AMER. ZOOL., 39:487-495 (1999)
Quantification of Stress in Lobsters: Crustacean Hyperglycemic
Hormone,
Stress Proteins, and Gene Expression1
ERNEST S. CHANG, 2 SHARON A. CHANG, RAINER KELLER, 3 P. SREENIVASULA REDDY, 4
MARK J. SNYDER, AND JEFFREY L. SPEES
Bodega Marine Laboratory, University of California, P.O. Box 247, Bodega Bay, California 94923
SYNOPSIS. Various methods for the quantification of stress in crustaceans have
been developed in our laboratory. An ELISA was developed for the crustacean
hyperglycemic hormone (CHH) from the lobster, Homarus americanus. It is sensitive to as little as 0.2 fmol of peptide. Increases in hemolymph CHH were observed following emersion. Significant levels of hemolymph CHH were also measured in lobsters that had been eyestalk-ablated. It was observed that these animals
continued to produce CHH, even though the heretofore only known source of CHH
had been removed. Portions of the central nervous system, from both intact and
eyestalk-ablated lobsters were observed to contain significant amounts of CHH. A
cDNA library was constructed from eyestalk neural tissue of H. americanus. With
the use of PCR, a 171 bp probe was isolated and purified. This probe was labeled
and used to examine levels of CHH expression in the central nervous system (CNS)
and in eyestalk neural tissue at different periods of the lobster molt cycle. CHH
mRNA is present throughout the CNS. In the eyestalk, it is undetectable in postmolt, low in intermolt, and high in premolt.
Stress proteins, also known as heat shock proteins (HSPs), are a highly conserved
class of proteins which show elevated transcription during periods of stress in
organisms as phylogenetically divergent as bacteria and humans. Using RT-PCR,
we have partially cloned the lobster HSP90 gene. A 380 bp probe was "P-labeled
and hybridized with northern blots of midgut gland total RNA from heat-shocked
lobsters. A 2 hr acute heat shock from 15°C (ambient water temperature) to 28°C
resulted in a 6.0-fold induction of HSP90 after 6 hr of recovery at 15°C. A northern
analysis of RNA isolated from the midgut glands of lobsters injected with 10 ng
of the molting hormone 20-hydroxyecdysone displayed a 2.1-fold induction of
HSP90 RNA 48 hr postinjection.
INTRODUCTION
Observations that eyestalk factors may
regulate blood glucose concentrations in
crustaceans were made several decades ago
by Abramowitz et al. (1944). They found
1
From the Symposium The Compleat Crustacean
Biologist: A Symposium Recognizing the Achievements
that injected eyestalk extracts were able to
elevate the blood glucose in crabs. They
also
localized this diabetogenic activity in
t h e
neurohemal sinus gland in the eyestalk.
T h e
hormone(s) responsible for this hyperglycemic effect has in the last 8 years been
identified, and is commonly referred to as
the crustacean hyperglycemic hormone(s)
( C H H ) Subsequent tO the first fully char-
of Dorothy M. Skinner presented at the Annual Meet-
ing of the Society for Integrative and Comparative Biology, 3-7 January 1998, at Boston, Massachusetts.
2
To whom all correspondence should be sent at Bodega Marine Laboratory, University of California, P.O.
Box 247, Bodega Bay, California 94923; phone: 707
875 2061; fax 707 875 2009. E-mail: eschang®
ucdavis.edu
3
Present address of Rainer Keller is Institut fur Zoophysiologie, Rheinische Friedrich-Wilhelms-Universi-
•
««
<-
,-.
actenzed hormone from Carcinus maenas
(Kegel et al., 1989), CHHs are now known
from a number of decapod crustaceans and
o n e i s o p o d s p e c i e s . Their amino acid seu
u
J *
• A U. D J
quences have been determined by Edman
degradation and, in some cases, also by
cloning of the precursor cDNAs (for reviews see Keller, 1992; Chang, 1993, 1995;
pDr;5se:t15addrensns' o ^ S r e ' e n i v a s u l a Reddy is De-
* > ^ l e i j n and Van Herp, 1995). A m o n g the
partment of Biotechnology, Sri Venkateswara University, Timpati, 517-602, India.
fully characterized CHHs are those from the
experimental animal of this present study,
^
487
488
E. S. CHANG ETAL.
the American lobster, Homarus americanus
(Chang et al., 1990; Tensen et al., 1991; De
Kleijn et al., 1995; Schooley and Chang,
unpublished). Hyperglycemia as a response
to various kinds of stress is well documented in decapod crustaceans; there is evidence that this response is mediated by the
release of CHH (for review see Keller et
al., 1985). In this paper we report on the
development of a sensitive ELISA for
CHH, the use of the ELISA as a tool for
the quantification of environmental stresses
in lobsters, the characterization of a probe
for the CHH gene(s), and its use in northern
blots.
