Comp. Biochem. Physiol., 1975, Vol. 5QA, pp. 561 to 564. Pergamon Press. Printed in Great Britain

THE COMPOSITION OF THE HAEMOLYMPH OF THE NEW ZEALAND CENTIPEDE. CORMOCEPHALUS

RUBRICEPS (NEWPORT)

JENNIFER J. BEDFORD AND JOHN P. LEADER

Department of Zoology, University of Auckland, Auckland, New Zealand

(Received 26 November 1973)

Abstract-l. The composition of the haemolymph of Cormocephalus rubriceps has been determined. The main cations, sodium, calcium. notassium and rnaanesium. show concentrations of 214.7. 4.8. 4.9, and 1.6 mM/l. respectively. _

2. The principal anions, chloride and phosphate, have concentrations of 112.9 and 0.7 mM/l. 3. Total ninhydrin-positive substances were 7.85 rg/rl, the bulk of which is made up of glycine,

alanine, glutamic acid and glutamine. 4. There was a high protein concentration and a moderate carbohydrate content in the haemo-

lymph. 5. These findings are then discussed in the light of proposed schemes of the evolution of the com-

position of insect haemolymph.

INl’BODUC’I’ION

FACTS and theories concerning phylogenetic relation- ships within the Arthropoda have been reviewed by Tiegs & Manton (1958) and Manton (1964, 1972). Much morphological and physiological evidence has been accumulated to substantiate the projected close evolutionary relationship of the Myriapoda and Hexapoda. Sutcliffe (1963) has also proposed a scheme of phylogenetic relationships within the Class Insecta based on present taxonomic evidence and on the chemical composition of the haemolymph. It is thus of interest to find that in the diplopod Iulus scandinavius the contributions of sodium and chloride are reduced to 29 and 25 per cent respectively (Sutcliffe, 1963). In this respect the haemolymph of this millipede is similar to that of some of the exopterygote insects. Little is known of the composition of the haemolymph of chilopods and diplopods, although some in- formation has been published concerning the free amino acids (Rajulu, 1970; Naire & Prabhu, 1971) and fatty acids (Oudejans et al., 1970). The present work reports the results of analysis of the major components of the haemolymph of the large New Zealand centipede, Cormocephalus rubriceps (New- port).

MATERIALS AND METHODS

Centipedes (Cormocephalus rubriceps), up to 2Ocm long, were collected from damp rotting logs on the West Coast near Auckland. Haemolymph analyses were carried out on freshly collected animals.

To collect the haemolymph for analysis the centipede was lightly anaesthetized with carbon dioxide, held tlat

on a piece of Paratilm and one antenna cut. The haemo- lymph was allowed to drip out, assisted by gentle pressure, and collected in a calibrated capillary tube. It was then cooled in an ice-bucket. It was generally possible to get at least O-2 ml haemolymph from each animal so that duplicate samples for all analyses could be made.

Total osmotic pressure was determined using the microcryoscopic method of Ramsay & Brown (1955); sodium and potassium ion concentrations on a flame photometer (Evans Electroselenium Ltd.) after suitable dilution; calcium and magnesium ion concentrations on an atomic absorption spectrophotometer (Techtron AA 100); and chloride ion concentration using a Buchler- Cotlove chloride titrator. Acid-soluble phosphate was determined using a modification (Bieleski, personal com- munication) of a standard calorimetric method (Fiske & Subbarow, 1925).

Total ninhydrin-positive substances (NPS) were measured by the method of Yemm & Cocking (1955) using glutamic acid as a standard. Individual ammo acids in deproteinized haemolymph were determined quantitatively by thin-layer electrophoresis and chromato- graphy (Bieleski & Turner, 1966) followed by scanning with a Vitatron (TLD 100) flying-spot densitometer at 505 nm.

Total protein in the haemolymph was determined using Folin’s reagent (Lowry et al., 1951). The principal proteins in the haemolymph were separated by flat sheet polyacrylamide gel electrophoresis using the method of Reid & Bieleski (1968) and staining with Coomassie Brilliant Blue R 250. After staining the gels were scanned with a Vitatron densitometer at 550 nm.

Acid and alkaline phosphatase in the haemolymph were located on the gels by running as above and then incubating the gels in-either acid (0.i M acetate, pH 5.2) or alkaline (0.1 M Tris. OH 8.5) buffer with naphthol AS-B1 phosphoric acid as the substrate for 2 hr at room temperature. They were then stained with a red violet LB salt (Burstone, 1962).

