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The chemical composition of haemolymph in insects and some other arthropods, in relation to their phylogeny
Comp. Biochem. Physiol., 1963, Vol. 9, pp. 121 to 135. Pergamon Press Ltd., London. Printed in Great Britain
THE
CHEMICAL COMPOSITION OF H A E M O L Y M P H INSECTS AND SOME OTHER ARTHROPODS, IN RELATION TO THEIR PHYLOGENY
IN
D. W. SUTCLIFFE Department of Zoology, University of Durham, King's College, Newcastle upon T y n e (Received 23 January 1963)
A b s t r a c t - - 1 . The aim of this study is to suggest that in pterygote insects a basic type of haemolymph is discernible, and that this basic type was modified in several ways and on a number of occasions during the evolution of the Insecta. 2. One organic and six inorganic components in insect haemolymph are considered with respect to their potential contributions to the total osmotic activity of the haemolymph--the concentration of each component is expressed as a percentage of the total osmolar concentration. 3. It is suggested that a high proportion of chloride is characteristic of the Exopterygota, whereas a low proportion of chloride is characteristic of the Endopterygota. 4. In the majority of exopterygote insects, sodium and chloride each account for major proportions of the total osmolar concentration. Potassium, calcium, magnesium, inorganic phosphate and free amino acids account for only minor proportions of the total. It is suggested that haemolymph with this composition represents the basic type of haemolymph in pterygote insects. This type of haemolymph is very similar to that found in modern representatives of phylogenetically related groups, viz. an apterygote insect, a chilopod and diplopod. Hence it is suggested that the basic haemolymph type found in modern pterygores represents the haemolymph of the ancestral stock of pterygote insects. 5. In endopterygote insects, with a monophyletic origin from the ancestral pterygote stock, the basic haemolymph type is modified by a reduction of chloride and an increase of organic acids, including a tendency to increase the proportion of free amino acids. 6. In certain exopterygote and endopterygote insects the haemolymph is radically modified in various ways. These are regarded as specializations. One type of specialization appears to be characteristic of the Lepidoptera and Hymenoptera, where inorganic ions contribute only a very minor proportion of the total haemolymph osmolar concentration; free amino acids, and, in some cases, other organic components, contribute the major proportion of the haemolymph total.
INTRODUCTION
INSECT haemolymph contains very high concentrations of organic compounds containing nitrogen, in particular a wide variety of free amino acids, a number of non-amino carboxylic acids, and various carbohydrates. The probable contribution of these and other organic components to the freezing-point depression (haemolymph~) is very variable, but in some insects they apparently account for nearly 121
122
D.W. SUTCLIFVE
all of the haemolympha, for example, 81 per cent in the honey-bee larva Apis mellifera and 86 per cent in the beetle larva Popillia japonica (Buck, 1953). The concentrations of inorganic components are also highly variable. The sodium concentration may be either very high or extremely low. The potassium concentration may be relatively high and exceed that of sodium. The concentrations of calcium and magnesium are also relatively high; in some instances both ions are present in very high absolute concentrations (see Buck, 1953; Duch~teau et al., 1953). Florkin (1949) has suggested that the high aminoacidaemia and high concentrations of magnesium are characteristic features of insect haemolymph. It is also stated that the chloride concentration is generally low, accounting for' only 10-15 per cent of the haemolymph (Buck, 1953 ; Wyatt, 1961), but Sutcliffe (1962, and in this paper) demonstrates that this view requires modification. For a comprehensive account of the composition of insect haemolymph see the review by Wyatt (1961). In the first attempt to explain the differences in concentrations of inorganic ions found in insect haemolymph, Bon6 (1944) suggested that a correlation exists between diet and the ratio of sodium-potassium concentration in the haemolymph; in herbivorous insects this ratio is less than unity, whereas in carnivorous insects the ratio is always greater than unity. Bon6 concluded that the correlation is, in general, applicable to all insects, although he pointed out a number of inconsistencies in his data. Later, following attempts to change the ratio of sodiumpotassium in several insects by feeding on selected diets, Bon6 (1947) concluded that " . . . the association observed between the sodium-potassium ratio in the coelomic fluid and the diet is not the result of the chemical composition of the food-stuff acting during the actual life of a given insect. The ionic composition of the internal medium, as found now, is a species characteristic, maintained by mechanisms of mineral regulation". Tobias (1948) independently came to the same conclusion, and regulatory mechanisms maintaining a specific haemolymph composition are now known in a variety of insects (reviewed by Shaw & $tobbart, 1963). In a recent study Sutcliffe (1962) showed that the supposed correlation with diet is not applicable to aquatic insects and earlier Duch~teau et al. (1953) found that the correlation is not applicable as a generalization of observations on terrestrial insects. Instead, these authors suggested that the occurrence of a high or low sodium concentration is largely correlated with systematic categories of the Insecta. Duchhteau et aL expressed the equivalent concentrations of each of four cations as percentages (indices) of the summed total of these cations in haemolymph of a wide variety of insects. In three species of Odonata, sodium contributed about 85 per cent of the cation total, and potassium, calcium and magnesium each contributed about 5 per cent. In striking contrast, in more than twenty species of Lepidoptera the sodium concentration was very low, contributing usually less than 10 per cent of the cation total, whereas magnesium provided 30-60 per cent, potassium 20-45 per cent, and calcium 10-30 per cent. Duch~teau et al. suggested that a high sodium index, exemplified by the Odonata,
THE C H E M I C A L C O M P O S I T I O N OF H A E M O L Y M P H
123
is a "primitive" or ancestral feature retained in other groups also considered to be "primitive" insects, whereas the low sodium index found in the Lepidoptera is an "advanced" or specialized feature characteristic also of certain other groups of insects regarded as phylogenetically recent in origin. The present paper is offered in support of this view, although the problem is approached in greater detail and in a manner different from that of Duch~teau et al.
METHODS One organic and six inorganic components are considered with respect to their potential contribution to the total osmotic activity of the haemolymph. Thus the concentrations of free amino acids, mM/l., and sodium, potassium, calcium, magnesium, chloride and inorganic phosphate, mg ions/l, are expressed as percentages of the total osmolar concentration, m-osm/l. The latter was obtained from the observed haemolymphA employing the relationship 1 osmole/1. = A 1.86°C. No allowance was made for incomplete dissociation of electrolytes as, for example, in a solution of sodium chloride, nor for chelation or "binding" between electrolytes and large molecules. The estimated osmotic effect of each component is therefore maximal. Indirect evidence of ion binding in haemolymph, particularly in the case of calcium and magnesium ions, is reviewed by Carrington & Tenney (1959) and Wyatt (1961). Some original estimations of various components in haemolymph of several insects are given in Table 1, together with data for a centipede, a millipede and a terrestrial isopod. Details of the methods employed are given by Sutcliffe (1962). That paper contains the raw data concerning aquatic insects. Data for terrestrial insects are drawn largely from Duch~teau et al. (1953) (also given in Buck, 1953) but also from original sources in the literature. All sources are indicated in Table 2, which embodies the main results of this study. The examples shown in Table 2 are selected to give a representative picture of the range of variation found in each order of the Insecta. Estimates of the possible contribution of free amino acids to the total osmolar concentration, given in Table 2, require special comment. Sutcliffe (1962) estimated the molar concentration of free amino acids by a modified version of the Folin colorimetric technique. Duch~teau & Florkin (1958) employed the microbiological technique, and give the concentrations of fifteen "apparent" free amino acids, in mg/100 ml. As an approximation, their results were converted to molar concentrations by dividing the total amount of the fifteen amino acids with 120. This value was chosen rather arbitrarily but is close to the mean molecular weight of those amino acids that appear to be most abundant in the insects investigated. In a few instances the molar concentrations were calculated from sources employing the micro-Kjeldhal technique and estimating either non-protein N or amino-N, in mg/1. These were converted to molar concentrations by division with 14. It
124
I2). W. SUTCLIFFE
should be noted that in both cases the estimated molar concentration is almost certainly too high, since the technique estimates excess nitrogen atoms derived from di-amino acids and any peptides that m a y be present. T A B L E 1 - - A N A L Y S E S ON HAEMOLYMPH OF SOME INSECTS AND OTHER ARTHROPODS
Concn. in raM/1. Group
Species
Haemolymph Aoc
K
C1
Insecta Orthoptera
aLocusta migratoria migratorioides bChorthippus parallelus Isoptera CCryptoterrnes havilandi Dermaptera aForficula auricularia Homoptera "yassid Megaloptera ISialis lutaria Neuroptera gOsmylusfulvicephalus Hymenoptera nNeodiprion sertifer Mecoptera ipanorpa communis Chilopoda JLithobius sp. Diplopoda klulus scandinavius Isopoda ~Oniscus asellus
0.841(4)
-
-
98(4) 119
0"730 0"736(5) + 0'035 0"679(6) + 0"09 0"765 0.805(2) 0.690 0.608(6) + 0.07 0.457(6) __0.05~ 1.093(3)
82 90 41 35 62(2) (5)_+2.4 17(6)+_5 34 154 61 256 -
-
Haemolymph from : (a) one adult in each of four samples ; (b) ten larvae in one sample ; (c) sixty larvae in one sample; (d) six adults in one sample, A on individuals; (e) eighteen adults in one sample, A on individuals; (f) eight adults in one sample; (g) seventeen adults in each of two samples; (h) one larva in each sample; (i) twenty-two adults in one sample; (j) twelve in one sample, A on individuals; (k) four in one sample, A on individuals; (l) eight in one sample, A on individuals. RESULTS F r o m consideration of T a b l e 2, and on the basis of all the data available at present, it appears that (a) the h a e m o l y m p h composition of pterygote insects m a y be categorized into several distinct types, enumerated below, (b) in the majority of cases one of these types is characteristic of the m e m b e r s of an order. I n type I (Fig. I(A)), exemplified by Aeschnagrandis larvae, sodium and chloride can account for major proportions of the total osmolar concentration. On the other hand, potassium, calcium, m a g n e s i u m and inorganic phosphate account for very minor portions, and free amino acids account for about 10 per cent of the total osmolar concentrations. T y p e I appears to be characteristic of the E p h e m e r optera, Odonata, Plecoptera, Dictyoptera and H e m i p t e r a - H e t e r o p t e r a .
