Mosquito diversity: From six field campaigns, 292 blood-engorged female mosquitoes, representing eight genera and 20 species, were collected (Table 1). The most abundant species was Aedes fulvus (15.8%), and the most diverse genus collected was Culex, with six species identified, followed by Uranotaenia with four species. By analyzing the obtained Cytochrome c Oxidase Subunit I (COI) sequences, we confirmed the identity of 10 species, supporting the morphological identification. Genetic distances, as determined by the Kimura-2-parameter (K2P) for intraspecies values, were under 2.4% for all species (see Supplementary Fig. S1). The maximum likelihood (ML) tree, built using the K2P distances between species, is shown in Fig. 2. Confirmation of species identity through barcodes was achieved for nine individuals with genetic identities to reference sequences above 97.6%. Reference sequences for Culex adamesi were not available in any published database, so we contributed the first sequences for this species. Also, species belonging to the genus Uranotaenia could not be morphologically identified and our barcodes showed only 92% of homology with Uranotaenia sapphirina, the closest species in the barcode database.
Table 1
Diversity and abundance of blood-engorged female mosquitoes collected from March 2017- April 2018 using four sampling methods in forest fragments in San Juan Carare, Colombia. The abundance of each species is calculated relative to the total number of engorged females collected (N = 292). The number of bloodmeals identified includes the result of Cyt-b and COI amplifications.
Species
|
Engorged female abundance
|
Percentage (%)
|
Bloodmeal source identified
|
Ae. fulvus
|
46
|
(15.8%)
|
33
|
Ae. serratus
|
39
|
(13.3%)
|
15
|
Cx. pedroi
|
35
|
(12.1%)
|
12
|
Mn. titillans
|
30
|
(10.2%)
|
4
|
Cx. spissipes
|
28
|
(9.6%)
|
5
|
Cq. albicosta
|
22
|
(7.5%)
|
3
|
Cx. nigripalpus
|
21
|
(7.2%)
|
11
|
Ps. albipes
|
20
|
(6.9%)
|
13
|
Cq. nigricans
|
16
|
(5.5%)
|
2
|
Cx. adamesi
|
10
|
(3.3%)
|
5
|
Cx. vomerifer
|
6
|
(2.1%)
|
0
|
Uranotaenia sp. 1
|
5
|
(1.7%)
|
5
|
An. neomaculipalpus
|
2
|
(0.7%)
|
1
|
An. triannulatus
|
2
|
(0.7%)
|
1
|
Culex sp. 1
|
2
|
(0.7%)
|
1
|
Culex sp.2
|
2
|
(0.7%)
|
0
|
Li. durhami
|
2
|
(0.7%)
|
1
|
Uranotaenia sp. 4
|
2
|
(0.7%)
|
0
|
Uranotaenia sp. 2
|
1
|
(0.3%)
|
0
|
Uranotaenia sp. 3
|
1
|
(0.3%)
|
0
|
Vertebrate diversity: During fieldwork, we recorded 90 bird species belonging to 23 families using mist net collections and visual observations in three habitat types: pasture, forest understory, and river edge. Avian families with the greatest observed local diversity were Tyrannidae (13 species) followed by Ardeidae (egrets), Psittacidae (parrots), Icteridae (New World blackbirds, cowbirds, orioles), Picidae (woodpeckers) and Thraupidae (tanagers) with five species each. The bird species most frequently detected were Smooth-billed Ani (Crotophaga ani), Great Egret (Ardea alba), Ruddy Ground-Dove (Columbina talpacoti), Bicolored Wren (Camphylorynchus griseus) and Bare-faced Ibis (Phimosus infuscatus). Mammal populations were assessed based on captures of 164 individuals belonging to 21 species of nine families. Chiroptera was the order with highest richness with 15 species, followed by Primates (four species), Didelphimorphia (one species) and Rodentia (one species). Additionally, nine species of reptiles and eight of amphibians were recorded (Supplementary Table S1).
