International Journal of Tropical Insect Science

https://doi.org/10.1007/s42690-021-00632-2

ORIGINAL RESEARCH ARTICLE

Potential toxic effects of aqueous leaf extracts of Calotropis gigantea

and Croton laccifera against Aphis craccivora
W. A. K. G. Thakshila1 · W. T. S. Dammini Premachandra1   · Christian Borgemeister2

Received: 20 January 2021 / Accepted: 26 August 2021

© African Association of Insect Scientists 2022

Abstract
Plant-derived insecticides represent one of the best alternatives to synthetic chemicals. In this study, aqueous leaf extracts
of Calotropis gigantea (Euphorbiaceae) and Croton laccifera (Apocynaceae) on ­1st ­(N1) and ­2nd instar ­(N2) nymphs, and
newly emerged apterous females (AF) of cowpea aphid Aphis craccivora (Koch) (Homoptera: Aphididae) was determined
for their contact, residual and systemic toxicities, using Yard-long bean plants. Compared to the untreated controls, both
leaf extracts affected survivorship of all aphid stages tested, as well as nymphal production of AF. Systemic and contact
toxicity had a great impact on A. craccivora while no strong toxicity was detected in residual bioassay with the two plant
extracts. Stronger effects were recorded in ­N1 compared to ­N2 and AF. In all three bioassays, C. gigantea outperformed C.
laccifera in terms of mortality and reduction of fecundity. Botanical insecticides on the basis of leaf extracts of C. gigantea
and C. laccifera are interesting candidates for integrated management of A. craccivora on Yard-long beans. Thus, further
large-scale field trials with these compounds are warranted.

Keywords  Aphids · Botanical insecticides · Fecundity · Mortality · Nymphs · Toxicity

Introduction
Yard-long bean (YLB), Vigna unguiculata (L.) Walp.sub-
species sesquipedalis (L.) (Fabaceae) is one of the most
economically important food legumes in South Asia (Singh
et al. 1997; Fery 2002). YLB’s immature pods are a popu-
lar green vegetable, containing proteins (Ano and Ubochi
2008), vitamins, minerals and fibres (Messina 1999; Singh
2005). They are extensively grown as part of low-land agri-
culture, often integrated with rice cultivation. In Sri Lanka,
YLBs are highly susceptible to various insect pests, among
them the cowpea aphid, Aphis craccivora Koch (Homoptera:
Aphididae) is particularly damaging under field conditions
(Edirisinghe 1994; Jayasinghe et al. 2015; Priyadarshani
et  al. 2016). It is a widespread pest on cowpea and
related legumes in India, the Philippines, tropical Africa
and Latin America (Singh and Jackai 1985; Pettersson et al.
* W. T. S. Dammini Premachandra
dammini@zoo.ruh.ac.lk
1
Department of Zoology, University of Ruhuna, Matara,
Sri Lanka
2
Center for Development Research (ZEF), University of Bonn,
Bonn, Germany
1998). Both nymphs and adults cause direct damage through
phloem sap feeding from buds, flowers, pods, leaves and
young shoots (Ofuya 1997; Berberet et al. 2009). The rapid
multiplication rates of A. craccivora within few days result
in high population build up which in turn cause yellowing,
curling and subsequent drying out of affected plant parts,
leading to great reductions in pod yield (Jackai and Daoust
1986; Srivastava and Singh 1986). Additionally, A. crac-
civora indirectly damages through transmission of over
30 plant viruses (Blackman and Eastop 2000; Bashir et al.
2002) and excretion of honey dew which induce develop-
ment of black sooty mould and leaf shedding (Kotadia and
Bhalani 1992).
Most often, growers rely on synthetic insecticides as the
primary means of control. However, its high reproductive
potential coupled with short generation times promotes
the rapid development of insecticide resistance, especially
under excessive and re-current applications (Georghiou
1990; Karunaratna et al. 1999). Moreover, synthetic insecti-
cides often cause environmental and health hazards (Boman
1980; Ambethar 2009). In addition to natural enemies, plant-
derived insecticides are among the most potent alternatives
to synthetic insecticides with minimum to no adverse effects

