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Department of Biology, University of Victoria, Victoria, British Columbia V8W 3N5, Canada; and Discipline of Anatomy and Histology, F13, University of Sydney, Sydney, NSW 2006, Australia
E-mail: lpage{at}uvic.ca
Cases where early development has changed substantially and rapidly, without altering later morphogenesis, can be informative about developmental organization and adaptation. I describe a surprising novelty of early embryogenesis in the arminacean nudibranch Madrella sanguinea from the southeastern coast of Australia. Fertilized eggs of this species segregated a large mass of red-pigmented cytoplasm to the vegetal pole, where much of it was extruded as a type of polar lobe during the first two cleavages. A packet of red material detached from the embryo before the gastrula stage, and these "red bodies" were abandoned when larvae hatched. Segregation of highly pigmented ooplasm and formation of a polar lobe are both unusual among opisthobranch gastropods. Discarding a portion of egg cytoplasm is extremely unorthodox among all animals. Nevertheless, hatched larvae of M. sanguinea underwent typical development for a planktotrophic nudibranch larva, and metamorphosis to a feeding juvenile was induced by the bryozoan prey of the benthic stage. The hypothesis that the red bodies contain a defensive allomone that is provisioned to eggs by the parent requires future testing.
The vital role of egg cytoplasm during early development is well illustrated by the many cases in which maternally derived morphogenetic determinants are essential for embryonic patterning. The determinants become differentially segregated within the ooplasm so that they are distributed into different blastomeres during embryonic cleavages (123). These determinants subsequently specify the phenotype of individual blastomeres, without input from neighboring cells. Current understanding of ooplasmic segregation and cell-autonomous specification of embryonic cell fate was greatly facilitated by early embryological studies on ascidians, where qualitative differences in regions of egg cytoplasm can be visualized as distinctive patterns of ooplasmic pigment that change after fertilization (3).
Among the spirally cleaving eggs of molluscs, embryonic patterning often results from a combination of regionally segregated morphogenetic determinants and inductive interactions between cells. For example, patellogastropods (true limpets) and euthyneuran gastropods (pulmonates and opisthobranchs) with equally cleaving eggs depend on regional segregation of morphogenetic determinants to specify the animal-vegetal axis (4), but an inductive interaction between micromeres and one of the macromeres later specifies the dorsoventral axis of the embryo (5,6). Among caenogastropods, ooplasmic segregation of morphogenetic determinants is important for specifying both these axial coordinates. Egg cytoplasm that will be uniquely distributed to the dorsal quadrant of caenogastropod embryos is transiently extruded as a polar lobe during early cleavages (1,5,6). A polar lobe has never been reported for eggs of patellogastropods, vetigastropods, or euthyneurans (6).
In addition to morphogenetic determinants for embryonic patterning, parents also package nutrients into eggs to provide energy for embryogenesis. Furthermore, recent research has revealed yet another category of maternally derived egg inclusions that has likely resulted from challenges to survival in a hostile external environment. This category includes sunscreens (7), photosynthetic symbionts (8), and secondary metabolites to protect from pathogens or predators (9).
Opisthobranch gastropods, particularly nudibranchs, contain many novel secondary metabolites that have a demonstrated or suspected role as feeding deterrents (101112). At least one species of nudibranch incorporates a defensive allomone into its benthic spawn mass (12), although the location within eggs or extraembryonic investments is unknown. I report here a type of early embryonic development that is very unusual for a nudibranch gastropod: it involves segregation of intensely pigmented egg cytoplasm into a type of polar lobe and eventual expulsion of this cytoplasm from the embryo proper. I hypothesize that the novelty may stem from parental provisioning of a defensive chemical to eggs.
Madrella sanguinea (Angas, 1864) is a small (15 mm) arminacean nudibranch that occurs along the eastern and southern coasts of Australia, including Tasmania (13). Both the nudibranch (Fig. 1A) and its obligate bryozoan prey, Mucropetraliella ellerii (MacGillivray, 1869) are a deep red-orange color (14). An adult of this species was collected from the rocky reef at the north end of Balmoral Bay in Sydney Harbour, Australia. Three egg masses deposited in the laboratory had a spiral configuration and consisted of a convoluted string of egg capsules in gelatinous material. Each capsule contained a single fertilized egg with a mean diameter of 74.5 µm (n = 15), only slightly less than the 78 µm reported previously for specimens collected just north of Sydney Harbour (15). In both cases, the small eggs developed into planktotrophic larvae. However, crawl-away juveniles emerging from egg capsules have been reported for a more southern population of Madrella sanguinea (14). Additional work is needed to determine if M. sanguinea, as currently described, incorporates a cryptic species pair or if poecilogeny occurs within a single species (14).
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During second cleavage, the macromere with most of the red cytoplasm failed to separate completely at its vegetal pole (Fig. 1E). A cytoplasmic bridge containing concentrated red pigment extended between the two sister macromeres, and this soon protruded prominently as a sort of polar lobe (Fig. 1F). At this point, each of the four macromeres, exclusive of the polar lobe, retained at least some red cytoplasm, but in differing quantity. Most pigment was within a macromere adjacent to the vegetal cross-furrow, while pigment within the other three macromeres progressively diminished in a counterclockwise direction, as viewed from the vegetal pole (Fig. 1F).
