Salivary amino acids in Lygus species (Heteroptera:Miridae)

Insect Biochem. Vol. 21, No. 7, pp. 759-765, 1991 Printed in Great Britain. All fights reserved

0020-1790/91$3.00+ 0.00 Copyright© 1991PergamonPress plc

SALIVARY AMINO ACIDS IN LYGUS SPECIES (HETEROPTERA: MIRIDAE) SEPPO LAUREMAand ANNA-LIISAVARIS Department of Agricultural and Forest Zoology, University of Helsinki, SF-00710 Helsinki, Finland (Received 3 January 1991; revised and accepted 23 May 1991)

Abstract--Free and protein-bound amino acids were investigated in the phytophagous bug Lygus rugulipennis and its salivary gland. Over 38 substances were separated. The total content of amino compounds in the insects was about 1400/~mol/g ft. wt (16% by weight), of which 97% was amino acid residues in proteins. The salivary glands, which comprise about 1.5% of the live weight of the insects, contain 3.5% of the total free amino acids and 1% of the proteins of the whole insect. Free and protein-bound amino acids comprise, respectively, about 1.4 and 11.6% of the fresh weight of the gland. The total concentration of free amino acids in the saliva was estimated to range from 0.5 to 2.2% by weight (ca. 0.1 M). The composition of free amino acids in the salivary gland of Lygus varies markedly. In four studied species (L. rugulipennis, L. gemellatus, L. pratensis, L. punctatus), the most abundant compounds were proline, arginine, lysine, leucine, glutamic acid, methionine sulphoxide and glycerophosphoethanolamine. In whole specimens of L. rugulipennis the predominant free amino acids were proline, alanine, taurine, glutamic acid, glutamine and methionine sulphoxide. The most abundant amino acids in proteins were glutamic and aspartic acid, glycine, alanine and leucine. The results indicate that the amino acid composition in the salivary glands of Lygus species does not differ markedly from that of the whole insect. The functions of salivary amino acids are discussed. Key Word Index: L ygus rugulipennis ; L. gemellatus ; L. pratensis ; L. punctatus ; salivary gland; amino acids; catalase

INTRODUCTION Lygus bugs are economically important insect pests on cultivated plants throughout the world (Leigh, 1976; Young, 1986; Holopainen and Vails, 1991). By means of their piercing and sucking mouthparts they feed on various plant tissues, flushing out contents of plant cells. In this "macerate-and-flush" method of feeding, the saliva and its enzymes have an important role (Flemion, 1958; Strong, 1970; Miles, 1972, 1987; Vails, 1972; Laurema et al., 1985). Besides the enzymes, the saliva of Lygus and other phytophagous bugs contains free amino acids (Kloft, 1960; Anders, 1961; Nuorteva and Laurema, 1961; Schiller, 1968a; Miles, 1972, 1987, 1989; Hoil, 1975; Heli6vaara and Laurema, 1988). Various functions have been proposed for these amino acids; associations have been suggested with the formation of galls (Anders, 1961; Henke, 1963; Sch/iller, 1968a, b) or other changes in the growth and metabolism of the infested plants (Kloft, 1960; Hori, 1975, 1976). Increases in free amino acids have often been found in the infested plants (Kloft, 1960; Hori, 1976; Laurema, 1989). According to Miles (1964b, 1972, 1987, 1989), amino acids in the saliva are associated with the formation of the so-called styler sheath and are necessary in the prevention of premature solidification of the sheath material. Salivary amino acids may also be excretory products in species which have an excess of nitrogenous compounds in their food (Anders, 1961; Sch~iller, 1960, 1968a; Miles, 1987). A special case is the amino acid tryptophan, which in

