environmental temperature fluctuations 
 can pose dramatic challenges 
 to cold - blooded organisms , such as insects , fish , and crustaceans , 
 since temperature changes can cause global perturbation that affects 
 the reaction rates of all the biological processes .
when the homeostasis 
 of life is challenged , complex responses , including physiological , 
 neurological , and behavioral , may co - mediate the response and rebuild 
 the balance to prolong the animals survival . using four congeneric species of crabs as study models ,
stillman 
 has suggested that the temperature acclimation capacity of these marine 
 invertebrates is crucial for their survival in the face of global 
 warming . by unveiling the underlying mechanism 
 of how organisms respond to increasing habitat temperature
, we can 
 obtain a better understanding of the direct impacts that climate changes 
 have on life .
it has been a lasting effort to probe the mechanism 
 of the thermal 
 perturbation response in invertebrates over the past decade . in the 
 lobster homarus americanus
, a large 
 spectrum of physiological parameters in the circulating hemolymph 
 has been observed to be influenced negatively by high temperature , 
 including glucose , total protein concentration , cholesterol , chloride 
 and calcium concentration , etc .
for example , the pyloric rhythm of the stomatogastric ganglion ( stg ) 
 from cancer borealis was shown to be 
 robust to temperature changes from 7 to 23 c , but the system 
  crashed  at high acute temperatures ( > 23 c ) . a recent study in cancer borealis and cancer pagurus showed that the 
 pyloric phases were maintained in intact animals at high temperature 
 ( 26 c ) , while pyloric frequency increased significantly . however , 
 the frequency range was more restricted than it was in vitro , which 
 might be caused by sensory feedback and neuromodulatory input .
temperature changes are also correlated with 
 cardiac performance in multiple organisms . in h. americanus , 

interestingly , 
 although the q10 values are similar in 
 vivo and in vitro , the heartbeat rates are faster in intact animals , 
 and less heart failures are observed at higher temperatures ( above 
 20 c ) compared to the in vitro preparations .
this is intriguing 
 because it may suggest that endogenous neural and/or hormonal signals 
 might protect the cardiac performance integrity in intact animals 
 from temperature ramp .
however , to date , there has been no direct 
 evidence regarding the involvement of neuromodulators in the regulation 
 of the temperature perturbation response . to address this knowledge 
 gap
, we employed a mass spectrometry - based 
 strategy to examine the quantitative changes of neuropeptides caused 
 by temperature elevation .
the jonah crab c. borealis was selected as an experimental model , which is an ideal system 
 for investigating neuromodulation due to its simple and well - defined 
 neural network .
the neural network of c. borealis includes two features : the stomatogastric 
 nervous system ( stns ) , which generates rhythmic motor patterns that 
 control the movement of the stomach , and the central nervous system 
 ( cns ) , which consists of the brain , thoracic ganglion , etc .
other 
 major neuroendocrine components are the pericardial organ ( po ) , which 
 surround the heart , and the paired sinus gland ( sg ) located in the 
 eyestalks .
neuropeptides from neurohemal organs , such as po , are released 
 into the hemolymph and pumped through the stomatogastric ganglion 
 ( stg ) to the brain , which can influence the output of the neural networks . 
 a large number of neuropeptides from c. borealis
have already been identified in previous studies using mass spectrometry , 
 facilitating us to further assess their functions . in this study , we measured 
 the neuropeptide changes in three different 
 neural organs in the nervous system of c. borealis in response to acute temperature elevation using dimethyl labeling . 

