A Review of the Dengue Mosquito, Aedes aegypti (Diptera:

Culicidae), in Australia
Deon Canyon (PhD)
Tropical Infectious and Parasitic Diseases Unit, School of Public Health and Tropical Medicine, James Cook
University, Townsville Qld 4811, Australia. Deon.Canyon@jcu.edu.au

Biological control with B.t.i.

While numerous biological control agents can kill mosquitoes, only one biological agent,
Bacillus thuringiensis subsp. israelensis (B.t.i.), has seen widespread operational use for the
control of mosquito larvae. B.t.i., a spore forming Bacilli, was discovered in a stagnant pond
located in the Nahal Besor Desert river basin near Kibbutz Zeelim in the north-western Negev
Desert of Israel (Margalit, 1990) in 1976 by Goldberg and Margalit (1977). de Barjac (in Nugud
and White, 1982) identified the new bacterial strain and designated it B. thuringiensis subsp.
israelensis serotype H14. Many studies, such as those by Lacey and Lacey (1981), Nugud and
White (1982), Lacey and Inman (1985), Kramer et al. (1988), Tietze et al. (1994), and Chui et al.
(1995), have evaluated and confirmed the larvicidal capacity of B.t.i. for many mosquito species
in different parts of the world.
Thomas and Ellar (1983) describe the mode of action of B.t.i. endotoxins. B. thuringiensis
produces a proteinaceous parasporal crystalline inclusion during sporulation. When this is
ingested by an insect, it solubilizes in the midgut and releases proteins called d-endotoxins.
These proteins disrupt internal membranes and result in death. In addition to B.t.i.s effectiveness
against arthropods, the World Health Organization (1995) reports an excellent safety record and a
very low mammalian toxicity: LD50 values for both oral and dermal toxicity are more than
30,000mg/kg.
Most studies on Bacillus thuringiensis resistance development have been done with
Lepidopteron populations and considerably fewer have been done on mosquitoes (Tabashnick,
1994). Such studies were undertaken because of the fear that resistance to B.t.i. would develop
quickly as it had to other insecticides. In an effort to examine the possibility of resistance
occurring and the speed at which it would occur in mosquito populations, Goldman et al. (1986)
attempted to artificially select wild and laboratory populations of Ae. aegypti for resistance to
B.t.i.. After 14 generations, one wild strain from Rio de Janeiro, Brazil had developed a slight,
but significant twofold increase in resistance. A study by Becker and Ludwig (1993) on B.t.i.
resistance in Ae. vexans (Meigen) field populations that had sustained 10 years of B.t.i. control,
revealed change in susceptibility. The future for B.t.i. is promising since the development of
resistance to B.t.i. in mosquitoes is by no means comparable to the speedy and encompassing
growth of resistance to organochorine, organophosphate, carbamate and synthetic pyrethroid
insecticides.

Memrias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276

Mem. Inst. Oswaldo Cruz vol.96 no.6 Rio de Janeiro Aug. 2001
doi: 10.1590/S0074-02762001000600020
SHORT COMMUNICATION
Evaluation of a New Tablet Formulation Based on Bacillus thuringiensis sorovar. israelensis for
Larvicidal Control of Aedes aegypti
Vol. 96(6): 859-860, August 2001
Maria Alice Varjal de Melo-Santos/+, Elizabeth Gomes Sanches*, Fernando Justino de Jesus*, Lda
Regis
Departamento de Entomologia, Centro de Pesquisas Aggeu Magalhes-Fiocruz, Caixa Postal 4742,
50670-420 Recife, PE, Brasil *Far-Manguinhos-Fiocruz, Rio de Janeiro, RJ, Brasil
The effect of sunlight on the efficacy and persistence of an experimental tablet formulation based on
Bacillus thuringiensis sorovar. israelensis (C4P1) was evaluated against Aedes aegypti larvae under
simulated field conditions. The initial mortality ranged from 93 to 100%, and the residual activity ( 70%
mortality) recorded in containers exposed to sunlight or shade were, respectively, 13-35 days and 40-54
days. The results suggest that C4P1 can provide long-term larvicidal effect and operational advantages.
Key words: Bacillus thuringiensis israelensis - Aedes aegypti - tablet formulation

Bacillus thuringiensis sorovar. israelensis (B.t.i) has been used since the 80's in several programs for
controlling black fly and mosquito species worldwide. It is considered highly toxic to the target organisms
and environmentally safe. Furthermore, no resistance has been detected on populations submitted to
long-term exposure to this pathogen (Regis & Nielsen-LeRoux 2000). B.t.i formulations have been
continuously improved to optimise their potency, stability (shelf life), ease of field application and residual
activity (Couch 2000). Trials under simulated field conditions have been considered a reliable
methodology to evaluate the efficacy and persistence of B.t.i based products because, under these
conditions, many factors involved in residual activity can be better controlled and investigated than under
real field conditions (Thiry et al. 1999, Regis et al. 2000).