THE CHEMICAL
Exopterygotes A
COMPOSITION
B
Ephemeroptera Orthoptera Dermaptera Odonata lsoptera Ptecoptera Dictyoptera Heteroptera Endopterygotes E •
,
125
OF HAEMOLYMPH
C
D
Phasmida
Homoptera
G
F
H
.
• •
° °
~
°
°
o,.,,
...
•
•
•
•
i?.'."; .,-..
Megaloptera Neuroptera Mecoptera Trichoptera Diptera
Lepidoptera Hymenoptera
~
Y
Coleoptera
FIG. 1. Osmotic effects of components illustrated as percentages of the total osmolar concentration of haemolymph in pterygote insects. Each block in the figure is visualized as two vertical sections, each section representing 50 per cent of the total osmolar concentration. The percentage contributions of cations are illustrated in the left-hand section, with sodium at the base (stippled), followed by potassium (black area), calcium (white area) and magnesium (vertical stripes). Anions are illustrated in the right-hand section, with chloride at the base (oblique stripes) followed by inorganic phosphate (fine stippling). Where possible, free amino acids are illustrated in equal proportions in both sections (coarse stippling). The large blank area in each block represents the proportion of the total osmolar concentration that must be accounted for by other components of the haemolymph. T y p e I I (Fig. I(B)), illustrated by Locusta migratoria migratorioides, represents the haemolymph of some Orthoptera, one dermapteran and one isopteran. It is essentially similar to type I, except that sodium and chloride contribute rather less to the total osmolar concentration, about 24 per cent and 22 per cent respectively, and a larger proportion of the total (approaching 30 per cent in Locusta) consists of unknown molecules. T y p e I I is not characteristic of all orthopterans.
126 TABLE 2--OSMOTIC
D.W.
SUTCLIFFE
EFFECT O F C O M P O N E N T S AS A P E R C E N T A G E OF T H E T O T A L O S M O L A R C O N C E N T R A T I O N O F H A E M O L Y M P H I N P T E R Y G O T E INSECTS
P e r cent of osmolar concn. O r d e r a n d species
Ephemeroptera Ephemeradanica 2c Odonata Aeschnagrandis ~° A . cyanea 2° Enallagma cyathigerum 2° Agrion virgo 2° Plecoptera Perla bipunctata 2c Dinocras cephalotes 2° Orthoptera Locusta migratoria migratorioides Chortophaga viridifasciata 4 .4nabrus simplex 1~ Gryllotalpa gryllotalpa Phasmida Carausius morosus 17c C. morosus 23 Dictyoptera Periplaneta americana 1 Isoptera Cryptotermes havilandi Dermaptera Forficula auricularia neteroptera Notonecta obliqua 2° C o r i x a p u n c t a t a ~° Rhodnius prolixus 17~ Megaloptera Sialis lutaria ~° Neuroptera Osmylus f u l v i cephalus
Haemolymph Stage - - A°C m-osm/l.
Na
K
Ca
Mg
L
0.504
271
38.0
6"6
L L
0"735 0"701
395 377
36"7 37"7
2"3 2.1
L A
0"620 0'790
333 425
41 "7 34"2
4.2 6'5
38.1 25-7
L
0"644
346
36"8
3'5
30"7
L
0"583
313
37'4
3"2
35.5
A
0"841
452
4"0 is 1"71°
3"01° 21"6
A A
0'890
479 450 ~s
24"2 xa • 22"8 4"9
0'8 3'3
4'4 0"3
---
A
0"840 is
38-26 52.01o
2"46 1.61o 3.11o
1.11o
__
451
--
CI
1"91°
0"6 0"7
28.4
1'91° 27"9 29.7
A A
0'570
306 306
2"9 4"9
9"1 5"9
2"6 2"5
23"9 17"3
30.4 33"0
A
0"89791
482
34"0
1"6
0'9
1'1
29-7
L
0"730
392
26'3
7"1
2-22a
4.5 °~ 21"0
A
0"736
396
24-2
3"3
4.29 • - -
22.7
A A
0,756 0"600
406 323
38"2 34-8
5"2 9"6
3.89b 2.39b . . . .
29.8 23"4
A
0"695
374
43"8
1 "6
--
339
32'2
1'5
2"2
433
21"2
9"2
L
A
0"805
5"6
. . . .
9"1
14.3
PO 4
Amino acids
-1"1 1.1
9"8 9.0
10'1 1"3
12"5
11"7 n
8"7
13"1 1"6
20"9 41-5
11'511
33"9 x 4"819
--
2"9
25"1
--
127
THE CHEMICAL COMPOSITION OF HAEMOLYMPH
TABLE 2
(cont.)--OSMOTICEFFECT
O F C O M P O N E N T S AS A P E R C E N T A G E O F T H E T O T A L O S M O L A R
CONCENTRATION OF H.zEMOLYMPH IN PTERYGOTE INSECTS
Per cent of osmolar concn. Order and species
Stage
Haemolymph A°C
Coleoptera Hydrophilus piceus Dytiscus marginalis 2° Popilliajaponica 1~ Hymenoptera Apis mellifera 5 Vespulagermaniea Mecoptera Panorpacommunfi Trichoptera Phryganeasp. 2° Limnephilus stigma 2° Anabolianervosa a° Lepidoptera Bombyx mori 7 Ephestia kuehniella 1° Nymphula nymphaeta ~° Prodenia eridania ~ Samia walkeri ~'12 Saturnia pyri 1° Teleapolyphemus ~ Diptera Gastrophilus intestinalis 14 Tipula montium ~° Aedes aegypti
m-osm/1.
[ Na
I
Amino K
Ca
Mg
C1
13"0
13"4
PO4
acids
A
0"647
348
35'0
4"0
6-9
17-511 9"211
A L
0"745 1"03
401 554
31-5 3"6
3"5 1'7
3'01° 1"4
4"71° 11"0 3"5 3"4
0"77. -0"9 30"7
L
0"860
462
1"1
5"2
0"9
1"7
7"2
2"2
L
0"880 lsa
473
10-26 8-7 e 5.51o 11.81o
A
0"690
371
25"4
10"2
--
9"2
L
0"455
245
28"2
2"9
L L
0"380 0'405
204 218
40"7 46"4
6-9 7"8
L
0"480
258
5"4
15'0
L
1"130lab
608
5"4
L L P P P
0"552 0'840 1.110 0"780 xsc 1"170
297 452 597 419 629
L
0"872
L L
0"443
2"91° 10"41° 15"1 . - - .
44.8 22.311
23"7
--
4"9 4"1
5"9 6"4
20"0 13"3
7"0
19"5
8"1
1"9
37"234
5"4
3"4
4"2
13"5 4"9 0'5 1'0 0"5
7'3 8"9 7'0 9"8 9"4
2"0 0"5 1"7 1"0
1"6 3"5
7"8 7"5 1"7
1"3 0-6
5'9
3'2
3"8
37"2 52'8 39"611 4"1
469
37"2
2"6
0"6
3'4
3'2
0-9
238 269 ~2
48"5 2'9 2"5 ~°~ 3"41°° 3"4 37"217b 1"517b. . . . 19"3~2
0"8
13"2 8.311 19"8
1Asperen & Esch, 1956; ~Babers, 1938; 3Barrat & Arnold, 1911; ~Barsa, 1954; SBishop et al., 1925; SBon6, 1944; 7Buck, 1953; aCarrington & Tenney, 1959; 8Clark, 1958, a data for Zootermopsis angusticoUis, b data for Notonecta lu'rbyii; l°Duchfiteau et al., 1953, a data for Tipula paludosa and T. oleracea; nDuchfiteau & Florkin, 1958 ; X~Gese, 1950; lSHoyle, 1954; 14Levenbook, 1950; 15Ludwig, 1951; lePepper et al., 1941; 17Ramsay, a 1952, b 1953, c 1955; lSRouschal, 1940, a data for adult Vespa crabro, b data for Ephestia elutella larva, c data for Saturnia pyri larvae; 1'Stevens, 1961 ; 2°Sutcliffe, 1962; SlTreherne, 1961 ; 22Wigglesworth, 1938; ~SWood, 1957; z4Wyatt et al., 1956; 26arbitrary, no data available.
