Amino acids in the haemolymph of diseased Popillia japonica (Newman) larvae

Purchased by Agricultural Research Service, U.S. Department of Agriculture, for Official Use J7.Ins. Phyriol., 1965, Vol. 11, pp. 671 to 682. Pergamon Press Ltd. Printed in Great Britain

AMINO ACIDS IN THE HAEMOLYMPH ,?OPILLIA JAPONICA (NEWMAN) ODETTE ROBERT

OF DISEASED LARVAE

:L. SHOTWELL, GLENN A. BENNETT, HARLOW H. HALL, D. STUBBLEFIELD, JOHN E. PETERS, CECIL H. VAN ETTEN and RICHARD W. JACKSON Northern Regional Research Laboratory,*

Peoria, Illinois

(Receizwd 17 November 1964)

Abstract-Free amino acid constituents in the haemolymph from diseased Popillia japonica (Newman) larvae were determined by ion-exchange chromatography, paper chromatography, enzymatic analysis, and amperometric titration. Haemolymph from larvae infected with BuciZZus popilliue, compared with that from healthy larvae, contained higher concentrations of glutamic acid, /3-alanine, aspartic acid, phenylalanine, threonine, serine, and lysine and lower concentrations of glycine, tyrosine, and histidine. Differences in the amino acid composition depend on the infective organism. The only amino acid to increase during infection with B. lentimorbus was glutamic acid. Amounts of histidine, proline, glycine, alanine, valine, isoleucine, tyrosine, and arginine decreased. The level of protein material in the haemolymph as well as the composition did not change markedly during infection with either B. popilliae or B. lentimorbus. Haemolymph contains little peptide of low molecular weight. Several additional amino acids were determined in haemolymph from healthy and diseased larvae. Milky disease apparently has no effect on the concentration of tryptophan, cystine, cysteine, glutamine, asparagine, and lanthionine.

INTRODUCTION

INVESTIGAT~NS are being continued on the production of spores in artificial media of Bacillus popilliae Dutky and B. lentimorbus Dutky which cause milky disease in Popillia japonica (Newman) larvae. Previously we reported on some amino acid constituents in haemolymph of healthy larvae (SHOTWELL et al., 1963), and these results are being used in the design of media for growth and sporulation studies of milky We have now examined the same constituents in haemolymph disease orgamsms. from diseased larvae in the hope that comparisons of healthy and diseased larvae would lead to the discovery of factors responsible for sporulation. Several additional amino acids have now been determined quantitatively in the haemolymph from both diseased and healthy larvae. * This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture. 671

672

ODETTEL. SHOTWELL et al.

