Anthropogenic landscape decreases mosquito biodiversity and drives malaria vector proliferation in the Amazon rainforest

viduals) matrixes were nonsignificant (P > 0.05) (S11 Fig in S1 File). As field collection localities were situated in areas with distinct vegetation classes (Fig 1), we tested their potential influence on Culicidae diversity and Ny. darlingi abundance using the Shannon-index. Results of a Tukey’s test showed that landscapes OTMFA had statistically lower Culicidae diversity than OTMF (P = 0.03). The sampling units in the Campinarana vege- tation class showed a significant difference compared with those in TMF (P = 0.03) and Fig 3. Culicidae species frequency displayed as a cloud for each of three ED categories (low, moderate and high) in forest fringe collections in rural areas of Amazonian Brazil. Font size is proportional to the frequency of Culicidae species. Edge density categories: Low (0–1.5 m / ha x 100); moderate (1.5–2.0 m / ha x 100); high (2.0–3.5 m / ha x 100). https://doi.org/10.1371/journal.pone.0245087.g003 PLOS ONE Anthropogenic landscape and mosquito biodiversity drives malaria vector proliferation PLOS ONE | https://doi.org/10.1371/journal.pone.0245087 January 14, 2021 13 / 25

OTMF (P = 0.01). Results of a Kruskal-Wallis test showed that differences in Ny. darlingi abundance between the sampling unit vegetation classes were statistically significant (chi- squared = 20.7; P < 0.001) (S12 Fig in S1 File). The Mann-Whitney-Wilcoxon test found sta- tistically significant differences between Ny. darlingi abundance in Anthropogenic (Atp) and OTMF (P = 0.04), OTMF and Campinarana (Cpn) (P < 0.001), and between OTMF and OTMFA (P = 0.002). Campinarana and TMF showed the highest abundance averages, with 204 and 234 specimens collected, respectively. Results of analysis employing GLMM (Table 3) revealed that increased diversity measured by the Shannon-index corresponded with a decrease of 226 specimens of Ny. darlingi Fig 4. Correlation matrix among Ny. darlingi abundance, PLAND, ED, richness, DW, diversity indices (richness, Shannon, Simpson and Berger-Parker), Amazonian vegetation classes (Atp: Anthropogenic; OTMFA: Open Tropical Moist Forest with Arecaceae dominance; TMF: Tropical Moist Forest; Cpn: Campinarana), and landscape structures (A: PLAND � 50% and ED < 0.015 m/ha; B: PLAND � 50% and ED � 0.015 m/ha; C: PLAND > 50% and ED � 0.015 m/ha). PLAND: percentage of forest cover; ED: edge density; DW: distance of household from water drainage network. https://doi.org/10.1371/journal.pone.0245087.g004 Table 2. Principal components dimensions showed in PCA. A) open land (PLAND � 50% and ED < 0.015 m/ha); B) fragmented open land (PLAND � 50% and ED > 0.015 m/ha); C) fragmented forested land (PLAND � 50% and

ED > 0.015 m/ha); and D) forest cover (PLAND > 50% and ED < 0.015 m/ha). Components Contributions Variables Dim.1 Dim.2 Dim.3 Dim.1 Dim.2 Dim.3 Shannon-index 0.84 48.2 45.0 Ny. darlingi (abundance) - 0.80 44.7 39.0 PLAND 0.74 - 0.63 46.7 ED 0.73 0.59 46.1 A - 0.79 - 1.41 B 0.34 1.09 C 0.93 D 1.06 R2 (P-value) 0.14 (0.01) 0.64 (0.00) 0.68 (0.00) Cumulative percentage of variance 36.1 65.0 87.1 https://doi.org/10.1371/journal.pone.0245087.t002 PLOS ONE Anthropogenic landscape and mosquito biodiversity drives malaria vector proliferation PLOS ONE | https://doi.org/10.1371/journal.pone.0245087 January 14, 2021 14 / 25