128
D.W. SUTCLIFFE
Inspection of Table 2 suggests that Gryllotalpa gryllotalpa is similar to type I, whereas the very low percentage of sodium in the mormon cricket, Anabrus simplex, is completely anomalous. The haemolymph composition in this insect closely resembles that of Lepidoptera and Hymenoptera (see below), including the very high concentration of amino-N, 187 raM/1. (Pepper et al., 1941). Type III (Fig. I(E)), exemplified by the larva of Sialis lutaria, represents the haemolymph of Trichoptera, Diptera, a megalopteran, a neuropteran and a mecopteran. Here the haemolymph contains a very low percentage of chloride relative to that of sodium. Sodium varies from 21 per cent of the total in adult Osmylus fulvicephalus (Neuroptera) to 48 per cent in Tipula montium larvae (Diptera), i.e. the range of sodium embraced by types I and II. Type III also resembles these with low percentages of potassium, calcium, magnesium and inorganic phosphate, but differs in the relatively high percentage of free amino acids, up to 25 per cent in Sialis larvae. Haemolymph in the majority of Coleoptera is also of type III except that magnesium possibly tends to account for a slightly higher proportion, about 10 per cent of the total (Fig. I(H)). However, in four coleopterans the haemolymph composition is that of type IV. The species concerned are Timarcha tenebricosa and Agelastica alni (Chrysomelidae), Tenebrio molitor (Tenebrionidae), and Popillia japonica (Scarabaeidae) (Fig. I(G)). It is worth noting that in other Scarabids the haemolymph appears to be type III (see data in Duch~teau et al., 1953 ; A in Rouschal, 1940, and Buck, 1953). Type IV (Fig. I(F)), illustrated by the larva of Prodenia eridania, is completely characteristic of the Lepidoptera and Hymenoptera. Here, both sodium and chloride account for only a minor percentage of the total osmolar concentration, about 10 per cent or less in each case. Potassium, calcium, magnesium and inorganic phosphate also account for small amounts, 10 per cent or less in each case, although magnesium may account for a larger proportion of the total in larvae of Bombyx mori if, in fact, the haemolympha is usually as low as 0.48°C (cf. Buck, 1953; Jeuniaux et al., 1961) and assuming that most of the magnesium is not bound to large molecules. A major proportion of the haemolymph total in type IV is accounted for by free amino acids, representing about 40 per cent in several lepidopterans and in the honey-bee, Apis mellifera. In fact, the concentration of free amino acids is very high in nearly all lepidopteran larvae and pupae (Duch~teau & Florkin, 1958; Florkin, 1959), with concentrations ranging between 10-20 mg/ml or roughly 83-166 mM/l. amino acids. In larvae of Bombyx mori, Jeuniaux et al. (1961) found a mean haemolympha = 0.60°C, of which 0.16°C (26.7 per cent) is accounted for by free amino acids (of. 37.2 per cent in Table 2). However, in the pupa of Telea polyphemus, Carrington & Tenney (1959) report only 36.5 mg per cent nonprotein N, or roughly 26 mM/l. as amino acid; this represents only 4 per cent of the haemolymph osmolar concentration. It is now possible to draw the following main conclusions. In haemolymph of
THE CHEMICAL COMPOSITION
OF HAEMOLYMPH
129
the Exopterygota (Imms, 1957) sodium and chloride both represent major proportions of the total osmolar concentration. On the other hand, in the Endopterygota chloride accounts for only a minor proportion, usually less than 10 per cent of the total, although in a few instances chloride is more important, e.g. 19 per cent of the total in larvae of Aedes aegypti. In most endopterygote orders, sodium accounts for a major proportion of the total osmolar concentration, but in the Lepidoptera, Hymenoptera and in some Coleoptera, sodium accounts for only a minor proportion. Also, it is suggested that a high proportion of chloride in haemolymph is characteristic of the Exopterygota, whereas a low proportion of chloride is characteristic of the Endopterygota. There appear to be three exceptions to the above generalizations. In Anabrus simplex sodium probably accounts for less than 10 per cent of the total (see Table 2). Unfortunately, Pepper et al. (1941) did not estimate the haemolymph chloride in this insect. Haemolymph of the stick insect, Carausius morosus, is unlike that of any other insect (Fig. I(C)) since it contains a very high proportion of magnesium phosphate (Ramsay, 1956), although Wood (1957) reported only 16mM/1. H~PO 4. However, although sodium represents less than 5 per cent, chloride accounts for at least 30 per cent of the total osmolar concentration, and in this respect it is a typical exopterygote insect. The third exception is the unidentified Jassid plant bug reported in this paper (Table 1, Fig. I(D)). Here, sodium represents 16 per cent and chloride only 11 per cent of the total osmolar concentration. In both respects this insect is atypical among the Exopterygota, resembling instead type IV characteristic of the Lepidoptera and Hymenoptera. At this point it is convenient to mention briefly other components found in haemolymph. Proteins, and non-protein compounds containing nitrogen (excluding free amino acids), probably contribute only a very small proportion of the total osmotic activity of haemolymph (see Buck, 1953 ; Wyatt, 1961). On the other hand, very high concentrations of certain carbohydrates are reported in some insects, sufficient to contribute as much as 10 per cent of the total osmolar concentration. In particular, the disaccharide trehalose possibly accounts for 8"5 per cent in adult Periplaneta americana (data in Treherne, 1960), 3-7 per cent in Bombyx mori larvae and 2.3 per cent in Telea polyphemus pupae (data in Wyatt, 1961). Using Treherne's (1958) estimate of the concentration of trehalose in adult Schistocerca gregaria, this represents 4.5 per cent of Locusta haemolymph. In some insects non-amino organic acids probably make noticeable contributions to the osmotic activity of haemolymph, e.g. citrate, 10-12 per cent in B. mori larvae, 4"5 per cent in Prodenia eridania larvae and 2.3 per cent in T. polyphemus pupae (data in Levenbook & Hollis, 1961); succinate 4.5 per cent and malate 5-5 per cent in Gastrophilus intestinalis larvae (data in Wyatt, 1961). Thus the total osmolar concentration of B. mori larvae is largely accounted for (see Table 2 and Wyatt, 1961). However, in many insects, in particular Lepidoptera, Hymenoptera and some Coleoptera, a major part of the haemolymph total apparently consists of unknown molecules, e.g. 50-60 per cent of the total in T. polyphemus, P. eridania and Popilliajaponica; and see Sutcliffe (1962).
130
D.W. SUTCLIFFE
DISCUSSION It must be emphasized that the recognition of at least three distinct types of haemolymph does not represent an attempt to classify rigidly the haemolymph composition of each species of insect, nor is it an attempt to classify orders of the Insecta. Several exceptions are already known and doubtless many more await discovery. Moreover, the method adopted here, assessing the osmotic contribution of each component, is slightly artificial, since we do not know the actual osmotic activities of the various components in haemolymph. Also, as noted in Table 2, analyses on one species were occasionally used to assess osmotic contributions in another species. The recognition of haemolymph types is, therefore, regarded merely as a convenient way of handling the data available at present. The aim of this study is to suggest that a basic type of haemolymph is discernible, and that this basic type was modified in several ways and on a number of occasions during the evolution of the Insecta. The views of morphologists and systematists concerning the relationships between pterygote insects are summarized in Jeannel (1949), Imms (1957) and Hinton (1958). We will consider only those orders for which analyses on haemolymph are available. Briefly, the Ephemeroptera and Odonata represent an ancient group, the Palaeoptera, which diverged from the Neoptera or main line of pterygote evolution at a very early date (Devonian, or earlier still). The remaining orders in the Exopterygota of Imms (1957) probably represent several distinct stocks arising out of the early Neoptera, whereas the Endopterygota ( = Oligoneoptera in Jeannel, 1949) apparently are derived from a single stock. This monophyletic group diverged along two lines, one leading to the "panorpoid complex" including the Mecoptera, Trichoptera, Lepidoptera and Diptera; the other line leading to the remaining endopterygotes considered here, i.e. the Megaloptera, Neuroptera, Coleoptera and Hymenoptera. Reverting to the results of this study, we see that haemolymph type I occurs in both of the modern palaeopteran orders, and occurs also in the Plecoptera, Dictyoptera and Heteroptera. According to Jeannel (1949) the latter three orders are modern representatives of three distinct stocks of neopteran Exopterygota. It is suggested, therefore, that type I represents the basic haemolymph composition of the ancestral stock of pterygote insects, and that this basic type is retained in the Palaeoptera and in many neopteran Exopterygota, with modification by a small reduction in sodium chloride (type II) in at least one dermapteran, one isopteran, and in some orthopterans, The stick insect, Carausius, and a Jassid homopteran represent considerable modifications of the basic type, and are considered below as specializations. It was suggested that a low proportion of chloride is characteristic of the Endopterygota. In view of their monophyletic origin, it seems reasonable to assume that the reduction in chloride was a single event that occurred very early in the evolution of the group, prior to the divergence of the "panorpoid complex". Apart from this, haemolyrnph in many endopterygotes retains much of the basic features of the ancestral pterygote stock, although there appears to be a marked
THE C H E M I C A L C O M P O S I T I O N OF H A E M O L Y M P H
131
tendency towards an increased proportion of free amino acids (type III). But in the Lepidoptera, Hymenoptera and in some Coleoptera, the haemolymph is radically modified (type IV). Here inorganic ions, particularly sodium and chloride, form a very minor proportion of the haemolymph, and the tendency towards an increased proportion of free amino acids (and other organic acids) is greatly enhanced. This specialization probably occurred independently on at least two occasions during the evolution of endopterygote insects, since it is believed that the Lepidoptera evolved from the "panorpoid" line. Also, there appears to be no close relationship between the Coleoptera and Hymenoptera (Jeannel, 1949; Imms, 1957). Duch~teau et al. (1953) recognized that in the Lepidoptera, Hymenoptera, Chrysomelidae (Coleoptera) and in Carausius, the haemolymph is specialized on the basis of a low percentage of sodium and high percentage of potassium and magnesium. From the viewpoint adopted here there is no striking tendency towards a greater increase in potassium and magnesium in these insects. This does not deny the fact that the absolute concentrations of these ions are often quite high but, since the total concentration of the haemolymph is also increased in these terrestrial insects, the proportions of potassium, calcium and magnesium relative to the total osmotic concentration are usually Similar to those in other insects. With regard to Bond's (1944) correlation, the relative osmotic effect of the potassium ion is very similar in all of the insects investigated; in most cases potassium contributes only 2-10 per cent of the total in both herbivorous and carnivorous insects. On the other hand, the very great reduction of sodium in these specialized insects is abundantly clear. At present, it is not possible to offer an adequate explanation for these specializations. Duch~teau et al. (1953) point out that all of the insects concerned feed on higher plants, and suggest that there is a correlation between the development of specialized haemolymph and the evolution of Angiosperms. The underlying reasons for this relationship are obscure, since already there is evidence that haemolymph is not specialized in some Scarabid beetles ("chafers") associated with Angiosperms. Moreover, it is certain that not all insects feeding on Angiosperms are specialized; locusts provide an obvious example. Hence it is difficult to accept the possibility that the specialized type of haemolymph is a special case of direct adaptation to the relative proportions of cations found in Angiosperms. Duch~teau et al. have discussed this point in detail, and no further comments are necessary. What is required now is more information on ion uptake and excretion in these insects (see Ramsay, 1955, 1956). However, it is possible that Bond's (1944). general conclusions were influenced by the fact that of his twenty-seven species, ten species with a low sodium-potassium ratio are regarded here as specializations. It is of interest to note that, with the possible exception of Carausius, the haemolymph composition in the specialized insects is extraordinarily similar to the composition of some types of tissue cells in other animals. Here the sodium concentration is usually very low, and organic molecules, including amino acids, sugars and various phosphorylated compounds are extremely abundant. The
132
D . W . SUTCLIFFE
similarity is particularly striking when the muscle fibre of Carcinus (Shaw, 1958) is compared with haemolymph of the Lepidoptera, Hymenoptera and specialized Coleoptera (cf. Figs. 2(H), I(F), I(G)). Is this in some way connected with special requirements of very rapidly developing tissues in the pupal instar ? We may now briefly compare the composition of haemolymph in pterygote insects with the blood composition of other arthropods. Analyses on representatives of modern classes within the Arthropoda are presented in Table 1 and Fig. 2. A ...°
D
B
%-%
!:T .'~ ".-.:~ • °°
~-%
"i": .."" ::y,.. ...•.
