The amino acid composition of the various types of cuticle of Limulus polyphemus

J. Insect Physiol., 1969, Vol. 15,pp. 495 to 507. Pergamon Press. Printed in Great Britain

THE AMINO ACID COMPOSITION OF THE VARIOUS TYPES OF CUTICLE OF

LIMULUS PETER KARLSON,l A. GLENN RICHARDS,2

POLYPHEMUS KALLIOPE E. SEKERI,i and PATRICIA A. RICHARDS2

‘Physiologisch-chemisches Institut der Philipps-Universitit, Marburg/Lahn, Germany, and *Department of Entomology, Fisheries and Wildlife, University of Minnesota, St. Paul, Minnesota (Recked

9 August 1968)

Abstract-Amino acid analyses are presented for various fractions of the carapace and separately for epicuticle and procuticle of arthrodial membrane of the horseshoe crab, Limulus polyphemus L. The carapace is characterized by high percentages of glycine, alanine, and tyrosine; the epicuticle is high in glycine and especially tyrosine; the procuticle of soft membrane is notably high in aspartic and glutamic acids. Of the common amino acids, tryptophan and cystine are absent. In contrast to other structural proteins but in general agreement with other reports on arthropod cuticle, the proteins contain high percentages of phenyl, dicarboxylic, diamino, and other acids which make bulky side groups on the peptide chains. In the carapace there are no striking differences in amino acid composition between outer, middle, and inner fractions although both dicarboxylic and diamino acids are somewhat higher in the dark outer portion. It follows that the events of sclerotization in this species are not reflected by amino acid composition, The data also give no support to the idea that tyrosine contributes to make a ‘self-tanning protein’ in cuticle. The data support previous suggestions that the epicuticle over sclerites is different from that over intersegmental membranes. Histological preparations show that the brown portion of the sclerotized carapace of Limulus is different from darkened sclerites of insects in that it can be stained with both acid fuchsin and orange G.

INTRODUCTION

cuticle is a highly complicated structure. By light microscopy several layers can be distinguished in hardened and darkened cuticles. The primary subdivision of the cuticle is into a relatively thin outer layer that is secreted first and lacks chitin, the epicuticle, and a much thicker inner portion which contains chitin, the chitin-protein cuticle or procuticle and its subsequent modifications (see LOCKE, 1964). In a number of insect groups where the development of the cuticle has been studied in detail, the cuticle has been shown to undergo a series of changes after being secreted, as diagrammed in Fig. 1. This is diagrammed pictorially in Fig. 2. According to this scheme, exocuticle, mesocuticle, endocuticle, THE ARTHROPOD

495

P. KARLSON, K. E. SEKERI, A. G. RICHARDS, AND P. A. RICHARDS

496

and arthrodial membrane are all differentiated from a common precursor by a special series of processes (for details of the chemistry of cuticle tanning see KARLSON and SEKERIS, 1962; BRUNET, 1965; SEKERIS and KARLSON, 1966; for details of the histological picture see RICHARDS,1967). __---/

----

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FIG. 1. Scheme of the minimal

number of stages involved in the sclerotization of the cuticles of insects, baaed largely on histological studies. The epicuticle is not included. For full discussion see RICHARDS(1967)

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FIG. 2. Pictorial representation of the sequence of steps for complete sclerotization of the chitin-protein portion of cuticle with changes beginning at the outer surface and proceeding inwardly. This seems to be the sequence for Limulus. For full discussion and other known sequences see RICH.IRDS(1967).

AMINO

.4CID COMPOSITION

OF TYPES OF CUTICLE

OF LINULUS

POLYPHEMUS

497

Chemically, uncalcified cuticles are composed of chitin which makes up 20 to 60 per cent of the dry weight and of proteins which make up most of the remainder. Lesser amounts of lipids, pigments, and other compounds may be present. While chitin is believed to be more or less the same compound wherever it is found in animals or plants, the proteins are diverse. Even within the cuticle of a single species extraction and fractionation have revealed a number of different proteins with varying ease of extraction (HACKMAN, 1964). All preceding chemical studies have, however, treated the cuticle as an entity. Indeed, in most arthropods it is virtually impossible to obtain separated samples of the various layers. A favourable exception is the very thick (l-3 mm) cuticle of the horseshoe crab, Limulus poZyphemus, which can be split mechanically into different layers that consist of histologically identifiable fractions of the cuticle. We have analysed these separable layers and present data here on the amino acid composition. Further studies are planned. MATERIALS Preparation

