Screening of Wolbachia

Endosymbiont Infection in Aedes

aegypti Mosquitoes Using
Attenuated Total Reflection Mid-
Infrared Spectroscopy

Aazam Khoshmanesh†, Dale Christensen†, David

Perez-Guaita†, Inaki Iturbe-Ormaetxe‡, Scott L.
O’Neill‡, Don McNaughton† and Bayden R.
Wood*†
*E-mail: bayden.wood@monash.edu.

†Centre for Biospectroscopy, School of Chemistry, Monash

University, Clayton, Victoria 3800, Australia

‡Institute of Vector-Borne Disease, Monash University, Clayton,

Victoria 3800, Australia

Analytical Chemistry

Vol. 89: , Issue. 10, : Pages. 5285-5293

Publication Date (Web): March 23, 2017

https://doi.org/10.1021/acs.analchem.6b04827

Dengue fever is the most common mosquito

transmitted viral infection afflicting humans,
estimated to generate around 390 million
infections each year in over 100 countries. The
introduction of the endosymbiotic bacterium
Wolbachia into Aedes aegypti mosquitoes has the
potential to greatly reduce the public health
burden of the disease. This approach requires
extensive polymerase chain reaction (PCR)
testing of the Wolbachia-infection status of
mosquitoes in areas where Wolbachia–A. aegypti
are released. Here, we report the first example of
small organism mid-infrared spectroscopy where
we have applied attenuated total reflection
Fourier transform infrared (ATR-FT-IR)
spectroscopy and multivariate modeling methods
to determine sex, age, and the presence of
Wolbachia (wMel strain) in laboratory
mosquitoes and sex and age in field mosquitoes.
The prediction errors using partial least squares
discriminant analysis (PLS-DA) discrimination
models for laboratory studies on independent
test sets ranged from 0 to 3% for age and sex
grading and 3% to 5% for Wolbachia infection
diagnosis using dry mosquito abdomens while
field study results using an artificial neural
network yielded a 10% error. The application of
FT-IR analysis is inexpensive, easy to use, and
portable and shows significant potential to
replace the reliance on more expensive and
laborious PCR assays.

Dengue is a systemic viral infection, which

causes a potentially life-threatening illness in
many patients. (1) It takes the form of four
serotypes (DENV1–DENV4), each of which can
cause the full range of symptoms. (2-4) Aedes
aegypti mosquitoes act as the primary vectors in
the transmission of the virus between humans,
resulting in an estimated 390 million infections
per annum globally. (5) These infections are
mostly limited to the tropical and subtropical
regions, where A. aegypti and to a lesser extent
Aedes albopictus occur. Dengue fever is an
expanding problem throughout the tropics of the
world as current control methods are proving to
be largely ineffective.

Wolbachia pipientis are small intracellular Gram-

negative bacteria found in up to 60% of insect
species, where they can induce a variety of
reproductive phenotypes and confer protection
against a variety of pathogens. Despite their
prevalence in nature, Wolbachia does not
naturally infect A. aegypti (the main dengue
vector); however, several Wolbachia strains have
been artificially introduced (6-8) to create
stable Wolbachia-infected A. aegypti that contain
high Wolbachia densities and have a reduced
ability to transmit dengue and other pathogens
including (9, 10) ZIKA. These strains are able
to spread through insect populations by means
of cytoplasmic incompatibility (CI), a form of
reproductive manipulation that favors
Wolbachia-infected female mosquitoes. The
ability to induce CI, together with their
pathogen-blocking abilities, have put these
bacteria at the forefront in the fight against
dengue and other pathogens transmitted by
mosquitoes. (11-14) As part of the Eliminate
Dengue Program (www.eliminatedengue.com),
Wolbachia-infected A. aegypti mosquitoes have
been released in several international field sites
with the aim of introducing Wolbachia into the
wild mosquito population and in turn reducing
dengue transmission. (15)

