Effects of sub-lethal concentrations of synthetic insecticides and Callitris glaucophylla extracts on the development of Aedes aegypti.

Abstract

Synthetic and botanical insecticides can have a profound effect on the developmental period, growth, adult emergence, fecundity, fertility, and egg hatch, resulting in effective control at sub-lethal concentrations. This paper investigated sub-lethal concentrations of fenitrothion, lambda-cyhalothrin, and Callitris glaucophylla Joy Thomps. & L.P. Johnson (Cupressaceae) extract to characterize their effects on the development of Aedes aegypti L. (Diptera: Culicidae) mosquito larvae. The LC25, LC50, and LC75 (four replicates) were used for each synthetic insecticide and the LC25 and LC75 (four replicates) were used for C. glaucophylla. Observations of larval mortality, duration of larval stage, pupal mortality, duration of pupal stage, adult emergence, sex ratio, and malformations were recorded over 14 days. A dose-response effect was observed for all insecticides. Although C. glaucophylla extract doses were higher than synthetic insecticide doses, the LC75 treatment outperformed synthetics by completely prohibiting adult emergence. Consequently, this botanical is recommended for field application either in combination with synthetic or natural insecticides or alone.

Full text

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Journal of Vector Ecology
295Vol. 30, no. 2
Effects of sub-lethal concentrations of synthetic insecticides and
Callitris glaucophylla extracts on the development of Aedes aegypti
Essam Abdel-Salam Shaalan1, Deon Vahid Canyon2?, Mohamed Wagdy Faried Younes3,
Hoda Abdel-Wahab1, and Abdel-Hamid Mansour1
1Zoology Department, Aswan Faculty of Science, South Valley University, Aswan, Egypt
2Anton Breinl Centre, School of Public Health, Tropical Medicine and Rehabilitative Sciences,
James Cook University, Townsville, Australia
3 Zoology Department, Faculty of Science, Menoufia University, Shebin El-Kom, Egypt
Received 9 March 2005; Accepted 28 July 2005
ABSTRACT: Synthetic and botanical insecticides can have a profound effect on the developmental period, growth, adult
emergence, fecundity, fertility, and egg hatch, resulting in effective control at sub-lethal concentrations. This paper investigated
sub-lethal concentrations of fenitrothion, lambda-cyhalothrin, and Callitris glaucophylla Joy Thomps. & L.P. Johnson
(Cupressaceae) extract to characterize their effects on the development of Aedes aegypti L. (Diptera: Culicidae) mosquito
larvae. The LC25, LC50, and LC75 (four replicates) were used for each synthetic insecticide and the LC25 and LC75 (four
replicates) were used for C. glaucophylla. Observations of larval mortality, duration of larval stage, pupal mortality, duration
of pupal stage, adult emergence, sex ratio, and malformations were recorded over 14 days. A dose-response effect was
observed for all insecticides. Although C. glaucophylla extract doses were higher than synthetic insecticide doses, the LC75
treatment outperformed synthetics by completely prohibiting adult emergence. Consequently, this botanical is recommended
for field application either in combination with synthetic or natural insecticides or alone. Journal of Vector Ecology 30 (2):
295-298. 2005.
Keyword Index: Botanical, phytochemical, insecticide, mosquito, sub-lethal.
INTRODUCTION
In toxicity studies, the gentle dose-response slope
observed for botanicals over 24 h renders many of them
unusable by economic standards despite them causing
significant mortality at sub-lethal concentrations. Besides toxic
larvicidal activity, botanical extracts have been shown to
induce pupicidal activity, effects on larval and pupal duration,
and often reduce adult emergence. For instance, an extract of
Callistemon lanceolatus induced concentration-dependent
mortalities in juvenile Culex quinquefasciatus (Mohsen et al.
1990), while an extract of Ipomoea carnea caused mortality
and disrupted the development and growth of Anopheles
stephensi (Saxena and Sumithra 1985). Neem oil and neem
seed kernel extract markedly reduced the percentage of
pupation and adult emergence of An. stephensi (Murugan et
al. 1996). Botanical extract-induced malformations,
particularly larval-pupal intermediates and half-ecdysed
adults, were common (Al-Sharook et al. 1991, Jayaprakasha
et al. 1997 and Karmegam et al. 1997).
Synthetic insecticides also induce sub-lethal effects,
however, these can be unpredictable. For instance, three
pyrethroids (d-phenothrin, d-allethrin, and tetramethrin)
reduced Aedes aegypti egg production, while only d-
phenothrin and d-allethrin reduced blood engorgement (Liu
et al. 1986). Topically applied dieldrin caused dose-dependant
effects on feeding and affected egg-laying capacity in Ae.
aegypti, but progeny were unaffected (Duncan 1963). Sub-
lethal concentrations of the botanical, Callitris glaucophylla,
induced significant larvicidal activity against Ae. aegypti
compared with fenitrothion and lambda-cyhalothrin (Shaalan
et al., unpublished data).
Thus, in the absence of highly toxic natural botanical
compounds, the growth or emergence inhibiting activity of a
botanical phytochemical may be essential to its uptake by the
insecticide industry. Indeed, the rational application of
exceptional phytochemicals may not only lead to new IPM
strategies but may inhibit the development of insect resistance
to existing synthetic insecticides.
This study investigated the effects of sub-lethal
concentrations of a liquefied refrigerant gas extract of C.
glaucophylla, fenitrothion, and lambda-cyhalothrin on the
development of Ae. aegypti mosquitoes and determined a
concentration that led to satisfactory control.
MATERIALS AND METHODS
Test mosquitoes
Aedes aegypti were obtained from a colony initiated from
mosquitoes collected in 2002 from Townsville, Australia. The
colony of mosquitoes were maintained at conditions of 27 ±
2 Cº and 70 % ± 5 R.H. under 14L - 10D cycles. Ae. aegypti
larvae were kept in plastic buckets half filled with tap water
and fed on goldfish flakes. Water in rearing containers was
refreshed every two days. Male and female adult mosquitoes
were maintained on a 10 % sugar solution while female adults
were also provided the opportunity to feed on rat blood.

