Larvicidal Efficacy of Jasminum sp.

(Oleaceae) Flower
Extracts against the Dengue and Chikungunya Vector
Aedes aegypti L. (Diptera: Culicidae)
Abstract

Dengue and chikungunya are transmitted by Aedes aegypti and for controlling these diseases, the vector mosquito has
to be controlled. Extensive use of synthetic and chemical insecticides has resulted in environmental hazards and also in
development of physiological resistance among vector mosquito species. Plant products are considered to be a potential
alternative approach as they are environmentally safe, target specific and biodegradable. In the present study, the
crude chloroform, methanol and aqueous flower extracts of Jasminum officinale, Jasminum auriculatum and Jasminum
grandiflorum were tested for the larvicidal efficacy against the third instar larvae of Aedes aegypti at concentrations of
62.5, 125, 250, 500, 1000, 2000, 4000 and 8000 mg/L. Mortality was recorded after 24 and 48 h. Amongst the extracts of
Jasminum species tested, the crude chloroform flower extract of Jasminum grandiflorum was found to be effective
showing 100% mortality at 1000 mg/L with LC50 value of 344.01 and 300.47 after 24 and 48 h respectively followed by
the crude methanolic flower extracts of Jasminum officinale and Jasminum auriculatum. Further investigations are
needed to elucidate the larvicidal activity of Jasminum grandiflorum crude chloroform flower extract against a wide
range of all stages of mosquito species and also the active ingredient(s) of the extract responsible for larvicidal activity
should be identified.

Keywords

Larvicidal efficacy; Aedes aegypti; Jasminum officinale; Jasminum auriculatum; Jasminum grandiflorum; Crude flower
extracts

Introduction

Human beings are being suffered from the menace of mosquitoes since time immemorial and it is believed that
mosquitoes are ranked as the most important pests causing human health concerns, since they are responsible for
transmission of dengue, dengue haemorrhagic fever, chikungunya, malaria, filarial fever and Japanese encephalitis that
cause severe public health problems [1]. Vector control is an important component in a disease control programmme. A
myriad of methods and strategies though available, may not contribute to the total control of vectors unless they are
used judiciously and in a sustained manner. The discovery and development of synthetic organic chemicals with
persistent residual action not only over shadow the use of herbal products against mosquitoes, but also become the
major weapon for mosquito control. But the extensive use of synthetic organic insecticides has resulted in
environmental hazards, ecological imbalance, harm to humans and animals and non target organisms being affected, in
addition to the physiological resistance of vectors [2]. This has necessitated the need for search and development of
environmental-safe, biodegradable and indigenous method for vector control. The flora of India has a rich aromatic
plant diversity with potential for development of natural insecticides for the control of mosquito and other pests [3].
Phytochemical insecticides have received much attention, in this regard, as they are considered to be more
environmentally biodegradable and considered safer than synthetic insecticides [4]. Therefore, the search for such
compounds has been directed extensively to the plant kingdom. Co-evolution has equipped plants with a plethora of
chemical defenses against insects. Aware of this effect, mankind has used plant parts or extracts to control
insects/mosquitoes since ancient time. Plant derived products have received increased attention from scientists as they
are a rich source of novel natural substances possessing insecticidal properties, safer to humans and ecosystem [5].
During the last decade, various studies on natural plant products against vector mosquito indicate them as possible
alternatives to chemical synthetic insecticides for mosquito control [6-13]. Therefore, in the present study, the crude
flower extracts of Jasminum officinale, Jasminum auriculatum and Jasminum grandiflorum were tested for the larvicidal
efficacy against the third instar larvae of Aedes aegypti.

Materials and Methods

Plant collection and extraction

Mature fresh flowers of Jasminum officinale, Jasminum auriculatum and Jasminum grandiflorum collected in and around
Chennai, Tamil Nadu, India were brought to the laboratory, shade dried at room temperature and powdered. Dried and
powdered flowers (1 kg) each was macerated with 3 L of chloroform, methanol and distilled water for a period of 96 h
each separately and filtered. The filtrate was then concentrated at reduced temperature on a rotary evaporator. The
crude chloroform, methanol and aqueous flower extracts of Jasminum officinale, Jasminum auriculatum and Jasminum
grandiflorum thus obtained were lyophilized and a stock solution of 1,00,000 mg/L prepared by adding adequate volume
of acetone was refrigerated at 40C until testing for bioassay
Test mosquitoes

Aedes immatures collected from various places in Chennai, Tamil Nadu, India were transported to the laboratory in
plastic containers. In the laboratory, the immature mosquitoes were transferred to enamel larval trays until adult
emergence. After emergence, the adult mosquitoes were identified upto species level and confirmed before rearing.
Cyclic generations of Aedes aegypti were maintained separately in two feet mosquito cages in an insectary. Mean room
temperature of 27 ± 2°C and a relative humidity of 70-80% were maintained in the insectary. The adult mosquitoes were
fed on ten per cent glucose solution. For continuous maintenance of mosquito colony, the adult female mosquitoes
were blood fed with laboratory reared albino mice. Ovitraps were placed inside the cages for egg laying. The eggs laid
were then transferred to enamel larval trays maintained in the larval rearing chamber. The larvae were fed with larval
food (dog biscuits and yeast in the ratio 3:1). The larvae on becoming pupae were collected, transferred to plastic bowls
and kept inside mosquito cage for adult emergence.

Larvicidal bioassay

Standard WHO [14] protocol with minor modifications was adopted for the study. The tests were conducted in glass
beakers. Aedes aegypti immature particularly early third instar larvae were obtained from laboratory colonized
mosquitoes of F1 generation. From the stock solutions, concentrations of 62.5, 125, 250, 500, 1000, 2000, 4000 and
8000 mg/L were prepared. Twenty healthy larvae were released into each 250 ml glass beaker containing 200 mL of
water and test concentration. Mortality was observed for 24 and 48 h after treatment. A total of three trials with three
replicates per trial for each concentration were carried out. Controls were run simultaneously. Treated control was
prepared by the addition of acetone to distilled water. Distilled water served as untreated control. The larval per cent
mortality was calculated and when control mortality ranged from 5-20% it was corrected using Abbott’s formula [15].
SPSS 11.5 version package was used for determination of LC50 and LC90 values [16]. One way ANOVA followed by
Tukey’s test was performed to determine the difference in larval mortality between concentrations.

Results

Results of the larvicidal effects of crude flower extracts of Jasminum species against Aedes aegypti are presented
in Tables 1 and 2. Among the plant species and extracts tested, the crude chloroform flower of Jasminum
grandiflorum was found to be effective followed by the crude methanol flower extracts of Jasminum officinale and
Jasminum auriculatum. One hundred per cent mortality was observed in Jasminum grandiflorum crude chloroform
flower extract at 1000 mg/L at 24 hours (Tables 3 and 4). The crude chloroform flower extract Jasminum grandiflorum
was found to be effective and promising with LC50 values of 344.01 and 300.47 mg/L after 24 and 48 h respectively
(Table 5).

Concentration (mg/L)

Plant species Solvents UC TC 62.5 125 250 500 1000 2000 4000 8000

0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 1.00 ±
Chloroform
0.00a 0.00a 0.00a 0.00a 0.00a 0.00a 0.00a 0.00a 0.00a 1.00b

Jasminum 0.00 ± 0.00 ± 0.33 ± 0.33 ± 0.66 ± 0.66 ± 0.66 ± 1.33 ± 3.66 ± 3.66 ±
Methanol
officinale 0.00a 0.00a 0.57a 0.57a 0.57a 0.57a 0.57a 1.15ab 1.15b 2.08b

0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.33 ± 0.66 ±
Aqueous
0.00a 0.00a 0.00a 0.00a 0.00a 0.00a 0.00a 0.00a 0.57a 1.15a

0.00 ± 0.00 ± 0.00 ± 0.33 ± 0.33 ± 0.66 ± 1.33 ± 1.66 ± 2.00 ± 2.33 ±
Chloroform
0.00a 0.00a 0.00a 0.57ab 0.57ab 0.57abc 0.57abc 1.52abc 0.00bc 0.57c

Jasminum 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 1.00 ± 4.00 ± 4.00 ±
Methanol
auriculatum 0.00a 0.00a 0.00a 0.00a 0.00a 0.00a 0.00a 1.00a 2.64b 1.00b

0.00 ± 0.00 ± 0.00 ± 0.66 ± 1.00 ± 1.33 ± 1.33 ± 1.66 ± 2.00 ± 2.00 ±
Aqueous
0.00a 0.00a 0.00a 0.57ab 1.00ab 0.57ab 0.57ab 0.57ab 1.00b 0.00b

20.00
0.00 ± 0.00 ± 6.66 ± 7.00 ± 7.33 ± 10.66 ± 20.00 ± 20.00 ± 20.00 ±
Chloroform ±
Jasminum 0.00a 0.00a 0.57b 1.00b 2.08b 2.08c 0.00d 0.00d 0.00d
0.00d
grandiflorum
0.00 ± 0.00 ± 0.00 ± 0.33 ± 0.33 ± 0.33 ± 0.66 ± 1.00 ± 6.33 ± 6.33 ±
Methanol
0.00a 0.00a 0.00a 0.57a 0.57a 0.57a 0.57a 1.00a 3.21b 0.57b
 Concentration (mg/L)

Plant species Solvents UC TC 62.5 125 250 500 1000 2000 4000 8000

0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 1.33 ± 1.66 ± 2.00 ± 2.33 ±
Aqueous
0.00a 0.00a 0.00a 0.00a 0.00a 0.00 0.57ab 0.57ab 1.00b 1.52b

UC: Untreated control; TC: Treated control. Values are mean of three replicates of three trials ± standard deviation.
Different superscript alphabets within the column indicate statistical significant difference in larval mortality between
concentrations at P<0.05 level by one way ANOVA followed by Tukey’s test.

Table 1: Larvicidal activity of crude flower extracts of Jasminum species against Aedes aegypti at 24 h.