certain conditions can be transformed into the phytohormone indoleacetic acid (IAA), and therefore is a potential cause of cecidogenesis in plant bugs (Miles, 1968, 1972, 1987). Lygus bugs are not cecidogenic, nor do they produce any well-defined stylet sheath (Flemion, 1958; Hori, 1971). Yet, according to Hoil (1975), larger amounts of free amino acids were found in the salivary glands of Lygus disponsi than in Eurydema rugosum, which can produce a sheath. Damage by Lygus can cause deformation of growth (Hoil, 1971; Vails, 1972; Tingey and Pillemer, 1977), but according to Strong (1970) these deformations arise from destruction of cells controlling the growth, rather than from any particular substance injected by the bugs. In order to re-evaluate the significance of salivary amino acids in the feeding process of hemipterous insects, a closer investigation of these compounds in Lygus species was performed. On account of the feeding mechanism and the mode of living of Lygus, it is suggested that amino acids can facilitate the action of salivary enzymes and protect them against inactivation caused by defensive mechanisms of the plants. Such a function may also exist in other phytophagous species, especially those that feed on mesophyll tissue. MATERIALS AND METHODS

The insects used in the experiments were collected with sweep nets from fields, meadows and heaths in the vicinity of Helsinki. If not immediately used, they were kept in cages on scentless mayweed (Tripleurospermum inodorum) in the

759

760

SEPPo LAUREMA a n d ANNA-LIISA VARIS

l a b o r a t o r y . The salivary g l a n d s o f a d u l t insects were dissected o u t as described by L a u r e m a et al. (1985). Salivary g l a n d s o f 15-75 insects were c o m b i n e d , lyophilised a n d h o m o g e n i s e d by g r i n d i n g a n d u l t r a s o n i c a t i o n in a small v o l u m e o f distilled water. The tissue a n d fluid c o n t e n t o f the g l a n d s were s e p a r a t e d o n a glass slide b y piercing the g l a n d s several times w i t h a needle. F o r the analysis o f free a m i n o acids in whole insects, 30 males or females were boiled for 1 rain in I0 ml o f 76% (w/w) ethyl alcohol a n d t r i t u r a t e d w i t h a m o r t a r a n d pestle. The clear s o l u t i o n was s e p a r a t e d b y c e n t r i f u g a t i o n for 10 rain at 7 0 0 g , a n d the residue w a s h e d w i t h 7 6 % alcohol in the centrifuge. The solutions were collected in a 25 m l volumetric flask. Aliquots o f the alcohol solution were concentrated in a r o t a r y e v a p o r a t o r at 50°C a n d treated w i t h 5 % (w/v) 5-suiphosalicylic acid (SSA) for 1 h at 4°C. The s o l u t i o n s were clarified b y c e n t r i f u g a t i o n for 15 m i n at 12,000g a n d the p H a d j u s t e d to 2.2 w i t h 0.3 M L i O H . A b o u t 160 #1 o f the final solution, c o r r e s p o n d i n g to 8 m g o r i g i n a l fresh weight, was used for analysis. The m e a n live weights o f the analysed males a n d females o f L. rugulipennis were 6.9 a n d 7.9 mg, respectively. The dry w e i g h t was a b o u t 4 3 % o f the fresh weight.

F o r the analysis o f free a m i n o acids in the salivary glands, the extracts in m o s t cases were directly treated w i t h SSA a n d L i O H , as described above. O f the final solution, a b o u t 160#1, c o r r e s p o n d i n g to 10-15 p a i r s o f salivary glands, was used for analysis. F o r the analysis o f a m i n o acid c o n s t i t u e n t s o f proteins, some samples o f salivary g l a n d s were e x t r a c t e d w i t h ethyl alcohol as in the case o f w h o l e insects. A l c o h o l - i n s o l u b l e residues o f salivary g l a n d s a n d w h o l e insects were h y d r o l y s e d w i t h 6 M H C I ( 0 . 5 m l / m g d r y wt) for 2 0 h at I I0°C in sealed tubes in a n i t r o g e n a t m o s p h e r e . A n a l y s e s o f a m i n o acids were p e r f o r m e d o n a n L K B - B i o c h r o m 4150 A l p h a a m i n o acid a n a l y s e r using s t a n d a r d p r o g r a m s r e c o m m e n d e d b y the m a n u f a c t u r e r . S o m e details were checked by thin-layer c h r o m a t o g r a p h y as described by H e l i 6 v a a r a a n d L a u r e m a (1988). The results are based o n analyses o f salivary g l a n d s o f a b o u t 700 specimens o f Lygus. C a t a l a s e was d e m o n s t r a t e d by i n c u b a t i n g whole salivary g l a n d s in 0 . 8 % NaC1 c o n t a i n i n g 0.03% h y d r o g e n peroxide (Aebi, 1974). In extracts o f salivary glands catalase was quantified w i t h acid K I a n d starch (Pearse, 1980).