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Journal of Vector Ecology
December 2005
296
Test insecticides
Technical grades of the organophosphorous insecticide
fenitrothion (96.8 %) and the pyrethroid insecticide lambda-
cyhalothrin (90.99 %) were provided by Nufarm Ltd (North
Victoria, Australia). Liquefied refrigerant gas extract of C.
glaucophylla was supplied by Michael Kennedy, Department
of Primary Industries, Queensland, Australia (details of
extraction awaiting IP protection). It is possible, but not
considered likely, that interaction with the compressed
refrigerant gas solvent caused or catalyzed chemical changes
in the extractive compounds, just as this could possibly happen
with conventional solvents and is known to happen during
steam distillation. The refrigerant was eliminated from the
extract by spontaneous distillation as the pressure was reduced.
Minute quantities could remain in the crude extract, but given
the volatility of the refrigerant, would be readily lost during
application of the extract to the test material during the
screening process. Any residual effect on extract activity has
not been evaluated.
Bioassays
Insect growth regulator testing instructions (WHO 1996)
were followed to investigate and determine the effects of sub-
lethal doses of test insecticides on Ae. aegypti larvae. Larvae
were subjected to two to three sub-lethal concentrations (LC25,
LC50, and LC75) in glass beakers. The sub-lethal concentrations
for fenitrothion were 0.0025 mg/l (LC25), 0.0044 mg/l (LC50),
and 0.0062 mg/l (LC75); for lambda-cyhalothrin they were
0.00004 (LC25), 0.00015 (LC50), and 0.00026 mg/l (LC75);
and for C. glaucophylla they were 2.6 mg/l (LC25) and 14.7
mg/l (LC75). All test chemicals were diluted in ethanol. One
ml of stock solution was added to 99 ml of de-ionized water.
Controls received 1 ml of ethanol only. Four replicates of 25
newly molted 4th instar larvae for each concentration were
conducted. For accurate determination of the sub-lethal
effects, the larval and pupal mortality as well as adult
emergence were recorded daily up to emergence of the adults
or death of the last larva or pupa. Due to the long duration of
the test, larvae were provided with food at 2-day intervals
during the test period.
From the overall results of the test, percentages of both
emerged and dead pupae and percentage of adult emergence,
sex ratio, larval duration, pupal duration, average
developmental period, and growth index were determined.
Growth index was calculated according to Saxena and
Sumithra (1985): Growth Index (GI) = percentage adult
emergence / average developmental period (days). Data
analyses were performed using a one-way ANOVA in SPSS
version 12.0.1 and significant differences were determined at
P<0.05.
RESULTS
Effects of sub-lethal concentrations of C. glaucophylla,
fenitrothion, and lambda-cyhalothrin on juvenile and adult
mosquitoes are shown in Tables 1 and 2.
Fenitrothion results indicated little significant difference
between controls and the LC25 dose and between the LC50
and LC75 doses. At the two higher doses, virtually all measured
variables were significantly different from controls. Larval
survival and adult emergence were reduced 10-fold, total
mortality ranged from 88-98 % and growth was significantly
limited.
Lambda-cyhalothrin results indicated more of a dose-
response with LC25s often being significantly different from
controls. Only LC50 and LC75 doses, however, significantly
reduced larval survival and adult emergence. Total mortality
was high at all doses and the GI was comparable to that
observed for fenitrothion.
C. glaucophylla results also showed clear dose-response
Insecticide
Fenitrothion
LC
Average larval
period (days)
Larval
mortality (%)
0.0±0.0a
13.0±8.1a
74.5±19.2b
92.0±5.2c
0.5±1.0a
7.0±3.5a
33.5±3.0b
69.5±9.3c
2.0±2.8a
9.5±4.4b
95.0±2.6c
Average pupal
period (days)
2.0±0.3b
1.9±0.2b
0.6±0.5a
0.2±0.3a
6.1±0.6c
1.8±1.0b
0.4±0.4a
0.1±0.1a
2.7±0.2c
1.1±0.7b
0.0±0.0a
Pupal
mortality (%)
6.5±3.8*
10.5±4.4
10.0±4.3
4.0±2.8
2.0±2.3a
53.5±18c
53.5±10c
23.0±6.2b
9.0±2.0a
25.0±12.5b
3.0±2.6a
Total average
development (days)
0 5.0±0.1c
4.3±0.8bc
2.8±1.6ab
1.3±0.5a
7.1±0.1d
6.6±0.2c
4.8±0.2b
1.7±0.5a
4.2±0.4c
3.4±0.6b
0.6±0.5a
6.98±0.3b
6.16±0.7b
3.31±2.0a
1.55±1.1a
13.20±0.6d
8.36±1.2c
5.27±0.4b
1.82±0.5a
6.9±0.6c
4.5±0.6b
0.38±0.5a
25
50
75
0
25
50
75
Lambda-
cyhalothrin
0
25
75
Callitris
glaucophylla

