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Veterinary Parasitology 162 (2009) 350–353
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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar
Short communication
Wolbachia infection in Australasian and North American populations of
Haematobia irritans (Diptera: Muscidae)
Bing Zhang a, Elizabeth McGraw b, Kevin D. Floate c, Peter James a,*,
Wayne Jorgensen a, Jim Rothwell d
a
Animal Research Institute, Department of Primary Industries and Fisheries, Brisbane, Australia
School of Integrative Biology, University of Queensland, Brisbane, Australia
Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada, T1J 4B1
d
School of Veterinary Science, University of Queensland, Brisbane, Australia
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 20 December 2008
Received in revised form 19 February 2009
Accepted 2 March 2009
Buffalo ﬂy (Haematobia irritans exigua) is a major pest of beef and dairy cattle in northern
Australia. Global warming is expected to increase the southern range of buffalo ﬂy.
Chemical control is compromised by resistance and may not be feasible in extensive
production systems and there is rapidly growing market preference for beef and dairy
products produced in low-chemical systems.
Wolbachia are vertically transmitted intracellular bacteria that can profoundly
inﬂuence host reproduction and ﬁtness and are of increasing interest for use in biocontrol
programs. To determine whether Australian ﬂies are infected with Wolbachia, buffalo ﬂies
were collected from 12 cattle herds around Australia and assayed by standard PCR for the
Wolbachia wsp gene. H. i. exigua from Indonesia and horn ﬂy (H. i. irritans) from Canada
were also tested. All H. i. exigua samples tested were negative for Wolbachia infection
whereas a very strong signal for Wolbachia was obtained from H. i. irritans.
Crown Copyright ß 2009 Published by Elsevier B.V. All rights reserved.
Keywords:
Haematobia irritans
Wolbachia
Diptera
1. Introduction
Buffalo ﬂy (Haematobia irritans exigua De Meijere) is one
of the main health problems of cattle in northern Australia,
recently estimated to cost the beef industry $78 m
annually (Sackett et al., 2006). In dairy cattle, infestations
above a threshold of 30 ﬂies were estimated to cause losses
in milk production and live weight gain of 2.6 ml and 0.14 g
per ﬂy per day, respectively (Jonsson and Mayer, 1999).
Buffalo ﬂy was introduced into the Northern Territory
in 1838 from Indonesia (Williams et al., 1985). Flies spread
south from Bundaberg in Queensland to Coffs Harbour in
NSW between 1974 and 1982, partly in response to a series
* Corresponding author at: Department of Primary Industries and
Fisheries, Locked Mail Bag No. 4 Moorooka, 4105 Qld, Australia.
Tel.: +61 7 3362 9409; fax: +61 7 3362 9429.
E-mail address: [email protected] (P. James).
of mild winters (Williams et al., 1985). Ongoing climate
change is expected to lead to increasing wetness in
northern Australia and increasing temperatures in southern Australia (White et al., 2003), which will increase the
range of buffalo ﬂy and numbers of cattle exposed to the
pest.
Buffalo ﬂy has traditionally been controlled mainly by
the application of pesticides, although ﬂy populations are
moderated by dung beetle activity and buffalo ﬂytraps can
also be used. However, resistance has reduced the
effectiveness of chemical controls (Farnsworth et al.,
1997), there are human and environmental health issues
associated with widespread use of insecticides and there is
growing market preference for beef and dairy products
produced in low-chemical systems.
Wolbachia (Rickettsiales: Rickettsaceae) are obligate,
intracellular bacteria found naturally in many arthropods
and some ﬁlarial nematodes (e.g. Floate et al., 2006). They
are able to rapidly invade and establish in insect populations
0304-4017/$ – see front matter . Crown Copyright ß 2009 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2009.03.012
B. Zhang et al. / Veterinary Parasitology 162 (2009) 350–353
through effects such as cytoplasmic incompatibility, feminization of males, male killing and induction of parthenogenesis (Stouthamer et al., 1999). Their wide-ranging effects
on reproduction and host ﬁtness have made Wolbachia the
subject of growing interest as potential biocontrol agents
(Werren and O’Neill, 1997; Zabalou et al., 2004; Floate et al.,
2006; Bourtzis, 2008). Such interest has led to testing for
Wolbachia in horn ﬂy (Haematobia irritans irritans (L.)). Horn
ﬂy, very closely related to buffalo ﬂy, is a major pest of cattle
in the Americas, Europe and northern Asia. To date, all
populations of horn ﬂy tested have been positive for
Wolbachia (Jeyaprakash and Hoy, 2000; Floate et al.,
2006), which suggests that infections may also be present
in the buffalo ﬂy. However, surveys of buffalo ﬂy populations
have not been previously performed.
Here, we report the results of the ﬁrst survey for
Wolbachia in buffalo ﬂies. Results will facilitate future
research on the potential use of these bacteria for use in
buffalo ﬂy biocontrol programs.
