See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/225045219

Lethal Effects of Aspergillus niger against Mosquitoes Vector of Filaria,

Malaria, and Dengue: A Liquid Mycoadulticide

Article  in  The Scientific World Journal · May 2012

DOI: 10.1100/2012/603984 · Source: PubMed

CITATIONS READS

12 1,149

2 authors:

Gavendra Singh Soam Prakash

Dayalbagh Educational Institute Dayalbagh Educational Institute
19 PUBLICATIONS   175 CITATIONS    68 PUBLICATIONS   1,068 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Nanogold and Nanosilver for vecto control View project

Researcher View project

All content following this page was uploaded by Soam Prakash on 03 June 2014.

The user has requested enhancement of the downloaded file.

The Scientific World Journal
Volume 2012, Article ID 603984, 5 pages
doi:10.1100/2012/603984
The cientificWorldJOURNAL

Research Article
Lethal Effects of Aspergillus niger against Mosquitoes Vector of
Filaria, Malaria, and Dengue: A Liquid Mycoadulticide

Gavendra Singh and Soam Prakash

Environmental and Advanced Parasitology and Vector Control Biotechnology Laboratories, Department of Zoology, Faculty of Science,
Dayalbagh Educational Institute, Dayalbagh, Agra 282005, India

Correspondence should be addressed to Soam Prakash, [email protected]

Received 20 October 2011; Accepted 9 January 2012

Academic Editor: Tatiana Scorza

Copyright © 2012 G. Singh and S. Prakash. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Aspergillus niger is a fungus of the genus Aspergillus. It has caused a disease called black mold on certain fruits and vegetables.
The culture filtrates released from the A. niger ATCC 66566 were grown in Czapek dox broth (CDB) then filtered with flash
chromatograph and were used for the bioassay after a growth of thirty days. The result demonstrated these mortalities with LC50 ,
LC90 , and LC99 values of Culex quinquefasciatus 0.76, 3.06, and 4.75, Anopheles stephensi 1.43, 3.2, and 3.86, and Aedes aegypti
1.43, 2.2, and 4.1 µl/cm2 , after exposure of seven hours. We have calculated significant LT90 values of Cx. quinquefasciatus 4.5,
An. stephensi 3.54, and Ae. aegypti 6.0 hrs, respectively. This liquid spray of fungal culture isolate of A. niger can reduce malaria,
dengue, and filarial transmission. These results significantly support broadening the current vector control paradigm beyond
chemical adulticides.

1. Introduction ing isolate selection, optimisation of production, and for-

mulation is required. While many successful laboratory eval-
Rapidly emerging insecticide resistance is creating an urgent uations of the efficacy of entomopathogenic fungi have
need for new active ingredients to control the adult mosqui- been conducted [2, 7–9]. Therefore, more research on fun-
toes [1]. A range of isolates belonging to the fungal species gal formulations and evaluating of various formulations,
Metarhizium anisopliae and Beauveria bassiana have been delivery techniques remains essential against mosquitoes.
shown to infect and significantly reduce the longevity of Aspergillus niger is a filamentous ascomycete fungus that
adult Anopheles mosquitoes, killing them within 14 days is ubiquitous in the environment and has been implicated
[2–4]. The Beauveria bassiana has infected mosquitoes of in opportunistic infections of humans [10]. A. niger is most
the insecticide resistant Anopheles arabiensis at two different widely known for its role as a citric acid producer [11]. With
temperatures [5]. The fungi have been applied by spraying on production of citric acid at over one million metric tons
mosquitoes with an oil formulation of infectious spores. The annually, A. niger citric acid production serves as a model
fungal spores begin pathogenic and invade the mosquitoes, fungal fermentation process. This organism is a soil saprobe
after which the fungus multiplies and kills its host within two with a wide array of hydrolytic and oxidative enzymes
weeks [2]. Similarly, the isolates of Metarhizium anisopliae involved in the breakdown of plant lignocellulose. A variety
ICIPE-30 and Beauveria bassiana I93-825 (IMI 391510) have of these enzymes from A. niger is important in the biotech-
reduced mosquito survival on immediate exposure and up nology industry. The A. niger is also an important model
to 28 days after application [6]. The critics have argued organism for several important research areas including the
that “slow acting” these fungal biopesticides is, therefore, study of eukaryotic protein secretion in general, the effects of
incapable of delivering mosquito control in different parts various environmental factors on suppressing or triggering
of the world. The entomopathogenic fungi can be integrat- the export of various biomass degrading enzymes, molecular
ed into control programmes additional information regard- mechanisms critical to fermentation process development,
2 The Scientific World Journal