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to consumers, growers and the environment (Goh et al. 2001;
Kim and Kim 2008).
Calotropis gigantea L. (Euphorbiaceae) and Croton lac-
cifera L. (Apocynaceae) are perennial shrubs native to South
and South-East Asia (Bangladesh, Cambodia, Burma, China,
India, Indonesia, Malaysia, Pakistan, Philippines, Sri Lanka
and Thailand) (Nasser et al. 2012; Nazar et al. 2013). In Sri
Lanka, it is predominantly an inhabitant of coastal areas.
Calotropis gigantea is bestowed with great medicinal values
(Kartikar and Basu 1994; Kumar and Kumar 2015;
Solohokara et al. 2015).
Published reports indicated that Croton laccifera is an
inhabitant in Indian Peninsula (Anonymous 2021) and Sri
Lanka. In Sri Lanka, it grows in both dry and wet regions,
especially in forests and bordering areas up to about 800 m
above sea level. It has been reported that Croton spp. caused
toxic effects against insects (Alexander et al. 1991; Silva
et al. 2017; Widanapathirana and Dasanayake 2013); how-
ever, aphicidal activity was not much reported (Bandara
1987; Bandara et al. 1988). Insecticidal and insect repel-
lent activities caused by C. gigantea against a variety of
insect species including aphid species have also been docu-
mented by several authors (Solunke and Deshpande 1991;
Arulprakash and Senthilkumar 2005; Devi et  al. 2018;
Prabhu et al. 2018). However, to date there is a dearth of
information on the potential impact of both C. gigantea and
C. laccifera on A. craccivora, and thus the present inves-
tigation aimed to determine the contact, residual and sys-
temic toxicity of aqueous leaf extracts of the two plants
against three developmental stages of A. craccivora using
microcosm experiments. The selection of the simple aque-
ous extraction of the botanicals was based on the inexpen-
siveness and simplicity in preparation that could facilitate
a potential future uptake by the small-scale farming com-
munity under Good Agriculture Practices (GAP) promoted
in Sri Lanka by the Department of Agriculture.
Materials and methods
Aphids
Aphis craccivora cultures were maintained on potted YLB
plants (cv. ‘Polon mae’) in screen cages in the Department
of Zoology, University of Ruhuna, Matara, Sri Lanka, at
the ambient temperature (30°C ± 2). The experiments were
conducted with first ­(N1) and second-instar-nymph ­(N2), and
freshly emerged apterous females (AF). Uniform aged ­N1
were obtained by allowing gravid females of A. craccivora
(≈ 600) to lay nymphs on excised YLB leaflets for 2 h, in a
sealed petri dish (8.5 × 1.5 cm), at the ambient temperature.
After 2 h, the adults were removed with a fine camel hair
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International Journal of Tropical Insect Science
paint-brush and new born ­N1 were collected for the subse-
quent experiments. In order to obtain N ­ 2, ­N1 were further
reared until the occurrence of first exuviae while AF were
obtained by rearing ­N1 until four moultings were performed.
In previous investigations, we recorded four nymphal stages
prior to the adults and the duration between each of the nym-
phal stages and from the last nymphal to the adult stage was
around 24 h (W.T.S. Dammini Premachandra, unpublished
data).
Plant extracts
Mature leaves of C. gigantea and C. laccifera were collected
from “Matara” located in southern Sri Lanka (Latitude: 5°
57′ 17.712'' N; Longitude: 80° 33′ 17.8416'' E) from the
plants before flowering. Leaves were thoroughly washed
using Sterile Distilled Water (SDW) and then macerated in
SDW (at a ratio of 1:2 [w/v]) in a kitchen blender. The mac-
erated suspension was left at ambient temperature for 24 h.
Thereafter, the suspension was stirred well and centrifuged
at 3,000 rpm for 30 min. The supernatant was separated as a
clear solution and used for the experiments (Premachandra
et al. 2014).
Bioassays
Experiment 1: direct contact toxicity
The effect of direct contact toxicity of aqueous leaf extracts
of C. gigantea and C. laccifera was determined against N ­ 1,
­N2 and freshly emerged AF under ambient temperature
conditions. In the AF bioassay, a group of 20 individuals
were placed with a fine camel hair paint-brush in a plastic
Petri dish (8.5 × 1.5 cm) lined with a filter paper. Thereaf-
ter, the aphids were sprayed topically with the two botani-
cals and SDW as untreated control, using a hand-held
sprayer. Immediately after spraying, treated AF were trans-
ferred to three-week old potted (11 cm × 7.5 cm × 8.5 cm)
YLB plants, and the pot was covered with a plexi-glass
cylinder (diameter 10  cm, 30  cm in height). The space
between the cylinder and the pot was sealed using model-
ling clay to prevent escape of the aphids. The top and four
side-holes (diameter 3  cm) at the upper and lower sec-
tions of the cylinder were covered with nylon screening
(64 µm pore size) to facilitate ventilation (Premachandra
et  al. 2003). Plants with treated and non-treated aphids
were arranged in a complete randomized design with five
replications, and mortality was recorded in 24 h intervals
for three days. Females were considered dead if they did
not move after a stimulation with a fine needle. In addition
to female mortality, number of new born N ­ 1 were counted
and nymphs laid at 24 h and 48 h subsequently removed
from the leaves. Assays with ­N1 and ­N2 were conducted