2. Materials and methods
2.1. Fly sample collection
Buffalo ﬂies were collected from 12 locations in
Australia (Fig. 1) and from Bali, Indonesia. A total of 134
buffalo ﬂies, 127 from Australia and 7 from Indonesia were
tested. Specimens were immediately stored in 20% DMSO/
0.25 M EDTA solution (Seutin et al., 1991). Horn ﬂies
(N = 70) were collected from three locations in southern
Alberta, Canada (Table 1), stored in 95% EtOH and held at
20 8C until tested.
2.2. Tests for Wolbachia
Buffalo ﬂies were tested for Wolbachia in laboratories
in Australia and Canada with the numbers tested in each
laboratory as indicated in Table 1. To conﬁrm the test
351
Fig. 1. Sites of buffalo ﬂy collection in Australia.
method, horn ﬂies were also tested in each laboratory
(Table 1). In Australia, genomic DNA was extracted from
individual ﬂies using Puregene Tissue Core Kits (Qiagen)
according to the manufacturer’s instructions. Extracted
DNA was tested for the presence of Wolbachia using
polymerase chain reaction (PCR) tests and wsp primers.
These primers amplify Wolbachia DNA (if present) to
detectable levels that can be visualized as bands of
characteristic size (ca. 600 bp) on agarose gels (Braig
et al., 1998). DNA from a single D. melanogaster known to
be infected was used as a positive control. PCR was
performed in a 20 ml volume containing 50 mM KCl,
10 mM Tris–HCl (pH 9.0), 2.5 mM MgCl2, 0.1 mM dNTPs,
0.5 mM primers, and 0.8 unit Taq DNA Polymerase
(Roche Molecular Biochemicals, Indianapolis, IN). The
thermal cycling regime was as follows: 3 min denaturation at 94 8C, 35 cycles of 0.5 min denaturation at 94 8C,
0.5 min annealing at 55 8C and 1 min extension at 72 8C,
followed by an extra 10 min extension step at 72 8C. The
Table 1
Summary of screening for Wolbachia infection in buffalo ﬂies and horn ﬂies.
Species
Source
Where tested
No. ﬂies testing positive
Buffalo ﬂy
Gatton, Qld
Berrimah Farm, NT
Douglas Daly, NT
Beatrice Hill Farm, NT
Australia
Australia
Australia
Australia
Canada
Australia
Australia
Canada
Australia
Australia
Australia
Australia
Canada
Australia
Australia
Australia
Canada
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Australia
Canada
Canada
Canada
4 of 5
50 of 50
5 of 5
10 of 10
Yeerongpilly, Qld
Mutdapilly, Qld
Rochedale, Qld
Pinjarra Hills, Qld
Duck Creek Research Farm, NSW
Warrawagine, WA,
Camballin, WA,
Ruby Plains, WA,
Gianyar, Bali, Indonesia
Horn ﬂy
Lethbridge Research Centre colony
Coaldale, AB
Onefour, AB
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
of
33
8
8
8
2
10
8
4
6
4
8
8
4
8
8
5
2
352
B. Zhang et al. / Veterinary Parasitology 162 (2009) 350–353
Fig. 2. Agarose gel electrophoresis analysis of PCR products: lane 1 = DNA
marker; lane 2 = positive control (wsp gene); lane 3–6 = horn ﬂy samples;
lane 7–12 = buffalo ﬂy samples.
to reduce ﬁtness traits such as survival, locomotion or
olfaction to suppress populations, limit dispersal or
curtail host-seeking behaviours (Fleury et al., 2000;
McGraw et al., 2002) Successful transfection of Wolbachia
across genus and species barriers and establishment of
stable infections in previously naı¨ve insect populations
has been reported (Bourtzis, 2008). Although our studies
found no evidence of Wolbachia infection in buffalo ﬂy,
the widespread occurrence of Wolbachia in most insect
orders and in closely related H. i. irritans suggests that H i.
exigua may be a competent host. Further studies are
planned to investigate the feasibility of transfection of H.
i. exigua with Wolbachia as a basis for novel control
approaches.
Acknowledgements
PCR reaction was run in a Mastercycler (Eppendorf,
Westbury, NY, USA). PCR products were separated by
agarose gel electrophoresis, and stained with ethidium
bromide (Fig. 2). DNA bands of ca. 600 bp were extracted
and puriﬁed from gels. Sequencing conﬁrmed the
presence of Wolbachia. Similar methods were used in
Canada, with speciﬁc details provided in Kyei-Poku et al.
(2003).
We acknowledge J. Kidd, L. Small, W. Ehrlich, G.
Everingham, P. Freeman, K. Puja, and M. Bullard for
collection of buffalo ﬂy samples, and P. Coghlin for
collection and testing of horn ﬂy samples in Canada. This
work was supported by Department of Primary Industries
and Fisheries, Queensland and University of Queensland.