MATERIALS AND METHODS Third instar P. japonica larvae* were injected with either B. popilliae or B. lentimorbus spores or vegetative cells (PRIDHAMet al., 1964). Haemolymph was collected as outlined by SHOTWELLet al. (1963) from larvae that became milky as determined by inspection on the seventh and fourteenth days after injection. Spores were removed from haemolymph by centrifugation, and the supernatant was freeze-dried immediately or following temporary storage in a frozen condition. The resulting solids were treated with 5% trichloroacetic acid (TCA) to separate amino acids and lower molecular weight peptides from proteins. TCA was removed by ether extraction. Each fraction was hydrolysed with acid (SHoTwELL& al., 1963). Total nitrogen was determined by micro-Kjeldahl method. Amino acids present in hydrolysed and unhydrolysed TCA-soluble fractions and in hydrolysed TCA-insoluble material (protein) from haemolymph samples were determined by ion-exchange chromatography. Analyses were made according to the automatic method of SPACKMANet al. (1958) with a model MS Beckman Spincot instrument. The basic amino acids were determined on both a 15 cm column at 50” and a 50 cm column at 30-50°C. The neutral and acidic amino acids were determined on the long column operated, as described by Spackman et al., at 30-50°C except for a 20 min forerun at 50°C. Paper chromatograms were made with the solvents and techniques described by SHOTWELLet al. (1963). Glutamine and asparagine in the haemolymph were separated by paper chromatography, water-phenol being used as solvent. Samples of haemolymph were diluted 1+ 2, and a total of 50h of diluted sample was applied at five different points on the line at the origin with a 5X micropipette (two applications at each point) so that the spots were adjacent. Amides were determined by alkaline hydrolysis of chromatogram sections in Conway units. The microtitration to determine ammonia formed by hydrolysis was carried out with an automatic microtitrator of 2.5 ml capacity (American Instrument Company). The procedures used were those developed by BUTLER(1951). Strips for use as blank controls were cut from areas of developed papers corresponding to regions containing glutamine and asparagine. For determination of L-tryptophan, cysteine, and cystine, both free and in proteins, samples of haemolymph were diluted l+-10 with distilled water. Proteins were separated by the addition of 3 ml of cold 6% perchloric acid to 3 ml of diluted haemolymph. After centrifugation, a 3 ml aliquot of the supernatant was treated with 2 ml of potassium monohydrogen phosphate to remove excess perchloric acid. The resulting solution, pH 7.5, was used to determine free amino acids. Samples of proteins after hydrolysis and solutions mentioned above were analysed for L-tryptophan content by enzymatic conversion of L-tryptophan to * The larvae were allowed to feed on roots of grass and clover for a period of 7 days before injection with milky disease organisms and until the haemolymph was collected. t The mention of brand or company names does not imply endorsement over other products of the same quality.

AMINOACIDSIN HAEMOLYMPH OF DISEASED POPILLIA

JAPONICA

LARVAE

673

indole which, after treatment with p-dimethylaminobenzaldehyde, was measured on a Beckm;an DB spectrophotometer. The method was that outlined by FRANK and DE Moss (1957). was prepared by cultivating Escherichia coli The enzyme, tryptophanase, (Migula) Calstellani et Chalmers strain T-3 (NRRL B-3055)” for 14 hr in twelve 3 1. Fernbach flasks at 37” Con a Gump rotary shaker operating at 200 rev/min. Inoculum for each flask, containing 490 ml of standard medium of BOEZI and DE Moss (1961) and 500 pg L-tryptophan/ml, was 10 ml of a 12 hr rotary-shaken culture of the organism in the same medium (95 ml/500 ml Erlenmeyer flask+ 10% preinoculum). The preinoculum was prepared by cultivating the growth from an agar slant of the organism in the medium (100 ml/500 ml Erlenmeyer) on a rotary shaker for 16 hr. The agar slant was made of the same medium plus 2% Difco agar. Tryptophanase-enriched cells were harvested by continuous-flow centrifugation in a Lourdes refrigerated centrifuge at 4”C, 15,000g. The cells were washed twice with 0.05 M potassium dihydrogen phosphate (pH 7.0), resuspended in phosphate buffer, and disintegrated in a pressure cell. Cell walls and debris were removed by centrifugation at 4°C. The resulting supernatant contained 63,000 units of tryptophanase. Th.e enzyme solution was stored frozen in small portions. Proteins were hydrolysed enzymatically for L-tryptophan determinations by the method of TOWER et al. (1962) except that hydrolysis was run 66 hr. Viokase was augmented by the addition of 2 ml of 13.6 pg Pronase (obtained from California Corporation for Biochemical Research) per ml M/l5 sodium dihydrogen phosphate buffer, pH 7<4 (NOMOTO et al., 1960). The degree of hydrolysis was checked with /&lactoglobul.in and bovine serum albumin. Sulphydryl and disulphide groups in free amino acids and low-molecularweight peptides and in proteins were determined by amperometric titration. The apparatus was similar to that used by ROSENBERG et al., (1950). A microvibrator stirrer was equipped with a platinum wire so that it could serve both as a stirrer and an electrode. The titrating vessels consisted of 5 ml Pyrex beakers attached to Petri The titrating solution was added from a Gilmont dishes with epoxy cement. microburette. The reference cell was the mercury-mercuric oxide-saturated barium hydroxide cell developed by SAMUELSON and BROWN (1935). The current was measured by a galvanometer of the swinging light-beam type having a sensitivity of 0.04 ~1A/mm division (resistance of 356 Q, 2.5 set period, and external damping resistance of 3000 a). Samples of haemolymph diluted l-+10 and corresponding samples from which the proteins had been removed by treatment with perchloric acid as described above were titrated. Sulphydryl and disulphide groups in proteins were found by difference. To deterrnine sulphydryl groups, 0.1-0.5 ml sample, 1 ml tris (hydroxmethyl) aminomethane (tris) buffer solution (4 ml 1.0 M tris with 3.4 ml 1.0 M nitric acid and * Obtained from James Hooke, University 43