AND METHODS

of cutick samples

All animals (Limulus polyphemus L.) were collected at the Marine Biological Laboratory, Woods Hole, Massachusetts, and shipped to Minnesota. Those used were all large females with a carapace diameter of 25 to 27 cm. The animals were cut open alive and eviscerated. For samples of sclerotized cuticle, the inner surface of the central portion of the carapace was scraped with a dull knife and then swabbed repeatedly with soft cloth to remove the bulk of the epidermal cells. The eye regions were cut out and discarded. The carapace was then cut. into pieces of convenient size (ca. 3 cm2), muscle attachment regions were separated from regions with no muscle attachments, and the pieces were immersed in 96% ethanol. The above procedures were done with such rapidity that the samples did not dry. They were then swabbed repeatedly in several changes of 96% ethanol at room temperature until the inner surface of the cuticle appeared to be completely clean. Using a razor blade the pieces were split manually (not cut) into outer, middle, and inner layers. Microtome sections of sample pieces showed that the splitting was approximately but not perfectly along the lines indicated in Fig. 3C. It was, of course, not possible to clean the large pore canals that traverse the cuticle of Limulus. However, both light and electron microscopy suggest that the pore canals of the cuticle of old individuals of Limulus do not contain cytoplasmic filaments. All samples contain some contamination from the chemically unknown contents of the ducts of dermal glands, but these are present in all samples and hence would not contribute to the recorded differences between samples. For samples of isolated epicuticle and procuticle advantage was taken of the fact that the procuticle of joints (commonly called arthrodial membrane) of Limulus swells in distilled water whereas the epicuticle does not. After several days at room temperature the swelling has become so great that the procuticle gradually tears itself off the epicuticle (the procuticle swells to five to ten times its original volume, and if the soaking is prolonged to several weeks the procuticle eventually 32

498

P. KARLSON, K. E. SEKERI, A.

G. RICHARDS, MD P. A. RICHARDS

disperses to give a milky fibrous suspension). For the preparations used, the membrane between abdomen and telson was cut free and soaked in distilled water, with several changes to fresh water, for 6 days. The resulting samples were stored either in 96”,/0ethanol or in distilled water plus an excess of chloroform. Samples were prepared both in 1963 and 1966. Those prepared in 1963 were air-dried after cleaning and shipped to Marburg dry; those prepared in 1966 were never allowed to dry. The samples analysed may be itemized as follows : 1. Scrapings from the outer surface of the carapace after cleaning in ethanol. It includes a mixture of epicuticle and the outermost parts of the exocuticle (Exo,). 2. Split-off outer portion of the carapace from areas with no muscle attachments. It includes epicuticle and exocuticle (Exe, + 1s*), see Fig. 3C. 3. Split-off middle portion of the carapace from the same pieces used for samples numbers 2 and 4. It consists of the outer part of the amber exocuticle (Exo,) with a little mesocuticle around the pore canals. 4. Split-off inner portion of the carapace from the same pieces used for samples numbers 2 and 3. It consists of the inner part of the amber exocuticle (Exo,), again with mesocuticle outlining the pore canals. 5. Entire cuticle from areas of the carapace with muscle attachments. It includes epicuticle, exocuticle (Exo,, c2’+1) and the embedded tonofibrillae. Because of the tonofibrillae these areas are not readily split into outer, middle, and inner fractions. 6. Epicuticle from the membrane between abdomen and telson tom free by swelling of the procuticle in distilled water. -4fter.the epicuticle was stripped off it was swabbed repeatedly on the inner surface to remove adhering fragments of procuticle; even so it is likely that small amounts of procuticle remain as contaminants but a small amount of contaminant would not significantly change the values given in the tables. Stored and shipped in water plus an excess of chloroform. 7. Procuticle from membrane between abdomen and telson. This is the procuticle from the pieces that supplied epicuticle for number 6. Eliminating the small sclerotized areas, this procuticle is of glassy transparency and invisible in water; on transfer to ethanol the pieces become milky white (see Fig. 3B). The sample listed in the tables as ‘1966A’ was transferred to ethanol for storage and shipping; the sample listed as ‘1966B’ was transferred to water pIus an excess of chloroform. Several pieces from each of lots 2-7 were sectioned with a freezing microtome, stained with Mallory’s triple connective tissue stain, and examined microscopically. Chemical procedures For amino acid analyses of entire pieces (Table l), the samples were dried, powdered, and extracted in a Soxhlet apparatus with petroleum ether to remove lipids. They were then hydrolysed with 6 N HCI at 110°C for 18 hr in sealed tubes filled with nitrogen. The hydrolysates were evaporated to dryness and then redissolved in a citrate buffer at pH 2.2.

