Maran, S.P.M. 1999.

Chosen reduviid predators I

I
- prey interaction : Nutritional and pheromonal I - ][ IEI
chemical ecology (Insecta: Heteroptera:
Reduviidae). Ph. D Thesis. Manonmaniam
Sundaranar University, Tirunelveli, India. pp. 111.

TOAINOLOCY

III. 1. INTRODUCTION

Venom is defined as a mixture of substances whichare produced in' specialized

glands of the venomous animal and injected with the aid of a stinging - piercing

apparatus into the body of its prey inorder to paralyse it (Ziothin, 1991b). Venomous

arthropods possess proper instrumentation for stinging - piercing and direct

introduction of toxic substances into the circulation and tissues of their prey. The

vast majority of venomous arthropods are predators and they feed exclusively on

living prey which comprises other arthropods, mainly insects (Zltkin, 1984). In

the process of predator - prey interaction, two devices are used; (a) a physical device,

the stinging apparatus enabling the direct introduction of the venom into the circulation

or close proximity to the critical target tissues of the prey and (b) the chemical

device, in the form of the active substances - the neurotoxins, which possess an

unique pharmacologically specific relation to these target tissues. A neurotoxin is

defined as a substance interferes with the functions of excitable tissues (Zlotlzin,

1988). The higher paralytic potency at nanomolar or lower concentrations, and

rapid action within seconds or minutes of neurotoxin is a pre - requisite for predators

employing venom for prey capture.

 Reduviid predators immobilize their prey by injecting venomous saliva into

them (Edwards, 1961; McMahan, 1982, 1983; Haridass and Ananthakrishnan,

1981a; Morrison, 1989; Ambrose, 1999; Ambrose and Maran, 1999 a; Maran

and Ambrose, 1999). Salivary venom of predatory hemipterans appears to he

typical in their overall action. The bite of tabanid (Diptera) larva (Beard, 1952),

asilid (Diptera) adult (Whitfield, 1925), lampyrid and neuropteran larvae (Blum,

1981) are reported to produce instantaneous paralysis in the victim.

Virtually nothing is known about the chemistry of the salivary venom

reduviids except the study of Edwards (1961) in P. rhadarnanthus and Zerachia

ct.aL, (1973 a,b) in Holotrichius innesi Horrvath. Edwards (1961) demonstrated

that the saliva of P. rhadamanthus is qualitatively similar to snake venom showing

most of the enzymes. However, the basis for the rapid paralytic activity of the

venom on prey has not been established and the investigation of the phenomenon

constitutes an important avenue for examining the comparative neuropharmacology

of insects.

The paralytic potential of reduviids, such as L. a//mis and H. nigroviolaceous

(Haridass and Ananthakrishnan, 1981a), A. pedestris (Morrison, 1989; Ambrose

and Maran, 1999 a) and C. brevipennis (Maran and Ambrose, 1999) was studied.

The research on the venom of reduviids might be limited due to difficulty in getting

large amount of venom owing to the smaller gland size. Our preliminary observation

on the predation of R. fuscipes, R. kumarii and •R. marginatus suggested that the

salivary venom is paralytic and toxic to their prey. The knowledge on paralytic

32
potency of these neurotoxins prompted its to estimate their quantity and protein

content, analyse their protein profile, paralytic dosage and paralytic duration to

three pests viz., Spodoptera litura Fabricius (Lepidoptera), Mylabris pustulata Thunberg

(Coleoptera) and Dysciercus cingulatus Fabricius (Heteroptera). These salivary toxins

through their selectivity may illustrate certain unique features in the physiology of

insect excitable system and thus may be employed as a valuable tool in the study of

insect neuropharmacology and as a potential model for the design of selective

insecticides in near future (Zlotkin, 1983).

III. 2. MATERIALS AND METHODS

Methodology of collection and maintenance of rediiviid predators is presented

in chapter I. The insect pests S. litura, M. pustulata and D. cingulatus were

collected from groundnut and cotton agroecosystems in and around Palaankottai.

III. 2. 1. QUANTIFICATION OF SALIVARY TOXIN

To findout the volume of saliva, the intact salivary glands of anaesthetised

R. fuscipes, R. kumarii and R. marginatus were dissected out in ice cold saline

(0.15 M) under stereoscopic microscope. Anterior and posterior lobes of the principal

salivary glands were separated and their salivary venom was measured by using the

microsvringe (gauge 26). T0 understand the impact of prey deprivation on the

volume of salivary toxin, it was measured in 1,2,3 and 4 day prey deprived

R. fuscipes and 2,4,6 and 8 day prey deprived R. kumarii and R. marginatus.

Since, R. fuscipes could not withstand more than 4 day prey deprivation, the

experiment was conducted only at 1,2,3 and 4 day prey deprivation, whereas it was

33
extended upto 8 days in R, kumarii and R. marginatus, which could withstand upto

15 day prey deprivation.

III. 2. 2. PROTEIN ESTIMATION

About 20 A l of salivary toxin from anterior and posterior lobes were

precipitated with 1 ml of 10% trichloroacetic acid (TCA) in an ice bath. After

centrifugation the precipitate was dissolved in 100 A l of IN NaoH and protein

concentrations was measured by following the method of Lowry etal., (1951) using

bovine serum albumin (BSA) as standard. Impact of prey deprivation on the

protein content in both the salivary lobes was also studied.

