116

Journal of Vector Ecology June 2021

Scientific Note

Next generation sequencing approach for simultaneous identification of mosquitoes

and their blood-meal hosts
Ephantus J. Muturi1*, Christopher Dunlap1, David P. Tchouassi2, and Jack Swanson3

USDA, Agricultural Research Service, National Center for Agricultural Utilization Research, Crop Bioprotection Research Unit,
1

Peoria, IL 61604, U.S.A., Ephantus.Muturi@usda.gov

2
International Centre of Insect Physiology and Ecology, Nairobi, Kenya
3
Illinois Department of Public Health, Division of Environmental Health, Springfield, IL 62761, U.S.A.

Accurate identification of mosquito species and their
blood-meal hosts is essential for understanding disease
dynamics and transmission cycles, establishing disease risk,
and designing effective and timely strategies for outbreak
prevention. Morphological identification of mosquitoes by
trained personnel is usually possible but damage to field-
collected specimens and occurrence of species complexes
complicates this process (Koekemoer et al. 2002, Versteirt
et al. 2015). Conventional identification of blood-meal
sources using serological methods which test for species-
specific proteins (Beier et al. 1988) or molecular approaches
that focus on species-specific amplification of putative hosts
(Kent 2009) is also challenging. These methods offer limited
resolution for the identification of blood-meal hosts not
previous associated with a given mosquito species and are
poorly designed for detection of mixed blood meals. These
biases may limit identification of new reservoir hosts and
undermine our ability to understand vector-borne disease
transmission cycles and to implement effective and timely
interventions. The use of universal primers designed to
identify mosquitoes and their blood-meal sources has helped
to reduce these biases but remain cumbersome and expensive
because each amplicon from individual samples must be
sequenced separately and in the case of mixed blood meals,
an additional cloning step may be required before sequencing
(Kent 2009).
Next-generation sequencing (NGS) is effective at
overcoming the drawbacks associated with conventional
approaches to mosquito identification and blood-meal
analyses. Each read from NGS is derived from a single source
DNA molecule facilitating identification of mixed blood
meals (Metzker 2010). Samples are amplified with the target
universal primer sets and the amplicons are individually
barcoded with unique index primer sets that make it possible
to pool the samples before sequencing. This setup increases the
resolution for species identification and significantly reduces
the cost of sequencing. Although many researchers have
harnessed the power of NGS to characterize the microbial
communities of mosquitoes and other disease vectors, only a
small number of studies have used this technology to identify
disease vectors or their blood-meal hosts (Logue et al. 2016,
Kieran et al. 2017, Abbasi et al. 2019, Gaithuma et al. 2020).
More importantly, none of these studies have harnessed the
power of NGS to simultaneously identify disease vectors and
their blood-meal hosts. In this study, we used next-generation
sequencing technology for simultaneous identification of
individual mosquitoes and characterization of their blood-
meal hosts. Combining mosquito identification and blood-
meal analysis in a single sequencing run could reduce the
cost of sequencing and improve disease surveillance and
control efforts by reducing the time required to produce an
actionable dataset.
The study was conducted using host-seeking field-
collected samples of Ae. albopictus, Aedes triseriatus and
Cx. pipiens that had been preserved dry for six months,
and a laboratory colony of Ae. aegypti (Rockefeller strain)
that was reared and maintained as previously described
(Muturi et al. 2019). Three to five-day old Ae. aegypti adults