AMINO

ACID

COMPOSITION

OF TYPES

OF CUTlCLE

OF

LIMULUS

POLYPHEMUS

499

Since carbohydrates often interfere with amino acid analyses of proteins it seemed desirable to remove the chitin fraction of the cuticle. Therefore other pieces from the same samples were extracted with 1 N NaOH at 40°C for 24 hr, and the extracted protein precipitated by the addition of HCl. The precipitate was dialysed and then lyophilized to 80 per cent dry. This protein was then hydrolysed in the same manner as the entire pieces (Table.2). Check determinations with other pieces showed that this procedure extracted approximately three-quarters of the alkali extractable material. Portions of the hydrolysate from each sample were put on top of the separation column of an amino acid analyser (‘Unichrom’, Beckman Instruments Inc.). The total hydrolysate applied to the column for each analysis was approximately 1 to 2 mg. The data are stated as moles per cent, i.e. the percentage of each individual amino acid in molar terms. This value is independent of the amount of hydrolysate applied to the column. Methionine is easily destroyed; hence estimates for it are given only from the NaOH extracts. To obtain information on tryptophan which is destroyed by acid hydrolysis, other pieces of the inner fraction of the carapace (Exo,) were subjected to alkaline hydrolysis and determinations made by a modification of the method of OPIENSKABLAUTH et al. (1963) by Professor L. M. Henderson of the Department of Biochemistry, University of Minnesota. Tests involving the quantitative recovery of added tryptophan showed that the method would have detected tryptophan if the protein contained 0.05 per cent or more. Since the determinations from our sample were all negative, tryptophan is presumably absent. Determinations of nitrogen and sulphur were made by the Mikroanalytisches Laboratorium in the Max Planck Institut fur Kohlenforschung, Miilheim (Ruhr). Some nitrogen determinations by micro-Kjeldahl were made at the University of Minnesota, and additional sulphur determinations by the Clark Microanalytical Laboratory, Urbana, Illinois. It must be remembered that since all values are in moles per cent, the absolute increase or decrease of any one amino acid has the effect of raising or lowering the per cent values of all other amino acids. RESULTS Material has not been available for examining the sequence of changes throughout a mouit cycle (Fig. 1). However, examination of sections from different body regions and from the carapace of a single young individual are consistent with the supposition that the cuticle of the carapace of Limuh goes through a series of changes such as diagrammed in Fig. 2. This is a sequence known from insect cuticles that become sclerotized through their entire thickness (RICHARDS, 1967). The three principle types of cuticle found on old individuals of Limuh are diagrammed in Fig. 3. There is one outstanding difference between the cuticle of the Limulus carapace and completely sclerotized insect cuticles. The brown exocuticle of the Limuh

P. KARLSON,

500

K. E. SEKERI, A. G. RICHARDS, AND P. A. RICHARDS

rnlddlJ

Em,

fraction

EXO:

c FIG.

3. The

types of cuticle found on large old female individuals of Limulus L. A. Section through small sclerite in the membrane at the base of the telson. Equivalent to Fig. 2C. B. Section of membrane at base of telson. Equivalent to Fig. 2A. C. Section of carapace of cephalothorax (some areas and some specimens have no black layer = no Exe,). Equivalent to Fig. 2F. Epi, epicuticle; Exo, exocuticle ; Meso, mesocuticle ; Endo, endocuticle.