I :(:,
°°.
i(:~:d , , , ,
':o¢,~
i'-~' ""
~~! -....
~ x
...
Petrobius
marifimus
~ x
Lithobius F
E
ii? ..°-.
~
lulus scandinovius
Tegenoria attica
G
=.....
~
.......
.,,,-.".'..;...
-.;.-,-'~....
.;'.~.~ ,.-~
Oniscus asellus
Asellus aquaticus
~::..-. ,.~
Astacus
Carcinus muscle fibre
FIG. 2. Osmotic effects of components illustrated as percentages of the total osmolar concentration of blood in: (A) an apterygote insect, (B) a chilopod, (C) a diplopod, (D) an arachnid, (E-G) crustaceans. (H) illustrates the osmotic effects of components in the muscle fibre of CarclnUs maenas. Conventions as in Fig. 1.
In all marine, brackish and fresh-water Crustacea-Malacostraca the blood composition is of the type where sodium chloride accounts for nearly all of the blood osmolar concentration, and potassium, calcium and magnesium each account for only about 1-3 per cent of the total (Fig. 2(G); data in Robertson, 1960; Lockwood, 1961). Organic molecules are present in extremely low concentrations and therefore contribute little to the blood total. Thus the blood composition in the terrestrial isopod Oniscus asellus (Table 1, Fig. 2(E)) is very similar to that of its marine-littoral relative Ligia oceanica (Parry, 1953) and of Asellus aquaticus (Fig. 2(F), Lockwood, 1959) found in fresh water. This type of blood also occurs in the arachnid Tegenaria atrica (Fig. 2(D) ; Croghan, 1959), in the chilopod Lithobius
THE C H E M I C A L C O M P O S I T I O N OF H A E M O L Y M P H
133
(Fig. 2(B)) and in the apterygote insectan Petrobius maritimus (Fig. 2(A); Lockwood & Croghan, 1959). Evidence concerning phylogenetic relationships within the Arthropoda is reviewed by Tiegs & M a n t o n (1958). T h e r e appears to be no close affinity between the Crustacea, Arachnida and Myriapoda, but the Insecta probably arose from a stock of myriapods. It is therefore of interest to find that in the diplopod lulus scandinavius (Table 1) the contributions of both sodium and chloride are reduced; sodium accounts for only 29 per cent and chloride 25 per cent of the blood osmolar concentration (Fig. 2(C)). In this respect the blood of lulus appears to be similar to that of some exopterygote insects, and it would be of great interest to know whether or not the blood of this and other diplopods contains high concentrations of free amino acids and other organic compounds. Hence it appears that the haemolymph of many modern exopterygotes, representing the basic haemolymph composition of the ancestral stock of pterygote insects, is very similar to that of modern representatives of phylogenetically related groups, i.e. Petrobius and myriapods. Also, as Duch~teau et al. (1953) pointed out, the haemolymph of these "primitive" exopterygotes is in many respects very similar to the blood of other arthropods, and similar to the blood of most vertebrates. Acknowledgements--Most of this study was carried out "during two visits to the Windermere Laboratory of the Freshwater Biological Association, and I wish to thank the Director for generously providing research facilities, and the members of staff for their willing cooperation in many ways. The investigation was initiated during the tenure of a D.S.I.R. Research Fellowship. REFERENCES ASPEREN K. VAN • ESCH I. VAN (1956) The chemical composition of the haemolymph in Periplaneta americana. Arch. nderl, zool. 2~ 342-360. BABE:~S F. H. (1938) An analysis of the blood of the sixth-instar southern armyworm, Prodenia eridania. J. Agric. Res. 57, 697-706. BAre,AT J. O. W. & ARNOLD G. (1911) A study of the blood of certain Coleoptera: Dytiscus marginalis and Hydrophilus piceus. Quart. J. Micr. So/., 56, 149-165. BARSA M. C. (1954) The behaviour of isolated hearts of the grasshopper Chortophaga viridifasciata and the moth Samia walkeri in solutions with different concentrations of sodium, potassium, calcium and magnesium. J. Gen. Physiol. 38, 79-92. BISHOP G. H., BaIGGS A. P. & RONZONI E. (1925) Body fluid of the honeybee larva: II. Chemical constituents of the blood and their osmotic effects. J. Biol. Chem. 66, 77-88. BON~ G. J. (1944) La rapport sodium-potassium dans le liquide ccelomique des insectes. I. Ses relations avec le rdgime alimentaire. Ann. Soc. zool. Belg. 75, 123-132. BON~ G. J. (1947) Regulation of the sodium-potassium ratio in insects. Nature, Lond. 160, 679-680. BUCK J. B. (1953) In Insect Physiology, Chapter 6 (Edited by ROEDERK. D.). Wiley, New York. CAm~INGTON C. B. & TENNEY S. M. (1959) Chemical constituents of haemolymph and tissue in Telea polyphemus Gram. with particular reference to the question of ion binding. J. Ins. Physiol. 3, 402-413. CLARK E. W. (1958) A review of literature on calcium and magnesium in insects. Ann. Ent. Soc. Amer. 51, 142-154.
134
D . W . SUTCLIFFE
CROGHAN P. C. (1959) The interstitial soil-water habitat and the evolution of terrestrial arthropods. Proc. Roy. Phys. Soc. Edinb. 27, 103-104. DUCH~.TEAU G. & FLORKIN M. (1958) A survey of aminoacidemias with special reference to the high concentration of free amino acids in insect haemolymph. Arch. int. Physiol. 66, 573-591. DgCH~,TEAtr G., FLORKIN M. & LrCLEaCQ J. (1953) Concentrations des bases fixes et types de composition de la base totale de l'h~molymphe des insectes. Arch. int. Physiol. 61, 518-549. FLORKIN, M. (1949) Biochemical Evolution. Academic Press, New York. FLORKIN M. (1959) T h e free amino acids of insect haemolymph. 4th Int. Congr. Biochem. X I I : Biochemistry of Insects. Pergamon, Oxford. GESE P. K. (1950) The concentration of certain inorganic constituents in the blood of the Cynthia pupa Samia walkeri Felder and Felder. Physiol. Zool. 23, 109-113. HINTON H. E. (1958) T h e phylogeny of the panorpoid orders. Ann. Rev. Entomol. 3, 181206. HOYLE G. (1954) Change in the blood potassium concentrations of the African migratory locust (Locusta migratoria migratorioides R. & F.) during food deprivation, and the effect on neuro-muscular activity. J . Exp. Biol. 31, 260-270. IMMS A. D. (1957) A General Textbook of Entomology, 9th ed. Methuen, London. JEANN~L R. (1949) Classification et phylog6nie des insectes, in Traitd de Zoologic, Tome IX, pp. 1-17. Masson, Paris. JEIJNIAUX CH., DUCH~,TEAu-BossoN GH. & FLOiaKIN M. (1961) Contributions/l la biochimie du ver a soie X X I I . Modifications de l'aminoacid6mie et de la pression osmotique de l'h6molymphe au cours du d6veloppement de Bombyx mori L. Arch. internat. Physiol. Bioch. 69, 617-627. LEVENBOOK L. (1950) T h e composition of horse bot fly (Gastrophilus intestinalis) larvae blood. Biochem. J. 47, 336-346. LEVrNBOOK L. & HOLLIS V. W. (1961) Organic acid in insects. I. Citric acid. J. Ins. Physiol. 6, 52-61. LOCKWOOD A. P. M. (1959) T h e osmotic and ionic regulation of Asellus aquaticus (L.). J. Exp. Biol. 36, 546-555. LOCKWOOD A. P. M. (1961) "Ringer" solutions and some notes on the physiological basis of their ionic composition. Comp. Biochem. Physiol. 2, 241-289. LOCKWOOD A. P. M. & CaOCHAN P. C. (1959) Composition of the haemolymph of Petrobius maritimus Leach. Nature, Lond. 184, 370-371. LUDWIG D. (1951) Composition of the blood of the Japanese beetle (Popillia japonica) larvae. Physiol. Zool. 24, 329-334. PARaY G. (1953) Osmotic and ionic regulation in the isopod crustacean Ligia oceanica. J. Exp. Biol. 30, 567-574. PEVPER J. H., DONALDSON F. T. & HASTINGS E. (1941) Buffering capacity and composition of the blood serum and regurgitated digestive juices of the mormon cricket (Anabrus simplex Hald.). Physiol. Zool. 14, 470-475. RAMSAY J. A. (1952) T h e excretion of sodium and potassium by the Malpighian tubules of Rhodnius. J. Exp. Biol. 29, 110-126. RAMSAY J. A. (1953) Exchanges of sodium and potassium in mosquito larvae. J. Exp. Biol. 30, 79-89. RAMSAYJ. A. (1955) T h e excretory system of the stick insect, Dixippus morosus (Orthoptera, Phasmidae). )t. Exp. Biol. 32~ 183-199. RAMSAY J. A. (1956) Excretion by the Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera, Phasmidae): calcium, magnesium, chloride, phosphate and hydrogen ions. J. Exp. Biol. 33, 697-708.
