The Small Heat Shock Protein p26 Aids Development of Encysting Artemia Embryos, Prevents Spontaneous Diapause Termination and Protects against Stress

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.

Stress tolerance during diapause and quiescence of the brine shrimp, Artemia

“…These variations suggest that the three known Artemia sHSPs, although similar in several ways, have different functions during diapause and quiescence, a proposal tested by RNAi. The injection of A. franciscana females with p26 double-stranded RNA (dsRNA) eliminates p26 from cysts but has no effect on the other identified sHSPs (King and MacRae 2012;King et al 2013). p26 knockdown by RNAi reduces cyst tolerance to desiccation, freezing, and heat, confirming the earlier deduction that p26 functions as a molecular chaperone in cysts (King and MacRae 2012).…”

“…The injection of A. franciscana females with p26 double-stranded RNA (dsRNA) eliminates p26 from cysts but has no effect on the other identified sHSPs (King and MacRae 2012;King et al 2013). p26 knockdown by RNAi reduces cyst tolerance to desiccation, freezing, and heat, confirming the earlier deduction that p26 functions as a molecular chaperone in cysts (King and MacRae 2012). Moreover, the loss of p26 slows embryo development and allows spontaneous diapause termination, indicating roles other than in stress tolerance.…”

“…Upon liberation from females, cysts undergo profound reduction in metabolism and enter diapause, a state of developmental arrest and greatly enhanced stress tolerance (Clegg 1967(Clegg , 1997Clegg and Jackson 1998;King and MacRae 2012;Duan et al 2014). Diapause occurs in animals other than Artemia, most commonly in insects, and initiates in anticipation of adverse environmental conditions signaled by day length, perhaps via changes in circadian clock gene expression (Nambu et al 2004;MacRae 2010;King and MacRae 2015;Meuti et al 2015).…”

“…The need for a termination cue differentiates diapause from quiescence, a hypometabolic, stress-tolerant state requiring only return to favorable growth parameters for termination (Drinkwater and Clegg 1991). Stress tolerance during diapause and quiescence in Artemia has been examined from several perspectives including the synthesis, composition, and structure of the cyst shell (Liu et al 2009;Dai et al 2011a;, the role of trehalose (Clegg 1965;Clegg and Jackson 1992;Yang et al 2013), the composition and activity of late embryogenesis abundant (LEA) proteins (Sharon et al 2009;Wu et al 2011;Toxopeus et al 2014), and the function of molecular chaperones such as small heat shock proteins (sHsps) and artemin (King and MacRae 2012;King et al 2013King et al , 2014.…”

Artemin, a Diapause-Specific Chaperone, Contributes to the Stress Tolerance of Artemia Cysts and Influences Their Release from Females

“…Diapause, which is most common in insects (Poelchau et al, 2013;Mizoguchi et al, 2013;Fan et al, 2013) but occurs in organisms as diverse as rotifers (Clark et al, 2012), tardigrades (Guidetti et al, 2011), crustaceans (Tarrant et al, 2008;King and MacRae, 2012;Clark et al, 2013), killifish (Meller et al, 2012) and mammals (Murphy, 2012;Cha et al, 2013), entails decreased metabolism and augmentation of stress tolerance (Denlinger, 2002;Koštál, 2006;MacRae, 2010;Hahn and Denlinger, 2011). Photoperiod, temperature and crowding induce diapause, an integral part of the life cycle of many animals (Xiao et al, 2010;Meuti and Denlinger, 2013).…”