ORIGINAL PAPER

The defensive role of latex in plants: detrimental effects on insects

Marcio V. Ramos • Thalles B. Grangeiro •

Eder A. Freire • Maurıcio P. Sales • Diego P. Souza •

Eliane S. Araujo • Cleverson D. T. Freitas

Received: 12 March 2009 / Accepted: 5 January 2010 / Published online: 23 January 2010

� Springer Science+Business Media B.V. 2010

Abstract The defensive role of the latex of Calotropis

procera has recently been reported. In this study, latex

proteins involved in detrimental effects on insects were

evaluated on another important crop pest. The latex was

fractionated to obtain its major protein fraction, which was

then used to evaluate its insecticidal properties against

Callosobruchus maculatus (Coleoptera: Bruchidae) in

artificial bioassays. Laticifer proteins (LP) were investi-

gated to characterize their action in such an activity. LP

was highly insecticidal at doses as low as 0.1% (W/W).

This effect was slightly augmented in F1 generation reared

in artificial seeds containing LP at similar proportions of

F0, but was fully reversed when F1 developed in LP-free

seeds. The insecticidal proteins were not retained in a

chitin column, and did not lose their insecticidal activity,

even after heat treatment or pronase digestion. However,

these samples inhibited papain (EC 3.4.22.2) activity and

gut proteases of C. maculatus larvae, and a reverse

zymogram showed the presence of protein bands resistant

to papain digestion. These activities were not observed in

unheated LP as they were probably masked by abundant

endogenous cysteine protease (EC 3.4.22.16) activity

present in unheated LP. LP was resistant to proteolysis

when assayed with C. maculatus gut extract. However, gut

proteins of C. maculatus were digested when incubated

with LP. These observations and the deleterious effects of

LP upon C. maculatus, reinforce the hypothesis that latic-

ifer fluids are involved in plant defense against insects and

indicate C. procera latex to be a source of promising

insecticidal proteins. The inhibitor of proteolysis present in

the latex seems to be resistant to heat and proteolysis and is

certainly involved in the detrimental effects observed.

Keywords Apocynaceae � Bruchidae �Calotropis procera � Plant defense � Proteases �Protease inhibitor

Introduction

A substantial number of plant species are reported to pro-

duce a milk-like endogenous fluid commonly named latex.

In these species, a tube-like network of specialized cells

called laticifers, whose cytoplasm contains water-soluble

components, rubber and other cytoplasmic organelles,

forms extensions of the phloem and holds the latex (Kek-

wick 2001). Frequently, latex fluids possess a poly-isoprene

fraction that is commonly named rubber. Moreover, plant

latices are described as rich in small secondary metabolites,

Handling Editor: Chen-Zhu Wang.

M. V. Ramos (&) � D. P. Souza � E. S. Araujo �C. D. T. Freitas (&)

Departamento de Bioquımica e Biologia Molecular da

Universidade Federal

do Ceara, Campus do Pici, Caixa Postal 6033, Fortaleza, Ceara

CEP 60451-970, Brazil

e-mail: vramos@ufc.br

C. D. T. Freitas

e-mail: cleversondiniz@hotmail.com

T. B. Grangeiro

Departamento de Biologia da Universidade Federal do Ceara,

Fortaleza, Brazil

E. A. Freire

Departamento de Bioquımica da Universidade Federal de

Campina Grande, Campina Grande, Brazil

M. P. Sales

Departamento de Bioquımica da Universidade Federal do Rio

Grande do Norte, Natal, Brazil

123

Arthropod-Plant Interactions (2010) 4:57–67

DOI 10.1007/s11829-010-9084-5

non-protein amino acids, enzymatic and non-enzymatic

proteins in their serum phase (Yeang et al. 2002; Taira et al.

2005). Recently, a vast number of cysteine proteases have

been described as occurring in many latex-producing plants

(Liggieri et al. 2004; Morcelle et al. 2004a, b). Chitinases,

lectins, lipases, oxidative and other hydrolytic enzymes

have also been described as occurring in latex fluids (Stirpe

et al. 1993; Azarkan et al. 1997; Giordani et al. 1992; Amani

et al. 2007; Fiorillo et al. 2007). These findings on the

biochemical composition of laticifer fluids support the

hypothesis of their defensive role (Taira et al. 2005).

Members of Asclepiadaceae, Sapotaceae, Apocynaceae

and Euphorbiaceae are among the most cited laticifer plants.

The shrub Calotropis procera (Ait.) R.Br. is a lactiferous

Apocynaceae species that is found in tropical and subtropical

regions. The abundance of endogenous Calotropis latex is

striking, and it can easily be collected from the green parts

without compromising the health of the plant. The latex of

C. procera has been reported to possess an expressive pro-

teolytic activity of the cysteine type (Dubey and Jagannad-

ham 2003). In another recent study, this activity was further

characterized in terms of biochemical and enzymatic

parameters. Serine and metaloproteases were not detected

and aspartic protease activities were barely visible (Freitas

et al. 2007). In addition, antioxidative activities of superoxide

dismutase and ascorbate peroxidase were detected, and

chitinolytic activity was found. In a first attempt to determine

the involvement of laticifer proteins of C. procera in plant

defense, the proteins were incorporated in artificial diets and

tested on different crop pests, including Diptera, Lepidoptera

and Hemiptera (Ramos et al. 2007). Furthermore, much

effort has been focused on plant-derived materials as

potential new sources of insecticidal proteins, safe to humans

and environmental friendly, which could be used as defen-

sive agents in economically important crops. Proteins in

C. procera latex may represent a new source of exploitation.

In this work, the latex of C. procera was processed and its

major protein fraction was assayed for insecticidal activity

on the cowpea weevil Callosobruchus maculatus (Coleop-

tera: Bruchidae). The deleterious effects of latex proteins

upon larval development, adult emergence and F1 genera-

tion are discussed in terms of enzymatic activities and the

presence of cysteine protease inhibitors within the fractions.