polyphams

carapace stains with both acid fuchsin and orange G. The mesocuticle of insects stains with either acid fuchsin or orange G but not with both when used in the Mallory procedure; the exocuticle of insects is completely refractory to staining. Hence this layer is labelled ‘ExoZ ’ instead of Exo, in Fig. 3C. Presumably something not found in insect cuticles is added to the brown cuticle of Limulus carapaces to permit this staining. But whatever it is does not produce any significant difference in amino acid analyses (Tables 1 and 2). Only some areas of some specimens have an outer black layer (Exo,). Many sections show only brown and amber layers under the epicuticle. The amber exocuticle (Exo,) is clearly divided by a sharp line into two parts but the material on both sides of this line appears to be the same. In some specimens the middle fraction of the cuticle is less clearly sub-layered into two, three or four parts (sections of twelve different pieces from three different animals examined). Perhaps these result from temporary interruptions in cuticle secretion (conceivably the inner fraction is post-exuvial). Of more interest, the numerous pore canals in the unstained amber layers are outlined by staining with acid fuchsin. By definition, then, the pore canals could be said to be lined with mesocuticle. The membrane at the base of the telson was studied because it supplies a relatively large piece. At least in terms of swelling in water the other joint membranes are similar. Small sclerites in this membrane show the common

7.05 4.83 5.02 3.10

5.15 340 4.49 3.94

4.36 4.02

4.46 4-12

1966.4

was stored

=

1966B

1966

4.37 3.13 8.30 2.80

4.94 2.24

4.50 2.58

1344 -

14.5 0.18

99.99 99.98

2.96 6,65

3.17 I.64

II*05 11.90 5.21 6.29

3.58 2.56 8.08 5.21

15.03 16.27 14.62 14.03 8.08 7.63 7.57 7.30 4.60 3.70

1963

1966

3.71 3.09 7.62 2,78

4.43 2.16

3.30 2.06

12.79 -

14.2 0.09

100~01 IOO~UO

3.82 1.76

2.65 2.06

12.06 II.12 4.90 7.62

6.48 2.35 7.95 2.84

13.92 17.71 15.69 14.73 12.45 8.24 ;4: jl.;;

1963

Inner fraction Epicuticle

Arthrodial

10.14 0.17

100.03

3.00 3.94

9.14 8.04

19.37 5.83

2.68 3.94 6.30 1.73

13.39 6.30 5.67 5.98 4.73

-

100~01

3.54 5.24

9.40 7.70

20.80 6.01

2.31 3.09 5.86 2.00

13.56 5.55 5.08 5.55 4.32

1966A 1966B

of chloroform.

0.23

99.99

2.46 1.43

5.83 3.99

7.37 4.30

4.81 3.89 8.49 1.84

16.38 13.51 8.70 12.38 4.61

1966

Entire with tonofibrillae

given as A and B. was stored in water with an excess

lOO*OO

3.74 2.49

7.79 4.52

determinations

13.5 0.13

in 96 “/6 ethanol,

12.95 Duplicate

I

* Procuticle

12.73 -

1OOtIO100.01

7.58 4.94

6.11 4.35

99.99 100.02

9.75’ 2.90 >

10.19 IO.73

Il.87 10.22 9.87 9.76 12.62

5.45 2.96 8.57 3.58

5.89 4.73 6.27 3.38

3.61 1.64 8.32 4.49

4.59 3.10 6.77 0.69

4.11 2.47 8.81 0.71

“9::;

;:g

1966B 17.13 14.33 6.70 7.32 2.80

1963 1966A 1500 18.63 12.49 9.27 6.79 3.47

16.80 16.07 10.93 II.60 6.23 7.24 6.46 6.08 2.70 2.98

1963A 1963B

Middle fraction

Carapace

ACID ANALYSESOF CUTICLE OF Limulus polyphemus

Outer fraction

I-AMINO

Values are in moles per cent.

y0 Nitrogen S: Sulphur

Totals

Aliphatic Glycine Alanine Valine Leucine Isoleucine Substituted aliphatic Serine Threonine Proline Histidine Phenyl Tyrosine Phenylalanine Dicarboxylic Aspartic acid Glutamic acid Diamino Arginine Lysine

Amino acids

Scrapings of outer surface

TABLE

$ i3

a7 :: 3

4.42 7.62

14.50 9.58

5.90 5.16

4.91 5.41 6.39 295

6.63 6.88 4.67

“8.;;

10.56 0.21

--

99.97 IOwOl

7.33 6.02

12.56 9.95

6.28 6.02

3.14 4.19 7.59 5.24

6.80 6.80 6.28 6.80 4.97

$ 2

% $

: I;;

3 g 0 q $?