561

562 JENNIFER J. BEDFORD AND JOHN P. LEADER

Total carbohydrate in the haemolymph was measured using the anthrone method of Young & Raisz (1952) and total reducing substances by hydrolysis of the haemo- lymph with alkali. Ten f~l of haemolymph was heated with 0.2 ml 4 N sodium hydroxide at 100°C for 10 min. Non-reducing substances were found by difference. Glucose was estimated using glucose oxidase (Raabo & Terkildsen, 1960). The carbohydrate composition of the haemolymph was also investigated using thin-layer chromatography. This was carried out on plates spread with silica gel-G buffered with 0.2 M sodium acetate, at 250 p thickness, and run in n-propanol-ethyl acetate- water (7 : 1 : 1) (Trevelyan er al., 1950) as a solvent. Other solvents were tried (Stahl, 1970) but did not prove as successful. The plates were sprayed with fresh 10% sulphuric acid in water or with anthrone reagent (10% sulphuric acid in a saturated ethanolic solution of anthrone-v/v (Wimer et al., 1970)). Samples were either deproteinized in 70% ethanol or extracted in methanol- chloroform-water (12 : 5 : 3) (Bieleski & Turner, 1966). Glucose and trehalose were also determined quantit- atively using gas-liquid chromatography (nitrogen as the carrier gas, SE-30 column). Samples were run at 180°C for the first 6 min and then programmed at 20”C/min up to 250°C for the remainder of the run on a Varian Aero- graph (Series 200) gas chromatograph.

RESULTS

1. The total osmotic pressure and the inorganic constituents of the haemolymph

The total osmotic pressure and the concentration of the measured ions in the haemolymph are shown in Table 1. The main contribution is from sodium and chloride ions. There is, however, a large inorganic anion deficit which is similar to that found in all but the apterygote insects. When all measured cations are taken into account this amounts to 70.8 mOsmole.

2. Organic constituents of the haemolymph

A. Amino nitrogen. When measured as NPS (expressed as glutamic acid equivalents) the total concentration of amino acids is 7.85 _+ 3.17 pg.g/tJ haemolymph. When determined separately (Table 2), however, after thin-layer electrophoresis and chromatography, the total concentration was found to be 5.46 pg/$. Part of this difference appeared to be nine unidentified compounds, also revealed by spraying with ninhydrin, which were possibly peptides as they did not move far in either the electrophoretic or chromatographic direction.

The most abundant amino acids were glycine, alanine, glutamic acid and glutamine, which together make up a little less than 50 per cent of the total concentration of the amino acids. Proline, which is often found in high concentrations in insects, was present in only trace amounts.

B. Protein. The total protein in the haemolymph, determined with Folin’s reagent, was 42*5+9.3 pg/pl haemolymph. Using polyacrylamide gel

Table 1. The total osmotic pressure (expressed as mM/I. sodium chloride) and measured concentration of the inorganic ions in the haemolymph of the centipede,

C. rubriceps

mM/l. haemolymph

Total osmotic pressure Cations : sodium

calcium potassium magnesium

Anions : chloride phosphate

Osmotic pressure as mosmoles Combined contribution of cations and

anions as mosmoles Anion deficit as mOsmoles

209.0 k 20.3 214.7+ 10.9

4.820.4 4.91-0.7 1.6kO.l

112.9&- 11.1 0.7

418.2 347.4

70.8

Table 2. The amino acid composition of the haemolymph of the centipede, C. rubriceps

Concentration

Amino acids rdrl mM/l.

haemolymph haemolymph

Alanine 0.42 Arginine 0.27 Cysteine 0.50 Glutamic acid and glutamine 1.72 Glycine 0.48 Histidine 0.21 Isoleucine 0.08 Leucine 0.04 Lysine 0.18 Methionine 0.32 Phenylalanine Present Serine 0.37 Taurine 0.28 Threonine 0.39 Valine 0.23 Peptides (unidentified) Present

4.71 1.55 4.12

11.69 6.39 1.35 0.61 0.30 1.23 2.14

3.52 2.24 3.27 1.96

Total 5.46 45.08 Total as NPS* 786 65.41

*Assuming a mean molecular weight of the amino acids as 120.

electrophoresis to separate the main protein com- ponents in the haemolymph there were found to be six bands present (Fig. 1). Three bands were found on the gel to show acid phosphatase activity (Fig. 1, c, d’, e’) and one large band showed alkaline phosphatase activity, corresponding to band e in Fig. 1.

