Studies on the salivary physiology of plant bugs: Oxidase activity in the salivary apparatus and saliva

J. Ins. Physiol., 1964, Vol. 10,

pp. 121 to 129. Pergamon Press Ltd. Printed in Great Britain

STUDIES ON THE SALIVARY PHYSIOLOGY OF PLANT BUGS: OXIDASE ACTIVITY IN THE SALIVARY APPARATUS AND SALIVA P. W. MILES Department

of Entomology,

Waite

Agricultural Research of Adelaide

Institute,

The

University

(Received 26 June 1963) Abstract-A

phenolase that will mediate the oxidation of DOPA and catechol occurs in the cells of the principal salivary duct and the accessory apparatus of the salivary glands of various Heteroptera: Pentatomorpha, and in the accessory apparatus only of a mirid, a ploiariid, and a nabid of the Cimicomorpha. Similar enzyme activity occurs in various parts of the salivary apparatus of the Heteroptera-Hydrocorisae and Homoptera. Whenever tested, the activity was found to be inhibited by cyanide and phenylthiourea, and to a lesser extent by azide. Ejected saliva from Pentatomorpha reacts variably with a sensitive amine reagent for oxidase and with DOPA. In Elasmolomus(Aphanus) sordidus(F.), this variability is at least partly dependent on diet.

INTRODUCTION

PREVIOUS papers

have reported that an oxidase which occurs in the accessory gland of the salivary apparatus of Oncopeltus fusciatus (Dall.) (MILES, 1960) and in the ejected saliva of Elusmolomus (Aphanus) sordidus (F.) (MILESand HELLIWELL, 1961) will bring about the oxidation of such general oxidase reagents as Nadi of sodium reagent and Burstone’s reagent (BURSTONE, 1960) in the presence azide at concentrations which effectively inhibit cytochrome oxidase. This paper is concerned with the distribution of the oxidase in the salivary apparatus of Heteroptera and Homoptera, its substrate specificity and sensitivity to inhibition, and the presence of the enzyme in ejected saliva. MATERIALS

AND

METHODS

The insects used in this study and their sites of collection were as follows: Pentatomidae : Pentatominae--5th instar larvae of Eumecopus australusiue Don. under bark of sugar gums (Eucalyptus cZudocuZyx F. Muell.), adults of Agonoscelis rutiliu F. on horehound (Marrubium vulgure L.), and adults of Nezuru viridulu L. on alfalfa, all at the Waite Institute. Pentatomidae : Asopinae-adults of Oechaliu schellembergi Guerin from alfalfa plots, Waite Institute. Scutelleridae-adults of Tectocoris Zineolu F. from a culture reared on cotton seed, Waite Institute. 121

122

P. W. MILES

Lygaeidae-adults of Elasmolomus (Aphanus) sordidus (F.) reared on peanuts, and adults of Phaenacantha sp. on alfalfa, both at Waite Institute. Also adults of an unidentified sp. swept from grass in Adelaide, South Australia. Pyrrhocoridae-adults of Dysdercus sidae Montz sent from North Queensland and fed on cotton seed at Waite Institute. Coreidae-adults of Mictis profana F. on citrus and of an unidentified species on pink gums (Eucalyptus fakiculosa F. Muell.) in Adelaide. Miridae-adults of Creontiades modestum Dist. .on alfalfa, Waite Institute. Ploiariidae-unidentified adults from alfalfa plots, Waite Institute. Nabidae-unidentified adult, one specimen only, from vegetable stall, Adelaide. Corixidae-unidentified adults of two species, from dam, Waite Institute. Notonectidae-unidentified adults from dam, Waite Institute. Nepidae-adults of Ranatra australiensis Hale from dam, Waite Institute. Stenocotidae-adults of Stenocotis depressa Walk. under bark of eucalypts, Waite Institute, Eurymelidae-adults of Eurymeloides pulchra Sign. under bark of eucalypts, Waite Institute. Flatidae-adults of Siphanta acuta Walk. from alfalfa plots and of Siphanta sp. from Spanish broom (Spartium junceum L.), Waite Institute. Aphidae-apterae and alatae of Aphis craccivora Koch. from culture on broad bean (Vicia faba F.), alatae of Aphis nerii B. de F. on narrow-leaved cotton bush (Asclepias fructicosa I~.), and alatae of Macrosiphum euphorbiae (Thomas) on glove amaranth (Gomphrena globosa I,.), all at Waite Institute. Salivary glands were dissected from live insects into Ringer’s solution (formula for Dytiscus and Periplaneta, PANTIN, 1948). All parts of the salivary apparatus were collected, although for ease and speed of removal the duct of the accessory gland of some Heteroptera was broken where it loops round the muscles of the sucking pump. The apparatus was washed quickly in a jet of distilled water and transferred to an incubating solution at 34°C.

t

I

1 cm. FIG. 1. Diagram of capillary pipette. Liquid is taken up by capillarity and discharged by blocking the side hole and squeezing the bulb which is made from bicycle valve tubing.