(a) (b)

Figure 1: The cultures of Aspergillus niger: (a) solid medium on Czapek dox agar (CDA), (b) liquid medium Czapek dox broth maintained
in the laboratory.

and mechanisms involved in the control of fungal morphol- The conical flasks, inoculated with A. niger, were incubated
ogy. These are encouraging characteristics which encourage at 25◦ C for 30 days (Figure 1).
for further research on mosquitoes control.
Mosquito vectors are solely responsible for transmitting
2.2. Preparation of Flash Chromatograph Columns and Fil-
diseases, such as malaria, dengue, chikungunya, Japanese en-
tration. In the Flash chromatograph, a plastic column was
cephalitis, yellow fever, and lymphatic filariasis [12]. Malaria
filled with silica gel, with the sample to be separated placed
is an important cause of death and illness in children and
on top of this support. The rest of the column is filled
adults, especially in tropical countries. Malaria is caused by
with an isocratic or gradient solvent which, with the help of
a parasite that is transmitted from one human to another by
pressure, enables the sample to run through the column and
the bite of infected Anopheles stephensi. Half of the world’s
become separated. Flash chromatography used air pressure
population is at risk from malaria. Each year almost 250 mil-
initially to speed up the separation. The culture filtrates were
lion cases occur, causing 860000 deaths [13]. Approximately
obtained by filtering the broth through Whatman no.1 filter
3.5 billion people live in dengue endemic countries which are
paper. These metabolites were further filtered with the flash
located in the tropical and subtropical regions of the world
chromatograph.
[14]. Lymphatic filariasis, commonly known as elephantiasis,
is so far a neglected tropical disease. The infection occurs
when filarial parasites are transmitted to humans through 2.3. Bioassays. The flash chromatograph purified culture
Culex quinquefasciatus. More than 1.3 billion people in filtrates were used for bioassays against laboratory reared Cx.
eighty-one countries worldwide are threatened by lymphatic quinquefasciatus, Ae. aegypti, and An. stephensi as per the
filariasis [15]. In the present investigation, we have reported standard procedures recommended by World Health Orga-
the lethal effects of purified culture filtrates of A. niger against nization with some modifications [16]. The freshly emerg-
An. stephensi, Cx. quinquefasciatus, and Ae. aegypti in the ed three-day-old sugar fed adults were used for the assay. The
laboratory. five different volumes of 1.6, 2.2, 2.7, 3.2, and 3.8 µL/cm2
of metabolites were sprayed in a cage (25 cm length ×
15 cm width × 5 cm depth) containing 25 mosquitoes. The
2. Materials and Methods exposed mosquitoes were kept under observation, and dead
mosquitoes were discarded daily. Each bioassay including
2.1. Collection and Culture of Aspergillus niger. The strain of
control was conducted in triplicate on different days. In the
Aspergillus niger (ATCC 66565) was obtained from Microbial
control cages deionized water was sprayed. Daily mortality
Type Culture Collection and Gene Bank (MTCC 2587) Insti-
counts were performs. The bioassays were carried out at
tute of Microbial Technology, Chandigarh, India. A. niger
room temperature with 75 ± 5% relative humidity. The nega-
was maintained on autoclaved Czapek dox broth (sucrose:
tive control was deionized water with 1% CDB while the po-
30.0 g, sodium nitrate: 3.0 g, dipotassium phosphate: 1.0 g,
sitive control was Gokilaht-S 5EC (d,d-trans-cyphenothrin).
magnesium sulphate: 0.05 g, potassium chloride: 0.05 g, fer-
rous sulphate: 0.01 g, deionized water: 1000 mL) and adjusts
pH 6.5. The broth was supplemented with 50 µg/mL chlo- 2.4. Statistical Analysis. The efficacy study of the filtrate
ramphenicol as a bacteriostatic agent. The colonies of A. niger metabolites of A. niger was assessed against Cx. quinquefas-
were grown on Czapek dox agar (CDA), solid medium plates ciatus, Ae. aegypti, and An. stephensi by probit analysis [17]
were transferred to each flask using an inoculation needle. with the statistical package IBM SPSS 19.0.
The Scientific World Journal 3

