Introduction

Malaria parasites of the genus Plasmodium, particularly in most of the medically important species, have undergone intensive studies, and they are well manageable as a result. Historically, descriptions of Plasmodium species infecting even-toed ungulates (order Artiodactyla), on the other hand, have appeared intermittently in literature (see review in Templeton et al.1). Among these, Plasmodium bubalis was discovered in Murrah buffalo (Bovidae: Bubalus bubalis) in India2 and was later reported in water buffaloes in several other countries (see for example Templeton et al.3; Kandel et al.4). Plasmodium traguli was found in mousedeer (Tragulidae: Tragulus javanicus) in Malaysia decades ago and has not appeared in literature since5,6. Plasmodium caprae was first recorded in African goats (Bovidae: Capra aegagrus hircus)7 and more recently in several countries outside Africa, including Thailand3,8. Among the various ungulate malaria parasites described thus far, at least three are endemic in Southeast Asia, suggesting the presence of mosquito vectors in this region. Most of the first discoveries of ungulate malaria occurred prior to the implementation of PCR in 1980, and thus vector identification efforts relied solely on morphological investigations. As a result, a comprehensive picture of these taxa’s transmission cycle could not be drawn.

According to Rattanarithikul et al.9 and the Walter Reed Biosystematics Unit10, at least 464 mosquito species have been recorded in Thailand, with 83 of these belonging to the Anophelinae subfamily. It is not surprising that vector studies on malaria of medical importance have gained greater attention and achieved greater accomplishments than others11,13. The majority of anopheline species in Southeast Asian countries are cryptic species complexes14,15,16. The Barbirostris complex, for example, includes at least six species, five of which are found in Thailand17,18. Misidentification is a common pitfall in vector studies, particularly when dealing with species complexes15. Such deceptive results could have an impact on vector control programs or mislead subsequent studies19,20,21.

Despite several limitations and difficulties, a study of mosquitoes feeding on infected mousedeer in Malaysia resulted in the successful incrimination of Anopheles umbrosus and Anopheles letifer as probable vectors of P. traguli22. After decades of inactivity, sporozoites of unknown malaria parasites were isolated from the salivary glands of Anopheles gabonensis and Anopheles obscurus in Gabon23. The cytochrome b sequences isolated from these sporozoites share the same clade with Plasmodium DNA detected from African ungulates. Plasmodium sporozoites were observed in the salivary glands of Anopheles punctipennis in a separate study conducted in the United States of America. Phylogenetic analysis revealed that their sequences were related to Plasmodium from white-tailed deer (Cervidae: Odocoileus virginianus)24. However, little is known about the vectors of P. bubalis and P. caprae, both of which are endemic in Southeast Asia. Anopheles minimus has been suspected of transmitting P. bubalis25. However, incrimination of this mosquito species remains controversial without clear evidence to support it. In Thailand and other countries, very limited research works on P. bubalis and its vector have been published3,4. Recently, a high prevalence of P. bubalis infection has been reported in Thailand; however, no information on the transmission or the probable vector has been provided26. We hypothesized that the mosquito vectors of P. bubalis are likely endemic species in the country. Therefore, we conducted this study aiming to identify the anopheline mosquito species transmitting P. bubalis in Thailand.

Results

P. bubalis detected in buffalo blood samples on a farm

Previous investigation in Thailand revealed that 35% of buffaloes on a farm located in Chachoengsao Province were infected with P. bubalis26. Thus, this study selected the same farm to identify the vector mosquitoes of P. bubalis. A total of 90 buffalo blood samples were collected in June 2020 (n = 45) and November 2021 (n = 45) from the farm. Mosquitoes were captured then underwent PCR screening for P. bubalis infection using primers targeting the cytb gene. Two buffalo blood samples (IDs THBuff20_37 and THBuff20_39) from the 2020 collection were positive (4.4%), indicating that P. bubalis infection occurred on this farm when mosquito samples were collected. In the 2021 collection, no blood or anopheline mosquito samples were positive.