T H E C H E M I C A L C O M P O S I T I O N OF H A E M O L Y M P H
135
ROBERTSON J. D. (1960) Osmotic and ionic regulation, in The Physiology of Crustacea, I, Chap. 9. Academic Press, New York & London. ROUSCHAL W. (1940) Osmotische Werte wirbelloser Landtiere und ihre okologische Bedeutung. Zeitschr. Wiss. Zool. Abt. A, 196-218. SHAW J. (1958) Osmoregulation in the muscle fibres of Carcinus maenas. J. Exp. Biol. 35, 920-929. SHAW J. & STOBBARTR. H. (1963) Osmotic and ionic regulation in insects. Adv. Ins. Physiol. 1, 315-399. STEVENS T. M. (1961) Free amino acids in the haemolymph of the American cockroach, Periplaneta americana L. Comp. Biochem. Physiol. 3, 304-309. SUTCLIFFE D. W. (1962) T h e composition of haemolymph in aquatic insects, ft. Exp. Biol. 39, 325-343. TIECS O. W. & MANTON S. M. (1958) T h e evolution of the Arthropoda. Biol. Rev. 33, 255337. TOBIAS J. M. (1948) Potassium, sodium and water interchange in irritable tissues and haemolymph of an omnivorous insect, Periplaneta americana. J. Cell. Comp. Physiol. 31, 125-142. T~HERNE J. E. (1958) T h e absorption and metabolism of some sugars in the locust, Schistocerca gregaria (Forsk.). J. Exp. Biol. 35, 611-625. TaEHERNE J. E. (1960) T h e nutrition of the central nervous system in the cockroach, Periplaneta americana L. T h e exchange and metabolism of sugars. J. Exp. Biol. 37, 513-533. TREHEaNE J. E. (1961) Sodium and potassium fluxes in the abdominal nerve cord of the cockroach, Periplaneta americana L. J. Exp. Biol. 38, 315-322. WIGGLESWOaTHV. B. (1938) T h e regulation of osmotic pressure and chloride concentration in the haemolymph of mosquito larvae. J. Exp. Biol. 15, 235-247. WOOD D. W. (1957) T h e effects of ions upon neuromuscular transmission in a herbivorous insect. J. Physiol. 138, 119-139. WYATT G. R. (1961) T h e biochemistry of insect haemolymph. Ann. Rev. Ent., 6, 75-102. WYATT G. R., LOUGHHEEDT. C. & WYATT S. S. (1956) T h e chemistry of insect haemolymph. Organic components of the haemolymph of the silkworm, Bombyx mori, and two other species, ft. Gen. Physiol. 39, 853-868.
THE
CHEMICAL COMPOSITION OF H A E M O L Y M P H INSECTS AND SOME OTHER ARTHROPODS, IN RELATION TO THEIR PHYLOGENY
IN
D. W. SUTCLIFFE Department of Zoology, University of Durham, King's College, Newcastle upon T y n e (Received 23 January 1963)
A b s t r a c t - - 1 . The aim of this study is to suggest that in pterygote insects a basic type of haemolymph is discernible, and that this basic type was modified in several ways and on a number of occasions during the evolution of the Insecta. 2. One organic and six inorganic components in insect haemolymph are considered with respect to their potential contributions to the total osmotic activity of the haemolymph--the concentration of each component is expressed as a percentage of the total osmolar concentration. 3. It is suggested that a high proportion of chloride is characteristic of the Exopterygota, whereas a low proportion of chloride is characteristic of the Endopterygota. 4. In the majority of exopterygote insects, sodium and chloride each account for major proportions of the total osmolar concentration. Potassium, calcium, magnesium, inorganic phosphate and free amino acids account for only minor proportions of the total. It is suggested that haemolymph with this composition represents the basic type of haemolymph in pterygote insects. This type of haemolymph is very similar to that found in modern representatives of phylogenetically related groups, viz. an apterygote insect, a chilopod and diplopod. Hence it is suggested that the basic haemolymph type found in modern pterygores represents the haemolymph of the ancestral stock of pterygote insects. 5. In endopterygote insects, with a monophyletic origin from the ancestral pterygote stock, the basic haemolymph type is modified by a reduction of chloride and an increase of organic acids, including a tendency to increase the proportion of free amino acids. 6. In certain exopterygote and endopterygote insects the haemolymph is radically modified in various ways. These are regarded as specializations. One type of specialization appears to be characteristic of the Lepidoptera and Hymenoptera, where inorganic ions contribute only a very minor proportion of the total haemolymph osmolar concentration; free amino acids, and, in some cases, other organic components, contribute the major proportion of the haemolymph total.
INTRODUCTION
INSECT haemolymph contains very high concentrations of organic compounds containing nitrogen, in particular a wide variety of free amino acids, a number of non-amino carboxylic acids, and various carbohydrates. The probable contribution of these and other organic components to the freezing-point depression (haemolymph~) is very variable, but in some insects they apparently account for nearly 121
122
D.W. SUTCLIFVE
all of the haemolympha, for example, 81 per cent in the honey-bee larva Apis mellifera and 86 per cent in the beetle larva Popillia japonica (Buck, 1953). The concentrations of inorganic components are also highly variable. The sodium concentration may be either very high or extremely low. The potassium concentration may be relatively high and exceed that of sodium. The concentrations of calcium and magnesium are also relatively high; in some instances both ions are present in very high absolute concentrations (see Buck, 1953; Duch~teau et al., 1953). Florkin (1949) has suggested that the high aminoacidaemia and high concentrations of magnesium are characteristic features of insect haemolymph. It is also stated that the chloride concentration is generally low, accounting for' only 10-15 per cent of the haemolymph (Buck, 1953 ; Wyatt, 1961), but Sutcliffe (1962, and in this paper) demonstrates that this view requires modification. For a comprehensive account of the composition of insect haemolymph see the review by Wyatt (1961). In the first attempt to explain the differences in concentrations of inorganic ions found in insect haemolymph, Bon6 (1944) suggested that a correlation exists between diet and the ratio of sodium-potassium concentration in the haemolymph; in herbivorous insects this ratio is less than unity, whereas in carnivorous insects the ratio is always greater than unity. Bon6 concluded that the correlation is, in general, applicable to all insects, although he pointed out a number of inconsistencies in his data. Later, following attempts to change the ratio of sodiumpotassium in several insects by feeding on selected diets, Bon6 (1947) concluded that " . . . the association observed between the sodium-potassium ratio in the coelomic fluid and the diet is not the result of the chemical composition of the food-stuff acting during the actual life of a given insect. The ionic composition of the internal medium, as found now, is a species characteristic, maintained by mechanisms of mineral regulation". Tobias (1948) independently came to the same conclusion, and regulatory mechanisms maintaining a specific haemolymph composition are now known in a variety of insects (reviewed by Shaw & $tobbart, 1963). In a recent study Sutcliffe (1962) showed that the supposed correlation with diet is not applicable to aquatic insects and earlier Duch~teau et al. (1953) found that the correlation is not applicable as a generalization of observations on terrestrial insects. Instead, these authors suggested that the occurrence of a high or low sodium concentration is largely correlated with systematic categories of the Insecta. Duchhteau et aL expressed the equivalent concentrations of each of four cations as percentages (indices) of the summed total of these cations in haemolymph of a wide variety of insects. In three species of Odonata, sodium contributed about 85 per cent of the cation total, and potassium, calcium and magnesium each contributed about 5 per cent. In striking contrast, in more than twenty species of Lepidoptera the sodium concentration was very low, contributing usually less than 10 per cent of the cation total, whereas magnesium provided 30-60 per cent, potassium 20-45 per cent, and calcium 10-30 per cent. Duch~teau et al. suggested that a high sodium index, exemplified by the Odonata,
THE C H E M I C A L C O M P O S I T I O N OF H A E M O L Y M P H
123
is a "primitive" or ancestral feature retained in other groups also considered to be "primitive" insects, whereas the low sodium index found in the Lepidoptera is an "advanced" or specialized feature characteristic also of certain other groups of insects regarded as phylogenetically recent in origin. The present paper is offered in support of this view, although the problem is approached in greater detail and in a manner different from that of Duch~teau et al.
METHODS One organic and six inorganic components are considered with respect to their potential contribution to the total osmotic activity of the haemolymph. Thus the concentrations of free amino acids, mM/l., and sodium, potassium, calcium, magnesium, chloride and inorganic phosphate, mg ions/l, are expressed as percentages of the total osmolar concentration, m-osm/l. The latter was obtained from the observed haemolymphA employing the relationship 1 osmole/1. = A 1.86°C. No allowance was made for incomplete dissociation of electrolytes as, for example, in a solution of sodium chloride, nor for chelation or "binding" between electrolytes and large molecules. The estimated osmotic effect of each component is therefore maximal. Indirect evidence of ion binding in haemolymph, particularly in the case of calcium and magnesium ions, is reviewed by Carrington & Tenney (1959) and Wyatt (1961). Some original estimations of various components in haemolymph of several insects are given in Table 1, together with data for a centipede, a millipede and a terrestrial isopod. Details of the methods employed are given by Sutcliffe (1962). That paper contains the raw data concerning aquatic insects. Data for terrestrial insects are drawn largely from Duch~teau et al. (1953) (also given in Buck, 1953) but also from original sources in the literature. All sources are indicated in Table 2, which embodies the main results of this study. The examples shown in Table 2 are selected to give a representative picture of the range of variation found in each order of the Insecta. Estimates of the possible contribution of free amino acids to the total osmolar concentration, given in Table 2, require special comment. Sutcliffe (1962) estimated the molar concentration of free amino acids by a modified version of the Folin colorimetric technique. Duch~teau & Florkin (1958) employed the microbiological technique, and give the concentrations of fifteen "apparent" free amino acids, in mg/100 ml. As an approximation, their results were converted to molar concentrations by dividing the total amount of the fifteen amino acids with 120. This value was chosen rather arbitrarily but is close to the mean molecular weight of those amino acids that appear to be most abundant in the insects investigated. In a few instances the molar concentrations were calculated from sources employing the micro-Kjeldhal technique and estimating either non-protein N or amino-N, in mg/1. These were converted to molar concentrations by division with 14. It
124
I2). W. SUTCLIFFE
should be noted that in both cases the estimated molar concentration is almost certainly too high, since the technique estimates excess nitrogen atoms derived from di-amino acids and any peptides that m a y be present. T A B L E 1 - - A N A L Y S E S ON HAEMOLYMPH OF SOME INSECTS AND OTHER ARTHROPODS
Concn. in raM/1. Group
Species
Haemolymph Aoc
K
C1
Insecta Orthoptera
aLocusta migratoria migratorioides bChorthippus parallelus Isoptera CCryptoterrnes havilandi Dermaptera aForficula auricularia Homoptera "yassid Megaloptera ISialis lutaria Neuroptera gOsmylusfulvicephalus Hymenoptera nNeodiprion sertifer Mecoptera ipanorpa communis Chilopoda JLithobius sp. Diplopoda klulus scandinavius Isopoda ~Oniscus asellus
0.841(4)
-
-
98(4) 119
0"730 0"736(5) + 0'035 0"679(6) + 0"09 0"765 0.805(2) 0.690 0.608(6) + 0.07 0.457(6) __0.05~ 1.093(3)
82 90 41 35 62(2) (5)_+2.4 17(6)+_5 34 154 61 256 -
-
Haemolymph from : (a) one adult in each of four samples ; (b) ten larvae in one sample ; (c) sixty larvae in one sample; (d) six adults in one sample, A on individuals; (e) eighteen adults in one sample, A on individuals; (f) eight adults in one sample; (g) seventeen adults in each of two samples; (h) one larva in each sample; (i) twenty-two adults in one sample; (j) twelve in one sample, A on individuals; (k) four in one sample, A on individuals; (l) eight in one sample, A on individuals. RESULTS F r o m consideration of T a b l e 2, and on the basis of all the data available at present, it appears that (a) the h a e m o l y m p h composition of pterygote insects m a y be categorized into several distinct types, enumerated below, (b) in the majority of cases one of these types is characteristic of the m e m b e r s of an order. I n type I (Fig. I(A)), exemplified by Aeschnagrandis larvae, sodium and chloride can account for major proportions of the total osmolar concentration. On the other hand, potassium, calcium, m a g n e s i u m and inorganic phosphate account for very minor portions, and free amino acids account for about 10 per cent of the total osmolar concentrations. T y p e I appears to be characteristic of the E p h e m e r optera, Odonata, Plecoptera, Dictyoptera and H e m i p t e r a - H e t e r o p t e r a .