The incubating solutions used to investigate oxidase activity were either Burstone’s reagent, or a mixture of equal parts of (a) 0.3% dihydroxyphenyialanine (DOPA), catechol or cresol, (b) Michaelis buffer or tris buffer, pH 7.4 (BURSTONE, 1960), and (c) either water or an inhibitor. The stock solutions of inhibitors were 0.005 M potassium cyanide, sodium azide, and phenylthiourea (PTU) in water;

STUDIES ON THE SALIVARY PHYSIOLOGY

OF PLANT BUGS

123

the cyanide and azide sohltions were adjusted to pH 7.4 with O-1 N HCI before final dilution to 0.005 M. Saliva was collected from insects in the following manner. An individual was inverted between thumb and forefinger, and a capillary pipette (Fig. l), drawn from 2 mm glass tubing, was used both to extend the rostrum and to collect any drops exuding from it. Great care was taken to avoid sucking up any of the repugnatorial fluid exuded from the underside of some of the bugs; only the drops of secretion forming at the tip of the rostrum were collected. If an individual had not begun to salivate within 2 min, it was discarded. The pipettes were calibrated to 1 or 2 ~1. When necessary, the same pipette was used to collect saliva from several successive insects until saliva had been collected to the mark. The saliva was then discharged from the pipette by blocking the side hole with a finger and squeezing the bulb, made from a piece of bicycle valve tubing knotted at one end. OXIDASE Behaviour

ACTIVITY

of the salivary

IN THE

apparatus

SALIVARY

in Burstone’s

GLANDS

AND DUCTS

reagent

apparatus of Eumecopus australasiae, Tectocoris lineola, Elasmolomus sordidus, and Dysdercus sidae were incubated in Burstone’s reagent for 4 hr at 34°C with and without inhibitors. The results are shown in Table 1. At

TABLE

least

ten

samples

~-REACTION* $ HR AT

of the

salivary

OF THE SALIVARY

34°C IN

GLANDS

Principal

Principal Inhibitor

OF HETEROPTERA

WHEN

BURSTONE’S REAGENT WITH AND WITHOUT salivary

FOR

duct and parts of

the accessory

gland

INCUBATED

INHIBITORS

apparatus

(all species)

Eumecopus

Tectocoris

Elasmolomus

Dysdercus -

None

(water)

KCN

+ + -

NaN,

-

+

+

+

+

+++

+++

++

+++

+++

*

f

+

PTU PTU

+ NaN,

* - None;

k uncertain;

+ -

++ _

+ slight

but

definite;

++ -

++ _

?

+ + strong ; + + + very

jI strong.

Cytochrome oxidase brings about the rapid oxidation of Burstone’s reagent, and a reaction occurred in all cells in the absence of inhibitors. Cyanide inhibited this activity in all parts of the gland to a similar extent. In the presence of azide, the reaction in the principal gland was suppressed, while oxidation still proceeded, although at a reduced rate, in the cells of the accessory apparatus and ducts (for morphology of some of the salivary glands, see Fig. 2). From these results, it seemed possible that an oxidase other than cytochrome oxidase was present-a conclusion subsequently confirmed by experiments with

124

P. W. MILES

Nevertheless, the Burstone reaction in the presence of more specific substrates. azide could conceivably have been due to an exceptionally high concentration of cytochrome oxidase and an attempt was made to test this possibility. PTU is a specific inhibitor of phenolases, but has no inhibitory effect on cytochrome oxidase. Thus, should oxidase activity in the presence of azide be due to phenolase, PTU and azide together should stop activity; whereas if the activity were due to particularly high concentrations of cytochrome oxidase, the addition of PTU should have no further inhibitory effect. Unfortunately, PTU appeared to hasten the oxidation of Burstone’s reagent, and there occurred a slight, although apparently uniform, staining of the entire apparatus in the presence of both inhibitors. Thus, although some confirmation of the presence of an oxidase other than cytochrome oxidase was obtained, the result did not show unequivocally that the cytochrome oxidase had been entirely inhibited by the azide. Substrate