International Journal of Tropical Insect Science
following the experimental protocol used for AF. Percent-
age cumulative mortality of treated and non-treated ­N1,
­N2 and AF were calculated. The whole experiment was
repeated five times.
Experiment 2: fresh residual contact toxicity
The effect of residual contact toxicity of aqueous leaf
extracts of C. gigantea and C. laccifera was determined
against ­N1, ­N2 and freshly emerged AF under ambient
temperature conditions. Leaves of three-week-old potted
(11  cm × 7.5  cm × 8.5  cm) YLB plants were sprayed on
both sides with the two botanicals and SDW as control
until runoff, using a hand-held sprayer and air dried for
30 min. Thereafter, individuals of ­N1, ­N2 and AF (20 per
leaflet) were separately placed on the treated and untreated
YLB plants. Subsequently, these plants, covered with a
plexi-glass cylinders (for details refer to the Experiment
1). The mortality of N­ 1, ­N2 and AF were recorded at 24,
48 and 72 h after placing them on YLB plants (for details
refer to the Experiment 1). Moreover, number of freshly
laid ­N1 were also counted and all the nymphs produced at
24 h and 48 h were removed from the YLB plant. Experi-
mental design, number of replications and repetitions were
similar to the Experiment 1.
Experiment 3: systemic toxicity
Seeds of YLB (var. ‘Polon mae’) were sown in plastic pots
(11 cm × 7.5 cm × 8.5 cm) containing a composed mix-
ture of cattle manure (50%), paddy straw (20%), green
manure (15%), poultry litter (10%), and coconut coir dust
(5%). When the plants were four weeks old, the soil was
drenched with 180 ml of the two botanical extracts and
SDW as control, separately. This amount was sufficient
to cause slight to no drainage. The YLB plants were not
watered for 2 d before drenching. One day after the soil
drench application, 30 ­N 1, ­N 2 and AF were positioned
separately on treated and untreated YLB plants. Subse-
quently, plants were placed in plexi-glass cylinders and
the mortality of ­N1, ­N2 and AF were recorded at 24, 48
and 72 h after placing them on plants (for details refer
to the Experiment 1). Five replications were used for the
treatment constituted of five replications and, both treated
and control YLB plants were arranged in a complete ran-
domized design. after placing them on YLB plants (for
details refer to the Experiment 1). Moreover, number of
freshly laid ­N1 were also counted and all the nymphs pro-
duced at 24 h and 48 h were removed from the YLB plant.
Experimental design, number of replications and repeti-
tions were similar to the Experiment 1.
Statistical analysis
Percentage cumulative mortality of treated and non-treated
­N1, ­N2 and AF were calculated with respect to three experi-
ments and were normalized using arcsin square root trans-
formation while number of nymphs produced by AF were
subjected to square root (X + 1) transformation, prior to anal-
ysis. The data from repeated trials were checked for homo-
geneity of variance using Brown and Forsythe's test and
then pooled for the further analysis if the assumptions were
not violated. Effects of the two botanicals on survival and
reproduction of the aphids were analysed separately for the
three bioassays. Dunnett's test was performed to compare the
cumulative aphid mortalities caused by the two botanicals
with the untreated controls, as well as nymphal production
of treated and untreated females, at each post-exposure time.
One-way-ANOVA was conducted to compare the mortali-
ties among the different life-stages with respect to a given
post-exposure time as well as mortalities of a given life-stage
across the three post-exposure times, for each botanical. The
same analysis was used to compare the nymphal production
of AF over three post-exposure times with respect to a given
botanical treatment. Moreover, aphid mortalities and prog-
eny production between the two botanical treatments were
compared using Student t test. Means were compared with
Tukey's range test and all analyses were performed using
SAS (SAS Institute 1999) at a significance level of 0.05.
Results
Experiment 1: direct contact toxicity
Both C. gigantea and C. laccifera leaf extracts caused sig-
nificantly higher mortality (P < 0.0001) in ­N1, ­N2 and AF,
compared to the respective untreated controls (Table 1).
Mortalities of ­N1 and ­N2 significantly (P < 0.0001) increased
over the post-exposure time (Table 1). At 72 h, percentage
reduction in survivorship of N ­ 1, ­N2 and AF was recorded
as 98, 78 and 58%, and 91, 70 and 39%, with respect to C.
gigantea and C. laccifera, respectively, relative to the con-
trols (Table 1). Of the two botanicals, C. gigantea caused
significantly higher aphicidal impact (P < 0.0001) com-
pared to C. laccifera. Moreover, significant differences
(P < 0.0001) in mortalities among ­N1, ­N2 and AF were
detected with each botanical extract following an ascending
order of AF < ­N2 < ­N1. The highest mortality in ­N1, ­N2 and
AF was recorded as 98.20, 78.20 and 58.40%, respectively,
with C. gigantea at 72 h (Table 1).
The contact toxicity caused by both extracts significantly
reduced (P < 0.0001) nymphal production of A. craccivora,
compared to the untreated controls, with a maximum
94% and 92% reduction in C. gigantea and C. laccifera,
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 International Journal of Tropical Insect Science

