere in the body cavity. Once raised to adulthood, offspring were crossed successfully *in vitro*.