Concentration (mg/L)

Plant species Solvents UC TC 62.5 125 250 500 1000 2000 4000 8000

0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.33 ± 0.33 ± 0.33 ± 0.33 ± 0.66 ± 2.00 ±
Chloroform
0.00a 0.00a 0.00a 0.00a 0.57a 0.57a 0.57a 0.57a 0.57ab 1.00b

20.00
Jasminum 0.00 ± 0.00 ± 0.33 ± 1.00 ± 1.00 ± 1.33 ± 1.33 ± 5.33 ± 19.66 ±
Methanol ±
officinale 0.00a 0.00a 0.57a 0.00a 1.00a 0.57a 1.15a 1.52b 0.57c
0.00c

0.00 ± 0.00 ± 1.00 ± 1.33 ± 1.66 ± 1.66 ± 2.00 ± 2.00 ± 2.00 ± 2.33 ±
Aqueous
0.00a 0.00a 1.00a 1.52a 1.15a 0.57a 0.00a 1.00a 1.00a 0.57a

0.00 ± 0.00 ± 0.66 ± 1.33 ± 1.66 ± 1.66 ± 2.00 ± 3.00 ± 4.00 ± 6.00 ±
Chloroform
0.00a 0.00a 0.57ab 0.57ab 0.57abc 0.57abc 1.00abc 1.00bc 1.00cd 2.00d

17.00
Jasminum 0.00 ± 0.00 ± 0.00 ± 0.33 ± 0.33 ± 0.33 ± 0.66 ± 2.33 ± 8.66 ±
Methanol ±
auriculatum 0.00a 0.00a 0.00ab 0.57abc 0.57bcd 0.57bcd 0.57bcd 0.57cd 3.78cd
1.00d

0.00 ± 0.00 ± 0.66 ± 1.66 ± 2.00 ± 2.33 ± 2.33 ± 2.66 ± 2.66 ± 3.66 ±
Aqueous
0.00a 0.00a 0.57ab 0.57abc 0.00bcd 0.57bcd 0.57bcd 0.57cd 1.15cd 0.57d

20.00
0.00 ± 0.00 ± 7.33 ± 8.66 ± 11.33 ± 11.66 ± 20.00 ± 20.00 ± 20.00 ±
Chloroform ±
0.00a 0.00a 0.57b 0.57bc 2.30bc 1.52c 0.00d 0.00d 0.00d
0.00d

Jasminum 15.33
0.00 ± 0.00 ± 1.33 ± 1.66 ± 2.66 ± 3.00 ± 3.33 ± 3.33 ± 7.66 ±
grandiflorum Methanol ±
0.00a 0.00a 0.57a 0.57a 0.57a 0.00ab 1.52ab 0.57ab 4.61b
1.52c

0.00 ± 0.00 ± 1.33 ± 1.66 ± 1.66 ± 1.66 ± 2.00 ± 2.66 ± 3.00 ± 3.66 ±
Aqueous
0.00a 0.00a 1.15ab 0.57abc 0.57abc 0.57abc 0.00abc 0.57bc 1.00bc 1.52c

UC: Untreated control; TC: Treated control. Values are mean of three replicates of three trials ± standard deviation.
Different superscript alphabets within the column indicate statistical significant difference in larval mortality between
concentrations at P<0.05 level by one way ANOVA followed by Tukey’s test.

Table 2: Larvicidal activity of crude flower extracts of Jasminum species against Aedes aegypti at 48 h.

Larval mortality (%)

Concentration (mg/L)

Plant species Solvents UC TC 62.5 125 250 500 1000 2000 4000 8000

Chloroform 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5.00

Jasminum officinale Methanol 0.00 0.00 1.65 1.65 3.30 3.30 3.30 6.65 18.30 18.30

Aqueous 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.65 3.30
 Larval mortality (%)

Concentration (mg/L)

Plant species Solvents UC TC 62.5 125 250 500 1000 2000 4000 8000

Chloroform 0.00 0.00 0.00 1.65 1.65 3.30 6.65 8.30 10.00 11.65
Jasminum
Methanol 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5.00 20.00 20.00
auriculatum
Aqueous 0.00 0.00 0.00 3.30 3.00 6.65 6.65 8.30 10.00 10.00

Chloroform 0.00 0.00 33.30 35.00 36.65 53.30 100.00 100.00 100.00 100.00
Jasminum
Methanol 0.00 0.00 0.00 1.65 1.65 1.65 3.30 5.00 31.65 31.65
grandiflorum
Aqueous 0.00 0.00 0.00 0.00 0.00 0.00 6.65 8.30 10.00 11.65

Table 3: Per cent larval mortality of Aedes aegypti against crude flower extracts of Jasminum species at 24 h.

Larval mortality (%)

Concentration (mg/L)

Plant species Solvents UC TC 62.5 125 250 500 1000 2000 4000 8000

Chloroform 0.00 0.00 0.00 0.00 1.65 1.65 1.65 1.65 3.30 10.00

Jasminum officinale Methanol 0.00 0.00 1.65 5.00 5.00 6.65 6.65 26.65 98.30 100.00

Aqueous 0.00 0.00 5.00 6.65 8.30 8.30 10.00 10.00 10.00 11.65

Chloroform 0.00 0.00 3.30 6.65 8.30 8.30 10.00 15.00 20.00 30.00
Jasminum
Methanol 0.00 0.00 0.00 1.65 1.65 1.65 3.30 11.65 43.30 85.00
auriculatum
Aqueous 0.00 0.00 3.30 8.30 10.00 11.65 11.65 13.30 13.30 18.30

Chloroform 0.00 0.00 36.65 43.30 56.65 58.30 100.00 100.00 100.00 100.00
Jasminum
Methanol 0.00 0.00 6.65 8.30 13.30 15.00 16.65 16.65 38.30 76.65
grandiflorum
Aqueous 0.00 0.00 6.65 8.30 8.30 8.30 10.00 13.30 15.00 18.30

Table 4: Per cent larval mortality of Aedes aegypti against crude flower extracts of Jasminum species at 48 h.

LC50 (mg/L) LC90 (mg/L)

Plant species Solvents 24h 48h 24h 48h

Chloroform 16537.93 11439.80 25195.10 14126.81

Jasminum officinale Methanol 12652.04 2339.06 21055.59 3738.17

Aqueous 30135.97 17013.18 55517.87 23589.38

Chloroform 17045.78 11001.98 28289.38 20180.45

Jasminum auriculatum Methanol 10593.40 5044.02 16353.63 8019.21

Aqueous 22427.85 18829.36 38694.80 35563.24

Chloroform 344.01 300.47 762.68 761.07

Jasminum grandiflorum Methanol 8880.63 5203.02 14467.53 9963.65

Aqueous 17839.42 15031.87 33425.77 24108.36

Table 5: Probit analysis of crude flower extracts of Jasminum species against Aedes aegypti.
Discussion

Vector control is facing a threat due to the emergence of resistance in vector mosquitoes to conventional synthetic
insecticides, warranting counter measures such as developmental of novel insecticides [17]. Mosquitoes in the larval
stage are attractive targets for pesticides because mosquitoes breed in water, and thus, it is easy to deal with them in
this habitat. Mosquito control approaches based on synthetic insecticides have created many problems like insecticide
resistance [18]. Natural products of plant origin with insecticidal properties have been tried in the recent past in order to
control a variety of insect pests and vectors. This has necessitated the need for a research and development of
environmentally safe, biodegradable indigenous method for vector control. Many researchers have reported on the
effectiveness of plant extract against mosquito larvae [19-21]. Phytoextracts are emerging as potential mosquito control
agents, and can be used successfully in mosquito management programmes [22]. The results of the present study
indicate the crude chloroform extract of Jasminum grandiflorum flowers to possess larvicidal activityagainst Aedes
aegypti.

The results of the present study corroborate with earlier reports of plant extracts tested against the larvae of Aedes
aegypti viz., the dichloromethane extract of aerial parts of Pterocaulon polystachium (LC50 149.2 ppm) [23], ethyl
acetate leaf extract of Sphaeranthus indicus (LC50 201.11 ppm) [24], hexane leaf extract of Abutilon
indicum (LC50 261.31 ppm) [25], ethyl acetate leaf extract of Leucas aspera (LC50 483.21 ppm) [26]. Kamaraj et al. [27]
have reported larvicidal efficacy of Cassia auriculata flower methanol extracts against the larvae
of Anopheles subpictus and Culex tritaeniorhynchus. Mathew et al. [28] reported the chloroform extract of Nyctanthes
arbortristis leaves to possess larvicidal activity against the Aedes aegypti with LC50 value of 526.3 ppm and its flower
methanol extracts with 679.4 ppm. The chloroform extract of Orthosiphon thymiflorus exhibited larvicidal activity with
LC50 value of 197.91 ppm against Aedes aegypti [29]. The chloroform leaf extract of Acalypha alnifolia when tested
against Aedes aegypti showed larvicidal activity with LC50 value of 182.58 ppm [30].

The preliminary screening of plant extracts against mosquitoes is a good means of evaluating the
potential mosquitocidal property present in it [13,31]. Natural insecticides of plant origin have been given importance
due to their ecofriendly nature and biodegradability as a substitute of synthetic insecticides for the control of vectors of
public health importance. Plants are the chemical factories and rich source of bioactive chemicals, some of which
have medicinal and pesticidal properties [32]. Different types of phytochemicals of plant either from the whole part or
from the specific parts come out with solvent during chemical extraction depending on the polarity of the solvent [33-
35]. The botanical extracts from the plant leaves, roots, seeds, flowers and bark in their crude form have been used as
conventional insecticides for centuries. The complex mixtures of phytocompounds can be used to develop
environmentally-safe vector and pest-managing agents. In conclusion, the results reported in the present study open the
possibility for further investigations of the efficacy of larvicidal properties of the crude chloroform extracts of Jasminum
grandifloruma against Aedes aegypti as a potential agent for combating mosquitoes. Further investigations are needed
to elucidate this activity against a wide range of all stages of mosquito species and also to identify the active
ingredient(s) of the extract responsible for larvicidal activity.

The larvicidal effects of black pepper (Piper nigrum L.) and piperine against insecticide resistant and susceptible strains
of Anopheles malaria vector mosquitoes

Abstract

Background

Insecticide resistance carries the potential to undermine the efficacy of insecticide based malaria vector control
strategies. Therefore, there is an urgent need for new insecticidal compounds. Black pepper (dried fruit from the
vine, Piper nigrum), used as a food additive and spice, and its principal alkaloid piperine, have previously been shown to
have larvicidal properties. The aim of this study was to investigate the larvicidal effects of ground black pepper and
piperine against third and fourth instar Anopheles larvae drawn from several laboratory-reared insecticide resistant and
susceptible strains of Anopheles arabiensis, An. coluzzii, An. gambiae, An. quadriannulatus and An. funestus.

Methods

Larvae were fed with mixtures of standard larval food and either ground black pepper or piperine in different
proportions. Mortality was recorded 24 h after black pepper and 48 h after piperine were applied to the larval bowls.

Results

Black pepper and piperine mixtures caused high mortality in the An. gambiae complex strains, with black pepper proving
significantly more toxic than piperine. The An. funestus strains were substantially less sensitive to black pepper and
piperine which may reflect a marked difference in the feeding habits of this species compared to that of the Gambiae
complex or a difference in food metabolism as a consequence of differences in breeding habitat between species.
Conclusions

Insecticide resistant and susceptible strains by species proved equally susceptible to black pepper and piperine. It is
concluded that black pepper shows potential as a larvicide for the control of certain malaria vector species.