Another potential indicator of environmental stress is the induction and/or elevated expression of stress proteins, also
known as heat shock proteins (HSPs). The
primary function of HSPs is to act as molecular chaperones, promoting the initial
folding of other proteins at the ribosome
and the refolding of unfolded proteins when
they are partially denatured (Nelson et al.,
1992). Environmental stresses such as
changes in temperature (Ritossa, 1962), oxygen (Ropp et al., 1983), and metal ion
concentration (Steinhert and Pickwell,
1988; Ryan and Hightower, 1994), induce
the synthesis of HSPs which act to prevent
protein aggregation and to maintain functional conformations. Most organisms exhibit increased production of three classes
of heat shock proteins: HSP90, MW of 8590,000; HSP70, MW of 68-72,000; and a
low molecular weight series of HSP2O-3O,
MW of 20-30,000 (McLennan and Miller,
1990; see recent reviews by Haiti, 1996;
Nover and Scharf, 1997). The enhanced
production of HSPs usually coincides with
a period of reduced "normal" protein synthesis. The recovery from heat shock and
other stressful events requires the protein
folding abilities of HSPs in all eukaryotes
(Lindquist and Craig, 1988; Becker and
Craig, 1994; Hartl, 1996). In the second
part of this paper we discuss our initial gene
expression studies with lobster HSP90, an
HSP isoform that is highly induced by elevated temperature and also by the steroid
molting hormone 20-hydroxyecdysone
(20E).
CHH:
A POSSIBLE CRUSTACEAN STRESS
HORMONE
Materials and methods
Animals and manipulations. Juvenile H.
americanus were raised in our culture facility as previously described (Chang and
Conklin, 1993; Conklin and Chang, 1993).
Molt stages of the lobsters were denned by
monitoring of previous and current molt
histories and examination of the exoskeleton (Aiken, 1973). Within each experiment,
full siblings were matched for sex, molt
stage, and weight (100-145 g). Lobsters
were starved for one day prior to the experiments. Experiments were initiated at
approximately the same time of day (0900
hr).
Hypoxic stress was achieved by emersion. Lobsters were placed into 2-liter jars
without any water. They were placed in an
incubator at ambient temperature (13°C).
Hemolymph (150 u,l) was sampled from the
same animals at various time points. Controls were matched animals that were left
immersed at 13°C and sampled at the same
time points.
Bilateral eyestalk ablation was achieved
by cutting the eyestalks at their bases at the
arthrodial membrane. We routinely achieve
>90% long term survival of the lobsters
following the operation.
ELISA. Details of the assay, including its
specificity, sensitivity, and reproducibility,
have been described elsewhere (Chang et
al, 1998). Briefly, lobster CHH-A (Tensen
et al., 1991) was purified by HPLC (Chang
et al., 1990) and injected into a rabbit to
produce the first antibody. The IgG fraction
was purified and used to coat the wells of
a multiwell plate. Non-specific binding sites
were blocked. Samples and standards were
incubated overnight at 4°C. The second antibody was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester.
Streptavidin-peroxidase was added and was
followed with 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid. The change in
absorbance at 405 run was measured in a
multiwell plate reader.
CHH gene expression. We constructed a
cDNA library made from lobster eyestalks.
X-organ/sinus gland complexes and sur-
QUANTIFICATION OF STRESS IN LOBSTERS
rounding neural tissue were dissected from
the eyestalks and frozen in liquid nitrogen.
RNA was isolated from the tissue homogenates using guanidinium thiocyanate, phenol, and chloroform (Chomczynski and
Sacchi, 1987). The mRNA was separated
from total RNA using the PolyATtract system (Promega). cDNA was made from the
purified mRNA and ligated to phage lambda arms using a ZAP-cDNA kit (Stratagene). The initial screening of the cDNA
library was achieved by PCR using a synthetic 27-mer (5'-TAT GAC CGC A AC
CTC TTC AAG AAG CTC-3') as the forward primer. It corresponds to amino acids
11 to 19 of CHH-A/B (De Kleijn et al.,
1995). The reverse primer was derived from
amino acids 62 to 67 (5'-GGA GAC GTA
CTC GTC GAT-3').