(P < 0.001), and an increase in PLAND, with edge density as the random effect, led to a decrease of 533 (P = 0.003). In model 2, the effects of ED categories at the forest fringe (ran- dom effect) on Ny. darlingi abundance and the Shannon-index in (β1 = -218; P < 0.001) and PLAND (β1 = -337; P = 0.04) were significant. Results of analysis considering model 3 were similar to those obtained with model 2 (Table 3). The path model for Ny. darlingi explained 24% of the variation in abundance and 28% of the variation in the Shannon-index (Fig 5). There were strong negative effects of vegetation class OTMFA and Campinarana on the Shannon-index (-0.53 and -0.50, respectively). Anthropogenic vegetation showed moderate negative and significant reciprocal effects for Table 3. Generalized linear mixed models results of Ny. darlingi abundance in function of the fixed effects of the Shannon-index, PLAND, ED, and house-water drainage network distance, with edge density (ED) categories as the random effect. SLR Model 1 † Model 2 ‡ Model 3 § β1 Standard Error t- value P- value β1 Standard Error t- value P- value β1 Standard Error t- value P- value β1 Standard Error t- value P- value Shannon- index -224.4 68.0 -3.2 0.001 -226.8 64.9 -3.4 < 0.001 -218.8 67.6 -3.2 0.001 -218.8 67.6 -3.2 0.001 PLAND� -343.8 176.1 -1.9 0.05 -533.3 178.9 -2.9 0.003 -337.3 166.5 -2.0 0.04 -337.3 166.6 -2.0 0.04 ED�� -9321.7 7784.5 -1.2 0.2 Distance house-water drainage -0.1 0.2 -0.7 0.4 -0.1 0.2 -0.7 0.4 -0.08 0.2 -0.4 0.6 -0.08 0.2 -0.4 0.7

SLR: Simple Linear Regression; � PLAND: forest cover percentage �� ED: Edge density † Model 1: ED categories at 500 m from peridomestic as random effect ‡ Model 2: ED categories at 500 m from forest fringe habitat as random effect § Model 3: landscape structure categories (A, B, C, D) as random effect. https://doi.org/10.1371/journal.pone.0245087.t003 Fig 5. Structural equation model diagram to describes the relationships between changes in Ny. darlingi abundance with each independent variable: Shannon-index, PLAND, ED, landscape structure categories (A: PLAND � 50% and ED < 0.015 m/ha; B: PLAND � 50% and ED � 0.015 m/ha; C: PLAND > 50% and ED � 0.015 m/ha; D: PLAND > 50% and ED < 0.015 m/ha), Amazonian vegetation class (Atp: Anthropogenic; OTMF: Open Tropical Moist Forest; OTMFA: Open Tropical Moist Forest with Arecaceae dominance; TMF: Tropical Moist Forest; Cpn: Campinarana) and DW. Dashed lines represent a reciprocal path, and solid line one direction. Red arrows (positive coefficient) and blue arrows (negative coefficient) represent the significant path coefficients (P < 0.05). https://doi.org/10.1371/journal.pone.0245087.g005 PLOS ONE Anthropogenic landscape and mosquito biodiversity drives malaria vector proliferation PLOS ONE | https://doi.org/10.1371/journal.pone.0245087 January 14, 2021 15 / 25