Background

Malaria is responsible for high levels of morbidity and mortality globally, with particular severity in sub-Saharan Africa
[1]. This region is home to several of the most efficient malaria vectors, the principal species being Anopheles
gambiae, An. coluzzii (formerly An. gambiae M form [2]), An. arabiensis and An. funestus. The former three species are
members of the Anopheles gambiae species complex and the latter is the nominal member and only major vector of
the Anopheles funestus species group [3, 4].

Insecticide based vector control strategies primarily directed against adult mosquitoes have significantly decreased
malaria incidence over the past decade, with indoor residual spraying of insecticides (IRS) and distribution of long-lasting
insecticide-treated bednets (LLINs) being pivotal [1]. Additionally, and especially in light of the increasing incidence of
insecticide resistance in target vector populations [5], larval source management (LSM) is playing an increasingly
important role in vector control [6].

Larval source management as a vector control technique has dramatically decreased since the shift toward the use of
synthetic mosquito adulticides, especially DDT and later pyrethroids – this despite distinct successes achieved by
larviciding in the early-to-mid 1900’s [7]. LSM is currently only recognized as supplementary to the core interventions of
IRS and LLINs by the World Health Organization (WHO), being employed in only 38 of the 97 countries with ongoing
malaria transmission [1]. Nevertheless, the WHO encourages the use of LSM where conditions are appropriate and it is
feasible to do so [8] and recent evidence, though sparse, does suggest that larviciding may have a reinvigorated,
valuable role to play in integrated vector management [7, 9, 10]. It is for these reasons that investigations into potential
options for use in larviciding are ongoing.

A tendency towards naturally occurring insecticides, particularly those of botanical origin, has gained increasing interest
in recent years. Plants have co-evolved with insects over many centuries, as have their defence mechanisms in response
to insect predation. Plant allelochemicals can be very advantageous as insecticidal actives compared to their synthetic
counterparts as they are biodegradable and in many instances show reduced or no adverse effects on non-target
organisms [11].

Piper nigrum L. (the fruit of which are used to produce white and black pepper) has a plethora of traditional and modern
day applications ranging from a US FDA recognized food additive to potential as an insecticide [12]. While assessing
spreading agents for Metarhizium anisopliae and Beauveria bassiana entomopathogenic fungal spores, Bukhari et al.
[13] observed 100 % mortality of Anopheles larvae exposed to white pepper, even in the absence of the fungal spores.
This has prompted an interest in the use of pepper as a possible larvicide for use in malaria vector control. In addition,
piperine, which is the principle alkaloid responsible for the pungency of pepper, has demonstrated toxicity against
fourth-instar larvae of the dengue and yellow fever vector Aedes aegypti [14, 15].

The aim of this study was to investigate the toxicity of P. nigrum (black pepper) and piperine when administered as a
food source to larvae of several insecticide resistant and susceptible strains of Anopheles species including An.
arabiensis, An. coluzzii, An. gambiae, An. quadriannulatus and An. funestus.

Methods

Mosquito strains

All of the mosquito strains used in this study are housed in the Botha De Meillon Insectary (BMDI) at the National
Institute for Communicable Diseases (NICD) in Johannesburg. All larvae were reared and bioassays conducted under the
standard insectary conditions [16]. Table 1 gives detailed information on each of the strains used.

Table 1 Anopheles species by laboratory strain and insecticide susceptibility profile fed on powdered formulations of
either Piper nigrum (black pepper) or piperine at the larval stage

Technical materials

Commercial ground black pepper was further homogenized in a Qiagen Tissue Lyser II, for 5 min, until a fine powdery
consistency was achieved. Analytical grade piperine in powder form (expiry date: December 2016) was purchased from
Sigma Aldrich (Saint Louis, MO). The technical materials were each proportionately mixed with standard larval food [16]
to obtain treatment mixtures ranging from 0 % pepper/piperine (control) to 100 % pepper/piperine (Table 2).

Table 2 Proportions of black pepper (Piper nigrum) or piperine to standard larval food used as treatment dosages
for Anopheles larval toxicity bioassays
Larval toxicity bioassays

Fifty third to fourth instar larvae from each Anopheles strain were gently introduced into plastic containers containing
500 ml distilled water (surface area = 150 mm × 215 mm). Fifty milligram (1 mg treatment mixture/larva) of each
treatment mixture, in addition to a control composed entirely of standard larval food, was tapped gently into each
container to allow for an even spread over the surface of the water. Mortality was recorded 24 h after the application of
the black pepper mixtures and 48 h after the piperine mixtures (little or no mortality was observed following 24 h larval
exposure to piperine). At the end of the period, larvae were gently prodded and non-responsive larvae were recorded as
dead [17]. Bioassays for each treatment mixture were replicated three times per strain.

Statistical analysis

Mortality was corrected using Abbott’s formula in replicates where control mortality exceeded 10 %. One-way ANOVA
and Tukey HSD post-hoc tests were used to determine (i) if the mortality in treated bioassays significantly differed from
that of the controls and at which doses in particular; (ii) if there were significant differences in response between
insecticide susceptible and resistant strains by species where pertinent; and (iii) if there were significant differences in
response between species of the Gambiae complex and Funestus group. For the latter, analysis excluded control
mortalities from the data. A Student’s t-test was used to determine if there were significant differences in response
between intoxication with black pepper and piperine. All statistics were conducted in IBM SPSS Statistics v22 with
significance set at 95 % confidence.

Results

For this study “dose”, which is represented by a percentage, refers to the proportion (Table 2) of black pepper or
piperine constituting 50 mg of treatment mixture administered to larvae.

Black pepper

Black pepper, when applied to the water, tended to spread over the surface quite evenly and effectively. Mortality was
induced in all species and strains by all treatments containing pepper (Fig. 1a–d). In general, mortality increased with
increasing proportions of pepper in the treatment mixture. Furthermore, the presence of pepper (even in low
quantities) consistently induced significantly higher mortalities in the An. gambiae complex larvae than in An.
funestus (Table 3). Dead An. gambiae complex larvae tended to cluster, pivoting around their tracheal gills (Fig. 2).

Fig. 1

Mean mortalities of laboratory-reared Anopheles larvae 24 h after being fed powdered black pepper (Piper nigrum) at
various concentrations. The dose value represents the percentage of black pepper in a 50 mg treatment mixture. 0 %
represents the control group which was fed standard larval food only and 100 % represents a treatment comprised of
black pepper only. A One-way ANOVA and Tukey HSD post-hoc comparisons were used for each strain to determine
significant differences in mortality at each dose compared to that of the relevant control at 95 %
confidence. a Anopheles arabiensis strains. b Anopheles gambiae (GAH) and An. coluzzii (SILC & SUA)
strains. c Anopheles quadriannulatus. d Anopheles funestus strains

Table 3 Tukey HSD post-hoc analysis of Anopheles larval mean mortalities 24 h post-exposure to black pepper (P.
nigrum)
Fig. 2

Clustering of dead Anopheles quadriannulatus larvae 24 h after being fed a powdered black pepper treatment mixture.
Similar clustering was observed among all strains of all An. gambiae complex species tested

Anopheles arabiensis

The treatment dose of 100 % black pepper achieved upwards of 98 % mortality in all three of the An. arabiensis strains
and all of the treatment doses induced significantly higher mortality than the control (0 % black pepper) for KGB
(ANOVA: P < 0.01; F = 211.51 at df = 17), SENN (ANOVA: P < 0.01; F = 62.05 at df = 17) and SENN-DDT
(ANOVA: P < 0.01; F = 148.30 at df = 17) (Fig. 1a). There were no significant differences in mean mortality between SENN-
DDT and either KGB or SENN (Table 3).

Anopheles gambiae and An. coluzzii

With the exception of GAH exposed to 10 % black pepper, all of the treatment mixtures achieved significantly higher
mortality against larvae of both GAH (An. gambiae) (ANOVA: P < 0.01; F = 21.31 at df = 17) and the An. coluzzii strains
SILC (ANOVA: P < 0.01; F = 86.25 at df = 17) and SUA (ANOVA: P < 0.01; F = 98.61 at df  = 17) (Fig. 1b). When exposed to
just the black pepper treatment (100 %), the mean mortalities of the respective strains each exceeded 95 %; even the
lowest treatment dose (10 %), with the exception of GAH at this dose, showed upwards of 70 % mean mortality. There
were no significant differences in the mean mortalities induced in GAH versus those of either SILC or SUA (Table 3).

Anopheles quadriannulatus

The SANGWE strain was particularly susceptible to black pepper. Significantly higher mean mortalities were achieved in
all pepper-inclusive treatments compared to the control, with approximately 90 % mortality observed even at the lowest
pepper dose (ANOVA: P < 0.01; F = 145.06 at df  = 17) (Fig. 1c).

Anopheles funestus

Other than the mortality of FANG exposed to the 100 % dose, which was less than in the 40 and 80 % doses, mortalities
among An. funestus larvae generally increased with increasing proportions of black pepper. Generally, ingestion of black
pepper did not cause more than 50 % mortality in either of the An. funestus strains. There was, however, significantly
higher mortality among FANG larvae in the 40–100 % treatment doses than in the control (ANOVA: P < 0.01; F = 6.70
at df  = 17) (Fig. 1d). For FUMOZ-R, although one-way ANOVA indicated a significant difference in mortality between the
doses (P < 0.05; F = 3.51 at df  = 17), none of the treatment mortalities differed significantly from the control. This is
likely due to the wide variation in response to pepper ingestion (Fig. 1d). While this suggests that FUMOZ-R is less
susceptible to black pepper than FANG, this was found to be statistically insignificant (Table 3).

Piperine

Unlike black pepper, the powdered piperine did not spread effectively and if not scattered carefully over the surface
area of water, it tended to clump. The larval clustering observed in the black pepper experiments did not occur in the
piperine trials. All mortality results were recorded 48 h post-feeding with piperine (Fig. 3). The mortalities induced in
SENN, SENN-DDT and GAH did not significantly differ from the mortalities in the An. funestus larvae, probably owing to
high levels of variation around the means, whereas the mortalities in the other Gambiae complex strains were
significantly higher than those recorded in the An. funestus strains (Table 4).

Fig. 3
Mean mortalities of laboratory-reared Anopheles larvae 48 h after being fed powdered piperine. The dose value
represents the percentage of piperine in a 50 mg treatment mixture. 0 % represents the control group which was fed
standard larval food only and 100 % represents a treatment comprised of piperine only. A One-way ANOVA and Tukey
HSD post-hoc comparisons were used for each strain to determine significant differences in mortality at each dose
compared to that of the relevant control at 95 % confidence. a Anopheles arabiensis strains. b Anopheles
gambiae (GAH& TONGS) and An. coluzzii (SUA) strains. c Anopheles quadriannulatus. d Anopheles funestus strains

Table 4 Tukey HSD post-hoc analysis of Anopheles larval mean mortalities 48 h post-exposure to piperine

Anopheles arabiensis

All of the treatment mixtures including and exceeding 40 % piperine induced significantly higher mortality than the
control in KGB (ANOVA: P < 0.01; F = 6.54 at df  = 17), SENN (ANOVA: P < 0.01; F = 33.77 at df  = 17) and SENN-DDT
larvae (ANOVA: P < 0.01; F = 46.63 at df  = 17) (Fig. 3a). However, the significantly higher mortalities achieved ranged
from approximately 20–95 % among the strains; and while this was seemingly indicative of the strains having
significantly different responses from each other, ANOVA showed otherwise for both KGB versus SENN-DDT and
SENN versus SENN-DDT (Table 4).