PCR was carried out in a 50 u.1 solution
containing DNA from 9 X 106 phage as
template, 10 pmol each of reverse and forward primers, 250 \LM of each dNTP, and
5 U Taq polymerase (Promega). The temperature profile was 94°C for 1 min, 68°C
for 2 min, and 72°C for 1 min for 30 cycles.
The resulting PCR product was analyzed by
0.8% agarose gel electrophoresis. A single
PCR product of the expected size (approx.
171 bp) was obtained. DNA sequencing of
the product confirmed that it coded for the
expected fragment of CHH-A (De Kleijn et
al., 1995). The insert was purified from the
gels with a Sephaglas kit (Pharmacia) and
randomly labeled with 32P-dCTP (USB).
Total RNA from various tissues (leg
muscle, abdominal muscle, hepatopancreas,
and sections of the central nervous system)
was extracted as above, quantified by UV
absorbance, fractionated on formaldehyde
1% agarose gels, transferred to nitrocellulose filters (S & S), and probed with the
radiolabeled insert according to standard
methods (Sambrook et al., 1989). Blots
were exposed to X-ray film and the developed film was scanned by densitometry.
Results and discussion
We observed that emersion, which results
in hypoxia, is a potent stimulator for the
elevation of hemolymph CHH. Figure 1
shows that it increases from resting values
of 4.0 fmol/ml to 168.1 fmol/ml after 4 hr.
489
200
0
1
2
3
Time of Emersion (hr)
FIG. 1. Effects of emersion on hemolymph CHH (circles, solid line). Lobsters (n = 7) were removed from
the water and placed into jars in an incubator at 13°C.
Hemolymph was withdrawn from the base of the legs
with a syringe and mixed with an anticoagulant buffer
(Chang et al., 1998). Insoluble material was pelleted
and 100 u.1 of the supernatants were loaded onto multiwell plates coated with the first antibody. The samples and standards were assayed as described in Materials and Methods. Means ± SD are shown. Control
data from immersed lobsters are represented by the
squares, dashed line (n = 8). Asterisks indicate significant differences from immersed controls at P < 0.01
(**) and at P < 0.001 (***). Modified from Chang et
al, 1998.
Our ELISA is sensitive enough to monitor
levels of CHH by repeated sampling of
small volumes of hemolymph from the
same animal. In these juvenile lobsters, the
handling and sampling per se did not stimulate CHH release into the hemolymph to
a significant degree. Therefore, the influence of some environmental stresses can be
studied without interference from this handling stress.
Our observations following emersion of
lobsters are in agreement with those of
Webster (1996) on the crab, Cancer pagurus. He found that emersion causes a significant increase of CHH in the hemolymph
15 min after emersion. Similarly, in our
lobsters, a significant increase was measurable after 20 min. Webster (1996) discussed
the physiological significance of this mechanism of endocrine metabolic adaptation for
C. pagurus, which may be subjected to
emersion and hypoxia in the intertidal zone.
Although the lobster is primarily subtidal,
they may occasionally experience local
hypoxic conditions. The observed response
to hypoxia may be a general phenomenon
490
E. S. CHANG ETAL.
among decapod crustaceans. We observed TABLE 1. CHH content in hemolymph, regions of the
that other stresses, such as temperature in- central nervous system, and various other tissues.*
Eyestalkcrease (Chang et al., 1998) and pH changes
Tissue
ntact
Ablated
(unpublished) resulted in elevated hemoLeft
eyestalk
799
t
924
n.d.
lymph CHH. Our results demonstrated that
518 t 555
n.d.
measurement of hemolymph CHH is a Right eyestalk
0.173
4.75 t 8.08
promising tool to monitor stress responses Brain
Subesophageal ganglion
0.071
0.983 t 1.76
in lobsters, and, more generally, to study Thoracic ganglia
0.300 t 0.195 0.048
0.181 t 0.084 0.003
the role of CHH in the metabolic regulation Abdominal ganglia
Testes
0.088 t 0.125 n.d.
of crustaceans.