J Am Mosq Control Assoc. 1995 Mar;11(1):72-6.

Comparative toxicity of selected larvicides and insect

growth regulators to a Florida laboratory population of
Aedes albopictus.
Ali A, Nayar JK, Xue RD.
University of Florida, IFAS, Central Florida Research and Education Center, Sanford 327719608, USA.
Five organophosphates (OPs) (chlorpyrifos, chlorpyrifos methyl, fenthion, malathion, and
temephos), 3 pyrethroids (bifenthrin, cypermethrin, and permethrin), and 2 microbial pesticides
(Bacillus thuringiensis serovar.israelensis [B.t.i.] and Bacillus sphaericus) were tested as
larvicides against a Florida Aedes albopictus population colonized in the laboratory. In addition,
3 insect growth regulators (IGRS) (diflubenzuron, methoprene, and pyriproxyfen) were
evaluated. All OPs, except for malathion, were highly effective as indicated by low LC90s
ranging from 0.0069 ppm (chlorpyrifos) to 0.026 ppm (fenthion); the larvae were considered
tolerant to malathion (LC90 = 1.043 ppm). LC90 values of pyrethroids were: 0.0175 ppm
(bifenthrin), 0.0079 ppm (cypermethrin), and 0.0031 ppm (permethrin). Commercial products of
B.t.i., Vectobac and Bactimos were considered economically effective against Ae. albopictus
larvae but products of B. sphaericus were ineffective (LC90s > 28 ppm). The IGRs showed
exceptional activity. Pyriproxyfen (LC90 = 0.000376 ppm), was 2.23 and 21.5 times more toxic
than diflubenzuron and methoprene, respectively. In general, toxicity ranking of chemicals and
microbials tested was: IGRs > pyrethroids > OPs > microbials.
PMID: 7616194 [PubMed - indexed for MEDLINE]

Effectiveness of aerial application of VectoBac G larvicide granules against mosquitoes in the

Olomouc region in spring 2006
Authors: J Chmela, L Maznek, Z Nakldal, L Peskov, R Halirov
Journal: Epidemiologie, mikrobiologie, imunologie: casopis Spolecnosti pro epidemiologii a
mikrobiologii Ceske lekarske spolecnosti J.E. Purkyne

In the spring 2006, a flood emergency occurred in the Olomouc region after a rapid snow thaw,
with the formation of multiple periodic pools and overflows persisting for long periods. In the
floodplain forests, as expected, mass occurrence of adult mosquitoes was observed, infesting the
nearby villages. The mosquito emergency continued until August and mosquitoes of both the
spring species, in particular, Ochlerotatus cantans, and summer species were implicated in it. To
control this emergency, aerial application of VectoBac G larvicide granules based on Bacillus
thuringiensis var. israelensis (BTI) at a dose of 6.4 kg/ha was used for the first time in the
Olomouc region. Its efficacy was close to 100 % for the treated water areas. The effect was
clearly evident in the communities situated in the vicinity of the treated breeding grounds.
Nevertheless, not all breeding grounds and blind stream meanders could be treated aerially. As a
result, mosquitoes from the untreated breeding grounds caused an emergency in the nearby
communities where insecticide aerosols had to be applied on a large scale, even repeatedly in
some cases. The treatment of the mosquito breeding grounds with BTI based larvicide granules
proved to be an effective and fully selective approach to controlling mosquitoes. Although the
technology is exacting and requires professional and organizational skills, it is expected that, in
the Czech Republic, this environmentally friendly approach to mosquito control will replace the
large scale use of non-selective chemical aerosol insecticides. The public health protection,
administrative and legislative authorities should be active in promoting the use of specific BTI
based larvicides for the treatment of mosquito breeding grounds, even if located in protected
areas, whenever it is needed for mosquito control in emergencies.

Journal of General Microbiology (1988), 134, 2551-2558. Printed in Great Britain 255 1

Comparative Toxicity of Bacillus thuringiensis var. israelensis

Crystal
Proteins in aiuo and in tritvo
By CHRIS N . CHILCOTT*T AND DAVID J . ELLAR
Department of Biochemistry, University of Cambridge, Tennis Court Road,
Cambridge CB2 l Q W, UK
(Received 19 January 1988; revised 9 May 1988)
Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q
column
to yield 130,65,28,53,30-35 and 25 kDa proteins. All the purified proteins killed Aedes
aegypti
larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated
mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays
against
a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally
cytotoxic with the lowest LCs0 values in vitro. By contrast, the activated forms of the 130
kDa
and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more
specific
than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity
profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and
Culex cell
lines.