The use of Wolbachia-infected A. aegypti for the

control of dengue involves the regular
monitoring of local mosquitoes in release areas
during and after the release. These mosquitoes
are collected in traps and assessed for the
presence of Wolbachia using molecular methods.
There are several different methods of screening
mosquitoes for Wolbachia. To date, these have
been based mainly on standard polymerase chain
reaction (PCR) assays (16, 17) that detect
Wolbachia DNA and are very sensitive and
reliable. The current best-practice screening
method involves quantitative polymerase chain
reaction (qPCR) assays. (15) This process
involves the application of DNA primers, which
target A. aegypti- and Wolbachia-specific genes,
together with fluorescently labeled hydrolysis
Taqman probes that allow the simultaneous
detection of both species using real time
fluorescence. These qPCR assays are able to
screen a high number of samples quickly, while
remaining robust and accurate, (18) but they
can be expensive due to the cost of real-time
PCR machines, primers, probes, and PCR
reagents, in particular the enzymes used in the
reaction, and require specialized training.

Alternative methods, such as near infrared

spectroscopy (NIRS), which spans the range
between 10​000 and 4000 cm–1 of the
electromagnetic spectrum, have been applied to
detect Wolbachia infection and identify species,
sex, and age grading in flies (19-22) as well as
identify two strains of Wolbachia in A. aegypti
(23) and malaria infection in mosquitoes.
(20) However, the interpretation of NIR
spectra is very difficult due to the broad nature
of the bands resulting from the overlap of many
overtone modes and the ubiquitous presence of
water absorbance. These challenges make
calibration procedures quite laborious and often
not transferable from one instrument to the
next. (24) Due to intrinsic band broadening,
the differences between NIR spectra of different
compounds are often very subtle, resulting in a
lower inherent molecular selectivity compared to
the mid-IR. Consequently, NIRS is a much less
sensitive technique and therefore unsuited to the
analysis of low-level components below a few
percent. (25)

Fourier transform infrared (FT-IR) spectroscopy

measures the molecular vibrations in the mid-
infrared region (4000–400 cm–1) of the
electromagnetic spectrum providing a snapshot
of the metabolic fingerprint of the biological
sample under investigation. The changes in this
fingerprint can be detected using multivariate
models, and these changes can be used as
predictive markers for the diagnosis of disease
and other conditions. FT-IR-based diagnostics
have a proven track record in detecting viral
infections in cells; (26-30) however, the
technique has never been applied to detecting
infections in mosquitoes.

Here, we show for the first time that attenuated

total reflection Fourier transform infrared
(ATR-FT-IR) spectroscopy, in combination
with multivariate modeling methods, has the
required ease of sample preparation, sensitivity,
and ability to become a fast and cheap method to
replace costly and time-consuming qPCR for
diagnosis of Wolbachia-infected mosquitoes as
well as for discrimination of 2 day and 10 day old
mosquitoes along with sex. It should be noted
that mosquito sex can also be distinguished by
eye (using the antennae flagellum anatomy, for
example) and the ATR measurements to
determine sex in this case were performed to
understand the chemical differences between
males and females. The system combines a
standard benchtop FT-IR spectrometer with a
diamond crystal ATR accessory (or silicon cell
designed for liquid samples), but portable units
are also available. The technique utilizes the
property of total internal reflection to generate
an evanescent wave, which penetrates on the
order of 3 µm into a sample, which is enough to
penetrate the abdomens of mosquitoes crushed
by the loading jig. The whole process of sample
deposition and spectral recording takes less than
2 min using a single pass ATR crystal enabling
approximately 3000 samples to be processed a
week (12 h day) with a single instrument. Our
results show real potential that a viable
spectroscopic alternative to current qPCR
methods can be developed, with efficiencies
made in time, cost, waste reduction, and training
of personnel, and represent a totally new
approach to assist in the elimination of dengue
fever.

Methods

Results and Discussion

Conclusions

Acknowledgment
B.R.W. is supported by an Australian Research
Council (ARC) Future Fellowship grant
FT120100926. We acknowledge Mr. Finlay
Shanks for instrumental support, the Eliminate
Dengue team in Cairns for providing field
mosquito samples, the Eliminate Dengue
insectary team at Monash for providing colony
samples, and the Eliminate Dengue diagnostics
team at Monash for validating the Wolbachia-
infection status of the mosquitoes by PCR.

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