Marsupial extension in terrestrial isopods ( Crustacea , Isopoda , Oniscidea )

In Oniscidea, the marsupium is a ventral pouch where the offspring develop independently of an external water source. The marsupium is formed by five pairs of overlapping oostegites that develop in the females during their reproductive period. In this study, ovigerous females of 35 species were dissected, their oostegites were extracted, and the intra-marsupial offspring were counted. Two marsupium forms were recognized: distended, in which the oostegites protrude distally in relation to the sternites; and non-distended, in which the oostegites are parallel to the sixth and seventh sternites. Armadillidium nasatum, A. vulgare, Pudeoniscus birabeni, Circoniscus gaigei and Cubaris murina, conglobating species with a non-distended marsupium, and Neotroponiscus daguerri and N. carolii, non-conglobating species with a distended marsupium, have a concavity on the ventral floor of the 6th and 7th pereionites, here called the marsupial extension. This is the first record of a marsupial extension which extends beyond the area formed by the oostegites in Oniscidea.


Introduction
Among the several adaptations that have allowed isopods to occupy terrestrial environments is the marsupium, an incubator pouch that allows the eggs to develop independently of an external source of water (Hoese, 1984).In Oniscidea, there are two types of marsupium (Hoese, 1984).The amphibious marsupium, found in Ligia spp., is open anteriorly and posteriorly.It of a thin translucent and impermeable cuticle (Patane, 1940;Hoese, 1984).Both the constitution and the way that the oostegites are organized and linked allow the expansion of the marsupial space to accommodate the offspring.In Crinocheta, the cotyledons are responsible for nutrition and oxygenation of the offspring inside the marsupium (Akahira, 1956;Hoese and Janssen, 1989).During comparative studies on morphological patterns of the cotyledons in several species of terrestrial isopods, a new marsupial configuration was observed, here called a marsupial extension.This study aimed to: (i) identify which species among those studied possess this marsupial extension, and (ii) to count the number of individuals housed inside this cavity.

Materials and Methods
Ovigerous females of 35 species from 11 families of terrestrial isopods, from the Collection of Crustaceans of the Zoology Department -UFRGS and from donations and collections, were analyzed.The females were fixed in Dubosc fixative (Humason, 1972), and were dissected and photographed before and after the removal of the oostegites and of the offspring contained in the marsupium.The offspring were counted and classified according to their stage of development, following Milatovič et al. (2010): egg with undifferentiated embryo (S1 to S8), differentiated embryo (S9 to S18, when the appendix, eyes and segmentation are visible in the vitelline membrane), and manca (S19, when the isopod is already free of the membrane).

Results and Discussion
Among the 35 terrestrial isopod species examined, seven species possessed the marsupial extension (Table I).The marsupial extension is a concavity on the sternites of the 6th and 7th pereionites.This concavity starts from the fifth sternite, where the last pair of oostegites closes the marsupium (Figs.1C,   1D).Inside this cavity there is a portion of the developing offspring, which are in the same developmental stage as the rest of the offspring in the marsupium.The proportion of the offspring in the extension is very significant, usually more than 10% and up to 25% of the total fecundity (Table II).For example, one female Armadillidium nasatum Budde-Lund, 1885 had 116 eggs in the marsupium and 23 eggs in the marsupial extension (Table II).The number of offspring inside the extension seems to be proportional to the total fecundity, which is in turn related to the female size (Quadros et al., 2009).
Among the species that have the marsupial extension, a different marsupial form was also noted.The most common marsupial form in Oniscidea is a distended type, i.e., in which the oostegites protrude distally in relation to the sternites (Figs.1A, 2A).Among the species examined, 30 have the distended marsupium (Table 1).On the other hand, the conglobating species A. nasatum, A. vulgare (Latreille, 1804), Circoniscus gaigei Pearse, 1979, Cubaris murina Brandt, 1833 and Pudeoniscus birabeni Vandel, 1963 have a nondistended marsupium, where the oostegites are parallel to the sixth and seventh sternites (Figs.1B; 2B; Table 1).A comparison of the internal space of the marsupia revealed that in the distended marsupium, the oostegites bend outwards when the growing embryos require more volume (Fig. 1A).However, in those species with a marsupial extension, the oostegites bend only slightly, and the sternites arch into the body cavity to provide more space for the embryos (Fig. 1B).This leads to a displacement of the female's internal organs (Fig. 1B).Possibly, this displacement results in compression of the organs, which in advanced stages may cause females to cease feeding.In fact, all females with a marsupial extension and non-distended marsupium examined showed a full gut only when they were carrying eggs with undifferentiated embryos (Fig. 2E).Females carrying embryos and mancas, when dissected, showed an empty gut, with no trace of food (Fig. 2F).Females with a distended marsupium, regardless of the intra-marsupial Table I.Configuration of the marsupium in terrestrial isopods and the presence of a marsupial extension and the conglobating ability.N = number of ovigerous females examined.
Another important point is that in species belonging to the ecomorphological category of the "rollers" (Schmalfuss, 1984), the lack of a large distention of the marsupium allows ovigerous females to conglobate.However, it is noteworthy that among the species that have a marsupial extension, two, Neotroponiscus carolii Arcangeli, 1936 and N. daguerrii (Giambiagi de Calabrese, 1939), are nonconglobating species.Information on the presence or absence of the marsupial extension in species in families and genera that were not considered in this study is necessary to understand the evolution of the conglobating ability, which is considered a convergence that evolved within the Tylidae, once in Synocheta, and several times within the Crinocheta (Schmidt, 2002).The marsupial form must   Table II.The distribution of the offspring in terrestrial isopods that have a marsupial extension.Fecundity is given as the total number of offspring per female and the number of offspring inside the marsupial extension (within parenthesis).The letters indicate the developmental stage: eg = egg with undifferentiated embryo; em = differentiated embryo; m = manca (see Material & Methods for details).also be considered in future studies on the phylogeny within Oniscidea.To date, no similar structure has been found in Peracarida and for a better understanding of the structure of this extension structure and its implications for the life history of these species, further studies are needed.