3. Results
Bourtzis, K., 2008. Wolbachia-based technologies for insect pest population control. Adv. Exp. Med. Biol. 627, 104–113.
Braig, H.R., Zhou, W., Dobson, S.L., O’Neill, S.L., 1998. Cloning and characterization of a gene encoding the major surface protein of the
bacterial endosymbiont Wolbachia pipientis. J. Bacteriol. 180 (9),
2373–2378.
Brownstein, J.S., Hett, E., O’Neill, S.L., 2003. The potential of virulent
Wolbachia to modulate disease transmission by insects. J. Invertebr.
Pathol. 84 (1), 24–29.
Farnsworth, W.R., Collett, M.G., Ridley, I.S., 1997. Field survey of insecticide resistance in Haematobia irritans exigua de Meijere (Diptera:
Muscidae). Aust. J. Entomol. 36, 257–261.
Fleury, F., Vavre, F., Ris, N., Fouillet, P., Bouletreau, M., 2000. Physiological
cost induced by the maternally transmitted endosymbiont Wolbachia
in the Drosophila parasitoid Leptopilina heterotoma. Parasitology 121,
493–500.
Floate, K.D., Kyei-poku, G.K., Coghlin, P.C., 2006. Overview and relevance
of Wolbachia bacteria in biocontrol research. Biocontrol Sci. Technol.
16, 767–788.
Jeyaprakash, A., Hoy, M.A., 2000. Long PCR improves Wolbachia DNA
ampliﬁcation: wsp sequences found in 76% of 63 arthropod species.
Insect Mol. Biol. 4, 393–405.
Jonsson, N.N., Mayer, D.G., 1999. Estimation of the effects of buffalo ﬂy
(Haematobia irritans exigua) on the milk production of dairy cattle
based on a meta-analysis of literature data. Med. Vet. Entomol. 13,
372–376.
Kyei-Poku, G.K., Floate, K.D., Benkel, B., Goettel, M.S., 2003. Elimination of
Wolbachia from Urolepis ruﬁpes (Ashmead) (Hymenoptera: Pteromalidae) with heat and antibiotic treatments: implications for host
reproduction. Biocontrol Sci. Technol. 13, 341–354.
Kyei-Poku, G.K., Giladi, M., Coghlin, P., Mokady, O., Zchori-Fein, E., Floate,
K.D., 2006. Wolbachia in wasps parasitic on ﬁlth ﬂies (Diptera:
Muscidae) with emphasis on Spalangia cameroni Perkins (Hymenoptera: Pteromalidae). Entomologia Experimentalis et Applicata
121, 123–135.
McGraw, E.A., Merritt, D.J., Droller, J.N., O’Neill, S.L., 2002. Wolbachia
density and virulence attenuation following transfer into a novel
host. Proc. Natl. Acad. Sci. U.S.A. 99, 2918–2923.
Sackett, D., Holmes, P., Abbott, K., Jephcott, S., Barber, M., 2006. Assessing the economic cost of endemic disease on the proﬁtability of
Australian beef cattle and sheep producers. Final report of project
AHW-087 Meat and Livestock Australia, Sydney, pp. 119 (ISBN
1741910021).
Seutin, G., White, B.N., Boag, P.T., 1991. Preservation of avian blood and
tissue samples for DNA analyses. Can. J. Zool.-Revue Canadienne de
Zoologie 69 (1), 82–90.
No infections of Wolbachia were detected in buffalo
ﬂies. However, Wolbachia were detected in each positive
control and in 69 of 70 horn ﬂies individually tested.
Results were consistent between laboratories. Previous
studies have identiﬁed only one isolate of Wolbachia
infecting the horn ﬂy (Jeyaprakash and Hoy, 2000; KyeiPoku et al., 2006).
4. Discussion
Wolbachia bacteria infect the reproductive cells of
an estimated 20–75% of arthropod species (Werren
et al., 1995; Jeyaprakash and Hoy, 2000; Werren and
Windsor, 2000; Floate et al., 2006), with infections
also reported in ﬁlarial nematodes (Werren and
O’Neill, 1997). Transmission occurs vertically from
infected females to their offspring in egg cytoplasm.
Although Wolbachia occurs in many Australian insect
species and is apparently widespread in North American
H. i. irritans populations (Jeyaprakash and Hoy, 2000;
Floate et al., 2006) no infections were detected in buffalo
ﬂies. Furthermore, the absence of Wolbachia in the
sample of H. i. exigua collected from Bali may suggest
that this is a more general characteristic of the
subspecies.
A number of options have been suggested for the use
of Wolbachia in insect biocontrol programs. These include
the use of cytoplasmic incompatibility induced by
Wolbachia to suppress pest populations, as a means of
spreading deleterious genes through a target population
(Stouthamer et al., 1999; Bourtzis, 2008), to reduce the
lifespan of insects and reduce disease transmission
(Sinkins and O’Neill, 2000; Brownstein et al., 2003) and
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