III. 2. 3. ELECTROPHORETIC ANALYSIS OF SALIVARY TOXIN

Qualitative analysis of proteins in the salivary toxin of R. Juscipes,

R. kumarii and R. marginatus was made by discontinuous sodium dodecyl sulphate -

polyacrylamide gel electrophoresis (SDS - PAGE). The separation gel contained

acrylamide his (44 0.8); buffer pH 8.8; 10% SDS; 1% Ammonium per sulphate

(APS); water and tetramethylene ethylene diamide (TMED), whereas the stacking

gel contained acrylamide : his (30 : 0.8); buffer pH 6.8; 10% SDS; 1% APS;

water and TMED. The salivary toxin treated with 2x treatment buffer and 50 l of

samples were loaded in wells individually. Sixty volt current was applied until the

marker dye entered the separating gel; the voltage was incresed to 120 V afterwards.

The fully run gels were stained in comassie brilliant blue and destained in acetic acid

methanol water solution. The bands thus obtained were read in densitometer.

34
III. 2. 4. PARALYTIC UNIT DETERMINATION

T0 determine the paralytic unit (PU) of salivary toxins of R. fuscipes,

R. kumarii and R. marginatus to the pests S. litura larva (245 - 255 mg),

M. pustulata 185 - 195 mg) and D. cingulatus (50 - 60 mg), a preliminary dosage

range finding test was conducted. From these tests, the dosage that could cause 0%

and 100% mortalities in pests were evaluated. Then different dosages of salivary

toxin (1.0, 1.5, 2.0, 2.5 and 3.0 Al for S. litura and 0.5, 1.0, 1.5, 2.0, 2.5 and

3.0 bLl for M. pustulata and D. cingulatus) in between the dose that could cause 0%

and 100% mortalities were injected into the body cavity of S. !itura through the

head shield and through the pleural membrane of the neck region for M. pustulata

and D. cingulatus using a microsvringe (gauge 26). Since, the entire body of the

S. litura caterpillar is softer than the head shield, the salivary toxin was injected

through the head shield. The arrest of wriggling movement in S. litura caterpillars

and the arrest of antennal twitching and leg movements in M. pustulata and

D. cingulatus were considered the positive responses of paralysis. Then, by following

the formula of Reed and Muench (1938).

1% above 50 - 50%\
PU(t1) Dose above 50% ( X (Dose above 50% - Dose under 50%)I

% above 50 - under 50

the paralytic unit was determined. The time taken for paralysis of the pests was

determined by injecting the PU dosage to these pests. The effect of prey deprivation

of these predators on the time taken (in seconds) for paralysis of these pests were

also studied.

35
III. 3. RESULTS

111. 3. 1. VOLUME OF SALIVA

Table 5 and Figures 4. a,b and c present the impact of prey deprivation on

the volume of saliva in both the anterior and posterior lobes of principal salivary

glands of R. fuscipes, R. kumarii and R. marginatus.

The volume of saliva was higher in the posterior lobes than in the anterior

lobes. Prey deprivation caused accumulation of saliva in both the lobes. The

anterior lobe of fed R. fuscipes had 2.80 ± 0.249 p! of saliva and its posterior lobe

contained 4.90 ± 0.433 tl. In R. kumarli, the anterior lobe contained 3.40 ±

0.305 bLl whereas its posterior lobe had 4.90 ± 0.276 A l. R. marginatus also exhibited

a similar trend, that the anterior lobe had lower volume (3.00 ± 0.297 pd) than the

posterior lobe (4.60 ± 0.339 gil). Among these three predators, R. kumarii had the

highest volume of saliva in the anterior lobe followed by R. marginatus and

R. fuscipes, whereas the posterior lobe of R. fuscipes and R. kumarii had equal

volumes of saliva (4.90 A l). R. marginatus had comparatively lesser volume of saliva

(4.60 ± 0.339 A l) in their posterior lobe.

Prey deprivation caused accumulation of saliva in both the lobes of principal

salivary gland. The accumulation was higher in the anterior lobe of R. kumari;

followed in R. marginatus and R. fuscipes. For instance, in the anterior lobe of 8

day prey deprived R. kumarii, the volume of saliva increased from 3.40 ± 0.314 tl

to 8.70 ± 0.472 .tl whereas it was from 3.00 ± 0.297 Al to 7.50 ± 0.542 tLl in

R. marginatus and from 2.80 ± 0.249 A l to 6.90 ± 0.314 tl in 4 day prey deprived

R. fuscipes (P < 0.001).

36
Table 5. Impact of prey deprivation on salivary quantity (Al) and protein content of saliva (gh.tl) of
R. fuscipes, R. kumarii and R. marginatus.