3. Results and Discussion 100

90
80

Lethality (%)
In the present observations, we have evaluated the lethal 70
60
effects of culture filtrates of A. niger against adult mosquitoes. 50
The lethal effects of A. niger with LC50 , LC90 , and LC99 values 40
30
of Cx. quinquefasciatus were 0.76, 3.06, and 4.751 µL/cm2 . 20
Moreover, in case of the An. stephensi it was observed as 10
0
1.43, 3.2, and 3.86. While in case of Ae. aegypti it was re- 0 1 2 3 4 5 6 7
corded as 1.43, 2.2, and 4.1 µL/cm2 . These values were calcu- Hours after exposures
lated after the exposure of seven hours along with their
probit quotations (Table 1). The entomopathogenic fungus Culex quinquefasciatus Anopheles stephensi
has been successfully reduceing mosquito vectors population Aedes aegypti Positive control
in laboratories and field trials [2–4, 18]. The fungal infections Figure 2: Effect of culture filtrates of Aspergillus niger against
for the mosquitoes become increasingly sick and are eventu- Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti after
ally killed, but the process can take up to a week or more. The exposure of 7 hours.
adult mosquitoes pick up the fungal spores when resting on
treated surfaces.
Unlike fast-acting chemical neurotoxins, fungal patho- 100
gens do not cause rapid mortality or immediate “knock- 95
down” but rather act over a number of days as the fungal 90

Lethality (%)
spores penetrate the insect cuticle and then proliferate within 85
the hemocoel [19]. The A. clavatus has been found highly 80
75
pathogenic against larvae of Ae. aegypti, Cx. quinquefasciatus, 70
and An. gambiae [20]. The mortality rates were 100% against 65
both Ae. aegypti, and Cx. quinquefasciatus, while against 60
An. gambiae it was 95% after 24 hours. The entomopatho- 3 4 5 6 7
genic fungus B. bassiana has used as an alternative vector Doses (µL/cm2 )
control tool against insecticide-resistant mosquitoes under
Culex quinquefasciatus Aedes aegypti
conditions typical of indoor resting environments [5]. A Positive control
Anopheles stephensi
range of fungal-based insecticide combinations was used to
test effects of timing and sequence of exposure. Both the Figure 3: Effect of culture filtrates of Aspergillus niger against Culex
laboratory-reared and field-collected mosquitoes were highly quinquefasciatus, Anopheles stephensi and Aedes aegypti at different
resistant to permethrin but susceptible to B. bassiana and M. concentrations.
anisopliae infection, inducing 100% mortality within nine
days. Simultaneous coexposure induced the highest morta-
lity, up to 70.362% for a combined Beauveria and permethrin variety of toxic metabolites, which vary from low molecular
exposure within a time range of one gonotrophic cycle (4 weight products of secondary metabolism to complex cyclic
days) [21]. In present investigation the lethal time effect of A. peptides and proteolytic enzymes [23]. A significant progress
niger with LT50 and LT90 values of Cx. quinquefasciatus 2.57, has been made in understanding enzymes involved with the
4.5, An. stephensi 1.58, 3.54, and Ae. aegypti 1.65, 6.0 hrs were penetration of host cuticle and the role of mosquitocidal
calculated (Table 1). At the first time for increase in percent toxins. The fungal metabolites can be more effective by joint
mortalities, a combination of an insecticide and an ento- action of numerous toxins and enzymes.
mopathogenic fungus has been tested against Ae. aegypti. It The A. niger is the best producer of extracellular lipase
can be an alternative to applications of high concentrations [24]. The present study shows that the A. niger purified fun-
of chemical insecticides. The Ae. aegypti could be controlled gal culture filtrates have enhanced their lethal effects against
by surface application of entomopathogenic fungi and that An. stephensi, Cx. quinquefasciatus, and Ae. aegypti. More-
the efficiency of these fungi increased by combining the fungi over, the presence of mycotoxin “ochratoxin” in A. niger can
with ultra-low concentrations of insecticides, resulting in be fast-acting metabolites for control of adult mosquitoes.
higher mortality following relatively short exposure times Ideally, all these new findings could be implemented with
[22]. a time application with its fast acting impact against An.
This study distinctly demonstrates that the A. niger cul- stephensi, Cx. quinquefasciatus, and Ae. aegypti populations.
ture filtrates have induced a higher impact on adult mosqui- This investigation can be further improved by implementing
toes with significant percent of mortalities (Figure 2). The enhanced fungus-based strategy to control the adult popula-
applied concentrations have affected Cx. quinquefasciatus, tion. In our laboratories, Trichophyton ajelloi, Chrysosporium
An. stephensi, and Ae. aegypti with relevant LC50 , LC90 and lobatum, C. tropicum, Lagenidium giganteum, Culicinomyces
LC99 values after exposure of seven hours (Figure 3). The clavisporus, and Fusarium oxysporum have so far been succes-
recorded lethal effects show the potential for integrated fun- sfully screened and were found pathogenic against larvae and
gus control measures to dramatically reduce malaria, filarial, adults of An. stephensi, Cx. quinquefasciatus, and Ae. aegypti.
and dengue vectors. The pathogenic fungi produce a wide These fungal strains have shown lethal effect after exposure of
 4