8

2

2

i2

1966A 19661% g I?

Procuticle*

membrane

so2

P.

KARLSON,

K. E.

SEKERI,

A. G. RICHARDS,AND P. A. RICHARDS

sclerotization picture diagrammed in Fig. 3A. These were not analysed chemically. The translucent parts of this membrane stain a pure blue with Mallory’s stain. Such areas (Fig. 3B) supplied the procuticle analysed. Measurements on sections from ten different pieces of whole carapace, stained dehydrated, and mounted in resin were: epicuticle 10 to 20 p, exocuticle, 2.5 to 35 p (when present), ‘exocuticles’ 440 to 500 TV,exocuticle, 730 to 860 p (outer portion 500 to 600 p*, inner portion 230 to 260 EL). These particular samples after dehydration, then, were 1.2 to 1.5 mm in total thickness. The membrane at the base of the telson seems to have a similar thickness in the fresh condition but it is only about half the thickness of the carapace after dehydration and mounting in resin. Incidentally, the epidermal cells are laden with dark pigment granules containing ommochromes ( LINZEN, 1967). Chemical The analytical data are presented in Tables 1 and 2. In overall comparison it is seen that the values for glycine and alanine of whole cuticle are high; combined they account for approximately 30 per cent of the total of all fractions from the carapace. Other outstandingly high percentages are shown for tyrosine except for the soft procuticle of arthrodial membrane which is high in the dicarboxylic acids. For hydrolysates of whole cuticle (Table 1) all fractions of the carapace are remarkably similar. Histidine is highest in exocuticle and lowest in epicuticle; phenylalanine, aspartic acid, glutamic acid, and lysine are relatively high in epicuticle and exocuticle. Areas with tonofibrillae (muscle attachments) are high in leucine and relatively low in histidine, tyrosine, arginine, and lysine. The epicuticle over soft membrane is very high in tyrosine and somewhat high in aspartic and glutamic acids, being correspondingly low in alanine, serine, and histidine. The soft chitin-protein portion of arthrodial membranes is notable for its very high content of dicarboxylic acids and somewhat high content of diamino acids; it is correspondingly low in glycine, alanine, and tyrosine. Hydrolysates of the NaOH extracts (Table 2) are considerably different, presumably due to the selective extraction of only part of the cuticular proteins. Glycine, serine, and arginine are much lower, tyrosine and phenylalanine somewhat lower. Correspondingly, the dicarboxylic acids (aspartic and glutamic), lysine, and valine are higher. The epicuticle is distinctive for its high content of serine and low content of methionine. Of more interest, the NaOH extracts show no considerable differences between any of the various fractions of the carapace, and great similarity between epicuticle and procuticle of the arthrodial membrane. Itemizing by individual amino acids using values given in Table 1, glycine is very high (14-20 per cent) in all samples except in the procuticle of soft membrane. Alanine is also high (10-16 per cent) except in soft membrane. Tyrosine is high (lo-20 per cent) except in areas with tonofibrillae and in the procuticle of soft membrane. Histidine is low in epicuticle, moderate elsewhere. The dicarboxylic acids are somewhat higher in the epicuticle and dark parts of the carapace, and much higher in the procuticle of arthrodial membrane, Lysine is low in the inner