of Illinois.

674

ODETTEL. SHOTWELLet al.

O-3 ml 1-O M potassium

chloride diluted to 30 ml, pH 7.4) (BENESCH et al., 1955) containing 8 M urea and 6 x 10~~ M ethylenediaminetetraacetate (EDTA), O-1 ml 1 M sodium carbonate, and water to give a volume of 1.6 ml were added to the titrating vessel. The mixture was titrated with standardized 0.001 M silver nitrate, and the end point was established by plotting on graph paper the current (PA) versus accumulated volumes (ml) of silver nitrate solution added. Disulphide groups were determined in the same manner except O-1 ml 1 M sodium sulphite was added instead of 1 M sodium carbonate. Urea was added to expose disulphide groups to action of sulphite and facilitate the reaction of silver ion with sulphydryl EDTA prevents metal-catalysed oxidation of sulphydryl groups in the groups. titration mixture. RESULTS The results of the ion-exchange determination of amino acids in haemolymph from larvae infected with B. popilliae obtained by use of the automatic analyser are shown in Table 1. Both the acid-hydrolysed and unhydrolysed TCA-soluble samples were analysed. Considerable amounts of histidine, proline, glutamic acid, glycine, alanine, valine, and fl-alanine occur free in the haemolymph from diseased larvae. It is difficult to make comparisons between haemolymph from healthy and diseased larvae because of biological variations in concentrations of amino acids. Studies on other insects have revealed large fluctuations in amounts of free amino acids both in haemolymph from single specimens and in pooled haemolymph samples (STEVENS, 1961). However, inspection of results obtained on several sets of samples did lead to conclusions in cases where differences between healthy and diseased larvae were large. Concentrations of glutamic acid and /Galanine are definitely higher in larvae infected with B. popilliae. Compare Table 1 with Table 1 in the previous paper by SHOTWELL et al. (1963). During the course of the disease there is also some increase in aspartic acid, phenylalanine, threonine, serine, and lysine. Amounts of glycine, These last three amino acids were shown to be tyrosine, and histidine decrease. among those essential for growth of B. popdiue by SYLVESTERand COSTILOW (1964). There are no obvious changes in arginine, proline, alanine, valine, methionine, isoleucine, or leucine. BENZ (1963) found that haemolymph of second-instar larvae of Melolontha melolontha Linnaeus infected with B. fribourgensis (Wille) had higher concentrations of /3-alanine, histidine, valine, and leucine and lower concentrations of serine, threonine, and alanine than did haemolymph of healthy larvae. His conclusions were based on paper chromatographic studies. Infection of larvae of Melolonthn with Rickettsiella mebnthonthae (Kreig) results in a loss of tyrosine in haemolymph. The TCA-soluble fraction of haemolymph from diseased P. japonica larvae contained eight unidentified substances that reacted with ninhydrin (Fig. 1). Since most of the unidentified peaks were not present in graphs obtained by chromatography of the hydrolysed TCA-soluble fraction, the peaks could be caused by peptides.