Titre du document / Document title

Efficacy of a new formulation of bacillus thuringiensis var israelensis (Bti) in laboratory and
field conditions of Kumaun foothills of Uttaranchal, India.

Auteur(s) / Author(s)
SHARMA S. N. ; SHUKLA R. P. ; MITTAL P. K. ; ADAK T. ; KUMAR A. ;

Rsum / Abstract
Efficacy of a new Bti formulation was tested in the laboratory and small scale field trials against
mosquito larvae were carried out from March to June, 2001 in different breeding habitats of
Kumaun foothill region of Nainital and Udham Singh Nagar districts, Uttaranchal state, India.
Laboratory tests revealed increased efficacy against Culex quinquefasciatus (LC50 = 0.035 mg/L)
followed by Aedes aegypti (LC50 = 0.0628 mg/L), An. culicifacies sp A (LC50 = 0.184mg/L) and
An. stephensi (LC50 - 0.2216mg/L). Results of field trials revealed 100% mortality of mosquito
larvae after treatment with Bti @ 0.5 gm/m2 surface area. The impact was similar on different
mosquito species breeding in different habitats. However, repeated treatments were required due
to reappearance of larvae in the breeding habitats within a week. No side effects of Bti was
observed during field trial on non- target organisms (NTOs). Environmental disturbances and
man made problems affected the spray impact of Bti.

Revue / Journal Title

Journal of communicable diseases ISSN 0019-5138 CODEN JCDSBF

Source / Source
2003, vol. 35, no4, pp. 290-299 [10 page(s) (article)]

Langue / Language
Anglais

Editeur / Publisher

Indian Society for Malaria and Other Communicable Diseases, New Delhi, INDE (1969)
(Revue)

Localisation / Location
INIST-CNRS, Cote INIST : 13630, 35400012713930.0100

REPORT FOR THE MINISTRY OF HEALTH

Environmental and health impacts of
Bacillus thuringiensis israelensis
Bacillus thuringiensis israelensis sporulating cell during
formation of the crystal (TEM by J-P Charles, Institut Pasteur).
Travis R. Glare and Maureen O'Callaghan
Biocontrol & Biodiversity,
Grasslands Division, AgResearch
PO Box 60, Lincoln
July, 1998

1. Summary
Mosquito vectors of human disease pose a constant threat to New Zealand and
recent interceptions of exotic mosquitoes with the potential to vector serious
mammalian diseases has highlighted the need for agents for use in control
and/or eradication programmes.
Bacillus thuringiensis (Bt) strains and varieties are pathogenic to a number of
pests, including Lepidoptera and Diptera. The discovery of B. thuringiensis
israelensis (Bti), a variety specific to Diptera (especially mosquitoes and
blackflies) in Israel in 1978, has led to the development of many products
based on this bacterium. These products have been used extensively in

mosquito and biting fly control programmes, especially in Africa, USA and
Germany.
There is a well documented history of environmental safety of Bt strains used
in pest control. The environmental safety of Bt, coupled with the nature of
toxicity and level of specificity for target hosts, has led to the use of Bt in many
pest control programmes in environmentally sensitive areas, including the
eradication of tussock moth in New Zealand.
Naturally occurring Bt strains have been isolated from the New Zealand
environment, including strains similar to Bti.
However, the aquatic
environment has not been sampled for Bt, so the natural occurrence in New
Zealand waterways is unknown.
The mode of action of Bti involves the synergistic interaction of four toxic
proteins. Bti rarely recycles in natural environments and the insect toxicity is
due to crystal proteins formed during sporulation.
Aspects of the environmental impact which need be considered for any
pesticide include mammalian and non-target safety, effect on the environment,
persistence and occurrence in the natural environment and possible host
resistance. For microbial-based pesticides, such as Bt, gene transfer must also
be considered.
A review of the literature on host range and effect on non-target organisms
indicates that Bti is relatively specific to the Nematocera suborder of Diptera,
in particular filter-feeding mosquitoes (Culicidae) and blackflies (Simuliidae).
It has also been shown to be pathogenic to some species of midges
(Chironomidae) and Tipulidae, although usually to a lesser extent than
mosquitoes and biting flies.