Anopheles gambiae and An. coluzzii

In GAH, only the 80 and 100 % doses induced significantly higher larval mortality than the control (One-way
ANOVA: P < 0.01; F = 7.95 at df  = 17) and in both TONGS (ANOVA: P < 0.01; F = 40.05 at df  = 17) and SUA
(ANOVA: P < 0.01; F = 108.22 at df  = 17), all dosages greater than and equal to 40 % piperine induced significantly higher
mortalities than the control (Fig. 3b). There were no significant differences in mortality induced by piperine between
either GAH and TONGS or GAH and SUA (Table 4).

Anopheles quadriannulatus

SANGWE larvae were more susceptible to the piperine than other An. gambiae complex strains (Table 4). Up to 100 %
mortality was induced in larvae exposed to 100 % piperine and even 10 % piperine induced more than 60 % mortality. All
of the piperine-inclusive doses induced significantly higher mortality than the control ( ANOVA: P < 0.01; F = 292.01
at df  = 17) (Fig. 3c).

Anopheles funestus

FANG larvae only showed significantly higher mortalities than that of the control in the 40 and 100 % piperine dosages
(ANOVA: P < 0.01; F = 5.14 at df  = 17). However, the highest mean mortality, which was induced by 100 % piperine, was
only 22 %. The mortalities induced by piperine in FUMOZ-R larvae were significantly higher than that of the control in all
dosages exceeding 10 % piperine (ANOVA: P < 0.01; F = 17.88 at df  = 17) although the highest mean mortality achieved
was low at only 24 % using 100 % piperine (Fig. 3d). There was no significant difference in the mortality induced by
piperine between FANG and FUMOZ-R (Table 4).

Black pepper versus piperine

The SILC and TONGS strains were excluded from the statistical analyses comparing mortalities induced by black pepper
versus piperine as neither were exposed to both. Mortality among the An. gambiae complex strains 24 h after being fed
black pepper was significantly higher than that induced by piperine 48 h post-feeding (Student’s t-test: P < 0.01; t = 8.34
at df  = 178). Although it appeared that black pepper induced higher mortality than piperine in the An. funestus strains
as well, the difference is not statistically significant at 95 % confidence (Student’s t-test: P = 0.07; t = 1.87 at df  = 58).

Discussion

Prior to this study, Anopheles larval toxicity caused by the ingestion of either black pepper or piperine had not been
explicitly investigated. To date, the evaluations of the insecticidal activity of P. nigrum has been focussed mainly on the
constituent alkaloids and their effects on a variety of insect species ranging from those of economic importance [18, 19]
to disease vectors such as Aedes aegytpi, An. stephensi and Culex quinquefasciatus [20, 21]. Of the compounds
comprising P. nigrum, piperine is the most recognized and is abundant in fully differentiated shoots of the plant [22, 23].

Commercial, ground black pepper and piperine showed marked toxic effects against late instar larvae of all the An.
gambiae complex strains tested. Piperine ingestion was evidently less toxic to Anopheles larvae than black pepper, but
did elicit larval mortality over 48 h and may serve as a synergist amongst the various amides constituting black pepper.
Black pepper and piperine induced mortality in insecticide susceptible and resistant An. gambiae complex strains with no
statistically significant differences between them. This suggests that insecticide resistance mechanisms play no role in
the detoxification of black pepper and piperine in these species.

The An. funestus strains, however, showed markedly lower sensitivities to black pepper and piperine. This may reflect
differences in their feeding habits and metabolism. Alternatively, their larval rearing conditions, which partially reflect
the characteristics of their natural breeding sites, may have influenced their relative sensitivities. This is because the
FANG and FUMOZ-R An. funestus strains are reared in water containing algae obtained from a nearby fish pond whereas
the An. gambiae complex larvae are reared in water containing no additives other than larval food. However, it is not
clear as to how or why this may have affected their responses to black pepper and piperine. One possibility is that food
sources in the algae reduced their propensity to feed on the pepper/piperine treatments, raising the question as to what
effect competing multiple food sources would have on the efficacy of such treatments in natural breeding sites.

Larviciding is one of four types of larval source management strategies, the others being habitat modification, habitat
manipulation and biological control. It is recommended mainly in areas where larval habitats are few, fixed and findable
[6]. Where implemented, LSM serves as a useful supplementary vector control measure, targeting mosquitoes at a stage
of their life-cycle where their mobility is limited to the body of water they inhabit. Currently employed larvicides must
comply with stringent criteria established by the WHO Pesticide Evaluation Scheme (WHOPES). These include
assessments of how hazardous they are to humans and the environment, their storage requirements and shelf life, the
susceptibility of local vectors and the associated costs of utilizing them [6]. In terms of these standards, plant-derived
products such as pepper and piperine may be particularly advantageous, depending on their efficacy and residual
activity under field conditions. These data give no indication of the persistence of ground black pepper and piperine over
time but it is envisaged that any pepper or piperine based control products would need to be formulated for increased
persistence. Formulated products would also need to ensure adequate dispersal across a water surface, especially in the
case of piperine which tended to clump.

A potential advantage of utilizing piperamides for larval control is that they act as neurotoxins, but in a manner distinct
from pyrethroids, and so represent a novel mode of action [22], something urgently required for alternative methods of
vector control [24]. They also have an inhibitory effect on enzymes and have shown synergistic effects when used in
conjunction with pyrethrum [25]. They have low mammalian toxicity and are not environmentally persistent, degrading
quickly under full sunlight [18, 26].

Conclusions

Piper nigrum in the form of finely ground black pepper is highly toxic to members of the An. gambiae species complex
including An. gambiae (sensu stricto), An. coluzzii, An. arabiensis and An. quadriannulatus. It appears markedly less toxic
to An. funestus. Similarly, piperine, proved substantially more toxic to members of the An. gambiae species complex
than to An. funestus, although piperine on the whole was less toxic than black pepper across all species and strains
tested. There were no differences in response between insecticide-resistant and their corresponding insecticide-
susceptible strains by species to intoxication by either black pepper or piperine, suggesting that insecticide resistance
mechanisms have no effect on their relative susceptibilities to these compounds/derivatives. An appropriately
formulated black pepper or derivative product may have potential as a larvicide for malaria vector control in settings
where larval source management adds benefit under the auspices of integrated vector control. Assessments of the other
piperamides present in black pepper may provide further insights into the larvicidal effects of this plant and its
derivatives.
Larvicidal activity of leaf extracts and seselin from Clausena
anisata (Rutaceae) against Aedes aegypti
Abstract

The Aedes aegypti mosquito is a vector of various diseases in both humans and livestock. Mosquito control focuses on
reducing the longevity as well as the population of mosquitoes to lessen their damage on human and animal health. It
entails several strategies such as environmental management, insecticide treatments, and molecular entomological
approaches. Environmental management centres on elimination of breeding sites, however mosquitoes can breed in
sites that cannot be eliminated. Resultantly, focus is turned onto mosquito larvae control. The objective of this study
was to evaluate the larvicidal activity of extracts and compounds from Clausena anisata against A. aegypti. The World
Health Organization guidelines for testing of mosquito larvicides were used. The acetone, dichloromethane and hexane
crude leaf extracts were evaluated in a preliminary screening for larvicidal activity at the concentrations of 12.5, 25, 50,
100 and 200 ppm. Batches of 25 third-instar larvae were transferred into cups each containing test solutions and larval
mortality was recorded 24 h and 48 h after exposure. Acetone was used as the solvent control whilst permethrin was
used as a positive control. Only the n-hexane extract caused mortality at the tested concentrations, thus it was further
tested at 40, 60, 80, 100, and 120 ppm and had LC50 values of 68.30 and 59.65 ppm after 24 h and 48 h respectively. A
stored hexane extract, of 2 months, was also evaluated under simulated field conditions to establish stability of extract.
It caused about 90% mortality when tested at 100 ppm. The n-hexane extract was subjected to open column
chromatography on silica gel to isolate the active compound. The isolated compound was identified as the
pyranocoumarin, seselin. Dose dependent mortality was observed in the larvae exposed to seselin. The LC50 values at
24 and 48 h were 13.90 and 9.96 ppm respectively. Results obtained from this study indicate a potential of the
incorporation of C. anisata extracts into the control of mosquito populations.

Keywords

Mosquito control

Biopesticides

Natural products

1. Introduction

The Aedes aegypti mosquito (Diptera: Culicidae) has been implicated in the mechanical transmission of various diseases
in livestock such as Rift Valley fever (Hoch et al., 1985), lumpy skin (Chihota et al., 2001) and anthrax (Turell and
Knudson, 1987). All these diseases are on the OIE, 2014 list of notifiable diseases. This list comprises of transmissible
diseases that have the potential for very serious and rapid spread, irrespective of national borders, that are of
serious socio-economic or public health consequence and that are of major importance in the international trade of
animals and animal product (OIE, 2014). Epidemiological evidence indicates that the incidence of these diseases is
highest during wet periods coinciding with periods of mosquito abundance and wanes with the onset of the dry season
(Magori-Cohen et al., 2012, Caminade et al., 2014). Although protection of livestock from these diseases can be achieved
by vaccination, vaccines and veterinary personnel are not always easily accessible in resource poor-communities. As
such other preventative methods such as minimising mosquito populations on farms are of paramount importance. A.
aegypti is also a principal vector of viruses that pose a threat to human health such as dengue, chikungunya, and yellow
fever viruses (Gubler, 1998). Dengue fever is regarded globally as the most important arthropod-borne viral disease. It is
endemic in at least 100 countries in Asia, the Pacific, the Americas, Africa, and the Caribbean, occurring every year
during a season when Aedes mosquito populations are high. About 50% of the world's population lives in areas where
there is a risk of dengue transmission (Murray et al., 2013). Thus the importance of controlling A. aegypti mosquito
populations cannot be over-emphasized.

Control of mosquito populations involves preventing wastewater that stands for longer than 4 days; keeping weeds
down around ponds, in ditches, and in shallow wetlands and irrigating properly so that all surface water is gone within
4 days (Lawler and Lanzaro, 2005). Mosquito larval control becomes important in cases where mosquito breeding sites
cannot be eliminated such as the drinking ponds, watering troughs and hoof prints. Mosquito control at larval stage has
the advantage of controlling the vector before it can acquire and transmit the disease.

