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First published online July 6, 2005
Journal of Experimental Biology 208, 2783-2798 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01680

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V-ATPase expression during development of Artemia franciscana embryos: potential role for proton gradients in anoxia signaling

Joseph A. Covi^* and Steven C. Hand

Division of Cellular, Developmental and Integrative Biology, Department of Biological Science, Louisiana State University, Baton Rouge, LA 70803, USA

View larger version (83K): [in a new window]   Fig. 1. (A) Alignment of the deduced amino acid sequence for the V-ATPase B-subunit from Artemia franciscana with mammalian brain-type (B2) isoforms (common name and GenBank accession numbers as follows: human, CAA44721 bovine, P31408; mouse, P50517; rat, NP_476561). (B) Alignment of the deduced amino acid sequence for the V-ATPase B-subunit from A. franciscana with mammalian kidney-type (B1) isoforms (human, NP_001683; bovine, P31407; mouse, NP_598918; rat, XP_232119). Sequence showing no differences between any of the species is represented by dots. White text on a black background represents mammalian sequence differing from A. franciscana but conserved among at least three of the four mammalian species (low variability). Black text on a blue background represents mammalian sequence differing both from Artemia and at least two mammalian sequences (high variability). Bold text on white background represents mammalian sequence with physiochemical properties similar to those of Artemia (potentially conserved). Gaps in the mammalian sequence are represented by a black dash on a grey background. The percent amino acid identity between aligned mammalian and A. franciscana sequence is enclosed in parentheses at the end of each sequence. Conserved NtpB domain used in phylogenetic analysis is bracketed by black and yellow arrow symbols above the A. franciscana sequence.

View larger version (35K): [in a new window]   Fig. 2. Phylogenetic relationship of amino acid sequences for the NtpB domain from invertebrate, mammalian brain-type (B2), mammalian kidney-type (B1) and yeast V-ATPase B-subunit isoforms. GenBank accession numbers are listed here in descending order with respect to the cladogram, beginning with A. aegypti (AAD27666 XP_312029, P31410, P31401, AAP37188 AAF08281 CAA44721 P31408, P50517, NP_476561, NP_001683, P31407, NP_598918, XP_232119, P16140, P31411). Scale represents the proportion of amino acid difference between sequences. Bootstrap values from 1000 replicates are listed at each node. Sequences are placed into two groups, specific and generalist, based on the highlighted bootstrap values and general functional repertoire of the inclusive isoforms. Percent amino acid identity with the A. franciscana NtpB domain is recorded in parentheses after each species name.

View larger version (34K): [in a new window]   Fig. 3. Profile of mRNA expression for the V-ATPase B-subunit, as observed by northern blot analysis of total RNA isolated via (A) CsCl cushion or (B) RNeasy affinity spin column. Data for the 3500 bp message are plotted as circles with a broken line. Data for the 2250 bp message from post-diapause embryos are plotted as diamonds and a solid line, while that for diapause embryos are plotted as a cross. Two of the 4 h samples for plot B were excluded from analysis, as they formed insoluble precipitates during the drying step preceding suspension in gel loading buffer. For northern blots, values are expressed as means ± S.E.M. (N=3). Shared lowercase letters above error bars indicate that no significant difference exists between time points, as determined by Tukey's test. An asterisk denotes that a significant difference was found between two time points, as determined by the Nemenyi test. (C) Relative abundance of four A. franciscana developmental stages during incubation at 22–23°C. For each time point, a total of 332–631 individuals was counted, and the data were plotted as a percentage of the total.

View larger version (47K): [in a new window]   Fig. 4. Western blot analysis of A. franciscana B-subunit fusion protein (F1, 4 µg NtA column purified; F2, 7 µg solublized inclusion body), V-ATPase expression in subcellular fractions of encysted embryos (M, mitochondrial pellet; H, heavy membrane pellet; V100, heavy microsomal vesicle pellet; S100, supernatant of 100 000 g spin; 40 µg per lane) and a Manduca sexta positive control (Ms, heavy membrane pellet; 1 µg per lane). The blot on the left was incubated with primary antibody raised against the native V-ATPase V1 complex purified from Manduca sexta midgut (Huss, 2001). All immunoreactive bands were identified by comparison with control blots incubated with secondary antibody only (blot not shown) and are listed in parentheses (together with their apparent Mr in kDa) to the left of the figure. The blot on the right (lane CM) is the control for mitochondrial lysates from A. franciscana (incubated with secondary antibody only) and is included to demonstrate that the banding in lane M is nonspecific.

View larger version (38K): [in a new window]   Fig. 5. Western blot analysis of V-ATPase expression in subcellular fractions of encysted embryos, employing three polyclonal antibodies raised against (A,B) the native V1 complex, (C,D) recombinant d-subunit of the V0 domain or (E) recombinant a-subunit of the V0 domain from Manduca sexta larvae. A biotinylated secondary antibody was used for blots in A, C, D and E. An alkaline phosphatase-conjugated secondary antibody was used for blot in B to allow visualization of subunit A (70 kDa), which is obscured in biotinylated blots by a strong non-specific band (ns) visible in blots A, C, D and E. Subcellular fractions from 0 and 8 h of development were run side by side (H, heavy membrane; V140, microsomal vesicles pelleted at 140 000 g; S140, post-ribosomal supernatant from 140 000 g spin; W, V140 pellet resuspended in buffer of low ionic strength and pelleted a second time; Ms, Manduca sexta heavy membrane preparation). All immunoreactive bands were identified by comparison with control blots incubated with secondary antibody only. Subunit designation (in parentheses) and apparent Mr (in kDa) for each band are listed to the left of each blot.

View larger version (15K): [in a new window]   Fig. 6. Percentage of A. franciscana embryos developing to the swimming naupliar stage when incubated in 35 artificial seawater in the presence of 0, 10, 20 and 30% ethanol. Data are means ± S.E.M. (N=5).

View larger version (18K): [in a new window]   Fig. 7. Concentration dependence for inhibition of hatching in A. franciscana with oligomycin. (A) Simultaneous observation of the relative abundance of four developmental stages – dechorionated encysted embryos (open diamond), emergence 1 (open square), emergence 2 (open triangle) and nauplii (solid circle) – calculated as a percentage of all individuals present. (B) Percentage of individuals reaching the swimming naupliar stage under the following conditions: chorion intact (control; solid line), dechorionated (long dash line) and dechorionated followed by blotted dry before placement in incubation medium (short dash line). Data are means ± S.E.M. (N=3).

View larger version (17K): [in a new window]   Fig. 8. Concentration dependence for inhibition of hatching in A. franciscana with bafilomycin. Observation of the relative abundance of four developmental stages – encysted embryos (open diamond), emergence 1 (open square), emergence 2 (open triangle) and nauplii (solid circle) – calculated as a percentage of all individuals present. Hatching studies were conducted using either (A) dechorionated embryos or (B) embryos with intact chorion.

View larger version (26K): [in a new window]   Fig. 9. Developmental time course of the relative abundance of three developmental stages – dechorionated encysted embryos (open diamond), emergence and emergence 2 stages combined (solid triangle) and nauplii (solid circle) – calculated as a percentage of all individuals present. Dechorionated embryos incubated in 35 Instant Ocean + 0.5% ethanol served as a control (A). Experimental treatments with 0.5 (B), 1.0 (C), 2.0 (D) and 4.0 µmol l–1 (E) bafilomycin in the presence of 0.5% ethanol began at hour 0. To determine whether or not embryos were permeable to bafilomycin prior to emergence, 4 µmol l–1 bafilomycin was added at hour 8 (F), as indicated with a broken line.

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