DOI: 10.1002/arch.

21384

ARTICLE

Chemicals isolated from Justicia adhatoda Linn

reduce fitness of the mosquito, Aedes aegypti L
Annamalai Thanigaivel1 Sengottayan Senthil-Nathan1
Prabhakaran Vasantha-Srinivasan1 Edward-Sam Edwin1
Athirstam Ponsankar1 Selvaraj Selin-Rani1
Venkatraman Pradeepa1 Muthiah Chellappandian1
Kandaswamy Kalaivani2 Ahmed Abdel-Megeed3
Raman Narayanan4 Kadarkarai Murugan5

1 Division of Biopesticides and

Environmental Toxicology, Sri Paramakalyani Abstract

Centre for Excellence in Environmental Extracts from Justicia adhatoda L. (Acanthaceae) strongly reduced
Sciences, Manonmaniam Sundaranar Univer- the fitness of the mosquito, Aedes aegypti Linn. The methano-
sity, Tirunelveli, Tamil Nadu, India
lic extracts inhibited several enzymes responsible for protecting
2 Post Graduate and Research Centre,

Department of Zoology, Sri Parasakthi College

insects from oxidative and other damage, including glutathione-S-
for Women, Tirunelveli, Tamil Nadu, India transferase, superoxide dismutase, cytochrome P450, and 𝛼- and 𝛽-
3 Department of Plant Protection, Faculty of esterases. They increased repellency (maximum repellency at 100
Agriculture, Saba Basha, Alexandria University, ppm) in host-seeking adult females using the “arm-in cage assay.”
Alexandria, Egypt
Histopathological examination showed the extracts led to serious
4 Department of Zoology, Sri Paramakalyani
midgut cell damage. Justicia adhatoda extracts led to reduced fecun-
College, Tirunelveli, Tamil Nadu, India
5 Thiruvalluvar University, Vellore, Tamil Nadu, dity and oviposition of gravid females compared to controls. The
India extracts led to substantially reduced A. aegypti survival. We infer
Correspondence that the extracts have potential to reduce pathogen transmission
Sengottayan Senthil-Nathan, Division of Biopes- by suppressing population growth of A. aegypti, and possibly other
ticides and Environmental Toxicology, Sri Para-
mosquito species.
makalyani Centre for Excellence in Environ-
mental Sciences, Manonmaniam Sundaranar
University, Alwarkurichi, Tirunelveli, Tamil Nadu KEYWORDS
627 412, India. botanical insecticide, deterrent, enzyme inhibition, histology,
Email: [email protected]; senthilkalaidr
metabolites, mosquito, oviposition
@hotmail.com
Grant sponsor: Indian Council of Medical
Research (ICMR), Government of India; Grant
number: 45/32/2013/BMS/TRM.

Arch. Insect Biochem. Physiol. 2017; 00: e21384 wileyonlinelibrary.com/journal/arch 

c 2017 Wiley Periodicals, Inc. 1 of 11
2 of 11 THANIGAIVEL ET AL.