2.31 2.72 5.03 4.21

1.36 2.10 9.03

Trace 7.75 2.72

1.86 7.55

2.35 99.97

14.69 11.84

12.87 7.05

99.93

5.57 4.63

9.16 2.43

3.59

8.44 12.11 8.70 5.85 3.40

1966B

1000J

4.15

Trace 5.79

14.52 9.28

7.64 4.58

1.53 2.84 5.57 2.73 -

7.53 11.57 10.81 6.33 5.13

Middle fraction 1966A

8.04 11.88 10.03 6.56 4.08

1966

Outer fraction

Carapace

100~00

4.28

Trace 5.66

16.23 8.68

7.17 5.16

1.13 1.51 7.30 0.0 3.14

8.93 12.96 6.16 7.04 4.65

1966

Inner fraction

100.25

2.50

2.37 6.97

1144 6.72

7.76 3.55

3.k

1.18 1.58 8.42

9.08 13.29 11.55 6.58 4.08

1966

Entire with tonofibriliae

99.99

0.92

Trace 5.25

18.06 13.55

7.56 7.56

1.66

7.74 2.03 7.46

6.64 8.48 4.88 4.70 3.50

1966

Epicuticle

99.96

4.35

Trace 6.96

18.61 13.50

7.30 6.60

2%

0.52 1.04 6.09

4.52 7.30 9.56 6.96 4.35

1966A,

100~10

3.82

Trace 6.94

19.62 14.06

7.12 6.25

2.26

0.52 1.04 6.94

3.99 6.25 9.03 7.99 4.17

1966A2,

Procuticlet

Arthrodial membrane

ACID ANALYSES OF 1 N NaOH EXTRACT OF THE CUTICLE OF Limuluspolyplrorurs*

Values are in moles per cent. * This extract represents approximately three-quarters of the material extractable with alkali. t Procuticles labelled A, and Aa were stored in 96% ethanol and are duplicates, B was stored in water plus chloroform.

Total

Aliphatic Glycine Alanine Valine Leucine Isoleucine Substituted aliphntics Serine Threonine Proline Tryptophan Hi&dine Phenyl Tyrosine Phenylalanine Dicsrboxyiic Aspartic acid Glutamic acid Diamino Arginine Lysine Sulphur-containing Methionine

Amino acids

TALILE %--AMINO

99.97

3.25

0.97 4.65

16.23 12.33

7.68 6.60

2.06

1.62 2.92 5.84

6.39 8.87 9.52 6.60 4.44

19663

504

P. KARLSON, K. E. SEKERI,-4. G. RICHARDS, ANDP. A. RICHARDS

fraction of the carapace and in areas with tonofibrillae, high in the procuticle of soft membranes. The other amino acids vary somewhat but without clear correlations being apparent. Tryptophan and cystine appear to be absent. Nitrogen determinations were made on some of the 1963 samples in Germany and on some of the 1966 samples in Minnesota. Samples cleaned in ethanol in 1963 gave values of 124.5 and 1295 per cent for the outer fraction, 1344 per cent for the middle fraction, and 12.79 per cent for the inner fraction. Samples cleaned in water in 1963 gave 12.73 per cent for the outer fraction, 10.58 per cent for the inner fraction, 10.14 per cent for the epicuticle of the telson membrane, and 10.56 per cent for the procuticle of this membrane. Samples cleaned in ethanol in 1966 gave slightly higher values, viz. whole carapace 14.1 per cent, outer fraction 13.5 per cent, middle fraction 14.5 per cent, and inner fraction 14.2 per cent, these values being averages of three or more determinations of each. The lipid content of Limuh cuticle is low. Extraction for 7.5 hr with petroleum ether in a Soxhlet apparatus resulted in removal of only O-7 per cent of the dry weight of samples that had been cleaned in ethanol. Incidentally, the hydrolysates of NaOH extracts also contain glucosamine: 7-7.4 per cent in the carapace fractions, 7.9-8.7 per cent in the procuticle of the telson membrane, and 10 per cent in the epicuticle of this membrane.

DISCUSSION Few studies have been made on the cuticle of Limulus or other arachnids. There is an old paper by LAFON (1943) which reports that the histological appearance is similar to what is seen in insects, and also that the cuticle of Limulus, unlike that of insects, contains 2.85 per cent sulphur. We cannot confirm this report on sulphur content. According to our analyses made in two different analytical laboratories the sulphur content is in the range of 0.10 to 0.28 per cent for whole cuticle (twelve analyses from six samples), our values being in accord with the determined methionine content. Despite being hard and dark-coloured, Limulus cuticle gives very little ash on incineration (0,095 per cent of the dry weight), less than that given by insect cuticles (RICHARDS 1956; HACKMAN, 1964; the first paper includes quantitative values for the various components in the ash). Limulus also has one of the lowest known values for chitin (28 per cent) of any uncalcified arthropod (RICHARDS,1949). The last-mentioned paper also points out that one cannot readily purify chitin from the carapace of Limuh (or cuticle of scorpions) but the nature of the NaOH and KMnO, resistant components is unknown. No conclusions can be drawn at the present time concerning the unexpected finding that the dark-brown outer fraction of the carapace stains with acid fuchsin and orange G. This region also gives a slightly but significantly lower nitrogen value in Kjeldahl analyses, and contains something not removed during routine purification procedures for chitin. There is no indication what is involved but it cannot be just the ommochrome pigments known to be present (LINZEN, 1967).