 Bti has not been reported to affect a large number of other invertebrate species
including most aquatic fauna. It is not toxic to bees. Fish are not affected,
either in the laboratory or after field application.
Environmental and health impacts of Bacillus thuringiensis israelensis
Bti is considered to pose little threat to mammalian safety. Per os inoculations
of animals and humans have not resulted in clinical symptoms. Concerns have
been raised that the solubilised -endotoxin of Bti activated in the laboratory
was toxic to mice when administered by injection and cytolytic to human
erythrocytes. However, solubilisation occurs at high pH (such as in insect
guts) and does not occur in mammalian guts.
The close genetic relationship between B. thuringiensis and the occasional
human pathogen, B. cereus has raised concerns about possible implication of
Bt in human gastro-intestinal illnesses and other health problems caused by B.
cereus. However, no such effect has been found after extensive field use. A
specific identification system for Bt strains would assist monitoring of future
applications.
Bti does not persist in the environment after application. Generally, reports of
activity after application show a decline in efficacy within days and little
residual activity after several weeks. The persistence of Bti after application is
dependent on the type of formulation/product used, with some formulations
(pellets/briquettes) designed specifically to enhance residual activity.
Some of the toxic proteins of Bt are encoded by genes residing on extrachromosomal DNA (plasmids) which can be exchanged between strains and
species by conjugation and/or transformation. While genetic transfer between
Bt and other soil bacteria has been demonstrated in the laboratory (in culture,
insects and sterile soils), it has not been shown in the field. No unexpected

pathogenic organisms have resulted from extensive Bt application, suggesting

that while gene transfer may have implications for genetically modified strains,
it is a lesser concern for wild-type strains.
Some insects, especially Lepidopterans, have become resistant following
constant application of Bt strains. However, resistance has not occurred after
application of Bti, possibly due to the complex mode of action, involving
synergistic interaction between up to four proteins. Use of a single protein
from Bti for mosquito control resulted in resistance after only a few
generations in the laboratory. However, use of Bti for over 10 years in Africa,
USA and Germany has not resulted in development of resistance.
Over 40 tons of Bti were applied in west Africa alone, without any reports of
safety or non-target concerns. The environmental threat posed by Bti would
appear to be significantly less than that posed by most other forms of mosquito
control which have a similar level of efficacy.

Journal of the American Mosquito Control Association 21(1):84-89. 2005

doi: 10.2987/8756-971X(2005)21[84:IOBTHF]2.0.CO;2
INTEGRATION OF BACILLUS THURINGIENSIS H-14 FORMULATIONS AND
PYRIPROXYFEN FOR THE CONTROL OF LARVAE OF AEDES AEGYPTI AND
AEDES ALBOPICTUS

Y. W. Lee,1, J. Zairi, H. H. Yap, and C. R. Adanan

Vector Control Research Unit, School of Biological Sciences, Universiti Sains Malaysia, 11800
Minden, Penang, Malaysia
1

To whom correspondence should be addressed.

ABSTRACT
Studies were carried out on the bioefficacy and residual activity of Bacillus thuringiensis
israelensis H-14 (Bti) (water-dispersible granules of VectoBac ABG 6511 and liquid
formulations of VectoBac 12AS) and pyriproxyfen (insect growth regulator, Sumilarv 0.5%) as
direct applications for control of larvae of Aedes aegypti and Aedes albopictus. Two dosages of
each Bti formulation (285 and 570 international toxic units [ITU]/liter) and the integration of
both Bti formulations and pyriproxyfen were used for residual tests with 45-liter earthen jars for
a period of 4 wk. In 1 test series, the treated water was replenished daily with 6 liters of seasoned
untreated water. In the 2nd test series, the water in the jars was topped up to the 40-liter level
during evaluation. Neither Bti formulation remained effective for a full week. Water-dispersible
Bti granules provided effective initial control activity against Ae. aegypti and Ae. albopictus for
both test designs (with replenishment and without replenishment of water). The higher dosage
(570 ITU/liter) for both Bti formulations was only partially effective at the end of 1 wk after
being diluted. After 1 wk, water-dispersible Bti granules provided greater larval mortality than
did liquid Bti formulation against both mosquito species when integrated with pyriproxyfen.
Pyriproxyfen (79.5 and 159 mg/liter) on its own showed low larvicidal activity but provided very
effective control of adult emergence. In this study, integration of Bti (285 and 570 ITU/liter) with
pyriproxyfen (79.5 mg/liter) extended the duration of partial larval control somewhat, but live
larvae persisted throughout the 4-wk test. The integration effect was more obvious when waterdispersible Bti granules were integrated with pyriproxyfen than when liquid Bti was used.
Integration of Bti with pyriproxyfen had a negative effect on adult emergence, which was
completely inhibited by pyriproxyfen after day 1. Daily replenishment of water increased Bti
activity and provided slightly better larval control. Aedes albopictus and Ae. aegypti were both
completely susceptible to the higher concentration of Bti and pyriproxyfen in both test designs
(with replenishment and without replenishment of water).
Keywords: Bacillus thuringiensis H-14, water-dispersible granules, liquid formulation,
pyriproxyfen, dengue vectors

Southeast Asian J Trop Med Public Health. 2008 Jul;39(4):601-9.