0.043 t 0.045 0.0003
To determine if all of the CHH was de- Hemolymph
Claw muscle
0.021 t 0.017 n.d.
rived from the eyestalk X-organ/sinus gland Abdominal muscle
0.0
n.d.
complex, a group of sibling lobsters had Hepatopancreas
0.0
n.d.
their eyestalks removed and they were sub* Sibling lobsters were either intact (n = 6 for hesequently sampled repeatedly for up to one molymph and neural tissues, n = 2 for other tissues)
year. Although some lobsters had undetect- or had been eyestalk-ablated one year previously (n =
able levels of CHH on some sampling days, 1). The tissues were weighed, homogenized, and assayed with an ELISA. Data are presented as pmol of
all lobsters displayed concentrations that CHH-immunoactivity
per gram of tissue ± SD. n.d.
were significantly above detection limits of means not determined.
the assay at several different times during
the experiment. A group of eyestalk-ablated
lobsters survived for over one year. These testes and claw muscle (Table 1) may be
animals still had resting levels of CHH of due to contamination by hemolymph.
We also examined temporal aspects of
1.8 ± 1.6 fmol/ml (mean ± SD; n = 3).
the
expression of the CHH gene(s). RNA
Moreover, upon emersion, significant elewas
obtained from eyestalk neural tissue of
vation of the CHH level was also observed
in these eyestalk-ablated animals, amount- lobsters at various stages of the molt cycle.
ing to 13.6 ± 8 . 1 fmol/ml after a 4 hr emer- We observed that there were trace levels of
sion period. This response was considerably CHH mRNA during intermolt, an increase
lower, however, than the response observed in early premolt (stage D,), very high levels
in intact animals, in which an increase to in late premolt (stage D3), and undetectable
168.1 ± 49.6 fmol/ml (mean ± SD; n = 5) levels during postmolt (stage A-B) (Fig. 2).
Although our probe partially codes for
was observed.
These data suggested that there were sites CHH-A (MIH), at this time we are unable
of CHH synthesis and release other than the to distinguish between the expression of
sinus gland. Likely candidates were other CHH-A and CHH-B.
portions of the central nervous system
STRESS PROTEINS
(CNS). We dissected portions of the CNS
from both intact and eyestalk-ablated lob- Materials and methods
sters. The data in Table 1 indicate that sigWithin each experiment, full sibling lobnificant amounts of CHH-immunoreactive sters were matched for sex, molt stage, and
material are present in the brain with lesser weight (137-185 g). Lobster midgut glands
amounts in the posterior portions of the (hepatopancreas) were dissected, frozen in
CNS. This conclusion is supported by a liquid N2, and stored at -70°C. Total RNA
northern analysis of various tissues and por- was isolated from the tissues with the RNAtions of the CNS. RNA that hybridizes with gents total RNA isolation kit (Promega).
our CHH probe is expressed in all of the RNA was quantified with a spectrophotomportions of the CNS examined (data not eter and equally loaded onto denaturing 1 %
shown). Our results are consistent with the agarose gels. Following a 15 min wash in
observations of De Kleijn et al. (1995) and diethylpyrocarbonate-treated water (to reSun (1995). No hybridization was observed move excess formaldehyde), gels were blotwith RNA from non-neural tissues. The low ted overnight onto nylon membranes
amounts of immunoactivity observed in the (MagnaGraph, MSI) and UV-crosslinked
QUANTIFICATION OF STRESS IN LOBSTERS
for lobster actin was added to check for
equal loading of RNA. All films were
scanned with a high resolution scanner interfaced with a computer running Adobe
Photoshop.
Another experiment involved the injection of mid-intermolt (stage C4) juvenile
male lobsters with either saline vehicle or
10 M-g of 20E. Levels of HSP90 mRNA in
the midgut gland were determined 48 hr
following the injection.
CO
0
CO
(0
491
1.9
0.9
C4 D^ D3 A-B
Molt Stage
FIG. 2. Expression of CHH gene(s) in lobster eyestalk neural tissue during different molt stages. Eyestalk neural tissue was dissected free of the exoskeleton and the retina. This tissue contained the X-organ/
sinus gland and surrounding neural tissue. RNA was
extracted and analyzed as described in Materials and
Methods. Equal amounts of RNA (30 u.g) were loaded
in each lane. Lane 1: intermolt (stage C4); lane 2: early
premolt (stage D,); lane 3: late premolt (stage D3); lane
4: postmolt (stage A-B). Size markers in kilobases are
shown on the left. The following numbers represent
quantification via scanning densitometry. The units are
arbitrary. The values are 55, 294, 527, and 21 for lanes
1—4, respectively. From Reddy el al., 1997.