Prey Saliva Protein content**

Predator deprivation
period
(days) Anterior lohc Posterior lohe Anterior lohe Posterior l0h

o 2.80 ± 0.249 4.90 ± 0.433 166.80 ± 11.81 88.20 ± 8.80

1 3.10 ± 0.347 5.50 ± 0.453 201.40 ± 10.74 100.20 ± 6.39

2 3.70 ± 0.395 6.30 ± 0.448 207.40 ± 13.50 114.40 ± 11.99

R. fuscipes

3 4.90 ± 0.480 6.30 ± 0.517 217.40 ± 17.15 115.40 ± 19.80

4 6.90 ± 0.314 7.80 ± 0.573 252.20 ± 19.45 122.24 ± 5.97

0 3.40 ± 0.305 4.90 ± 0.276 203.80 ± 31.97 113.60 ± 10.11

2 4.60 ± 0.426 6.40 ± 0.305 223.20 ± 14.50 153.60 ± 13.85

4 6.10 ± 0.314 7.80 ± 0.249 289.40 ± 26.78 194.20 ± 13.27

R. kumarn

6 7.30 ± 0.229 8.70 ± 0.366 367.40 ± 24.81 201.20 ± 27.47

8 8.70 ± 0.472 9.90 ± 0.546 413.80 ± 8.43 201.40 ± 5.83

0 3.00 ± 0.297 4.60 ± 0.339 182.33 ± 15.14 96.80 ± 7.09

2 4.40 ± 0.266 6.20 ± 0.249 184.40 ± 20.37 114.00 ± 10.32

4 6.00 ± 0.596 8.20 ± 0.592 232.80 ± 20.30 145.40 ± 7.28

R rnarginatus
6 6.60 ± 0.498 8.70 ± 0.558 285.40 ± 25.68 159.00 ± 9.28

8 7.50 ± 0.542 10.50 ± 0.763 299.40 ± 26.22 166.20 ± 11.68

* n = 10 ± SE
** n = 6 ± SE
 (a)
rA I

0 2

Prey deprivation period (days)

Fig. 4. Impact of prey deprivation on the salivary quantity in the ante-

rior and posterior lobes of principal salivary gland of a) R. fuscipes
b) R. kumarii and c) R. marginatus
 Regarding the posterior lobes, the increase in the volume of saliva was the

highest in R. marginatus followed in R. kumarii and R. fuscipes. For instance in the

posterior lobe of 8 day prey deprived R. marginatus, the volume of saliva increased

from 4.60 ± 0.339 Al to 10.50 ± 0.763 Al, whereas it was from 4.90 ± 0.276 l

to 9,90 ± 0.546 tLl in R. kumarii and from 4.90 ± 0.433 t1 to 7.80 ± 0.573 t1

in 4 day prey deprived R. fuscipes (P < 0.001).

III. 3. 2. PROTEIN CONTENT OF SALIVA

The protein contents of saliva in the anterior and the posterior lobes of

R. fuscipes, R. kumarii and R. margincitus are presented in Table 5; Figure 5. It

was uniformly higher in the anterior lobe than in the posterior lobe, irrespective of

their volume. Among these three predators, R. kumarii had the highest protein in

the anterior lobe (203.80 ± 31.97,aglpJ) followed by R. marginatus (182.33 ±

15.14 g/l) and R. fuscipes (166.80 ± 11.81 gIl). As observed for the anterior

lobe, the posterior lobe of R. kurnarii also had the highest protein (113.60 ± 10.11

g/l) followed b y R. marginatus (96.80 ± 7.09 gIl) and R. fuscipes (88.20 ±

8.80 gIl).

Prey deprivation caused increase in the amount of protein in both the lobes

of all of these three predators. R. kumarii showed the highest increase in the protein

content followed by R. marginatus and R. fuscipes. For instance, 8 day prey deprivation

increased the protein content from 203.80 ± 31.97 tg/tl to 413.80 ± 8.43 g/l

(P< 0.00 1), whereas it was from 182.33 ± 15.14 AgItA to 299.40 ± 26.22 tg/tl

in R. marginatus and from 166.80 ± 11.81 g/l to 252.20 ± 19.45 tL910 in 4

day prey deprived R. fuscipes (P < 0.001).

37
 (a) Anterior Lobe

0 1 2 3

F
0L

2 4 6 8
0

0 2 4

Prey deprivation period (days)

Fig. 5. Impact of prey deprivation on the protein content in the anterior and
posterior lobes of principal salivary gland of a) R. fu.cipc
b) R. kumarii and c) R. marginatus
 The highest increase of protein content in the posterior lobe as a function

of prey deprivation was observed in R. kumarii followed in R. marginatus and

R. fuscipes. For instance in the posterior lobe of 8 day prey deprived R. kumarii, the

protein content was increased from 113.60 ± 10.11 tgIjil to 201.40 ± 5.83 tg/

pi (P < 0.001) whereas it was from 96.80 ± 7.09 tg/l to 166.20 ± 11.68 bLgl

122.24 ±
Al in R. marginatus (P < 0.001) and from 88.20 ± 8.80 A glAl to

5.97 tgIl in 4 day prey deprived R. fuscipes (P < 0.001).

M. 3. 3. PROTEIN PROFILE OF SALIVA

The densitometric electropherogram indicates that the total number of protein

ma rginatus
fractions in the principal salivary gland of R. fuscipes, R. kumarii and R.

6 illustrates the
were 9,8 and 8, respectively (Table 6; Figures 6 a, b and c). Table

percentage composition of the protein fractions in the principal salivary gland extract.

was observed in peak 1 and the

In R. fuscipes, the highest relative area (49.7%)

(31.50%) in
lowest (3.10%) in peak 4. R. kumarii showed the highest relative area

had the highest relative

peak 8 and the lowest (3.60%) in peak 3. R. marginatus

area of 20.10% in peak 8 and the lowest (5.30%) in peak 4.