OTMFA (r2 = -0.35), Campinarana (r2 = -0.28) and FC (r2 = -0.32). The results of path analysis are detailed in S3 Table in S1 File. The model showed a plausible causal direction on Ny. dar- lingi abundance that involves land use change, represented by negative effects of (1) Anthropo- genic vegetation on FC, (2) Campinarana and OTMFA on the Shannon-index (mosquito community diversity). These effects can increase Ny. darlingi abundance. The forest loss pro- cess (deforestation), particularly in Campinarana and OTMFA, can decrease the diversity of the mosquito community and therefore increase Ny. darlingi abundance. The Path analysis results are presented in S4 Table in S1 File. The canonical correspondence analysis (Fig 6) demonstrated that the four landscape cate- gories were pooled according to species composition of the 79 sampling units. In addition, Ny. darlingi abundance was associated with Culicidae communities represented by the landscapes A, C, and partially D, with the highest positive values associated with A. The components CCA1 and CCA2 explained 50% and 27% respectively, and together they explained 77% of the variation species-landscapes and 5.6% of the total data variation. Landscape B sampling units span the field collections, and landscape A was grouped on the CCA1 axis but separated out well on the CCA2 axis, suggesting that the lower forest cover habitat component is an impor- tant determinant of landscape A and B mosquito sample assemblages. The sampling units of landscape A were distinct, separating best both on the CCA1 and CCA2, suggesting that open

habitat component was most important in determining landscape A mosquito assemblage. Discussion Based on our results, it is possible to construct a network of anthropogenic changes and their effects on Culicidae diversity and Ny. darlingi in the natural tropical rainforest landscape. Here, the forest cover loss causes habitat loss and fragmentation, increasing the forest edge effects and changing the Culicidae species composition. Mosquito communities are strongly impacted by the mechanism associated with changes in land-use. Some species can readily invade new niches that emerged because of forest fragmentation and increased forest edges. High percentage of forest cover and increased Culicidae diversity can lead to a decrease of 533 and 226 mosquitoes, respectively. Deforestation leads to biodiversity loss [72], decreasing the dilution effect, and increasing diffuse competition. In these settings, the reduction of shading, raising the local temperature around human dwellings and in the neighboring larval habitats, reduces the larva to adult development time, and shifts primary vector species [3, 24, 35]. Therefore, habitat fragmentation is a key factor in the dynamics of this species because the increase in forest edge density facilitates the species dispersion and proliferation, creating a serious public health problem. In Brazil, 99% of the 193,838 reported malaria cases in 2018 were in the Amazon biome, and more than 89% (173,006) were caused by P. vivax and 10% (19,283) by P. falciparum [73].

Mosquitoes of the subfamily Anophelinae are responsible for transmitting Plasmodium between infectious and susceptible humans. In endemic areas across the Amazon basin, malaria is associated with activities that require forest clearing, such as infrastructure construc- tion, agricultural expansion, mining, logging, and increased urbanization [28, 74]. Increased malaria incidence is a trade-off of poorly planned or unplanned land occupation for socioeco- nomic development [32, 75–77]. It is well known that anthropogenic change in natural forest environments is a major driver of increased malaria incidence in subtropical and tropical endemic countries [30, 39, 78]. Recently, Chaves et al. (2018) demonstrated that increased malaria incidence is associated with complex mechanisms of change associated with deforestation, forest fragmentation, habitat degradation, geopolitical and social elements that facilitate malaria propagation. In addition, PLOS ONE Anthropogenic landscape and mosquito biodiversity drives malaria vector proliferation PLOS ONE | https://doi.org/10.1371/journal.pone.0245087 January 14, 2021 16 / 25

Chaves and collaborators [4] showed that an increase in the abundance of small patches of non-forest areas can intensify malaria transmission. Results of the current study provided strong statistical support for the mechanisms positively associated with anthropogenic modifi- cation in the Amazonian tropical rain forest landscape, changes in mosquito community, increased abundance of Ny. darlingi and intensification of malaria transmission. The socioeco- logical determinants were not tested, however, despite being a corollary to mosquito bites, Fig 6. Canonical correspondence analysis of mosquito assemblages and landscape structure classifications at each sampling unit. Open landscape (A) is represented by black circles, fragmented open land (B) is represented by red, fragmented forest (C) is represented by green, and forested (D) is represented by blue circles. The length of the arrow representing the strength of the relationship. The yellow triangle represents Ny. darlingi. A: PLAND � 50% and ED < 0.015 m/ha; B: PLAND � 50% and ED � 0.015 m/ha; C: PLAND > 50% and ED � 0.015 m/ha; D: PLAND > 50% and ED < 0.015 m/ha. https://doi.org/10.1371/journal.pone.0245087.g006 PLOS ONE Anthropogenic landscape and mosquito biodiversity drives malaria vector proliferation PLOS ONE | https://doi.org/10.1371/journal.pone.0245087 January 14, 2021 17 / 25