(UV Stratalinker 1800, autocrosslink
mode). Blots were then pre-hybridized (2
hr) in 5X SSPE buffer (Sambrook et al.,
1989), 50% (w/v) deionized formamide, 5X
Denhardt's, 1% SDS, and 100 (xg/ml
sheared salmon sperm DNA. A 380 bp partial clone for lobster HSP90 was isolated by
RT-PCR using an HSP90 forward primer
based upon previously published sequence
data (Blackman and Meselson, 1986) and
an oligo-dT15 reverse primer (3' RACE).
This partial clone was 32P-labeled (Prime-It
RmT, Stratagene) and added directly to the
prehybridization solution. The probe was
allowed to hybridize overnight at 42°C. The
following morning, blots were washed
twice with 2X SSPE (2 min, and again for
10 min) and then placed on film for 2 days
at -70°C. Following exposure of the film,
the blots were stripped with several washes
(0.1X SSC (Sambrook et al., 1989), 1%
SDS, 65°C) until background was minimal
and prehybridized, hybridized, and washed
as above, except that a partial cDNA probe
Results and discussion
The effects of acute heat shock (2 hr at
28°C, then returned to 15°C) were determined on midgut gland HSP90 mRNA levels (Fig. 3). Within 2 hr of recovery, HSP90
mRNA was induced 5.5-fold versus control
tissue. It increased by 6.0-fold after 6 hr of
recovery and was still elevated by 1.8-fold
after 24 hr of recovery. Despite the wealth
of information on stress protein responses
in terrestrial organisms, we know little
about HSP responses in marine invertebrates. Most of the existing data have been
generated from work on bivalve molluscs.
In marine mussels {Mytilus edulis), four different HSP70 isoforms were detected by
western blotting using a human HSP70 antibody; elevations in two of these forms resulted from heat stress (Smerdon et al.,
1995). Also in mussels, HSP70 isoforms
have been shown to increase with heat
stress during tidal emersion (Hofmann and
Somero, 1995, 1996a, b). Likewise, an in
vitro study with oyster hemocytes showed
HSP70 induction during heat stress (Tirard
et al., 1995). An association of HSP26 with
metabolic and other stresses in Anemia
franciscana embryonic development was
recently demonstrated (Jackson and Clegg,
1996). Various HSP70 isoforms also appear
to be involved in the stress responses of
other crustaceans such as amphipods and
crayfish (Rochelle et al., 1991; Werner and
Nagel, 1997). From our review of the literature, we believe this is the first demonstration of the involvement of HSP90 in
marine invertebrate thermal challenge.
Following the injection of 20E, we observed that ecdysteroid treatment results in
a 2.1-fold induction of HSP90 expression
in midgut gland 48 hr postinjection (Fig. 4).
492
E. S. CHANG ETAL.
HSP90
actin
e
JZ
m
x:
C\J
CD
"o
o
0
CO
.c
OJ
u.
j->
c
o
O
CO
C\J
j£
CVJ
r.
CM
o
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C\J
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FIG. 3. Heat shock-induced HSP90 gene expression in H. americanus. Autoradiogram (northern blot) of RNA
isolated from midgut gland (total RNA, 15 u.g loaded per lane). Juvenile male intermolt lobsters were removed
from a flow-through culture system with ambient (15°C) seawater and subjected to an acute heat shock (28°C)
for 2 hr and then returned to 15°C for recovery. Lane 1: control (no heat shock); lane 2: 2 hr recovery; lane 3:
6 hr recovery; lane 4: 24 hr recovery. The northern blot was hybridized with a 32P-labeled cDNA probe specific
for HSP90. The lower panel shows hybridization of a lobster actin cDNA probe with actin RNA (control for
equal loading). The following numbers represent quantification via scanning densitometry. The units are arbitrary. For the HSP90 blot, the values are 91, 504, 548, and 163 for lanes 1-4, respectively. For the actin blot,
the values are 75, 66, 56, and 49 for lanes 1—4, respectively.
Other results (Spees et al., in prep.) indicate
that HSP90 expression is significantly higher in this tissue during the mid-premolt
stages. Similar to the H. americanus HSP90
response, Drosophila HSP27 is induced by
ecdysteroids during larval and pupal development (Huet et al., 1996). In seemingly
different tasks from those related to thermal
stress, members of the HSP90 and HSP70
(and possibly others) are also involved in
the activities of different types of intracellular receptors. For instance, both HSP70
and HSP90 are required for stable steroid
hormone receptor complexes (Hutchinson
et al., 1994; Jakob, 1996). HSP90 also
binds to and stabilizes the aryl hydrocarbon
receptor protein in a state capable of interacting with its substrates (Whitelaw et al.,
1995; Jakob, 1996). Our data imply that
lobster HSP90 may be involved in midgut
gland ecdysteroid responses or perhaps in
ecdysteroid receptor interactions.