III. 3. 4. PARALYTIC UNIT (PU)

Tables 7 to 15 present the detailed protocol of collection of experimental

data and the calculation of paralytic units. The preliminary range finding tests

R. fuscipes, R. kumarii and R. rnarginatus

showed that 0.5 and 3.5 1A of saliva of

S. litura, M. pustulata and

could cause 0% and 100% mortalities in the test insects

D. cingulatus

38
Table '6. Electrophoretic pattern of salivary gland complex of R. fuscipes, R. kumarii and
R. marginatus.

Peak number Relative Area (%)

R. fuscipes R. kumarii R. marginatus

1 49.7 18.30 18.90

2 6.40 18.4 12.00

3 6.60 3.60 6.50

4 3.10 6.00 5.30

5 4.10 5.10 11.80

6 2.00 8.40 17.90

7 3.20 8.70 7.30

8 7.10 31.50 20.10

9 17.80 - -
 (a)
(A - min = 0.49; A • max = 1.20)
I

CH
. ..........................

LN
fl 91 97 9 lOs 1*1 t2 Is'3 II ll I' 119
I • 1l',tt. ......................................Start •
r.Su • ,iø v.,,,, •

(b)
(A - mm = 0.77; A - max = 1.22)
•1 . ..., ..
...............................
..............................

IJ I ...........
'I
H'

fJ j i •i
1
\, .1
..........

• flh11,&. .................................> tI9 • • tilap I:, V1pp • I

(c)
(A - min 0.67; A - max 1.21)

3,.....

I
,iJ \f

FIE

'.7 71 S1 .4 7 9 97 7? 19 i.
I . .. . till ............................................ . 90 III Ill .i (, I . I. 1' 10.7 7?
IW3 ........... • I

fIUEC 6. I )cll j oiiictv j c scan of S1)S- PAGI: cicctro [)JIorcsc

(l saliva ()I
(a) R. fzcipes (h) R. kumarii and (c) R. ';largi;Ia/:ls.


Table. 7. Summary of calculation of paralytic unit (PU in pl) of R. fuscipes saliva to S. litura
caterpillar (N = 10)

Volume Positive Negative Positive I

Positive Negative Percentage
injected Response Accumulated Accumulated Total
Response
(pJ)

1.0 1 9 1 27 1/27 4

1.5 2 8 3 18 3/21 14

2.0 4 6 7 10 7/17 41

2.5 7 3 14 4 14/18 78

3.0 9 1 23 1 23/24 95

3.5 10 0 33 0 33/33 100

PU = 0.212 d / caterpillar


Table. 8. Summary of calculation of paralytic unit (PU in A l) of R. fuscipes saliva to M. pustu/aa

(N = 10)

Volume Positive I
Positive Negative Positive Negative
injected Percentage
Response Response Accumulated Accumulated iota!
(al)

0.5 0 10 0 34 0/34 0

1.0 1 9 1 24 1/25 4

1.5 3 7 4 15 4/19 21

2.0 5 5 9 8 9/17 53

2.5 7 3 16 3 16/19 84

3.0 10 0 26 0 26/26 100

PU = 0.195 ILl I insect



Table. 9. Summary of calculation of paralytic unit (PU in 1) of R. fuscipes saliva to D. cinguiatus

(N = 10)

Volume
Positive Negative Positive Negative Positive I
injected Percentage
Response Response Accumulated Accumulated Total
(pJ)

0.5 2 8 2 22 2/24 8

1.0 3 7 5 14 5/19 26

1.5 5 5 10 7 10/17 59

2.0 8 2 18 2 18/20 90

2.5 10 0 28 0 28/28 100

PU = 0.137 fLl / insect



Table. 10. Summary of calculation of paralytic unit (PU in tl) of R kwnarii saliva to S. litura
caterpillar (N = 10)

Volume Positive Negative Positive I

I Positive Negative Percentage
injected Response Accumulated Accumulated Total
Response

1.0 2 8 2 23 2/25 8

1.5 3 7 5 15 5/20 25

2.0 5 5 10 8 10/18 55

2.5 7 3 17 3 17/20 85

10 0 27 0 27/27 100
3.0

PU 0.191 l / caterpillar


Table. 11. Summary of calculation of paralytic unit (PU in Al) of R. kumarii saliva to M. pustulata
(N = 10)

Volume
Positive Negative Positive Negative Positive I
injected Percentage
Response Response Accumulated Accumulated Total
l)

0.5 1 9 1 27 1/28 4

1.0 2 8 3 18 3/21 14

1.5 5 5 8 10 8/18 44

2.0 7 3 15 5 15/20 75

2.5 8 2 23 2 23/25 92

3.0 10 0 33 0 33/33 100

PU = 0.160 pJ I insect


Table. 12. Summary of calculation of paralytic unit (PU in A I) of R. kumarii saliva to D. cingviatus
(N = 10)

Volume
Positive Negative Positive Negative Positive I
injected Percentage
Response Response Accumulated Accumulated Total
(pi)

0.5 1 9 1 23 1/24 4

1.0 3 7 4 14 4/18 22

1.5 5 5 9 7 9/16 56

2.0 8 2 17 2 17/19 89

2.5 10 0 27 0 27/27 100

PU = 0.141 pJ I insect


Table. 13. Summary of calculation of paralytic unit (PU in A l) of R. raarginatus saliva to S. lituro
caterpillar (N = 10)

Volume
Positive Negative Positive Negative Positive
injected Percentage
Response Response Accumulated Accumulated Total
(t1)