1 INTRODUCTION

Aedes aegypti Linn is a vector of deadly pathogens, including yellow fever, dengue, and, most recently, the Zika virus
(Thanigaivel et al., 2012). Overuse of chemical insecticides leads to insecticide resistance, issues related to human
health, and reduced water quality (Pimentel, 2009; Revathi, Chandrasekaran, Thanigaivel, Kirubakaran, & Senthil-
Nathan, 2013). Almost 40 years ago Djerassi, Shih-Coleman, & Diekman (1974) recognized the need to develop new
insect pest management technologies, such as plant extracts having insecticidal properties that degrade fairly quickly
(Pradeepa, Sathish-Narayanan, Kirubakaran, & Senthil-Nathan, 2014; Pradeepa, Sathish-Narayanan, Kirubakaran,
Thanigaivel, & Senthil-Nathan, 2015; Senthil-Nathan, 2013, 2015). Use of plant extracts as larvicides and insect growth
regulators against A. aegypti have been reviewed by Pontual, Napoleao, Assis, & Ranilson (2012).
Justicia adhatoda L. is part of the Acanthaceae plant family, commonly known as adhatoda, pavettai, or vasaka (Adha-
toda vasica). The plant is a small evergreen, subherbaceous bush that grows commonly in open plains of India, Sri Lanka,
Burma, and Malaysia, especially in the lower Himalayas (up to 1,300 m above sea level). Adhatoda leaves have been
used extensively in ayurvedic medicine for over 2,000 years primarily for respiratory disorders (Jain et al., 2014). The
leaf extract is safe for human consumption and the oil has low mammalian toxicity. Leaf extracts have been used to
treat malaria, dysentery, and diarrhea (Chakraborty & Brantner, 2001). Mulla et al. (2010) showed that extracts have
potent anti-inflammatory activity, and A. vasica was traditionally used by midwives at the time of delivery because of
its uterotonic activity. The plant is suggested for first-aid medicine in primary health care in most developing countries
where it grows and has been used to treat adults and children over long periods of time (weeks to months) without
restriction (Chakraborty & Brantner, 2001).
Because of its potent biological activity, we posed the hypothesis that J. adhatoda leaf extracts exert strong negative
influences on several aspects of A. aegypti biology, including survival, oviposition, enzyme inhibition, and adult repel-
lency under laboratory conditions. Here, we report on the outcomes of experiments designed to test our hypothesis.

2 MATERIALS AND METHODS

2.1 Mosquito culture

The A. aegypti culture is maintained in the Biopesticides and Environmental Toxicology Laboratory (BET Lab), SPK Cen-
tre for Excellence in Environmental Sciences without exposure to pesticides. The mosquito larvae were fed with ground
dog biscuits and yeast at 3:1 ratio. The feeding was sustained until the larvae entered the pupal stage. The pupae were
collected from the culture trays and moved to plastic containers (9 × 9 cm2 ) containing 400 ml of water. The plastic
jars were kept in a 90 × 90 × 90 cm3 mosquito cage for adult emergence. Mosquito larvae were maintained at 27 ±
2°C, 75–85% relative humidity, under a photoperiod of 14-h light:10-h dark. A 10% sugar solution was provided to
newly emerged adults for 3 days before blood feeding. The adult females were allowed to blood feed on a chicken (one
chicken per day, exposed on the dorsal side) for 5 days. After blood feeding, enamel trays with water from the culture
trays were placed in the cage as oviposition substrates.

2.2 Collection and preparation of plant extracts

Justicia adhatoda leaves were collected from trees of natural forests in Kolli hills, Namakkal District, Tamil Nadu, India
and shade dried. They were mechanically crushed and extracted in a Soxhlet apparatus using methanol (60–80°C, AR
grade Worli Road, Mumbai, Maharashtra 400030, India) until exhaustion, which yielded a greenish yellow viscous pasty
mass (extract). Solvent from the extract was separated in a vacuum rotary evaporator under reduced pressure of 22–
26 mm Hg at 40°C and the concentrate was further evaporated to complete dryness at room temperature. The crude
residue was stored at 4°C in a brown bottle for GC-MS analysis and further toxicity test using the standard procedures
of WHO (2005).
THANIGAIVEL ET AL. 3 of 11

2.3 Leaf extract treatments

Selected concentrations of leaf extracts, indicated in Results, were mixed thoroughly with 200 ml rearing food in 250
ml glass jars. Ten larvae (fourth instar) were transferred into the experimental jars, where they fed on the J. adhatoda
extracts for 2 days prior to enzyme assays.

2.4 Preparation of whole body homogenates for enzyme assay

The treated and control fourth instar larvae were washed with double distilled water, and the adhering water was
removed from the surface by blotting with tissue paper. The larvae were separately homogenized in Eppendorf tubes
using a hand homogenizer in 500 𝜇l of ice-cold sodium phosphate buffer (20 mM, pH 7.0) to assess enzyme activity.
The homogenates were centrifuged (8,000 × g at 4°C) for 20 min and supernatants were used for the further analyses
(Napoleão et al., 2012). The final homogenates were held on ice until used in assays.