Table 1. Effects of sub-lethal concentrations of fenitrothion, lambda-cyhalothrin, and Callitris glaucophylla extract on
juvenile Aedes aegypti development and mortality. Early 4th instar larvae were exposed continuously until adulthood.
Values for the same insecticide followed by a different letter within the same column are statistically different (P<0.05).
* Group not significant.

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Journal of Vector Ecology
297Vol. 30, no. 2
in most measured categories. The LC75 produced exceptional
results, killing all larvae and pupae and preventing any
emergence.
Overall, there was no significant difference in the sex of
emergent mosquitoes and malformations, for the most part,
were uncommon and not related to dose. Mortality
experienced by emergent adults was not significant except in
the C. glaucophylla LC25 dose where a third perished.
DISCUSSION
The tested synthetic insecticides and botanical extract
induced a wide range of sub-lethal effects on larval mortality,
larval duration, pupicidal activity, pupal duration, adult
emergence, sex ratio, adult mortality, and malformation.
The insecticide concentrations estimated to cause 25, 50,
and 75 % larval mortality in 24 h did not produce expected
mortalities with LC25 doses consistently causing lower
mortality and LC75 doses causing higher mortality in
fenitrothion and C. glaucophylla. The Probit estimation of
LC25 and LC75 doses are less accurate unless large data sets
are used, which is why publications typically report LC50s
and not LC90s. Pupal mortality did not exhibit a linear
relationship with the applied sub-lethal concentrations but was
more associated with lower doses. Pupal mortality was lower
than controls at the LC75 dose in fenitrothion and C.
glaucophylla, but lambda-cyhalothrin was more effective
against pupae. Adult mortality results did not present anything
of note. Dead adults were mostly half-ecdysed adults. Total
mortality was consistently positively correlated with
insecticide concentrations and the duration of exposure
(Marcard et al. 1986).
Beside immediate toxic larvicidal effects, all insecticides
significantly reduced the average larval period compared to
controls and, to a large extent, with each other. Larvae were
observed to pupate faster as their environment increased in
toxicity. This is clearly a self-preservation mechanism since
the pupal form is less susceptible to the environment. All
concentrations markedly disrupted pupal duration except for
the fenitrothion LC25 dose. Consequently, the average
development period (a factor in the Growth Index formula)
was consistently negatively correlated with insecticide
concentrations and the duration of exposure. This effect can
vary, however, with some researchers showing no effect on
the larval and pupal developmental periods (Saxena et al.
1993, Sharma and Saxena 1994) and other researchers
showing prolongation of the larval and pupal developmental
periods (Karmegam et al. 1997, Saxena and Yadav 1983,
Zebitz 1984, Saxena and Sumithra 1985, Mwangi and
Rembold 1988, Robert and Olson 1989, Mohsen et al. 1990a,
b ; Pushpalatha and Muthukrishnan 1995, Pushpalatha and
Muthukrishnan 1999). In another study, Melia volkensii was
observed to prolong the lifespan of An. arabiensis larvae but
not the pupal period (Mwangi and Mukiama 1988).
Conversely, Supavarn et al. (1974) reported on 11 of 36
botanicals that significantly inhibited pupal development while
only a few botanicals affected larval development.
Successful adult emergence is conversely proportional
with the insecticide concentration and larval mortality. Of note,
C. glaucophylla completely inhibited adult emergence
compared to fenitrothion (2 – 8 %) and lambda-cyhalothrin
(2 – 4 %) at the LC75 dose. This effect is expected since several
studies have shown that botanical extracts either reduce or
inhibit adult emergence. For instance, Descurainia sophia
inhibited Cx. quinquefasciatus emergence (Mohsen et al.
1990b) and Tagetes erectes significantly reduced adult
emergence in An. stephensi (Sharma and Saxena 1994).
Changes in the sex ratio of emergent adults tended
towards favoring females, however, results were not
significantly different. This is not always the case, since Robert
Insecticide
Fenitrothion