The unusual polar lobe generated at second cleavage did not re-incorporate into the macromeres. It remained attached to the vegetal pole of the embryo as a spherical packet of red-colored cytoplasm ("red body") at least through the period when micromere quartets were generated (Fig. 1G). The red body separated completely from the embryo by the gastrula stage. Red pigment remaining within the gastrula was concentrated at two internal foci (Fig. 1H) and was later localized within the right and left digestive glands as the larval body differentiated (Fig. 1I). Planktotrophic veliger larvae hatched after 6.5 days at 17–18 °C, at which time the red bodies were abandoned within the collapsed egg mass (Fig. 1J).
Several aspects of embryogenesis by M. sanguinea from Sydney Harbour are unusual relative to other opisthobranchs. Many opisthobranchs have pigmented eggs (e.g., 12, 16), but segregation of pigmented from nonpigmented ooplasm has not, to my knowledge, been reported. Although some scaphopods, bivalves, and gastropods extrude a polar lobe during early cleavages, opisthobranchs do not. Nevertheless, asymmetric early cleavages have been reported for several opisthobranchs (5). One of these, the anaspidean Phyllaplysia taylori, is interesting because each egg capsule includes a viable egg and a so-called nutritive body, which is ingested by the developing snail prior to hatching (17). Loss of cytoplasm from eggs, as occurs in M. sanguinea, is highly unorthodox among metazoans. However, Gibson (18) reported that 18% of Pleurobranchea maculata embryos blebbed-off fragments that were ingested by older embryos. Larvae of M. sanguinea did not ingest their red bodies.
I cultured larvae of M. sanguinea through metamorphosis to confirm that events observed during embryogenesis were normal. Larval morphology at hatching and during later development was typical for planktotrophic nudibranch veligers (16,19). Notable events included development of eyespots, retraction of the mantle fold, and development of a propodium that allowed crawling beginning at 15 days post-hatching (Fig. 1K). Metamorphosis was induced by Mucropetraliella ellerii, and juveniles began feeding on this bryozoan within a day of metamorphic shell loss. Red pigment appeared initially within the digestive gland of feeding juveniles (Fig. 1L) and spread to epidermal tissue within a week of metamorphosis.
Materials manufactured by organisms and placed within their eggs or egg investments carry an energetic cost that should translate into reduced fecundity. Since the red bodies sequestered from eggs of M. sanguinea represent a substantial proportion of total egg volume, the material may enhance fitness in a trade-off against fecundity. However, the benefit of this material is perplexing because embryos segregate and discard much of it during early embryogenesis and larvae abandon the red bodies when they enter the pelagic realm. The red material is probably not a sunscreen, because such a role would be better performed by material that remained distributed throughout the embryo rather than becoming sequestered into an extraembryonic mass and two restricted internal sites. It is also unlikely that the primary role is to make eggs cryptic when deposited on the red bryozoan, because the close color match is disrupted when the red pigment is sequestered into small packets separate from the embryos. However, in light of the presence of defensive allomone in the egg mass of another nudibranch species (12), I suggest that the red-pigmented cytoplasm may incorporate a predator deterrent. Unlike sunscreens or camouflaging pigment, a defensive allomone could be advantageous without remaining within embryos. If a predator punctured an egg capsule, it is unlikely that the delicate embryo inside would survive the attack, regardless of where the allomone was located. However, if the predator was repelled, then the other 1000–3000 embryos within an individual egg mass (15), all of them siblings, would survive. Kin selection could therefore account for selection of this trait.
Regardless of the function of the red-pigmented material within eggs of M. sanguinea, its intense color allows clear visualization of its segregation and distribution among macromeres. The red ooplasm began streaming toward the vegetal pole soon after release of the 1st polar body, which is consistent with results of experimental work showing that morphogenetic determinants within equally cleaving gastropod eggs become segregated along the animal-vegetal axis between release of the 1st and 2nd polar bodies or between the 2nd polar body release and 1st cleavage (4). However, unequal distribution of red cytoplasm within the four macromeres resulting from second cleavage is not consistent with the notion that the initial four macromeres of equally cleaving gastropod eggs are identical prior to later induction of the dorsoventral axis by micromeres. This non-identity among macromeres may not affect which of the macromeres later becomes the D macromere, but it may result in some subtle but unknown phenotypic difference among the resulting larvae. Unequal distribution of ooplasm among macromeres in M. sanguinea may be yet another novelty of early embryogenesis in this species. Alternatively, it may be a case where a vivid color marker of egg cytoplasm, as occurs in some ascidian eggs (3), has revealed a phenomenon that has not yet been resolved through experimental manipulations on equally cleaving eggs of gastropods.
Embryogenesis of M. sanguinea adds to the growing list of known cases in which early development has been substantially altered without affecting final body form (20). Indeed, the capacity of young embryos to tolerate at least some perturbations may have allowed parents to provision eggs with assorted materials to promote offspring survival. The hypothesis that the red-pigmented cytoplasm within eggs of M. sanguinea incorporates a predator deterrent could be tested by isolation and analysis of chemicals within abandoned red bodies, together with predator feeding experiments using the chemical isolates.
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