AMINO

ACID

COMPOSITION

OF

TYPFS

OF

CUTICLE

OF LIhflJLUS

POLYPHEMUS

505

The most striking finding in the present study is the considerable degree of similarity in amino acid composition of the various fractions. It follows that the differences between epicuticle and the various kinds of exocuticle (perhaps also arthrodial membrane but see below) are not reflected by amino acid determinations. It seems that the important differences must be sought at the molecular and intermolecular levels and perhaps in non-protein components. Since exocuticle is currently viewed as modified procuticle (Figs. 1 and 2) this finding is not to be considered surprising. Duplicate determinations on different aliquots of 1966 samples agree well. Determinations on equivalent samples from 1963 and 1966 agree less closely. This difference could represent either or both sampling errors (different areas of one carapace not being identical) and variation between different individual specimens. It would seem from the values given in Table 1 that the procuticle of soft arthrodial membrane is considerably different from that of the carapace. But, as stated in the section on Materials and Methods, the samples of carapace were cleaned in 96% ethanol while those from the arthrodial membrane were cleaned in water. It is well known that insect cuticles, especially soft cuticles, contain considerable amounts of water-soluble protein (see HACKMAN,1964; KARLSON et al., 1969). Presumably the same will be true for Limulus cuticles because we have nitrogen determinations on both ethanol-cleaned and water-cleaned samples of the carapace. All nitrogen determinations on arthropod cuticles are low because of the presence of chitin which contains only 6.9 per cent nitrogen. The outer portion of the carapace cleaned in ethanol gave an N value of 12.95 per cent; another sample cleaned in water gave 12-45 per cent, i.e. little change if any. But inner fractions of the carapace cleaned in ethanol gave an N value of 12.79 per cent whereas ones cleaned in water gave only 10.58 per cent. This implies that a considerable amount of protein (cu. 30 per cent) is removed from the inner layers of Limultrs cuticle by water at room temperature. The N value of lo-58 per cent for water-cleaned exocuticle, is almost identical with the N value of 10.56 per cent for water-cleaned It follows that one should be cautious in procuticle of arthrodial membrane. comparing the tabulated values for arthrodial membrane procuticle with values from the carapace samples. There are few quantitative determinations of amino acids from arthropod cuticles to compare with ours. The exuvia (shed skins) of the orthopteran Anabr~ simplex should be compared only with our Limulus carapace samples because endocuticle and mesocuticle are digested by the moulting fluid. By microbiological assay these exuvia gave values rather similar to ours from Limulus carapaces but with lower percentages of glycine and histidine and considerably higher percentages of proline and cystine (JOHNSONet al., 1952). Values by LIPKE et al. (1965) for the exuvia of the cockroach Periplaneta americana are considerably different but their preparative procedure was also quite different. Lipke et al. report great or significant changes in the percentages of proline, tyrosine, and phenylalanine as sclerotization proceeds-no comparable change is indicated by our various fractions of Limdus. Compared to our NaOH extracts, the extracts of larval and