Mosquito larvae can be controlled by substances added directly to the water. These substances may be organisms that
consume them such as larvivorous fish (Chandra et al., 2013), biological compounds that poison them or cause fatal
infections that are specific to them (Bacillus thuringiensis israelensis) (Ramírez-Lepe and Ramírez-Suero, 2012),
chemicals that disrupt their development or physiology such as the insect growth regulator, methoprene (Lawler and
Lanzaro, 2005, Karunamoorthi, 2011), or oils and films that suffocate them (Bukhari et al., 2011). Biological compounds
tend to be more expensive than chemical controls but they affect fewer non-target organisms (Gillette, 1988). Chemical
controls are typically very effective against mosquitoes but they are toxic to non-target organisms and inaccessible due
to high cost and limited availability in nearby markets, particularly in resource-poor communities. In addition, in cases
where the chemicals are continuously used mosquitoes develop resistance. Resistance to temephos, the commonly
used larvicide, has been widely reported (Grisales et al., 2013). Oils and films also suffocate non-target aquatic life and
cause bird feathers to mat, and matted feathers cannot keep young birds warm and dry. Thus, against the facts
presented, there is a need for a continual search of larvicides that can be used on farms.

Many plant extracts have been tested against various species of mosquitoes, focusing on larvicidal action (Shaalan et al.,
2005). The use of plant extracts against noxious insects has the advantage that the closely related compounds within
these complex mixtures often act synergistically (Isman, 1997). The exposure of a target organism to a group
of phytochemicals, rather than to a single active principle, lowers the probability for that organism to develop resistance
or behavioural desensitization. Clausena anisata is one of the ethnomedicinal plants that have been reportedly used
traditionally to repel or kill mosquitoes (Okunade and Olaifa, 1987, Mavundza et al., 2011). It has also been reported to
have insecticidal and repellent activities against various insects in ethnoveterinary medicine (Chavunduka, 1976) and in-
vitro laboratory studies (Boeke et al., 2004, Ndomo et al., 2008). It contains compounds that interfere with larval feeding
and the neuroendocrine control mechanisms in the blowfly (Mukandiwa et al., 2012, Mukandiwa et al., 2013). We
therefore investigated the biological activity of C. anisata extracts and the isolated compound against Aedes
eagypti mosquito larvae.

2. Materials and methods

2.1. Collection of plants

The leaves of C. anisata (Wild) Hook. f. ex. Benth were collected in autumn from the Pretoria National Botanical Garden,
South Africa and dried at room temperature in a well-ventilated room. The plant species was identified by tree name
tags and were authenticated by the Guide at the National Botanical Garden. The voucher specimen of the plant species,
numbered PMDN317, is kept at the Medicinal Plant Collection Herbarium of the Department of Paraclinical Sciences,
University of Pretoria, South Africa. Collection, drying and storage guidelines of the plant material followed were as
outlined by McGaw and Eloff (2010).

2.2. Extraction

Dried leaf material was ground to fine powder (c. 1 mm diameter) using an IKA-WERKE M20 mill (GMBH & Co.,
Germany). Three different extractants were used, namely: acetone, dichloromethane and hexane (all technical grade,
Merck). To prepare the extracts, 25 g of the leaf material was shaken vigorously for 1 h in 250 ml of the respective
extractants on an orbital shaker (Labotec®, model 20.2, South Africa). The extracts were allowed to settle, centrifuged at
2000 g for 10 min and the supernatant filtered through Whatman No. 1 filter paper into pre-weighed glass vials. The
extraction process was repeated 3 times for three samples of the plant material. The extracts were dried in a stream of
cold air at room temperature and the mass extracted with each solvent was determined. The dried extracts were
reconstituted in acetone for use in the bioassays.

2.3. Test organism

A. aegypti mosquito eggs were obtained from the Pesticide Trial Section of the South African Bureau of Standards
(SABS). The eggs were placed in distilled water to hatch. The emerging larvae were reared and tested at 28 ± 2 °C
temperature, ≥ 45 ± 10% relative humidity, and a 12:12 (light:dark) photoperiod and were fed tropical fish flakes

2.4. Larvicidal bioassay

2.4.1. Laboratory evaluation

The larvicidal activity of the plant extracts was evaluated according to the World Health Organization guidelines for
laboratory and field testing of mosquito larvicides (WHO, 2005). The acetone, dichloromethane and hexane crude
extracts were evaluated in a preliminary screening at the concentrations of 12.5, 25, 50, 100, and 200 ppm. Batches of
25 third-instar larvae were transferred to a small disposable test cups, each containing 100 ml of distilled water. Then
one ml of aliquots of the plant extracts at the concentrations ranging from 1.25 to 20 mg/ml was added, producing final
concentrations ranging from 12.5 to 200 ppm. The acetone and dichloromethane extracts had no larvicidal activity at all
the concentrations tested. The larvae exposed to these extracts underwent the subsequent developmental stages
successfully. Therefore, only the activity of the hexane extract was further evaluated at 40, 60, 80, 100, and 120 ppm to
enable the determination of the LC50 value. Four replicates were set up for each concentration and the tests were
repeated 3 times on different days. Permethrin (0.1 ppm; technical grade dissolved in acetone) was used as a positive
control. It was selected as a positive control because it is based on plant compounds, the pyrethrins from
the chrysanthemum flower, and it has been reported to be among the pyrethroids with best activity against mosquito
larvae (Mulla and Schaefer, 1980). Acetone was used as the solvent control.
The twelve fractions of the crude hexane extract (Section 2.4) and the isolated compound (Section 2.5) were also
evaluated for larvicidal activity in the same manner as described above. The fractions were tested at 50 ppm. Only one
concentration was used as this step was only meant to guide further fractionation of the extract. The isolated compound
was tested at concentrations of 10, 20, 30, 40 and 50 ppm. Larval mortality was recorded 24 h and 48 h after exposure.
Moribund larvae were counted and added to dead larvae for calculating percentage mortality. Dead larvae are those
that cannot be induced to move when they are probed with a needle in the siphon or the cervical region whilst
moribund larvae are those incapable of rising to the surface or not showing the characteristic diving reaction when the
water is disturbed (WHO, 2005).

After data collection at 48 h food was added into the test containers and every other day thereafter for 7 days. During
this period observations were made on the development of the larvae to pupae stage.

2.4.2. Small-scale simulated field trial

According to WHO guidelines larvicides that show promise in laboratory studies (Phase I) should be subjected to Phase
II. In Phase II, field trials of formulated products are performed on a small scale against target mosquitoes, preferably in
representative natural breeding sites or, where such trials are not feasible, under simulated field conditions. For this
phase of the study, 2 l beakers, filled to half capacity with water were placed on 2 different sites on the grounds of the
University of Pretoria. We decided not to use water from the field, because there could be too many variables between
different water bodies that would hinder repeatability of the results. At this stage we needed to develop a proof of
concept, i.e. that the extract is effective against mosquito larvae. The water was allowed to age for 24 h, after which,
batches of 100 laboratory-reared third instar larvae of A. aegypti were released into each container with larval food.
After 2–3 h of larval acclimation, 1 ml of the hexane C. anisata extract dissolved in acetone at 100 mg/ml was added into
one of the beakers per site to a final concentration of 100 ppm. The selection of the test concentration was guided by
the results from the laboratory assay; the 100 ppm concentration caused larval mortality of over 90%. The containers
were covered with nylon mesh to prevent other mosquitoes or other insects from laying eggs and to protect the water
from falling debris. Two replicates of the treatment and two controls were used in each test. The containers were
examined after 48 h and live larvae were counted to score post-treatment larval mortality. The test was repeated four
times. The extract used in this study was intentionally 2 months old, to get an indication of the stability of the extract.
The extract was stored in closed glass jar in a cupboard at room temperature with an average minimum of 15 °C and
maximum of 32 °C and humidity of ≥ 25%. The isolated compound was also evaluated under simulated field conditions in
the same manner described above. It was evaluated at 25 ppm, which was selected as a concentration most likely to
cause 90% mortality based on the laboratory assay.

2.5. Fractionation of the crude n-hexane extract

Three (3) grammes of the crude n-hexane extract was fractionated by column chromatography using silica gel (Kieselgel
60, 70–230 mesh, 0.063–0.200 mm, Merck), with a gradient solvent of n-hexane: ethyl acetate 100:0, 98:2, 95:5, 90:10,
85:15, 80:20 to 70:30 (hexane:ethyl acetate). The resulting fractions were combined based on thin layer
chromatography (TLC) analysis to give a total of 12 fractions.

2.6. Isolation of an active component from the n-hexane fraction

From the bioactivity assays of the different fractions mentioned above, only 2 fractions, 1/11 and 12/13, caused larval
mortality with Fraction 12/13 being the most active. It was therefore subjected to repeated column chromatography.
Open column chromatography was undertaken on silica gel (Kieselgel 60, 70–230 mesh, 0.063–0.200 mm, Merck) using
n-hexane:ethyl acetate at 100:0, 98:2, 96:4, 94:6, 98:2, and 90:10 (hexane: ethyl acetate) and led to the isolation of
Compound A.

2.6.1. Structural analysis of the isolated active compound

Spectroscopic techniques,1HNMR, 13C NMR and 2DNMR(HMBC, HSQC, COSY, DEPT), were used for the elucidation of
the structure of isolated active compound using a Bruker ARX-400 nuclear magnetic resonance (NMR) spectrometer (in
deuterated chloroform (CDCl3)). Chemical shifts were reported with reference to the respective residual solvents or
deuterated solvent peaks. The structure of the isolated compound was confirmed by comparison of its NMR data with
that in literature. ESI-MS were obtained on Waters Synapt HDMS spectrometer.

2.7. Data analysis

Data from the larvicidal assays for all replicates was pooled together for analysis. The data was analysed using the
pharmacology software Kinetica version 5 (Thermo Scientific). The concentration required to kill 50% of the larvae
(LC50) was determined using the Sigmoid Emax model (Hill).

3. Results
3.1. Larvicidal activity of crude hexane extract of C. anisata

3.1.1. Larvicidal activity under controlled conditions

Larval mortality was observed for only the larvae exposed to the hexane extract of C. anisata in the preliminary
screening of the extracts. Therefore further evaluation of the hexane extract was conducted at concentrations selected
based on the observations from the preliminary screening. Mortality increased with the increase in concentration of the
extract from 40 ppm to 120 ppm with the LC50 values after 24 h and 48 h being 68.30(60.293–73.736) and 59.67
(53.148–67.092) ppm respectively (figures in parenthesis are the lower confidence limit (LCL) and the upper confidence
limit (UCL)). Mortality was also time dependent, being higher at 48 h compared to at 24 h (Fig. 1). Larvae exposed to the
hexane extract at 40 and 60 ppm that did not die completed the rest of the developmental stages. However, those
exposed to 80 ppm and above, all eventually (> 48 h) died at larval stage.