Impact of larviciding with a Bacillus thuringiensis

israelensis formulation, VectoBac WG, on dengue
mosquito vectors in a dengue endemic site in Selangor
State, Malaysia.
Lee HL, Chen CD, Masri SM, Chiang YF, Chooi KH, Benjamin S.
Medical Entomology Unit/Infectious Diseases Research Center, Institute For Medical Research,
Jalan Pahang, Kuala Lumpur, Malaysia. leehl@imr.gov.my
The field bioefficacy of a wettable granule (WG) formulation of Bacillus thuringiensis
israelensis (Bti), VectoBac WG (Bti strain AM65-52) against dengue vectors, Aedes aegypti and
Ae albopictus; was evaluated in a suburban residential area (TST) and in a temporary settlement
site (KB) in the state of Selangor, Malaysia. Pre-control ovitrap surveillance of the trial sites
indicated a high population of both types of Aedes mosquitoes. The populations were monitored
continuously by weekly ovitrapping. Bti was sprayed biweekly at a dosage of 500 g/ha by using
a mist-blower. The spray application was targeted into outdoor larval habitats. If required, Bti
formulation was also applied directly into indoor water-holding containers at 8 g/1,000 l. Based
on ovitrap surveillance, a significant reduction in Aedes populations was evident 4 weeks after
initiating the first Bti treatment. The ovitrap index (OI) and the larvae density decreased
drastically in both trial sites. In TST, the indoor OI was significantly reduced from 57.50 +/7.50% to 19.13 +/- 5.49% (p<0.05), while the outdoor OI decreased from 38.89 +/- 11.11% to
15.36 +/- 5.93%. In KB, similarly, the OI was significantly reduced by more than half, from
66.66 +/- 6.67% to 30.26 +/- 2.99% (p< 0.05). In all cases, the reduction in OI was paralleled by
reduction in larval density.

Journal of Economic Entomology 99(5):1590-1595. 2006

doi: 10.1603/0022-0493-99.5.1590
Long-Lasting Effects of a Bacillus thuringiensis Serovar israelensis Experimental
Tablet Formulation for Aedes aegypti (Diptera: Culicidae) Control

Gemma Armengol1,2, Johana Hernandez1, Jose G. Velez1, and Sergio Orduz1,3

1

Unidad de Biotecnologa y Control Biolgico, Corporacin para Investigaciones Biolgicas,

Medelln, Colombia.
2

Current address: Department of Animal Biology, Vegetal Biology and Ecology, Universitat
Autnoma de Barcelona, Barcelona, Spain. Corresponding author, e-mail:
gemma.armengol@uab.es.
3

Escuela de Biociencias, Facultad de Ciencias Bsicas, Universidad Nacional de Colombia sede

Medellin, Medellin, Colombia.

Abstract
Dengue is a growing public health problem in many tropical and subtropical countries
worldwide. At present, the only method of controlling or preventing the disease is to eliminate its
vector, Aedes aegypti (L.) (Diptera: Culicidae). In the current study, an experimental larvicide
tablet formulation XL-47 based on Bacillus thuringiensis serovar israelensis (Bti) and containing
4.8% of technical powder was developed. This formulation was evaluated against Ae. aegypti in
three different sets of experiments, under field-simulated conditions: two experiments were
indoors and under partial sunlight exposure and one experiment was outdoors with sunlight
exposure. Larvae were added throughout the experiment two times per week, and the residual
larvicidal activity was recorded daily. Pupal formation was reduced in the containers with Bti by
>80% in relation to the containers without treatment for 12 wk; to our knowledge, this is the
longest period of control reported for a Bti tablet formulation outdoors under sunlight exposure.
Moreover, samples from the top, middle, and bottom of the water column were collected to
perform bacterial plate counts and toxicity assays. The Bti population and the active ingredient of
the tablet formulation remained mainly at the bottom of the containers and mosquito larvae
reached the formulation by diving and shredding the tablets material. In conclusion, the
experimental tablet formulation XL-47 showed an inhibition of pupal formation that lasted for
long periods under sunlight exposure.
Received: October 25, 2005; Accepted: May 25, 2006

Keywords: Bacillus thuringiensis serovar israelensis, Aedes aegypti, tablet formulation,

biological control