Table 1  Mean (± SE) percentage cumulative mortality of three life-stages of Aphis craccivora induced by contact toxicity of aqueous leaf
extracts of Calotropis gigantea and Croton laccifera 
Life stage Mean (± SE) percentage cumulative mortality
Calotropis gigantea Croton laccifera Untreated control
24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

1st instar- 79.20 cA 93.40 cB 98.20 cC 69.80 cA 82.60 cB 90.80 cC 1.20 aA 1.40 aA 1.80aA
nymph (± 0.99) (± 0.85) (± 0.49) (± 1.37) (± 1.50) (± 0.90) (± 0.44) (± 0.46) (± 0.57)
2nd instar 64.20 bA 70.00 bB 78.20 bC 55.00 bA 62.20 bB 70.00 bC 1.80 aA 2.00 aA 2.40aA
nymph (± 0.80) (± 1.00) (± 0.81) (± 1.19) (± 0.91) (± 1.12) (± 0.48) (± 0.65) (± 0.66)
Female 55.40 aA 56.80 aA 58.40 aA 37.60 aA 38.80 aA 40.00 aA 0.60aA 1.60 aA 1.80aA
(± 0.91) (± 0.90) (± 1.31) (± 1.05) (± 0.72) (± 0.58) (± 0.33) (± 0.47) (± 0.48)

Means followed by the same lowercase letters within columns with respect to a given botanical and uppercase letters within the rows with
respect to a given life stage are not significantly different (P =0.05; Tukey test; SAS Institute 1999). Data were subjected to arcsine square-root
transformation before the analysis; non-transformed percentage mortalities are presented in the table

respectively (Table 4). The suppressive effect of C. gigantea
was significantly (P < 0.0001) greater than that of C. laccif-
era. However, for both extracts nymphal production did not
differ (P > 0.05) over post-exposure time (Table 4).
Experiment 2: residual toxicity
Calotropis gigantea leaf extracts significantly reduced
(P < 0.0001) nymphal production, with a maximum reduc-
tion of 25% compared to the untreated controls (Table 4). No
significant residual toxicity of C. laccifera compared to the
untreated control was recorded. Nymphal production signifi-
cantly (P < 0.0001) increased with increasing post-exposure
time for both botanicals (Table 4).

