Mitochondrial genome of Plasmodium vivax/simium detected in an endemic region for malaria in the Atlantic Forest of Espírito Santo state, Brazil: do mosquitoes, simians and humans harbor the same parasite?

Background The transmission of malaria in the extra-Amazonian regions of Brazil, although interrupted in the 1960s, has persisted to the present time in some areas of dense Atlantic Forest, with reports of cases characterized by particular transmission cycles and clinical presentations. Bromeliad-malaria, as it is named, is particularly frequent in the state of Espírito Santo, with Plasmodium vivax being the parasite commonly recognized as the etiologic agent of human infections. With regard to the spatial and temporal distances between cases reported in this region, the transmission cycle does not fit the traditional malaria cycle. The existence of a zoonosis, with infected simians participating in the epidemiology, is therefore hypothesized. In the present study, zoonotic transmission of bromeliad-malaria in Espírito Santo is investigated, based on the complete mitochondrial genome of DNA extracted from isolates of Plasmodium species which had infected humans, a simian from the genus Allouata, and Anopheles mosquitoes. Plasmodium vivax/simium was identified in the samples by both nested-PCR and real-time PCR. After amplification, the mitochondrial genome was completely sequenced and compared in a haplotype network, including all sequences of P. vivax/simium mitochondrial genomes sampled from humans and simians from all regions in Brazil. Results The haplotype network demonstrates that humans and simians from the Atlantic Forest share the same haplotype, but some isolates from humans are not identical to the simian isolate. In addition, the plasmodial DNA extracted from mosquitoes revealed sequences different from those obtained from simians, but similar to two isolates from humans. Conclusions These findings reinforce the hypothesis that in the Atlantic Forest, and especially in the state with the highest frequency of bromeliad-malaria in Brazil, the same parasite species is shared by humans and simians, at least in part. The difference between the sequences of mosquitoes and simians raises two hypotheses: (1) two distinct transmission cycles for human malaria exist in the study area, one of them involving simians and the other exclusive to human hosts, or (2) there is only one transmission cycle involving humans and simians, but the identification of variations among simians was not possible due to a lack of other samples.


Background
In Brazil, malaria occurs originally across the entire national territory. However, the Amazon region reports 99% of all the cases in the country [1]. Since the 1940s, the national control program has kept malaria transmission restricted to the Northern area.
Thus, in the 1960s and 1970s, the extra-Amazonian region came close to complete disease elimination. Nevertheless, residual transmission persisted in areas of the dense Atlantic Forest [2]. In the Atlantic Forest, malaria presents a very low incidence, and the cases are mainly related to Plasmodium vivax, presenting few clinical symptoms [1][2][3][4].
The low incidence and the territorial dispersion of the reported cases provide evidence in favor of the existence of an unrecognized reservoir of the parasites. This in turn raises hypotheses regarding the participation of asymptomatic carriers or local simians in the transmission [5]. The genetic similarity between the P. vivax, that infects humans, and the parasites that infect simians in the Atlantic Forest, named Plasmodium simium, reinforces the hypothesis of zoonosis. In fact, most studies indicate that both species are genetically identical [5][6][7][8]. However, in a recent study, Brasil et al. [9] suggested the possibility of using some single nucleotide polymorphisms (SNPs) in the differentiation of both species.
In the extra-Amazonian region, the term bromeliad-malaria refers to the disease whose vector, recognized as Anopheles (Kerteszia) cruzii [10], depends on bromeliads as breeding sites. Molecular and serological evidence presented by different studies has demonstrated that bromeliad-malaria is closely dependent on human activities carried out close to the forest environment [11][12][13]. In addition, the occurrence of the disease is sparse, and the outbreaks are rare [14]. Considering the characteristics presented above and the fact that the parasites harbored by local simians are genetically indistinguishable from those found in human blood, the hypothesis of a zoonotic scenario for bromeliad-4 malaria is strongly supported [15][16][17][18][19][20][21]. However, even with a variety of scientific investigations corroborating the zoonoses hypothesis, a considerable debate remains regarding the direction of parasite transference. For instance, by comparing the genetic variability in studies based on the Duffy binding protein of erythrocytes collected from simians of the species Alouatta guariba, Costa (2014) [22] has suggested that the simian parasite originated from its human counterpart. This hypothesis is additionally supported by Rodrigues et al. [23] based on limited genetic variability between P. simium and P. vivax.
In order to investigate the zoonotic transmission of bromeliad-malaria, this study presents the molecular characterization of P. vivax/simium based on the sequencing of the mitochondrial genome of parasites isolated from both human and simian hosts, and, unprecedentedly, from Anopheles mosquitoes in an endemic area of the Brazilian Atlantic Forest.