Materials and methods

General experimental procedures

Acrylamide, bis-acrylamide, 2-mercaptoethanol, dithio-

threitol (DTT), Iodoacetamide (IAA) and sodium dodecyl

sulfate (SDS) were purchased from GE Healthcare, Brazil.

N-benzoyl-DL-arginine b-naphthylamide hydrochloride

(BANA), transepoxysuccinyl-L-leucylamido (4-guanidio)-

butane (E-64), ethylenediaminetetraacetic acid (EDTA), 5-

bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium

(BCIP/NBT), azocasein, porcine pancreatic a-amylase (EC

3.2.1.1), papain (EC 3.4.22.2), chitin practical grade from

Crab Shells, proteases from Streptomyces griseus (pron-

ase), goat anti-rabbit IgG conjugated to alkaline phospha-

tase and molecular weight markers were obtained from

SIGMA chemical Company, Brazil. Other chemicals used

were of analytical grade.

Laticifer proteins

Laticifer proteins (LP) were extracted from crude latex by

sequential centrifugation and dialysis. The crude latex was

obtained from wild C. procera plants in the vicinity of the

city of Fortaleza, State of Ceara, Brazil. The plant material

was identified by a local taxonomist and the voucher

N.32663 was deposited at the Prisco Bezerra Herbarium of

the Universidade Federal do Ceara, Brazil. Following an

incision at the shoot apices of the plant, the latex was col-

lected in distilled water to obtain a diluted solution of 1:2 (V/

V). For some experiments, latex was collected mixed with

IAA or E-64 to inhibit endogenous cysteine protease. Cen-

trifugation of the mixture was performed at 4�C and 5,0009g

for 10 min. The pellet was discarded and the soluble phase

was submitted to dialysis against distilled water at 8�C for

60 h using a dialysis membrane with a cut-off of 8,000 Da.

The centrifugation step was repeated under the same con-

ditions as described above. After having been cleaned and

freed of rubber, the new supernatant was freeze dried and

stored at room temperature until further analyses. This

material was named LP to refer to major laticifer proteins.

Insects

Cultures of Callosobruchus maculatus were maintained on

commercial seeds of cowpea [Vigna unguiculata L.

(Walp)]. Insects were reared and maintained in a growth

chamber at 27 ± 2�C, 60–70% relative humidity, and at a

photoperiod of 12 h light/12 h dark. For the bioassays,

1-day-old adults were sexed and isolated in glass recipients

to assist fertilization. Five couples of insects were equally

distributed in the pots containing 10 seeds. After 48 h, the

insects were discarded and the number of eggs laid on the

seeds was counted and adjusted to a minimum of five and a

maximum of eight per seed.

Seeds of Vigna unguiculata L. (Walp)

Seeds from two cultivars of V. unguiculata L. Walp were

initially used to prepare artificial seeds. The cultivar CNC

0434 was kindly supplied by the Brazilian Agency for

58 M. V. Ramos et al.

123

Agricultural Research (EMBRAPA). Another cultivar named

EPACE-10, found in local markets, was also used. Both

cultivars were used in the first set of bioassays to determine

the insecticidal activity of LP. After this stage, only the

commercial genotype (EPACE-10) was used to prepare

artificial seeds. Seeds were first de-hulled, fragmented and

triturated in a coffee grinder to produce fine flour, which was

used to make artificial seeds. All assays with the two cultivars

were performed concurrently, under similar conditions to

permit comparison between the two sources of beans.

Artificial seeds

Artificial seeds were made after mixing fine flour of

V. unguiculata seeds from each cultivar with lyophilized LP

to give mixture ratios of 0.05, 0.1, 0.25, 0.5 and 1.0% of LP

(W/W). The mixtures were encapsulated using gelatin

capsules (size N. 01) used in commercial pills. The final

weight of the artificial seeds was about 580 mg. Forty seeds

were prepared for each LP concentration and divided into

four groups of 10 seeds. The seeds were stored for 48 h in a

growth chamber before initiating the assays. The control

experiments were performed with artificial seeds containing

only the seed flour of cultivars, corresponding to 0% of LP.

Experimental design

Three different bioassays were performed, two of which

were done concurrently. The first was performed to evaluate

larval development. The seeds were then opened 20 days

after oviposition. The number of living larvae was deter-

mined and correlated to the total of eggs hatched to express a

percentage of survival. The individual larval weight was

determined for each replicate corresponding to different LP

concentrations. The second assay followed the same pro-

cedure as above, but the larvae were allowed to complete

development until insects emerged. The number of adults

and the day of emergence were recorded to express a ratio of

emergence and time of development, respectively. Finally,

in the third bioassay, the insects that emerged from the seeds

containing different percentages of LP (F0) were stimulated

to fertilize and oviposit in a new set of seeds containing

either the same percentage of LP as the seeds from which the

insects had emerged or free of LP, representing controls. The

percentage of adults that emerged and time of emergence

(days) were recorded (F1) and compared to those of the

insects that had emerged from seeds without LP (F1*).

