AMER. ZOOL., 32:470-484 (1992)
Proteins of the Crustacean Exoskeleton'2
DOROTHY M. SKINNER AND S. SINDHU KUMARI
Biology Division, P.O. Box 2009, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8077 and
Biophysics Department, East Tennessee State University, Johnson City, Tennessee 37614
AND
JACK J. O'BRIEN 3
Biology Division, P.O. Box 2009, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8077 and
Department of Biological Sciences, University of South Alabama, Mobile, Alabama 36688
DEDICATION TO D. E. BLISS
Dorothy Bliss was one of my scientific associates to whom I owe one
of the greatest debts.
Two years prior to my arrival as a graduate student at the Harvard
Biological Laboratories, the race had been run between the Harvard and
Yale groups to discern the role of the X-organ sinus gland complex in the
control of molting. It had resulted in the classic 1952 paper on the Bermuda land crab Gecarcinus lateralis (The neurosecretory system of
brachyuran Crustacea. Biological Bulletin, 103:157-169), coauthored by
Bliss and her (and later, my) mentor at Harvard, Dr. John Welsh.
In addition to the classic demonstration of the X-organ sinus gland
complex, Dorothy had spent considerable time inspecting G. lateralis
burrows in both Bimini and Bermuda, observing the behavior of these
animals in the field, and analyzing their stomach contents. By the time I
arrived, Dorothy had established for the land crabs favorable laboratory
conditions for housing, substratum, diet, and lighting conditions (constant
darkness). For Dorothy, determining the latter meant spending hours in
a dark room with a single red light bulb in a gooseneck lamp, measuring
the length of regenerating limb buds of proecdysial specimens. In consultation with Jim Durand, Dorothy developed the limb-regenerate index
that is so useful in following crabs through proecdysis. (Bliss, D. E., 1956.
Neurosecretion and the control of growth in a decapod crustacean. In K.
G. Wingstrand (ed.), Bertil Hanstrom. Zoological Papers in Honor of His
Sixty-fifth Birthday, Nov. 20,1956, pp. 56-75. Zool. Inst., Lund, Sweden).
Dorothy and I overlapped for three of the four years I spent as a graduate
student with Professor Welsh prior to her departure for New York where
she became Curator of Invertebrates at the Museum of Natural History.
A major segment of the debt I owe to Dorothy was the introduction to
Gecarcinus lateralis, the animal that has provided me with an exceptionally favorable experimental system for a lifetime of investigation in both
of my areas of research: the phenomenology and control of molting, and
the structure, organization and function of satellite DNAs, ubiquitous
components of eukaryotic genomes. Gecarcinus' attributes in the first area
1
From the Symposium on The Compleat Crab presen ted at the Annual Meeting of the American Society
of Zoologists, 27-30 December 1990, at San Antonio,
Texas.
2
"The submitted manuscript has been authored by
a contractor of the U.S. Government under contract
No. DE-ACO5-84OR21400. Accordingly, the U.S.
Government retains a nonexclusive, royalty-free license
to publish or reproduce the published form of this
contribution, or allow others to do so, for U.S. Goveminent purposes."
3
Present address: Department of Biological Sciences, University of South Alabama, Mobile, Alabama
36688.
470
PROTEINS OF THE CRUSTACEAN EXOSKELETON
471
are self-evident, while in the second, it is a treasure because its genome
contains two satellite DNAs, one with the most simple sequence yet
described (poly d[A-T], a repeat unit of two base pairs), the other with
the most complex sequence yet described (a repeat unit of approximately
2,000 bp). Given the land crab's cyclic life style and terrestrial habitat,
its usefulness as a subject for research on molting and its control might
have been anticipated but who could have predicted the gems in its
genome? Their presence makes G. lateralis an ideal single-organism package for studies on DNA satellites.
The title of a recent biography of Barbara McClintock is "A Feeling
for the Organism." The title is equally descriptive of Dorothy Bliss.
DOROTHY M. SKINNER
SYNOPSIS. We describe here some of the components of the exoskeleton
of the decapod crustacean with emphasis on the constituent proteins,
including both structural and enzymatic. All four layers, but particularly
the inner three, of the exoskeletons of four brachyurans contain high
concentrations of proteins <31 kDa; the innermost membranous layer is
especially rich in such proteins. A number of crab exoskeletal proteins
resemble insect cuticle proteins in size (Mr) and isoelectric point (pi). A
further similarity is the cross reactivity of crab exoskeletal proteins with
four different antibodies against cuticular proteins of two species of insects.
One of the small Mr exoskeletal proteins in the Bermuda land crab Gecarcinus lateralis has a similar distribution as a protein of similar size in the
cuticle of the tobacco horn worm Manduca sexta. The partial dissolution
of an old exoskeleton and formation of the two outer layers of a new
exoskeleton are major events in readying a crustacean for the increase in
size that occurs at each molt. Expressing both parallel and sequential
activation of a number of genes, a single layer of epidermal cells that
bounds a crustacean such as G. lateralis synthesizes specific proteins at
different stages of the intermolt cycle as the outermost epicuticle and
exocuticle are formed during proecdysis and as the endocuticle and membranous layer are formed during metecdysis. Finally, two sets of proteinases isolated from integumentary tissues of land crabs degrade the same
exoskeletal proteins in vitro as are degraded in vivo during proecdysis.