Table 1. Free amino acids and some related compounds in the salivary glands of four species of Lygus and in total extracts of L. rugulipennis Salivary glands

Compound o -Phosphoserine Glycerophosphoethanolaminc Taurine o-Phosphocthanolamine Urea Aspartic acid Methionine sulphoxide Threonine Serine Asparagine Glutamic acid Glutamine Sarcosine ~t-Aminoadipic acid Proline Glycine ct-Alanine Citrulline ct-Aminobutyric acid Valine Pipecolic acid Cystine Methionine Cystathionine Isoleucine Leucine Tyrosine fl -Alanine Pbenylalanine fl-Aminoisobutyric acid ),-Aminobutyric acid Ethanolamine Ammonia Ornithine Lysine Histidine Tryptophan Arginine Total

May n = 40 29 443 + + 17 303 60 146 + 310 304 636 202 266 + 61 47 58 25 109 117 69 357 144 + 200 86 + 220 4209 (560)

L rugulipennis June Aug. n = 65 n = 60

92 580 + 165 158 39 491 688 617 806 625 + 20 1095 578 1169 . 686 + + 402 4439 1387 1012 + 791 _+ _+ 289 256 19 1609 471 + 2163 16,647 (2282)

Oct. n=150

94 1142 + + 27 551 433 570 380 884 459 23 905 535 868 .

52 658 120 + 414 50 472 100 160 + 374 257 . 887 153 350 + . 75

. 451 . . 272 48 279 887 803 512 167 1016 + 770 230 + 863 13,169 (1755)

Total extract L. pr.

L. pu.

n=30

n=30

n=89

79 585 125 + 205 344 477 679 537 819 572 -

78 484 104 . 226 230 554 784 655 910 676 + . 628 640 1132 -

66 190 72 + . 31 810 + 180 + 533 496 -

.

. 805 547 1028 .

. .

L. ge.

. . 97 + 55 101 79 +35 _+ 610 1730 20 174 101 -+ 278 7402 (765)

555 . . 339 + 432 1110 739 . 508 + + 256 219 5 1438 266 + 1534 14,203 (1943)

.

L. rugulipennis ~;c~ 79 n=30 n=30

. 102 .

.

. 653 + 21 182 183 _ 1772 306 + 1765 15,665 (2148)

96 6206 1693 725 + 1954 3591 -

7184 1648 5413 + + 794

8047 898 497 I + + 727

+ 357 + 231 267 930

+ 220 + 149 286 757

253 + _+ 2403 + 403 2040 + 2235 40,333 (5017)

220 + + 745 + 405 1877 + 2561 40,674 (5361)

.

. 380 550 1264 813

138 2187 1124 934 + 3482 2286 .

1479 270 480 -

675

125 686 3735 +

.

.

.

302 1398 4324 +

56 + + 126 374 . 106 534 461 + 308 230 ± 383 7287 (913)

.

The concentrations are expressed as picomoles:per salivary gland of one insect or in the case of total extract as nanomoles per gramme flesh weight. The numbers in parentheses indicate the total concentrations in nanograms per salivary glands or micrograms per gramme fresh weight. The concentrations in salivary glands are means of analyses of males and females. The salivary glands of L. rugulipennis were studied in four different months, whereas the analyses of salivary glands of other species and the total extracts of L. rugulipennis are from specimens collected in October. Abbreviations: L. ge. ffi L. gemellat~; L. pr. = L. prutensis; L. pu. = L. pw)ctatus. 4- = present in small amount; + = present as a trace; - = not present in detectable amount,

Salivary amino acids in Lygus species RESULTS

Investigation of whole insects of L. rugulipennis indicated that the total content of alcohol-soluble amino compounds was about 40/~mol/g fr. wt (0.5% by weight). The predominant compounds were proline, alanin¢, taurine, glutamine, glutamic acid and methionine sulphoxide. There were some differences between the sexes; e.g. the concentration of methionine sulphoxide was higher in females than in males [Table 1; Fig. l(a)]. The free amino acids in the salivary glands of different populations of L. rugulipennis and three other species of Lygus varied remarkably in the total content and relative amounts of different compounds. The total content of non-protein amino compounds in different samples of salivary glands ranged from 4 to 16 nmol per insect. Predominant compounds were praline, arglnine, lysine, leucine, glutamic acid, methionine sulphoxide and glycerophosphoethanolamine.