Bloodmeal analysis: From the blood-engorged female mosquitoes, we amplified DNA sequences in 154 of the 292 samples analyzed, using the primers targeted to vertebrate COI (33.7% of the samples) and Cyt-b (66.3%) genes. Vertebrate species identified included 17 mammals, four birds, three reptiles, and two amphibians. From these, 20 were sylvatic and five domestic, besides human blood meals. From the total number of sequences, 21.9% did not have enough resolution for specific identification, probably as a result of mixed bloodmeals in the sample. Vertebrate bloodmeals detected, most frequently derived from the howler monkey (Alouatta seniculus, 27.7%), which was fed upon by Aedes fulvus and Psorophora albipes. Aedes fulvus was the mosquito species with the largest number of vertebrate species (N = 7, 26 %) detected in its diet. Mammals provided the highest proportion of bloodmeals (Fig. 3b) and eight species from the orders Pilosa, Didelphimorphia, Rodentia and Cingulata were only detected with this method. Domestic animals were less common sources for bloodmeals, and included chicken, pig, horse, water buffalo and duck. These species were mainly found in the diet of Cx. nigripalpus, Cx. adamesi and Mansonia titillans. Human blood was detected in eight species: Ae. serratus, An. neomaculipalpus, An. triannulatus, Psorophora albipes, Coquillettidia venezuelensis, Cx. pedroi, Cx. spissipes and Cx. nigripalpus. Bird species were only detected in Culex and Mansonia mosquitoes. Overall, species belonging to the class Mammalia were more often used by the mosquitoes found in the area, followed by birds, reptile and amphibian blood in this decreasing order (Fig. 3b). A bipartite network constructed from bloodmeal identifications shows the total number of vertebrate species belonging to each class (Fig. 3a).
Flavivirus detection: Flavivirus amplification was detected in 17 pools (24.3%), and 25 individuals (10%) within the pools were positive for the detection of the flavivirus NS5 gene. Retesting these 25 RNA samples for detection of the Zika virus envelope gene yielded negative results. The cDNA obtained in each reaction was revealed by agarose gel electrophoresis, but we did not obtain unique bands or viral sequences through Sanger sequencing in any of the samples. Sequencing the amplicons of the NS5 targets failed to detect known flaviviral sequences.
Network analysis: The mosquito-vertebrate network comprised 41 different species interactions (Fig. 3a). Modularity was high (Q = 0.59, z = 13.4, p < 0.05), indicating significant specialization of mosquito choice of vertebrates utilized for bloodmeals. We obtained eight modules after 50 iterations (Fig. 4). Five of the modules were associated with a single mosquito species, meaning they showed a very different behavior compared to other species. The remaining modules grouped two, three or five species of mosquitoes. Regarding vertebrates, interestingly, we found one host module composed of domestic and synanthropic species, three exclusively of mammalian species, one of only reptile species and two only of amphibians. All species of marsupials (D. marsupialis, P. opposum, and M. caucae) were in the same module with the primate Alouatta seniculus and the rodent Zygodontomys brevicauda. Overall, vertebrates with similar traits in terms of habitat use, were grouped together.
The specialization index at the community level (H2′) had a value of 0.55, indicating aggregated patterns of mosquito feeding behavior and preferences for certain vertebrates. In addition, the analysis of nestedness confirmed that mosquito-feeding behavior is not significantly nested (NODF = 6.84, p = 0.62). At the species level, the degree of mosquito specialization varied among species when comparing the d and COV indexes. The species Culex sp.1 and Uranotaenia sp.1 had the highest values for both indexes (d = 1, COV = 1) suggesting those were the species with the most exclusive interaction types and narrow host range. Anopheles neomaculipalpus and An. triannulatus had a low value of index d = 0.21, but high value for the COV index = 1 (Table 2) as human blood was the only bloodmeal detected on these mosquitoes, but also in other species. Aedes fulvus, Ae. serratus Cx. adamesi, Cx. nigripalpus, and Cx. pedroi had the highest values for “species strength”, denoting greater diversity in the detected interactions. Lastly, the value of betweenness centrality (BC) for host species varied from 0 to 0.39 (Fig. 3a).
Table 2
Index values of specialization by mosquito species recognized in San Juan, Carare, Colombia. The Specialization index (d) and the Coefficient of interaction variation (COV) are estimated with scaled values between 0 to 1.
Species
|
d
|
COV
|
Species strength
|
Aedes fulvus
|
0.62
|
0.66
|
5.28
|
Aedes serratus
|
0.49
|
0.55
|
3.14
|
Anopheles neomaculipalpus
|
0.21
|
1
|
0.06
|
Anopheles triannulatus
|
0.21
|
1
|
0.06
|
Coquillettidia albicosta
|
0.45
|
0.55
|
1.02
|
Coquillettidia nigricans
|
0.27
|
0.69
|
0.22
|
Culex nigripalpus
|
0.54
|
0.56
|
3.15
|
Culex adamesi
|
0.74
|
0.41
|
3.57
|
Culex sp. 1
|
1
|
1
|
1
|
Culex pedroi
|
0.61
|
0.51
|
3.46
|
Culex spissipes
|
0.45
|
0.47
|
1.63
|
Limatus durhami
|
0.49
|
1
|
0.17
|
Mansonia titilans
|
0.52
|
0.78
|
0.71
|
Psorophora albipes
|
0.35
|
0.86
|
0.48
|
Uranotaenia sp.1
|
1
|
1
|
1
|