1.0 1 9 1 23 1/24 4

1.5 3 7 4 14 4/18 22

2.0 5 5 9 7 9/16 56

2.5 8 2 17 2 17/19 89

3.0 10 0 27 0 27/27 100

PU = 0.191 Al I caterpillar


Table. 14. Summary of calculation of paralytic unit (PU in ,al) of R. marginatus saliva
to M. pustulata (N 10)

Volume
Positive Negative Positive Negative Positive /
i njected
Response Response Accumulated Accumulated Percentage
(al) Total

0.5 0 10 0 34 0/34 0

1.0 1 9 1 24 1/25 4

1.5 2 8 3 15 3/18 17

2.0 5 5 8 7 8/15 53

2.5 8 2 16 2 16/18 89

3.0 10 0 26 0 26/26 100

PU = 0.199 Al I insect


Table. 15. Summary of calculation of paralytic unit (PU in JA) of R. marginatus saliva to
D. cinguiatus (N = 10)

Volume Negative Positive I

Positive Negative Positive
IflJCctCd Percentage
Response Response Accumulated Accumulated Total
(tI)

0.5 0 10 0 28 0/28 0

1.0 3 7 3 18 3/21 15

1.5 4 6 7 11 7/18 41

2.0 6 4 13 5 13/18 72

2.5 9 1 22 1 22/23 96

3.0 10 0 32 0 32/32 100

PU = 0.165 Al/ insect

 Injection of saliva of predators into S. litura caterpillars caused body wriggling

and caterpillars became flaccid and motionless afterwards. M. pustulata and

D. cingulatus wriggled body and groomed antennae and forelegs with their mouth

parts. Initially restless victims fell upside d0 and motionless. Spreading Of wings

was also observed in M. pustulata and D. cingulatus.

Table 16 and Figure 7 show a comparative account of paralytic units

R. fuscipes, R. kumarii and R. marginatus to S. litura, M. pustulata and D. cingulatus.

To paralyse S. litura caterpillars, R. fuscipes had to inject 0.212 Al of toxin

whereas R. kumarii and R. marginatus could paralyse it with 0.191 gl toxin.

R. fuscipes had to inject 0.195 jJ of toxin to paralyse the coleopteran pest

M. pustulata, whereas R. kurnarii and R. marginatus c ould paralyse it with 0.160 Al

and 0.199 A l toxin, respectively. R. fuscipes could paralyse the hemipteran pest

D. cingulatus with 0.137 of toxin, whereas it was paralysed with 0.141 and

0.165 A l of saliva of R. kurnarii and R. marginatus , respectively.

III. 3.5. PARALYTIC DURATION

Tables 17 to 19 show the paralytic durations for the pests S. litura,

M.pustulata and D. cingulatus by the saliva of R. fuscipes, R. kumarii and

R. marginatus. The saliva of prey deprived predators could paralyse the prey more

rapidly than that caused by fed ones. Moreover, the saliva in the anterior lobe

caused paralysis more rapidly, whereas the saliva in the posterior lobe caused paralysis

after a prolonged duration of injection.

39
 Table 16. Paralytic units of £ fuscipes, R. kumarii and R. marginatus salivary
venom to S. litura, M. pustvlata and D. cingu/atus.

Paralytic unit (PU = /.d/insect) of predators

Prey species
R. fuscipes R. kumarii R. marginatus

S. litura 0.212 0.191 0.191

M pustulata 0.195 0.160 0.199

D. cing ula tus 0.137 0.141 0.165

 Im-
0 1
zi
4-. Q
C/) ca
0
U)
U) E
CC
ci
:
E
•E_ '
U)
ocii
U)
C
U
a-
Q)
CO
LC
(l3esuI,IrI) lun OflAIUJUd
III. 3. 5. 1. SALIVA OF R. fuscipes

Saliva from the anterior lobe of fed R. fuscipes took 119.50 ± 1.82,

121.10 ± 2.79 and 96.30 ± 1.76 seconds to paralyse S. litura, M. pustulata and

D. cingulatus, respectively. Four day prey deprivation significantly (P < 0.001)

reduced the paralytic durations for these three pests (63.50 ± 0.98, 77.10 ± 1.35

and 64.10 ± 1.06 seconds for S. litura , M. pustulata and D. cingulatus, respectively)

by the saliva of the anterior lobe.

Saliva from the posterior lobe of fed R. Juscipes took 402.00 ± 4.87,

407.00 ± 6.15 seconds to paralyse S. litura, M. pustulata and D. cingulatus,

respectively. Four day prey deprivation significantly (P < 0.001) reduced the

paralytic durations for these three pests (199.20 ± 3.25, 229.40 ± 7.09 and

174.00 ± 2.85 seconds for S. litura , M. pustulata and D. cingulatus, respectively)

by the saliva of the posterior lobe (Table 17; Figures 8 a, b and c).

M. 3. 5. 2. SALIVA OF R. kumarii
Saliva from the anterior lobe of fed R. kumarii took 86.60 ± 1.46, 82.80

± 0.77 and 79.10 ± 2.04 seconds to paralyse S. litura, M. pustulata and

D. cingulatus , respectively. Eight day prey deprivation significantly (P < 0.001)

reduced the paralytic durations for these three pests (57.20 ± 0.98, 55.70 ± 1.15

and 50.90 ± 1.92 seconds for S. litura, M. pustulata and D. cingulatus, respectively)

by the saliva of the anterior lobe.