Pronase digestion and heat treatment

To investigate the protein nature of insecticidal molecules in

LP two distinct protocols were tested. Heat treatment and

enzymatic digestion of LP by pronase were performed in an

attempt to eliminate insecticidal activity by destroying

protein structure and activity and thus investigating the

protein nature of insecticidal molecules. LP [100 mg in

50 ml of 50 mM PBS buffer (pH 7.5)] was submitted to in

vitro enzymatic digestion using pronase for 3 and 24 h.

Enzyme was added to the samples treated for 24 h at a time

corresponding to zero, 12 and 18 h. The enzyme/sample rate

was 1:100 (W/W). After 3 or 24 h of digestion at 37�C, the

samples were heated at 60�C for 10 min to inactivate the

pronase. The samples were then freeze dried. The product of

the pronase digestion was added to artificial seeds at 0.1%

(W/W), and the weight and survival of the larvae were

determined after 20 days of oviposition. Pronase was chosen

rather than a defined proteolytic enzyme to digest LP more

extensively because it is a non selective mixture of enzymes.

LP (100 mg) in 50 ml of water was heated at 98�C for

30 min, and then immersed in ice. The samples were

submitted to centrifugation at 25�C and 5,0009g for

10 min. The pellet and supernatant were separated and

lyophilized individually. Both materials were added to

artificial seeds at 0.1% (W/W) and tested for the effects on

the larvae of C. maculatus, as already described. Enzymatic

activities were also investigated in both LP samples.

Polyacrylamide gel electrophoresis

LP and pronase-digested LP samples were submitted to

electrophoresis in 12.5% polyacrylamide gels (12 9 11 9

0.02 cm), which were prepared as described by Laemmli

(1970) with minor modifications. The gel was run at 20 mA

and 25�C and proteins stained with coomassie brilliant blue

(R-350) solution in water/acetic acid/methanol (5:1:4, V/V/

V). Protein profile was observed after washing the gels with

the same solution without the dye.

Cysteine protease activity

Enzymatic assays to quantify cysteine protease (EC

3.4.22.16) activity in LP and LP derived fractions were

performed using BANA as the substrate. Aliquots of 50 or

100 ll of the LP [1 mg/ml in 50 mM PBS (pH 6.0)] were

pre-incubated with 40 ll of an activation solution of 3 mM

DTT and 2 mM EDTA for 10 min at 37�C and 200 ll of

BANA added [1 mM in 50 mM PBS (pH 6.0)]. After

30 min, the reaction was stopped by adding 500 ll of 2%

HCl in ethanol and 500 ll of 0.06% 4-(Dimethyl-amino)

cinnamaldehyde. After 40 min, the resulting color was

measured by spectrophotometry at 540 nm (Abe et al. 1992).

Effect of cysteine protease inhibitors (E-64 and IAA)

LP (100 mg) was dissolved in 50 ml of water containing

3 mM DTT and 2 mM EDTA, maintained for 10 min at

The defensive role of latex in plants 59

123

25�C, and 2 ml of E-64 (0.2 mM in water) was then added.

After 30 min, the mixture was submitted to dialysis at 8�C

for 48 h in distilled water to remove free E-64. In another

protocol the latex was collected in water containing 20 mM

Iodoacetamide and LP was obtained as previously descri-

bed. Residual cysteine protease activity was estimated by

using BANA as the substrate. Both LP-treated materials

were added to artificial seeds at different amounts and

tested for the effects on the development of C. maculatus.

Affinity chromatography on chitin

LP or heat treated LP (SLP) was fractionated on a chitin

column according to Freitas et al. (2007). Samples (10 mg/

ml) were dissolved in 150 mM NaCl and insoluble mate-

rials were removed by centrifugation at 10�C for 10 min

and 10,0009g. The soluble LP or SLP was chromato-

graphed on a chitin column (2 9 15 cm) previously

equilibrated in 150 mM NaCl. The column was first

washed with starting solution to elute unbound materials.

The bound proteins were washed out of the column by

adding 1 M acetic acid solution. The absorbance of the

column flow, collected in fractions of 2 ml, was measured

in a spectrophotometer at 280 nm. Pooled fractions corre-

sponding to unbound proteins (P-I) and bound proteins (P-

II) were submitted to dialysis in distilled water followed by

lyophilization. Fractions P-I and P-II were added to artifi-

cial seeds at 0.1% (W/W) and used in bioassays.

Chitinase activity

Chitinase (EC 3.2.1.14) activity was measured by a col-

orimetric assay essentially as in (Boller 1992). The assay

mixture contained 100 ll of LP or heat treated LP or P-I or

P-II (1 mg/ml) in 50 mM sodium acetate buffer (pH 5.2)

and 2 mg of colloidal chitin in a total volume of 500 ll.

The mixtures were incubated at 37�C for 60 min, then

boiled for 5 min and centrifuged (10,0009g for 20 min).