a number of neuropeptides were found to be decreased in two neuroendocrine 
 organs , po and sg , indicating that these two organs were actively 
 involved in the temperature perturbation response . in addition , the 
 neuropeptidome changes of the brain and circulating fluid hemolymph 
 were also examined .
a temperature stress marker peptide was discovered 
 in the hemolymph and de novo sequenced .
methanol , acetonitrile , formic 
 acid , acetic acid , and edta disodium salt were purchased from fisher 
 scientific ( pittsburgh , pa ) .
borane pyridine , formaldehyde , and deuterium 
 formaldehyde were from sigma - aldrich ( st . louis , mo ) .
2,5-dihydroxybenzoic 
 acid ( dhb ) used as maldi matrix was obtained from mp biomedicals , 
 inc .
acidified methanol was prepared using 90% methanol , 
 9% acetic acid , and 1% water .
jonah 
 crabs cancer borealis were purchased 
 from the fresh lobster company ( gloucester , ma ) and maintained without 
 food in artificial seawater at 1213 c for at least 1 
 week before experiments . to increase the ambient temperature ,
crabs 
 were placed in a bucket filled with artificial seawater at 1213 
 c , and the bucket was then placed in a water bath preheated 
 to around 50 c . the temperature within the bucket was increased 
 to around 23 c in 15 min .
the neural tissues , including 
 po , sg , and brain , were dissected in chilled ( approximately 10 c ) 
 physiological saline ( composition : 440 mm nacl ; 11 mm kcl ; 13 mm cacl2 ; 26 mm mgcl2 ; 10 mm hepes acid ; ph 7.4 ) .
hemolymph samples were collected by inserting 
 a 22-gauge needle attached to a 3 ml plastic syringe through the junction 
 of the thorax and abdomen into the pericardial chamber .
all the crabs 
 used in this study were male , and animals with similar shell color , 
 size , and weight were paired for each comparison group .
briefly , 
 a mixture of 0.45 ml of acidified methanol and 0.3 ml of edta solution 
 ( 20 mm aqueous solution ) was spiked into 0.75 ml of freshly obtained 
 hemolymph to extract peptides and precipitate large proteins .
edta 
 was added to prevent cation - triggered hemolymph clotting during the 
 sample preparation process .
the samples were centrifuged at 16  000 g for 10 min , after which the supernatant was collected 
 followed by ultrafiltration through a 10 kda mwco tube by centrifugation 
 at 15  000 g .
the low mass filtrate was concentrated 
 to dryness using a savant sc 110 speedvac concentrator ( thermo electron 
 corporation , west palm beach , fl ) and was resuspended in 80 l 
 of 0.1% fa in water .
after sonication for 10 min , the sample was desalted 
 by c18 micro spin column ( argos , elgin , il ) according to 
 the product manual and eluted in 6 l of 0.1% fa in 50% acetonitrile 
 ( v / v ) .
tissue extractions 
 were obtained by homogenizing the neural tissues in 200 l of 
 cooled acidified methanol .
the undissolved tissue pellets were removed 
 by centrifugation at 16  000 rcf for 10 min .
the supernatants 
 were dried in a savant sc 110 speedvac concentrator ( thermo fisher 
 scientific , waltham , ma ) and resuspended in 20 l of 0.1% formic 
 acid , after which the samples were desalted by a c18 ziptip 
 ( millipore , bedford , ma ) according to the product instruction .
a 3 l 
 aliquot of tissue extract from neural tissues was first mixed with 
 0.7 l of borane pyridine ( c5h8bn , 120 
 mm in 10% methanol ) , after which 0.5 l of formaldehyde ( fh2 , 15% in h2o ) for control samples or 0.5 l 
 of deuterium formaldehyde ( fd2 , 15% in h2o ) 
 for temperature stress samples was added .
the mixtures were then placed 
 in a 37 c water bath for 20 min for the labeling reaction to 
 complete .
samples from control and stressed animals were mixed with 
 a 1:1 ratio before mass spectrometry analysis . a model 4800 maldi - tof / tof 
 ( applied biosystems , framingham , ma ) equipped with a 200 hz , 355 nm 
 nd : yag laser was used for neuropeptide quantification analysis .
acquisitions 
 were performed in positive ion reflectron mode , and instrument parameters 
 were set using the 4000 series explorer software ( applied biosystems , 
 framingham , ma ) .
mass spectra were obtained by averaging 900 laser 
 shots covering mass range m / z 5004000 , 
 and ms / ms were acquired by 1 kv collisionally induced dissociation 
 ( cid ) using air as collision gas . to elucidate the primary sequence 
 of the derivatized stress marker peptide , sustained off - resonance 