were sugar-starved for 24 h and then artificially fed on
blood from one of the following host species: bovine, sheep,
horse, rabbit, porcine, or a 1:1:1:1 mixture of bovine, sheep,
horse, and rabbit blood. The blood-feeding apparatus used
in this study is described in detail elsewhere (Muturi et al.
2019). All blood-fed females were sorted by blood-meal
type and immediately preserved at -20° C in conical flasks.
Five samples of each mosquito species and blood meal type
(for Ae. aegypti only) were processed. DNA from individual
mosquitoes was extracted using DNeasy blood and tissue kit
(Qiagen, Valencia, CA). Each Ae. aegypti sample was PCR-
amplified using two primer sets targeting cytochrome b gene
for identification of host blood meal source and cytochrome
oxidase 1 gene for mosquito species identification (Table 1).
Illumina adapter overhang nucleotide sequences were added
to the gene‐specific primer sequences as provided in the
Illumina library preparation protocol. Since Ae. albopictus, Ae.
triseriatus, and Cx. pipiens were not blood-fed, they were only
PCR-amplified using the primer set targeting cytochrome
oxidase 1 gene for mosquito species identification. Whole
blood for bovine, horse, sheep, rabbit, porcine, and an equal
mixture of bovine, horse, sheep, and rabbit blood were also
amplified using the primer set targeting cytochrome b gene
for identification of the host blood-meal source. PCR cycling
conditions were an initial denaturation at 94° C for 2 min,
then 35 cycles of 94° C for 30 s, 55° C for 30 s, and 72° C for 90
s, followed by a 4-min final extension at 72° C for mammalian
cytochrome b gene and an initial denaturation step at 95° C
for 5 min. This was followed by 40 cycles of 94° C for 30 s, 40°
C for 45 s, and 72° C for 1 min, and a final extension at 72˚
Vol. 46, no.1 Journal of Vector Ecology 117
Table 1. DNA sequences for primer sets used for amplification of insect mitochondrial gene and mammalian cytochrome B gene
for blood meal identification.
Blood Mam-F 5’-TGAYATGAAAAAYCATCGTTG-3’
Mammalian
meal ID Mam-R 5’-TGTAGTTRTCWGGGTCKCCTA-3’
Mosquito COI-F 5’-TGATCAAATTTATAAT-3’
MtDNA COI
ID COI-R 5’-GGTAAAATTAAAATATAAACTTC-3’
Overhang F 5’ -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
adapters R 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’
C for 10 min for insect-specific cytochrome oxidase 1 gene.
PCR reactions were conducted in 25 µl volume containing 2.5
µl genomic DNA, 5 µl each of forward and reverse primers (1
µM), and 12.5 µl of 2x KAPA HiFi HotStart ready mix. PCR
products were purified using AMPure XP beads (Beckman
Coulter, CA, U.S.A.) and a second PCR reaction conducted to
attach dual indices and Illumina sequencing adapters using
the Nextera XT index kit. For each mosquito sample, a unique
set of dual index primers was used for each amplicon PCR
primer set. The final barcoded samples were purified using
AMPure XP beads and normalized to 4 nM. The samples were
then pooled and simultaneously sequenced on an Illumina
MiSeq instrument.
The sequences were processed using the CLC genomic
workbench version 20 (Qiagen Inc., Valencia, CA). Reads
with less than 50 base pairs and those that did not map to any
sample based on the unique barcodes used were discarded.
The remaining sequences were trimmed, and the paired-end
reads merged. The sequences were assembled de novo and
operational taxonomic units (OTUs) accounting for less than
0.23% (<100 sequences) of the total sequences were discarded
as they may have resulted from off-target amplifications. For
each sample, all unique sequences from each primer set were
compared against all DNA sequences present in the NCBI
nucleotide database using BLASTn. The best match for each
DNA sequence was recorded considering only sequences
with ≥97% sequence similarity over the entire sequence
length. This clustering threshold was chosen on the basis it