AMINO

ACIDS IN HAEMOLYMPH

OFDISEASED

POPILLIAJAPONICA

LARVAE

67.5

The amino acid composition of haemolymph obtained from larvae infected with B. Zentinwwbm (Table 2) differs from that of haemolymph obtained from larvae Amino acids occurring in highest concentrations in infected with B. popilliae. haemolymph from larvae infected with B. lentimorbus are histidine, proline, glutamic The only amino acid whose concentration increases acid, -glycine, and lysine. during the course of infection is glutamic acid. The following amino acids appear in TABLET-AMINO-ACIDCOMPOSITION*

1 Amino acids

2 Hydro-

Unhydro(mgFl:rnl)

Lysine Histidine Arginine Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Valine Cystine Methionine lsoleucine Leucine Tyrosine Phenylalanine B-Alanine ‘Ammonia Total nitrogen accounted for (per cent) Total amino acid (me/l00 ml) Totagoluble nitrogen (mg/lOO ml) Total solids concentration in haemolymph (mg/lOO ml)

Popilliajaponica

OFTCA~-SOLUBLEMATERIALINHAEMOLYMPHFROM LARVAE INFECTED WITH Bacilluspopilliae

Unhydro-

(m:Segdg ml)

1:; 111 :s 3:; 353 226 117 121 24 37 50 44 0 33 186 78 31.8

Hydro-

(mpliY1soeodrnl) (m

%$137 242 132 131

39 59 Trace 28

E 393 1226 293 146 173

:: 466 428 105 162 129 0

60 62 Trace 71 33 436: 510 126 182 141 0

is

:; 31 0

:;

2;; 293

:3 36

:; 130

3604

Unhydroml)

;; 76 75 Trace

63.9

2013

3

0

46.4

61.6

1626

1896

(mg$CZml)

Hydro(rn$tt

197:

196: 39 :: 71 121 492 204 124

205 134 76 Trace Trace 20 11 40

1:: 2: 19 26

25: 13

23: 207

34.4

67.4

1373

1884

982

469

690

11370

9840

8760

* As determined by a Beckman-Spinco t 5 o/0 Trichloroacetic acid.

haemolymph frorr diseased glycine, tyrosine, histidine, Quantities of lysine, aspartic alanine, and /3-alanine remain acid-labile, ninhydrin-reacting B. lentimorbus. The total amount of free larvae averages 1800 mg/lOO

ml)

automatic analyser.

larvae in lesser amounts than from healthy larvae: proline, alanine, valine, isoleucine, and arginine. acid, threonine, serine, methionine, leucine, phenylunchanged. There are also a number of unidentified substances in haemolymph of larvae infected with amino acids in haemolymph of healthy ml. There is no reduction of total amino

P. japonica acids when

ODETTEL. SHOTWELLet al.

676

larvae are infected with B. popilliae although it has been postulated that B. popilliae will sporulate only in a deficient medium (STEINKRAUS and PROVVIDENTI, 1958). It is possible, of course, at the time that sporulation is initiated there is a temporary shortage of amino acids. Our analyses were of haemolymph in ,Y

Glycina s

0.4 05- -

1

Gluiomic

60

80

100

120

140

160

180

200

Effluent,

380

400

420

440

460

480

500

520 Effluent,

220 ml I50

540

Alonine

240 260 cm column

280

300

320

340

360

560

600

620

640

660

680

mL 150cm

580

Ammonia

:

column n V

I.0

0.5 -

acid

n

fl

0.4 3 0.36 o-Amino+-butyric 9 0.2 b

acid?

Ethanolamine?

Histidine

Effluent,

mC 50cm

column

FIG. 1. Chromatographic analysis (automatic analyser) of haemolymph solids from larvae infected with Bacillus popilliae soluble in 5 y0 trichloroacetic acid. Solid lines plot absorption at 570 rnp; broken lines at 440 mp. Question marks by names of amino acids indicate that their presence in haemolymph has not been confirmed by a second analytical technique. of which sporulation had already taken place. BENZ (1963) found that sporulation B. fribourgensis occurred in haemolymph of Melolontha when concentrations of amino acids were still quite high. P. japonica larvae infected with B. lentimorbus have considerably less total amino acid (900 mg/lOO ml) than healthy larvae.