The differential expression of HSPs during the molt cycle may provide insights into
the responses of potential target tissues to
ecdysteroids. The regulation of synthesis,
circulating titers, and metabolism of ecdysteroids during the molting cycle of lobsters
have been well characterized in our laboratory (Bruce and Chang, 1980; Chang and
Bruce, 1981; Snyder and Chang, 1991 a, b,
c). During the intermolt stages, titers of 20E
493
QUANTIFICATION OF STRESS IN LOBSTERS
2
3
4
HSP90
actin
c
o
O
oo
o
C\J
FIG. 4. HSP90 gene expression in H. americanus induced by injection of 20-hydroxyecdysone (20E) into
juvenile intermolt males. Autoradiogram (northern blot) of RNA isolated from midgut gland (total RNA, 30 u.g
loaded per lane). Lanes 1 and 2: control (saline vehicle injection); lanes 3 and 4: 10 |j.g 20E injection. All
lobsters were sacrificed and sampled 48 hr postinjection. Each lane represents an individual animal. The blot
was probed as described in the legend for Figure 3. The lower panel shows hybridization of a lobster actin
cDNA probe with actin RNA (control for equal loading). The following numbers represent quantification via
scanning densitometry. The units are arbitrary. For the HSP90 blot, the values are 106, 150, 284, and 262 for
lanes 1-4, respectively. For the actin blot, the values are 92, 106, 162, and 209 for lanes 1-4, respectively.
are low, representing an average of less
than 10 ng/mJ of hemolymph (recalculated
from Snyder and Chang, 1991a). In the data
reported here, injections of 10 (xg of 20E
resulted in calculated mean hemolymph
20E levels close to those found in late premolt (600 ng/ml in stage D3; Aiken, 1973).
The observed increase in HSP90 expression
may thus represent the normal tissue responses found during premolt preparations.
We are currently investigating these relationships in greater detail.
part by the California State Resources
Agency (to M. J. S. and E. S. C.) and NSF
grant 96-31128 (to M. J. S.). The views expressed herein are those of the authors and
do not necessarily reflect the views of
NOAA or any of its sub-agencies. The U.S.
Government is authorized to reproduce and
distribute for governmental purposes. R. K.
was a Bodega Marine Laboratory Distinguished Research Fellow and P. S. R. was
supported by the Rockefeller Foundation.
REFERENCES
ACKNOWLEDGMENTS
This paper is funded in part by a grant
from the National Sea Grant College Program, National Oceanic and Atmospheric
Administration, U.S. Department of Commerce, under grant number NA66RG0477,
project number R/A-108 through the California Sea Grant College System, and in
Abramowitz, A., F. Hisaw, and D. Papandrea. 1944.
The occurrence of a diabetogenic factor in the
eyestalks of crustaceans. Biol. Bull. 86:1-4.
Aiken, D. E. 1973. Proecdysis, setal development and
molt prediction in the American lobster. J. Fish.
Res. Board Can. 30:1334-1337.
Becker, J. and E. A. Craig. 1994. Heat-shock proteins
as molecular chaperones. Eur. J. Biochem. 219:
11-23.
494
E. S. CHANG ETAL.
Blackman, R. K. and M. Meselson. 1986. Interspecific
nucleotide sequence comparisons used to identify
regulatory and structural features of the Drosophila hsp 82 gene. J. Mol. Biol. 188:499-515.
Bruce, M. J. and E. S. Chang. 1980. Ecdysteroid titers
of juvenile lobsters following molt induction. J.
Exp. Zool. 214:157-160.
Chang, E. S. 1993. Comparative endocrinology of
molting and reproduction: Insects and crustaceans.
Annu. Rev. Entomol. 38:161-180.
Chang, E. S. 1995. Physiological and biochemical
changes during the molt cycle in decapod crustaceans: An overview. J. Exp. Mar. Biol. Ecol. 193:
1-14.
Chang, E. S. and M. J. Bruce. 1981. Ecdysteroid titers
of larval lobsters. Comp. Biochem. Physiol. 70A:
239-241.
