Skip to main content
Download PDF

Diversity and prevalence of Leucocytozoon in black flies (Diptera: Simuliidae) of Thailand

Parasites & Vectors volume 17, Article number: 475 (2024) Cite this article

Abstract

Background

Leucocytozoonosis, a parasitic disease of birds, is caused by haemosporidian protozoan parasites of the genus Leucocytozoon, which infect diverse avian species, including poultry. These parasites are transmitted by several black fly species, but knowledge of the factors determining the diversity and prevalence in these vectors, which is crucial for fully understanding disease epidemiology, is largely unexplored. In this study, we investigated factors associated with the prevalence and diversity of Leucocytozoon species in black flies from Thailand.

Methods

Adults of two black fly taxa (Simulium asakoae Takaoka and Davies complex and S. khelangense Takaoka, Srisuka and Saeung) were collected using sweep nets at nine locations in northern and northeastern regions of Thailand. Specimens were identified morphologically and the results corroborated by DNA barcoding. Molecular methods using specific primers for amplification of the mitochondrial cytochrome b (cyt b) gene of Leucocytozoon were used to detect the parasite in black flies. Species and lineages of Leucocytozoon were determined using the MalAvi database of malaria parasites and related haemosporidians in avian hosts. Regression analysis was used to examine relationships between Leucocytozoon diversity and prevalence, black fly abundance and habitat characteristics.

Results

A total of 11,718 adult black flies were collected, of which 4367 were members of the S. asakoae complex and 7351 were S. khelangense. For molecular detection of Leucocytozoon, we randomly selected 300 individual female black flies of the S. asakoae complex and 850 females of S. khelangense pooled into groups of five individuals (= 170 pools). A total of 34 of the 300 specimens of the S. asakoae complex and 118 of the 170 pools of S. khelangense were positive for Leucocytozoon. Fifty-four lineages (haplotypes) were identified, all of which belonged to those reported in domestic chickens, Gallus gallus, with one exception that was identified in S. khelangense and found to be closely related to the Leucocytozoon lineages reported in owls; this is the first record of the latter lineage in Asian black flies. Among these haplotypes, nine and 45 were exclusively found in the S. asakoae complex and S. khelangense, respectively. No lineage was shared between these black fly taxa. Analysis of similarity (ANOSIM) revealed significant Leucocytozoon lineage composition between the two black flies. Phylogenetic analysis found that Leucocytozoon lineages in the S. asakoae complex and S. khelangense are largely isolated, agreeing with the ANOSIM result. The overall prevalence of Leucocytozoon in the S. asakoae complex was 11.3% and ranged from 9% to 13% in each collection. Leucocytozoon prevalence in S. khelangense was 21%, varying from 13% to 37% in each collection. The Shannon H′ index indicated greater Leucocytozoon diversity in S. khelangense (H′ = 3.044) than in the S. asakoae complex (H′ = 1.920). Regression analysis revealed that Leucocytozoon diversity was positively related to black fly abundance and negatively related to maximum air temperature.

Conclusions

The results of this study show that the prevalence and diversity of Leucocytozoon lineages in the S. asakoae complex and S. khelangense from Thailand were associated with the abundance of these black flies and with air temperature. The Leucocytozoon lineages identified also showed some degree of black fly taxon specificity, possibly related to different abundance peaks of these vectors. The environmental conditions that favor the development of black flies are possibly a driver of Leucocytozoon prevalence, diversity and vector–parasite co-evolution.

Graphical Abstract

Background

Haemosporidian parasites of the genus Leucocytozoon infect diverse avian hosts globally [1]. At least 45 morphological species are currently recognized, but much greater diversity has been documented (1542 lineages in the MalAvi database; http://130.235.244.92/Malavi/, accessed 31 October 2024) based on the mitochondrial cytochrome b (cyt b) sequence [2]. Leucocytozoon infections can cause a disease known as leucocytozoonosis, which is typically more severe in poultry than in wild birds [1, 3, 4]. Leucocytozoonosis can reduce growth rate and egg production and even lead to death, causing significant economic losses to the poultry industry [3].