761

Besides the compounds shown in Table 1, thin-layer chromatography revealed the presence of some polyamines, which cannot be detected with the standard programs of the analyser. In the salivary glands samples from males and females of the same population, the compositions were generally similar, but the amounts were higher in females. In six populations the total amounts in females were on the average 1.45 _+ 0.226 (X =1:SD) times as great as in males. In insects of the same population the amino acid composition of the salivary glands resembled that of the whole insect [Fig, l(a), (b)]. No significant effect of the food plant on the salivary amino acids was found in specimens of L. rugulipennis kept for 1 month on scentless mayweed (Tripleurospermum inodorum), heather ( Calluna vulgaris ) or mangold (Beta vulgaris ). Attempts to demonstrate the location of amino acids in the salivary gland suggested that the greater part occurred in the posterior lobe of the principal

10

(a) whole insect

1

Male Female

0

6

0~ ¢-

0

L

[

l~

QpeTauAspMeoThr Ser Glu Gin Pro Qly Ala Val CysMet lie Leu TyrPheLys His Arg

0,8

(b) salivary gland

1

Tissue Content

0,6 m

t. >

(. 0,4 U) u)

o

~ 0,2

j 1

o GpeTauAspMeoThr Ser GIu Gin Pro Gly Ala Val CysMet lie Leu Tyr Phe Lye His Ar,

Fig. 1. Free amino acids in whole insects (a) and salivary glands (b) of L. rugulipennis. Concentrations in salivary glands are means of males and females. Solid bars represent the salivary gland tissue and open bars its fired content. Gpe -- glycerophosphoethanolamine; Tau = taurine; Meo -- methionine s~lphoxide.

762

SEPPO L A ~

and ANNA-LII~AVARIS

gland. When the glands were pierced with a needle, about 2/3 of the free amino acids, and half of the proteins were found in the exuding fluid. However, there was some variation in the distribution of individual compounds between the "content" and "tissue" of the gland [Fig. l(b)]. The total amino acid content of proteins in L. rugulipennis was about 1370/~mol/g fr.wt (15.5% by weight). In the salivary glands of one insect the amino acid contents in proteins were about 104 nmol. The most abundant amino acids in proteins were glutamic and aspartic acid, glycine, alanine and leucine. The amino acid composition of proteins in the salivary gland resembled that of the whole insect. The compositions of proteins were also similar in the "tissue" and "content" of the gland (Fig. 2). As estimated from the dimensions of the gland in 0.8% NaC1, the total volume of a pair of salivary glands in L. rugulipennis ranged from 0.05 to 0.24#1, averaging 0.109-1-0.039#1 (X_+SD; n = 43). In the larger species the volumes were:

(a) whole insect

150

L. gemeilatus 0.135 + 0.039/zl (n = 11), L. pratensis 0.150_+ 0.030#1 (n = 5) and L. punctatus 0.160_+ 0.050 #l (n = 14). The salivary glands of females were on the average 1.33 times as large as those of males. The principal gland constitutes about 91% (69-97%) and the accessory gland 9% (3-31%) of the total volume of the gland. In the principal gland the posterior lobe was on the average 1.54 (0.71-4.55) times as large as the anterior lobe. (The volumes of the ducts were not included in the calculations.) If the live weight of L. rugulipennis is assumed to average 7.4 mg, its salivary glands comprise 1.5% of the weight of the whole insect. About 3.5% of the total free amino acids and 1% of the proteins of the insect can be estimated to occur in its salivary glands. The total concentration of free amino acids in the salivary gland ranges from 0.04 to 0.16 mol/1, and is at the same level or higher than in the whole insect. On a fresh weight basis, the free amino acids comprise 0.5-2.2 and the proteins ca. I 1.6% of the glands. The larger amount of amino acids in the salivary glands

