Masami Wakahara

A TADPOLE-INDUCED POLYPHENISM IN THE SALAMANDER HYNOBIUS RETARDATUS

Abstract.— Larvae of the salamander Hynobius retardatus have two distinct morphs: normal and broad‐headed, cannibal morphs. We performed three experiments to differentiate among the following hypotheses: The broad‐headed morph is induced to allow: (1) feeding on nutritious conspecifics; (2) exclusion of strong competitors for food or space; or (3) feeding on large, tough prey when smaller prey items are unavailable. When newly hatched larvae were reared with a heterospecific, Rana pirica (an anuran amphibian) tadpoles, the broad‐headed morph was induced more frequently compared with those reared with conspecifics. The phenotype expressed depended on the size of the tadpoles: The broad‐headed morph occurred more frequently with small and the normal morph with large tadpoles. Metamorphosis occurred sooner in larvae fed conspecifics compared with those fed heterospecific tadpoles, and the mean growth rate of larvae fed conspecifics was significantly faster than that of those fed tadpoles, suggesting that the heterospecific tadpoles were less nutritive than the conspecifics. These results do not support the hypotheses that the broad‐headed morph evolved for consuming conspecifics because of their better balance of nutrients or for excluding strong competitors for food or space. We tentatively conclude that the morph evolved to eat large, tough prey, including both conspecifics and heterospecific tadpoles. Because H. retardatus usually spawns very early in the spring in small ponds partially covered with ice and snow, newly hatched larvae may starve from the lack of proper food owing to extremely low water temperatures. Thus, the broad‐headed morph of H. retardatus may represent a cold‐habitat adaptation to overcome the severe circumstance when the only food items available are relatively large conspecifics or heterospecific tadpoles.

Heterochrony and Neotenic Salamanders: Possible Clues for Understanding the Animal Development and Evolution

Abstract A synthesis of developmental genetics with evolutionary genetics is now making possible to understand significant evolutionary changes in multicellular organisms. The key concept for unifying the two must be heterochrony. Heterochrony causes evolutionary modifications due to changes in timing and/or rate of development. The heterochrony is conventionally categorized into three patterns as neoteny (retardation in somatic development), progenesis (acceleration in gonadal development), and direct development (acceleration in somatic development, resulting in lack of larval or tadpole stages). A lot of species showing neoteny are known in urodeles, but not in anurans. Neotenic urodeles are also divided into three categories; permanent or obligate, “inducible” obligate, and facultative neotenies. Hynobius retardatus, a specific population of which had been reported to show neoteny but is believed to be extinct at present, has become to be used for experimental analysis of heterochronic expression of several adult characters during its ontogeny. Gonadal maturation and a transition of globin subunits from larval to adult types have been shown to occur independently on the morphological metamorphosis in H. retardatus. Mechanisms underlying the heterochrony, including morphogenetic clock, heterochronic genes in Drosophila and C. elegans, temporal colinearity in Hox gene complex in mice, and atavistic transformation induced by altered expression of Hox genes are discussed in terms of current molecular biology.

Spermatogenesis is extraordinarily accelerated in metamorphosis-arrested larvae of a salamander,Hynobius retardatus

Laboratory experiments were conducted to induce neoteny inHynobius retardatus, which had been reported to propagate in larval forms like axolotl. A large number of newly hatched larvae were reared in an aqueous solution of thiourea (TU) and sodium perchlorate (SPC) in order to arrest the metamorphosis. Gonadal development in the metamophosis-arrested larvae was compared with that in normally metamorphosing and metamorphosed controls. Metamorphosis-arrested male larvae produced morphologically mature spermatozoa approximately 4 months after hatching, when the gonads in the controls began to differentiate into testes, or to show the premeiotic proliferation of germ cells. Possible endocrine controls of these phenomena are discussed.