2.5 Carboxylesterase assays

The activity of 𝛼- and 𝛽-carboxylesterase was evaluated using the method of Agra-Neto et al. (2014). The larval extracts
(20 𝜇l; 84 𝜇g protein) in 0.1 M phosphate potassium buffer (pH 7.2) were prepared. The preparations were mixed with
500 𝜇l buffer (0.3 mM 𝛼- or 𝛽-naphthyl acetate in 0.1 M phosphate potassium at pH 7.2 containing 1 % acetone). The
reaction mixture was incubated for 20 min at 30°C, then 0.1 ml of a mixture containing 0.3 % Fast Blue B and 3.3 %
sodium dodecyl sulfate (SDS) was added. After centrifugation (3,000 × g, 28°C), the supernatant absorbance at 590 nm
was recorded. One unit of enzyme activity was defined as the amount of enzyme required to generate 1 𝜇mol of 𝛼- or
𝛽-naphthol per minute.

2.6 Superoxide dismutase activity

The SOD activity was carried out using SOD determination kit purchased from Sigma–Aldrich (Bangalore,
Karnataka, India). In the presence of superoxide anion, the water-soluble tetrazolium salt WST-1 [2-(4-iodophenyl)-
3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] reduced to a water soluble formazan dye,
highest absorbance at 440 nm. The amount of superoxide anion is linearly proportional to absorbance, the SOD activ-
ity was quantified following the decrease in the color development at 440 nm (inhibition of WST-1 reduction).
The assay used xanthine oxidase for generation of superoxide anion by oxidation of xanthine. The whole larval
extract (30 𝜇l; 72 𝜇g protein) in 0.05 M phosphate buffer pH 7.0 was mixed to a solution (30 𝜇l) containing xanthine
oxidase and its substrate, followed by addition of WST-1 solution (250 𝜇l). The assay was incubated for 3 min at 28°C,
and, thus, the absorbance at 440 nm was determined. One unit of SOD activity was defined as the amount of enzyme
required to inhibit the increase of absorbance at 440 nm by 50%.

2.7 Glutathione-S-transferase activity

Glutathione-S-transferase (GST) activity was assayed according to Larson, Lorch, Pridgeon, Becnel, and Clark (2010).
Fourth instars were homogenized in 250 𝜇l of 50 mM sodium phosphate buffer (pH 7.2), then centrifuged at 15,000 ×
g at 4ο C for 20 min. The Sigma–Aldrich (Catalog 0410) GST assay kit was used to evaluate the conjugation of the thiol
group of glutathione to the 1-chloro-2, 4-dinotrobenzene (CDNB) substrate. A total of 20 𝜇l of homogenate was added
to each well before 200 𝜇l of solution containing Dulbecco’s phosphate buffer (Sigma–Aldrich), glutathione reduced
(4 mM), and CDNB (2mM). Rodriguez, Bissett, Fernandez, Lauzan, & Soca (2001) determined the saturation concen-
tration of CDNB is 50 mM and the optimum time for reading is 3 min. The 96-well flat-bottom UV microplate (catalog
3635, Corning, Haryana) was immediately loaded onto a Synergy HT microplate reader (BioTek, Mumbai, Maharash-
tra, India). After a 1-min lag time, the absorbance was read at 340 nm and the samples were read each 60 sec for 3
min. Protein concentrations were verified using an albumin standard and bicinchoninic acid protein assay kit (Catalog
4 of 11 THANIGAIVEL ET AL.

TP0l00, Sigma–Aldrich). The activity of GST was expressed as micromolar per milligram protein per minute substrate
conjugated.