Table 1: Adulticidal activities of culture filtrates of Aspergillus niger against Culex quinquefasciatus (Say), Anopheles stephensi (Liston), and Aedes aegypti (Lin.).
Concentrations Lethality (%) LC50 LC90 LC99 LT50 LT90
(µL/cm2 ) Culex quinquefasciatus Anopheles stephensi Aedes aegypti (µL/cm2 )c (µL/cm2 )c (µL/cm2 )c (hrs)d (hrs)d
0.0 0.0 0.0 0.0 Culex quinquefasciatus 0.76 3.06 4.75 2.57 4.5
1.6 73.3 75 85 y = 0.81 + 8.58x (0.11–1.36) (2.48–3.64) (4.15–5.49) (1.32–3.82) (3.29–5.85)
2.2 80 80 90 Anopheles stephensi 1.43 3.2 3.86 1.58 3.54
2.7 86.6 85 90 y = 0.14 + 8.82x (0.86–2.0) (2.62–3.77) (3.29–4.33) (0.41–2.75) (2.37–4.41)
3.2 89 90 95 Aedes aegypti 1.43 2.2 4.1 1.65 6.0
3.8 93.8 95 95 y = 0.19 + 9.23x (0.816–1.92) (1.77–2.93) (3.8–4.91) (0.61–2.69) (4.96–7.04)
Gokilaht-S 5ECa 100 100 100
Deionized water +CDb 00 00 00
a
Positive control.
b Negative control.
c Lethal concentrations with the corresponding 95% confidence level.
d Lethal time with the corresponding 95% confidence level.
The Scientific World Journal
The Scientific World Journal 5