INTRODUCTION
As is generally true of all arthropods,
crustaceans are limited in both form and
size by the physical constraints of their exoskeletons. This essay is in large part restricted
to the proteins found in the four-layered
exoskeleton of decapod crustaceans consisting (from outside in) of an epicuticle,
exocuticle, endocuticle and membranous
layer (Fig. 1). Periodic shedding of the old
exoskeleton and synthesis of a replacement
allow for growth and metamorphosis of
external features (reviewed in Drach, 1939;
Skinner, 1985a, b; Stevenson, 1985; Chang,
1989).
j n e integumentary tissues that underlie
the exoskeleton are comprised of an inner
and an outer sheet of epidermis separated
by a layer of connective tissue containing
storage cells, tegumental glands and hemolymph sinuses (Fig. 1; Skinner, 1962). The
first sign of proecdysis in integumentary tissues is the separation of the epidermis from
the old exoskeleton; this occurs as the membranous layer of the old exoskeleton is
degraded. The process has been designated
apolysis (Jenkin and Hinton, 1966) and the
stage in the intermolt cycle in which apolysis occurs, D,. After the epidermal cells
enlarge, they synthesize and secrete a new
D. M. SKINNER ET AL.
PROECDYSIS
" 2 EARLY
^ 2 LATE
ECDYSIS METECDYSIS
/
\
A
B
EPIDERMIS-!
t
FIG. 1. Diagram of cross sections of the exoskeleton and underlying integumentary tissues of G. lateralis during
an intermolt cycle, t In Do, structure of integumentary tissue is indistinguishable from that in C4. ep, epicuticle;
ex, exocuticle; en, endocuticle; ml, membranous layer; t, tegumental gland; cl, cell of Leydig; s, hemolymph
sinus; lp, lipoprotein cell (after Skinner, 1962).
epicuticle and exocuticle in stage D 2 (Skinner, 1962; Stringfellow and Skinner, 1988).
Throughout these processes and continuing
until ecdysis, the inner layers of the old exoskeleton are degraded. In stages Dj^, the
size of the epidermal cells decreases to that
seen in anecdysis. Concomitant with these
events in the exoskeleton, missing appendages are regenerated (Bliss, 1956; Skinner,
1962, 1985a), and muscle in the chelae
undergoes atrophy (Skinner, 1966; Mykles
and Skinner, 1982, 1990). In some marine
Crustacea, such as lobsters, and in other
Crustacea that inhabit environments deficient in calcium, such as land crabs, calcium
removed from the calcified layers of the old
exoskeleton during proecdysis is deposited
by the epithelium that lines the cardiac
stomach to form gastroliths (Travis, 1960).
In G. lateralis, the four gastroliths, which
together can weigh as much as a gram in
late proecdysis, are solubilized within 24 hr
after ecdysis. The calcium is then available
for hardening of the exoskeleton.
After ~ 7 5 % of the old exoskeleton is
degraded, it dehisces at thin sutures whose
strategic placement facilitates exuviation.
At ecdysis, the animal emerges from the old
exoskeleton clad in a newly synthesized soft
exoskeleton comprised of epicuticle and
exocuticle. Following ecdysis, the new exoskeleton is thought to be hardened by tanning agents that move from the hemolymph
into the exoskeletal matrix (Vacca and Fingerman, 1975a, b). There is little if any synthesis of new exoskeleton during stage A of
metecdysis while the initiation of stage B is
heralded by the appearance of endocuticle
lamellae. Endocuticle synthesis continues
through the early stages of C. The completion of the formation of membranous layer
signals the onset of anecdysis, C4. The coordinately controlled set of metabolic events
that precedes the emergence of the animal
in its new exoskeleton is presumably governed by activation of a number of genes
that are quiescent at other stages in the
intermolt cycle. Some of the exoskeletal
proteins described here should serve as
favorable subjects in analyses of the hormonal control of proecdysis.
BACKGROUND
Major components of the crustacean exoskeleton are protein, chitin, and Ca++ salts.
Exoskeletal proteins of a crab Scylla serrata
PROTEINS OF THE CRUSTACEAN EXOSKELETON
(Hackman, 1974) and a crayfish Astacus
leptodactylus (Vranckx and Durliat, 1980,
1986) have been electrophoresed on polyacrylamide gels and the amino acid compositions of mixtures of exoskeletal proteins
have been determined (Hackman, 1974).
With the exception of proteins of A. leptodactylus, some of which were as large as 400
kDa (Vranckx and Durliat, 1986), most of
the crustacean exoskeletal proteins were < 31
kDa (Hackman, 1974; Welinder, 1975).
Prior investigations of the exoskeletons of
six species of crabs, shrimps, crayfish, and
lobster by Welinder (1974, 1975) allowed
him to designate them as "soft" on the basis
of their proportions of chitin and protein
(% dry weight following decalcification). The
exoskeletons had high chitin contents (~6372%) and proteins that were (i) rich in acidic
amino acids, (ii) poor in non-polar amino
acids, and (iii) highly soluble. The epicuticle, rich in lipids, is thought to protect the
inner exoskeletal layers from solubilization
in the animals' aquatic habitats. Although
such characteristics relegate the exoskeleton
of marine crabs, such as Cancer pagurus, to
the group of "soft" exoskeletons, these exoskeletons are heavily calcified and therefore
physically very hard. Another type of
arthropod exoskeleton, designated "hardened," had a low chitin content (25-35%),
and proteins that were (i) rich in non-polar
amino acids and (ii) less soluble; such exoskeletons were found in insects and the
horseshoe crab Limulus polyphemus (Welinder, 1975).