Species composition of mosquitoes collected from buffalo farm by morphology

A total of 1,571 female mosquitoes were collected from a farm in Chachoengsao. Morphological examination indicated that anopheline mosquitoes accounted for 8.53% (n = 134), while Culex spp. accounted for 74.6% (n = 1172), Aedes spp. accounted for 13.05% (n = 205), Mansonia spp. accounted for 0.38% (n = 6), and unidentifiable due to body part destruction accounted for 3.44% (n = 54) (Fig. 1A). Among 134 anopheline mosquitoes, 5 different Anopheles groups were identified including Barbirostris, Hyrcanus, Funestus, Ludlowae, and Jamesii groups; 1 mosquito was unable to be identified in any group due to missing wings and legs (Fig. 1B).

Figure 1
figure 1

Chart illustrating the percentage of mosquitoes, according to morphological identification. (A) Percentages of each genus of mosquitoes collected in this study. (B) Anopheles mosquito groups.

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Identification of P. bubalis DNA from mosquito salivary gland samples

For a total of 133 identified anopheline mosquitoes, salivary glands with the head and thorax were carefully separated from the rest of the mosquitoes’ bodies, then the salivary glands and midguts were stained with 0.1% mercurochrome dye and examined under a microscope. However, no oocysts and sporozoites were found. Then, one to three samples consisting of the salivary glands, head, and thorax were combined based on the group and, finally, 51 pooled samples were prepared (Table 1). DNA was extracted from the samples and PCR screening was performed for Plasmodium cytb, 18S rRNA, and cox1 genes. The number of each pool was as follows: Barbirostris group (n = 35, 23 pools), Hyrcanus group (n = 81, 19 pools), Ludlowae group (n = 14, 7 pools), Funestus group (n = 1, 1 pool), and Jamesii group (n = 2, 1 pool). Out of 51 pools of anopheline mosquitoes, 3 pools were PCR positive for Plasmodium. These samples were the Barbirostris group (IDs THMosqBuff20_P6_3, THMosqBuff20_P8_2) and Hyrcanus group (ID THMosqBuff20_P20_3) (Table 1). The minimum infection rates (MIR) were 5.7% (0.015–0.186) in the Barbirostris group mosquito and 2.5% (0.004–0.128) in the Hyrcanus group mosquito (Table 2). Additionally, for those of non-anopheline mosquitoes, a total of 22 pools (Culex spp. n = 24, 8 pools); Aedes spp n = 24, 8 pools; and Mansonia spp. n = 6, 6 pools) were tested. Plasmodium bubalis was not detected in any Culex spp., Aedes spp., or Mansonia spp. pools.

Table 1 Summary of P. bubalis’s PCR screening results of anopheline mosquitoes collected from the buffalo farm.
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Table 2 Minimum infection rates of Plasmodium in collected mosquitoes.
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Analysis by the BLASTN program using cytb and cox1 sequences obtained from 3 pools against non-redundant nucleotide collection revealed that they were 100% identical to P. bubalis type I (accession no. LC090213). Analysis by the BLASTN program using putative P. bubalis’s 18S rRNA sequences did not identify any sequences in the database with 100% identity. The maximum identity was 92% with 18S rRNA sequences of Plasmodium falciparum (accession no. LR131366) as well as those of other Plasmodium species. Because no 18S rRNA sequences derived from any ungulate malaria parasites were available in the GenBank™ database, we used two buffalo-derived samples (IDs THBuff20_37 and THBuff20_39) for PCR-amplification with the same universal primers for Plasmodium 18S rRNA and sequences were determined. Sequences derived from 3 mosquito samples showed 100% identity with the sequences from 2 buffalo samples, further supporting the presence of P. bubalis in the mosquitoes.

Phylogenetic analyses using the cytb (789 bp), cox1 (254 bp), and 18S rRNA (351 bp) genes revealed that Plasmodium sequences from this study belong to the same cluster as P. bubalis type I isolates previously reported from Thailand (Fig. 2, Suppl. Figure 2, Suppl. Figure 3). The current findings indicated that all Plasmodium sequences obtained from mosquitoes in this study were P. bubalis type I.