OF DISEASED POPILLIA

AMINO ACIDS IN HABMOLYMPH

JAPONICA

LARVAE

677

Comparing the total quantities of amino acids before and after hydrolysis of the TCA-soluble portion of haemolymph and taking into consideration the amount of glutamine and asparagine present, concentrations of peptides average about 30 mg/lOO ml. The amount is extremely variable, but no differences between healthy

TABLE

~-AMINO-ACID

COMPOSITION*

Popillia japonica

OF TCAt-SOLUBLE

LARVAE INFECTED WITH

MATERIAL

IN HABMOLYMPH

FROM

Bacillus lentimorbus

1

2

Amino :acids Unhydrolysed (mg/lOO ml) Lysine Histidine Arginine Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine p-Alanine Ammonia Total nitrogen accounted for (per cent) Total amino acid (mg/lOO ml) Total soluble nitrogen (mg/lOO ml) Total solids concentration in haemolymph (mg/lOO ml)

Hydrolysed (mg/lOO ml)

Unhydrolysed (mg/lOO ml)

Hydrolysed (mg/lOO ml)

105 145 28 7 8 42 265 174 50 70 0 2 20 21 0 8 22 54

130 160 21 47 17 9 57 767 190 66 77 48 6 27 30 Trace 12 25 218

Trace 1 18 11 48 7 51 24

65 173 44 60 19 44 166 509 225 110 78 90 4 34 16 5 9 69 280

30.5 967

59.1 1689

23.6 934

62.3 1884

* As determined by a Beckman-Spinco t 5 o/0 Tricholoroacetic acid.

48 148 60 4 9 41 142 28 173 83 62

661

778

7600

8020

automatic analyser.

and diseased larvae are obvious. The amino acid composition of the protein material in the haemamlymph from diseased larvae is given in Table 3. Changes in the quantity and composition of protein during the course of infection with B.popilliae are not marked. There appear to be some increases in amounts of glutamic acid, methionine, leucine, tyrosine, and phenylalanine in proteins. There was less

678

ODETTE

L. SHOTWELLet al.

histidine and arginine. Infection with B. lentimo,~busdid not cause obvious changes in either the quantity or the composition of the protein. Ion-exchange chromatographic studies have now led to a number of possible identifications for ninhydrin-reacting substances not previously reported. Concentrations of these substances in hydrolysed and unhydrolysed haemolymph TABLE ~-AMINO ACIDCOMPOSITION* OF PROTEINSIN HAEMOLYMPH OF DISEASED Popillia japonica LARVAE y0 total nitrogen in protein Amino acids

Lysine Histidine Arginine Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ammonia Total nitrogen accounted for (per cent) Amount of TCA-insoluble material (g/l00 ml) Total nitrogen in hydrolysed TCAinsoluble material (g/100 ml)

Infected with Bacillus popilliae 1

2

8.1 3.8 4.9 11.5 5.9 4.0 4.6 10.7 3.4 4.1 6.0 0 2.2 4.9 7-8 7.5 6.4 7.6

7.4 5.7 5.4 4.4 5.7 4.4 5.6 10.4 3.1 3.5 6.6 0 2.? 5.3 8.8 13.6 6.9 9.0

103.8:

109.0:

Infected with Bacillus lentimorbus 1

2

10.6 8.8 9.8 6.8 5.3 3.2 3.7 5.6 3.2 3.4 5.0 Trace 1-o 3.4 5-6 5.9 3.3 9.1

9.1 8.5 10.2 7.6 4.1 3.4 3.6 5.9 3.6 3.2 4.4 0.5 1.0 3.2 5.4 5.6 3.3 12.2

9.0 9.7 10.2 7.8 4.2 3.3 4-o 5.5 3.3 3.2 4.7 o-2 1.2 3.4 5-5 5.7 4,2 7.7

93.7

94.8

92.8

3

3.05

3.91

2-28

2.64

2-57

0.371

0.454

0.218

o-335

0.290

* As determined by a Beckman-Spinco automatic analyser. t Insoluble in 5% trichloroacetic acid. $ Based on averages from several columns.

samples from healthy and diseased larvae are presented in Table 4. Two-dimensional paper chromatograms of haemolymph solids had ninhydrin-reacting spots with R, values corresponding to lanthionine. Quantities of lanthionine were greater in acid-hydrolysed TCA-soluble fraction than in the unhydrolysed fraction. There

AM:[NO ACIDS IN HAEMOLYMPH

OF DISEASED POPILLIAJAPONICA

may be a diff’erence in concentration between diseased and healthy larvae. has been found in haemolymph from Bombyx (WYATT, 1961). Small could be present in all samples of haemolymph from P. juponica larvae

TABLE

~-POSSIBLE

IDENTIFICATIONS

HAEZMOLYMPH

Ninhydrinreacting substances

OF HEALTHY

Infective organism

OF AND

NINHYDRIN-REACTING

Ornithine

Ethanolamine

Glutathione -

None B. popilliae B. lentintorbus None B. popilliae B. lentimorbus None B. popilliae B. lentimorbus None B. popilliae B. lentimorbus

SUBSTANCES*

In TCAt-soluble material (mg/l 00 ml)

35, 97, 67 21, 30, 39 27,48 6, 8, 7 8, 9, 11 19, 7 0 22, 10, 0 2, 0 Trace, 4, 14 2, 12, 0 0, 0

* As determined by a Beckman-Spinco t 5% Trichloroacetic acid.

Ornithine quantities according

PRESENT

IN

DISEASED POpilk2 jU~O?liCU LARVAE

Unhydrolysed Lanthionine

679

LARVAE

In TCA-insoluble material (% of total N)

Hydrolysed 147, 167, 171 56, 18, 126 60, 87 10, 11, 8 13, 20, 14 22, 9 0, 12, 0 51, 13, 15 2, 0 -

-

0 0 0

0.1, 0.1, 0.1 0.2, 0.02, Trace Trace, Trace 1.0, 0, 1.0 1.0, 0.3, 0.4 Trace, 1.0 -

-

automatic analyser.

to ion-exchange studies, but these results were not confirmed by paper chromatography because of the relatively low concentrations of ornithine. Ethanolamine was not detected in unhydrolysed haemolymph from healthy larvae but is present in all other samples. A small peak at the position where glutathione is eluted was observed on graphs obtained from the automatic analyser (Fig. 1). No peak corresponding to oxidized glutathione was detected. Results of paper chromatographic and ion-exchange studies indicated traces of cystathionine in samples of haemolymph from healthy and diseased larvae. Cystathionine occurs in high concentrations in the haemolymph of several species of silk worms but is not present in larvae of other Lepidoptera (KONDO, 1962). There are pe,aks that could result from small quantities of methionine sulphoxide in graphs obtained on the automatic analyser of haemolymph from healthy larvae, and those infected with B. popilliae but not B. lentimorbus. Galactosamine could be present in hydrolysates of proteins from all samples of haemolymph. Evidence was obtained by ion-exchange studies of the presence of traces of a-amino-n-butyric acid in haemolymph from diseased larvae but not from healthy larvae. It occurs in haemolymph of Bombyx larvae (WYATT, 1961). Evidence for the presence of all

ODETTEL. SHOTWELLet al.