Compared with untreated controls, both C. gigantea and C.
laccifera significantly affected (P < 0.0001) the survivorship
of all three tested developmental stages of A. craccivora
(Table 2) with significantly (P < 0.0001) greater effects
for ­N1 compared to ­N2 and AF. As in experiment 1, the
residual effect of C. gigantea was significantly (P < 0.0001)
stronger than that of C. laccifera in particular with respect
to ­N1 (53.20%) and N­ 2 (43%) mortality (Table 2). Aphicidal
effects did not differ significantly over post-exposure time.
The maximum reduction in survivorship in ­N1, ­N2 and AF
was recorded as 52, 42 and 25, and 34, 26 and 15% for C.
gigantea and C. laccifera, respectively, 72 h after exposure
(Table 2).
Experiment 3: systemic toxicity
Soil drenching treatment with both plant extracts caused sig-
nificantly (P < 0.0001) higher mortality in all the three tested
life- stages of A. craccivora compared to the untreated con-
trols (Table 3). Aphid mortalities significantly (P < 0.0001)
increased with post-treatment time, and similar to the contact
and residual toxicities, a significant increase (P < 0.0001) in
aphicidal impact was detected in the order of < AF < ­N2 < ­N1.
In ­N1 C. gigantea and C. laccifera caused 100% and 92.96%
mortality 72 h after soil drenching, respectively (Table 3).
Mortality in N­ 2 ranged from 71.60 to 91.28% and 61.20 to
79.12% for C. gigantea and C. laccifera, respectively. Soil

Table 2  Mean (± SE) percentage cumulative mortality in three life-stages of Aphis craccivora caused by residual toxicity of aqueous leaf
extracts of Calotropis gigantea and Croton laccifera and untreated controls at three post-exposure times

Life stage Mean (± SE) percentage cumulative mortality

Calotropis gigantea Croton laccifera Untreated control
24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

Nymph 1 50.60 cA 52.20 cA 53.20 cA 33.60 cA 34.60 cA 35.20 cA 01.20 aA 01.40 aA 01.60 aA
(± 1.24) (± 1.33) (± 1.38) (± 0.94) (± 0.99) (± 0.93) (± 0.44) (± 0.26) (± 0.56)
Nymph 2 41.80 bA 42.60 bA 43.00 bA 24.20bA 25.40 bA 26.00bA 00.40 aA 00.60 aA 00.60 aA
(± 1.04) (± 0.78) (± 1.12) (± 0.94) (± 1.04) (± 1.04) (± 0.27) (± 0.33) (± 0.28)
Female 24.60 aA 25.00 aA 25.60 aA 14.60 aA 15.00 aA 15.60 aA 00.20 aA 00.40 aA 00.40 aA
(± 0.99) (± 1.00) (± 0.97) (± 0.95) (± 1.04) (± 1.05) (± 0.20) (± 0.28) (± 0.28)

Means followed by the same lowercase letters within columns with respect to a given botanical and uppercase letters within the rows with
respect to a given life stage are not significantly different (P =0.05; Tukey test; SAS Institute 1999). Data were subjected to arcsine square-root
transformation before the analysis; non-transformed percentage of mortalities are presented in the table

13
International Journal of Tropical Insect Science

Table 3  Mean (± SE) percentage cumulative mortality in three life-stages of Aphis craccivora caused by systemic toxicity of aqueous leaf
extracts of Calotropis gigantea and Croton laccifera and untreated controls at three post-exposure times
Life stage Mean (± SE) percentage cumulative mortality
Calotropis gigantea Croton laccifera Untreated control
24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

1st instar- 80.16 cA 90.40cB 100.00cC 74.56 cA 84.72 cB 92.96 cC 01.20 aA 01.80 aA 02.20 aA
nymph (± 1.34) (± 1.01) (± 0.00) (± 1.07) (± 1.06) (± 1.14) (± 0.60) (± 0.70) (± 0.71)
2nd instar 71.60 bA 82.60 bB 91.28 bC 61.20 bA 68.92 bB 79.12 bC 01.40 aA 02.20 aA 02.60 aA
nymph (± 0.54) (± 0.90) (± 0.80) (± 0.98) (± 1.01) (± 0.86) (± 0.68) (± 0.77) (± 0.82)
Female 67.60 aA 76.40 aB 84.80 aC 42.80 aA 62.96 aB 72.92 aC 00.20 aA 00.40 aA 00.80 aA
(± 1.00) (± 1.39) (± 0.84) (± 0.69) (± 1.24) (± 1.21) (± 0.20) (± 0.28) (± 0.37)