Figure 2
figure 2

Phylogenetic positions of Plasmodium detected from Anopheles mosquitoes in this study. The phylogenetic tree was inferred by Bayesian inference method using partial cytb sequences (789 bp). Haemoproteus columbae was used to root all sequences. At the nodes, Bayesian posterior probabilities (PP ≥ 0.65) are indicated. Plasmodium sequences obtained in this study are highlighted in red. The length for the substitutions/site (0.02) is indicated.

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Molecular identification of anopheline mosquitoes collected from a buffalo farm

To identify the species of anopheline mosquitoes collected from buffalo farms by molecular analysis, cox1, cox2, and ITS2 gene sequences were determined for three Plasmodium-positive Anopheles mosquito pools, as well as 15 additional Plasmodium-negative pools in this study. The obtained sequences were initially assessed by the BLASTN program against a non-redundant nucleotide collection for species identification. Based on the sequence of DNA barcoding region for mosquito identification, several studies have suggested an evolutionary divergence of 2–3% as a threshold for intraspecific variation27,28,29. Thus, sequences with the highest identity (minimum ≥ 97%) are listed in Supplementary Table 2. BLASTN analysis of some cox1 sequences obtained in this study was unable to reach this threshold, indicating the limitation of this approach due to the insufficient collection of mosquito sequences in the database. Nonetheless, analysis of all 3 genes of 1 Funestus group pool was matched to An. varuna. All 3 gene sequences of one Ludlowae group mosquito hit An. vagus. An. peditaeniatus was hit by two Hyrcanus group pools with all 3 gene sequences including a P. bubalis sequence-positive pool (THMosqBuff20_P20_3), and by one Hyrcanus group pool with cox2 and ITS2 sequences. An. pseudojamesi was hit by one Jamesii group pool.

The ITS2 sequences of 9 pools of Barbirostris group mosquitoes showed 98.8–100% identity to An. campestris or An. wejchoochotei. The Cox2 sequences of 9 pools of Barbirostris group mosquitoes showed 99–100% identity to An. campestris. However, there were no An. wejchoochotei cox2 sequences available in the database, which limited the assessment of the cox2 sequence with An. wejchoochotei. BLASTN search using 8 cox1 sequences (1,416 bp) hit An. donaldi with ~ 97% identity and one cox1 sequence (333 bp) showed 99.1% identity to An. campestris. Because all An. wejchoochotei cox1 sequences deposited in the database are much shorter than the 8 sequences in this study, we aligned our cox1 sequences with An. wejchoochotei cox1 sequences (AB971335, AB971336, AB971337, AB971338, AB971339, and AB971340) from the morphologically well-described samples18 and found they were matched with > 99% identity (Supplementary Fig. 1). Because An. wejchoochotei sequences were reported in 2015 from morphologically defined samples18, and the identities of "An. campestris" from which DNA sequences were deposited to the database before this report were not clear, it was impossible to distinguish An. campestris and An. wejchoochotei molecularly at the time. Thus, we concluded that P. bubalis-positive anopheline mosquitoes from the Barbirostris group (THMosqBuff20_P6_3 and THMosqBuff20_P8_2) were either An. campestris or An. wejchoochotei; one from the Hyrcanus group (THMosqBuff20_P20_3) was An. peditaeniatus.

Discussion

The current study aimed to identify potential P. bubalis vectors in Thailand. An. wejchoochotei or An. campestris, An. peditaeniatus, An. varuna, An. vagus, and An. pseudojamesi were molecularly confirmed on a farm where P. bubalis was detected from water buffaloes, and P. bubalis DNA sequences were detected from An. wejchoochotei or An. campestris, and An. peditaeniatus. According to Rattanarithikul et al.9 and the Walter Reed Biosystematics Unit10, all of these anopheline mosquitoes have previously been recorded in districts throughout Thailand as well as across Southeast Asian countries. An. wejchoochotei is found in Thailand and Cambodia, whereas An. peditaeniatus can be found in Thailand, Cambodia, Indonesia, Malaysia, Myanmar, the Philippines, and Vietnam9,17,18,30,31,32,33. An. wejchoochotei and An. peditaeniatus were recently found to harbor human Plasmodium species in Cambodia13.