For each 300 ll of the supernatants, 100 ll of 0.6 M

potassium tetraborate was added and the amount of

N-acetyl-glucosamine liberated was determined (Reissig

et al. 1955). Enzymatic activity was estimated from a

calibration curve (Boller 1992). The controls included

enzyme and substrate blanks as well as internal standards.

Analysis of LP digestion by larval homogenates

of C. maculatus

The gut epithelium and the content of the digestive tube

were homogenized without removing the gut musculature

and perivisceral fat body, similar to that described in the

literature (Gomes et al. 2005). Fifty larvae of C. maculatus

(18–20 days aged), with no previous starving period, were

dissected under ice-cold saline to remove the entire

digestive tube. The larvae were dissected, and the guts

were homogenized in 1 ml 50 mM acetate buffer (pH 5.6)

and centrifuged at 5,0009g for 20 min at 4�C. The

supernatant was recovered and the total proteolytic activity

was determined using 1% azocasein as substrate (Xavier-

Filho et al. 1989). Laticifer proteins [400 lg in 50 mM

acetate buffer (pH 5.6)] were incubated with the larvae

extract (345 ll) in order to obtain a rate of 1 mAU/lg of

Protein (gut enzyme activity/LP). One mille-unit (mAU) of

azocaseinolytic activity was defined as the amount of

enzyme capable of increasing absorbance by 0.001 OD, at

pH 5.6 and 37�C for 1 h. The digestion was performed at

37�C for 2, 4 and 8 h. The reaction was stopped by

immersing the tubes in ice-cold water. Products of diges-

tion were detected by SDS-PAGE followed by protein gel

blot analysis. Rabbit anti-serum IgG against LP was used to

reveal LP digestion.

Detection of endogenous cysteine protease inhibitory

activity in LP

The possible existence of cysteine protease inhibitors in LP

was initially checked by papain (EC 3.4.22.2) activity

using BANA as the substrate and LP as the inhibitor.

However the abundant endogenous cysteine protease

activity of LP interfered in the assays. LP was then

substituted by heat treated LP (HT-LP). Heat treated LP

was shown to completely lose its endogenous proteolytic

activity. The heat treated LP was centrifuged and only the

soluble fraction (SLP) was recovered and freeze dried

before testing. SLP samples (6 mg/ml) were dissolved in

50 mM PBS (pH 6.0) and different aliquots were pre-

incubated with papain or gut proteases of C. maculatus

larvae at 37�C for 30 min. Additional steps were as

described above.

Detection of cysteine protease inhibitors by reverse

zymogram

Samples of LP treated with or without b-mercaptoethanol

and proteins on peaks, obtained after passing LP through

the chitin column, were applied to gels of sodium dodecyl

sulfate polyacrylamide (SDS-PAGE) copolymerized with

0.1% gelatin. The electrophoresis was performed as

reported (Laemmli 1970). After the runs, the gels were

immersed in 100 ml of 50 mM PBS buffer (pH 6.0) con-

taining papain (1 mg), DTT (3 mM), EDTA (2 mM) and

incubated at 37�C for 14 h. The embedded gelatin and

other proteins susceptible to papain proteolysis were

digested, thereby removing stainable proteins, except

where putative inhibitor bands were located. The gels were

then stained and washed to remove any excess of

60 M. V. Ramos et al.

123

coomassie brilliant blue (R-350) as already cited. Protein

bands expected to correspond to protease inhibitors were

detected in gel as blue bands on a clear background. The

reverse zymography was compared to gels without gelatin,

according to (Ohashi et al. 2003).

a-amylase inhibitory activity

The possible presence of a-amylase inhibitors in LP was

investigated using LP as an inhibitor for porcine pancreatic

a-amylase activity (EC 3.2.1.1) on soluble starch. LP

(4 mg/ml) was dissolved in 50 mM acetate buffer (pH 5.5)

containing 20 mM NaCl and 0.1 mM CaCl2. Aliquots of

100 and 200 ll were pre-incubated with an enzyme solu-

tion of porcine pancreatic a-amylase at 37�C for 15 min.

Aliquots of 2 ml of 1% soluble starch were added. After

60 min, for each 250 ll of the reaction, 2.5 ml of lugol

(1 mM Iodine and 24 mM potassium iodide) were added

and the resulting colour in the tubes was measured by

absorbance at 565 nm.

Statistic analysis

Data corresponding to the performance of larvae and adult

emergence in F0 and F1 assays and effects of diets con-

taining LP, treated LP or derived fractions of LP on larval

weight were analyzed by one-way [analysis of variance]

ANOVA. The Student–Neuman–Keul’s test was used to

identify the means that differed where the ANOVA test

was significant. A P value of \0.05 was considered to be

significant.

Results and discussion

Effect of laticifer proteins on larval development

Larvae of C. maculatus grown in artificial seeds containing

LP at increasing percentages were greatly affected. For lar-

vae fed on control seeds, survival and weight reached

73.63 ± 6.48% and 4.64 ± 0.31 mg, respectively, while

larvae developed in seeds containing 0.1% (W/W) LP

exhibited a marked negative performance on survival and

weight (Fig. 1a, b). The lethal dose capable of reducing

larval survival by 50% (LD50) was calculated as 0.14% (W/

W) and the amount of LP that provoked a 50% reduction in

weight gain (ED50) was estimated to be 0.13% (W/W). These

values were far lower than those previously observed for

third instars Ceratitis capitata (Diptera: Tephritidae) fed on

diets containing LP (LD50 = 4.61% and ED50 = 3.07%).

