Discussion
In this paper, we describe a method of mosquito species identification
based on a traditional endpoint PCR and a precise amplicon size
detection, avoiding the need for sequencing. We found that ITS2 sequence
size differences between species are detectable by capillary
electrophoresis. This enabled us to assign species-specific profiles for
nine Anopheles species (seven species from theNyssorhynchus subgenus and two from the Anophelessubgenus) present in French Guiana. Identification is based on ITS2
amplicon size information combined with a simple morphological
observation, the color of the fifth hind tarsus. This additional piece
of data is quick and easy to acquire, by eye or under a
stereomicroscope, when collecting mosquitoes in the field and does not
require any advanced knowledge in taxonomy.
This species identification method has the advantage of being simple and
relatively quick to set up, and can be used in routine in a laboratory
avoiding the requirement for sequencing. During the three steps of
method development, we were not able to sequence our samples on-site and
we had to send samples in 96-well plates between French Guiana and
France for sequencing. For 7.8, 9.2 and 1.4 % of samples, at steps 1, 2
and 3 of method development respectively, it was not possible to obtain
definitive results as forward and reverse sequencing outcomes were
different. Moreover, we observed that 3.0 and 3.7 % of sample
sequencing results, at steps 1 and 2 of method development respectively,
were erroneous. We were able to detect these errors because of
contradictory results between sequencing and our method, whether because
of fragment size not matching the putative species or because of
conflicting leg color. Without the implementation of the method, these
errors would have gone unnoticed. In case of doubt after sequencing, we
amplified the ITS2 region again and sent the sample for sequencing to
check the initial result. Hence, sequencing can be a source of error,
especially when samples need to be shipped, as cross-contaminations may
occur during plate processing, shipment and sequencing. In our case,
plates were sealed and packaged carefully but we noticed that plate caps
that have undergone temperature changes during PCR and storage in the
freezer are slightly easier to open. Moreover, we used adhesive PCR
plate seals for one shipment of two plates (one of step 1 and one of
step 2), which resulted in more misidentifications and uncertain
results. During subsequent shipments (steps 2 and 3), we used plate caps
that were replaced with new ones just before shipping and observed a
relatively low error rate, yet did not investigate this further.
In our method, missing or erroneous leg color is the major source of
problem. It rarely led to incorrect identification (1.2 % samples
species at step 2), but more often led to uncertainties (10, 12 and 0.27
% at steps 2, 3 and Routine, respectively), which represents the
majority of method uncertainties (12, 16 and 0.54 % samples, at steps
2, 3 and routine, respectively). Wrong leg color specifically led to 4.3
and 8.2 % indeterminations at steps 2 and 3 respectively, indicating
that efforts to ensure correct annotation of this information at the
time of capture can significantly lead to improvement in determination
rates. A simple double check by a colleague may be the key, as these
data are usually collected in a repetitive way, sometimes at night, in a
non-usual environment. Nonetheless, this problem cannot be completely
solved, as some mosquito samples lack both hind legs. One could decide
to exclude them from the study, yet we were able to identify the species
of 54/94 mosquitoes (steps 1, 2 and 3) with missing fifth hind tarsus
color information, and of 16/17 in routine. The fifth hind tarsus color
element is therefore a piece of data that should not be neglected, but
even if it is missing, identification remains possible in many cases.
The second key point for the reliability of our method is, obviously,
the proper use of the capillary electrophoresis device and its
components. The channels of the capillary electrophoresis cartridge are
prone to clogging if they are used incorrectly and the results can be
erroneous in these cases. The only recommendation on this point is to
follow the supplier’s instructions carefully and check results
individually, in order to detect any aberrant results. In case of
aberrant results, we either excluded the problematic channel or replaced
the cartridge.
While the current method has been developed on Anopheles species
from French Guiana, this approach may be extended to species
identification of individual fieldwork specimens in any taxon. Widening
the scale besides Anopheles (Nyssorhynchus ), our method
clearly discriminated our Anopheles samples from four mosquitoes
of the Culex and Psorophora genera during step 1, and we
expect development steps to require much less specimens if the species
of interest are more distant from each other. More testing would be
needed to determine whether all mosquito genera have a significantly
different profile from one another and whether, within these genera,
species identification is possible. Our tool may then facilitate
identification of a broad range of mosquitoes, and be particularly
advantageous during epidemics in order to target species that are
potential vectors of arboviruses or parasites. It could be compatible
with sampling by non-experts combined with photographs during
collaborative work or citizen science projects. Finally, when
presence/absence information are sufficient, pools of different samples
sharing the same morphological trait (in our case, the color of
Ta-III5) could be processed.
To specify the interest of our method with currently available methods,
several aspects can be considered: the possibility to go back and check
results, the level of local diversity and the cost-effectiveness. Our
method allows to easily check results as DNA extracts can be stored for
a long time. They can be used for a second similar analysis by PCR and
capillary electrophoresis as well as for sequencing of ITS2 or any other
sequence, which may notably apply to population genetics studies. The
other methods of molecular biology have similar advantages, while
morphological observations and audio recordings may not, unless samples
have been properly stored.
In terms of diversity, we detected nine different Anophelesspecies, including four dominant ones. With a higher local diversity,
too many overlaps between intervals would reduce the efficiency of the
method with the current experimental conditions. The latter can be
improved, for instance by running capillary electrophoresis straight
after PCR, which could allow to deal with a slightly higher diversity.
Sequencing would remain the best option in case of high species
diversity. With a lower diversity of only two or three species,
multiplex PCR with species-specific primers would remain the simplest
and cheapest method. Hence, our species identification method is a
reliable alternative for locally moderate species diversity.
Considering costs, capillary electrophoresis requires an initial
equipment investment, yet the processing cost of each sample is much
lower with the proposed method than with sequencing. When considering
the initial cost of the device and of capillary electrophoresis reagents
on one hand, shipping and sequencing cost of samples on the other hand
(and ignoring the cost of labour and our interest in the other
applications of the device), our investment on the capillary
electrophoresis apparatus would be repaid after 4,000 to 5,000 samples.
Beyond that, more than 5 \euro per sample can be saved. Hence,
episodic needs may rather be addressed with sequencing, while our
approach seems reasonable for a laboratory requiring steady species
identification. Alternatively, even with very low consumable cost per
sample [42] , the MALDI-TOF approach requires a 10-fold
higher investment and maintenance budget than capillary electrophoresis.
It would thus require a much higher number of samples to be more
cost-effective than our method.
In sum, we introduce PCR and capillary electrophoresis combined with
simple morphological observation as a convenient method to discriminate
species of different field-collected samples. We specifically deployed
it to identify nine Anopheles species found in French Guiana,
mainly from the Nyssorhynchus subgenus. After deployment, this
method allows to save time and money and to keep control over
experimental schedule.