Saliva from the posterior lobe of fed R. kumarii took 292.30 ± 1.07,

263.90 ± 10.45 and 281.00 ± 1.98 seconds to paralyse S. litura, M. pustulata and

40

C) 10 N 10 cc
- '0 C N cc
- 10 10 10 N
(I)
0 +1 + +1 +1 +1
+1
C, C C, C C
- C
I
co '0
C) '-0 N 0' 11
cc m 0' N
C
10 10 '0 — '0
N 0 ' C 0
½
o +1 + +1 +1
C, C C C C
C) c) N cc 0 —
C16
a' cc
d
cc N 10
- C) C 10 N
-
o'
N 'Q
- C
½
o +1 +1 +1 +1 +1
C)
C, C, C, C, C
+4 C N cc 0'
LO N
c-n c-n N N
½
C) 0' 0' C '-0
0 N 10 c-n
1) N N —
a)
In
0 +1 +1 +1 +1 +1
a)
"-4
'-4 C 0 0 0 0
E +4
4-' C c-n cc N N
N C a' cc N
U) —
C)
C)
C) N 0' '-0 10
0 - cc 0' '0 — N
0 N 4 c-fl c-fl
½
3 +1 +1 +1 +1 +1
C C C C C
½ C) C — - — - N
+4
N c-n '-0 c-fl 0'
o C 10
0
0 -e
0
4-' cc 10 cc
C)
cc 10
'U
PU
C)
-e 0 +1 +1 +1 +1 +1
C, C,
a, a,
+4
10 LI) 10
PU 10 N N c-n
4- — C, Cl cc 'C
0
44
C)
C)
PU
C
0-
N
U
C — N c-fl
C)
'- 0
C)
H
 (a)

I U

0 I 2 3

Prey deprivation period (days)

Fig. 8. Impact of prey deprivation on time talen for paralysis of

(a) S. litura (b) M pustulata and (c) D. cingulatus in R. Juscipes

CO (0' CO CO (fl
- 0' 4J) N 0' 10
LU — It) (fl '1) N
tJ)
C +1 +1 +1 +1 +1
+1
C) 0 (D 0 C)
0) C) —4 C)
)1) 11) '-4 —4
CO N C) 00 10
N N N '- —
C)
C) - it) N It) cfl
C) N N C
4.4
o +1 +1 +1 +1 +1
C) C) C) C) C)
+4
0) 1-4 0' '0 '4 C'
C) cci d
N '0 '0 '-0 '-0
-e
C
C)
U) CO '0 (N 4!)
N '0 '-4 '-0
- C) (fl
--4
4.)
o +1 +1 +1 +1+1
C) C) 0 0 C)
N CO It
(f C' C 0' 0
- 0' N '-0
N N — -
04)
(3' 10
0 - N '0 '00' —
C)
0.)
In
+1 +1 +1 +1 +1
In C)
0) C) C) C) C)
4.4 CO co N N
- CO N '0 0 10
In
4-'
C) N - (fl N
C - C) CO '0 CO
0 - 4!) N -
-'
4 +1 +1 +1
0
- C) C) C) C) C)
C) cfl C) N (fl
N N It) -'zF .j4
0 it) N N
ol
N N N — 0' —4
'04 '0 C' '-0 CO
I
- N N 0' 0'
- — . '- C) C) C)
4.4
+1 +1 +1 +1 +1
0
C) C) C) C) C)
+4 '-0 co It) N
L6 o
CO N 10 '0 4!)
0-.
• riO
Co
C) N '0 CO
C)
'- 0
 (a)

0
C)

C)

ri

Prey deprivation period (days)

Fig. 9. Impact of prey deprivation on time taken for paralysis of

(a) S. litura (b) M. pustulata and (c) II cingulatus in R. kumarii
D. cingulatus, respectively. Eight day prey deprivation significantly (P < 0.001)

reduced the paralytic durations for these three pests (174.40 ± 1.87, 160.40 ±

2.52 seconds for S. litura, M. pustulata and D. cingulatus, respectively) by the saliva

of the posterior lobe (Table 18 ; Figures 9 a, b and c).

M. 3. 5. 3. SALIVA OF R. marginatus

Saliva from the anterior lobe of fed R. marginatus took 84.10 ± 0.87,

77.10 ± 1.46 and 83.70 ± 1.51 seconds to paralyse S. litura, M. pustulata and

D. cingulatus, respectively. Eight day prey deprivation significantly (P < 0.001)

reduced the paralytic durations for these three pests (55.00 ± 1.14, 57.80 ± 1.63

seconds for S. litura, M. pustulata and D. cingulatus, respectively) by the saliva of

the anterior lobe.

Saliva from the posterior lobe of fed R. marginatus took 314.50 ± 1.73,

304.70 ± 7.04 and 281.00 ± 4.24 seconds to paralyse S. litura, M. pustulata and

D. cingulatus, respectively. Fight day prey deprivation significantly (P < 0.001)

reduced the paralytic durations for these three pests (186.70 ± 1.17, 181.70 ±

3.57 and 162.20 ± 7.51 seconds for S. litura, M. pustulata and D. cingulatus,

respectively) by the saliva of the posterior lobe (Table 19; Figures 10 a, b and c).