irradiation collision - induced dissociation ( sori - cid ) was performed 
 on a maldi - ftms ( varian , lake forest , ca ) equipped with a 7.0 t actively 
 shielded superconducting magnet .
the ftms instrument consisted of 
 an external high - pressure maldi source with a 355 nm nd : yag laser 
 ( laser science , inc .
, franklin , ma ) to create ions that were accumulated 
 in the external hexapole storage trap before being transferred through 
 a quadrupole ion guide to the icr cell .
all mass spectra were collected 
 in positive ion mode , and detection was performed in broadband mode 
 from m / z 108.00 to 2500.00 . for 
 sori - cid ,
an arbitrary waveform with a 10 da isolation window 
 was introduced to isolate the ion of interest , and ions were excited 
 with sori burst excitation ( 2.648 v , 25003000 ms ) .
a pulse 
 of nitrogen gas was introduced through a pulse valve from 2500 to 
 2750 ms to induce collision activation .
the neuropeptide identification 
 was based on mass matching with 
 previously characterized neuropeptides from c. borealis .
de novo sequencing of the stress marker 
 peptide was done manually without the assistance of any software . 
 for relative quantification of neuropeptides ,
each labeled mixture 
 sample was spotted on the maldi plate twice , and two replicate spectra 
 were acquired for each spot , resulting in four replicate spectra for 
 each sample .
peak lists were extracted from the crude spectra without 
 any postacquisition processing using the data explorer software ( applied 
 biosystems , framingham , ma ) .
the peak pairs generated from the known 
 neuropeptides were selected for quantitative analysis . the relative 
 abundance ratio for each neuropeptide between the temperature - stressed 
 sample and the control sample
was determined by dividing the heavy 
 labeled peak intensity with the light labeled peak intensity .
student s t test was performed to 
 evaluate the differences of each peptide , and a p value < 0.005 was considered to be statistically significant .
it is rather difficult to study the 
 expression of each individual 
 neuropeptide in different physiological conditions using traditional 
 biological assays , such as western blotting , because neuropeptide 
 families usually contain a large number of isoforms sharing similar 
 amino acid sequences that could cause antibody cross - reactivity .
to 
 solve such a problem , a number of mass spectrometry - based methods 
 have been developed , leading to the discovery and mapping of numerous 
 neuropeptides in different animals .
it is our ultimate goal to utilize such tools to elucidate the functions 
 of neuropeptides in the regulation of neural circuits .
mass spectrometry - based 
 quantitation methods have also been used to accurately measure the 
 quantitative changes of neuropeptide levels in animals under different 
 conditions .
for example , label - free peptidomics was employed to study 
 the neuropeptide alterations in the tree shrew hypothalamus during 
 volatile anesthesia .
another method , 
 dimethyl labeling , has been reported as a fast and simple reaction 
 that can be applied for differential proteomic and peptidomic analyses . in our previous work ,
dimethyl labeling was 
 used to study the involvement of neuropeptides in feeding . in this current study
, we used a similar strategy 
 to examine the quantitative changes of neuropeptides in three different 
 neural organs from c. borealis induced 
 by acute temperature elevation .
the po is a major neurohemal organ 
 surrounding the heart , which can release hormones into the circulating 
 hemolymph and regulate the functions of heart and other distant organs . to determine the variability of neuropeptide 
 expression between different animals
, we initially compared two groups 
 of control crabs . a representative maldi - tof / tof spectrum of a dimethyl - labeled 
 mixture of two control extracts from po is shown in figure 1a , and the peak pairs of labeled neuropeptides are 
 indicated with their amino acid sequences .
however , as shown 
 in figure 1b , the intensities of many detected 
 neuropeptides were significantly reduced in the temperature stress 
 sample .
maldi - tof / tof mass spectra of dimethyl - labeled mixtures of ( a ) 
 two control pericardial organ extracts and ( b ) pericardial organ extracts 
 from control ( 12 c ) versus temperature - stressed ( 23 c ) 
 animals . the h2-labeled dimethylated peaks ( control )
are 
 indicated with closed circles , and the d2-labeled dimethylated 
 peaks ( stressed ) are indicated with open circles .
the labeling reaction 
 resulted in 4 da mass differences per label between each peak pair . 