specificity

of the oxidase in the accessory apparatus

DOPA, catechol, p-cresol, and mixed isomers of cresol were compared substrates for oxidases in the glands of Eumecopus, Tectocoris, Elasmolomus, Dysdercus. Differential staining of the gland with the oxidation products of substrates within two hours at 34°C was taken as evidence of oxidase activity. reaction occurred in the principal gland, and the results for the ducts and accessory gland are summarized in Table 2. DOPA gave a quicker reaction TABLE ~-SUBSTRATE

Substratet DOPA Catechol Cresols

as and the No the and

SPECIFICITY* OF THE PHENOLASE IN THE SALIVARY APPARATUS OF HRTEROPTERA

Eumecopus (2 hr) ++ ++ -

Tectocoris (1 hr) ++ ++ -

Elasmolomus (1 W ++ + -

Dysdercus (1 hr) ++ + -

* - No reaction; + slight but definite reaction; + + strong reaction. t Equal volumes of 0.3% solution and tris buffer, pH 7.4.

appeared to differentiate the degree of activity in different parts of the ducts and accessory gland to a greater extent than did catechol. The cresols had no effect. The parts that stained when incubated in DOPA and catechol corresponded with the parts that reacted in Burstone’s reagent in the presence of azide. These results indicate the presence of a polyphenol oxidase in the salivary apparatus. Effect of pH on the DOPA-oxidase

reaction

In Eumecopus, Tectocoris, Elasmolomus, and Dysdercus, the rate of reaction of the glands with DOPA increased with pH from rather slow at pH 7.0 to very rapid at pH 9.0. At the same time, the spontaneous oxidation of the substrate also became rapid above pH 8.0, giving rise to general staining of the gland. For this

STUDIES ON THE SALIVARY PHYSIOLOGY

125

OF PLANT BUGS

reason, all reactions were carried out at pH 7.4 to avoid, as far as possible, staining by non-enzymatically oxidized substrate. Because the oxidation of substrates was sensitive to pH, it was found necessary to take particular care that cyanide and azide solutions, added as inhibitors, had first been adjusted to pH 7.4, otherwise the alkalinity of the solutions tended to mask their inhibitory effects.

Inhibition of DOPA-oxidase The action is summarized TABLE

of inhibitors on the DOPA-oxidase activity in the accessory gland in Table 3. It is clear that the salivary phenolase of Tectocoris,

3-DOPA-0x1~~s~

OF THE ACCESSORY Inhibitor

(0.0015

ACTIVITY*

IN

PRINCIPAL

DUCT

AND

Elasmolomus

Dysdercus

(1 br)

(1 br)

(1 br)

++

++

+

f

+

++

reaction;

f

++

++ -

+

+

-

-

PTU

uncertain;

+

Elasmolomus, and Dysdercus is not as sensitive although

-

slight but definite;

some inhibition

PARTS

PRESENCE OF INHIBITORS

Tectocoris

NaN,

by cyanide,

SALIVARY

THE

(2 br)

KCN

inhibition

IN

Eumecopus

M)

No

THE

OF SOME HETEROPTRRA

GLAND

None

* -

actiwity

+I + +

strong.

to inhibition by azide as it is to by the former certainly occurred.

Site of actiwity During this investigation, the location of phenolase activity in the salivary apparatus appeared to be much the same in species of the same family, but variation between families was noted. All the insects listed in Materials and Methods were tested and some typical results are indicated in Fig. 2. The salivary glands of all the pentatomids collected, including the carnivorous Oechalia, and of Tectocoris in the closely related Scutelleridae, reacted similarly, although the salivary glands of the latter family have complex digitations of the lobes of the principal gland. Again the lygaeids and the related pyrrhocorid reacted similarly, although the latter family has an additional median lobe on the principal gland (see BAPTIST, 1941). Of the three aphids tested, Macroszphum differed somewhat from the two Aphis spp. Of particular interest was the discovery that the cells of the principal salivary duct of all the Heteroptera: Pentatomorpha (LESTON et al., 1954) that were tested reacted strongly with DOPA; whereas in the Cimicomorpha tested (Miridae, Ploiariidae, Nabidae) phenolase was found only in the accessory apparatus. Occasionally signs of phenolase activity within the Zumenof the principal salivary duct were found in the mirid Creontiades.