Richards (1951) described "hard" crustacean exoskeletons as being high in protein
and/or calcium and "soft" exoskeletons as
being richer in chitin and low in calcium.
The distinguishing features of rigid vs. flexible insect cuticles are specific types of proteins (Cox and Willis, 1985, 1987a). Flexible insect cuticles, such as abdominal
intersegmental membranes, are rich in proteins with acidic pis (pH 4.4-5.0), while rigid
cuticles, such as larval tubercles, head capsules or pupal wings, are missing such proteins (Cox and Willis, 1985, 1987a, b; Willis, 1987). From analyses of mixtures of
proteins it was concluded that soft crustacean exoskeletons are also rich in acidic
473
amino acids (Welinder, 1975). It is apparent
that Welinder's "soft" and "hardened"
would be equivalent to Willis' "flexible" and
"rigid" descriptive terms for insect cuticles.
GENERAL STRUCTURE AND COMPOSITION OF
THE EXOSKELETON
The thicknesses of the four layers of the
exoskeleton of G. lateralis are: epicuticle,
~7 fim; exocuticle, 30 nm; endocuticle, 200400 nm; and membranous layer, 20-30 nm
(Skinner, 1962); the four layers account for
15, 8, 75, and 2% of the dry weight of the
exoskeleton, respectively. Protein concentration and percent organics increase progressively in each deeper layer (O'Brien et
ai, 1991). Afifthexoskeletal layer, the ecdysial membrane, appears early in proecdysis
according to Travis (1960) and is shed as
the lining of the exuviae of insects and decapod crustaceans.
STRUCTURAL DIFFERENCES AMONG THE
EXOSKELETONS OF DIFFERENT
CRUSTACEAN SPECIES
Although reviews often treat the four
cuticle layers of decapods as characteristic
of all crustacean exoskeletons, many crustaceans may be missing one or more of the
four layers. For example, exoskeletons of
parasitic barnacles (Sacculina carcini, Loxothylacus panopei, and Ulophysema oeresundense) consist of only a thin epicuticle
(Hubert et ai, 1979; Bresciani and Jespersen, 1985). The exoskeletons of small uncalcified crustaceans such as the copepod Calanus finmarchicus (Raymont et ai, 1974)
and the cladoceran Daphnia magna (Halcrow, 1976) are described as having only
"inner" and "outer" layers. Exoskeletons of
other crustaceans have been described as
having three layers (epi-, exo-, and endocuticle) with no mention of a membranous
layer. These include cirri of the barnacle
Balanus balanoides (Koulish and Klepal,
1981), branchial carapaces of early postlarval lobsters Homarus americanus (Arsenault et ai, 1984), and gills of the brown
shrimp Penaeus aztecus (Foster and Howse,
1978). In contrast, there is no epicuticle on
the gills of the brachyuran Ocypode platytarsis, the shrimp Metapenaeus monoceros,
474
D. M. SKINNER ET AL.
B
i— EPICUTICLE-"
—ENDOCUTICLE—"-MEMBRANOUS LAYER
t-EXOCUTICLE-H
Ca. T.n. P. p. G.I. Ca. T.n. Pp. G. I.
Ca. T.n. Pp.
G.I.
Co. T.n. Pp. Gl.
kDa
kDa
-54
48
-88
-70
-55
-28
^25
5
A?23
'22
72
-14
-11
FIG. 2. Exoskeletal proteins from the four layers of the exoskeletons of four species of anecdysial brachyurans.
(A), (B) proteins electrophoresed on 9-18% ID SDS-polyacrylamide gels and stained with silver (Wray et al.,
1981). Cancer antennarius, C. a.; Taliepus nuttalli, T. n.; Pugettia producta, P. p.; G. lateralis, G. 1. Panel (A)
epicuticle and exocuticle; Panel (B) endocuticle and membranous layer. (From O'Brien et al., 1991.) Panel (C)
Laser densitometer scans of selected lanes of panels (A) and (B). Approximate Mr (kDa) adjacent to protein
peak. Densitometer tracings of photographs of gels of membranous layer proteins are shown in Figure 3.
the anomuran Emerita asiatica, and the isopod Ligia exotica. The absence of a lipidrich epicuticle as an external layer may
account for the high permeability of the gills
of these organisms to water (Mary and
Krishnan, 1974). No membranous layer was
observed in the three-layered exoskeleton
ofEuphausia superba (Buchholz etal, 1989
as cited in Buchholz and Buchholz, 1989).
It is in the heavily calcified exoskeletons
of decapod crustaceans, such as the spiny
lobster Panulirus argus (Travis 1954, 1955)
and crabs G. lateralis (Skinner, 1962) and
Uca pugnax (Green and Neff, 1972), that a
membranous layer is well developed, at least
in post-larval stages. Although investigators
often use the same terms to identify crustacean exoskeletal layers, it is unclear
whether the innermost "endocuticle" of an
uncalcified crustacean exoskeleton is
475
PROTEINS OF THE CRUSTACEAN EXOSKELETON
homologous to the calcined "endocuticle"
or to the innermost uncalcified membranous layer of the decapod exoskeleton. The
relationships and homologies of these layers
in different crustacean species await analyses of (at least partial) amino acid sequences
of the proteins that comprise them as has
been done for a number of cuticle proteins
of several insects (reviewed in Willis, 1987,
1989). Whatever the particular composition
of the exoskeleton of a species of Crustacea,
at each molt, that exoskeleton is partially
degraded and replaced by a newly synthesized exoskeleton.