THE CHEMICAL
Exopterygotes A
COMPOSITION
B
Ephemeroptera Orthoptera Dermaptera Odonata lsoptera Ptecoptera Dictyoptera Heteroptera Endopterygotes E •
,
125
OF HAEMOLYMPH
C
D
Phasmida
Homoptera
G
F
H
.
• •
° °
~
°
°
o,.,,
...
•
•
•
•
i?.'."; .,-..
Megaloptera Neuroptera Mecoptera Trichoptera Diptera
Lepidoptera Hymenoptera
~
Y
Coleoptera
FIG. 1. Osmotic effects of components illustrated as percentages of the total osmolar concentration of haemolymph in pterygote insects. Each block in the figure is visualized as two vertical sections, each section representing 50 per cent of the total osmolar concentration. The percentage contributions of cations are illustrated in the left-hand section, with sodium at the base (stippled), followed by potassium (black area), calcium (white area) and magnesium (vertical stripes). Anions are illustrated in the right-hand section, with chloride at the base (oblique stripes) followed by inorganic phosphate (fine stippling). Where possible, free amino acids are illustrated in equal proportions in both sections (coarse stippling). The large blank area in each block represents the proportion of the total osmolar concentration that must be accounted for by other components of the haemolymph. T y p e I I (Fig. I(B)), illustrated by Locusta migratoria migratorioides, represents the haemolymph of some Orthoptera, one dermapteran and one isopteran. It is essentially similar to type I, except that sodium and chloride contribute rather less to the total osmolar concentration, about 24 per cent and 22 per cent respectively, and a larger proportion of the total (approaching 30 per cent in Locusta) consists of unknown molecules. T y p e I I is not characteristic of all orthopterans.
126 TABLE 2--OSMOTIC
D.W.
SUTCLIFFE
EFFECT O F C O M P O N E N T S AS A P E R C E N T A G E OF T H E T O T A L O S M O L A R C O N C E N T R A T I O N O F H A E M O L Y M P H I N P T E R Y G O T E INSECTS
P e r cent of osmolar concn. O r d e r a n d species
Ephemeroptera Ephemeradanica 2c Odonata Aeschnagrandis ~° A . cyanea 2° Enallagma cyathigerum 2° Agrion virgo 2° Plecoptera Perla bipunctata 2c Dinocras cephalotes 2° Orthoptera Locusta migratoria migratorioides Chortophaga viridifasciata 4 .4nabrus simplex 1~ Gryllotalpa gryllotalpa Phasmida Carausius morosus 17c C. morosus 23 Dictyoptera Periplaneta americana 1 Isoptera Cryptotermes havilandi Dermaptera Forficula auricularia neteroptera Notonecta obliqua 2° C o r i x a p u n c t a t a ~° Rhodnius prolixus 17~ Megaloptera Sialis lutaria ~° Neuroptera Osmylus f u l v i cephalus
Haemolymph Stage - - A°C m-osm/l.
Na
K
Ca
Mg
L
0.504
271
38.0
6"6
L L
0"735 0"701
395 377
36"7 37"7
2"3 2.1
L A
0"620 0'790
333 425
41 "7 34"2
4.2 6'5
38.1 25-7
L
0"644
346
36"8
3'5
30"7
L
0"583
313
37'4
3"2
35.5
A
0"841
452
4"0 is 1"71°
3"01° 21"6
A A
0'890
479 450 ~s
24"2 xa • 22"8 4"9
0'8 3'3
4'4 0"3
---
A
0"840 is
38-26 52.01o
2"46 1.61o 3.11o
1.11o
__
451
--
CI
1"91°
0"6 0"7
28.4
1'91° 27"9 29.7
A A
0'570
306 306
2"9 4"9
9"1 5"9
2"6 2"5
23"9 17"3
30.4 33"0
A
0"89791
482
34"0
1"6
0'9
1'1
29-7
L
0"730
392
26'3
7"1
2-22a
4.5 °~ 21"0
A
0"736
396
24-2
3"3
4.29 • - -
22.7
A A
0,756 0"600
406 323
38"2 34-8
5"2 9"6
3.89b 2.39b . . . .
29.8 23"4
A
0"695
374
43"8
1 "6
--
339
32'2
1'5
2"2
433
21"2
9"2
L
A
0"805
5"6
. . . .
9"1
14.3
PO 4
Amino acids
-1"1 1.1
9"8 9.0
10'1 1"3
12"5
11"7 n
8"7
13"1 1"6
20"9 41-5
11'511
33"9 x 4"819
--
2"9
25"1
--
127
THE CHEMICAL COMPOSITION OF HAEMOLYMPH
TABLE 2
(cont.)--OSMOTICEFFECT
O F C O M P O N E N T S AS A P E R C E N T A G E O F T H E T O T A L O S M O L A R
CONCENTRATION OF H.zEMOLYMPH IN PTERYGOTE INSECTS
Per cent of osmolar concn. Order and species
Stage
Haemolymph A°C
Coleoptera Hydrophilus piceus Dytiscus marginalis 2° Popilliajaponica 1~ Hymenoptera Apis mellifera 5 Vespulagermaniea Mecoptera Panorpacommunfi Trichoptera Phryganeasp. 2° Limnephilus stigma 2° Anabolianervosa a° Lepidoptera Bombyx mori 7 Ephestia kuehniella 1° Nymphula nymphaeta ~° Prodenia eridania ~ Samia walkeri ~'12 Saturnia pyri 1° Teleapolyphemus ~ Diptera Gastrophilus intestinalis 14 Tipula montium ~° Aedes aegypti
m-osm/1.
[ Na
I
Amino K
Ca
Mg
C1
13"0
13"4
PO4
acids
A
0"647
348
35'0
4"0
6-9
17-511 9"211
A L
0"745 1"03
401 554
31-5 3"6
3"5 1'7
3'01° 1"4
4"71° 11"0 3"5 3"4
0"77. -0"9 30"7
L
0"860
462
1"1
5"2
0"9
1"7
7"2
2"2
L
0"880 lsa
473
10-26 8-7 e 5.51o 11.81o
A
0"690
371
25"4
10"2
--
9"2
L
0"455
245
28"2
2"9
L L
0"380 0'405
204 218
40"7 46"4
6-9 7"8
L
0"480
258
5"4
15'0
L
1"130lab
608
5"4
L L P P P
0"552 0'840 1.110 0"780 xsc 1"170
297 452 597 419 629
L
0"872
L L
0"443
2"91° 10"41° 15"1 . - - .
44.8 22.311
23"7
--
4"9 4"1
5"9 6"4
20"0 13"3
7"0
19"5
8"1
1"9
37"234
5"4
3"4
4"2
13"5 4"9 0'5 1'0 0"5
7'3 8"9 7'0 9"8 9"4
2"0 0"5 1"7 1"0
1"6 3"5
7"8 7"5 1"7
1"3 0-6
5'9
3'2
3"8
37"2 52'8 39"611 4"1
469
37"2
2"6
0"6
3'4
3'2
0-9
238 269 ~2
48"5 2'9 2"5 ~°~ 3"41°° 3"4 37"217b 1"517b. . . . 19"3~2
0"8
13"2 8.311 19"8
1Asperen & Esch, 1956; ~Babers, 1938; 3Barrat & Arnold, 1911; ~Barsa, 1954; SBishop et al., 1925; SBon6, 1944; 7Buck, 1953; aCarrington & Tenney, 1959; 8Clark, 1958, a data for Zootermopsis angusticoUis, b data for Notonecta lu'rbyii; l°Duchfiteau et al., 1953, a data for Tipula paludosa and T. oleracea; nDuchfiteau & Florkin, 1958 ; X~Gese, 1950; lSHoyle, 1954; 14Levenbook, 1950; 15Ludwig, 1951; lePepper et al., 1941; 17Ramsay, a 1952, b 1953, c 1955; lSRouschal, 1940, a data for adult Vespa crabro, b data for Ephestia elutella larva, c data for Saturnia pyri larvae; 1'Stevens, 1961 ; 2°Sutcliffe, 1962; SlTreherne, 1961 ; 22Wigglesworth, 1938; ~SWood, 1957; z4Wyatt et al., 1956; 26arbitrary, no data available.