24, 42, and 72 hours. Consequently, A. niger has found effec- of aspergillosis,” Clinical Infectious Diseases, vol. 33, no. 11, pp.
tive in very short time. This unique property of this fungus 1824–1833, 2001.
requires further field testing in different climatic zones. [11] J. Magnuson and L. Lasure, “Organic acid production by
filamentous fungi,” in Advances in Fungal Biotechnology for In-
dustry, Agriculture, and Medicine, J. Tkacz and L. Lange, Eds.,
Conflict of Interests pp. 307–340, Plenum; Kluwer Academic, New York, NY, USA,
2004.
The authors do not have any conflict of interests. [12] J. A. Rozendaal, Vector Control: Methods for Use by Individuals
and Communities, WHO, Geneva, Switzerland, 1997.
[13] World Health Organization, “World Malaria Report,” 2010.
Acknowledgments [14] World Health Organization, “Lymphatic filariasis,” 2011,
http://www.who.int/mediacentre/factsheets/fs102/en/.
The authors thank M/s. Sumitomo Chemicals India Pvt. Ltd.
[15] World Health Organization, “Guidelines on the quality, safety
for supply of Gokilaht-S 5EC. They thanking Prof. V. G. and efficacy of dengue tetravalent vaccine (live attenuated),”
Das, Director, Dayalbagh Educational Institute, Agra for his WHO/DRAFT, (DEN) 1–93, May 2011.
encouragements. They are also thankful to the University [16] World Health Organization, “Guidelines for testing mosqui-
Grants Commission, New Delhi, of Major Research Project to adulticides for indoor residual spraying and treatment of
(MRP/Soam Prakash) for the financial support 2010–2012 mosquito nets,” WHO/CDS/NTD/WHOPES/GCDPP/2006.3,
and to DST-FIST program (2003–2008) for providing labo- 2006.
ratory facilities. G. Singh is indebted to UGC, New Delhi, for [17] D. J. Finney, Probit Analysis, Cambridge University Press,
an award of Postdoctoral Fellowship (2009–2011). Cambridge, UK, 3rd edition, 1971.
[18] S. S. Mohanty and S. Prakash, “Effects of culture media on lar-
vicidal property of secondary metabolites of mosquito patho-
References genic fungus Chrysosporium lobatum (Moniliales: Monili-
aceae),” Acta Tropica, vol. 109, no. 1, pp. 50–54, 2009.
[1] S. Blanford, W. Shi, R. Christian et al., “Lethal and pre-lethal [19] M. B. Thomas and A. F. Read, “Can fungal biopesticides con-
effects of a fungal biopesticide contribute to substantial and trol malaria?” Nature Reviews Microbiology, vol. 5, no. 5, pp.
rapid control of malaria vectors,” PLoS ONE, vol. 6, no. 8, 377–383, 2007.
article e23591, 2011. [20] F. Seye, O. Faye, M. Ndiaye, E. Njie, and J. M. Afoutou, “Patho-
[2] S. Blanford, B. H. K. Chan, N. Jenkins et al., “Fungal pathogen genicity of the fungus, Aspergillus clavatus, isolated from the
reduces potential for malaria transmission,” Science, vol. 308, locust, Oedaleus senegalensis, against larvae of the mosquitoes
no. 5728, pp. 1638–1641, 2005. Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus,”
[3] E. J. Scholte, B. N. Njiru, R. C. Smallegange, W. Takken, and B. Journal of Insect Science, vol. 9, no. 53, 2009.
G. J. Knols, “Infection of malaria (Anopheles gambiae s.s.) and [21] M. Farenhorst, B. G. J. Knols, M. B. Thomas et al., “Synergy
filariasis (Culex quinquefasciatus) vectors with the entomo- in efficacy of fungal entomopathogens and permethrin against
pathogenic fungus Metarhizium anisopliae,” Malaria Journal, west african insecticide-resistant anopheles gambiae mosqui-
vol. 2, no. 1, article 29, 2003. toes,” PLoS ONE, vol. 5, no. 8, article e12081, 2010.
[4] E. J. Scholte, K. Ng’Habi, J. Kihonda et al., “An entomopatho- [22] A. R. Paula, A. T. Carolino, C. O. Paula, and R. I. Samuels, “The
genic fungus for control of adult African malaria mosquitoes,” combination of the entomopathogenic fungus Metarhizium
Science, vol. 308, no. 5728, pp. 1641–1642, 2005. anisopliae with the insecticide Imidacloprid increases viru-
[5] C. K. Kikankie, B. D. Brooke, B. G. J. Knols et al., “The infecti- lence against the dengue vector Aedes aegypti (Diptera: Culici-
vity of the entomopathogenic fungus Beauveria bassiana to dae),” Parasites and Vectors, vol. 4, no. 1, article 8, 2011.
insecticide-resistant and susceptible Anopheles arabiensis mos- [23] A. L. Demain and A. Fang, “The natural functions of sec-
quitoes at two different temperatures,” Malaria Journal, vol. 9, ondary metabolites,” in Advances in Biochemical Engineering
no. 1, article 71, 2010. Biotechnology: History of Modern Biotechnology, Th. Scheper,
[6] L. L. Mnyone, M. J. Kirby, D. W. Lwetoijera et al., “Tools Ed., vol. 69, pp. 1–39, Springer, Heidelberg, Germany, 2000.
for delivering entomopathogenic fungi to malaria mosquitoes: [24] G. Falony, J. C. Armas, J. C. D. Mendoza, and J. L. M.
effects of delivery surfaces on fungal efficacy and persistence,” Hernández, “Production of extracellular lipase from Aspergil-
Malaria Journal, vol. 9, no. 1, article 246, 2010. lus niger by solid-state fermentation,” Food Technology and Bi-
[7] S. Prakash, G. Singh, N. Soni, and S. Sharma, “Pathogenicity of otechnology, vol. 44, no. 2, pp. 235–240, 2006.
Fusarium oxysporum against the larvae of Culex quinquefascia-
tus (Say) and Anopheles stephensi (Liston) in laboratory,” Para-
sitology Research, vol. 107, no. 3, pp. 651–655, 2010.
[8] G. Singh and S. Prakash, “Efficacy of Lagenidium giganteum
(Couch) metabolites for control Anopheles stephensi (Liston) a
malaria vector,” Malaria Journal, vol. 9, supplement 2, article
P46, 2010.
[9] G. Singh and S. Prakash, “Evaluation of culture filtrates of
Culicinomyces clavisporus: mycoadulticide for Culex quinque-
fasciatus, Aedes aegypti and Anopheles stephensi,” Parasitology
Research, vol. 110, no. 1, pp. 267–272, 2012.
[10] J. R. Perfect, G. M. Cox, J. Y. Lee et al., “The impact of
culture isolation of Aspergillus species: a hospital-based survey

View publication stats