Discussion
This study provides evidence that carrion flies are the superior method
for landscape scale biodiversity surveillance compared to both camera
traps and the other iDNA sampling methods (carrion fly iDNA identified
62 vertebrate species, sandfly iDNA detected 49 species, mosquito iDNA
detected 16 species, and camera traps identified 28 species). Previous
work has found that iDNA from carrion flies produced complementary
results when compared to more traditional methods of biomonitoring such
as camera traps (Gogarten et al., 2020; Rodgers et al., 2017). We found
a similar pattern to Rodgers et al. (2017) in their comparison of
carrion flies to existing camera trap data in that even with a small
sample size and effort, carrion flies used as iDNA samplers were able to
profile most of the diversity that had been found with long-term camera
trapping while also revealing new species diversity. However, with our
larger sampling effort, we were able to show that carrion flies were
much more effective at describing diversity at the landscape scale and
species richness likely would have increased for each iDNA sampler if we
had increased our sampling effort, particularly at the site level,
whereas species richness had fully saturated by 1,500 camera-days (Fig.
2 and Fig. 4).
While camera traps revealed lower gamma diversity than carrion flies and
sandflies, they had the greatest median species richness at sites (alpha
diversity) compared to each of the iDNA samplers. This points to camera
traps reliably revealing the same suite of species from across sites
resulting in higher alpha diversity but lower overall gamma diversity
when compared to iDNA samplers. The missing species richness exhibited
by camera traps at the landscape scale of our study is likely due to
camera traps preferentially targeting larger-bodied mammals (Burton et
al., 2015; O’Brien & Kinnaird, 2011). We found support for this as
camera traps revealed the greatest diversity of carnivore species and
ungulate species while missing most of the iDNA-described diversity of
the arboreal species and the smaller-bodied mammals.
The improved biodiversity detection with carrion flies over other iDNA
sources and camera traps comes with additional logistical advantages.
Carrion flies can be trapped with inexpensive, homemade traps compared
to the more expensive camera traps or the UV LED CDC light traps used to
capture sandflies and mosquitoes. These homemade traps also did not
require batteries compared to the UV LED CDC light traps which require
6V batteries that need to be changed approximately every 24 hours. Our
trap design (Appendix S1: Fig. S2) was effective at attracting and
trapping carrion flies while at the same time preventing individuals
from accessing the bait. Using a blocking primer for the species used as
bait is also an option when utilizing carrion flies as an iDNA source.
Carrion flies are thus much more amenable to sampling in remote
landscapes where transporting and shipping pre-made traps or bulky
equipment can be difficult. We also identified the most species with
carrion flies compared to the other iDNA samplers despite a much lower
sampling effort of 3 traps per site compared to 27 traps per site for
mosquitos and sandflies, which resulted in much fewer individual carrion
flies (n = 1,759) compared to the total number of sandflies (n = 56,774)
and mosquitos (n = 4,776). Carrion fly samples had the greatest
amplification efficiency of non-human vertebrates (Table 1) despite only
5 individual flies coextracted compared with 15 mosquitos and 50
sandflies, although amplification efficiency reflected all captured
sandflies (not only blood-fed females) and did not include human DNA
which was highest in mosquito samples.
Although carrion flies were the most effective iDNA sampler, we wanted
to examine whether the gamma and alpha diversity described varied
substantially among iDNA sources to assess the effectiveness of
sandflies and mosquitos as biodiversity samplers. We found that the
overlap in vertebrate communities from each iDNA source was generally
supported at the landscape scale, however, the different iDNA sources
detected different communities of species at each site, and the same
sites were typically distant in ordination space. Although carrion flies
were the most effective sampler for detecting gamma diversity, we found
carrion flies were less redundant with mosquitos and sandflies for
detecting local biodiversity and carrion flies missed 85% and 67% of
the species occurrences from across all sites in the sandfly data and in
the mosquito data respectively, although most of these misses
corresponded to the dominant species in either the sandfly or mosquito
data as shown in Appendix S1: Fig. S4. This points to needing a
substantially larger sampling effort at the site level for iDNA methods,
particularly carrion flies, to saturate species detections.
Aside from the effect of sampling effort on diverging alpha diversity
profiles, some of the mismatch is explained by the differing feeding
ecologies of each insect group. We detected substantial taxonomic biases
in vertebrate detections among iDNA sources that were likely driven by
differences in the host preferences of the common mosquito and sandfly
species. Aside from domestic dog, we found evidence of contrasting
feeding preferences for vector taxa that were consistent with to their
known relationships with host species. Sandflies overwhelmingly targeted
armadillos (Dasypus novemcinctus and Dasypus kappleri )
which aligns with our understanding of both the relationship between
armadillos and sandflies (Akhoundi et al., 2016; Alexander, 2000;
Lainson et al., 1979) as well as armadillos as an important host species
for the life cycle of Leishmania parasites (Lainson et al., 1979;
Lainson & Shaw, 1989), which are transmitted by female sandfly vectors.
Kocher et al. (2017) reported the first direct evidence of
sandfly-armadillo interaction using metabarcoding of sandflies, and our
findings overwhelmingly support this finding. Mosquitos also exhibited
strong host preferences for humans, which even after rigorous quality
control to eliminate contamination was the dominant iDNA data source
representing 80% of the total reads. While many species of mosquito are
opportunistic feeders, there are a number of species that target humans
including Aedes aegypti which is the main vector for dengue,
yellow fever, and chikungunya and is found throughout tropical and
sub-tropical regions worldwide (McBride, 2016; Zwiebel & Takken, 2004).
Interestingly, our metabarcoding data is corroborated by previous
studies spanning the Amazon region that show upwards of 80% of A.
aegypti bloodmeals contain human DNA (McBride, 2016). Compared to the
targeted feeding behavior of sandflies and mosquitos (as well as leeches
shown by Drinkwater et al. 2019), the feeding ecology of carrion flies
may offer the least biased view of the diversity and relative biomass of
species across a landscape because their feeding behavior is likely
driven by both the presence of carrion and scat (Calvignac-Spencer et
al., 2013; Rodgers et al., 2017). Given these findings, the feeding
ecologies of each insect group should be an important consideration
because choice of iDNA sampler can lead to mismatches in the vertebrate
diversity profiles, especially with smaller sampling sizes.
By exploring the biases associated with each sampling type, we found
evidence that carrion flies are significantly more effective in
describing landscape level biodiversity when compared to diversity found
with camera traps and other iDNA samplers, even with the least sampling
effort and smallest sample size. We found that camera traps predictably
were biased towards larger-bodied mammals and away from arboreal and
smaller-bodied species, thus missing much of the biodiversity described
with iDNA samplers, but also had the highest species richness at the
site-level. Sandflies and mosquitos show feeding behavior targeted at
armadillos and humans, respectively. Although sandflies and mosquitos
were inferior samplers for large-scale biodiversity surveillance
compared to carrion flies, they add value to existing
host-vector-pathogen surveillance efforts. This is particularly relevant
in forest frontiers where vectors maintain enzootic sylvatic cycles
which can cause disease spillover into humans and/or domestic animals if
they are bitten by these infected vectors (Figueiredo, 2007; Vasconcelos
et al., 2001). Additionally, our findings support the known ecological
relationships between these vector taxa and their hosts thus further
validating metabarcoding results within the larger realm of ecological
research. With the advances in high-throughput sequencing and the
steadily decreasing expense of metabarcoding, iDNA should be used as an
effective and efficient method for biomonitoring.