was below the observed/expected sequencing accuracy and
above the 5% intraspecies sequence divergence of cytb, which
includes the majority of species.
A total of 1,589,755 reads was retained and compared
against all DNA sequences present in the NCBI nucleotide
database using BLASTn. Overall, 95.42% of sequences were
correctly assigned to the respective vertebrate host species
blood-meal source (Figure 1). About 1.56% of the sequences
did not match any sequences in the GenBank, 1.78% of the
sequences were not assigned to any vertebrate host species at
≥97% sequence similarity and 1.25% of the sequences were
assigned to Ae. aegypti, the mosquito species used in this study.
Additionally, 0.01% of the sequences from bovine blood were
assigned to wild yak, Bos mutus. Correct identification of all
vertebrate host species in mosquito samples fed on mixed
blood meals was also achieved. In four out of five samples, all
four vertebrate host species were detected, while all vertebrate
host species except bovine were detected in one of the samples.
Reference
Kim et al. 2009
Kambhampati and Smith 1995
Provided by Illumina
Provided by Illumina
The percentage (± SE) of off-target amplification sequences
identified in mosquitoes fed on bovine, horse, porcine, rabbit,
sheep, and mixed blood were 7.82 ± 2.69, 7.81 ± 2.07, 4.23 ±
2.05, 2.22 ± 0.73, 4.19 ± 1.68, and 1.19 ± 0.85, respectively,
and did not differ significantly (F = 2.314, df = 5, 30, P = 0.07).
For mosquito identification, a total of 4,285,228 reads
were retained for downstream analysis. One mosquito
sample from a mixed-blood treatment (Ae. aegypti) and
one sample from Ae. triseriatus had no sequences greater
than 0.23% (<100 sequences) of the total sequences and
therefore could not be correctly identified. However, all the
other samples were correctly identified, except that samples
that were morphologically identified as Ae. triseriatus were
molecularly identified as Ae. hendersoni (Figure 2). A number
of sequences (6.14%) from one Ae. aegypti sample were
molecularly identified as Aedes mascarensis (Figure 2).
This study evaluated the use of NGS for simultaneous
identification of vectors and their blood-meal hosts and
is one of the few studies that have harnessed the power of
NGS technology to simultaneously study multiple aspects
of vector biology (Abbasi et al. 2019, Gaithuma et al. 2020).
Our assay was accurate in identifying the blood-meal sources
including mixed blood meals in a single mosquito. This is in
contrast with Sanger sequencing technology which requires
an additional expensive and labor-intensive cloning step for
amplified products to facilitate detection and identification of
mixed blood meals in a single mosquito. For one of the five
mosquito samples that had taken mixed blood meals, we were
unable to detect bovine blood. Although the mixed blood
meal was composed of equal amounts of blood from each of
the four host species, fewer sequences were detected for horse
compared to bovine, rabbit, or sheep. This is not surprising
because it is not uncommon for DNA amplification efficiency
to vary among DNA sequences for different vertebrate
host species (Logue et al. 2016). Additionally, host DNA is
degraded after a blood meal, and although the mosquito
samples were sorted and preserved a few hours after blood
feeding, it is possible that the rate of host DNA degradation
varied among mosquito samples. Although we added equal
amounts of blood for each vertebrate host in the mixed blood
meals and made every effort to mix them thoroughly, it is
possible that the mixing was not homogeneous leading to
differential ingestion of host blood meals by the mosquitoes.
A few sequences intended for identification of host blood
meal source either matched perfectly with Ae. aegypti, failed
to align with any of the target hosts, or were identical to a
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Journal of Vector Ecology