Means followed by the same lowercase letters within columns with respect to a given botanical and uppercase letters within the rows with
respect to a given life stage are not significantly different (P =0.05; Tukey test; SAS Institute 1999). Data were subjected to arcsine square-root
transformation before the analysis; non-transformed percentage of mortalities are presented in the table

Table 4  Mean (± SE) number of nymphs produced by apterous females of Aphis craccivora treated with aqueous leaf extracts of Calotropis
gigantea and Croton laccifera with respect to three toxicities
Treatment Contact toxicity Residual toxicity Systemic toxicity
24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

Calotropis 19.00aA 20.72aA 21.56aA 38.60aA 108.92aB 240.80aC 11.88aA 14.84aA 14.92aA
gigantea (± 0.71) (± 0.97) (± 0.73) (± 2.69) (± 3.61) (± 5.28) (± 0.61) (± 1.04) (± 1.15)
Croton lac- 27.40bA 28.52bA 29.76bA 47.20bA 129.68bB 309.84bC 20.88bA 22.60bA 24.00bA
cifera (± 0.83) (± 1.16) (± 1.23) (± 2.81) (± 2.92) (± 2.37) (± 1.01) (± 0.86) (± 1.52)
Untreated 51.56cA 132.56cB 354.56cC 49.68bA 131.84bB 319.00bC 50.68cA 152.16cB 348.20cC
control (± 2.60) (± 2.40) (± 2.03) (± 2.68) (± 3.48) (± 3.46) (± 1.48) (± 2.35) (± 3.03)

Means followed by the same lowercase letters within columns with respect to a given botanical and uppercase letters within the rows with
respect to a given life stage are not significantly different (P =0.05; Tukey test; SAS Institute 1999). Data were subjected to arcsine square-root
transformation before the analysis; non-transformed percentage of mortalities are presented in the table

drenching with C. gigantea caused 85% reduction in survi-
vorship in AF against 73% in C. laccifera (Table 3).
Systemic effect of both extracts significantly affected
(P < 0.0001) the nymphal production of Aphis craccivora
relative to untreated controls (Table 4). C. gigantea was
found to be stronger compared to C. luccifera presenting
77–96% reduction. No significant differences in progeny
production (P < 0.05) was detected over post- exposure time
(Table 4).
Discussion
To the best of our knowledge, this the first detailed analysis
of contact, residual and systemic toxicities of aqueous leaf
extracts of C. gigantea and C. laccifera against different
developmental stages of A. craccivora.
We could show that both C. gigantea and C. laccif-
era leaf extracts caused high mortality in A. craccivora
and greatly inhibited nymphal production in all three
bioassays, though with varying degrees of effective-
ness (Tables  1, 2 and 3). Variations in effectiveness of
different bioassays for a given botanical extract can be
due to functioning of multiple active ingredients in a
synergistical manner (Miresmailli and Isman 2014). Of
the three tested developmental stages of A. craccivora,
both extracts induced aphicidal toxicity in the ascending
order of AF < ­N2 < ­N1 across all the bioassays (Tables 1,
2 and 3). The higher susceptibility of ­N1 might be due
to their thinner integuments compared to those of older
life-stages enabling easy penetration of active ingredients
(Premachandra et  al. 2005). Both plant extracts caused
high mortality via contact and systemic toxicity, resulting
in 90–100% reduction in survivorship of N ­ 1, over the SDW
control. For the N
­ 2, with 91% the highest reduction in sur-
vivorship was recorded in the systemic treatment of C.
gigantea. Sammour et al. (2011) reported 90% reduction
in survivorship in N
­ 2 of A. craccivora with 1% neemix (an
oil product of Azadirachta indica) via its contact toxicity,
72 h post-treatment. Similarly, Koul (1999) showed that
neem seed extracts systemically caused lower survival rate
in nymphs of cabbage aphid, Brevicoryne brassicae (L.)
(Homoptera: Aphididae) compared to adults. The diverse
active ingredients of botanical insecticides can act as