peaks are annotated with their corresponding neuropeptide sequences . to further verify the observation , 
 15 pairs of control and stressed 
 crabs were compared , where each single crustacean contributed a set 
 of paired po . in the quantification experiments ,
three families of 
 neuropeptides were detected in the po , including rfamide - related peptide 
 ( farp ) , ryamide , and b - type allatostatin ( ast - b ) ( table 1 ) .
table 1 and figure 2 show the average abundance ratio between control and stressed 
 animals for each individual neuropeptide .
peptides from both the rfamide 
 and ryamide families were significantly reduced in the po after acute 
 temperature shock ( p < 0.005 ) .
the ratios between 
 the temperature stress and control samples ranged from 0.34 to 0.68 , 
 although different isoforms in the same family were observed to have 
 varying degrees of change . gahknylrfa ( m / z 1104.61 ) showed the largest level of decrease upon temperature 
 change ( 0.34 , p < 0.001 ) , while gnrnflrfa ( m / z 1022.56 ) was the only rfamide peptide 
 with no significant change upon temperature elevation . both of the 
 two observed ryamides , fvggsrya ( m / z 784.41 ) and sgfyanrya ( m / z 976.46 ) , 
 were reduced in the po at high temperature .
two isoforms of ast - b 
 were detected in the po extract in this study , although neither of 
 them showed significant changes .
it is interesting that these neuropeptide 
 families respond differently to the thermal challenge , indicating 
 that they may play distinct roles in response to temperature changes .
neuropeptides with significant changes 
 are indicated with an asterisk ( * p  0.005 , 
 * * p < 0.001 ) .
farp , fmrfamide - related peptide ; 
 ast - b , b - type allatostatin .
ratios / c : ratio of neuropeptide 
 signal intensities between stressed ( 23 c ) and control ( 12 c ) 
 crabs .
. ratios of peptide levels between stressed ( 23 c ) 
 and control ( 12 c ) were determined using dimethyl labeling ( columns , average of ratios calculated from 15 groups of 
 comparison ; bars , standard deviation ; * p < 0.005 ; * * p
the peptide hormones released 
 by the po have rapid and direct access 
 to the heart , which suggests that heart is a primary target of the 
 pericardial hormones .
rfamides are present in many different organisms 
 with a large diversity of amino acid sequences . in crustaceans , 

in an electrophysiological study , it was observed 
 that the application of flrfamide peptides can increase the output 
 activity of the cardiac ganglion that drives the contractions of the 
 heart .
it was also reported that the heart beat rate in 
 lobster h. americanus increased with 
 environmental temperature in vivo and in vitro , while its contraction 
 amplitude was reduced .
therefore , it 
 is quite likely that farps play a key role in this heart functional 
 change in response to temperature stress .
farps are also reported 
 to alter the arterial hemolymph flow and regulate the circulatory 
 system .
these processes may be important 
 for the animal to manage oxygen regulation under a stressful environment . 