2.8 Cytochrome P450 activity

Larvae exposed to crude leaf extracts were cleaned in double distilled water and dissected. Larvae were placed in 40
mM sodium phosphate buffer (pH 7.2) and chilled before dissection. The heads, last abdominal segments, and diges-
tive tracts were isolated. Carcasses were used for determining the cytochrome P450 activity. The assays were based
on the measurement of ethoxycoumarin-O-deethylase activity in the body walls. The methods of Boyer, David, Rey,
Lemperiere, & Ravenel (2005) were modified to determine the effect on the cytochrome P450 activity. Black, round-
bottom 96-well microplates were filled with 100 𝜇l of a 0.4 mM 7-ethoxycoumarin solution containing 50 mM sodium
phosphate buffer (pH 7.2). Individual larval carcasses were placed in each well and incubated for 4 h at 30ο C. The reac-
tion was stopped with the addition of 50 𝜇l glycine buffer (1 mM, pH 10.4) and 50 𝜇l ethanol. Larval carcasses remained
at the bottom of the well and were not detached before reading. Six wells containing 100 𝜇l of phosphate buffer, 50 𝜇l
glycine buffer, and 50 𝜇l ethanol served as control. The fluorescence of the reaction medium was measured from the
top of the wells using a Synergy HT microplate reader (BioTek Instruments, India) with 400 nm excitation and 480 nm
emission filters. The production of 7-hydroxycoumarin (7-OH) was expressed as 𝜇mol 7-OH/mg larvae/min.

2.9 Oviposition deterrence index

Selected concentrations of J. adhatoda extracts, indicated in Section “Results,” were mixed thoroughly with 200 ml
rearing food in 250 ml glass jars. The gravid females were divided equally between treated and control jars. During the
tests, the groups of females were kept separate for 48 h in cages (25 × 25 × 30 cm). Post egg counts, the oviposition
deterrence index (ODI; Hwang, Schultz, Axelord, Krame, & Mulla, 1982) was calculated by using the formula (1):

Nt − Ns
ODI = × 100, (1)
Nt + Ns

where Nt is the total number of eggs in test solutions and Ns is the total number of eggs in control.

2.10 Fecundity
Adults were used in fecundity experiments, which developed from the control and experimental larvae exposed to indi-
cated concentrations of leaf extract. Sets of females (five females/set) were mated in cages of 30 × 30 × 30 cm3 at each
concentration. Three days post blood meal, eggs were collected daily from the water in small plastic containers (ovit-
raps) in the cages. The fecundity was calculated by the number of the eggs laid in the ovitraps divided by the number of
mated females. Adult mortality was also recorded.

2.11 Repellent bioassay

Adults which developed from the control and experimental larvae exposed to indicated concentrations of leaf extract
were tested for repellency at 25, 50, 75, and 100 ppm (World Health Organization [WHO], 2009). One hundred gravid
females (5–7 days post emergence, maintained on 10% sucrose solution) were starved for 24 h without a blood meal.
An area 3 × 10 cm2 on each forearm of three human volunteers was marked with a permanent marker, dried, and rinsed
once with water, then presented to caged adult females. The remaining skin area was covered with a paper sleeve.
The forearms had no contact with lotions or perfumed soaps on the day of the assay. As a blank control, methanol
was placed on one forearm of the same volunteer with the same process as the test repellents, whereas the other
forearm was untreated. The mosquitoes were tested during daytime from 08:00 to 16:00 h. The control and treated
arms were introduced simultaneously into the mosquito cage and the mosquitoes were activated by gently tapping
the sides of the cages. Each test concentration was repeated three times. The volunteers conducted their test for each
THANIGAIVEL ET AL. 5 of 11

concentration by inserting the treated and control arm into the cages at the same time for one full minute, every 5 min.
The mosquitoes that landed on the hand were recorded and then shaken off before blood imbibition. The percentage
of repellency was calculated by the following formula (2).

[ ]
% Repellency = (Ta − Tb) ∕Ta × 100, (2)

where Ta is the number of mosquitoes in the control group and Tb is the number of mosquitoes in the treated group.