Chang, E. S. and D. E. Conklin. 1993. Larval culture
of the American lobster (Homarus americanus).
In J. P. McVey (ed.), CRC handbook of marieulture, Vol. 1, pp. 489-495. CRC Press Inc., Boca
Raton.
Chang, E. S., R. Keller, and S. A. Chang. 1998. Quantification of crustacean hyperglycemic hormone
by ELISA in hemolymph of the lobster, Homarus
americanus, following various stresses. Gen.
Comp. Endocrinol. 111:359-366.
Chang, E. S., G. D. Prestwich, and M. J. Bruce. 1990
Amino acid sequence of a peptide with both moltinhibiting and hyperglycemic activities in the lobster, Homarus americanus. Biochem. Biophys.
Res. Commun. 171:818-826.
Chomczynski, P. and N. Sacchi. 1987. Single-step
method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Analyt.
Biochem. 162:156-159.
Conklin, D. E. and E. S. Chang. 1993. Culture of juvenile lobsters (Homarus americanus). In J. P
McVey (ed.), CRC handbook of mariculture. Vol.
1, pp. 497-510. CRC Press Inc., Boca Raton.
De Kleijn, D. P. V. and F. Van Herp. 1995. Molecular
biology of neurohormone precursors in the eyestalk of Crustacea. Comp. Biochem. Physiol.
112B:573-579.
De Kleijn, D. P. V., E. P. H. De Leeuw, M. C. Van Den
Berg, G. J. M. Martens, and F. Van Herp. 1995
Cloning and expression of two mRNAs encoding
structurally different hyperglycemic hormone precursors in the lobster Homarus americanus.
Biochim. Biophys. Acta 1260:62-66.
Haiti, F. U. 1996. Molecular chaperones in cellular
protein folding. Nature 381:571-580.
Hofmann, G. E. and G. N. Somero. 1996a. Interspecific variation in thermal denaturation of proteins
in the congeneric mussels Mytilus trossulus and
M. galloprovincialis: Evidence from the heatshock response and protein ubiquitination. Mar.
Biol. 126:65-75.
Hofmann, G. E. and G. N. Somero. 1996b. Protein
ubiquitination and stress protein synthesis in Mytilus trossulus occurs during recovery from tidal
emersion. Mol. Mar. Biol. Biotech. 5:175-184.
Hofmann, G. E. and G. N. Somero. 1995. Evidence
for protein damage at environmental temperatures.
Seasonal changes in levels of ubiquitin conjugates
and HSP70 in the intertidal mussel Mytilus trossulus. J. Exp. Biol. 198:1509-1518.
Huet, E, J.-L. Da Lage, C. Ruiz, and G. Richards.
1996. The role of ecdysone in the induction and
maintenance of HSP27 transcripts during larval
and prepupal development of Drosophila. Dev.
Genes Evol. 206:326-332.
Hutchinson, K. A., K. D. Dittmar, M. J. Czar, and W.
B. Pratt. 1994. Proof that HSP70 is required for
assembly of the glucocorticoid receptor into a heterocomplex with HSP90. J. Biol. Chem. 269:
5043-5049.
Jackson, S. and J. S. Clegg. 1996. Ontogeny of low
molecular weight stress protein p26 during early
development of the brine shrimp Artemia franciscana. Dev. Growth Differ. 38:153-160.
Jakob, U. 1996. HSP90—news from the front. Front
Biosci. 1:309-317.
Kegel, G., B. Reichwein, S. Weese, G. Gaus, J. PeterKatalinic and R. Keller. 1989. Amino acid sequence of the crustacean hyperglycemic hormone
(CHH) from the shore crab, Carcinus maenas.
FEBS Lett. 255:10-14.
Keller, R. 1992. Crustacean neuropeptides: Structures,
functions and comparative aspects. Experientia
48:439-448.
Keller, R., P. P. Jaros, and G. Kegel. 1985. Crustacean
hyperglycemic neuropeptides. Am. Zool. 25:207—
221.
Lindquist, S. and E. A. Craig. 1988. The heat-shock
proteins. Annu. Rev. Genet. 22:631-677.
McLennan, A. G. and D Miller. 1990. A biological
role for the heat shock response in crustaceans. J.
Therm. Biol. 15:61-66.
Nelson, R. J., T. Ziegelhoffer, C. Nicolet, M. WernerWashburne, and E. A. Craig. 1992. The translation
machinery and 70 kd heat shock protein cooperate
in protein synthesis. Cell 71:97-105.