MEMBRANOUS LAYER
25i|23
8-
CRUSTACEAN EXOSKELETAL PROTEINS
We have devised methods to separate the
individual layers of the brachyuran exoskeleton (O'Brien et ai, 1991); these permit
the identification of particular proteins in a
particular exoskeletal layer. Proteins were
extracted from all four layers either separately or combined, in guanidine thiocyanate (5 M), a chaotropic agent which is a
strong denaturant (Chirgwin et al., 1979;
Stringfellow and Skinner, 1988). Proteins in
exocuticle (Fig. 2A), endocuticle and membranous layer (Fig. 2B) of anecdysial specimens of four brachyurans (the rock crab
Cancer antennarius, the southern shieldbacked kelp crab Taliepus nuttalli, the
shield-backed kelp crab, Pugettia producta,
and the land crab G. lateralis; O'Brien et
ai, 1991) are predominately <31 kDa (see
Fig. 2C for scans of selected lanes of Fig.
2A and B). The small Mr proteins were qualitatively indistinguishable in scans of the
membranous layers of all four species (Fig.
3). Although these small proteins form only
a few very intense bands on ID gels, some
of them separate into numerous spots
(charge trains) on 2D gels because of differences in net charge.
Some major proteins with similar M,s and
pis occur in all four exoskeletal layers of G.
lateralis; others in only one or two layers.
Proteins with MrS and pis similar to crab
exoskeletal proteins also occur in insect
cuticles. Many proteins of the crustacean
exocuticle and endocuticle are similar to
each other in Mr and pi; they are also very
similar to proteins of the membranous layer.
Epicuticle from pigmented regions of the
exoskeleton contains more unique exoskel-
DISTANCE (cm)
FIG. 3. Densitometer tracings of membranous layer
proteins of the four species of brachyurans as described
in Figure 2C: Gecarcinus lateralis, Cancer antennarius,
Taliepus nuttalli, Pugettia producta. In each scan note
qualitative identity of Mr of most of the protein bands
ranging from 31 to 10 kDa in all four species. Definitive
evidence for the identity of the similarly-sized proteins
would require determination of at least part of the
amino acid sequence of each protein.
etal proteins (on both ID and 2D gels) than
any other layer; this includes five proteins
(designated the epicuticular quintet; O'Brien
et ai, 1991) that range in size from 54 to
42 kDa and three proteins of 22.1, 21, and
476
D. M. SKINNER ET AL.
19.7 kDa, designated the ~20 kDa trio.
Unlike the epicuticular quintet, no member
of the trio stains with either silver or Coomassie blue or can be iodinated; all members
do react with antibodies to cuticular proteins of insect larvae. The epicuticle also has
three times as many proteins that bind Ca++
in vitro as any of the other layers (O'Brien
et al, 1991). In calcium binding analyses,
proteins were separated by SDS-PAGE,
transferred to nitrocellulose membranes,
washed with appropriate buffers, incubated
with 45Ca++, rinsed to remove unbound
45
Ca ++ , dried, and autoradiographed
(Maruyama et al., 1984).
INVESTIGATOR-INITIATED CONTROL OF
MOLTING
In Crustacea, proecdysis is regulated by
increasing titers of 20-hydroxyecdysone
(20HE) in the absence of a molt inhibiting
hormone (MIH) that is present during anecdysis (Freeman and Costlow, 1979; Skinner,
1985a, b; Quackenbush, 1986; Webster,
1986;Fingerman, 1987; Chang, 1989; Chang
etal, 1990;Jegla, 1990). Hormonal control
of molting is discussed in detail by P. M.
Hopkins (1992) elsewhere in this volume.
Unstimulated, large specimens (60 to 100
g) of G. lateralis molt approximately once
per year. In the study of intermolt cyclerelated characteristics of exoskeletal proteins, it has been convenient to be able to
control molting in the laboratory. Precocious molts can be triggered by removal of
MIH by ablating eyestalks (Zeleny, 1905a,
b\ Smith, 1940; Skinner and Graham, 1972;
Holland and Skinner, 1976; Kleinholz,
1976), a surgical treatment that many species of crustaceans do not survive. Land
crabs, and at least 15 other species of
brachyurans (reviewed in Skinner, 1985a),
can be propelled into a precocious molt if
they lose more than four limbs. These can
be walking legs or a combination of chelae
and walking legs (Skinner and Graham,
1970, 1972). Once initiated, proecdysis may
be interrupted if one or more limbs or limb
buds are lost prior to a critical stage, after
which there is no "turning back" (Skinner
and Graham, 1972; Holland and Skinner,
1976). We suggest the critical point is the
separation of the epidermis from the old
exoskeleton at apolysis. The interruption of
proecdysis is not caused by a release of MIH
since it also occurs in animals without eyestalks (Holland and Skinner, 1976). We have
proposed the existence of two limb autotomy factors, LAFan to account for the triggering of a precocious molt in anecdysial
crabs that lose many limbs and LAFpro to
account for the interruption of proecdysis
in crabs that lose a limb before a critical
period (Skinner, 19856). A similar delay in
the time to ecdysis has been described in
premolt megalopae of the mud crab Rhith-
ropanopeus harrisii that lose a limb
(McConaugha and Costlow, 1987), evidence, the authors say, for the presence of
LAFpro.
