Abstract
Artemia franciscana embryos enter diapause as encysted gastrulae, a physiological state of metabolic dormancy and enhanced stress resistance. The objective of this study was to use RNAi to investigate the function of p26, an abundant, diapause-specific small heat shock protein, in the development and behavior of encysted Artemia embryos (cysts). RNAi methodology was developed where injection of Artemia females with dsRNA specifically eliminated p26 from cysts. p26 mRNA and protein knock down were, respectively, confirmed by RT-PCR and immuno-probing of western blots. ArHsp21 and ArHsp22, diapause-related small heat shock proteins in Artemia cysts sharing a conserved α-crystallin domain with p26, were unaffected by injection of females with dsRNA for p26, demonstrating the specificity of protein knock down. Elimination of p26 delayed cyst release from females demonstrating that this molecular chaperone influences the development of diapause-destined embryos. Although development was slowed the metabolic activities of cysts either containing or lacking p26 were similar. p26 inhibited diapause termination after prolonged incubation of cysts in sea water perhaps by a direct effect on termination or indirectly because p26 is necessary for the preservation of diapause maintenance. Cyst diapause was however, terminated by desiccation and freezing, a procedure leading to high mortality within cyst populations lacking p26 and indicating the protein is required for stress tolerance. Cysts lacking p26 were also less resistant to heat shock. This is the first in vivo study to show that knock down of a small heat shock protein slows the development of diapause-destined embryos, suggesting a role for p26 in the developmental process. The same small heat shock protein prevents spontaneous termination of diapause and provides stress protection to encysted embryos.
Highlights
Embryos of the crustacean Artemia franciscana develop ovoviviparously, yielding swimming nauplii upon release from females, or they develop oviparously, producing encysted gastrulae known as cysts [1,2]
Appendages normally visible on adult Artemia and poorly resolved in the upper portion of Figure 1C are obscured in the lower magnification Figures 1B, D, E because animals were immobilized on the surface of cold agarose and both animals and agarose were partially dried with a Kimwipe prior to injection
Artemia cysts, one of the most stress tolerant life forms known among animals, accumulate at least three small heat shock proteins (sHSPs), with p26 in abundance [1,13,15,16,17], as well as other molecular chaperones such as artemin [18,19]. sHSPs are abundant in resting eggs of the rotifer Brachionus plicatilis [40,41] and the sHSP Hsp22 increases during the diapause of Calanus finmarchicus, a marine copepod [42]
Summary
Embryos of the crustacean Artemia franciscana develop ovoviviparously, yielding swimming nauplii upon release from females, or they develop oviparously, producing encysted gastrulae known as cysts [1,2]. A phylogenetically widespread state of dormancy divided into several potentially overlapping phases including initiation, maintenance and termination [3,4,5,6,7]. Molecular chaperones synthesized in diapause-destined embryos of Artemia, and which potentially contribute to cyst stress tolerance, include the small heat shock proteins (sHSPs) p26, ArHsp and ArHsp22 [1,13,14,15,16,17], as well as artemin, a ferritin homologue [18,19]. The sHSP a-crystallin domain contributes to dimerization, oligomerization and chaperone activity [21,22,23,27,28]. The sHSP amino-terminus is involved in subunit dynamics, oligomerization and substrate binding while the flexible carboxylterminal extension, characterized by the I/V-X-V/I motif, modulates oligomerization, chaperoning and solubility [22,23,26,31,32]
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