Maternal effects on phenotypic plasticity in larvae of the salamander Hynobius retardatus

Maternal effects are widespread and influence a variety of traits, for example, life history strategies, mate choice, and capacity to avoid predation. Therefore, maternal effects may also influence phenotypic plasticity of offspring, but few studies have addressed the relationship between maternal effects and phenotypic plasticity of offspring. We examined the relationship between a maternally influenced trait (egg size) and the phenotypic plasticity of the induction rate of the broad-headed morph in the salamander Hynobius retardatus. The relationship between egg size and the induction of the broad-headed morph was tested across experimental crowding conditions (densities of low conspecifics, high conspecifics, and high heterospecific anuran), using eggs and larvae from eight natural populations with different larval densities of conspecifics and heterospecifics. The broad-headed morph has a large mouth that enables it to consume either conspecifics or heterospecifics, and this ability gives survival advantages over the normal morph. We have determined that there is phenotypic plasticity in development, as shown by an increase in the frequency of broad-headed morph in response to an increase in the density of conspecifics and heterospecifics. This reaction norm differed between populations. We also determined that the frequency of the broad-headed morph is affected by egg size in which larger egg size resulted in expression of the broad-headed morph. Furthermore, we determined that selection acting on the propensity to develop the broad-headed morph has produced a change in egg size. Lastly, we found that an increase in egg size alters the reaction norm to favor development of the broad-headed morph. For example, an equal change in experimental density produces a greater change in the frequency of the broad-headed morph in larvae developing from large eggs than it does in larvae developing from small eggs. Population differences in plasticity might be the results of differences in egg size between populations, which is caused by the adaptive integration of the plasticity and egg size. Phenotypic plasticity can not evolve independently of maternal effects.

Heterochronic Expression of Several Adult Phenotypes in Normally Metamorphosing and Metamorphosis-Arrested Larvae of a Salamander Hynobius retardatus

Abstract Heterochronic expressions of several larval and adult phenotypes in Hynobius retardatus, which had been reported to show neotenic reproduction, were described in normally metamorphosing animals (controls) and goitrogen-induced, metamorphosis-arrested larvae. External gills, dorsal tailfins and Leydigs cells in the epidermis were completely diminished in the controls during and after metamorphosis, but fully remained in the metamorphosis-arrested larvae. Two types of dermal glands, mucous and serous glands, however, behaved differently from Leydigs cells, even though they constituted the same skin. The dermal gland cells in the controls appeared at a premetamorphic stage, gradually increased in number during the metamorphosis and fully developed after the metamorphosis. Those in metamorphosis-arrested larvae appeared much later than in the controls. Thus, aged, metamorphosis-arrested larvae had skin which consisted of larval type epidermis (Leydigs cells) and adult type dermis (mucous and serous glands). Contrary to these, a transition of globin subunits from larval to adult types occurred practically on the same time schedule in both the controls and metamorphosis-arrested larvae. These observations suggest that there are at least three types of organs or cells which behave differently during and after metamorphosis in Hynobius retardatus.

Spatio-temporal expression of a DAZ-like gene in the Japanese newt Cynops pyrrhogaster that has no germ plasm

To investigate the germ cell specification in urodeles, we cloned a DAZ-like sequence from the Japanese newt Cynops pyrrhogaster, Cydazl, and raised antibodies specific to Cydazl. Cydazl is a homologue of the human DAZ (deleted in azoospermia), DAZL, and Xenopus dazl genes, which are involved in gametogenesis or germ cell specification. During gametogenesis, expression of Cydazl mRNA and Cydazl protein was detected at first in the small previtellogenic oocytes in females but was not localized as seen in Xenopus and was restricted to secondary spermatogonia prior to meiosis in males. During early embryogenesis, maternal stores of the Cydazl transcript and protein were present in the entire embryos, not localized in any specific region. The zygotic expression was detected in hatching larvae (stage 50) by RT-PCR analysis whereas specific cells expressing Cydazl could not be determined by in situ hybridization at this stage. Strong expression of Cydazl and Cydazl were detected in primordial germ cells (PGCs) that had entered the gonadal rudiment at late stage 59. These results suggest that Cydazl does not function early in development, for the specification of germ cells, but functions later for differentiation of germ cells in the developing gonads during embryogenesis and for meiotic regulation, supporting the previous idea of an intermediate germ cell formation mode in urodeles.