LC Adult mortality (%) Adult emergence (%) Emergent females
54.0±9.4c
39.0±8.2b
Emergent males
39.5±7.7b
33.5±6.4b
7.5±12.4a
0.5±1.0a
Malformations
0.0±0.0*
2.5±1.9
3.3±3.6
1.0±2.0
Growth Index
6.72±0.5b
5.86±0.6b
1.21±1.3a
0.47±0.7a
0 0.0±0.0*
1.5±1.9
0.5±1.0
0.5±1.0
95.5±3.8c
72.5±13.3b
11.5±17.8a
2.5±3.8a
25
50
75
4.0±5.4a
2.0±4.0a
Lambda-
cyhalothrin



0 4.0±5.7b
13.5±3.0ab
7.25±6.7a
0.5±1.0a
94.0±6.9c
26.5±15.9b
6.0±49a
1.5±1.9a
55.5±8.9c
20.0±10.5b
5.0±4.2a
1.0±1.2a
43.0±14.1b
6.5±6.8a
1.0±2.0a
0.5±1.0a
0.0±0.0a
0.0±0.0a
0.5±1.0a
5.6±3.0b
7.1±0.3 c
3.0±1.5b
1.1±0.9a
0.7±0.9a
25
50
75
Callitris
glaucophylla


0 7.5±5.3a
30.5±19.2b
0.0±0.0a
81.5±5.3c
35.0±26.1b
0.0±0.0a
45.0±2.6c
22.5±16.0b
0.0±0.0a
36.5±3.0c
12.5±10.1b
0.0±0.0a
0.0±0.0*
0.0±0.0
2.0±2.3
11.8±1.2b
7.7±6.0b
0.0±0.0a
25
75

Table 2. Effects of sub-lethal concentrations of fenitrothion, lambda-cyhalothrin, and Callitris glaucophylla extract on
adult Aedes aegypti development and mortality. Early 4th instar larvae were exposed continuously until adulthood.
Values for the same insecticide followed by a different letter within the same column are statistically different (P<0.05).
*Group not significant.
Growth Index = Adult emergence (%) / Average developmental period (days).

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Journal of Vector Ecology
December 2005
298
and Olson (1989) found a change in the sex ratio towards
more males in Cx. quinquefasciatus after sub-lethal exposure
propoxur and resmethrin.
The percentage of malformations was very low for all
insecticides. The only observed abnormalities were larval-
pupal intermediates, half-ecdysed adults, and adults with
malformed wings. These morphogenetic abnormalities are
commonly caused by botanical extracts and are thought to
result from a disturbance to growth regulating hormones
(Zebitz 1984; Mwangi and Mukiama 1988; Pereira and
Gurudutt 1990; Saxena et al. 1993).
The growth indices of larvae treated at LC50 and LC75
doses were markedly shorter than controls and LC25 doses for
all insecticides with negligible difference between controls
and LC25 doses. Similar results were obtained by Saxena and
Sumithra (1985) and Saxena et al. (1993), who found that the
GI of mosquitoes treated with Annona squamosa alkaloids
was longer in controls.
In conclusion, for most measured developmental effects,
the response was dose and exposure duration dependant.
Significant developmental effects were observed for
fenitrothion, lambda-cyhalothrin, and the botanical, C.
glaucophylla. The latter induced responses at a LC75 dose
that were exceptional and worthy of consideration for field
trials pending non-target assessment.
Acknowledgments
We are grateful to Dr. Michael Kennedy, Department of
Primary Industries, Queensland, Australia, for providing us
with the C. glaucophylla extract.
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