Fig. 1. Larvicidal activity of the different concentrations of hexane extract of Clausena

anisata against Aedes eagypti larvae.

3.1.2. Larvicidal activity under small-scale simulated field conditions

The hexane extract was still active after 2 months of storage resulting in 89% ± 8.61 mortality of exposed larvae when
tested at 100 ppm. The larvae that did not die also failed to develop to the next life stage.

3.2. Isolation and identification of larvicidal compounds

The total amount of SFr. 12/13 extracted from 3.0 g of the crude hexane extract was 820 mg thus the fraction yield was
27.3%. The structure of the isolated active compound was elucidated by NMR and MS as the pyranocoumarin, seselin,
chemically called 2′, 2′-dimethylpyranocoumarin. A detailed description of this compound is available elsewhere
(Mukandiwa et al., 2013). The total amount isolated from 3 g of crude hexane extract was 473 mg thus an isolation
efficiency of 15.77%. Based on the starting plant material from which the crude hexane extract was obtained (25 g), the
compound percentage yield was 1.89%.

3.3. Mosquito larvicidal activity of Seselin

Dose dependent mortality was observed in the larvae exposed to seselin (Fig. 2). The LC50 values at 24 and 48 h were
13.90 and 9.96 ppm respectively. Larvae exposed to less than 10 ppm did not die and successfully underwent the rest of
the developmental stages.

Fig. 2. Larvicidal activity of different concentrations (ppm) of seselin against Aedes eagypti mosquito larvae.
Exposure to seselin at 25 ppm resulted in 100% mortality when evaluated under simulated field conditions similar to
what was observed under laboratory conditions at the same concentration.

4. Discussion

The aim of this study was to establish the larvicidal activity of C. anisata against the A. eagypti mosquito and identify the
compound(s) responsible for the observed activity. This work adds to the current efforts worldwide to discover new
mosquito control agents. Plant bioactive chemicals are generally considered as nontoxic, easily available at affordable
prices, biodegradable and show broad-spectrum target-specific activities against different species of vector mosquitoes
(Ghosh et al., 2012).

The hexane extract of C. anisata had promising LC50 values of 68.30 and 59.65 ppm after 24 h and 48 h respectively. In a
previous research by Mavundza et al. (2013), the ethanolic extract of C. anisata had an LC50 value of 112.7 ppm
against Anopheles arabiensis mosquitoes. Our findings and those of previous researchers indicate that
C. anisata certainly contains mosquito larvicidal compounds. The observed difference in activity can be attributed to the
different mosquito species and extract types of the C. anisata leaves used in the 2 studies. The fact that mosquito larvae
exposed to 80% and higher concentrations of the crude hexane extract that did not die failed to complete the
subsequent life stages suggests that the extract may also act as an insect growth regulator.

In this study, we went further than just screening the plant for larvicidal activity to isolate and identify the larvicidal
compound in C. anisata. In addition to the isolated seselin the researchers hypothesize that there may be more active
compounds in the extract. Several studies have focused on the larvicidal activities of various plant species in preliminary
screenings (Hardin and Jackson, 2009, Ghosh et al., 2012) but only a few have gone further to determine the active
principles. As a result few botanicals have moved from laboratory to field use. The observed activity of seselin against A.
eagypti is comparable to what other researchers have reported for other isolated plant compounds against the same
species which have LC50 values that range from 0.25 to 14.7 ppm (Ghosh et al., 2012). The identified compounds include
lapachol, (E)-6-hydroxy-4, 6-dimethyl-3-heptene-2-one, α-terpinene, N-methyl-6β-(decal′, 3′, 5′-trienyl)-3-β-methoxy-2-
β-methylpiperidine, Methyl-p-hydroxybenzoate, β-sitosterol, and Pipernonaline. This clearly supports the notion that
bioactive constituents of plants have potential to be employed as larvicides useful in controlling mosquito vectors.
Although inferior to methoprene and temephos which have LC50 values ranging between (0.00278 to 0.0195 ppm)
(Braga et al., 2005, Silva and Mendes, 2007) and (0.006 to 0.038 ppm) (Loke et al., 2010, Lek-Uthai et al., 2011)
respectively, these compounds could still be important as alternatives considering that resistance to both compounds
has been reported already (Grisales et al., 2013).

Seselin has been previously identified as the antifeedant compound in C. anisata deterring blowfly larval feeding
(Mukandiwa et al., 2013). It has been isolated from plants particularly those belonging to the Rutaceae family (Keating
and O'Kennedy, 1997, Borges et al., 2005). It has several activities including vasodilatory (Lima et al., 2006); antitumor
and anti-HIV (Huang et al., 1994); antifungal (Bandara et al., 1991, Cardenas-Ortega et al., 2007); ovicidal
against Tetranynchus urticae (red spider mite) (Tanaka et al., 1985); weak to moderate cytotoxicity (Gunatilaka et al.,
1994); peripheral anti-inflammatory and antinociceptive (Lima et al., 2006); inhibits phytohemagglutinin-stimulated cell
proliferation in human blood mononuclear cells (Tsai et al., 2008); inhibitory activity in both indole acetic acid oxidase
and peroxidase enzyme systems (Goren and Tomer, 1971) and autotoxicity in citrus trees (Singh et al., 1999). However
this is the first report on seselin having mosquito larvicidal activity.

The crude hexane extract of C. anisata at 100 ppm resulted in mortalities of 90.00% ± 7.53 and 93.33% ± 6.46 after 24
and 48 h respectively thus only 0.1 mg of the extract per ml of water was required to cause over 90% mortality.
Accordingly, 1 g of the crude hexane extract (which requires 31.25 g of plant material) will kill mosquito larvae in 10 l of
water. Thus it may be feasible to use the crude hexane C. anisata extract as a larvicide on farms as part of an integrated
pest management system. The 2 month-old extract was able to give similar results in the simulated field study as a fresh
extract in the laboratory study. This is noteworthy in light of the fact that one of the major drawbacks to the use of plant
extracts is that they are unstable under environmental conditions and quickly lose their activity. A more detailed stability
study will be conducted in further studies in which differently aged extracts will be tested. Safety evaluation of C.
anisata in animal drinking water will also be a focus of our next study.

5. Conclusion

Results obtained from this study have initiated on-going investigations into the incorporation of C. anisata into the
control of mosquito populations, with a view to developing an environmentally acceptable product of value in
integrated vector control. The use of a plant extract that reduce mosquito populations at the larval stage can provide
many associated benefits to vector control.
Mosquito larvicidal activity of Aloe vera (Family: Liliaceae) leaf extract and Bacillus
sphaericus, against Chikungunya vector, Aedes aegypti
Abstract

1. Introduction
A recent estimate shows that more than 50 million people are at risk of dengue virus exposure worldwide. Annually, there
are 2 million infections, 500,000 cases of dengue hemorrhagic fever, and 12,000 deaths (Guha-Sapir and Schimme,
2005). Aedes aegypti is generally known as a vector for an arbo-virus responsible for dengue fever, which is endemic to
Southeast Asia, the Pacific island area, Africa, and the Americas. This mosquito also acts as a vector of yellow fever in
Central and South America and West Africa. However, Dengue fever has become an important public health problem as
the number of reported cases continues to increase, especially with more severe forms of the disease, dengue hemorrhagic
fever, and dengue shock syndrome, or with unusual manifestations such as central nervous system involvement
(Pancharoen et al., 2002).
A. aegypti is a cosmotropical species that proliferates in water containers in and around houses. Secondary vectors
include Aedes albopictus, an important vector in Southeast Asia and that has spread to the Americas, western Africa and
the Mediterranean rim, Aedes mediovittatus in the Caribbean, and Aedes polynesiensis and Aedes scutellaris in the
western Pacific region. A. aegypti breeds in many types of household containers, such as water storage jars, drums, tanks,
and plant or flower containers (Muir and Kay, 1998; Honorio et al., 2003; Harrington et al., 2005; Murugan et al., 2011).
Mosquito control, in view of their medical importance, assumes global importance. In the context of ever increasing trend
to use more powerful synthetic insecticides to achieve immediate results in the control of mosquitoes, an alarming
increase of physiological resistance in the vectors, its increased toxicity to non-target organism and high costs are
noteworthy (WHO, 1975). Most of synthetic chemicals are expensive and destructive to the environment and also toxic to
humans, animals and other non-target organisms. Besides, they are non-selective and harmful to other beneficial
organisms. Some of the insecticides act as carcinogenic agents and are even carried through food chain which in turn
affects the non-target organism. Therefore alternative vector control strategies, especially effective and low cost are
extremely imperative (Piyarat et al., 1974; Kalyanasundaram and Das, 1985).
The use of different parts of locally available plants and their various products in the control of mosquitoes has been well
established globally by numerous researchers. The larvicidal properties of indigenous plants have also been documented
in many parts of India along with the repellent and anti-juvenile hormones activities (Singh and Bansal, 2003). Almost all
tropical regions of the world are experiencing the resurgence and reoccurrence of one of the world’s most deadly diseases,
i.e., malaria, filariasis, dengue, and Chikungunya in world and India is no exception. Traditionally, plants and their
derivatives were used to kill mosquitoes and other household and agricultural pests. In all probability, these plants used to
control insects contained insecticidal phytochemicals that were predominantly secondary compounds produced by plants
to protect themselves against herbivorous insects (Shaalan et al., 2005; Preeti Sharma et al., 2009).
Aloe vera is a perennial plant belonging to the family of Liliaceae, of which there are about 360 species (Klein and
Penneys, 1988). Taxonomists now refer to Aloe barbadensis as A. vera (Coats and Ahola, 1979). Aloe is one of the few
medicinal plants that maintain its popularity for a long period of time. The plant has stiff, graygreen lance-shaped leaves
containing clear gel in a central mucilaginous pulp. A. vera gel has hypoglycemic (Rajasekaran et al., 2004), wound
healing (Pandarinathan et al., 1998) and anti-inflammatory effects (Davis et al., 1991). The A. vera (L.) Burm. f., plant
(synonym = Aloe barbadensis Miller) is commonly referred to as A. vera and belongs to the lily family (family: Liliaceae,
tribe Aloinae). This species is one of the approximately 420 species of aloe (Burdock, 1997).
Since, 1986 A. vera has been used as a traditional medicine and as an ingredient in many cosmetic products; it has gained
high importance for its diverse therapeutic properties. The plant, being succulent, contains 99.5% water and the remaining
solid material contains over 75 different ingredients including vitamins, minerals, enzymes, sugars, anthraquinones or
phenolic compounds, lignin, tannic acids, polysaccharide, glycoproteins, saponins, sterols, amino acids, and salicylic
(Reynolds and Dweck, 1999). A. vera provides nutrition, shows anti-inflammatory action and has a wide range of
antimicrobial activity (Reynolds and Dweck, 1999).
Bacillus sphaericus is a naturally occurring soil bacterium that can effectively kill mosquito larvae present in water. B.
sphaericus has the unique property of being able to control mosquito larvae in water that is rich in organic matter. B.
sphaericus is effective against Culex spp. but is less effective against some other mosquito species. Commercially
available formulations of B. sphaericus are sold under the trade name Vectolex. When community mosquito control is
needed to reduce mosquito-borne disease, the Department of Health favors the use of larvicide applications targeted to the
breeding source of mosquitoes (Meisch, 1990).
Bacterial larvicides have been used for the control of nuisance and vector mosquitoes for more than two decades. The
discovery of bacterium like B. sphaericus, which is highly toxic to dipteran larvae, has opened the possibility of its use as
a potential biolarvicide in mosquito eradication program worldwide (Kalfon et al., 1984). The mosquitocidal activity of
the highly active strain of B. sphaericus resulted in their development as commercial larvicides. This is now used in many
countries in various parts of the world to control vector and nuisance mosquito species (Wirth et al., 2001).
Indeed, source reduction is one of the key components in the malaria vector control program since the target is
exceptionally specific unlike adult control. Innovative vector control strategy like use of phytochemicals as alternative
sources of insecticidal/larvicidal agents in the fight against the vector-borne diseases has become inevitable. Above and
beyond, in recent epoch, around the globe phytochemicals have gained massive attention by various researchers because
of their bio-degradable and eco-friendly values (Karunamoorthi and Ilango, 2010). In this context, the purpose of the
present investigation is to explore the larvicidal properties of A. vera leaf extract and bacterial insecticide, B.
sphaericus against Chikungunya vector, A. aegypti, under the laboratory conditions. Therefore, this study provides first
report on the mosquito larvicidal activity combined effect of A. vera leaf extract and B. sphaericus against A. aegypti as
target species.