P. W. MILES

126

In the Hydrocorisae, results were more variable within species. Sometimes the accessory vesicle stained strongly, sometimes on one side more than the other. In most specimens, the ducts of the accessory gland stained and also the duct of the principal gland to a lesser extent. It is perhaps noteworthy that one of the nepids tested was an individual that was moribund after starvation for a week, and in the

_L 1 mm.

B

Pd

:: f

CL

bb I

lmm

4

2.

PL

mm.

F

FIG. 2. Salivary glands of various Heteroptera and Homoptera, showing the sites of phenolase activity (shaded or black according to degree) as indicated by incubation at 34°C for 2 hr in 0.2% DOPA buffered to pH 7.4; semidiagrammatic, with especially the width of ducts exaggerated. A, Ranatra australiensis (Nepidae) ; B, unidentified sp. (Corixidae); C, unidentified sp. (Notonectidae); D, unidentified sp. (Nabidae); E, Creontiades modesturn (Miridae); F, AgonosceZis rutibiz (Pentatomidae) ; G, Elasmolomus sordidus (Lygaeidae); H, Mictis projana (Coreidae); I, Stenocotis depressa (Stenocotidae); J, Eurymeloides pulchra (Eurymelidae); K, Siphanta acuta (Flatidae); L, Macrosiphum euphorbiue; and M, Aphis ne-rii (Aphidae). Phenolase was occasionally demonstrated in the lumen of ducts when the surrounding cells showed no activity, as indicated in diagrams E and K.

STUDIES

ON

THE

SALIVARY

PHYSIOLOGY

OF

PLANT

BUGS

127

salivary apparatus of this specimen no activity was detected other than a slight darkening of the duct of the accessory gland on one side. Although few species of Homoptera were tested, these showed so much variation in site of activity that no generalizations emerged. Nevertheless, all showed strong activity in some part of the salivary apparatus. Material that stained with oxidized DOPA was found in the lumen of the ducts of Siphanta acuta, Aphis nerii, and A. craccivora. OXIDASE

ACTIVITY

IN THE SALIVA

Ease of collection of saliva Attempts to collect saliva from plant bugs were successful only with a few species. Elusmolomus adults taken from a laboratory culture usually salivated readily, and up to 1.0 ~1 could occasionally be taken from one individual. Nezara adults and larvae also yielded quantities of saliva (DAY and IRZYKIEWICZ, 1954); Mictis and Eumecopus from the field were less reliable, and little or no success was reported below indicate the possibility obtained with other species. Investigations that the nutritional status of the insects may have a bearing on the readiness with which they salivate. Evidence of oxidase activity The saliva of all the bugs examined exuded as a clear, colourless liquid. The saliva of Elasmolomus sordidus brings about the oxidation of Burstone’s reagent (MILES and HELLIWELL, 1961). It was found that the saliva of this insect would itself occasionally blacken in the collecting pipette, at the air/liquid interface. Batches of saliva showing this activity would also blacken when spotted onto filter paper that was kept damp, e.g. by hanging over water in a sealed test-tube. This blackening appeared to be enzymatic, since it was prevented by mixing the saliva as soon as collected with an equal volume of O-005 M cyanide or PTU. Azide at the same concentration did not prevent such darkening. It was soon apparent that the power of Elasmolomus saliva to darken or to bring about the oxidation of Burstone’s reagent was very variable. Nevertheless, 1 ~1 of the saliva would usually cause the darkening of a 20 ~1 drop of 0.1 per cent DOPA solution on a white tile within 2 hr, when the tile was incubated at 34°C in a water-saturated atmosphere and the colour of the mixture was compared with a drop of DOPA solution alone and with a drop of water to which a similar volume of saliva had been added. 1 r_tl quantities of the more active samples of saliva would bring about noticeable oxidation of 3 ml DOPA solution buffered to between pH 7 and 8. This enabled readings to be taken in a spectrophotometer against a blank containing buffered DOPA solution alone. A peak of absorption at 460 rnp was recorded. The saliva collected from Nezara never blackened, nor did it produce measurable darkening in millilitre amounts of DOPA solution. However, it did appear to produce oxidation in Burstone’s reagent and DOPA solution when 1 ~1 of saliva was added to 10 ~1 of reagent on a white tile. The saliva of Mictis and Eumecopus sometimes gave a positive reaction with Burstone’s reagent.