increasing vector abundance and putting settlers at risk of malaria [79], the socioeconomic conditions from localities were similar (i.e., subsistence rural practices in low populational density (< 1,500 people per km2), medium-low Human Development Index (HDI ~ 0.6), lack of basic sanitation and precarious housing). However, the landscape structure differed in each sampling unit of our study design, verifying that landscape (without socioeconomic confusion variables) operates as a regulator of the mosquito community and malaria risk. Relative to the landscape habitat of the primary malaria vector, females will seek more shaded and safer larval habitats (realized niche) for egg laying [34]. In this context, moderate forest cover (i.e., a range between 45 ¬ 65%, partially shaded) increases the occurrence of larval habitats and adult resting places. On the other hand, low forest cover (full sunlight larval habi- tats) might pressure the species to disperse, because the females require appropriate water bod- ies (partially shaded) to deposit eggs. Some authors have demonstrated that the microclimate, pH, microbiota and other factors affect the productivity of Ny. darlingi larval habitats [41, 80, 81]. Results of the present study demonstrated that changes in the natural landscape structure, defined by the measurement of three metrics, significantly impacted adult productivity. Adult abundance is likely associated with an increase in the presence of suitable larval habitats and vertebrate hosts for blood feeding. Similarly, a positive relationship between clearing natural

forest and the presence of Ny. darlingi was found in French Guiana [82]. Results of our study indicate that deforestation and forest clearing for human occupation and changes in land-use create new ecological and environmental conditions, which drive changes in the composition and community structure of plants [83, 84] and animals [85, 86], including insects of the family Culicidae. The anthropogenic changes in the tropical rainforest environment are linked to processes of ecological succession that can be initiated by a distur- bance in a community [87]. Forest streams can be impacted by deforestation, i.e., once a tree is cut its trunk or branches can block the water flow, increasing resting places for adults, habitat for female oviposition, and microhabitat for immature development (S13 Fig in S1 File) [88]. Following deforestation, blocked water flow, and readily available blood meals, mosquitoes can proliferate. The introduction of new sources of blood for mosquitoes, and biotic compo- nents of malaria ecology such as ecological interactions among vector-host species also affect the dominance of vector species [56]. Humans and domestic animals, as well as local weather conditions are important factors in the changing landscape due to environmental disturbance [89, 90]. These factors together contribute to a dynamic process of change, dominated by fast- growing, resilient, generalist species that will be replaced by more competitive species as suc- cession proceeds or new anthropogenic changes interfere in the natural succession process

[91–93]. Medeiros-Sousa and colleagues [94] found strong evidence that biodiversity loss increases the risk of pathogen transmission. This can be observed in mosquito communities, for example, when a taxonomic group increases or decreases its species assemblages in a chain of ecological succession. In our study, Culicidae diversity (β diversity) responded inversely to Ny. darlingi vector abundance across changes in PLAND metrics (S14 Fig in S1 File), a process that can influence diffuse competi- tion and transmission dynamics of vector-borne pathogens [10]. This ecological process was used to explain malaria dynamics in the tropical rain forest, where access to vertebrate blood sources can be mediated by the threshold tolerance of a host to mosquito bites, and defensive host behavior [3]. The increased β diversity in higher PLAND can increase diffuse competition and decrease in malaria risk. In S15 Fig in S1 File landscapes with less than 25% of PLAND had fewer rare species (rich- ness) according to the collector curve effort. This evidence reinforces the hypothesis that forest cover can drive the dominance of Ny. darlingi. Therefore, if we define risk as the chance of a susceptible host being bitten by an infected vector, landscapes with lower forest cover PLOS ONE Anthropogenic landscape and mosquito biodiversity drives malaria vector proliferation PLOS ONE | https://doi.org/10.1371/journal.pone.0245087 January 14, 2021 18 / 25