Study area
Espírito Santo is a Brazilian state located in the Southeast region, which harbors large areas of dense Atlantic Forest. The fieldwork for collecting samples of anopheline mosquitoes and monkeys was concentrated in Valsugana Velha, district of Santa Teresa, and the main area with reports of malaria in this municipality. Santa Teresa is located 78 km from the capital of Espírito Santo, Vitória ( Figure 1). The landscape in the region is irregular, with a mountainous relief reaching an altitude of 655 meters above sea level, and average temperatures that vary between 15.3 °C and 21.0 °C. Four human blood samples were collected from the inhabitants of Santa Teresa, and 18 from other municipalities of Espírito Santo, also covered by the Atlantic Forest. and four Anopheles cruzii) [24].

DNA extraction and confirmation of the infection
Plasmodial DNA from human and simian blood samples was extracted by the QIAamp Blood DNA Mini Kit, while the plasmodial DNA from the mosquitoes was extracted by the DNAeasy Blood and Tissue Kit, both following the instructions of the manufacturer (Qiagen). Infection was confirmed in all samples by nested-PCR [25,26] and real-time PCR (adaptation from Rubio et al. [27]) with primers designed to amplify the 18S RNA subunit gene. Positive and negative controls were used in all reactions.

Amplification and sequencing of the plasmodial complete mitochondrial genome
The complete mitochondrial genome (6 kb) of P. vivax/simium from the 22 samples of human blood was amplified and sequenced following the protocol proposed by Rodrigues et al. [28]. A new protocol had to be developed in order to perform the 6 amplification of the plasmodial DNA extracted from simian and mosquito samples.

Data analysis
The sequences of the complete mitochondrial genome were aligned by the program ClustalX (version 2.1) and edited manually in the program MEGA (version 7.0). The genetic p-distance between sequences was also calculated in MEGA. Number of haplotype, haplotype diversity (Hd) and nucleotide diversity (Pi) were calculated using 7 DNAsp (version 5). The haplotype network was generated by median-joining [29] in the program Network, version 4.6 (Fluxus Technologies, www.fluxus-engineering.com), with standard parameters. Two different datasets were used for the haplotype network analysis: one including all 29 P. vivax/simium mitochondrial genome sequences from Atlantic Forest processed in the present study (n=29); and another also including all 149 Taking the GenBank accession number NC_007243.1 as the reference, the SNPs at positions 4134 and 4468 were observed in all sequences sampled from human, simian and mosquito mtDNA, in order to verify if they were useful in distinguishing between P. simium and P. vivax. As suggested by Brasil et al. [9], P. vivax should present T/A, and P. simium C/G in positions 4134 and 4468, respectively.

Results
The network comprising only samples from our study (n=29) is shown in Figure 2 (Table 2).
[ Table 2. SNPs of the Plasmodium vivax/simium mitochondrial genome from human, simian and mosquito samples from Espírito Santo, Brazil] The genetic divergence within the haplotypes sampled from Atlantic Forest was very low, with only 7 SNPs identified in a total of 5,590 bp (maximum p-distance 0.1%) 9 ( Table 2). Among these, 3 SNPs were found in the non-coding region of the mitochondrial genome, 1 SNP within CYTB gene sequence (synonymous mutation) and 3 SNPs within COX1 gene sequence (1 synonymous and 2 nonsynonymous mutations).
COX3 sequences were conserved among all samples from Atlantic Forest. Haplotype (Hd) and nucleotide (Pi) diversity were also low for samples from Atlantic Forest (Table 3). Although simian isolates represent larger and more geographically widespread samples than mosquitoes', haplotype and nucleotide diversity was lower in the former than in the latter (Table 3).
[ Table 3. Number of haplotypes, nucleotide diversity and haplotype diversity in The two SNPs suggested by Brasil et al. [9] were not useful to distinguish between P. simium and P. vivax, at least for the samples of the present study. This is because some haplotypes had sequences different from those proposed as distinctive between P. simium and P. vivax. More specifically, they showed a combination of these sequences (Table 2: Hap4, Hap5, Hap6). The phylogenetic tree ( Figure 3B) also shows that isolates sampled from humans, mosquitoes and simians were not reciprocally monophyletic and some of these sequences clustered together with high statistical support (BPP ≥ 95%).
The haplotype network showed a reticulate relationship between haplotypes, with no evidence of isolation of any haplotype, and with only one or two mutation steps connecting all of the sequences from the samples of the Atlantic Forest region.