C. Carbohydrates. The total carbohydrate in the haemolymph (Table 3) is moderately low by arthro- pod standards. There is a lot of reducing substance present which is not glucose. Much of the non- reducing component is tentatively identified as trehalose both from the retention time on the gas

Composition of haemolymph of the New Zealand centipede 563

Fig. 1. Densitometer trace of the soluble proteins in the haemolymph of C. rubriceps. Band e shows alkaline phosphatase activity, and bands c, 8 and e1 show acid phosphatase activity.

Table 3. The carbohydrate composition of the haemo- lymph of C. rubriceps

mg/lOO ml haemolymph

Total carbohydrate (anthrone-positive substances as glucose equivalents)

2342 54

Non-reducing carbohydrate (anthrone- positive substances after alkaline hydrolysis)

140

Glucose (by glucose oxidase) Glucose (by gas chromatography) Trehalose (by gas chromatography)

Less than 5 621

93&8

chromatograph and stability to alkaline hydrolysis. The large amount of non-glucose reducing sub- stance(s) is currently being further investigated.

DISCUSSION

In accounting for the data available to him, Sutcliffe (1963) tentatively proposed that the insects could be divided into four main groups with respect to their haemolymph composition. While recogniz- ing the paucity of information, and noting the likelihood of numerous exceptions, he suggested that “primitive” insects (exopterygotes) had in general a haemolymph rich in inorganic ions and poor in dissolved organic compounds. In general the more “advanced” insects showed a tendency to reduce and replace the inorganic components of the haemolymph with organic substances, largely amino acids, and with an increasing contribution from unidentified organic compounds.

Duchateau er al. (1953) pointed out that the haemolymph of “primitive” exopterygotes is in many respects similar to the blood of other arthropods

and similar to the blood of most vertebrates. This statement is, however, based on very few analyses, and it might be expected that further information on the composition of the haemolymph might show, as Sutcliffe (1963) suggested, specific or group special- izations for a particular mode of life superimposed upon a more basic pattern.

Current views on the evolution of the insects agree in placing their origin near that of the Myria- poda (Manton, 1964,1972). It is scarcely surprising, therefore, that the haemolymph of C. rubriceps should show superficial similarities to that of the exopterygote insects. Both sodium and chloride are present in high concentrations, while potassium, calcium, magnesium and inorganic phosphate account for only a small proportion, and amino acids for about 10 per cent of the total osmolar con- centration. These features comprise the “primitive” haemolymph type of Sutcliffe’s (1963) grouping, characteristic of the Ephemeroptera, Odonata, Ple- coptera, Dictyoptera and Hemiptera-Heteroptera. In general, therefore, the haemolymph composition of C. rubriceps complies with Sutcliffe’s proposition.

An unusual feature of this centipede is the occur- rence of large amounts of cysteine in the haemo- lymph. Rajulu (1970) suggested that the occurrence of this amino acid in high concentration in the haemolymph might be a characteristic of the Myriapoda, but with so few analyses available, such a generalization would seem premature.

A persistent problem in the analysis of insect haemolymph is the large contribution made to the total osmolarity by unidentified organic substances. This is also the case with Cormocephalus. These substances must be largely negatively charged, since known inorganic ions generally account for almost all of the cationic contribution. This

564 JENNIFER J. BEDFORD AND JOHN P. LEADER

so-called anion deficit cannot be accounted for by amino acids, present in large amounts, since these carry no net charge at the normal pH of arthropod haemolymph (6.8-7.2, see Wigglesworth, 1972). More likely contributors to the anion balance sheet are organic acids. These are known to be present in high concentrations in the haemolymph (Leven- book & Hollis, 1961), many are divalent, and at the pH of the haemolymph will be fully dissociated. The analysis of these compounds, however, presents many difficulties, particularly for the small volumes generally available. Once this difficulty can be overcome, then further analyses will permit a clearer picture to be drawn of the functional evolu- tion of the haemolymph.

Acknowledgements-We wish to thank Dr. E. G. Bollard and Mr. N. A. Turner of the Plant Diseases Division, D.S.I.R., for the use of their atomic absorption spectrophotometer; Dr. R. L. Bieleski of the same address for help with the phosphate determinations; Dr. J. M. A. Brown of the Botany Department, University of Auck- land, for the use of their gas chromatograph; and the University of Auckland Research Committee. for con- tributions towards the cost of apparatus for the analysis of arthropod haemolymph carbohydrates. This work was carried out while one of us (J. J. B.) held a University Grants Committee Postdoctoral Fellowship.

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Key Word Index-Centipede; carbohydrates; amino acids; haemolymph; inorganic ions.