2. Materials and methods

2.1. Collection of eggs and maintenance of larvae

The eggs of A. aegypti were collected from National Centre for Disease Control field station of Mettupalayam, Tamil
Nadu, India, using an “O”-type brush. These eggs were brought to the laboratory and transferred to 18 × 13 × 4-cm
enamel trays containing 500 mL of water for hatching. The mosquito larvae were fed pedigree dog biscuits and yeast at
3:1 ratio. The feeding was continued until the larvae transformed into the pupal stage.

2.2. Maintenance of pupae and adults

The pupae were collected from the culture trays and transferred to plastic containers (12 × 12 cm) containing 500-mL of
water with the help of a dipper. The plastic jars were kept in a 90 × 90 × 90-cm mosquito cage for adult emergence.
Mosquito larvae were maintained at 27 ± 2 °C, 75–85% relative humidity, under a photoperiod of 14:10 (light/dark). A
10% sugar solution was provided for a period of 3 days before blood feeding.

2.3. Blood feeding of adult A. aegypti

The adult female mosquitoes were allowed to feed on the blood of a rabbit (a rabbit per day, exposed on the dorsal side)
for 2 days, to ensure adequate blood feeding for 5 days. After blood feeding, enamel trays with water from the culture
trays were placed in the cage as oviposition substrates.

2.4. Collection of plant and preparation of extract

A. vera was collected in and around Bharathiar University Campus, Coimbatore, India. The voucher specimen has been
deposited and kept in our research laboratory for further reference. A. vera plant was washed with tap water and shade
dried at room temperature. An electrical blender powdered the dried plant materials (leaves). From the powder, 300 g of
the plant materials was extracted with 1 L of organic solvents of petroleum ether for 8 h using a Soxhlet apparatus (Vogel,
1978). The extracts were filtered through a Buchner funnel with Whatman number 1 filter paper. The crude plant extracts
were evaporated to dryness in a rotary vacuum evaporator. One gram of the plant residue was dissolved in 100 mL of
acetone (stock solution) and considered as 1% stock solution. From this stock solution, different concentrations were
prepared ranging from 80, 160, 240, 320 and 400 ppm, respectively.

2.5. Microbial bioassay

B. sphaericus was obtained from T. Stanes & Company Limited, Research and Development Centre, Coimbatore, Tamil
Nadu, India. The organism was grown in a liquid medium containing (in grams per liter of distilled water): FeSO4·7H2O,
0.01; MnSO4, 0.1; MgSO4·7H2O, 0.2; CaCl2, 0.08; K2HPO4, 0.025; yeast extract, 2; peptone, 4; and D-glucose, 1 and
casein, 5. Solutions of yeast extract, peptone casein, D-glucose, K2HPO4 and CaCl2 were separately prepared, sterilized,
and added before inoculation. The pH of the medium was adjusted to 7.1 before sterilization. The required quantity of B.
sphaericus was thoroughly mixed with distilled water and prepared at various concentrations ranging from 25, 50, 75, 100
and 125 ppm, respectively.

2.6. Larval toxicity test

A laboratory reared colony of A. aegypti larvae was used for the larvicidal activity. Twenty-five individuals of first,
second, third, and fourth instars larvae were kept in a 500 mL glass beaker containing 249 mL of dechlorinated water and
1-mL of desired concentration of A. vera leaf extracts and B. sphaericus were added. Larval food was given for the test
larvae. At each tested concentration, two to five trials were made and each trial consists of five replicates. The control was
setup by mixing 1 mL of acetone with 249 mL of dechlorinated water. The larvae exposed to decholorinated water
without acetone served as control. The control mortalities were corrected by using Abbott’s formula (Abbott, 1925).

Corrected mortality=Observed mortality in treatment−Observed mortality in control100−Control mortality×100Percentage

mortality=Number of dead larvaeNumber of larvae introduced×100
The LC50 and LC90 were calculated from toxicity data by using probit analysis (Finney, 1971).
2.7. Statistical analysis
All data were subjected to analysis of variance; the means were separated using Duncan’s multiple range tests by Alder
and Rossler (1977). The average larval mortality data were subjected to probit analysis, for calculating LC50 and LC90,
values were calculated by using the Finney (1971) method. SPSS (Statistical software package) 9.0 version was used.
Results with P < 0.05 were considered to be statistically significant.
Go to:

3. Results
Larval mortality of A. aegypti after the treatment of petroleum ether A. vera was observed. Table 1 provides the results of
larval mortality of A. aegypti (I–IV instars) after the treatment of A. aegypti at different concentrations (80–400 ppm).
Thirty-four percent mortality was noted at I instar larvae by the treatment of A. vera at 80 ppm, whereas it has been
increased to 89% at 400 ppm of A. vera leaf extract treatment. Similar trend has been noted for all the instars of A.
aegypti at different concentrations of A. vera treatment. The LC50 and LC90 values were represented as follows; LC50 value
of I instar was 162.74 ppm, II instar was 201.43 ppm, III instar was 253.30 ppm, and IV instar was 300.05 ppm,
respectively. The LC90 value of I instar was 442.98 ppm, II instar was 518.86 ppm, III instar was 563.18 ppm and IV instar
was 612.96 ppm, respectively (Fig. 1).

Mosquito % of larval mortality LC50 (LC90) 95% confidence limit χ2 (df = 4)

larval

instars

Concentration of AVLE (ppm) LFL UFL

80 160 240 320 400 LC50 (LC90) LC50 (LC90)

I 34a 53a 64a 71a 89a 162.74 128.19 189.79 2.87⁎

(442.98) (395.17) (516.81)

II 29ab 48b 56b 64b 81b 201.43 168.33 229.81 2.27⁎

(518.86) (455.12) (623.39)

III 24bc 36c 47c 58c 75c 253.30 225.36 282.81 0.66⁎

(563.18) (493.94) (675.91)

IV 19c 28d 41d 50d 68d 300.059 271.32 336.01 0.67⁎

(612.96) (534.46) (742.76)

Figure 1

Larvicidal activity of A. vera leaf extract against A. aegypti expressed as LC50 and LC90.

Table 1
Larvicidal activity of A. vera leaf extract against A. aegypti.
Control – nil mortality, LFL – lower fiducidal limit, UFL – upper fiducidal limit, χ2 – Chi-square value, df – degrees of freedom.

Within a column means followed by the same letter(s) are not significantly different at 5% level by DMRT. Each value is five replicates.

Significant at P < 0.05 level.

⁎

Table 2 shows the results of larval mortality of A. aegypti (I–IV instars) after the treatment of B. sphaericus at different
concentrations (25–125 ppm). Twenty-seven percent mortality was noted at I instar larvae by the treatment of B.
sphaericus at 25 ppm, whereas it has been increased to 85% at 125 ppm of B. sphaericus treatment and 12% mortality was
noted at pupae by the treatment of B. sphaericus at 25 ppm and it has been increased to 60% at 125 ppm. Similar trend has
been noted for all the instars of A. aegypti at different concentrations of B. sphaericus treatment. The LC50 and LC90 values
were represented as follows: LC50 value of I instar was 68.21 ppm, II instar was 79.13 ppm, III instar was 93.48 ppm, and
IV instar was 107.05812 ppm, respectively. The LC90 value of I instar was 149.15 ppm, II instar was 164.67 ppm, III
instar was 183.84 ppm, and IV instar was 201.09 ppm, respectively (Fig. 2).

Figure 2

Larvicidal activity of B. sphaericus against A. aegypti expressed as LC50 and LC90.

Table 2
Larvicidal activity of bacterial insecticide, B. sphaericus against A. aegypti.
 Mosquito % of larval mortality LC50 (LC90) 95% confidence limit χ2 (df = 4)

larval

instars

Concentration of B. LFL UFL

sphaericus (ppm)

25 50 75 100 125 LC50 (LC90) LC50 (LC90)

I 27a 39a 50a 67a 85a 68.21 60.35 75.58 2.05⁎

(149.2) (134) (171.8)

II 21ab 35b 46a 59b 78b 79.13 71.36 87.28 1.07⁎

(164.7) (146.8) (192.2)

III 16bc 28c 41ab 50c 69c 93.48 85.12 103.71 0.84⁎

(183.8) (162.1) (218.6)

IV 12c 23d 35b 44d 60d 107.05 97.36 120.48 0.55⁎

(201.1) (175.3) (243.7)

Control-Nil mortality, LFL – lower fiducidal limit, UFL – upper fiducidal limit, x2 – Chi-square value, df – degrees of freedom.

Within a column means followed by the same letter(s) are not significantly different at 5% level by DMRT. Each value is five replicates.

Significant at P < 0.05 level.