Third instars Anticarsia gemmatalis (Lepidoptera: Nocuti-

dae) was also affected when exposed to diets containing LP;

however these effects were less evident (Ramos et al. 2007).

In the same study, the insecticidal action of LP was also

investigated against Dysdercus peruvianus (Hemiptera:

Pyrrhocoridae) and Spodoptera frugiperda (Lepidoptera:

Noctuidae). LP was ineffective on the latter even when LP

was present at 1% (W/W) and only partially active on the

former at doses higher than 1% (W/W).

Effect of laticifer proteins on adults

The percentage of emergence and mean developmental

time of C. maculatus adults reared on control seeds were

estimated as 60.5 ± 5.6% and 30.8 ± 1.47 days, respec-

tively. These values were altered to 30.5 ± 5.1% and

35.9 ± 1.16 days for insects grown in seeds containing

0.1% (W/W) LP (Fig. 1c, d). Diets containing 0.5% (W/W)

LP delayed the mean developmental time of insects by a

period of 20 days and severely affected the emergence of

adults, which was reduced to 2%. In seeds containing 1%

(W/W) LP, no insects emerged, even 120 days after ovi-

position. The performance of C. maculatus grown in both

cultivars tested was almost identical. As a result, in further

assays only the commercial source of seeds was used.

0

20

40

60

80

100

0 0.1 0.2 0.3% LP (w/w)

Su

rviv

al (

%)

A

0

1

2

3

4

5

6

0 0.1 0.2 0.3

% LP (w/w)

Wei

gh

t (m

g)

B

0

20

40

60

80

0 0.1 0.2 0.3

% LP (w/w)

Ad

ult

Em

erg

ence

(%

)

C

15

30

45

60

0 0.1 0.2 0.3

% LP (w/w)Mea

n D

evel

op

men

tal

Tim

e (D

ays)D

Fig. 1 Effects of dietary laticifer proteins from Calotropis proceraon Callosobruchus maculatus. Larval survival (a), weight (b), adult

emergence (c), and mean developmental time (d) were determined for

insects reared in artificial seeds with increasing concentrations of

laticifer proteins; (205 B n B 258). Figure inserted shows larvae of

20-day reared in seeds containing increasing amounts of LP. Bar:

5 mm. Artificial seeds were prepared using cowpea seed flour from

genotype CNC 0434 (open diamond) and from one commercially

available (filled circle)

The defensive role of latex in plants 61

123

Effect of laticifer proteins on F1 generation

The cumulative effects of LP on the life cycle of insects

were investigated when adults (aged 2 days) that had

emerged from the seeds containing different percentages of

LP (F0 insects) were stimulated to fecundate and then

allowed to lay eggs in a new set of artificial seeds con-

taining either similar contents of LP or on seeds made only

of V. unguiculata L. (Walp) seed flour, thus producing the

generations F1 and F1*, respectively. With the exception of

adults reared in control seeds, the performance of F1 insects

as measured by the percentage of adult emergence (Fig. 2a)

and mean developmental time (Fig. 2b), was worse when

compared to the parental generation F0, regardless of the

LP concentration used. However, the detrimental effects of

LP on these biological parameters of the life cycle of the

insects were fully reversed when insects born from LP-

containing seeds were stimulated to oviposit in control

seeds to produce the next generation (F1*). Thus, the per-

centage of adult emergence and mean developmental time

of F1* insects were comparable to those reared for both

generations in control seeds. These results indicate the

interference of LP upon insect development as observed in

the first set of bioassays. However, insects born in seeds

containing LP produced healthy eggs capable of generating

healthy larvae and adults that recuperated their growth

performance (F1*) when newly developed in LP-free seeds.

These observations suggest that laticifer proteins represent

an antinutritional source for larval development but do not

interfere in adult insects in terms of reproduction. As far as

we are aware this is the first study to evaluate the effects of

laticifer proteins on insect generations. Additional studies

will be required to support the preliminary findings

reported here.

Effect of pronase digestion and heat treatment of LP

on larval performance

Both enzymatic and heat treatment of LP were performed

in an attempt to destroy proteins found in LP and thus,

correlate this event with a possible decrease of insecticidal

activity. However, as shown in (Fig. 3), LP was only par-

tially digested by pronase after 3 or 24 h of incubation.

Pronase-treated LP (24 h) was added to artificial seeds at

0.1% (W/W) and larval survival and weight were deter-

mined 20 days after oviposition. The weight of the larvae

reared in seeds containing 0.1% (W/W) pronase-treated LP

was reduced (1.20 ± 0.37 mg) and this value was com-

parable to the weight of larvae from seeds containing 0.1%

undigested LP (1.36 ± 0.36 mg). Both values were sig-

nificantly lower in comparison to the weight of larvae

reared in control seeds (4.39 ± 0.5 mg, P \ 0.05). Thus,

insecticidal compounds resisted proteolysis.