2.12 Histological studies

The leaf extract treated and control larvae were fixed overnight in Bouin’s solution, dehydrated and mounted in paraffin
wax blocks. Larval tissue blocks were sectioned on a microtome (Leica, Germany), mounted on the glass slides, and
stained with hematoxylin and eosin for the examination under a bright field microscope (Senthil-Nathan, Hisham, &
Jayakumar, 2008). The sections were observed and photographed under light microscope (Optika vision lite 2.0 ML)
connected to a computer and midgut cells of the treated and untreated larvae were photographed. The site of action in
the midgut of treated larvae were observed and compared with control.

2.13 Statistical analysis

Data from mortality experiments were analyzed by analysis of variance (ANOVA of arcsine, logarithmic, and square-
root transformed percentages) and data were expressed as means of five replicates. Significant differences between

R
treatment groups were analyzed by Tukey’s multiple range test (significance at P < 0.05) using Minitab 17 program.
For enzyme activity, Microcal Software (Sigma plot 11) was used. The lethal concentrations required to kill 50% (LC50 )

R
of larvae in 24 h were calculated by Probit analysis with a dependability interval of 95% using the Minitab 17 program.

3 RESULTS

3.1 Enzyme assay

The J. adhatoda leaf extracts inhibited 𝛼-carboxylesterase activity of fourth instar larvae tested at 24 h. The activity
decreased over time and then stabilized with protein reduction of 0.62 log detected at the lowest treatment concen-
tration (2.5 ppm). Exposure to 100 pm led to protein reduction to 0.34 log (Figure 1a). Similarly, 𝛽 carboxylesterase
activity decreased significantly (R2 = 0.610) with 1.8 and 0.55 log protein reduction rates at 2.5 and 100 ppm, respec-
tively (Figure 1b).
The SOD activity was significantly decreased at 12.5 U/mg at 100 ppm and the inhibitory rate was minimum 29
U/mg at 2.5 ppm (R2 = 0.811; Figure 1c).
GST enzyme activity responded in a dose-dependent manner. The enzyme ratio significantly increased to 0.73
𝜇mol/mg/min at 100 ppm compared to 2.5 ppm, which remained at 0.36 𝜇mol/mg/min (R2 = 0.965; Figure 1d). Simi-
larly, cytochrome P450 enzyme activity increased significantly posttreatment. The enzyme activity was higher (11.25
𝜇mol/mg/min) at 100 ppm treatments compared to treatments at 2.5 ppm (5.98 𝜇mol 7-OH/mg larvae/min; R2 = 0.874;
Figure 1e).

3.2 Oviposition deterrence index

The ODI significantly increased at 100 ppm (F4,20 = 41.62; P < 0.0001; Figure 2). There was no difference at 10 ppm or
controls (P < 0.489).
 6 of 11

2.5 2.5 2.5

Control Control
5 ppm 40 5 ppm
a 10 ppm 10 ppm
2.0 25 ppm 2.0 25 ppm 2.0
50 ppm 50 ppm
30 a
1.5 ab 1.5 1.5
b
bc
20 b
1.0 1.0 a 1.0
c ab
a c bc

SOD activity (U/mg)

c
b c c
GST activity (µmol/mg/min)

0.5 b 0.5 10 d 0.5

c c

β -Carboxylestrase (mM/min/mg/protein)

α-Carboxylestrase (mM/min/mg/protein)
0.0 0.0 0 0.0
a b c d

Control
5 ppm
20 10 ppm
25 ppm
50 ppm
15 a

b
10 bc
bc

larvae/min)
c

CYP450 activity (µmol 7-OH/mg

0
e

F I G U R E 1 (a) 𝛼, (b) 𝛽 Carboxylestrase, (c) SOD, (d) GST (e) CYP450 enzyme activity of fourth instar larvae of Aedes aegypti after treatment with methanolic leaf extract of Justicia
adhatoda; the data were fitted on polynomial (regression) model, whereas vertical bars indicate standard error (±SEM)
THANIGAIVEL ET AL.
THANIGAIVEL ET AL. 7 of 11