680

these amino acids is shown in Fig. 1. Taurine was not detected in the haemolymph of P. juponica larvae although it is commonly present in insects. Glutamine and asparagine are not separable by the automatic analyser as used, but both amides were detected initially by paper chromatography. Results of glutamine and asparagine determinations are shown in Table 5. Glutamine is a major component of the amino acid pool of haemolymph of P. juponica larvae and occurs in quantities comparable to those in haemolymph of Bombyx mori larvae (WYATT, 1956) and of Prodeniu eridunia larvae (LEVENBOOK, 1962). Much lower TABLE ~-AMINO

ACIDS IN HAEMOLYMPH POpi&

Amino acids

* t : 0

FROM

317,335,152,426 .52,47,43, 28 15, 17, 16, 16 0 180,132,98,91

HEALTHY

AND

DISEASED*

LARVAE

Free amino acids (mg/lOO ml) Healthy

Glutamine t Asparagine t L-Tryptophan 1 Cysteines Cystine$

j@O?IiCU

Diseased 44,348,408,388 34,40,46,49 21, 20, 16, 19 0 55,48,98,96

Bound amino acids (% total N) Healthy 1.1, l*l,O.S, 1.1 1.1,1.3,0.6,0.8 1.0, 1.0, 1.0, 1.0 0~1,0*04,0~2,0*2 2.0, 1.0, l-4,1.3

Diseased 1.1, 1.0,1.2 1.5, 1.2,l.l 0.7,0.8,0.7 0~1,0~1) 0.2 1.4, 2*3,3.1

Infected with B.popilliae. Paper chromatographic separation followed by alkaline hydrolysis in Conway units. Conversion by tryptophanase to indole. Amperometric titration.

levels of glutamine are in haemolymph of Schistocercagregaria Fijrsk (BENASSI et al., 1961). The ratio of glutamine to glutamic acid in the haemolymph of P. juponica larvae is 9.6, which is typical of insect haemolymph (LEVENBOOK, 1962). Asparagine is present in P. japonica larvae haemolymph in amounts similar to those in Bombyx mori (WYATT, 1956). BENASSI et al. (1961) did not detect asparagine in haemolymph of S. gregaria. No differences in concentrations of glutamine and asparagine in haemolymph from healthy and diseased P. juponica larvae were noted. Small amounts of bound asparagine and glutamine were found to be present by analyses of enzymatic hydrolysates. Tryptophan could not be determined by ion-exchange chromatography because of the acidic solutions used, but it has been detected on paper chromatograms (Table 5). Tryptophan was found by BENASSI et al. (1961) in haemolymph of of B. mori. Amounts S. gregaria, but not by WYATT et al. (1956) in haemolymph of L-tryptophan in healthy and diseased P. japonica larvae were the same. Enzymatic hydrolysis and determination revealed small amounts of L-tryptophan in haemolymph proteins. Values for cysteine and cystine reported in Table 5 were obtained by amperometric titration assuming that the sulphydryl groups titrated were those of cysteine and that the disulphide groups were in cystine. Titration of the non-protein

AMINOACIDSIN HAEMOLYMPH OF DISEASEDPOPILLIA

JAPONICA

LARVAE

681

fraction of haemolymph did not give any evidence of free sulphydryl groups although very small quantities of glutathione were detected by ion-exchange chromatography. Cysteine could be present only in negligible amounts. If the disulphide groups can be assumed to be due to cystine as they are in Table 5, there is rather a high concentration of free cystine in haemolymph of P. japonica larvae that may decrease on infection with B. popilliae. Neither cysteine nor cystine has been reported in appreciable concentrations in haemolymph of the American cockroach (STEVENS, 1961) or silkworm (WYATT, 1956). Cysteine and cystine account for a small part of the protein nitrogen in the haemolymph of P. japonica larva. The proportion of protein nitrogen that is in cysteine and cystine does not change when larvae are infected with B. popilliae. Acknowlea’gements-We thank GRANT ST. JULIAN, Jr. for providing us with haemolymph from healthy and diseased P. juponica larvae. We also wish to thank the Plant Pest Control Division, U.S.D.A.,

for supplying larvae. REFERENCES

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