In this study, we detected P. bubalis’s DNA in salivary gland samples, but oocysts and sporozoites were not observed under a microscope. This was most likely due to the low infection rate of the parasite in the water buffaloes, which resulted in a low parasite burden in the mosquitoes3. P. traguli oocysts and sporozoites have been discovered in An. umbrosus and An. letifer by microscopic examination in a historic mousedeer study in Malaysia22. The successful observation of P. traguli in mosquitoes may be due to a relatively higher infection rate in mousedeers than P. bubalis in water buffaloes because the P. traguli detection rate in the mousedeer blood samples was high (≥ 37%)22.

Furthermore, nucleotide sequence analysis using Bayesian Inference (BI) confirmed that Plasmodium parasites isolated from An. wejchoochotei or An. campestris and An. peditaeniatus in this study were genetically identical and were grouped to previously described P. bubalis type I isolated from buffaloes26, suggesting that these mosquito species were plausible vectors for P. bubalis.

Taai and Harbach18 described An. wejchoochotei for the first time, while Reid34 recorded An. campestris in 1962. It should be noted that mosquitoes from Thailand that have since been identified as An. wejchoochotei were initially referred to as An. campestris-like by Harrison and Scanlon35 due to their resemblance to An. campestris. Both are members of the Barbirostris complex group and cannot be distinguished solely by the morphology of the adult mosquitoes; morphological information of the larva is required. Previous research suggested that cox1, cox2, and ITS2 are reliable genetic markers for distinguishing cryptic species within the complex group of anopheline mosquitoes36,37. A recent study in Sulawesi, Indonesia, used approximately 700 bp of the cox1 gene to distinguish members of mosquito species complexes38. Furthermore, cox1 and ITS2 sequences have been used to identify cryptic mosquito species39. Based on the cox1 barcode region, an evolutionary divergence of 0.5% (range 0.0–3.9%) was proposed as a threshold for intraspecific variation28. Consequently, we carried out an investigation into the cox1, cox2, and ITS2 markers of anopheline mosquitoes in this study. We found that sequences from Plasmodium-positive mosquitoes (THMosqBuff20_P6_3 and THMosqBuff20_P8_2) showed high similarity with either An. campestris or An. wejchoochotei sequences in the GenBank™ database. The conflicting species discrimination of the previously deposited sequences between An. campestris and An. wejchoochotei (formerly, An. campestris-like) will be solved by molecular analysis of the morphologically confirmed An. campestris samples in the future.

The Barbirostris and Hyrcanus groups belong to the Myzorhynchus series of Anopheles mosquitoes, which contains most vectors of human malaria except for An. punctipennis, which belongs to the Anopheles series. An. umbrosus and An. letifer, suspected vectors of P. traguli, and An. gabonensis and An. obscurus, the vectors of African ungulate malaria parasites, also belong to the Myzorhynchus series. Thus, Myzorhynchus series mosquitoes appear to have a dominant role in the transmission of ungulate malaria parasites.

Conclusions

An. wejchoochotei or An. campestris and An. peditaeniatus were identified as vectors of P. bubalis type I.

Methods

Study site, mosquito collection, dissection, and DNA extraction

This study was conducted on a buffalo farm in Chachoengsao province of Thailand (Fig. 3A). To investigate mosquito composition and identify the probable vector of P. bubalis, we carried out a survey of Murrah dairy buffaloes in Chachoengsao Province (13°28′53.98"N 101°27′35.23"E) for 14 consecutive nights in June 2020 and 2 nights in November 2021. The Murrah dairy buffalo farm is located 1 km away from the Nong Mai Kaen community. The area is surrounded by rubber trees with small ponds to wallow the water buffaloes (Fig. 3B).

Figure 3
figure 3

modified from Google Earth Pro version 7.3.4.8248. The red triangle indicates blood sample collection sites, while the yellow triangle indicates mosquito sampling sites.