128
D.W. SUTCLIFFE
Inspection of Table 2 suggests that Gryllotalpa gryllotalpa is similar to type I, whereas the very low percentage of sodium in the mormon cricket, Anabrus simplex, is completely anomalous. The haemolymph composition in this insect closely resembles that of Lepidoptera and Hymenoptera (see below), including the very high concentration of amino-N, 187 raM/1. (Pepper et al., 1941). Type III (Fig. I(E)), exemplified by the larva of Sialis lutaria, represents the haemolymph of Trichoptera, Diptera, a megalopteran, a neuropteran and a mecopteran. Here the haemolymph contains a very low percentage of chloride relative to that of sodium. Sodium varies from 21 per cent of the total in adult Osmylus fulvicephalus (Neuroptera) to 48 per cent in Tipula montium larvae (Diptera), i.e. the range of sodium embraced by types I and II. Type III also resembles these with low percentages of potassium, calcium, magnesium and inorganic phosphate, but differs in the relatively high percentage of free amino acids, up to 25 per cent in Sialis larvae. Haemolymph in the majority of Coleoptera is also of type III except that magnesium possibly tends to account for a slightly higher proportion, about 10 per cent of the total (Fig. I(H)). However, in four coleopterans the haemolymph composition is that of type IV. The species concerned are Timarcha tenebricosa and Agelastica alni (Chrysomelidae), Tenebrio molitor (Tenebrionidae), and Popillia japonica (Scarabaeidae) (Fig. I(G)). It is worth noting that in other Scarabids the haemolymph appears to be type III (see data in Duch~teau et al., 1953 ; A in Rouschal, 1940, and Buck, 1953). Type IV (Fig. I(F)), illustrated by the larva of Prodenia eridania, is completely characteristic of the Lepidoptera and Hymenoptera. Here, both sodium and chloride account for only a minor percentage of the total osmolar concentration, about 10 per cent or less in each case. Potassium, calcium, magnesium and inorganic phosphate also account for small amounts, 10 per cent or less in each case, although magnesium may account for a larger proportion of the total in larvae of Bombyx mori if, in fact, the haemolympha is usually as low as 0.48°C (cf. Buck, 1953; Jeuniaux et al., 1961) and assuming that most of the magnesium is not bound to large molecules. A major proportion of the haemolymph total in type IV is accounted for by free amino acids, representing about 40 per cent in several lepidopterans and in the honey-bee, Apis mellifera. In fact, the concentration of free amino acids is very high in nearly all lepidopteran larvae and pupae (Duch~teau & Florkin, 1958; Florkin, 1959), with concentrations ranging between 10-20 mg/ml or roughly 83-166 mM/l. amino acids. In larvae of Bombyx mori, Jeuniaux et al. (1961) found a mean haemolympha = 0.60°C, of which 0.16°C (26.7 per cent) is accounted for by free amino acids (of. 37.2 per cent in Table 2). However, in the pupa of Telea polyphemus, Carrington & Tenney (1959) report only 36.5 mg per cent nonprotein N, or roughly 26 mM/l. as amino acid; this represents only 4 per cent of the haemolymph osmolar concentration. It is now possible to draw the following main conclusions. In haemolymph of
THE CHEMICAL COMPOSITION
OF HAEMOLYMPH
129
the Exopterygota (Imms, 1957) sodium and chloride both represent major proportions of the total osmolar concentration. On the other hand, in the Endopterygota chloride accounts for only a minor proportion, usually less than 10 per cent of the total, although in a few instances chloride is more important, e.g. 19 per cent of the total in larvae of Aedes aegypti. In most endopterygote orders, sodium accounts for a major proportion of the total osmolar concentration, but in the Lepidoptera, Hymenoptera and in some Coleoptera, sodium accounts for only a minor proportion. Also, it is suggested that a high proportion of chloride in haemolymph is characteristic of the Exopterygota, whereas a low proportion of chloride is characteristic of the Endopterygota. There appear to be three exceptions to the above generalizations. In Anabrus simplex sodium probably accounts for less than 10 per cent of the total (see Table 2). Unfortunately, Pepper et al. (1941) did not estimate the haemolymph chloride in this insect. Haemolymph of the stick insect, Carausius morosus, is unlike that of any other insect (Fig. I(C)) since it contains a very high proportion of magnesium phosphate (Ramsay, 1956), although Wood (1957) reported only 16mM/1. H~PO 4. However, although sodium represents less than 5 per cent, chloride accounts for at least 30 per cent of the total osmolar concentration, and in this respect it is a typical exopterygote insect. The third exception is the unidentified Jassid plant bug reported in this paper (Table 1, Fig. I(D)). Here, sodium represents 16 per cent and chloride only 11 per cent of the total osmolar concentration. In both respects this insect is atypical among the Exopterygota, resembling instead type IV characteristic of the Lepidoptera and Hymenoptera. At this point it is convenient to mention briefly other components found in haemolymph. Proteins, and non-protein compounds containing nitrogen (excluding free amino acids), probably contribute only a very small proportion of the total osmotic activity of haemolymph (see Buck, 1953 ; Wyatt, 1961). On the other hand, very high concentrations of certain carbohydrates are reported in some insects, sufficient to contribute as much as 10 per cent of the total osmolar concentration. In particular, the disaccharide trehalose possibly accounts for 8"5 per cent in adult Periplaneta americana (data in Treherne, 1960), 3-7 per cent in Bombyx mori larvae and 2.3 per cent in Telea polyphemus pupae (data in Wyatt, 1961). Using Treherne's (1958) estimate of the concentration of trehalose in adult Schistocerca gregaria, this represents 4.5 per cent of Locusta haemolymph. In some insects non-amino organic acids probably make noticeable contributions to the osmotic activity of haemolymph, e.g. citrate, 10-12 per cent in B. mori larvae, 4"5 per cent in Prodenia eridania larvae and 2.3 per cent in T. polyphemus pupae (data in Levenbook & Hollis, 1961); succinate 4.5 per cent and malate 5-5 per cent in Gastrophilus intestinalis larvae (data in Wyatt, 1961). Thus the total osmolar concentration of B. mori larvae is largely accounted for (see Table 2 and Wyatt, 1961). However, in many insects, in particular Lepidoptera, Hymenoptera and some Coleoptera, a major part of the haemolymph total apparently consists of unknown molecules, e.g. 50-60 per cent of the total in T. polyphemus, P. eridania and Popilliajaponica; and see Sutcliffe (1962).
130
D.W. SUTCLIFFE
DISCUSSION It must be emphasized that the recognition of at least three distinct types of haemolymph does not represent an attempt to classify rigidly the haemolymph composition of each species of insect, nor is it an attempt to classify orders of the Insecta. Several exceptions are already known and doubtless many more await discovery. Moreover, the method adopted here, assessing the osmotic contribution of each component, is slightly artificial, since we do not know the actual osmotic activities of the various components in haemolymph. Also, as noted in Table 2, analyses on one species were occasionally used to assess osmotic contributions in another species. The recognition of haemolymph types is, therefore, regarded merely as a convenient way of handling the data available at present. The aim of this study is to suggest that a basic type of haemolymph is discernible, and that this basic type was modified in several ways and on a number of occasions during the evolution of the Insecta. The views of morphologists and systematists concerning the relationships between pterygote insects are summarized in Jeannel (1949), Imms (1957) and Hinton (1958). We will consider only those orders for which analyses on haemolymph are available. Briefly, the Ephemeroptera and Odonata represent an ancient group, the Palaeoptera, which diverged from the Neoptera or main line of pterygote evolution at a very early date (Devonian, or earlier still). The remaining orders in the Exopterygota of Imms (1957) probably represent several distinct stocks arising out of the early Neoptera, whereas the Endopterygota ( = Oligoneoptera in Jeannel, 1949) apparently are derived from a single stock. This monophyletic group diverged along two lines, one leading to the "panorpoid complex" including the Mecoptera, Trichoptera, Lepidoptera and Diptera; the other line leading to the remaining endopterygotes considered here, i.e. the Megaloptera, Neuroptera, Coleoptera and Hymenoptera. Reverting to the results of this study, we see that haemolymph type I occurs in both of the modern palaeopteran orders, and occurs also in the Plecoptera, Dictyoptera and Heteroptera. According to Jeannel (1949) the latter three orders are modern representatives of three distinct stocks of neopteran Exopterygota. It is suggested, therefore, that type I represents the basic haemolymph composition of the ancestral stock of pterygote insects, and that this basic type is retained in the Palaeoptera and in many neopteran Exopterygota, with modification by a small reduction in sodium chloride (type II) in at least one dermapteran, one isopteran, and in some orthopterans, The stick insect, Carausius, and a Jassid homopteran represent considerable modifications of the basic type, and are considered below as specializations. It was suggested that a low proportion of chloride is characteristic of the Endopterygota. In view of their monophyletic origin, it seems reasonable to assume that the reduction in chloride was a single event that occurred very early in the evolution of the group, prior to the divergence of the "panorpoid complex". Apart from this, haemolyrnph in many endopterygotes retains much of the basic features of the ancestral pterygote stock, although there appears to be a marked
THE C H E M I C A L C O M P O S I T I O N OF H A E M O L Y M P H
131
tendency towards an increased proportion of free amino acids (type III). But in the Lepidoptera, Hymenoptera and in some Coleoptera, the haemolymph is radically modified (type IV). Here inorganic ions, particularly sodium and chloride, form a very minor proportion of the haemolymph, and the tendency towards an increased proportion of free amino acids (and other organic acids) is greatly enhanced. This specialization probably occurred independently on at least two occasions during the evolution of endopterygote insects, since it is believed that the Lepidoptera evolved from the "panorpoid" line. Also, there appears to be no close relationship between the Coleoptera and Hymenoptera (Jeannel, 1949; Imms, 1957). Duch~teau et al. (1953) recognized that in the Lepidoptera, Hymenoptera, Chrysomelidae (Coleoptera) and in Carausius, the haemolymph is specialized on the basis of a low percentage of sodium and high percentage of potassium and magnesium. From the viewpoint adopted here there is no striking tendency towards a greater increase in potassium and magnesium in these insects. This does not deny the fact that the absolute concentrations of these ions are often quite high but, since the total concentration of the haemolymph is also increased in these terrestrial insects, the proportions of potassium, calcium and magnesium relative to the total osmotic concentration are usually Similar to those in other insects. With regard to Bond's (1944) correlation, the relative osmotic effect of the potassium ion is very similar in all of the insects investigated; in most cases potassium contributes only 2-10 per cent of the total in both herbivorous and carnivorous insects. On the other hand, the very great reduction of sodium in these specialized insects is abundantly clear. At present, it is not possible to offer an adequate explanation for these specializations. Duch~teau et al. (1953) point out that all of the insects concerned feed on higher plants, and suggest that there is a correlation between the development of specialized haemolymph and the evolution of Angiosperms. The underlying reasons for this relationship are obscure, since already there is evidence that haemolymph is not specialized in some Scarabid beetles ("chafers") associated with Angiosperms. Moreover, it is certain that not all insects feeding on Angiosperms are specialized; locusts provide an obvious example. Hence it is difficult to accept the possibility that the specialized type of haemolymph is a special case of direct adaptation to the relative proportions of cations found in Angiosperms. Duch~teau et al. have discussed this point in detail, and no further comments are necessary. What is required now is more information on ion uptake and excretion in these insects (see Ramsay, 1955, 1956). However, it is possible that Bond's (1944). general conclusions were influenced by the fact that of his twenty-seven species, ten species with a low sodium-potassium ratio are regarded here as specializations. It is of interest to note that, with the possible exception of Carausius, the haemolymph composition in the specialized insects is extraordinarily similar to the composition of some types of tissue cells in other animals. Here the sodium concentration is usually very low, and organic molecules, including amino acids, sugars and various phosphorylated compounds are extremely abundant. The
132
D . W . SUTCLIFFE
similarity is particularly striking when the muscle fibre of Carcinus (Shaw, 1958) is compared with haemolymph of the Lepidoptera, Hymenoptera and specialized Coleoptera (cf. Figs. 2(H), I(F), I(G)). Is this in some way connected with special requirements of very rapidly developing tissues in the pupal instar ? We may now briefly compare the composition of haemolymph in pterygote insects with the blood composition of other arthropods. Analyses on representatives of modern classes within the Arthropoda are presented in Table 1 and Fig. 2. A ...°
D
B
%-%
!:T .'~ ".-.:~ • °°
~-%
"i": .."" ::y,.. ...•.
