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First published online March 21, 2005
Journal of Experimental Biology 208, 1297-1308 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01525

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Oxygen and water flux across eggshells of Manduca sexta

H. Arthur Woods^1,*, Roger T. Bonnecaze² and Brandy Zrubek¹

¹ Section of Integrative Biology, The University of Texas at Austin, Austin, TX 78712, USA
² Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712, USA

View larger version (45K): [in a new window]   Fig. 1. (A) Schematic of an insect eggshell, drawn from published information on Manduca and Drosophila. Features are not to scale. From left to right, the layers are: chorion, trabecular layer (TL), crystalline chorionic layer (CCL), wax layer, vitelline envelope (VE), serosal cuticle (SC), serosal membrane (SM), serosa, embryo, yolk. From the VE outwards, all layers are in place at oviposition, having been secreted by maternal follicular cells. By contrast, the SC and SM are secreted from the serosa, which is derived from the blastoderm. The SC is secreted first, starting at about 12 h after oviposition (at 24°C), followed by the SM from 23–44 h after oviposition (Lamer and Dorn, 2001). The embryo develops closely apposed to the serosa (rather than deep within the yolk), both before and after katatrepsis. The yolk itself becomes cellularized in the first 12 h after oviposition (Lamer and Dorn, 2001). (B) Schematic of gas flux model. The model terms represent the characteristics of five different layers or sets of layers. From left to right they are: boundary layers around the egg or its substrate, chorion, trabecular layers, a combination of the CCL, wax layer and VE, and all remaining interior layers.

View larger version (134K): [in a new window]   Fig. 2. Aeropyle density and cross-sectional area of eggs of Manduca sexta. (A) Light microscope view (400x, immersed in oil) of the surface of a chorion removed from the egg and washed in saline. Polygons are the imprints of the follicular cells that deposited the chorion (Orfanidou et al., 1992). White arrows point to two aeropyles; about 13 aeropyles are visible in this field. (B) SEM view of the outer surface of a chorion. Polygons are not visible, but dimples (see A) are. A single aeropyle is in the upper left quadrant. (C) Close up view of a single aeropyle. (D) Histogram of aeroyple cross-sectional areas. Data represent 10 aeropyles from each of five eggs (N=50). Mean cross-sectional area (±S.E.M.) is 0.775 ± 0.054 µm2.

View larger version (13K): [in a new window]   Fig. 3. Control trace demonstrating successful identification of a thermal conversion factor for switching between N2 and He using a single mass-flow controller. The stream flowing through the CO2 analyzer was composed of 5 ml min–1 of a span gas (505 p.p.m. CO2 in N2), 21 ml min–1 O2 and 74 ml min–1 of either He or N2 carrier. Using the identified conversion factor (1.37), we were able to switch between carrier gases without affecting the reading from the CO2 analyzer (25 p.p.m. CO2, which is 5% of 505 p.p.m.).

View larger version (22K): [in a new window]   Fig. 4. CO2 emission (at 37°C) by batches of Manduca sexta eggs either 1.5 d (A and B) or 3 day old (C and D). Each batch contained 210–450 eggs. Eggs were exposed, in series, to either 21 or 9.5% O2 with either He or N2 making up the balance. Only variation in O2 availability affected metabolic rates; swapping carrier gases (which altered the diffusion coefficient of O2 in the air filled parts of the chorion and trabecular layer) had no effect.

View larger version (18K): [in a new window]   Fig. 5. Water loss (at 25°C) from batches of 2 day old Manduca sexta eggs. Each batch contained 40 eggs. The first few minutes of each trace represent a baseline reading (air stream directed through an empty chamber). Air streams were then redirected through the egg-containing chamber (black arrow) followed by another baseline reading (colored arrows). During the second baseline, batches were extracted for 1 min in 100% ethanol or 2:1 (v:v) chloform:methanol, blotted dry and returned to their chambers. A control batch was not extracted. The air stream was then directed again through the egg-containing chamber (black asterisk), after which a final baseline reading (colored asterisks) was taken.

View larger version (34K): [in a new window]   Fig. 6. (A) CO2 (black) and water vapour emission (blue) by three batches of eggs from laying to hatching (27°C). Each batch contained 40 eggs. (B) Calculated drop in PO2 (in kPa) across the wax layer using permeabilities calculated from developmental data in panel A (solid) or and from assuming a constant permeability equivalent that calculated for eggs of age 32 h (see text for details). Because PO2 at sea level is approximately 21 kPa, this value sets the upper bound on possible PO2.