Discussion
Plasmodium vivax is a ubiquitous protozoan with a cosmopolitan distribution, causing infections in a number of populations across different continents. Its South American counterpart, P. simium, is the etiological agent of malaria in simians inhabiting the Atlantic Forest [30]. Some studies have suggested that P. vivax and P. simium are the same species, based on their genetic similarities [5][6][7][8]. In some areas, including the one of the present study, both simians and humans are infected by this agent, making the hypothesis of zoonosis plausible. The results of the present study, uncovering a haplotype diversity in a situation of low genetic divergence in Espírito Santo, indicate a heterogeneity of the isolates obtained from different host species, and reinforce the understanding that P. vivax and P. simium are the same species with small genetic variations [31]. These results corroborate the findings of Camargos et al. [32], and Rodrigues et al. [23], whose phylogenetic analyses of samples from different world regions indicated a recent transfer of the parasite from humans to simians of the New World.
Among the eight haplotypes identified in the study area, two were common to humans and simians, based both on the sequences deposited on GenBank, as well as those obtained from the local simian. This finding represents evidence of parasite transmission from one species to another. At the same time, such a sharing could not be confirmed for the remaining haplotypes, as the sequences were clearly distinctive. Four of the haplotypes obtained from humans were exclusive to the study area, and the two remaining ones were compatible with those previously considered from the Amazonian region [23].
The inclusion of samples obtained from mosquito vectors for the comparison of mitochondrial sequences had never been performed before, despite being previously 1 1 suggested by Ramasamy [34] and Brasil et al. [9]. Interestingly, our results were not consistent with Rodrigues et al. [23], who suggested that two SNPs were distinct between malaria from the Atlantic Forest (C/G) and from the Amazonian region (A/T).
Rather, plasmodial DNA extracted from mosquito vectors from our Atlantic Forest study area revealed the nucleotides A/T at these loci. These same SNPs were also proposed by Brasil et al. [9] as distinctive between P. vivax and P. simium. However, we demonstrate here that said SNPs were not able to distinguish the two lineages in Espírito Santo, as they were not fixed in at least three samples from humans in the study area (Table 2). Furthermore, the sharing of haplotypes between isolates from different hosts, and the lack of monophyly among samples isolated from humans and simian have been shown in the present work.
The mosquitoes responsible for the transmission of malaria in the Atlantic Forest system belong to the Kerteszia subgenus, with the species Anopheles cruzii [24,35,36] being the most prominent. Specimens of species of the subgenus Nyssorhynchus are also captured in the region, occasionally infected by P. vivax/simium recovered from the blood contained in their abdomens [24]. Despite the possibility of the mosquitoes of the subgenus Nyssorhynchus being infected by feeding on human blood, their role as vectors is improbable. Haplotype 8 was obtained from humans and from one pool of Anopheles (Nyssorhynchus) strodei captured close to dwellings, suggesting that the mosquitoes were infected accidentally, by the humans. Haplotype 8 has sequences identical to those previously considered specific to the Amazonian region. Two other haplotypes (Hap7 and Hap7a), obtained from other mosquitoes, despite being closely related to those from the Amazonian region, also have distinctive SNPs, making them exclusive for the mosquitoes of the study area ( Figure 2, Figure 3, Table 2).
The study has some limitations. The parasite DNA was obtained from humans, the The presence of sequences identified in isolates obtained from mosquitoes and shared by two isolates from humans, but different from those obtained from simians in Espírito Santo, raises two hypotheses. The first hypothesis is that this is a zoonotic cycle and there are variations in the SNPs among the simians, whose identification was not possible due to the lack of additional simian samples. The second hypothesis is that there are two distinct transmission cycles for human malaria in the study area, one of them involving simians and the other exclusive to human hosts. In order to clarify such questions, future studies should include more samples from simians and vectors, all obtained in the same period.

Conclusions
Sequencing of the complete mitochondrial genome of P. vivax/simium in an area of the Atlantic Forest in Brazil uncovered eight haplotypes, two of which were shared by humans and simians. Interestingly, the other six haplotypes were distinctive, harboring sequences either unique for human infections in the Atlantic Forest or identical to those 1 3 of the Amazonian region. Such results indicate the possibility of a zoonotic cycle, but due to the differences between plasmodial DNA sequences from simians and mosquitoes, an additional cycle involving only humans cannot be ruled out.

Ethics approval and consent to participate
The collection of human blood samples was performed in a previous study [14] and this 1 4 material has remained stored since then. In the previous study, blood samples were collected only after obtaining signed informed consent. The collection of samples of simian blood and mosquito specimens [24] had authorization from the Brazilian environmental agency (IBAMA/SISBIO; Number 2508929).

Consent for publication
Not applicable.

Availability of data and material
Some datasets analyzed during the current study are not publicly available due to the volume of data, and in deference to other colleagues whose data is not yet published, but are available from the corresponding author upon request.

Competing interests
The authors declare no competing interests.   Tables   Table 1. Sequence of primers for amplifying and sequencing of the complete mitochondrial genome of P. vivax/simium.