Control
160 160

Number of eggs laid by the female

a 10 ppm

Deterrence index in percentage

20 ppm
140 140
50 ppm
120 75 ppm 120
100 a b 100
80 bc 80
c
60 b 60
c
40 c 40
d
20 d 20
0 0
Oviposition deterrence index Fecundity

F I G U R E 2 Oviposition deterrence index and number of eggs laid by the female Aedes aegypti after treatment with
Justicia adhatoda; mean (±SEM) followed by the same letter in the above bars indicate no significant difference (P<0.05)
in a Tukey’s test

100
Control
Treated

80
Survival (%)

60

40

20

0
10 20 30 40
Time (Days)

F I G U R E 3 Survival rate of Aedes aegypti treated with Justicia adhatoda; survivorship curves differ at the 𝛼 = 0.05
confidence interval according to log-rank statistics

3.3 Fecundity analysis

The leaf extract (100 ppm, but 10 ppm) reduced fecundity compared to controls (F4,20 = 29.74; P < 0.0001; F4,20 =
29.74; P < 0.642; Figure 3). Adult (𝜒 2 = 10.519, df = 1, P = 0.001) survivorship was significantly reduced posttreatment
at 100 ppm (Figure 4).

3.4 Repellent assay

Leaf extracts influenced female repellency in a dose-related manner. Significant differences obtained between 100
ppm (F6, 28 = 3.35, P < 0.013) from all other concentrations, 75 ppm (F6, 28 = 19.49, P < 0.0001), 50 ppm (F6, 28 = 8.09,
P < 0.0001), and 25 ppm (F6, 28 = 12.74, P < 0.0001; Figure 4). The efficacy of repellency decreased with time.

3.5 Gut histological study

The midgut histology examination revealed that cellular organelles were severely damaged 100 ppm (Figure 5b) rel-
ative to controls (Figure 5a). The peritrophic matrix (PM) was completely collapsed in the extract-treated larvae and
intact in control larvae.
8 of 11 THANIGAIVEL ET AL.

25 ppm
100 50 ppm
75 ppm

Repllencey in percentage (%)

100 ppm
90

80

70

60

50

40

0 30 60 90 120 150 180 210

Time in minutes

FIGURE 4 Repellency of methanolic leaf extract of Justicia adhatoda against Aedes aegypti

F I G U R E 5 Cross-section through midgut of fourth instar Aedes aegypti treated with Justicia adhatoda leaf extract
(a) treated compared with (b) control; (epi) vacuolated gut epithelium, (lu) gut lumen, (pm) peritrophic matrix

4 DISCUSSION

The data presented in this article strongly support our hypothesis that J. adhatoda leaf extracts exert strong nega-
tive influences on several aspects of A. aegypti biology. There are five elements of relevant data. First, dietary expo-
sure to the leaf extracts led to dose-dependent decreases in activities of 𝛼- and 𝛽-carboxyesterases and of SOD and
increases in GST and CYP450 activities. Second, exposure to the leaf extracts led to dose-dependent increases in
oviposition deterrence and to similar decreases in fecundity. Third, exposure to the extracts led to sharply decreased
survival, which approached zero within 15 days compared to about 30 days in control insects. Fourth, the presence
of leaf extracts on human arms repelled fasted adult females, with optimal repellency at about 50 ppm. Finally, larvae
exposed to dietary leaf extracts suffered substantial damage, including midgut epithelial cells and peritrophic matrix.
Taken together, these elements amount to very strong support of our hypothesis.
Many botanical insecticides and extracts affect insect metabolism by inhibiting or stimulating the activity of diges-
tive enzymes (Napoleao et al., 2012; Senthil-Nathan, 2007, 2013; Senthil-Nathan, Kalaivani, Choi, & Paik, 2009;
Senthil-Nathan, Kalaivani, Murugan, & Chung, 2005; Senthil-Nathan et al., 2006). Our results show the leaf extracts
THANIGAIVEL ET AL. 9 of 11