(A) Map depicting a buffalo farm in Chachoengsao for sample collection in Thailand. (B) The landscape of mosquito sampling sites in a buffalo farm in Chachoengsao. The images were obtained and

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CDC light traps with dry ice were set overnight at less than 1.5 m above ground level. Peripheral nets were placed surrounding the buffalo stable. Mosquitoes on the peripheral net were captured from 7.30 PM to 11.30 PM using tube aspirators (10 mm in diameter × 200 mm in length). The mosquitoes were then brought to the laboratory for morphological and molecular analysis. All anopheline mosquitoes were identified into group/species levels using taxonomic keys9,40, while non-anopheline mosquitoes were identified up to only genus level according to the pictorial identification key of important disease vectors in the WHO Southeast Asia41. Anopheline mosquitoes were carefully dissected within three days after collection to obtain the salivary glands of each mosquito. A 26G and ½ inch-long sterile needle was used to dissect individual mosquitoes, which was changed after each dissection to prevent cross-contamination. In addition, 0.1% mercurochrome dye was used to stain oocysts on the midgut wall and sporozoites in the salivary glands, and samples were examined under a microscope at 1,000-times magnification. Salivary glands, which were still attached to the head and thorax, were kept in 0.2 mL of 1 × PBS at 4 °C for further DNA extraction for mosquito species identification and malaria parasite detection.

DNA samples from mosquitoes were extracted using NucleoSpin® Tissue (Macherey–Nagel, Düren, Germany) according to the manufacturer’s guidelines with a minor modification in the elution step (elution volume reduced to 30 μL). Previous studies suggested that it is possible to detect higher infectivity in mosquito pool samples42,43. Thus, adult female mosquitoes were grouped based on their morphology. Mosquito pools were made following morphological identification and were subsequently confirmed by molecular identification. Each pool was made up of one to three mosquitoes from the same groups depending on sample availability.

Blood collection from buffaloes, DNA extraction, and microscopic examination

To evaluate the malaria infection status in buffaloes, we carried out a survey of Murrah dairy buffaloes on a farm in Chachoengsao in June 2020 and November 2021, during which mosquitoes were captured (n = 45 and n = 45, respectively). These blood samples were drawn from the jugular vein using 21G needles and BD vacutainers containing acid citrate dextrose (ACD). It should be noted that P. bubalis have been detected from buffaloes on this farm in our previous surveys3,26. DNA was extracted as described above.

Anopheline mosquito’s cox1, cox2, and ITS2 gene amplification

Three genes of anopheline mosquito comprising cox1, cox2, and ITS2 were amplified by PCRs using KOD FX Neo Polymerase (Toyobo, Japan) according to the manufacturer's protocol. The AnplCOXIF(5’-GGATCCCTTCAGCCATTTAATCGCG-3’) and AnplCOXIR primers (5’-TCGAGCTTAAATTCATTGCACTAATCTGCC-3’) were designed to amplify the cox1 region with 1,584 bp-long products. The Cox2 region was amplified by Anplcox2F-Anplcox2R primers (5’-GGATCCAGATTAGTGCAATGAATTTAAGC-3’) and (5’-CTGCAGGATTTAAGAGATCATTACTTGC-3’) to generate a total of 792 bp-long products. For the ITS2 region, PCR amplification was carried out using ITS2A and ITS2B primers, as previously described44. The PCR product size of the ITS2 region varied depending on the mosquito group (~ 1,500 bp for Barbirostris complex, ~ 562 bp for Hyrcanus, ~ 697 bp for Ludlowae, ~ 518 bp for Funestus, and ~ 555 bp for Jamesii).