Multiple autotomy is effective in triggering precocious molts in R. harrisii, decreasing the duration of an intermolt cycle from
40 to 16 days. Interestingly, however, as an
apparent consequence of parasitization by
the rhizocephalan Loxothylacus panopei,
mud crabs do not molt and cannot be
induced to molt even by multiple limb
autotomy (O'Brien and Skinner, 1990); parasitization by rhizocephalans can mimic the
effect of LAFpro. Parasitized crabs with limb
regenerates have been observed in the field
(Hartnoll, 1967; O'Brien and Skinner, 1990).
It seems possible that rhizocephalans that
invade animals already in proecdysis,
including those bearing regenerating limb
buds, continue to mimic the effects of
LAFpro.
THE ROLE OF THE EPIDERMIS IN THE
TURNOVER OF THE EXOSKELETON
Marked cytological changes and hypertrophy in the epidermis correlate with both
the degradation of the old exoskeleton and
formation of the new epicuticle and exocuticle. Consequently the epidermis has been
presumed to be the site of synthesis of these
proteins (Skinner, 1962; Green and Neff,
1972) as it is in insects (Roberts and Willis,
1980; Willis et al, 1981; Riddiford, 1982;
Silvert et al, 1984). In early experiments,
integumentary tissues from G. lateralis in
metecdysis (stage B) that were incubated in
vitro synthesized layers of endocuticle (Mayo
and Skinner, 1972; and see Fig. 2 in Stringfellow and Skinner, 1988). Autoradiograms
477
PROTEINS OF THE CRUSTACEAN EXOSKELETON
of sections of integumentary tissue removed
from the branchiostegites of G. lateralis at
different stages of the intermolt cycle and
incubated in vitro with radiolabeled amino
acids corroborate the role of the epidermis
in the synthesis of the exoskeleton (Stringfellow and Skinner, 1988). Proteins extracted
from such tissues were analyzed by a number of methods. From their patterns of synthesis at different stages in G. lateralis,
proteins from the exoskeleton plus integumentary tissues may be classified into five
groups (Table 1; and see Stringfellow and
Skinner, 1988).
Group I integumentary proteins have a
wide range of Mr and are present throughout
the intermolt cycle; most of them are synthesized at a relatively constant rate
throughout the cycle. Accordingly, they are
designated "housekeeping" proteins. A
similar set of such housekeeping integumentary proteins (Group II) is distinguished by the increase in their synthesis
from two- tofivefoldduring proecdysis when
formation of the epicuticle and exocuticle
of the new exoskeleton is in progress. That
at least some of the proteins that are highly
radiolabeled during proecdysis are indeed
components of the exoskeleton is suggested
by the presence of protein bands of similar
sizes (31-14 kDa; Stringfellow and Skinner,
1988) in extracts of epi- and exocuticles
recovered from anecdysial G. lateralis (Fig.
2A; and see O'Brien et ai, 1991). Group III
integumentary proteins are synthesized
intermittently from very early proecdysis,
when degradation of the old exoskeleton
begins, and throughout the remainder of
proecdysis. Groups IV and V first appear at
stages in the intermolt cycle that suggest that
they too are structural components of the
new exoskeleton. Group IV integumentary
proteins, synthesized only at the time of epiand exocuticle formation, have a wide range
of sizes. Again, some proteins of the sizes
in Group IV (~200 and ranging from 7 1 16 kDa) are seen in extracts of epicuticles
and exocuticles from anecdysial exoskeletons (O'Brien et ai, 1991). The very large
proteins of Group V are synthesized in stage
B of metecdysis at the time when the formation of the endocuticle begins; extracts
from endocuticles of anecdysial G. lateralis
TABLE 1. Classification of proteins from integumentary tissues and exoskeleton ofG. lateralis.
Group
I:
II:
III:
IV:
V:
Characteristics
Synthesized at relatively constant rate
throughout the intermolt cycle: "housekeeping"
Synthesized as I, except synthesis increases
at least 2 x during one stage of the intermolt cycle
Synthesized during degradation of the old
exoskeleton (specific exoskeletal proteinases?)
Synthesized during epi- and/or exocuticle
and endocuticle formation (pro- or metecdysis) or both
Synthesized during endocuticle formation
(metecdysis)
are rich in proteins of these sizes (177, 164,
155 kDa).
CHARGE TRAINS IN ANECDYSIAL
EXOSKELETAL LAYERS
Proteins that are approximately the same
size but differ by net charge form charge
trains in isoelectric focusing. All four layers
of the exoskeleton from anecdysial crabs had
proteins with acidic pis and some of the
proteins from the three internal layers
formed charge trains (see Fig. 4A and B)
similar to those of proteins extracted from
flexible insect cuticle (Andersen et ai, 1986;
Cox and Willis, 1987a, b). Membranous
layer extracts had the most. From the positions of some of the charge trains in 2D gels,
it appears that membranous layer proteins
are comprised of more neutral than acidic
amino acids (compare Figs. 4A and B). These
data agree with those of Welinder (1975)
who reported fewer acidic amino acids in
proteins extracted from the membranous
layer than from epicuticle + exocuticle or
endocuticle.
