organisms exposed to different environmental stressors , including temperature changes , trace metals and ultraviolet light , respond by rapidly synthesising a suite of highly conserved proteins called heat shock or stress proteins . under stressful conditions , such as extreme temperature ,
some proteins lose their higher order structures and related functions , and hsps are synthesized to promote refolding of these denatured proteins .
heat shock proteins ( hsps ) work as molecular chaperones to protect the organism under stressful conditions and repair stress - damaged proteins to enable normal functions to continue . 

hsp40 and hsp70 function as co - chaperones and are typically involved in folding , assembly and transport of proteins , working together to minimize protein aggregation .
memory formation is a vital process that enables an individual to adapt its behavior to current or future conditions .
short - term memory ( stm ) , lasting seconds to minutes , is a result of functional changes in pre - existing synapses . whereas , long - term memory ( ltm ) , lasting hours to days , involves gene activation and the synthesis of new proteins .
memory formation is dynamic , and environmental stressors can change the way an animal is able to learn and form memory , either by enhancing or blocking memory formation , depending on the nature of the stress and timing relative to the learning period .
the great pond snail lymnaea stagnalis is widely used as a model system to study learning and memory because of its relatively simple neuronal system and easily recordable set of behaviors that can be altered through training .
a range of environmental stressors have been shown to both enhance and reduce the ability of l. stagnalis to learn and form memories .
in particular , stress associated with thermal stimulus has previously been shown to enhance memory formation in l. stagnalis . 

l. stagnalis lives in shallow , often stagnant , bodies of water that can be exposed to a range of temperature fluctuations depending on weather conditions .
the stress associated with sudden 1  h duration exposures to 30c pond water was found to enhance memory formation . here , we investigate time - course gene expression profiles of hsp40 and hsp70 in the central nervous system of l. stagnalis .
activation of these heat shock proteins may be involved in the enhancement of ltm formation . to examine whether thermal stress induces expression of hsp40 and hsp70 in l. stagnalis
, we subjected a group of snails to a thermally stressful condition ( 30c for 1  h ) and sampled central nervous system tissue for gene expression analysis at 6  time intervals following the termination of thermal stress ( 0  h , 0.5  h , 1  h , 2  h , 4  h , 8  h ) . both hsp40 and hsp70
were rapidly induced in the central nervous system within 30  min of the end of thermal stress ( fig .  1 ) .
hsp40 reached maximum induction before hsp70 , between 1 and 2  h following termination of thermal stress ( fig . 
the induction of hsp70 was greater than that of hsp40 , with over 4  times the relative fold change ( fig .  1 ) .
hsp70 expression began to decline approximately 4  h following the end of thermal stress ( fig . 
a critical exponential curve applied to the change in gene expression over time was a good fit to the data for hsp40 ( r = 0.99 , p = 0.0202 ; fig . 
figure 1.heat shock protein 40 ( hsp40 ) expression ( a ) and heat shock protein 70 ( hsp70 ) expression ( b ) in the central nervous system of lymnaea stagnalis following exposure to thermal stress .
critical exponential curve fitted to the data ( hsp40 , r = 0.99 , p = 0.0202 ; hsp70 , r = 0.88 , p = 0.1767 ) . 
 
