Toward the definition of
Aedes albopictus
and
A. koreicus
pathobioma from an area of recent invasion
in northern Italy
Discussion & Conclusions
Results
Methods
Background
Fausta Rosso*, Valentina Tagliapietra, Davide Albanese, Massimo Pindo, Frederic Baldacchino, Daniele Arnoldi, Claudio Donati, Annapaola Rizzoli
Fondazione Edmund Mach, Research and Innovation Centre, Via E. Mach 1, 38010 San Michele all'Adige, Italy
*fausta.rosso@fmach.it
Pathobiome 2018, Ajaccio, Corse, France March 18-20, 2018
After rarefaction (without replacement), a total of 2153 OTUs were clustered belonging to 106 orders, 157 families and 217 genus. Ae. albopictus and Ae.
koreicus showed high variations at individual level although they shared 75.98% of
the reads in their gut microbiota. The core was dominated by species of the genus
Pseudomonas (64.63% and 63.38% in Ae albopictus and Ae. koreicus,
respectively), followed by Wolbachia (9.8% and 0.002%), Janthinobacterium (4.7%
and 4%), Ralstonia (2.1% and 2.3%), Enterobacter (0.01% and 1.5%) and
Providencia (0.0005% and 1.2%) plus other minor ones (Stenotrophomonas, Streptococcus, Candidatus Portiera, Delftia and Gluconobacter). There were no
specific OTUs belonging to Ae. koreicus, while 3 OTUs were specific to Ae.
albopictus, corresponding to 9.81% of the reads, and with 2 out of 3 belonging to the
genus Wolbachia.
α-diversity was significantly higher in Ae. albopictus compared to Ae. koreicus according to the number of observed OTUs, the Chao1 estimator and Shannon entropy (P=0.0027, 8.4x10-4and 9.5x10-4, respectively). Alsoβ-diversity revealed a
significantly different gut microbiota as resulted from a PERMANOVA test (9999 permutations) (P<10-4, P<10-4 and P=9x10-4 for unweighted UniFrac, weighted
UniFrac and Bray Curtis, respectively).
In the Province of Trento, Northern Italy, two invasive alien species of mosquitoes,
Aedes albopictus and Ae. koreicus, are well established. The former was recorded
the first time in 1996 (Roiz et al., 2010), and the latter arrived fifteen years later, in 2011 (Capelli et al., 2011). In our territory, these two species overlapped and adapted to colonize man-made type of habitats such as garden and cemetery flower pots, abandoned containers, manholes, water recipents, etc.. The common developmental environment is probably the reason of the large core microbiota shared by the two species. Nonetheless, the 15-year time difference of arrival, could explain the higher richness of Ae. albopictus compared to Ae. koreicus. Of particular interest is the contrasting presence of Wolbachia and representatives of the Acetobacteraceae family, most probably Asaia, in the two species.
Wolbachia is known to inhibit the horizontal transmission or reduce the vector
competence of some mosquito-borne pathogens like Plasmodium spp., dengue, yellow fever, West Nile fever and chikungunya viruses (Blagrove et al. 2012, 2013; Moreira et al. 2009; van den Hurk et al. 2012; Bian et al. 2010, 2013; Glaser and Meola 2010; Mousson et al. 2012; Novakova et al. 2017). Asaia spp. has been considered a potential vector of antiplasmodial factors through paratransgenesis (Favia et al. 2008). Previous studies found a contrasting presence of these two symbionts in other mosquito species (Anopheles gambiae, An. stephensi (Hughes et al., 2014), Ae. aegypti (Ruang-Areerate et al., 2006)) and our findings add Ae.
koreicus to this list. The epidemiological consequences of this aspect need to be
further elucidated.
This study provides the first description of the highly diverse community of micro-organisms occurring in the midgut of two invasive mosquito species of public health concern in northern Italy. It also provides the basis for the realization of experimental studies aiming at assessing the consequences of the variation of gur microbiota composition on the vectorial capacity for pathogens carried by these two
Aedes mosquitoes species. Aedes mosquitoes are highly successful invasive species globally and they also
pose public health concerns as vectors of several viruses of medical importance. Next-generation sequencing technology (NGS) has recently helped to unveil the complex, dynamic and extended role of microbiome community. In particular midgut microbiota of mosquitoes is paramount for their fitness, physiology, food and blood digestion, metabolism, immunity, adaptation to new environmental conditions, and in turn affects their vectorial competence and capacity to transmit pathogens.
We show some preliminary analysis on the characterization of the microbial community of Aedes albopictus and Aedes koreicus using a metagenomic approach.
Ae. albopictus Ae. albopictus+Ae. koreicus
Distribution map of Ae. albopictus and Ae. koreicus in the Province of Trento (Italy)
Ae. koreicus mosquito gut from an
adult female after dissection and extraction (24x) (foto D. Arnoldi)
BG-Sentinel trap (Biogen AG) for the collection of adult stages of
mosquitoes
Ae. koreicus adult female(foto by N. Busatto)
Ae. albopictus adult female(foto by N. Busatto)
Midgut samples from 30 Ae. albopictus and 30 Ae. koreicus non-fed females. DNA extraction using Qiamp DNA Investigator Kit (Qiagen).
PCR amplification of the V3-V4 hypervariable region of the 16S ribosomial RNA targeting 460-bp fragment using the bacterial primer pair 341F (5’ CCTACGGGNGGCWGCAG 3’)
and 806 R (5’ GACTACNVGGGTWTCTAATCC 3’) with overhang Illumina adapters.
Pyrosequencing and pre-processing of the reads using the MICCA pipeline (http://www.micca.org, version 1.6).
Removing of reads with less than 60% similarity to the Greengenes database (May 2013 version, clustered at 85% identity) using VSEARCH version 1.9.5. Picking and clustering of high quality reads at 97% identity using the “de novo greedy” Operational Taxonomic Unit (OTU) implemented in MICCA. Classification of the representative sequences using
the consensus classifier against the Greengenes database (May 2013 version). Sequences alignment using NAST.
Downstream analyses were performed using phyloseq, vegan and picante libraries in R environment. Samples rarefaction (without replacement) at 45.000 reads per sample. PCR products purification and Illumina sequencing adapters Nextera XT Index Primer (Illumina) attached by PCR. Barcoded libraries sequenced on an Illumina Miseq (PE3000)
platform (Miseq Control Software 2.0.5. and Real Time Analysis software 1.16.18).
Literature cited:
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Blagrove, M. S. C. et al. 2013. PLoS Negl Trop Dis 7 (3):e2152. Capelli, G. et al. 2011. Parasit Vectors 4 :188.
Glaser, R. L. et al. 2010. PloS One 5 (8):e11977. Hughes, D. T. et al. 2014. PNAS 111(34):12498-503 Moreira, L. A. et al. 2009. Cell 139 (7):1268–78.
Mousson, L. et al. 2012. PLoS Negl Trop Dis 6 (12):e1989. Novakova, E. et al. 2017. Front Microbiol 8:526. Roiz, D. et al. 2010. Vector Borne Zoonotic Dis 10 (8):811–16. Ruang-Areerate, T et al. 2006. PNAS. 103(33):12534-9. van den Hurk A. F. et al. 2012. PLoS Negl Trop Dis 2012;6(11):e1892