P.

128

W.

MILES

Influence of diet on oxidase actiwity in the saliva In an attempt to discover means of producing more uniform batches of saliva, an ad hoc experiment on the feeding of Elasmolomus in relation to salivary oxidase was carried out. Batches of twenty adults which had moulted within the previous 8 hr were confined in clear plastic containers with either food (fresh peanuts in broken shells) or no food, and either water (from a cotton dental roll emerging from a water-filled plastic vial) or no water, in all combinations. The containers were kept at 25°C. After a week, it was found that no saliva could be collected from the batches given no water. Of those that had been given water, the saliva produced darkening in a drop of DOPA solution only when the insects had been given food as well. Inhibition of oxidase in the ejected saliva Saliva was collected from a well-established colony of ElasmoZomus that had been kept plentifully supplied with peanuts and water. As soon as 1 ~1 of saliva had been collected, it was discharged into 5 ml of ice-cold tris buffer, pH 7.4. The process was continued until 5 ~1 of saliva, collected from about twenty salivating adults, had been added to the one lot of buffer. Four 1 ml aliquots were pipetted into 1 cm cuvettes containing 1 ml 0.3% DOPA in tris buffer, pH 7.4, and 1 ml buffer or 1 ml 0.006 M inhibitor in buffer. Spectrophotometric readings were taken immediately against blanks of the same composition except for the TABLE &OPTICAL BUFFERED TO pH 3000),

DENSITY AT 460 7.4,

AFTER

rnp OF SOLUTIONS CONTAINING 0.1

INCUBATION

ElasmolomusSALIVA

WITH

WITH AND WITHOUT INHIBITORS (FINAL CONCENTRATION:

34°C. Inhibitor

PER CENT DOPA,

(FINAL DILUTION:

0.002

M),

1 IN

FOR 1 HR AT

MEASURED AGAINST CONTROLS CONTAINING NO SALIVA Time

0

Time

1 hr

Adjusted

value after 1 hr

None

0.004

0.120

0.116

Azide

0.003

0.084

0.081

Cyanide

0.000

0.023

0.023

PTU

o-004

0.004

0.000

saliva, i.e. a separate blank for every mixture containing the saliva. The readings were repeated at the end of 1 hr after incubation at 35°C in the dark. The results are recorded in Table 4. In a parallel experiment, samples of saliva diluted with buffer alone to 1 in 3000 never showed any measurable increase in optical density with time. CONCLUSION

These experiments show that a phenolase that will bring about the oxidation of diphenols occurs in the salivary glands of all the Heteroptera and Homoptera tested. Where the ejected saliva could be collected, an enzyme with similar substrate affinity and similar sensitivity to inhibition was found in it. Even where

STUDIESON THE SALIVARYPHYSIOLOGYOF PLANTBUGS

129

saliva was not collected, the discovery of oxidized DOPA in the lumen of the principal salivary duct of glands incubated in DOPA (in a mirid, a flatid, and two species of aphid) suggests that here, also, a phenolase is secreted by the living insect. The significance of the salivary phenolase in relation to the sheath material secreted by Heteroptera: Pentatomorpha and by Homoptera has already been in fuller foreshadowed in a previous paper (MILES, 1960) an d will be considered detail elsewhere. REFERENCES BAPTIST B. A. (1941) The morphology and physiology of the salivary glands of Hemiptera-Heteroptera. Quart.J. micr. Sci. 82, 91-139. B~JRSTONEM. S. (1960) Histochemical demonstration of cytochrome oxidase with new amine reagents. J. Histochem. Cytochem. 8, 63-70. DAY M. F. and IRZYKIEWICZH. (1954) On the method of transmission of non-persistent phytopathogenic viruses by aphids. Aust.J. biol. Sci. 7, 251-272. LESTON D., PENDERCASTJ. G., and SOUTHWOODT. R. E. (1954) Classification of the terrestrial Heteroptera (Geocorisae). Nature, Lond. 174, 91-94. MILES P. W. (1960) The salivary secretions of a plant-sucking bug, Oncopeltus fasciatus (Dall.) (Heteroptera: Lygaeidae)--III. Origins in the salivary glands. J. Ins. Physiol. 4, 271-283. MILES P. W. and HELLIWELL A. (1561) Oxidase activity in the saliva of a plant-bug. Nature, Lond. 192, 374-375. PANTIN C. F. A. (1948) Microscopical Technique for Zoologists. Cambridge University Press.