13

growth regulators, interfering with the hormone regula-
tion of insects which in turn cause varying susceptibilities
of different life stages (Schmutterer 1990).
The high aphicidal impact induced by the systemic activ-
ity of the two tested botanicals suggests that the active ingre-
dients containing in the extracts were taken up by the YLB
plant roots and efficiently transported to the leaves via the
phloem network on which the aphids feed. Previous studies
reported systemic toxicity of botanical insecticides against
A. craccivora (Busch and Teusch 1992; Dimetry et al. 1995;
Dolma et al. 2017). For instance, Sammour et al. (2011)
recorded 72 h post-treatment a 95% and 88% reduction in
survivorship of females and N ­ 2 of A. craccivora, respec-
tively, following a systemic treatment of broad bean plants
with 0.5% neemix. In our study, C. gigantea and C. lac-
cifera were able to induce 91% and 85%, and 79% and 93%
reduction in ­N2 and AF, respectively, corroborating results
of these earlier studies.
Even though the residual toxicity caused less aphicidal
impact, 24 h after the initial exposure a C. gigantea treat-
ment still sufficed to induce a 50% reduction in the survi-
vorship of ­N1 A. craccivora. Likewise, Ahmed et al. (2020)
reported lower effectiveness in residual compared to direct
contact toxicity with ethanolic extracts of Citrullus colo-
cynthis (L.) (Cucurbitaceae), Cannabis indica (L.) (Canna-
baceae) and Artemisia argyi (L.) (Asteraceae) against Brevi-
coryne brassicae L. Apparently botanicals with high contact
toxicity can act as a neurotoxin (Yazdgerdian et al. 2015) and
have a greater ability to penetrate the insects' cuticle to gen-
erate impact than when absorbed by the digestive tract via
feeding (Akhtar and Isman 2007). Moreover, low residual
activity can be caused by reduced insect feeding on treated
plant surfaces, leading to lower intake of toxicants (Gökçe
et al. 2007). Finally, according to Schmutterer (1988) the
residual toxicity of botanicals is also dependent upon the
nature of the treated plant surface.
Over time, we recorded a significant increase in nymphal
mortality in both contact and systemic experiments, and for
the AF only in the latter (Tables 1 and 3). The extent of
aphicidal effects in Brevicoryne brassicae by aqueous leaf
extracts of Lantana camara L. (Verbenaceae) increased over
post-exposure time (Myumi and Maunga 2018). Interest-
ingly, for all the three tested life-stages of A. carccivora in
our residual treatment as well as for the AF in our contact
bioassay no such trends were observed. Such discrepancies
can result from variations in the mode of action of active
ingredients of botanicals as well as the varying relative
susceptibility of the different life stages of the test insects
(Brader et al. 1992; Hampton et al. 2002). Moreover, we
noted that of the overall aphid mortalities recorded over
the total 72 h time period, the greatest proportion occurred
within 24 h of the initial exposure, implying the rapid effec-
tiveness of both tested leaf extracts.
13
International Journal of Tropical Insect Science
Contact and systemic treatments of both C. gigantea and
C. laccifera considerably reduced nymphal production of A.
craccivora (Tables 1 and 3), with maximum efficacy asso-
ciated with the systemic activity which curtailed 91% and
87% nymphal production by C. gigantea and C. laccifera,
respectively. Similar levels of population reduction A. carc-
civora was reported by Sammour et al. (2011) after systemic
treatment of broad bean seedlings with 5% neemix. Effec-
tiveness of plant-derived insecticides for curtailing nymphal
production in different aphid species has been shown by sev-
eral researchers (Tang et al. 2002; Sammour, et al. 2011;
Shannag 2014). Vimala et al. (2010) demonstrated that the
active ingredients contained in neem-based insecticides can
block neuro-secretory cells causing disruption of adult matu-
ration and egg production which in turn can negatively affect
the reproductive potential of aphids.
In terms of aphicidal impact and inhibition of repro-
duction of A. craccivora as a whole C. gigantea showed
higher toxicity than C. laccifera in all three bioassays
(Tables 1–3; Fig. 1). In Sri Lanka, Bandara (1987) con-
ducted a direct contact bioassay with dichloromethane and
methanolic flower extracts of C. gigantea. However, both
extracts failed to induce significant mortality in A. crac-
civora 48 h after the treatment. Yet we proved strong toxic