Nover, L. and K.-D. Scharf. 1997. Heat stress proteins
and transcription factors. Cell. Mol. Life Sci. 53:
80-103.
Reddy, P S., G. D. Prestwich, and E. S. Chang. 1997.
Crustacean hyperglycemic hormone gene expression in the lobster Homarus americanus. In S. Kawashima and S. Kikuyama (eds.). Advances in
comparative endocrinology, Vol. 1, pp. 51—56.
Moduzzi Editore, Bologna.
Ritossa, R. 1962. A new puffing pattern induced by
temperature shock and DNP in Drosophila. Experientia 18:571-573.
Rochelle, J. M., R. M. Grossfeld, D. L. Bunting, M.
Tytell, B. E. Dwyer, and Z.-Y. Xue. 1991. Stress
protein synthesis by crayfish CNS tissue in vitro.
Neurochem. Res. 16:533-542.
Ropp, M., A. M. Courgeon, R. Calvayrac, and M.
Best-Belpomme. 1983. Possible role of the super
oxide ion in the induction of heat shock and specific proteins in aerobic Drosophila melanogaster
cells during return to normoxia after a period of
anaerobiosis. Can. J. Biochem. Cell Biol. 61:456461.
Ryan, J. A. and L. E. Hightower. 1994. Evaluation of
heavy-metal ion toxicity in fish cells using a com-
QUANTIFICATION OF STRESS IN LOBSTERS
bined stress protein and cytotoxicity assay. Environ. Toxicol. Chem. 13:1231-1240.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: A laboratory manual, 2nd Ed.,
Vol. 1. Cold Spring Harbor Laboratory, Cold
Spring Harbor.
Smerdon, G. R., J. P. Chappie, and A. J. S. Hawkins.
1995. The simultaneous immunological detection
of four stress-70 protein isoforms in Mytilus edulis. Mar. Environm. Res. 40:399-407.
Snyder, M. J. and E. S. Chang. 1991a. Ecdysteroids in
relation to the molt cycle of the American lobster,
Homarus americanus. I. Hemolymph titers and
metabolites. Gen. Comp. Endocrinol. 81:133—145.
Snyder, M. J. and E. S. Chang. 1991b. Ecdysteroids in
relation to the molt cycle of the American lobster,
Homarus americanus. II. Ecdysteroid metabolite
excretion. Gen. Comp. Endocrinol. 83:118-131.
Snyder, M. J. and E. S. Chang. 1991c. Metabolism and
excretion of injected [3H]-ecdysone in adult female lobster, Homarus americanus. Biol. Bull.
180:475-484.
Steinert, S. A. and G. V. Pickwell. 1988. Expression
of heat shock proteins and metallothionein in mussels exposed to heat stress and metal ion challenge. Mar. Environ. Res. 24:211—214.
495
Sun, P. S. 1995. Expression of the molt-inhibiting hormone-like gene in the eyestalk and brain of the
white shrimp Penaeus vannamei. Mol. Mar. Biol.
Biotechnol. 4:262-268.
Tensen, C. P., D. P. V. De Kleijn, and F. Van Herp.
1991. Cloning and sequence analysis of cDNA encoding two crustacean hyperglycemic hormones
from the lobster Homarus americanus. Eur J.
Biochem. 200:103-106.
Tirard, C. X, R. M. Grossfeld, J. F. Levine, and S.
Kennedy-Stroskopf. 1995. Effect of hyperthermia
in vitro on stress protein synthesis and accumulation in oyster haemocytes. Fish Shell. Immunol.
5:9-25.
Webster, S. G. 1996. Measurement of crustacean hyperglycaemic hormone levels in the edible crab
Cancer pagurus during emersion stress. J. Exp.
Biol. 199:1579-1585.
Werner, I. and R. Nagel. 1997. Stress proteins HSP60
and HSP70 in three species of amphipods exposed
to cadmium, diazinon, dieldrin and fluoranthene.
Environ. Toxicol. Chem. 16:2393-2403.
Whitelaw, M. L., J. McGuire, D. Picard, J.-A. Gustafsson, and L. Poellinger. 1995. Heat shock protein HSP90 regulates dioxin receptor function in
vivo. Proc. Natl. Acad. Sci. U.S.A. 92:4437-4441.
Corresponding Editor: Louis E. Burnett, Jr.