⁎

The considerable larval mortality after the combined effect of B. sphaericus and A. vera extract against all the larval
instars in A. aegypti is provided in (Table 3 and Fig. 3). The concentration at 20 + 10 ppm combined treatment of B.
sphaericus and A. vera for I instar larval mortality was 41%. The LC50 and LC90 values were represented as follows:
LC50 value of I instar was 54.80 ppm, II instar was 63.11 ppm, III instar was 74.66 ppm and IV instar was 95.10 ppm. The
LC90 value of I instar was 145.29 ppm, II instar was 160.14, III instar was 179.74 ppm and IV instar was 209.98 ppm,
respectively.
Figure 3

Combined treatment of larval mortality of A. vera leaf extract and B. sphaericus against A. aegypti at 24 h.

Table 3
Combined treatment of larvicidal activity of A. vera leaf extract and bacterial insecticide, B. sphaericus against A. aegypti.

Mosquito % of larval mortality LC50 (LC90) 95% confidence limit χ2 (df = 4)

larval

instars

Concentration of AVLE (ppm) + B. LFL UFL

sphaericus (ppm)

20 + 10 40 + 20 60 + 30 80 + 40 100 + 50 LC50 (LC90) LC50 (LC90)

I 41a 56a 69a 82a 94a 54.80 41.63 64.65 4.73⁎

(145.29) (131.78) (165.23)

II 38a 52a 61b 79ab 90a 63.11 50.52 72.79 4.11⁎

(160.14) (144.29) (184.28)

III 33b 45b 57b 74b 83b 74.66 62.93 84.30 3.48⁎

(179.74) (160.35) (210.42)

 Mosquito % of larval mortality LC50 (LC90) 95% confidence limit χ2 (df = 4)

larval

instars

Concentration of AVLE (ppm) + B. LFL UFL

sphaericus (ppm)

20 + 10 40 + 20 60 + 30 80 + 40 100 + 50 LC50 (LC90) LC50 (LC90)

IV 27c 36c 48c 62c 75c 95.10 84.57 105.73 3.16⁎

(209.98) (184.25) (252.87)

Open in a separate window

Control – nil mortality, LFL – lower fiducidal limit, UFL – upper fiducidal limit, χ2 – Chi-square value, df – degrees of freedom.

Within a column means followed by the same letter(s) are not significantly different at 5% level by DMRT. Each value is five replicates.

Significant at P < 0.05 level.

⁎

Go to:

4. Discussion
Mosquitoes in the larval stage are attractive targets for pesticides because mosquitoes breed in water, which makes it easy
to deal with them in this habitat. The use of conventional pesticides in the water sources, however, introduces many risks
to people and the environment. Natural pesticides, especially those derived from plants, are more promising in this aspect.
Aromatic plants and their essential oils are very important sources of many compounds that are used in different respects
(Amer and Mehlhorn, 2006a).
Recent studies on the larval and pupal mortality of Anopheles stephensi after the treatment of methanol extract
of Clerodendron inerme leaf extract showed 22% mortality at I instar larvae as a result of treatment at 20 ppm; in contrast,
it was increased to 81% at 100 ppm of C. inerme leaf extract of larval and pupal mortality of A. stephensi (I–IV instars)
after the treatment of methanol extract of Acanthus ilicifolius at different concentrations (20–100 ppm). A 23% mortality
was noted at I instar larvae by the treatment of A. ilicifolius at 20 ppm, whereas it was increased to 89% at 100 ppm of A.
ilicifolius leaf extract treatment (Kovendan and Murugan, 2011).
The isolated compound saponin from ethyl acetate extract of Achyranthes aspera was effective against the larvae of A.
aegypti and Culex quinquefasciatus with LC50 value of 18.20 and 27.24 ppm, respectively (Bagavan et al., 2008). The
neem formulation, Neem Azal, produced an overall mortality or inhibition of emergence of 90% (EI90, when third-instar
larvae were treated) at 0.046, 0.208, and 0.866 ppm in A. stephensi, C. quinquefasciatus, and A. aegypti, respectively
(Gunasekaran et al., 2009). Fraction A1 of ethanol from Sterculia guttata seed extract was found to be most promising; its
LC50 was 21.552 and 35.520 ppm against C. quinquefasciatus and A. aegypti, respectively (Katade et al., 2006a,b).
With A. barbadensis the larvicidal activity increases with increase in the exposure period from 24 to 48 h with decrease in
LC50 values from 15.31 to 11.01 ppm (carbon tetrachloride extract), 25.97 to 16.60 ppm (petroleum ether extract) and
144.44 to 108.38 ppm (methanol extract). Similar trend was also observed in case of Cannabis sativa with LC50 values
88.51 to 68.69 ppm (carbon tetrachloride extract), 294.42 to 73.32 ppm (petroleum ether extract) and 160.78 to 71.71 ppm
(methanol extract) on increase in the exposure period. Further, Barnard and Rui De (2004) observed the repellent activity
of A. vera against A. albopictus and Culex nigripalpus.
The leaf extract of Acalypha alnifolia with different solvents – hexane, chloroform, ethyl acetate, acetone, and methanol –
were tested for larvicidal activity against three important mosquitoes such as malarial vector, A. stephensi, dengue
vector, A. aegypti and Bancroftian filariasis vector, C. quinquefasciatus and highest larval and pupal mortality were found
in the leaf extract of methanol Carica papaya against the first to fourth instar larvae and pupae of values LC50 = 51.76,
61.87, 74.07, 82.18 and 440.65 ppm, respectively (Kovendan et al., 2012b,c). In the present results, the LC50 and
LC90 values of A. vera against first to fourth instars larvae were 162.74, 201.43, 253.30 and 300.05 ppm; the LC90 values
of 442.98, 518.86, 563.18 and 612.96 ppm, respectively.
However, in our case, early detection of resistance against B. sphaericus could be better for the management of resistance
development. Experts of resistance management have been involved in resistance detection with generations of
mosquitoes. Moreover, the detection of resistance in an early stage could be a better approach to control mosquitoes.
Laboratory- and field-collected C. quinquefasciatus exposed to B. sphaericus strain 2362 for 35 generations in the
laboratory showed a level of resistance 43- and 12-fold than that of potential generation, respectively (Rodeharoen and
Mulla, 1991). B. sphaericus, a spore-forming, entamopathogenic bacterium, has been shown to possess potent larvicidal
activity against several species of mosquito larvae (Davidson, 1983; Yousten and Wallis, 1987). A flowable concentrate
of B. sphaericus (Neide) strain 2362 was applied against Anopheles gambiae Giles s.l. mosquito larvae in small plot field
trials in Bobo-Dioulasso area, Burkina-Faso. Third and fourth instar larvae were controlled for 10–15 days with a dosage
of 10 g/m2, 3–10 days with 1 or 0.1 mg/m2, and 2 days with 0.01 g/m2 (Nicolas et al., 1987).
B. sphaericus showed a good control over A. stephensi which may be due to the presence of Bin and mosquitocidal toxins
(Mtxs). As a consequence of the specific toxicity to mosquito larvae of Bin and Mtxs produced during the sporulation and
vegetative stages, respectively, some toxic strains have been widely used for many years as bio-pesticides in the field of
mosquito control programs (Bei et al., 2006). The soil bacterium showed varied mortality rate related to the larval stages
and concentrations. The younger larval stages were much susceptible than the later ones. Active strains of B.
sphaericus are known to produce considerable quantities of at least two sets of proteinaceous mosquito larvicidal factors
at the onset of stationary phase (Souza et al., 1988; Baumann et al., 1991; Porter et al., 1993). Larvicidal activity was
observed in all strains of B. sphaericus from Amazonia in differentiated toxicity levels (Eleiza de et al., 2008). In the
present study, B. sphaericus treatment reduced the larvicidal properties of microbial insecticides development of growth
control.
Singh and Prakash (2009) have reported that six different concentrations were used in laboratory bioassays (05, 10, 20,
30, 40, and 50 mg/l) for A. stephensi. Similarly, in the case of C. quinquefasciatus, six statistically significant different
concentrations were used (0.01, 0.04, 0.05, 0.10, 5.0, and 10.0 mg/l) of B. sphaericus. It was recorded after exposure of
24 h. The percentages of mortalities were different for the different instars of C. quinquefasciatus and were used in
laboratory bioassays (05, 10, 20, 30, 40, and 50 mg/l) for A. stephensi. Similarly, in the case of C. quinquefasciatus, six
statistically significant different concentrations were used (0.01, 0.04, 0.05, 0.10, 5.0, and 10.0 mg/l) of B. sphaericus. It
was recorded after exposure of 24 h. The percentages of mortalities were different for the different instars of C.
quinquefasciatus and A. stephensi. Bioassay studies of B. sphaericus have been carried out in different parts of the world,
including India, on mosquitoes in laboratories and fields (Rodrigues et al. 1998). B. sphaericus against the first to fourth
instar larvae and pupae had the following values: I instar was 0.051%, II instar was 0.057%, III instar was 0.062%, IV
instar was 0.066%, and for the pupae was 0.073%, respectively. B. sphaericus, an obligate aerobe bacterium, showed that
it has good and effective mosquito control properties and also can act as an eco-friendly, biopesticide for further vector
control programs. In a previous study, B. sphaericus, the bacterial pesticide was isolated from the soil samples and used to
control the malarial vector, A. stephensi (Kovendan et al., 2012a). In the present results, the LC50 and LC90 values of B.
sphearicus were 68.21, 79.13, 93.48, and 107.05 ppm; The LC90 values of 149.15 164.67, 183.84 and 201.09 ppm,
respectively.
Vector control is one of the most powerful weapons in the process of managing vector populations to reduce/interrupt the
transmission of disease. As a result, vector control remains considered to be a cornerstone in the vector-borne disease
control program due to lack of reliable vaccine, drug resistance parasites and insecticide resistance of insect vectors
disease (Karunamoorthi, 2011). In previous study, B. sphaericus and Leucas aspera first to fourth instars larvae and pupae
against A. stephensi the LC50 and LC90 values were represented as follows: LC50 values of 2.03%, 2.04%, 2.05%, 2.05%
and 2.07%; the LC90 values of 2.10%, 2.11%, 2.12%, 2.13% and 2.16%, respectively (Kovendan et al., 2012a). In the
present results, the LC50 and LC90 values of A. vera leaf extract and B. sphaericus against first to fourth instars larvae were
54.80, 63.11, 74.66 and 95.10 ppm; the LC90 values of 145.29, 160.14, 179.74 and 209.98 ppm, respectively.

5. Conclusion
This result clearly reveals that both the leaf extract of A. vera and bio-control agent B. sphaericus could serve as a
potential larvicidal agents against the dengue vector A. aegypti and they have demonstrated a synergist act too. This
approach could not only improve the bio-efficacy of B. sphaericus but also substantially reduce the possibilities of
physiological resistance development in mosquito population. Therefore, the present strategy should be promoted in the
dengue vector control program. The mode of action and larvicidal efficiency of the A. vera extract under the field
conditions should be scrutinized and determined. Besides, further investigation regarding the effect on non-target
organism is extremely important and imperative in the near future.