effects with aqueous leaf extract of C. gigantea implying
varying insecticidal efficacies among the different parts
of the same plant species. Such variations can be associ-
ated with chemical constituents present in plants extraction
process, solvent type and developmental stage of the insect
species assessed (Hampton et al. 2002, Gökce et al. 2007;
Sayed et al. 2020). Plants produce an array of secondary
metabolites to avoid pest attacks (Baser et al.1998; Ahmed
2007; Camaroti et  al. 2018). These compounds, singly
or collectively, affect feeding, growth, development and
oviposition of insects (Schmutterer 1990; Mulla and Su
1999). Previous research work indicated that aqueous leaf
extracts of C. gigantea contain of phenolic compounds,
alkaloids, tannins, saponins and terpenoids (Kumar et al.
2012; Kortbeek et al. 2018) and these metabolites effec-
tively caused toxic effects on different aphids species like
Myzus persicae (Sulzer), Aphis gossypii Glover, Lipaphis
erysimi (Kaltenbach), Eriosoma lanigerum (Hausmann)
and Acyrthosiphon pisum (Harris) (Isman 2000; Park et al.
2011; Ateyyat et al. 2012; Ge et al. 2015). Also, it has been
reported that the genus Croton spp. is rich with diterpe-
nes and alkaloids (Salatino et al. 2007; Ndunda, 2014).
Bandara et al. (1988) indicated that the leaf extracts of C.
laccifera grown in Sri Lanka contained sitosterol and 5
hydroxy-3,7,4–trimethoxyflavone which possessed insec-
ticidal activity (Widanapathirana and Dassanayake 2013).
According to Kanimozhi (2006), 5% aqueous leaf extract
of C. gigantea cause 60% mortality in Aphis gossypii,
and hot and cold water extracts C. procera (Aiton) W. T.
International Journal of Tropical Insect Science
Aiton, a close relative of C. gigantea, supressed A. crac-
civora population under the economic threshold level on
field grown country beans Lablab purpureus L., indicating
promising aphicidal activity (Das et al. 2008). Further, C.
gigantea extracts were found to be lethal to the pink mealy
bug Maconellicoccus hirsutus (Green) (Hemiptera: Pseu-
dococcidae), causing 93% mortality 72 h after exposure
(Devi et al. 2018). For C. laccifera, previous research in
Sri Lanka on A. craccivora indicated that the kauranoids
and hardwickiic acid contained in the plant roots caused
61% and 62% mortality, respectively, in females 24 h after
topical exposure (Bandara et  al. 1987; Hampton et  al.
2002; Gökce et  al. 2007). Subsequently, Bandara et  al.
(1990) found that steam distillate leaf extract (2.85:14 w/w
Leaves: steam distillate) of C. laccifera and Croton aro-
maticus caused 34% and 73% mortality in female A. crac-
civora, respectively, 48 h after direct application. Moreo-
ver, paddy farmers in Sri Lanka use detached branches of
C. laccifera as a bunch to sweep paddy fields to combat the
paddy bug infestations (Widanapathirana and Dasanayake
2013). Finally, powdered leaves of C. laccifera are repel-
lent for the rice weevil Sitophilus oryzae (L.) (Coleoptera:
Curculionidae) (Gunarathna and Karunarathne 2009).
In conclusion, since C. gigantea displayed remark-
able efficacy with multiple modes of action in our study,
we believe that there will be a high potential to incor-
porate this botanical as a promising low-cost candidate
in integrated management programs of A. craccivora in
Sri Lanka and possibly beyond. Especially its high sys-
temic toxicity for adults and strong contact toxicity for
early nymphs warrant further studies to explore the field
efficacy, persistence and potential impact on aphid natural
enemies.
Acknowledgements  The authors are grateful to the Department of
Zoology, University of Ruhuna, Matara, Sri Lanka, for providing space
to conduct the experiments.
Author contribution  Ms. W.A.K.G. Thakshila performed the experi-
ments; Analyzed and interpreted the data; Wrote the paper.; W.T.S.
Dammini Premachandra: Conceived and designed the experiments;
Analyzed and interpreted the data; Contributed reagents, materials,
analysis tools or data; Wrote the paper.; Christian Borgemeister: Con-
ceived and designed the experiments; Wrote the paper.; All authors
read and approved the manuscript.
Funding  The authors did not receive support from any organization
for the submitted work.
Data availability  If necessary willing to provide data.
Declarations  novel prenylated quinolin-2-ones from East Asian Zanthoxylum
Conflict of interest  The authors declare no conflict of interest.
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