0

20

40

60

80

100

Ad

ult

Em

erg

ence

(%)

0% 0.25 %0.1 %0.05 % 0.5 %Fo F1 Fo F1 F1* Fo F1 F1* Fo F1 F1* Fo

aa

a

bb

a

d

c

b

b

a

d

A

0

15

30

45

60

75

Mea

n D

evel

op

men

tal T

ime

(Day

s)

0% 0.25 %0.1 % %5.00.05 %Fo F1 Fo F1 F1* Fo F1 F1* Fo F1 F1* Fo

a

dd

cc

b

aaaa

d,e

e

B

Fig. 2 Evaluation of cumulative effects of Calotropis proceralaticifer proteins on Callosobruchus maculatus life cycle. Percentage

of adult emergence (a) and mean developmental time (b) were

determined for C. maculatus reared in seeds containing increasing

amounts of LP (F0). The F0 adults were then used to establish a new

generation in seeds containing either similar percentages of LP (F1) or

ones without any LP (F1*). Data corresponding to the performance of

larvae and adult emergence in F0 and F1 assays were analyzed

separately for different LP content (%) by one-way [analysis of

variance] ANOVA. The Student–Neuman–Keul’s test was used to

identify the means that differed where the ANOVA test was

significant. A P value of \0.05 was considered to be significant

A B C 97

66

30

20

14

kDa Fig. 3 Polyacrylamide gel elec-

trophoresis of native Calotropisprocera laticifer proteins. LP

(A) and LP treated with pronase

at 37�C for 3 (B) and 24 (C) h.

Twenty micrograms of proteins

were loaded in each well.

Molecular weight markers (GE

Healthcare)

62 M. V. Ramos et al.

123

Following the same trend the insecticidal potency of

laticifer proteins of C. procera upon C. maculatus was not

substantially affected (P [ 0.05), even after heat treatment

at 98�C for 30 min (Fig. 4a). Laticifer proteins involved in

the deleterious effects seemed to resist high temperatures.

Active proteins remained soluble when heat treated LP was

centrifuged to remove insoluble materials. As observed in

Fig. 4a, larvae grown in artificial seeds prepared with

soluble LP fraction (SLP) exhibited negative performance

equally seen in larvae grown in unheated LP-containing

seeds. These results (Figs. 3, 4a) support the hypothesis

that proteins resistant to proteolysis and to high tempera-

tures affect insecticidal properties of LP. The protein nat-

ure of LP has been continuously studied and characterized

in some extent (Freitas et al. 2007; Oliveira et al. 2007).

Furthermore, tannins and polysaccharides, high molecular

weight molecules, which are sometimes toxic to insects,

were not detected in LP (data not shown). It was decided

therefore to further investigate protein activities which may

be involved in insecticidal action.

Correlation between enzymatic activities and

insecticidal action

Latex of Calotropis procera presents strong cysteine pro-

tease activity as first described by Dubey and Jagannadham

(2003), and this activity could be, at least in part, respon-

sible for the insecticidal effects of LP observed in this

study. This hypothesis was tested treating a LP solution

with specific cysteine protease inhibitors (E-64), or alter-

natively, the latex was collected in water containing

Iodoacetamide (IAA), as described in methods. As stated

before, serine and metaloproteases were not detected in LP

and aspartic protease activities were barely visible (Freitas

et al. 2007). Endogenous cysteine protease activity of LP

treated with E-64 or IAA was inhibited by 92% and 100%,

respectively (Fig. 5a), and both treated materials (freeze

dried) were assayed for insecticidal action on C. maculatus.

The treatment of LP with IAA or E-64 did not reduce its

insecticidal effectiveness (Fig. 5b). The weight of the lar-

vae reared in seeds containing 0.1% LP treated with the

cysteine protease inhibitors were: 1.13 ± 0.1 mg (LP ?

IAA) and 2.0 ± 0.2 mg (LP ? E-64). These values were

not significantly different when compared to the weight of

larvae reared in seeds with native LP (1.77 ± 0.5 mg,

P [ 0.05). Moreover neither heat-treated LP nor its derived

fractions (supernatant and pellet) exhibited cysteine pro-

tease activity, but both were still insecticidal. It was,

therefore, thought that cysteine proteases were not involved

in the insecticidal properties of LP upon C. maculatus

under the experimental conditions. However, gut extracts

of 20 day-old larvae of C. maculatus were unable to digest

LP in vitro. On the contrary, as shown in (Fig. 6), LP was

highly efficient in digesting proteins of the gut extract a

few minutes after the mixture was done. The defensive role

of cysteine protease in latex has already been propounded

in a relevant publication (Konno et al. 2004). In addition,

other authors have given important evidence showing the

accumulation of cysteine protease in maize genotypes in

response to feeding by Lepidoptera larvae (Pechan et al.

2000, 2002). Cysteine proteolytic activity largely pre-

dominates in LP, but this activity was lost after heat

treatment. In view of these observations, it is concluded

that the involvement of cysteine proteases in the insecti-

cidal action of LP was not confirmed by the bioassays

performed, regardless of the ability of LP to digest, in vitro,

Fig. 4 Effect of heat treatment on the insecticidal properties of

laticifer proteins (a) and inhibition of proteolysis of purified papain

and gut proteases of C. maculatus larvae by the heat stable laticifer

proteins (b). Callosobruchus maculatus larvae were reared in LP-free

seeds (C-control) as well as in seeds with heat-treated LP (HT-LP).