on the other hand , the ryamide peptide family was first discovered 
 in the pericardial organ of c. borealis , and its physiological effects are largely unknown .
recently , two peptides with a similar ryamide motif were 
 identified in drosophila and believed 
 to be involved with feeding ; however , it is unclear 
 whether they also have cardiac effects .
another major neuroendocrine organ 
 in crustaceans is the sg , which is a paired organ located in the two 
 eyestalks , and the quantitative changes between temperature stress 
 and control preparations were also determined .
since the total amount 
 of neuropeptides was lower in the sg compared to po , four sg tissues 
 were used for each extract .
overall , five groups of samples were analyzed , 
 from which six neuropeptides were detected ( figure 3 and table 2 ) . as shown in figure 3 , four orcokinin peptides were detected to be reduced 
 at high temperature ( p < 0.005 ) , specifically 
 nfdeidrsgfgfa ( m / z 1474.7 ) , nfdeidrsgfgfv 
 ( m / z 1502.7 ) , nfdeidrssfgfv ( m / z 1532.7 ) , and nfdeidrssfgfn ( m / z 1547.7 ) . the ratios of these four peptides 
 between the stress and control samples were similar , between 0.7 and 
 0.8 .
the other two observed neuropeptides , proctolin and yramide , 
 did not show significant changes .
. ratios of peptide levels between stressed ( 23 c ) 
 and control ( 12 c ) were determined using dimethyl labeling ( columns , average of ratios calculated from five groups of 
 stress experiments for each neuropeptide ; bars , standard 
 deviation ; * , student s t test , p < 0.005 )
. neuropeptides with significant changes 
 are indicated with asterisk ( * p  0.005 , * * p < 0.001 ) .
ratios / c : ratio of neuropeptide 
 signal intensities between stressed ( 23 c ) and control ( 12 c ) 
 crabs .
the sg is known to be 
 involved in various forms of environmental 
 stresses , such as temperature changes , salinity changes , pollution , 
 and hypoxia .
it was found that crustacean hyperglycemic hormones ( chhs ) were 
 released from the sg system during stress , resulting in subsequent 
 elevation of the blood glucose level .
although we were unable to detect 
 and quantify the presence of chhs in this work , we found that neuropeptide 
 orcokinins were decreased in the sg under temperature elevation .
generally , 
 orcokinins are myotropic peptides that have excitatory effects on 
 different tissues .
have reported that orcokinins are 
 measurable in the hemolymph with an approximate concentration of 10 m , indicating that 
 orcokinins may be released into hemolymph and function as a hormone 
 in addition to its role as a locally acting neurotransmitter . in this 
 study , we demonstrated that the orcokinin levels were reduced in the 
 sg upon temperature stress , implicating that orcokinins may be involved 
 in the stress response via a hormonal route .
to evaluate the neuropeptide expression changes 
 in the cns , c. borealis brain extracts 
 were also investigated .
two brains were used for each extract sample , 
 and 10 comparison groups were conducted . compared to the po and the 
 sg , a larger number of neuropeptides were detected that reside from 
 eight different families , including farp , ryamide , yramide , cancer borealis tachykinin - related peptide ( cabtrp ) , 
 crustacean cardioactive peptide ( ccap ) , proctolin , sifamide , and orcokinins . 
 as shown in table 3 ,
no significant changes 
 were observed for any of these neuropeptide families in the brain .
cabtrp , cancer borealis tachykinin - related peptide ; ccap , crustacean cardioactive peptide . 

ratios / c : ratio of neuropeptide signal intensities between 
 stressed ( 23 c ) and control ( 12 c ) crabs .
the results from po and 
 sg indicate that endocrine hormonal regulation 
 may play a very important role in response to acute temperature elevation . 
 to the authors knowledge , this is the first direct demonstration 
 that neuropeptides , including rfamides , ryamides , and orcokinins , 
 are involved in the physiological regulation of the temperature stress 
 response .
in addition , the mass spectrometry - based methods show unique 
 advantages for comprehensive study of multiple neuropeptides simultaneously , 
 leading to valuable insights into their physiological functions .
it 
 should be noted that the neuropeptides in the brain are not observed 
 to be significantly changed in this study .
it is possible that the 
 neuropeptide synthesis in the brain is not strongly affected by temperature , 
 or it may also be due to the short time course of the temperature 
 elevation experiment
. a longer term temperature perturbation study 
 will be conducted in the future to further explore the roles played 
 by the central nervous system and its interaction with other neuroendocrine 
 organs .
it has been reported that a number of physiological variables were 
 changed in the hemolymph in response to temperature elevation .
to better understand the temperature response 
 system in the crab , we analyzed the peptidomic complements of the 
 hemolymph from animals undergoing an acute temperature ramp . in our 
 previous study , we developed an optimized sample preparation method 
 for peptide extraction from the crude hemolymph samples .
this method was utilized here to extract peptides 
 from the c. borealis hemolymph . as 
 shown in figure 4 ,
a peptide peak with m / z 1137.6 ( smp1137 ) with the highest peak 
 intensity was repeatedly observed in the temperature stress hemolymph 
 samples ( n = 5 ) .
it should be noted that this ion 
 was also detected from the control hemolymph in some cases but at 
 much lower peak intensities ( figure 4a ) .
mass spectral 
 comparison of processed hemolymph samples from ( a ) 
 control ( 12 c ) and ( b ) temperature - stressed ( 23 c ) crabs 
 acquired using maldi - tof / tof .
the dominant peak ( m / z 1137.6 ) observed at high temperature is indicated 
 with an asterisk . to identify this peptide , 
 cid fragmentation
was performed on a 
 maldi - tof / tof instrument to acquire peptide sequence information . 