~B

Male Female

~. ~o0 o) E

o

E c

50

[I

Asp Thr Ser Glu Pro Qly Ale Vel Cys Met lle Leu Tyr Phe Lye His Arg

5[

(b) salivary gland

~l

Tissue

QIcN

i

C

1

0

Asp Thr Set Glu Pro Gly Ale Val Cye Met lie Leu Tyr Phe Lye His Arg

GleN

Fig. 2. Amino acids in protein hydrolysates of whole insects (a) and salivary glands (b) of L mg=/ipennis. Cys = half-cystine + cysteic acid; Met = methionine + methionine su[phoxides; OlcN = g]ucosamine. The analyses of amino acids shown in Figs 1 and 2 were made on insects collected in October.

Salivary amino acids in Lygus species of females can be explained by the larger size of their glands. Incubation of salivary glands of Lygus species in dilute H202 resulted in most cases in generation of gas bubbles from the cut ends of the tracheoles, and variable amounts of the gas also accumulated in the interior of the gland, especially in the accessory duct and the posterior lobe of the principal gland. The discharge of gas was completely inhibited by 0.5 mM sodium azide. This observation, which was made with all four studied species of Lygus, indicates the presence of catalase in their salivary glands. Catalase was also demonstrated in extracts of the salivary gland, and found to occur in almost equal amounts in the gland tissue and its fluid content. Attempts to demonstrate the presence of peroxidase or phenoloxidase gave negative results. DISCUSSION

An important pool of free amino acids in insects is the haemolymph (Chen, 1985; Mullins, 1985). From the haemolymph amino acids and many other substances are transported to the salivary glands and saliva as well as other organs. For that reason variations in the composition of the hemolymph can affect the saliva (Nuorteva and Laurema 1961; Miles, 1967, 1987). The food plant, however, was not found to have any effect on the salivary amino acids in the present study. According to Sch~iller (1963), races of Viteus vitifolii feeding on the same cultivar of vine differ in the amino acids of their saliva. In Lygus the amino acids in the salivary glands do not seem essentially different from those of the whole insect. In some other insects differences have been found between the amino acids in the haemolymph and those in the salivary glands or saliva (Schiiller, 1963; May and Madel, 1975; Chen, 1985). In accordance with the observations of Kloft (1960) and Miles (1967), the bulk of free amino acids was found in the content of the salivary gland, which indicates that they occur in the saliva. The total content of free amino acids in Lygus was somewhat lower than generally found in bemipterous insects (Heli6vaara and Laurema, 1988). However, the estimated total concentration of free amino acids in the saliva (ca. 0.1 M) was at the same level than in the hemolymph of insects in general. According to Hori (1975), the amounts of amino acids in the salivary glands of L. disponsi were twice as large as the highest levels in the present study. In the saliva of Viteus the concentration can be as high as 7% (Anders, 1961). In other species of aphid, the concentrations are lower (Sch/iller, 1968a). Much lower amounts of amino acids have been reported from the saliva of some other insects (May and Madel, 1975; Patel et aL, 1981). As compared with the general level of free amino acids in leaves and stems of higher plants (ca. 0.01 mol/kg fr. wt; Hegarty and Peterson, 1973), the concentration in Lygus saliva is ten times as high. According to Flemion (1958), the volume of saliva remaining in the feeding track of L lineolaris ranges from 0.05 to 0.25 #1, and that of the imbibed fluid from 0.2 to 2.0/~1. If these estimates are correct, the amount of salivary amino acids remaining in the

763

plant approximately equals the amount of free amino acids which the insect can extract from the average plant tissue. Thus, the salivary amino acids cannot have any direct effect on their level in the plant. Moreover, the insect may not acquire any net supply of free amino acids by feeding on the plant tissue. This would be profitable to the insect only if the amino acids have some particular functions in its saliva. In the feeding process amino acids can affect the function of salivary enzymes in several ways. As ampholytes, amino acids have an effect on the pH and other physicochemical properties of the saliva. According to Hori (1973), free amino acids, especially glutamine, are activators of the salivary amylase of