Cox and Willis (1987a) described three
groups of proteins from flexible insect cuticle: (1) those with acidic pis, (2) those that
formed charge trains, and (3) those that
formed elongated vertical streaks. Crab exoskeletal proteins showed similarities to the
first two groups of insect proteins; but not
to the third. This is not unexpected since
the second dimension slab gels on which the
crab proteins were electrophoresed lacked
478
D. M. SKINNER ET AL.
138
118
B
138
88
118
88
I 55
28
28
25
24
16
16
23
16
14
14
24
14
14
11
104
10
10
FIG. 4. Exoskeletal proteins analyzed on 2D gels. Autoradiograms of iodinated proteins (McConahey and
Dixon, 1980) from two layers of anecdysial exoskeletons of G. lateralis. SDS gel for second dimension was 918% polyacrylamide, 0-10% glycerol. Mrs (kDa) of proteins are indicated adjacent to spots. (A) Exocuticle, 3 x
106 cpm, 15.2 Mg protein; (B) As (A) except membranous layer, 13.2 ng protein. Autoradiogram (A) exposed 48
hr; (B) exposed 42 hr. (From O'Brien et al., 1991.)
urea; urea can cause streaking (Cox and Willis, 1987a).
Welinder (1975) observed that the exoskeletons of all crustaceans examined were
"soft" arthropod cuticles even those that are
physically hard due to calcification. Our data
indicate that many crustacean exoskeletal
proteins are acidic, similar to those of flexible insect cuticle, thus supporting Welinder's categorization of crustacean exoskeletons as "soft" cuticle.
PROECDYSIS IN VITRO
Attempts were made to mimic physiological conditions by incubating anecdysial
integumentary tissues of A. leptodactylus
(Traub et al., 1987) and G. lateralis (Paulson
and Skinner, 1991) in vitro with concentrations of 20HE characteristic of different
intermolt cycle stages. Synthesis of several
proteins was stimulated in both systems. In
G. lateralis, synthesis of five of 30 protein
bands analyzed was stimulated as much as
eightfold at hormone concentrations ranging from 10~9 to 10~7, the latter characteristic of proecdysis in G. lateralis (Soumoff
and Skinner, 1988). The affected proteins
were 41, 24, 18, 16, and - 1 3 (or 14) kDa.
Consider the effects of 20HE on the pattern of synthesis of one of these proteins, a
~ 24 kDa protein stimulated most by a low
level of 20HE, namely, 10~9 M (Paulson and
Skinner, 1991). Its synthesis would be
expected to increase early in proecdysis as
ecdysteroid titers started to rise. When proteins have been iodinated, a large amount
of a ~24 kDa protein can be seen in membranous layer (Fig. 4B; O'Brien et al., 1991),
which is synthesized during stage C3 when
titers of ecdysteroids have passed their peak
and returned to low levels in vivo (Soumoff
and Skinner, 1988). This stimulation of synthesis of specific proteins by low levels of
20HE in vitro might be mimicking the
response in vivo to the hormonal milieu of
late metecdysis. Synthesis of the 18,16, and
13 kDa proteins, which was maximally
stimulated at 10~7 M in vitro (Paulson and
Skinner, 1991), increased later in proecdysis
(Stringfellow and Skinner, 1988) as predicted. Charge trains of 24, 16, and 14 kDa
demonstrate the presence of numerous pro-
479
PROTEINS OF THE CRUSTACEAN EXOSKELETON
TABLE 2.
Small M, proteins in exoskeletal layers o/G. lateralis during anecdysis, degraded in vivo during
proecdysis.
EPI
kDa
25
23
16
13
11
10
AN
AN
ML'*
ENDO
EXO
EX
EX
AN
EX
o
o
+
+
AN
EPI, epicuticle; EXO, exocuticle; ENDO, endocuticle; ML, membranous layer, * ML is not present in exuvia;
AN, anecdysial exoskeleton; EX, exuviae. Number of + indicates relative amount of protein present. Degree of
proecdysial degradation is indicated by relative difference in number of + in AN and EX; o indicates complete
degradation; blank indicates not seen (or not present).
teins (O'Brien et al., 1991). Since the titer
of 20HE changes with time, the response
pattern also changes. Thus the time-dependent fluctuations in these responses might
reflect different 20HE titers and result in the
large standard errors and broad range of
sensitivity that were reported (Paulson and
Skinner, 1991).
ENZYMES INVOLVED IN THE
DEGRADATION OF THE EXOSKELETON
We have isolated two sets of proteinases
from integumentary tissues of G. lateralis
that degrade the same exoskeletal proteins
in vitro as are degraded in vivo during proecdysis (Tables 2 and 3; and see O'Brien and
Skinner, 1987, 1988). Two cysteine proteinases with alkaline pH optima (ACPs;
O'Brien and Skinner, 1987) and two proteinases with acid pH optima (APs; O'Brien
and Skinner, 1988) have been partially purified. The ACPs and APs degrade specific
exoskeletal proteins when assayed in vitro;
a number of the proteins degraded during
treatment in vitro by the integumentary proteinases are also missing from extracts of
exuviae (Tables 2 and 3). These data suggest
that the integumentary proteinases are active
in vivo during proecdysis.
