Fig 1.
Post-diapause life history stages of A. franciscana.
Light micrographs of A. franciscana obtained with a Nikon AZ100 microscope. (a) Cysts prior to emergence (0 h); (b) emerged nauplius 1 (E1); (c) emerged nauplius 2 (E2); (d) emerged nauplius 3 (E3); (e) instar 1 nauplius; (f) early instar 2 nauplius. Early and late (not shown) instar 2 nauplii are morphologically similar. ES, eye spot. Magnification bar a, 100 μm; b, c, 250 μm; d—f, 100 μm.
Table 1.
Primers used for PCR.
Fig 2.
Nucleotide and amino acid sequences for the full length open reading frame of ArHsp40-2. J-domain, underlined in black; HPD motif, boxed in blue; G/F rich region, underlined in blue; DIF motif, boxed in red; C-terminal domain, underlined in red. The number of nucleotides and amino acid residues is indicated on the right.
Fig 3.
Sequence alignment of ArHsp40-2 with type 2 Hsp40s from other organisms.
The amino acid sequence of ArHsp40-2 from A. franciscana was aligned with type 2 Hsp40s from the organisms listed in Table 2. The domains of ArHsp40-2 are over-lined in black and labeled in red. SBD, substrate binding domain. Identical amino acid residues are indicated by an asterisk, similar residues by a colon and semi-conserved residues by a period. The number of amino acid residues in each sequence is indicated on the right.
Fig 4.
Sequence alignment of ArHsp40 and ArHsp40-2.
The amino acid sequences of ArHsp40 and ArHsp40-2 were compared by CLUSTAL OMEGA (http://www.ebi.ac.uk/clustalw2). J-domain, over-line black; HPD motifs (red boxes); G/F rich region, over-line red; DIF motifs, blue boxes; CXXCXGXG motifs of the zinc binding domain in ArHsp40 only, green over-line; C-terminal domain, blue over-line. Identical amino acid residues are indicated by an asterisk, similar residues by a colon, semi-conserved residues by a period, and no residue by a dash. The number of amino acid residues in each sequence is indicated on the right.
Fig 5.
Protein modeling of ArHsp40 and ArHsp40-2.
The structures of ArHsp40 (a) and ArHsp40-2 (b), displayed with the amino-terminus to the left, were generated by PyMol https://pymol.org/2/ software. J-domain, orange; HPD motif, pink; G/F rich region, green; zinc binding domain (ZBD), cyan (ArHsp40 only); carboxyl-terminal substrate binding domain, magenta, contains double β-barrel structures termed substrate binding domains 1 and 2 (SBD1, SBD2), followed by the dimerization domain.
Table 2.
Comparison of ArHsp40-2 with type 2 Hsp40s from other organisms.
Fig 6.
Specificity of antibodies raised to ArHsp40 and ArHsp40-2.
Protein extracts from A. franciscana and recombinant ArHsp40s were resolved in SDS polyacrylamide gels, blotted to nitrocellulose and stained with antibodies. Lane 1, 1 μg of recombinant ArHsp40; 2, 1 μg of recombinant ArHsp40-2; 3, 20 μg of protein extract from A. franciscana cysts; 4, 20 μg of protein extract from A. franciscana instar 1 nauplii. Blots were probed with Anti40-type 1 (a) and Anti40-type 2 (b).
Fig 7.
ArHsp40-2 mRNA was developmentally regulated during post-diapause development of A. franciscana.
The amount of ArHsp40-2 mRNA determined by qPCR was normalized to α-tubulin mRNA in post-diapause A. franciscana. Bar 1, 0 h cysts; 2, 5 h cysts; 3, 10 h cysts/E1; 4, E2/E3; 5, instar 1 nauplii; 6, early instar 2 nauplii; 7, late instar 2 nauplii. The experiment was performed in duplicate with RNA samples from 3 replicates of each life history stage. The amount of ArHsp40-2 mRNA in life history stages indicated by bars labeled with asterisks are significantly different from the amount of ArHsp40-2 mRNA in 0 h cysts, **, P<0.01. Error bars represent standard error of 3 replicates per experiment.
Fig 8.
ArHsp40-2 was developmentally regulated during post-diapause development of A. franciscana.
Cell-free homogenates from post-diapause A. franciscana were resolved in SDS-polyacrylamide gels and either stained with Colloidal Coomassie blue (a) or blotted to nitrocellulose and probed with either Anti40-type2 (b) or anti-Y (c). The antibody-reactive protein bands were quantified with Image Studio Software and the ratio of ArHsp40-2:tyrosinated α-tubulin was determined. Lane 1, 0 h cysts; 2, 5 h cysts; 3, 10 h cysts/E1; 4, E2/E3; 5, instar 1 nauplii; 6, early instar 2 nauplii; 7, late instar 2 nauplii. The amount of ArHsp40-2 in life history stages indicated by bars labeled with asterisks are significantly different from the amount of ArHsp40-2 in 0 h cysts, ***, P<0.005. Error bars represent standard error of 3 replicates per experiment.
Fig 9.
ArHsp40-2 was induced by heat shock in post-diapause A. franciscana nauplii.
Cell-free homogenates from heat shocked instar 1 nauplii were resolved in SDS-polyacrylamide gels, blotted to nitrocellulose and probed with either Anti40-type2 (a) or anti-Y (b). The antibody-reactive protein bands were quantified with Image Studio Software and the ratio of ArHsp40-2:tyrosinated α-tubulin was calculated (c). Lane C, no heat shock; HS, 1 h at 39°C; 2R, 4R, 6R, 8R, recovery at 27°C for 2, 4, 6 and 8 h respectively. The amount of ArHsp40-2 in life history stages indicated by bars labeled with asterisks are significantly different from the amount of ArHsp40-2 in 0 h cysts, **, P<0.01; ***, P<0.005. Error bars represent standard error of 3 replicates per experiment.