The ultrastructure of book lung development in the bark scorpion Centruroides gracilis (Scorpiones: Buthidae)

Frontiers in Zoology The ultrastructure of book lung development in the bark scorpion Centruroides gracilis (Scorpiones: Buthidae) Roger D Farley 0 0 Department of Biology, University of California , Riverside, California, 92521 , USA Background: Near the end of the nineteenth century the hypothesis was presented for the homology of book lungs in arachnids and book gills in the horseshoe crab. Early studies with the light microscope showed that book gill lamellae are formed by outgrowth and possibly some invagination (infolding) of hypodermis (epithelium) from the posterior surface of opisthosomal limb buds. Scorpion book lungs are formed near the bilateral sites of earlier limb buds. Hypodermal invaginations in the ventral opisthosoma result in spiracles and sac-like cavities (atria). In early histological sections of embryo book lungs, widening of the atrial entrance of some lamellae (air channels, air sacs, saccules) was interpreted as an indication of invagination as hypothesized for book gill lamellae. The hypodermal infolding was thought to produce the many rows of lamellar precursor cells anterior to the atrium. The ultrastructure of scorpion book lung development is compared herein with earlier investigations of book gill formation. Results: In scorpion embryos, there is ingression (inward migration) of atrial hypodermal cells rather than invagination or infolding of the atrial hypodermal layer. The ingressing cells proliferate and align in rows anterior to the atrium. Their apical-basal polarity results in primordial air channels among double rows of cells. The cuticular walls of the air channels are produced by secretion from the apical surfaces of the aligned cells. Since the precursor cells are in rows, their secreted product is also in rows (i.e., primordial air channels, saccules). For each double row of cells, their opposed basal surfaces are gradually separated by a hemolymph channel of increasing width. Conclusions: The results from this and earlier studies show there are differences and similarities in the formation of book lung and book gill lamellae. The homology hypothesis for these respiratory organs is thus supported or not supported depending on which developmental features are emphasized. For both organs, when the epithelial cells are in position, their apical-basal polarity results in alternate page-like channels of hemolymph and air or water with outward directed hemolymph saccules for book gills and inward directed air saccules for book lungs. - Background At the end of the ninteenth century and in the early twentieth century numerous papers were published comparing the development of book gills in the horseshoe crab with the development of book lungs in arachnids, especially spiders. As reviewed by Farley [1], this work was done with the hypothesis that these respiratory structures are homologous, e.g., the internal book lungs in the opisthosoma were derived by insinking of the anlage that had previously resulted in external book gills of an aquatic ancestor. There have been diagrams and much discussion about how an ancient ancestor with lamellate gills like extant horseshoe crabs could have given rise to arachnid book lungs [2-12]. Recent investigations of horseshoe crab, scorpion and spider embryos report similar patterns of gene expression at the bilateral opisthosomal sites where book gills or book lungs eventually form [13-17]. In early studies with the light microscope and histological sections, the air sacs (air channels, lamellae, saccules) of developing spider and scorpion book lungs were suggested to be infoldings of the hypodermis from the spiracular invagination (primordial atrium) posterior to opisthosomal limb buds. This process was thought to be similar to the small amount of invagination that may occur along with outgrowth folds for book gill development at the posterior surface of branchial appendages in horseshoe crabs [2-10,18-22]. Slight widening of the air sac entrance at the atrial wall was interpreted as indications of hypodermal infolding. The presumed infoldings were thought to result in the parallel rows of lamellar precursor cells anterior to the atrium. In the spider species they examined, Montgomery [23] and Janeck [24] reported that the initial widenings of the air sac entrance are transitory, and the air sacs are formed from aligned cells in a cluster derived from the hypodermis. More recently for the spider Cupiennius salei [25], the segment polarity gene engrailed-1 is reported to be expressed as five stripes at the site where lamellae originate posterior to the second opisthosomal limb buds. The primordial site is invaginated and covered ventrally by the posterior folding and flattening of the preceding limb bud, as reported in earlier histological studies [8,23]. Also in C. salei, the developmental gene pdm/ nubbin is expressed in a striped pattern possibly related to lamellar formation [15]. In his diagram of histological sections of scorpion embryos, Brauer [19] showed some small folds in the atrial wall. This was considered as evidence of hypodermal invagination like that proposed for book gills [17,22,26] although the presumptive folds were not actually shown to be related to the formation of book lung lamellae. As pointed out earlier [1], lamellate respiratory organs are important for our understanding of evolutionary history and taxonomic relationships, but modern procedures are needed for a more detailed comparison of cell activity during book gill and book lung development. The main objective herein is to use transmission electron microscopy (TEM) to examine cell ultrastructure during formation of scorpion book lungs. The results can then be used where relevant and helpful for evolutionary hypotheses and further comparative studies. The scanning electron microscope (SEM) was used in recent developmental investigations of the respiratory organs in the scorpion [27] and horseshoe crab [1]; the present investigation is a continuation of that effort. The SEM study of book lung development in scorpions [27] provides an overview of the process, but the SEM is limited in the resolution of cell detail. Also, tissue preparation requires dissection and/or fracturing to expose components for viewing. This has potential for cell damage and/or loss, with emphasis on the surface features of the tissue or organ. In the present study, whole book lungs were removed, and sections were cut at successive stages of development in embryos and first and second instars. Book lung formation in scorpions is a slow and gradual process [27]. It begins in the embryo with the appearance of a spiracle and a sac-like invagination (primordial atrium) just inside the spiracle. Lamellar development continues through birth and the first molt that occurs 1-2 weeks after the newborn first instars (pronymphs) climb up on the mothers back. The book lung gradually becomes a functional respiratory organ with about 50 lamella (...truncated)