The ACPs were more effective in digesting membranous layer proteins than were
the APs. With a few exceptions, almost all
of the membranous layer proteins smaller
than 24 kDa were at least partially digested
by the ACPs. Of the four proteolytic activities, ACPI was the most active; it digested
all proteins smaller than 24 kDa including
the 10 kDa protein that was not attacked
by the other proteinases. Because the ACPs
shared characteristics with proteinases present in extracts of membranous layer (O'Brien
and Skinner, 1987), such as pH optima close
to the pH of crab hemolymph (Skinner et
al., 1965), they may play a more important
role in the extracellular digestion of exoskeleton during proecdysis than do the APs.
Nevertheless, both AP activities digested
some of the proteins in extracts from the
membranous layer. The 23 kDa protein was
particularly sensitive; it was digested by both
ACPs and APs. An 11 kDa protein was
digested to a greater degree by AP II than
by AP I. AP II also has characteristics of a
cysteine proteinase, as do the two ACPs to
which the 11 kDa protein was also very susceptible (O'Brien and Skinner, 1987).
Since apolysis can be readily induced in
anecdysial brachyurans by chilling (O'Brien
et al., 1986), and since synthesis of proteinases during the period spent at 0°C should
be minimal, the enzymes responsible for
apolysis may be constitutive. Alternatively,
TABLE 3. Small Mr proteins in the membranous layer
o/G. lateralis degraded in vitro by ACP I, ACP II, AP
I, and/or AP II.
kDa
ACPI
ACP II
API
APII
24
no
no
yes
yes
yes
yes
23
yes
yes
partial
yes
16
partial
partial
yes
13
yes
partial
no
yes
11
partial
yes
partial
yes
10
no
no
no
No, not degraded; yes, totally degraded; partial, partially degraded.
Data from O'Brien and Skinner, 1987, 1988.
480
D. M. SKINNER ET AL.
there may be cold-induced structural
changes in the chitin-protein complexes that
occur in epidermal cells whose extensions
run through the exoskeleton in pore canals
or in the fiber canal system described by
Compere and Goffinet (1987).
In addition to protein, chitin is a major
constituent of the crustacean exoskeleton
(Stevenson, 1985). Chitinase activity has
been demonstrated in integumentary tissues
of two species of crabs (C. pagurus and Maia
squinado) at all stages of the intermolt cycle
(Jeuniaux, 1959); such activity has also been
isolated from green crabs Carcinus maenas
(Lunt and Kent, 1960). Calcium-dependent
proteinases, biochemically distinct from
proteinases extracted from integumentary
tissues, appear to be responsible for the
proecdysial atrophy of chela muscle in both
G. lateralis (Skinner, 1966; Mykles and
Skinner, 1983; Mykles, 1992) and H. americanus (Mykles and Skinner, 1986).
A CRUSTACEAN EXOSKELETAL PROTEIN
WITH CHARACTERISTICS SIMILAR TO
THOSE OF AN INSECT CUTICLE PROTEIN
A 28 kDa protein is one of the principal
proteins confined to the innermost membranous layer in G. lateralis; a protein of
similar size (27 kDa) is found only in the
innermost layers of the M. sexta last instar
larval cuticle (Wolfgang and Riddiford,
1986). There is strong cross reactivity of a
polyclonal antibody against larval cuticle
proteins (LCP) of M. sexta (Riddiford, 1982;
antibody the kind gift of L. M. Riddiford)
to a 28 kDa protein in crab membranous
layer (Kumari and Skinner, 1992). The
lamellae of the crab endocuticle are quite
thick (~10 nm); those of the membranous
layer are only 0.1-0.3 x as thick. Similarly,
the outer endocuticular layers in the insect
cuticle are thick (~ 1 ^m) and those of the
inner endocuticle are 5 to 10-fold thinner.
At the time when the 27 kDa protein is
synthesized in M. sexta larvae, the thickness
of the newly synthesized innermost layers
of the insect cuticle decreases (Wolfgang and
Riddiford, 1986). Insects have not been
described as having a membranous layer.
However, the morphological similarities of
the thickness of the lamellae, the cross-reactivity of M. sexta anti-LCP with the 28 kDa
crab protein, and the presence in abundance
of a similarly-sized protein in the innermost
layers of the cuticles of these two classes of
arthropods, leads to the conjecture that the
innermost layer of insect cuticles may be
closely related to the membranous layer of
crustaceans. Hormonal control of the synthesis of the 27 kDa protein has also been
investigated in M. sexta; the protein is synthesized at the larval to pupal transition at
a time following a burst of 20HE and absence
of juvenile hormone (Wolfgang and Riddiford, 1986). The pattern of hormone titers
that control the appearance of the 28 kDa
protein in crab exoskeleton has not been
established. Although this membranous
layer protein is synthesized in C3 when the
titers of 20HE generally are low, Hopkins
(1988) has detected a small peak of 20HE
during Do in a fiddler crab Uca pugilator.