heat shock protein 40 ( hsp40 ) expression ( a ) and heat shock protein 70 ( hsp70 ) expression ( b ) in the central nervous system of lymnaea stagnalis following exposure to thermal stress .
critical exponential curve fitted to the data ( hsp40 , r = 0.99 , p = 0.0202 ; hsp70 , r = 0.88 , p = 0.1767 ) .
these results demonstrate that exposure to an acute thermal stress of 30c was sufficient to increase the synthesis of hsps above constitutive levels in l. stagnalis .
the temporal changes in expression of hsps observed in the current study were similar to those recorded previously in other organisms .
bahrndorff , et  al . observed a peak in the expression of hsp70 in orchesella cincta within 2  h of exposure to thermal stress followed by a sharp decrease after 6  h. here , expression of hsp70 peaked approximately 4  h after the end of thermal stress , followed by a sharp decline ( fig . 
altered gene activity and protein synthesis are known to be required for ltm formation in l. stagnalis .
specifically , molluscan insulin - related peptide ii and protein kinase c are thought to play a role in the ltm formation of conditioned taste aversion .
the observed increase in the synthesis of hsps in the central nervous system of l. stagnalis , following exposure to thermal stress , suggests that these proteins may play a role in ltm formation in relation to a thermal stimulus .
l. stagnalis were subjected to a thermally stressful condition ( 30c for 1  h ) and central nervous system tissue was sampled at 6  time intervals following the end of the thermal stress ( 0  h , 0.5  h , 1  h , 2  h , 4  h , 8  h ) .
immediately upon sampling , snails were euthanized by placing in ice water for 15  min , the central nervous system was dissected and then completely submerged in rnalater ( qiagen ) in a 1.5  ml micro - centrifuge tube .
central nervous system tissue was pooled from 2 snails for each replicate at each time point , thus , there were 3 replicates per time point .
rna was extracted from the samples using the rneasy mini kit ( qiagen ) following the manufacturer 's protocol , with initial sonication ( 510  s ) , additional tissue disruption with a qiashredder column ( qiagen ) , and a 15  min dnase treatment .
rna was eluted in 30 l of rnase - free water and the concentration and quality of total rna was determined using a nanodrop 1000 spectrophotometer ( nanodrop technologies ) . only samples that met quality criteria ( 260/280 ratio > 2.0 ) were used for further analyses .
all samples were diluted to 50  ng l and 400  ng of rna was used to synthesize cdna following the manufacturer 's protocol for the improm - ii reverse transcription system ( promega ) , with hexanucleotide primers and deoxynucleotide mix ( sigma - aldrich ) .
cdna was synthesized under the following conditions : annealing at 25c for 5  min , extending at 42c for 60  min , and heat - inactivating transcriptase at 70c for 15  min ( geneamp pcr system , 9700 , applied biosystems ) .
heat shock protein primers ( table  1 ) were selected using primer blast ( ncbi ) .
the amplicons were designed to span 1 intron junction and were checked to avoid secondary structure , self - annealing sites , complementarity , and potential hairpins using oligocalc ( northwestern university , usa ) .
elongation factor 1-alpha primers ( table  1 ) were taken from van nierop , et  al . 

table 1.lymnaea stagnalis gene specific primers for heat shock protein genes ( hsp40 and hsp70 ) and a housekeeping gene ( elongation factor 1- , elf-1 ) .
num.forward ( 5-3)reverse ( 5-3)product ( bp)annealing temperature ( c)hsp40dq278442.1atgttaaacctggatggaaggcagggcaggcacgttttgcggtgttt7958hsp70dq206432.1tgctggccgaagcggagaagcctcaagctggttcctggccg7860elf-1dq278441.1accacaactggccacttgatcccatctcttgggcctctttct8554 
  lymnaea stagnalis gene specific primers for heat shock protein genes ( hsp40 and hsp70 ) and a housekeeping gene ( elongation factor 1- , elf-1 ) .
reference numbers from ncbi , product length in base pairs ( bp ) to conduct quantitative reverse transcriptase pcr ( qrt - pcr ) , lyophilized primers ( eurofins mwg operon ) were reconstituted to 100 mol with rnase - free water and mixed with sybr green jumpstart taq readymix ( sigma - aldrich ) to give a final reaction concentration of 375 nmol in a 20 l total volume .
fluorescence was detected ( stepone real - time pcr system , applied biosystems ) over 40 cycles with cycling conditions of denaturing at 94c for 15  s , annealing at 55c for 1  min and extension at 72c for 1  min . for analysis ,
a standard curve of cdna template ( from a known sample ) was run on each plate for each gene to allow for within experiment plate normalization .
the efficiency of qrt - pcr was calculated ( e = 10  1 ) from the standard curve for each plate . only efficiencies
between 0.85 and 1.2 were used for further analysis , and comparative quantification , using the efficiency corrected method , was used to determine fold - changes in the genes of interest normalized to elf-1. the expression of elf-1 in the central nervous system of l. stagnalis was not affected by thermal stress , thus , the use of elf-1 as a housekeeping gene was justified .
this work was funded in part by the faculty of science and technology , plymouth university .
 organisms exposed to environmental stressors respond by rapidly synthesising a suite of highly conserved proteins called heat shock proteins ( hsps ) . 
 environmental stress can also enhance and/or block memory formation , with long - term memory formation requiring gene activation and protein synthesis . 
 thermal stress in the pond snail lymnaea stagnalis can enhance memory formation , and , in this study , the effect of thermal stress on hsp gene expression in the nervous system was investigated . 
 time - related expression profiles for hsp40 and hsp70 indicated rapid ( < 30  min ) induction for both transcripts . for hsp40 , induction was < 20 fold relative to control and expression returned to control levels within 8  h , whereas hsp70 induction was > 100 fold and expression did not return to control levels within 8  h. 
Abbreviations
None
Materials and Methods
Disclosure of Potential Conflicts of Interest
Funding