Specification and Establishment of Dorsal‐Ventral Polarity in Eggs and Embryos of Xenopus laevis

Dorsal‐ventral (D‐V) polarization in Xenopus eggs and embryos is achieved by passing through a series of complicated phenomena such as initial specification of the polarity before first cleavage, establishment of polarity during cleavage stages resulting in an acquisition of a unique developmental capacity by each blastomere, regional differentiation of mesoderm, and finally neural induction by Spemanns organizer. In order to gain an insight into basic mechanisms which govern D‐V polarization, experimental modifications or perturbations of the body axis of embryos, including physical or chemical treatments of eggs, altered orientation of eggs under the normal gravity, centrifugation, manipulation of blastomeres, cytoplasmic withdrawal, and bisection or partial ligation of fertilized eggs are reviewed: all data are consistent with the concept that a cytoplasmic activity which becomes localized in the dorsal side of the egg is responsible or indispensable for the establishment of the D‐V axis. The cytoplasmic activity is tentatively called “anterodorsal structure‐forming activity.” A model which explains the specification, establishment, and realization of D‐V polarity in Xenopus laevis is proposed.

Mechanical vibrations from tadpoles' flapping tails transform salamander's carnivorous morphology

Some prey or predator organisms exhibit striking rapid morphological plastic changes with distinct morphology under the condition of predator or prey presence. Remote chemicals propagating from the inducing agents are the prevalent induction cues for most examples of induction of distinct morphs. Sonic and visual cues, as well as chemical cues, are known as triggers for induction of behavioural plasticity. Here we show that hydraulic vibration originating from flapping tails of anuran tadpoles is a key cue in relation to induction of a distinct carnivorous morphology, a broad-headed morph, in larval salamander Hynobius retardatus, which is able to efficiently capture and handle prey. This result was further supported by the fact that simple mechanical vibrations of tail-like vinyl fins were able to induce the morph without any biological cues. Induction of the morph triggered by hydraulic vibration provides a novel concept for understanding the proximate mechanisms of induction of morphological changes.

Adaptable Larval Life Histories in Different Populations of the Salamander, Hynobius retardatus, Living in Various Habitats

Abstract Several variations in larval life histories were described in a salamander Hynobius retardatus living in Hokkaido, Japan, which had been reported to propagate in larval forms in a specific environment of Lake Kuttara like the axolotl. In almost all populations living in lower land Hokkaido, spawning was observed in early spring, and hatched larvae metamorphosed by August or September. In some populations living in the similar ponds but supplied with a mountain stream or spring water, however, larvae could not complete their metamorphosis by late autumn in the first year. All the larvae passed winter season under snow and then metamorphosed between late May and mid June in the second year. In some specific populations in cold, mountainous ponds, larvae could not metamorphose during the first and second years and metamorphosed in the third year. Thus, three age-groups of larvae, which were 2-year- and 1-year-overwintered larvae, and larvae under the age of one, were concurrently observed in one pond. Body size at the completion of the metamorphosis in the 2-year-overwintered larvae was significantly larger than that in the metamorphosing or metamorphosed larvae under the age of one. When hemoglobin (Hb) transition from larval to adult types in each population was examined, adult globin subunits were expressed in the overwintered larvae, even though they had not completed their morphological metamorphosis, suggesting that the expression of some adult phenotypes was independent of morphological metamorphosis.

Modification of Dorsal-Ventral Polarity in Xenopus laevis Embryos Following Withdrawal of Egg Contents before First Cleavage

When fertilized Xenopus laevis eggs were pricked just beneath the marginal zone with a thick glass needle prior to the first cleavage, a small amount of cytoplasm escaped into the exudate. Those eggs were placed in a poly L‐lysine‐coated plastic dish filled with 10% Ficoll solution. The location of the sperm entrance site (SES) of each egg was marked by scratching the surface of the plastic dish. The pricked embryos were anchored to the dish through poly L‐lysine, and developed, therefore, without changing their original position. Consequently, development of the dorsalventral polarity was conveniently monitored with respect to the location of the SES. Embryos which developed from eggs pricked on the side opposite the SES showed modification of the dorsal‐ventral polarity: Semi‐quantitative studies showed that an exudation approximately 1.5–12.5% of the whole egg contents from the presumptive dorsal side caused a reversal of the dorsal‐ventral polarity. That is, the dorsal lip of the blastopore formed on the same side of the SES, whereas the dorsal lip formed on the side opposite the SES in the normal control and sham‐operated embryos. Half of the embryos which had larger cytoplasmic exudates more than 12.5% of the whole egg contents failed to form the dorsal lip by the time all controls and the embryos with smaller exudates showed normal dorsal lip formation. When eggs were pricked on the SES side, the normal topographic relationship between the SES and future dorsal lip side was reinforced.