I :(:,
°°.
i(:~:d , , , ,
':o¢,~
i'-~' ""
~~! -....
~ x
...
Petrobius
marifimus
~ x
Lithobius F
E
ii? ..°-.
~
lulus scandinovius
Tegenoria attica
G
=.....
~
.......
.,,,-.".'..;...
-.;.-,-'~....
.;'.~.~ ,.-~
Oniscus asellus
Asellus aquaticus
~::..-. ,.~
Astacus
Carcinus muscle fibre
FIG. 2. Osmotic effects of components illustrated as percentages of the total osmolar concentration of blood in: (A) an apterygote insect, (B) a chilopod, (C) a diplopod, (D) an arachnid, (E-G) crustaceans. (H) illustrates the osmotic effects of components in the muscle fibre of CarclnUs maenas. Conventions as in Fig. 1.
In all marine, brackish and fresh-water Crustacea-Malacostraca the blood composition is of the type where sodium chloride accounts for nearly all of the blood osmolar concentration, and potassium, calcium and magnesium each account for only about 1-3 per cent of the total (Fig. 2(G); data in Robertson, 1960; Lockwood, 1961). Organic molecules are present in extremely low concentrations and therefore contribute little to the blood total. Thus the blood composition in the terrestrial isopod Oniscus asellus (Table 1, Fig. 2(E)) is very similar to that of its marine-littoral relative Ligia oceanica (Parry, 1953) and of Asellus aquaticus (Fig. 2(F), Lockwood, 1959) found in fresh water. This type of blood also occurs in the arachnid Tegenaria atrica (Fig. 2(D) ; Croghan, 1959), in the chilopod Lithobius
THE C H E M I C A L C O M P O S I T I O N OF H A E M O L Y M P H
133
(Fig. 2(B)) and in the apterygote insectan Petrobius maritimus (Fig. 2(A); Lockwood & Croghan, 1959). Evidence concerning phylogenetic relationships within the Arthropoda is reviewed by Tiegs & M a n t o n (1958). T h e r e appears to be no close affinity between the Crustacea, Arachnida and Myriapoda, but the Insecta probably arose from a stock of myriapods. It is therefore of interest to find that in the diplopod lulus scandinavius (Table 1) the contributions of both sodium and chloride are reduced; sodium accounts for only 29 per cent and chloride 25 per cent of the blood osmolar concentration (Fig. 2(C)). In this respect the blood of lulus appears to be similar to that of some exopterygote insects, and it would be of great interest to know whether or not the blood of this and other diplopods contains high concentrations of free amino acids and other organic compounds. Hence it appears that the haemolymph of many modern exopterygotes, representing the basic haemolymph composition of the ancestral stock of pterygote insects, is very similar to that of modern representatives of phylogenetically related groups, i.e. Petrobius and myriapods. Also, as Duch~teau et al. (1953) pointed out, the haemolymph of these "primitive" exopterygotes is in many respects very similar to the blood of other arthropods, and similar to the blood of most vertebrates. Acknowledgements--Most of this study was carried out "during two visits to the Windermere Laboratory of the Freshwater Biological Association, and I wish to thank the Director for generously providing research facilities, and the members of staff for their willing cooperation in many ways. The investigation was initiated during the tenure of a D.S.I.R. Research Fellowship. REFERENCES ASPEREN K. VAN • ESCH I. VAN (1956) The chemical composition of the haemolymph in Periplaneta americana. Arch. nderl, zool. 2~ 342-360. BABE:~S F. H. (1938) An analysis of the blood of the sixth-instar southern armyworm, Prodenia eridania. J. Agric. Res. 57, 697-706. BAre,AT J. O. W. & ARNOLD G. (1911) A study of the blood of certain Coleoptera: Dytiscus marginalis and Hydrophilus piceus. Quart. J. Micr. So/., 56, 149-165. BARSA M. C. (1954) The behaviour of isolated hearts of the grasshopper Chortophaga viridifasciata and the moth Samia walkeri in solutions with different concentrations of sodium, potassium, calcium and magnesium. J. Gen. Physiol. 38, 79-92. BISHOP G. H., BaIGGS A. P. & RONZONI E. (1925) Body fluid of the honeybee larva: II. Chemical constituents of the blood and their osmotic effects. J. Biol. Chem. 66, 77-88. BON~ G. J. (1944) La rapport sodium-potassium dans le liquide ccelomique des insectes. I. Ses relations avec le rdgime alimentaire. Ann. Soc. zool. Belg. 75, 123-132. BON~ G. J. (1947) Regulation of the sodium-potassium ratio in insects. Nature, Lond. 160, 679-680. BUCK J. B. (1953) In Insect Physiology, Chapter 6 (Edited by ROEDERK. D.). Wiley, New York. CAm~INGTON C. B. & TENNEY S. M. (1959) Chemical constituents of haemolymph and tissue in Telea polyphemus Gram. with particular reference to the question of ion binding. J. Ins. Physiol. 3, 402-413. CLARK E. W. (1958) A review of literature on calcium and magnesium in insects. Ann. Ent. Soc. Amer. 51, 142-154.
134
D . W . SUTCLIFFE
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135
ROBERTSON J. D. (1960) Osmotic and ionic regulation, in The Physiology of Crustacea, I, Chap. 9. Academic Press, New York & London. ROUSCHAL W. (1940) Osmotische Werte wirbelloser Landtiere und ihre okologische Bedeutung. Zeitschr. Wiss. Zool. Abt. A, 196-218. SHAW J. (1958) Osmoregulation in the muscle fibres of Carcinus maenas. J. Exp. Biol. 35, 920-929. SHAW J. & STOBBARTR. H. (1963) Osmotic and ionic regulation in insects. Adv. Ins. Physiol. 1, 315-399. STEVENS T. M. (1961) Free amino acids in the haemolymph of the American cockroach, Periplaneta americana L. Comp. Biochem. Physiol. 3, 304-309. SUTCLIFFE D. W. (1962) T h e composition of haemolymph in aquatic insects, ft. Exp. Biol. 39, 325-343. TIECS O. W. & MANTON S. M. (1958) T h e evolution of the Arthropoda. Biol. Rev. 33, 255337. TOBIAS J. M. (1948) Potassium, sodium and water interchange in irritable tissues and haemolymph of an omnivorous insect, Periplaneta americana. J. Cell. Comp. Physiol. 31, 125-142. T~HERNE J. E. (1958) T h e absorption and metabolism of some sugars in the locust, Schistocerca gregaria (Forsk.). J. Exp. Biol. 35, 611-625. TaEHERNE J. E. (1960) T h e nutrition of the central nervous system in the cockroach, Periplaneta americana L. T h e exchange and metabolism of sugars. J. Exp. Biol. 37, 513-533. TREHEaNE J. E. (1961) Sodium and potassium fluxes in the abdominal nerve cord of the cockroach, Periplaneta americana L. J. Exp. Biol. 38, 315-322. WIGGLESWOaTHV. B. (1938) T h e regulation of osmotic pressure and chloride concentration in the haemolymph of mosquito larvae. J. Exp. Biol. 15, 235-247. WOOD D. W. (1957) T h e effects of ions upon neuromuscular transmission in a herbivorous insect. J. Physiol. 138, 119-139. WYATT G. R. (1961) T h e biochemistry of insect haemolymph. Ann. Rev. Ent., 6, 75-102. WYATT G. R., LOUGHHEEDT. C. & WYATT S. S. (1956) T h e chemistry of insect haemolymph. Organic components of the haemolymph of the silkworm, Bombyx mori, and two other species, ft. Gen. Physiol. 39, 853-868.