as shown in figure 5a , the fragmentation was 
 quite complete , and the majority of the b- and y - ions were observed . 
 to obtain complementary information for amino acid sequence assignment , 

formaldehyde could react 
 with all the primary amine groups in the peptide , leading to differentiation 
 of the lysine residue ( k ) as compared to glutamine ( q ) or the combination 
 of glycine and alanine ( ga or ag ) .
a 56 
 da mass shift was observed after the dimethylation derivatization , 
 suggesting the presence of a lysine residue in the sequence .
the fragmentation 
 spectrum further confirmed the position of the lysine ( k ) residue 
 in the primary sequence . to further validate the deduced sequence , 

a peptide standard was synthesized and analyzed using the same activation 
 methods ( figure 5b and figure 6b ) , which showed a fragmentation pattern similar to that of 
 the putative smp1137 peptide from the hemolymph .
ms / ms spectra acquired 
 by maldi - tof / tof using collision - induced 
 dissociation for ( a ) putative smp1137 from the hemolymph sample and 
 ( b ) synthetic peptide standard with the proposed sequence at a concentration 
 of 1 m . the presence of b- and y - ions is indicated by horizontal 
 lines below ( b - ions ) or above ( y - ions ) the corresponding amino acid 
 residues in the peptide sequence .
ms / ms spectra acquired by maldi - fticr for ( a ) dimethyl - labeled 
 putative smp1137 from the hemolymph sample and ( b ) dimethyl - labeled 
 synthetic standard with the predicted sequence at a concentration 
 of 1 m . the asterisk indicates the two dimethylated sites , 
 including the n terminus and the lysine ( k ) residue .
the presence 
 of b- and y - ions is indicated by horizontal lines below ( b - ions ) or 
 above ( y - ions ) the corresponding amino acid residues in the peptide 
 sequence . to the best of the authors 
 knowledge , this peptide does 
 not belong to any known neuropeptide families , and it is possibly 
 a fragment from a certain protein in the hemolymph .
notably , lorenzon 
 et al . reported that the protein concentration in the lobster hemolymph 
 increased significantly with the body temperature .
it has also been reported that heat shock proteins , whose 
 primary function was to promote initial folding of other proteins 
 at the ribosome and the refolding of unfolded proteins when they were 
 partially denatured , were elevated in crustacean hemolymph when the 
 animal undergoes stressful conditions .
however , we performed a blast search using the sequence of smp1137 
 against the protein database , with no match of any relevant proteins 
 found , suggesting that the smp1137 could be a fragment cleaved from 
 an unknown protein that is related to temperature stress response . 

future experiments will be performed to identify the origin of smp1137 
 and to determine whether this peptide has any physiological activities . 

in addition , it is also of great interest to investigate whether smp1137 
 is related to other types of environmental stresses , such as salinity 
 changes , pollution , and hypoxia , or it is a unique signature for temperature 
 change .
in this study , quantitative 
 peptidomics was employed to study the 
 neuropeptide changes in the jonah crab cancer borealis associated with acute temperature elevation .
the results showed 
 that neuroendocrine organs , including the pericardial organ and the 
 sinus gland , were actively involved with the temperature perturbation 
 response .
the neuropeptides released by these two organs may be involved 
 with temperature change response via hormonal regulation .
this study 
 provided direct evidence that neuropeptides may play an important 
 role in the regulation of biological changes in crustaceans in response 
 to environmental perturbation .
 temperature changes influence the 
 reaction rates of all biological 
 processes , which can pose dramatic challenges to cold - blooded organisms , 
 and the capability to adapt to temperature fluctuations is crucial 
 for the survival of these animals . in order to understand the roles 
 that neuropeptides play in the temperature stress response 
 , we employed 
 a mass spectrometry - based approach to investigate the neuropeptide 
 changes associated with acute temperature elevation in three neural 
 tissues from the jonah crab cancer borealis . at high temperature , 
 members from two neuropeptide families , including 
 rfamide and ryamide , were observed to be significantly reduced in 
 one of the neuroendocrine structures , the pericardial organ , while 
 several orcokinin peptides were detected to be decreased in another 
 major neuroendocrine organ , the sinus gland . 
 these results implicate 
 that the observed neuropeptides may be involved with temperature perturbation 
 response via hormonal regulation . furthermore , 
 a temperature stress 
 marker peptide with the primary sequence of sfrrmggkaq ( m / z 1137.7 ) was detected and de novo sequenced in 
 the circulating fluid ( hemolymph ) from animals under thermal perturbation . 