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puparial cuticles of the fly Calliphora erythrocephala are higher in serine, threonine, histidine, and arginine, and lower in aspartic acid, glutamic acid, and e-specially tyrosine (UsoN et al., 1969). In the Decapod crustacea, where calcification largely replaces tanning, the cuticular amino acids of the lobster Homarus and the crab Callinectes have been reported as closely resembling our values for the procuticle of arthrodial membranes (DUCHATEAU and FLORKIN, 1954) but more recent analyses on the specialized structures termed gastroliths in the crab Orconectes gave quite different results (TRAVIS et al., 1967). It does not seem profitable to speculate on the differences in these reports. But it is notable that all reports on the amino acids of cuticular proteins of arthropods give high values for phenyl, dicarboxylic, and diamino acids. In contrast to simple fibrous proteins such as silk fibroin, the peptide chains of arthropod cuticles must be relatively lumpy due to the many bulky side-groups. Two questions of interest to cuticle specialists are whether or not the epicuticle is alike all over the arthropod, and whether or not the procuticle of membranes is the same as endocuticle under sclerites. Differences have been suggested on the basis of staining reactions (RICHARDS, 1952, 1967) but these have not been documented with satisfying data. Presumably the epicuticle, like sclerotized exocuticle, with not differ significantly whether cleaned in ethanol or water. Assuming this to be the case, the epicuticle on arthrodial membranes is definitely not the same in amino acid composition as the epicuticle on the carapace (scrapings from outer surface, Table 1). The procuticle of the arthrodial membrane of LimuEus is the most distinctive of all the fractions analysed but, unfortunately, old individuals of Limulus do not supply endocuticle with which to compare it. One point remaining is the oft-mentioned possibility of tyrosine being a ‘selftanning protein’ in the cuticle. Our determinations showing similar amounts of tyrosine in outer, middle, and inner fractions of the carapace give no support to this suggestion. Acknowledgements-This work was supported by funds from the Deutscher Forschungsgemeinschaft and a grant from the National Science Foundation (No. GB-4059). We also thank Professor L. M. HENDEMON of the Department of Biochemistry, University of Minnesota, for the tryptophan determinations. (Paper No. 6648, Scientific Journal Series, Agricultural Experiment Station, University of ,Minnesota, St. Paul, Minnesota.)

REFERENCES BRLWETP. (1965) The metabolism or aromatic compounds. Biochem. Sot. Symp. 25,49-77. DUCHATEAUG. and FLORKIN M. (1954) Sur la composition de l’arthropodine et de la s&roprotkine cuticulaires de deux crustaces dCcapodes (Homn~us vulgaris Edwards, Callinectes sapidrts Rathbun). Physiol. camp. oecol. 3, 365-369. HACKM~ R. H. (1964) Chemistry of the insect cuticle. In The PhysioIogy of Insecta {Ed. by ROCKSTEIN M.) 3,471-506. Academic Press, New York. JOHNSONL. H., PEPPERJ. H., BANNINGM. N. B., HASTINGSE. and CLARK R. S. (1952) Composition of the protein material in the exuviae of the mormon cricket, An&us simplex Hald. Physiol. Ziiol. 25,250-2X

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P. and SEKERISC. E. (1962) N-acetyldopamine as sclerotizing agent of the insect cuticle. Nature, Lond. 195, 183-l 84. KARLSON P., SEI~ERIK. E. und MARMARA~V. J. (1969) Die Aminosiiurezusammensetzung verschiedener Proteinfraktionen aus der Cuticula von Calliphora erythrocephala in verschiedenen Entwicklungsstadien. J. Insect Physiol. To be published. LAFON M. (1943) Sur la structure et la composition chimique du tegument de la limule (Xiphosurapolyphemus). Bull. Inst. Oceanogr. 850, l-1 1. LIN~FN B. (1967) Zur Biochemie der Ommochrome. Naturzuissenschaften 54,259-267. LIPKE H., GRAINCERM. M., and SIAKOTOSA. N. (1965) Polysaccharide and glycoprotein formation in the cockroach. J. biol. Chem. 240,.594-600. LOCKE M. (1964) The structure and the formation of the integument in insects. In The Physiology oflnsecta (Ed. by ROCK~TEINM.) 3, 379-470. OPIENSKA-BLAUTH J., CHAFWINSKIM., and BERBECH. (1963) A new, rapid method of determining tryptophan. Analyt. Biochem. 6,69-76. RICHARDS A. G. (1949) Studies on arthropod cuticle-III. The chitin of Limulus. Science, N.Y. 109,591-592. RICHARDS A. G. (1952) Studies on arthropod cuticle-VII. Patent and masked carbohydrate in the epicuticle of insects. Science, N. Y. 115,206-207. RICHARDS A. G. (1956) Studies on arthropod cuticle-XI I. Ash analyses and microincineration. J. Histochem. Cytochem. 4,140-152. RICHARDSA. G. (1967) Sclerotization and the localization of brown and black colors in insects. Zool.Jb. (Anat.) 84,25-62. SE~~ERIS C. E. and KARLSONP. (1966) Biosynthesis of catecholamines in insects. Pharmac. Rev. 18,89-94. TRAVISD. F., FRANCOISC. J., BONARL. C., and GLIMCHERM. J. (1967) Comparative studies on the organic matrices of invertebrate mineralized tissues. J. Ultrastruct. Res. 18, 519550. KARLSON