have antifeedant and toxic effects on Spodoptera littoralis larvae (Sadek, 2003). Saxena, Tikku, Atal, & Koul (1986)
reported that J. adhatoda leaf extracts had five alkaloids with bioactivity against four major insect pests. We infer that
one or more of these alkaloids exert the negative effects reported here on mosquito larvae.
Detoxifying enzymes are involved in the development of insecticide resistance (Jagadeshwaran & Vijayan, 2009;
Lumjuan et al., 2011). The activities of some detoxifying enzymes, 𝛼- and 𝛽- carboxylesterase and SOD were reduced,
while GST and CYP450 activity were increased in test larvae. SOD, which is expressed in many tissues, including
mosquito anal gills (Nivsarkar, Kumar, Laloraya, & Laloraya, 1991), catalyzes the dismutation of superoxide radical into
hydrogen peroxide. We found the leaf extract treatments led to reduced SOD activity, which may expose larvae to the
sequelae of increased oxidative damage to cells and cell components. We infer the J. adhatoda extract may operate at
several points in mosquito larvae, including reduced responses to cellular oxidative reactions. Among their other roles,
GSTs are detoxification enzymes in insects, acting in survival of insects exposed to xenobiotics and in metabolic resis-
tance to insecticides (Clark, 1990). GST and CYP450 activities decreased after leaf extract treatments. Although activ-
ities of some CYP450s are upregulated after exposure to plant allelochemicals (Caballero, López-Olguín, Ruiz, Ortego,
& Castañera, 2008), we interpret our data to show the leaf extracts exert differing influences on mosquito biochemical
defense mechanisms.
In the light of our findings that the leaf extracts influenced enzyme activities, oviposition, and repellency, it is not
surprising to see the extracts were strongly deleterious to larval survival. The idea that plant extracts may be devel-
oped into new mosquito management technologies heavily depends on increased mortality following exposure to the
extracts. We note the kinetics of larval mortality show survival decreased to less than 50% within 10 days’ exposure.
Seen from the perspective of crop protection, surviving more than 10 days of exposure would not indicate reasonable
outcomes. The situation is otherwise for mosquitoes because adult, rather than larval, mosquitoes are responsible for
disease transmission. We infer that severe reductions in larval survivorship are needed for a necessary basis for con-
tinued research into the influence of plant products on mosquito larvae.
The leaf extract acted as an oviposition repellent and/or deterrent and as a repellent on human skin. The efficacy of
the ovicide to act on the embryo inside the egg shell depends on its penetration and exposure period (Broadbent & Pree
1984; Grosscurt, 1977; Kuppusamy & Murugan, 2012). The oviposition deterrent properties against adult mosquitos
have been observed for various plant extracts (Autran et al., 2009) including Pelargonium citrosa, (Jeyabalan, Arul, &
Thangamathi, 2003) and Melia azedarach (Senthil-Nathan et al., 2006). Extracts of many plants have been studied as
mosquito repellents including Zanthoxylum limonella and Citrus aurantifolia (Das, Baruah, Talukdar, & Das, 2003; Katz,
Miller, & Hebert, 2008). We infer that insects sense and avoid chemicals with potentially harmful effects, as seen in
most organisms, including bacteria (Topp & Gallivan, 2007).

ACKNOWLEDGMENTS
This research was supported by Indian Council of Medical Research (ICMR), Government of India (grant no.
45/32/2013/BMS/TRM). Also authors would thank Dr. W. B. Hunter for his thorough review of the manuscript.

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How to cite this article: Thanigaivel A, Senthil-Nathan S, Vasantha-Srinivasan P, et al. Chemicals isolated
from Justicia adhatoda Linn reduce fitness of the mosquito, Aedes aegypti L Arch. Insect Biochem. Physiol.
2017;00:e21384. https://doi.org/10.1002/arch.21384