Figure 1. Identification of Aedes aegypti blood meals taken from known vertebrate hosts using NGS. None, indicates that sequences could not be assigned to
any known hosts, None_97, indicates that sequences could not be assigned to any known host at 97% sequence identity.
June 2021
 Vol. 46, no.1
Journal of Vector Ecology

Figure 2. Identification of mosquito species using NGS. Albo, Aedes albopictus, Cp, Cx. pipiens, Tris, Aedes triseriatus.
119
120 Journal of Vector Ecology June 2021
different bovine species (Bos mutus). These primer-template
mismatches are commonly encountered in blood meal
analysis studies and could be addressed through use of a
hierarchical approach or species-specific primer sets (Logue
et al. 2016). Also, we used the NCBI nucleotide database
as the reference platform, and it is possible that incorrectly
annotated sequences or pseudogenes from this database
may have yielded spurious results. Thus, when using next
generation sequencing to identify the blood-meal sources of
field-collected mosquito samples, low abundance sequences
that do not match any known host at ≤97% sequence similarity
should be interpreted with caution to avoid misinterpreting
them as presenting new unidentified host species.
This study focused on identification of Ae. aegypti
bloodmeals from five mammalian hosts including bovine,
porcine, sheep, rabbit, and horse. We recognize that
mosquitoes feed on other groups of vertebrate hosts that
were not considered in this study, such as birds, reptiles, and
amphibians. Although a more detailed study encompassing
these vertebrate host species will be quite informative, our
results suggest that next-generation sequencing is likely to
be equally accurate in classifying blood-meal hosts from
these additional groups and may increase the probability of
detecting novel blood-meal hosts. Additionally, our approach
was relatively simple and assumed detection success of host
profiles based on their presence in engorged mosquitoes,
which could affect our conclusions. In nature, blood meals
in mosquitoes and other hematophagous arthropods vary in
size and degree of partial digestion depending on time post-
ingestion. Mosquitoes may also feed on different hosts in a
disproportionate manner, yet our mixed blood meals were
constituted in a 1:1 ratio. Mosquitoes also feed on multiple
hosts sequentially rather than simultaneously as provided
in this study. These factors can affect successful profiling of
blood meals in hematophagous insects in nature (Kent 2009)
and defining their impact on blood-meal identification is
necessary. Our data using the present approach, nonetheless,
demonstrates the effectiveness of NGS to reliably identify
multiple hosts species from individual engorged mosquitoes,
which remains a challenge using conventional DNA-based
approaches (Kent 2009). Future work is required to validate
our protocol by analyzing wild engorged specimens of
different arthropods and incorporating multiple markers or
gene targets.
Cytochrome oxidase I (COI) gene is commonly
used for mosquito identification with a high success rate,
although it remains difficult to identify a few closely related
species (Shahhosseini et al. 2018, Versteirt et al. 2015).
Our results show that next-generation sequencing of PCR
amplicons targeting COI gene was accurate at identifying
the four mosquito species examined in this study. However,
mosquito samples that were morphologically identified as
Ae. triseriatus were instead identified as Ae. hendersoni by the
COI marker. The two mosquito species are morphologically
indistinguishable as adults since they belong to the same
species complex and occur in sympatry in the eastern
United States (Reno et al. 2000). The discrepancy between
the morphological and molecular identification of the two
mosquito species is therefore not surprising. Some sequences
from Ae. aegypti were also identified as Ae. mascarensis.
This is also not unexpected since Ae. mascarensis is a close
relative of Ae. aegypti and evidence of retrogression between
the two species has been documented (Hilburn and Rai 1982,
Soghigian et al. 2020). Overall, the findings of this study
demonstrate that Miseq sequencing of the COI marker gene
offers a rapid and reliable methodology for identification of
mosquito species, but additional investigations are needed to
improve taxonomic differentiation of closely related species
in regions where they occur in sympatry.
In summary, our results demonstrate that the NGS
approach described in this study can be used for simultaneous
identification of mosquito species and their blood-meal hosts,
including mixed blood meals that are difficult to identify using
conventional approaches. However, further improvements
may be needed for identification of sibling species. To
confirm the validity of this method and make further
improvements, additional studies should be conducted using
different species of field caught blood-engorged mosquitoes.
Once fully developed, this approach may reduce the cost of
sequencing and improve ability to tackle emerging mosquito-
borne diseases by facilitating timely identification of potential
vectors and their blood-feeding patterns. The method
could then be developed further to facilitate simultaneous
identification of mosquito species, detection of their vectored
pathogens/parasites, and characterization of their blood-
feeding and sugar-feeding patterns, and their symbiotic
microbiota.
Acknowledgments
We thank Paige Pierson and Heather Walker for their
invaluable technical support. This research was supported
in part by the U.S. Department of Agriculture, Agricultural
Research Service, and National Science Foundation
(grant number DEB 1754115). Mention of trade names or
commercial products in this publication is solely for the
purpose of providing specific information and does not imply
recommendation or endorsement by the U.S. Department
of Agriculture. USDA is an equal opportunity provider and
employer.
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