The heat-treated fraction (98�C for 30 min) was centrifuged

and the supernatant (SLP) and precipitate (P) were assayed against

C. maculatus in bioassays. All fractions were tested at 0.1% (W/W).

The Inhibitory activities of the soluble fraction (SLP) upon papain

(open square) and gut proteases (filled triangle) of C. maculatuslarval were also determined. Each point represents the average of

three replicates. Data corresponding to the larvae weight were

analyzed by one-way [analysis of variance] ANOVA. Student–

Neuman–Keul’s test was used to identify the means that differed

where the ANOVA test was significant. A P value of \0.05 was

considered to be significant

Fig. 5 Proteolytic activity of Calotropis procera laticifer proteins

and inhibition of proteolysis by specific cysteine proteinase inhibitors

(E-64 and IAA) (a). Effect of these fractions on Callosobruchusmaculatus larval weight (b)

The defensive role of latex in plants 63

123

proteins of the gut extract of C. maculatus. Although this

proteolytic activity could somehow contribute to the neg-

ative performance of insects growing in seeds containing

LP, it is now clear that another heat-stable LP compound

must be involved in the harmful effects observed on larvae.

In another study, Freitas et al. (2007) showed that

C. procera LP exhibited a chitinolytic activity (6.1 nKat/ml)

that was fractionated into two peaks, P-I (non retained,

1.9 nKat/ml) and P-II (retained, 4.5 nKat/ml) upon affinity

chromatography on a chitin column. This procedure was

repeated here (Fig. 7a, b). In the present study when these

two fractions (P-I and P-II) were tested at 0.1% (W/W) on

C. maculatus (Fig. 7c, d), only P-I exhibited significant

(P \ 0.05) detrimental effects on larval survival

(40.17 ± 6.76%) and weight (0.36 ± 0.06 mg). For larvae

reared on seeds containing P-II or on control seeds, sur-

vival reached 80.17 ± 7.20 and 77.25 ± 9.3% and weight

was determined as 4.35 ± 0.22 and 4.45 ± 0.11 mg

(P [ 0.05), respectively. The binding ability of LP on the

chitin column was significantly reduced after heat treat-

ment, suggesting that structural integrity of the proteins

was essential for the chitin-binding capacity (Fig. 7a).

Furthermore, the chitinolytic activity in LP, P-I and P-II

was completely lost after heating at 98�C for 30 min. As

heat-treated LP (SLP) still exhibited insecticidal activity

(Fig. 4a), the chitinolytic activity in C. procera latex does

not explain the deleterious effects caused by LP upon

C. maculatus.

In the same study (Freitas et al. 2007), the inhibitory

activity of alpha amylase was not observed in LP, even

though the sample concentrations used were higher than

those normally assayed. Trypsin inhibitory activity has

been previously demonstrated to be absent in LP as well as

carbohydrate-binding proteins (lectins) with agglutinating

properties (Freitas et al. 2007). In view of these conclu-

sions, additional efforts were made to correlate insecticidal

activity of LP with other proteins found in the latex.

A cysteine protease inhibitor may be involved

in insecticidal properties

First attempts to detect endogenous cysteine protease

inhibitory activity in LP by using colorimetric assays failed

because the endogenous proteolytic activities of the latex

overloaded the measurements. However, following an

interesting strategy described by Ohashi et al. (2003) the

presence of cysteine protease inhibitors in LP was

investigated.

Inhibitors of cysteine protease activity in LP were

detected using SDS-PAGE reverse zymography for papain

inhibition, as shown in (Fig. 8). Two very close bands

corresponding to papain inhibitors were detected with

T=0 T=8 T=0 T=8 A B C B C A B C B C

Fig. 6 Polyacrylamide gel electrophoresis (left) and the protein gel

blot analysis (right) of Calotropis procera laticifer proteins. LP

(8 lg) was incubated with gut extract (30 lg) of Callosobruchusmaculatus. LP was detected using anti-LP rabbit IgG as the primary

antibody and goat anti-rabbit IgG conjugated with alkaline phospha-

tase as the secondary antibody. Bound secondary antibodies were

detected by adding 5-bromo-4chloro-3-indolyl phosphate/nitro blue

tetrazolium as the substrate. The samples are as follows: Gut extracts

(A); LP (B); Gut ? LP (C). The mixture Gut ? LP was analyzed at 0

and 8 h of incubation at 37�C (pH 5.6)

Fig. 7 Affinity chromatography of LP and heat treated LP (SLP) on

chitin (a), chitinolytic activity of LP, PI and PII (b) and effects of

dietary laticifer proteins (LP) from Calotropis procera on Calloso-bruchus maculatus larvae survival (c) and weight (d). Control (0%

LP); LP, laticifer proteins; P-I, peak non-retained; and P-II, peak

retained, on chitin column. All assays were performed at 0.1% (W/

W). Figure inserted shows larvae of 20-day reared in control seeds or

0.1% PI. Bar: 5 mm Data corresponding to the larval weight and

survival were analyzed by one-way [analysis of variance] ANOVA.