L. disponsi. Digestive enzymes of phytophagous animals are also subject to denaturation by defensive mechanisms of plants. In as far as these mechanisms are based on reactions of amino acid residues in proteins, free amino acids may be able to interfere with the defences and protect the proteins from denaturation. The best known biochemical defensive mechanisms in higher plants are based on quinones and tannins (e.g. Felton et al., 1989; Schultz, 1989). Quinones, at least, also react with free amino acids (Henke, 1963; Pierpoint, 1969; Igarashi and Yasui, 1985), and therefore such reactions must occur in the saliva and gut of phytophagous insects. The significance of these reactions in the insects' digestive processes remains to be elucidated. As the function of peroxidase in the production of quinones requires hydrogen peroxide (Matheis and Whitaker, 1984), catalase in the insects' saliva could contribute to prevent of harmful effects of quinones. In higher animals, free amino acids are not generally effective against tannins (Butler, 1989), although at greater concentrations they can reduce the binding of tannins to proteins (Takechi and Tanaka, 1987). The saliva of higher animals often contains special proline-rich proteins, which bind tannins (Butler, 1989). The saliva of Lygus, however, does not seem to be rich in proline-containing proteins [Fig. 2(b)]. According to Miles (1964a, 1972, 1987) the saliva of most bemipterous insects contains polyphenol oxidase (phenolase). In fact, at least some aphids seem to use their salivary phenolase and/or peroxidase for detoxification of harmful plant phenolic compounds (Miles and Peng, 1989). In the saliva of Lygus, however, these enzymes are hardly detectable (Hori, 1974; Laurema et al., 1985; this study). Therefore, Lygus species evidently use some other means of protection against harmful phenols. Phenolases can also oxidize tyrosine to dihydroxyphenylalanine (DOPA) and its quinone. However, although tyrosine is relatively abundant in Lygus, DOPA was not detected in the salivary glands. The marked scarcity of salivary phenolase in Lygus also makes the conversion of tryptophan to IAA improbable. According to Strong (1970), IAA is not synthesized in the salivary apparatus of L. hesperus. A characteristic component in Lygus is methionine sulphoxide. In the Hemiptera, methionine sulphoxide has been reported from some aphids, a psyllid, and a heteropterous bug (Sch~iller, 1968a; Laurema, 1989; Heli6vaara and Laurema, 1988). In connection with

764

SEPPO LAUREM.,,and ANNA-LIISAVARIS

the present study, it was also found in the salivary glands of the mirid bugs Stenodema calcaratum and Trigonotylus ruficornis. Enzymes reducing methionine sulphoxide to methionine occur in animals and plants (Brot and Weissbach, 1988). The opposite reaction is catalysed by hydrogen peroxide, phenolic compounds and phenolase (Igarashi and Yasui, 1985). Methionine and its hydroxy analog can reduce the detrimental effect of condensed tannins in the diet of some higher animals (Butler, 1989). Some phospholipids can effectively bind tannins (Takechi and Tanaka, 1987). It is not known, however, whether their deacylated derivatives, such as glycerophosphoethanolamine have any significance in this respect. The abundance of proline and alanine is typical of insects, and is connected with their energy metabolism (Mullins, 1985; Heli6vaara and Laurema, 1988). Since proline readily condenses with quinones (Pierpoint, 1969; Rzepecki and Waite, 1989), its abundance in the saliva suggests that this secondary amino acid can have significance in the interaction of phytophagous Hemiptera with their host plants. In contrast to the free amino acid pool, the composition of proteins in insects is more uniform, and there is hardly anything unusual in Lygus in this respect. Synthesis of proteins does not seem to determine the composition of the free amino acid pool in Lygus. Acknowledgements--We wish to thank Mrs Silja M/ikel/i for aid in the collection and preparation of the insects and Miss Kirsti Noro for assistance in the performance of the amino acid analyses. This work was supported financially by the Finnish Cultural Foundation. REFERENCES

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