percentages (< 25%) represent higher malaria risk in terms of human presence and a higher density of a competent vector species. Overall, the results of the analyses showed that PLAND is a predictor variable for Ny. dar- lingi dominance in endemic areas of the Brazilian Amazon. Abundance of the mosquito vec- tors decreases with increase in the percent forest cover. Despite we have not directly tested the source-sink dynamics, the relationship between vector abundance and forest cover can also receive a contribution from this ecological range, as described by various authors [51, 52, 95]. In a malaria landscape where Ny. darlingi is the dominant vector, the source habitats are those that export individuals of the species, whereas the sink habitats import individuals, thereby sustaining the population in the heterogeneous landscape. The forest edge represents the source, whereas inside and nearby human dwellings represent the sink habitat for Ny. darlingi, with recolonization from sources due to a continuous process of increasing forest edge length by forest clearing and habitat fragmentation for human occupation and changes in land use. The establishment of a new Ny. darlingi population in a sink habitat is facilitated by the high reproductive success of the species, and rapid population growth in the Amazonian landscape. In addition, high temperature increases the development rate of immature stages as demon- strated by Chu et al. [35]. Populations in sink areas tend to occupy low-quality habitats that do

not support them for long, whereas those in source areas, with high-quality habitat, persist lon- ger and contribute to the recolonization of other fragments through dispersal of a fraction of the individuals [96]. Ecological factors that maintain Ny. darlingi in the peridomestic habitat (S7A Fig in S1 File) and the forest fringe (S7B Fig in S1 File) were impacted by PLAND and ED variables. The rela- tionship between niche requirements and the patterns of landscape structure are supported by several studies of Ny. darlingi bionomics [21, 34, 97]. Therefore, considering the ecological determinants of the presence of Ny. darlingi, a low edge density can represents a source habitat because the availability of habitats for females to lay their eggs increases [34]. In contrast, a high edge density represents a sink habitat because the occurrence of habitat suitable for ovi- position decreases [88]. In the peridomestic habitat, this mechanism is reversed, with Ny. dar- lingi the most abundant species. Likely, the anthropophilic/opportunistic blood-feeding behavior of the species facilitates the occurrence of part of the population in close contact with humans and domestic animals in an anthropogenic landscape. In Iquitos, Peruvian Amazon, the Ny. darlingi population is opportunistic, with a high proportion of the population feeding on humans (42.5%), and an unexpectedly 25.1% of individuals blood-feeding on avian hosts [98]. Similar results were found for Ny. darlingi collected in eastern Amazonian Brazil [99],

where the availability of host species influences the blood feeding preference of the mosquito. In conclusion, our study demonstrated that changes in Brazilian Amazon landscapes decrease overall Culicidae diversity and allow Ny. darlingi to become dominant if forest cover percentage decreases and edge density is low. Fragmented forest habitats inhabited by vulnera- ble people in precarious houses are the sources of this species, whereas continuous forested habitats or completely deforested habitats are sinks. The PLAND showed an empirical influ- ence on blood-feeding activities and ED on oviposition behavior both lead to high Ny. darlingi abundance in the peridomestic habitat. Considering the key role of Ny. darlingi in malaria transmission cycle, our finding is highly relevant for understating heterogeneities in the dynamics of malaria epidemiology. Supporting information S1 File. (PDF) PLOS ONE Anthropogenic landscape and mosquito biodiversity drives malaria vector proliferation PLOS ONE | https://doi.org/10.1371/journal.pone.0245087 January 14, 2021 19 / 25

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