PCR detection of Plasmodium’s cytb, 18S rRNA, and cox1 genes

DNA samples from buffalo blood underwent nested PCR screening for Plasmodium using primers targeting cytb gene DW2 (5’-TAATGCCTAGACGTATTCCTGATTATCCAG-3’) and DW4 (5’-TGTTTGCTTGGGAGCTGTAATCATAATGTG-3’) as the outer primers and NCYBINF (5’-TAAGAGAATTATGGAGTGGATGGTG-3’) NCYBINR (5’-CTTGTGGTAATTGACATCCA-ATCC-3’) for the inner primers, as previously described45. Subsequently, Plasmodium-positive samples were further confirmed using primer sets targeting the 18S rRNA and cox1 genes. The first amplification of the 18S rRNA gene was carried out using Plasmodium universal primers, rPLU5 (5’-CCTGTTGTTGCCTTAAACTTC-3’) and rPLU6 (5’-TTAAAATTGTTGCAGTTAAAACG-3’), as previously described by Snounou et al.46. New inner primers were designed based on the conserved region of the 18S rRNA gene among the genus PlaSSUF1 (5’-CTTAGTTACGATTAATAGGAGTAG-3’) and PlaSSUR1 (5’-TCCTACT-CTTGTCTTAAACTAG-3’) for forward and reverse directions, respectively, for the second amplification. In addition, PCR targeting the Plasmodium’s cox1 gene was conducted using the following primers: Cox1-F3-2 (5’-ATTATGTAATTGCACATTTCCATTTTG-3’) and Pbucox1-4B3 (5’-CCAAATAAAGTCATTGTWGAACC-3’). Each PCR amplification was carried out in a reaction volume of 12.5 μL, consisting of 2 × PCR buffer KOD FX Neo, 2.0 mM of dNTP, 0.4 μM of each primer, 1.0 Unit of KOD FX Neo DNA Polymerase (Toyobo, Japan), 1 μL genomic DNA as a template, and additional sterile distilled water up to 12.5 μL. The cycling conditions and product size of each PCR assay are described in Supplementary Table 1. Subsequently, 5 μL of PCR products were run on 1.5% agarose gel electrophoresis before being stained by Red Safe (Intron Biotechnology, Korea) and visualized under a UV transilluminator. The PCR products of positive samples were scaled up to 50 μL for purification and sequencing. Gel purification was carried out using NucleoSpin® Gel and PCR clean up (Macherey–Nagel, Düren, Germany) according to the manufacturers' protocols. Purified PCR products were sequenced in both directions. DNA samples extracted from mosquitoes were subjected to PCR screening for P. bubalis in the same way as mentioned in blood samples. Additionally, Plasmodium’s cytb-positive samples underwent PCR confirmation using primers targeting the 18S rRNA and cox1 genes, which were subsequently subjected to sequencing.

Sequence analyses

The chromatogram files of all target genes were edited manually using BioEdit software version 747. Low-quality sequences were excluded, resulting in a total of 41 mosquito pools being used for molecular analysis of each gene. Once the alignment was completed, sequences were compared to published sequence data in the GenBank™ database using the BLASTN program. The alignment of multiple sequences obtained from this study and additional sequences from the GenBank™ were made using the ClustalW via BioEdit version 7.

The ClustalW implemented in BioEdit version 7 was used to align sequences obtained in this study and additional sequences from GenBank™ database. MrBayes v3.2.750 was used to create phylogenetic trees using the Bayesian Inference (BI) method and the Markov chain Monte Carlo method. BI phylogenetic analysis was performed using two independent runs of four chains, each for 10 million generations. As a result of burn-in, the first 25% of trees were discarded. Tracer v1.751 was used to assess the mixing and convergence of runs, as well as effective sample sizes (EES > 200). FigTree v1.4.4 was used to visualize the trees (available at http://tree.bio.ed.ac.uk/software/figtree/).

Statistical analysis

To evaluate the infection rate of positive mosquitoes, the minimum infection rate (MIR) was calculated for each species in which Plasmodium DNA was detected. If Plasmodium was detected from a mosquito pool, it was assumed that the pools contained at least one infected mosquito. Therefore, MIR was calculated as (number of positive pools/total number of analyzed mosquitoes) × 100, as previously described48,49. The MIR was calculated using the Wilson confidence interval method for binomial proportions, and the results were expressed as a percentage with a 95% confidence interval (CI).

Ethics statement and biosafety

This study has been reviewed and approved by Chulalongkorn University Animal Care and Use Committee (Approval No. 1931027). All protocol in this study was performed according to the Institutional Biosafety Committee of Chulalongkorn University (No. 2031033).