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The structural basis for water exchange between the female cockroach (Blattella germanica) and her ootheca

Donald E. Mullins^*, K. June Mullins and Keith R. Tignor

Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0319, USA

View larger version (35K): [in a new window]   Fig. 1. Microparabiotic chamber constructed from glass tubing, designed to allow for measurement of water-soluble materials across small pieces of oothecal cuticle. (A) Exploded view of the chamber. (B) Assembled view of the chamber. See Materials and methods section and Fig. 4 for details. Scale bar, 1 cm.

View larger version (141K): [in a new window]   Fig. 2. Scanning electron microscopy images of Blattella germanica oothecae. (A) Distal end of an ootheca. Scale bar, 0.25 mm. (B) Proximal end of an ootheca showing the `escutcheon-shaped' vaginal imprint (arrow). (C) Magnification of the ventro-lateral escutcheon region (arrow indicates the `pore-field' area). (D) Magnification of `pore-field' (arrow). (E-G) Increasing magnification of the pore field area shown in D (H,I) Pores (arrows) revealed on the internal surface of the oothecal covering after chorion removal using trypsin. The pore sizes are approximately 1-2 µm in diameter.

View larger version (81K): [in a new window]   Fig. 3. Confocal images of the oothecal pore-field area. These images were generated by optically scanning through a ventro—lateral oothecal section of the pore-field area (Fig. 2D-I). Prior to viewing, 1 µl of fluorescein dye (5 mg ml-1) was applied to the tissue preparation to show fluorescent contrast on the oothecal surfaces, enabling visualization of the pores (as represented by the black holes) through the oothecal matrix. These images were gathered at 0.26 µm intervals from the exterior to the interior surfaces of the oothecal covering. (A-L) The optical sections taken, beginning below the surface (A), through the matrix, to the field above the inside surface (L). Scale bars, 5 µm. (M-Q) Selected images obtained from projection of the confocal stack, which show the three-dimensional aspect of the covering. Scale bars, 2 µm.

View larger version (34K): [in a new window]   Fig. 4. Fluorescent dye used for detection of buffer solution leaks in microparabiotic chambers. 30 µl of buffer was delivered to the internal (left) side of the oothecal tissue, and 30 µl of buffer containing a radiolabeled material and fluorescein (50 µg ml-1) was delivered to the external (right) side of the oothecal tissue prior to incubation. After various incubation times (0.5 h, 2 h, 6 h or 24 h), the microparabiotic chamber was viewed under ultraviolet light (256 nm) to detect leaks. (A) No detectable leakage. (B) Leak detected. Sample replication not used. Scale bar, 1 cm.

View larger version (26K): [in a new window]   Fig. 5. Comparison of movement of various water-soluble materials across the escutcheon region of the proximal end of 0-1-week-old oothecae using microparabiotic chambers (Figs 1, 4). Comparisons of the rate of isotope movement from the exterior side to the interior side of the oothecal surface were measured using 1 µl samples taken at 0.5 h, 2 h, 6 h and 24 h incubation times. Radioactivity recovered on the inside of the oothecal covering is reported as log% of the initial radiolabel placed on the exterior side of the covering. Molecular weights (Da) of the isotopes used are shown in parentheses. The number of replications (N) used for data analysis is shown for each material. Statistical comparisons of the means were done using ANCOVA (analysis of covariance); different letters associated with the curves indicate significant differences at the P=0.05 level.

View larger version (95K): [in a new window]   Fig. 6. Photograph demonstrating the degree of sclerotization of the oothecae; the distal end of the ootheca is darker (more sclerotized) than the proximal end (less sclerotized). This photograph also shows the close structural relationship between the gravid female and her oothecae. Scale bar, 1 mm.

View larger version (90K): [in a new window]   Fig. 7. The female vestibulum after removal of a 1-week-old ootheca, showing the extent of membranous surfaces that may be involved in water transport. Scale bar, 1 mm.

View larger version (78K): [in a new window]   Fig. 8. Light micrographs of the proximal end of an empty ootheca obtained using a Wild MP400 Photomakroscop equipped for dark-field illumination. 5-10µl water was applied to the chorion (inner surface), and observations were made as the water evaporated from the specimen. The presence of water is revealed by the birefringent or light areas, where the water was retained. Note that the hexagon-shaped structures, characteristic of the chorion, contain water in their internal (ring-like) structures as well as along the exterior or planar surfaces. (A) Scale bar, 1 mm. (B) Scale bar, 0.5 mm.