Student–Neuman–Keul’s test was used to identify the means that

differed where the ANOVA test was significant. A P value of \0.05

was considered to be significant

64 M. V. Ramos et al.

123

apparent molecular mass around 28 kDa, with or without

treatment of LP with b-mercaptoethanol. This protein

profile was also detected in LP samples that were treated

with either pronase or heat, as well as in the P-I fraction

obtained in the affinity chromatography on chitin. All these

fractions exhibited insecticidal effects on C. maculatus. It

is important to note that P-I from the chitin column

exhibited inhibitory activity of papain. This result rein-

forces the findings that insecticidal activity of P-I is

probably due the presence of the inhibitor of proteolysis

rather than chitinase activity, which was destroyed upon

heat treatment. In view of this, the inhibitory activity was

further investigated using colorimetric assays. However

this time, the heat treated LP was used as the source of the

cysteine protease inhibitor (and free of endogenous cys-

teine protease activity); purified papain (EC 3.4.22.2) and

gut proteases of C. maculatus larvae as a source of

enzymes and BANA as the substrate. As a result, hydro-

lyses of BANA produced by papain and gut proteases of

C. maculatus larvae was inhibited by heat treated LP in a

dose dependent manner (Fig. 4b). In the light of these

results it is now possible to suggest that an endogenous

inhibitor of cysteine protease activity contributes to the

insecticidal activity of LP. It is also interesting to note that

these results emphasize the co-existence of cysteine pro-

teases and an inhibitor of this proteolytic activity in the

same laticifer fluid.

The proteolytic properties of laticifer fluids are widely

known (El Moussaoui et al. 2001; Morcelle et al. 2004a, b;

Domsalla and Melzing 2008). However, the co-existence of

proteolytic inhibitory activity is still rare. A Kunitz-type

trypsin inhibitor was recently reported in Carica papaya

latex and in the latex of Hevea brasiliensis isoinhibitors of

the potato-protease inhibitor I family (Sritanyarat et al.

2006; Azarkan et al. 2006). However, in these cases, the

inhibitors were of the serine type. The occurrence of pro-

teins with inhibitory activity of papain in latex of Carica

papaya was earlier described (Monti et al. 2004). The

crucial role played by these molecules in latex is still

unclear, but their genes would appear to encode suitable

molecules for transgenic programs devoted to protect

plants against crop pests.

Conclusions

The proteins found in the laticifer fluid of C. procera

exhibited very important deleterious effects on the growth

and development of C. maculatus. This result corroborates

with others reported recently that showed detrimental

effects of LP on other insects, and supports that laticifer

proteins are implicated in the negative performance of

insects reared on artificial diets. It is not clear whether the

endogenous proteolytic activity of cysteine proteases found

in LP contributes to the deleterious effects observed upon

larval development. The in vitro proteolysis induced by LP

upon gut larval protein extract suggests there are detri-

mental effects on larvae. However, heat treated LP devoid

of proteolytic activity was still insecticide. This result

confirmed that although cysteine protease activity may be

involved in insecticidal activity, it is not the main activity

concerned. Further assays revealed that an endogenous

inhibitor that was capable of inhibiting purified papain and

proteolytic activity of gut extract of C. maculatus co-exists

with cysteine protease activity in the latex. Papain inhibi-

tory activity was only detected by a reverse zymogram or

in LP preparations free of the cysteine protease activity.

This inhibitory activity was found in all LP preparations

capable of affecting performance of C. maculatus. Finally,

the inhibitor is currently being purified by affinity chro-

matography on a papain-Sepharose column and it will

certainly be assayed for insecticidal effect. In view of this

and based on experimental evidence, this work supports the

hypothesis that laticifer proteins are implicated in defen-

sive activities of latex-producing plants. An endogenous

inhibitor of cysteine proteases is present in the latex of

C. procera. As far as we are aware, this inhibitor is directly

involved in the deleterious effects observed in C. macul-

atus insects and could probably be involved in the delete-

rious effects of LP observed in other crop pests already

evaluated. It is now clear that latex of C. procera accu-

mulates proteins that are capable of protecting the plant

against Coleoptera rather than solely Lepidoptera as

A B C D E F G H I J K L M

Fig. 8 Detection of papain inhibitors in LP using SDS-PAGE and

reverse zymogram of gelatinolysis inhibition. Molecular weight

markers (GE Healthcare) (A); bovine serum albumin (B), reverse

zymography of bovine serum albumin (C); LP (D), reverse zymog-

raphy of LP (E); LP treated with 2-mercaptoethanol (F), reverse

zymography of LP treated with 2-mercaptoethanol (G); LP after

incubation with pronase for 24 h (H), reverse zymography of LP after

incubation with pronase (I); P-I, peak non-retained on chitin column

(J), reverse zymography of P-I (K); P-II, peak retained on chitin

column (L), reverse zymography of P-II (M). Twenty micrograms of

protein were loaded in each well. Arrows show the protein band

corresponding to the inhibitor which was not affected by the papain

activity

The defensive role of latex in plants 65

123

observed earlier. It is worth noting that this new protease

inhibitor could become an important insecticidal protein to

be purified and better characterized in terms of its structural

and functional aspects. These proposals are now the subject

of continuous investigation.

Acknowledgements To the Conselho Nacional de Desenvolvi-

mento Cientıfico e Tecnologico (CNPq), Fundacao Cearense de

Amparo a Pesquisa (FUNCAP), MCT/PADCT, FINEP and Program

RENORBIO. M.V.R. is grantee of the International Foundation for

Science (IFS 3070-3). The authors are in debt with Mr. Brian Stephen

Currey who critically reviewed the language of the manuscript.

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