III. 4. DiscussioN

Arthropods have reached an incomparable degree of perfection in the

employment of chemicals in order to immobilize or to deter their prey (Zlotkin and

Gordon, 1985). Reduviid predators kill their prey by injecting saliva containing

41
 cc cc cc
N Cfl 10
LL N 0' 0' N
(I) +1 +1 +1 +1 +1
0
+1 C C C C C
C N 0' N
'-4 10 N L6 N
r.1
N N N -
C
• 1 —
LI?
—
N
N
N
N
ol
C
CT)
LO
'-4
'C
14
o +1 +1 +1 +1 +1
o C C C C C
N 'C '-i Co C
C r- cc
cc Co N 'C 10
o ' N — 0' N
- C co '0 10
N 'I) 4 4 N
0 +1 +1 +1 +1 +1
C C C C 0
0 N C 'C N
0 - N —
C 0' It— cc
ts N N N —
N
-3 'C N N 0'
0 00 It
14
0 + +1 +1 +1 +1
0 C C C C C
LI) 0' 11) 10
-< N N 'C 'C 10
0 N C 0' N N
-
-
N N N N '-4
N .-4
+1 +1 +1 +1 +1
0
C C 0 C C
0 10
0 C — N 'C
-4 cc 'C N 10
N N N N
N '-0 N N -
co '-0 N 'C —
14
+1 +1 +1 +1 +1
C C C C C
cc N C' C
'C
10 N 'C 10 LI)
0
- rI,
-4 "S
CL I C N '0 10
.- 0
H ej
 (a) 1-1--l- I

0 2 4 6 8

(b)
300

250

200
U
U
00

V) 100

0 2 4 6 8

300

250

200

ISO

100

50

0 I I I I
6
-- I
0 2 4 5

Prey deprivation period (days)

Fig. 10. Impact of prey deprivation on time taken for paralysis of

(a) S. litura (b) M. pustulata and (c) D. cingulatus in R. marginatus
paralytic toxins and histoly-tic enzymes through stylet mouth parts (McMahan, 1982,

1983). Presumably, most predatory insects salivate to liquefy, degrade, fragment or

lubricate their food prior to ingestion. in some insects this occurs without immediate

or apparent toxic action to prey. in some others, toxic action is evident and the prey

is locally or systemically inactivated. Such toxic action is found most conspicuously

in representatives of reduviids (Edwards, 1961; Haridass and Ananthakrishnan,

1981 a; Morrison, 1989; Ambrose and Maran, 1999 a; Maran and Ambrose,

1999; present observation) pentatomids, nabids and other bugs (Beard, 1963). In

the present observation, saliva of R. Juscipes , R. kumarii and R. marginatus caused

rapid paralysis of tested insect pests S. litura , M. pustulata and D. cingulatus at

low range concentration, a characteristic feature of neurotoxins (Zlotkin, 1991a)

which are proteins of low and medium molecular weight (Z1otki, 1984). According

to the site of action of these neurotoxins in the nervous system, Dolly (1988)

classified them into (a) ion channel toxins, which modify ion conductance, (b) pre -

synaptic toxins, which affect neurotransmitter release and (c) post - synaptic toxins,

which block neurotransmitter receptors. Further studies are necessary to categorize

the saliva of reduviids.

According to Morgan (1969) and Keegan et al., (1960), the venom may

vary qualitatively and quantitatively from species to species and may also vary even

within an individual at different times of the year or under different environmental

conditions or even within the age. In this present observation, the highest volume

of saliva in both the lobes of R. kumarii and the lowest in R. fuscipes observed could

be attributed to the size of the animal as well as the size of salivary glands.

42
R. kumarii was the largest predator in size and weight than R. marginatus and

R. Juscipes . Similarly the largest principal salivary gland was observed in R. kumarii

followed in R. marginatus and R. fuscipes. Further, the increase in venom volume

th increased prey deprivation period was similar to the observation in A. pedestris

(Ambrose and Maran, 1999 a). This might be due to that the saliva is getting

accumulated during prey deprivation as the predators need not spent the saliva for

prey paralysis.

Hodgen and Frank (1980; cited in Margaret and Phanuel, 1988) suggested

that the proteins play an important role in venom toxicity and the role assumes

differential importance in various species of venomous animals. in R. fuscipes,

R. kuniarii and R. marginatus, a higher protein content was observed in the anterior

lobe than that in the posterior lobe irrespective of their volume, suggesting that the

toxicity of saliva was borne in the anterior lobe contents, than that in the posterior

lobes. A more similar observation was made by Haridass and Ananthalzrishrian

(1981 a) in L. a/fminis and H. nigroviolaceous; Morrison (1989) and Ambrose and

Maran (1999 a) in A.. pedestris and Maran and Ambrose (1999) in C. brevipennis.

But Edwards (1961) opined that the toxicity was borne both in the anterior and

posterior lobes besides the digestive enzymes secreted by the posterior lobe. The

histological observations by Haridass and Ananthakrishnan (1981a) and Morrison

(1989) and in the present study (refer chapter I) on the salivary glands of reduviids

undoubtedly revealed that both the anterior and posterior lobes are histologically

different and hence they also vary in their secretory activity. Eventhough the

posterior lobe contents caused paralysis and mortality after a long duration of injection

43
in the tested insect pests, it might be due to the corrosive nature of the digestive

enzymes. Thus the quantitative variation of proteins found in the anterior as well

as in the posterior lobe contents of R. Juscipes , R. kumarii and R. marginatus could

be attributed to their interspecific variations. Ambrose and Maran (1999 b) found

out protein content of salivary gland variations even within the polymorphic forms

of a species. The intraspecific quantitative variation in the amount of protein in

different lobes of principal salivary glands reflect their differential functions and

divisions of labour. The increase in protein content in both the lobes as a function

of prey deprivation is similar to the observations for A. pedestris (Ambrose and

Maran, 1999 a) and C. brevipennis (Maran and Ambrose, 1999), since prey

deprivation caused accmulation of saliva, ultimately the protein content.