Since the duration of the intermolt cycle is
only ~40 days in U. pugilator (Skinner and
Graham, 1972), and D o is relatively long
(i.e., ~30 days in a ~55 day proecdysial
period in G. lateralis), the small peak of
20HE may coincide with or even trigger
synthesis of the 28 kDa protein in the land
crab. The parallels are intriguing but the
relationships between the insect and crab
~27 kDa proteins will not be definitively
established until at least part of the amino
acid sequences of the two have been determined.
EPITOPE SHARING AMONG CUTICLE
PROTEINS OF A CRAB
(G. LATERALIS) AND TWO INSECTS
MELANOGASTER)
(M. SEXTA AND D.
As the next step in determining whether
crustacean exoskeletal proteins and insect
cuticle proteins are related, we are using
immunochemistry (Kumari et al., 1990). In
brief, proteins from the four exoskeletal layers of crabs at different stages of the intermolt cycle are analyzed on Western blots.
Proteins are electrophoresed on ID gels,
blotted on PVDF membranes (Graddis,
1990) and fixed with 0.5% glutaraldehyde
which prevents the loss of small Mr proteins
(Karey and Sirbasku, 1989). The membranes are then exposed to polyclonal antibodies against insect cuticle proteins. The
antibodies are against larval cuticle proteins
481
PROTEINS OF THE CRUSTACEAN EXOSKELETON
(LCP) and pupal cuticle proteins (PCP) of
M. sexta (Riddiford, 1982; gifts of L. M.
Riddiford) and against third instar larval
(L3) cuticle proteins L3CP 1 + 2 (both 17.5
kDa; different pis; Snyder et ai, 1981) or
L3CP 3 (9 kDa) + 4 (11 kDa) of D. melanogaster (Silvert et ai, 1984; gifts of W. J.
Wolfgang). The D. melanogaster antisera
cross react with all of the major larval cuticle proteins of D. melanogaster and some
of M. sexta (Wolfgang, personal communication).
As evidence of specificity, in our immunochemical analyses neither insect antibody
cross reacts with all of the small (<31 kDa)
crab exoskeletal proteins. Furthermore, M.
sexta anti-PCP reacts hardly at all with any
crab exoskeletal protein at any stage of the
intermolt cycle whereas M. sexta anti-LCP
reacts intensely with several of the major
crab exoskeletal proteins in all stages. Recognition of different protein bands by different antibodies and differential response
of crab exoskeletal proteins to different types
(larval and pupal) of antibodies strengthen
the argument for specificity (Kumari and
Skinner, 1992).
The absence of reactive small Mr proteins
from immunoblots shown in Stringfellow
and Skinner (1988) may be due to their loss
from nitrocellulose membranes particularly, as was the case in those analyses, when
the proteins have not been fixed to the
membranes with glutaraldehyde. As
described above, we now use PVDF membranes and, after electrotransfer, fix the proteins with 0.5% glutaraldehyde.
Only the data describing cross reactions
of anti-Drosophila L3CP 3 + 4 are included
here. This antibody reacts quite intensely
with a number of anecdysial epicuticular
proteins (from Mr 9.7 to 229 kDa; Fig. 5)
and early proecdysial exoskeletal proteins.
The reactive proteins include both the epicuticular quintet and trio; the antibody also
reacts with proteins <39 kDa in the other
three exoskeletal layers (Fig. 5). Reactive
proteins from late proecdysial animals and
exuviae are almost completely restricted to
the epicuticle; they, too, include the quintet
and trio (data not shown).
In general, larval cuticle antibodies cross
react more than pupal cuticle antibodies.
P
kDa
X
N
ML
kDa
FIG. 5. Proteins from the four layers of the exoskeleton of anecdysial G. lateralis cross react with antibody
against D. melanogaster third instar larval cuticular
proteins 3 + 4 (L3CP 3 + 4). Autoradiogram of immunoblot. P, epicuticle; X, exocuticle; N, endocuticle; ML,
membranous layer. Proteins were electrophoresed on
a 9-18% gel, transferred to a PVDF membrane, fixed
with glutaraldehyde, blocked with BLOTTO, and
exposed to anti-L3CP 3 + 4. The membrane was washed
in phosphate buffer-saline (PBS), exposed to iodinated
protein A, washed in PBS plus 0.1% Triton, washed
again in PBS, dried, and exposed to X-ray film (Kodak
XAR-5) for 92 hr at -80°C. (After Kumari and Skinner, 1992.)
Finally, although we have not ruled out the
possibility that some of the immunoreactive crab exoskeletal proteins are glycosylated, as observed for some insect cuticle proteins (Cox, 1987; Cox and Willis, 19876),
the pattern of interactions of crab exoskeletal proteins with polyclonal antibodies
against insect cuticle proteins suggests that
some exoskeletal proteins may be evolu-
482
D. M. SKINNER ET AL.
tionarily conserved in members from these
two arthropod classes. Amino acid analyses
of the reactive crab exoskeletal proteins and
comparison with amino acid sequences of
similarly-sized insect cuticle proteins should
indicate whether the epitopes common to
both classes of arthropods are amino acid
sequences or structural characteristics
(Kumari and Skinner, 1992).
ACKNOWLEDGMENTS
We thank the Bermuda Biological Station
for providing specimens of G. lateralis.
Research in D.M.S.'s lab has been supported by grants from the National Science
Foundation, the Muscular Dystrophy
Foundation, NIH training grants and the
DOE under contract DE-ACO5-84OR21400 with Martin Marietta Energy Systems, Inc.
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