ArHsp40 and ArHsp40-2 contribute to stress tolerance and longevity in Artemia franciscana, but only ArHsp40 influences diapause entry

Abstract

Embryos of the crustacean develop either ovoviviparously or oviparously, yielding swimming larvae (nauplii) or encysted gastrulae (cysts), respectively. Nauplii moult several times and become adults whereas cysts enter diapause, a state of dormancy characterized by exceptionally low metabolism and high stress tolerance. Synthesis of molecular chaperones such as the J-domain proteins ArHsp40 and ArHsp40-2 occurs during embryo development and post-diapause growth of and they influence development and stress tolerance. To further investigate J-domain protein function, ArHsp40 and ArHsp40-2 were each knocked down by RNA interference. Reductions in ArHsp40 and ArHsp40-2 had no effect on adult survival, time to release of cysts and nauplii from females and first-brood size. However, knockdown of both J-domain proteins reduced the longevity and heat tolerance of nauplii, with the loss of ArHsp40 having a greater effect. The knockdown of ArHsp40, but not of ArHsp40-2, caused approximately 50% of cysts to abort diapause entry and hatch without exposure to an exogenous signal such as low temperature and/or desiccation. Cysts lacking ArHsp40 that entered diapause exhibited decreased stress tolerance as did cysts with reduced ArHsp40-2, the latter to a lesser degree. The longevity of nauplii hatching prematurely from cysts was less than for nauplii arising by other means. The results expand our understanding of Hsp40 function in stress tolerance and development, especially during diapause, and they provide the first example of a molecular chaperone that influences diapause entry.

Mechanisms of Desiccation Tolerance: Themes and Variations in Brine Shrimp, Roundworms, and Tardigrades

“…Two J-domain proteins have been identified in Artemia: ArHsp40 and ArHsp40-2 (Jiang et al, 2016;Rowarth and MacRae, 2018b). Knockdown of either of the genes encoding these proteins reduces the percentage of cysts that are able to hatch following freezing and desiccation (Rowarth and MacRae, 2018a). Developmentally, ArHsp40 and ArHsp40-2 reach their peak expression levels in embryos exiting diapause (Jiang et al, 2016;Rowarth and MacRae, 2018b).…”

Cyst viability and stress tolerance upon heat shock protein 70 knockdown in the brine shrimp Artemia franciscana

“…The hatching percentage obtained was higher than those with their Hsp40 reduced by RNAi. Approximately 20 and 40% of cysts hatched, respectively, upon ArHsp40 and ArHsp40-2 knockdown (Rowarth and MacRae 2018). In another case, only 5 to 6% of cysts hatched upon p26 and group 1 LEA protein knockdown under the same experimental condition as used in this study (King and MacRae 2012;Toxopeus et al 2014).…”

“…These results showed that knockdown of Hsp70 had no apparent effect on the cyst morphology. Likewise, knockdown of a type 1 J-domain protein, ArHsp40, and type 2 J-domain protein, ArHsp40-2, had no apparent effect on brood size and time required to release cysts and nauplii in A. franciscana (Rowarth and MacRae 2018).…”

“…Hsp70 binds substrate proteins in the absence of ATP to reduce the occurrence of non-functional protein formation (King and MacRae 2015) and suppressing Hsp70 expression may disrupt the normal cellular function during diapause termination. It is reported that during stress, ArHsp40 and ArHsp40-2 deliver substrates to Hsp70, providing protection against desiccation, heat, and oxidation (Rowarth and MacRae 2018). Hsp70 may be a part of Hsp110-Hsp70-Hsp40 protein disaggregation system (Kityk et al 2018), and with cooperation of sHsp p26, they protect partially denatured proteins from irreversible denaturation (King and MacRae 2012).…”