Although the saliva of R. fuscipes , R. kumarii and R. ma rginatus exhibited

almost a similar mode of action. on pests, electrophoretic studies indicated that they

are qualitatively different. Common protein fractions were not found among them.

Edwards (1961) found a mixture of six proteins which include trypsin like proteolytic

fractions in the saliva of P. rhadamanthus. The saliva of A. pedestris has 6, 11 and

2 protein fractions in anterior and posterior lobes and accessory glands, respectively

(Morrison, 1989). Thus this qualitative variation in the proteins could be attributed

to their interspecific as well as inter I intragenetic variations, a promising tool in the

molecular biosystematics.

The paralytic units observed for R. fuscipes, R. kumarii and R. marginatus

to S. litura, M. pustulata and D. cingulatus were comparatively very lesser than

44
those observed for A. pedestris (Ambrose and Maran, 1999 a) and C. brevipennis

(Maran and Ambrose, 1999) on M. pustulata and D. cingulatus . Since protein play

an important role in venom toxicity (Hodgon and Frank, 1980) higher protein

content of the venom could cause paralysis very rapidly even with low dose of saliva.

A. pedestris and C. brevipennis have comparatively lesser quantity of protein in

their saliva (Ambrose and Maran, 1999 a; Maran and Ambrose, 1999) than that

observed for the three Rhynocoris species. Hence lesser dose of saliva of these

Rhynocoris species could cause rapid paralysis in the tested insect pests than those

observed for A. pedestris and C. brevipennis . The higher amount of protein in the

saliva and lower paralytic units of harpactorine reduviids and lower protein and

higher paralytic units in peiratine (C. brevipennis) and reduvijne (A. pedestris) reduviids

could be attributed to their non - tibiaroliate and tibiaroliate conditions, respectively.

Miller (1939) reported that the degree of development of tibial pad is correspondingly

related to the degree of toxicity of saliva injected by the predators into their prey.

Observation of Livingstone and Ambrose (1979, 1984) revealed that though the

tibiaroliate reduviicls are provided with powerful tibial pads, and take lesser time to

grab the prey, they take relatively longer time to completly paralyse their prey. But

in the non - tibial pad group of reduviids, such as harpactorine reduviids, though the

time taken for pinning is too long, the paralysis time is relatively very brief. This

suggests that the saliva of non - tibial pad group of reduviids is more toxic than that

of the tibial pad group.

The three Rhoco5 predators took varied time to paralyse the three pests.

Since V instar caterpillar of S. litura are larger than M. pustulata and D. cingulatus,

45
predators took longer time to paralyse it. Since prey deprivation caused accumulation

of saliva, ultimately protein, the prey deprived predators could cause rapid paralysis

than the fed ones. Because daily fed predators invest their saliva continually to

paralyse their prey and they d0 not have accumulated saliva. So they could cause

slower (normal) paralysis than the prey deprived ones. The higher paralytic potential

of anterior lobe could be attributed to their higher protein content.

The penetration of such salivary toxins into the target tissue is facilitated

by the lytic enzymes viz., hyaluronidase and phospholipase (Edwards, 1961).

Hyaluronidase is a spreading factor for venoms (Edwards, 1961; Mommsen, 1978

a, b; Foelix, 1982; Schmidt, 1982), which separate the cells in intact tissues and

reduces the viscosity of hyaluronic acid and prey fluids and phospholipases are

considered as a widerspread constituent of animal venomwhich digest the

in the cell membranes, disrupting neuron and muscle cells (Beard, 1963; Schmidt,

1982). Thus it was concluded that the phospholipases and hyalitronidases act to

disrupt the intracellular matrix, abolish the excitability of nerve and muscle and lead

to general lysis in the prey (Edwards, 1961).

Since the saliva of reduviids is involved in extra oral digestion (EOD) and

causes paralysis of prey, it is difficult to resolve the differences between the digestive

enzyme and envenomation (Edwards, 1961; Foelix, 1982; Schmidt, 1982). Schmidt

(1982) also stated that, since the venom of H. innesi reversibly paralyse the nerve

muscle preparation, it is not a digestive enzyme, instead strictly a venom. But

Cohen (1993) and Ambrose and Maran (1999 b) observed the presence of endo and

46
exopeptidase in the salivary glands of Z. renardii and R. margincztus. The preceeding

chapter (chapter VI) discusses salivary digestive enzymes.

Thus it may be concluded that the venom of reduviids possess neurotoxins,

which represents a chemical adaptation 0f these predators to paralyse their prey

altering the structure and function of the nervous system of the pre Since these

neurotoxins are analogous to the synthetic insecticides, they can be utilized to

model the efficient insecticides. Hence these agriculturally significant neurotoxins

have to be studied further on the background of recent advances in protein

biotechnology, which may include the mass production of toxins, their design and

modification via recombinant DNA technology.

47