The majority of Leucocytozoon species are transmitted by black flies (Diptera: Simuliidae), with the exception of one species, L. (Akiba) caulleryi, which is transmitted by biting midges (Ceratopogonidae: Culicoides) [5, 6]. To date, 48 black fly species are known to be vectors or potential vectors of Leucocytozoon [3, 6,7,8,9,10,11,12,13], but knowledge of these vector species represents only a fraction of the vast diversity of Leucocytozoon genetic lineages [14]. Similarly, among the 2407 extant species of the world’s black flies [15], numerous species have not yet been investigated for their involvement in parasite transmission, and most studies so far have focused on black flies in North America [3].

Vectors are crucial components of vector-borne disease epidemiology. Therefore, knowledge of the factors related to the transmission of pathogens by vectors is central to effective disease control and prevention. Key factors in the successful transmission of haemosporidian parasites to susceptible hosts are the environmental conditions that determine vector abundance and distribution, both of which can influence the prevalence and diversity of the parasites [16]. Factors that relate to these vector population characteristics include rainfall, elevation, season and temperature [16]. For example, a high prevalence of Leucocytozoon in wild birds in the highlands of Neotropical Colombia is associated with elevation [9]. Seasonal changes in temperature affect the emergence of black fly vectors and are related to the prevalence of Leucocytozoon in vertebrate hosts [17]. However, factors associated with the prevalence and diversity of Leucocytozoon in black fly vectors remain largely unexplored [16].

In Thailand, the prevalence of Leucocytozoon is high in domestic chickens (up to 89%) [18] compared with wild bird species (2–8%) [19, 20]. At least three black fly taxa (Simulium asakoae Takaoka and Davies complex, S. chumpornense Takaoka and Kuvangkadilok and S. khelangense Takaoka, Srisuka and Saeung) are potential vectors of [10, 13, 21, 22], and there is some degree of association between vector species and parasitic lineages [13]. The prevalence rates of Leucocytozoon in black flies vary geographically and seasonally [10, 13, 22]. For example, in one study, the majority (91%) of Leucocytozoon detected in S. khelangense (= S. chumpornense in Pramual et al. [22]) were from black flies collected during the dry season (March–May) and at the beginning of the rainy season (June), with only 9% collected in the late rainy season (September) [22]. This finding reinforces the possibility that environmental factors related to season might affect the prevalence and distribution of Leucocytozoon.

In the study reported here we investigated the prevalence and diversity of Leucocytozoon in two black fly taxa, the S. asakoae complex and S. khelangense. Adult black flies were collected from various localities that had not been investigated previously in Thailand. We tested whether the diversity and prevalence of Leucocytozoon, based on mitochondrial cyt b lineages, are associated with black fly abundance and habitat characteristics. We also explored whether Leucocytozoon lineages are associated with specific black fly taxa.

Methods

Specimen collection and identification

Eleven collections of wild adult black flies were carried out at nine sampling sites in northern and northeastern Thailand from August 2019 to February 2024 (Table 1; Fig. 1). Specimens were collected with a sweep net (39-cm-diameter hoop with a 3-part telescopic handle and total extended length of 120 cm). The sweep net was moved back and forth in a figure eight motion 0.5–2.0 m above the ground. Specimens were collected during times when adult black flies were actively searching for hosts, specifically early in the morning (06:00–08:00 h) or late afternoon (16:00–18:00 h) [23, 24]. Duration of collection times for each sampling location varied from 30 min to 4 h and involved between one and four collectors each collection. The adult black flies collected were placed in plastic vials containing 80% (v/v) ethanol and stored at − 20 °C until they were sorted out from other collected insects under a stereomicroscope in the laboratory. Only female black flies were used in subsequent analyses. The specimens were examined under a stereomicroscope for the presence of a blood meal and then used for host blood-source identification. Species were identified morphologically using known keys and descriptions of black flies in Thailand [25, 26].

Table 1 Sampling locations, black fly taxa, number of specimens used for molecular detection of Leucocytozoon parasites and number of positive detections
Full size table
Fig. 1
figure 1

Sampling locations of black flies of the Simulium asakoae complex (blue-filled circles) and Simulium khelangense (black-filled circles, used for molecular detection of Leucocytozoon parasites. Details on each sampling site are given in Table 1

Full size image

Molecular analysis

PCR-based identification of black flies

Females of S. khelangense are difficult to identify based on morphological characteristics; therefore, we emplyed a molecular method based on cytochrome c oxidase I (COI) to facilitate species identification. Prior to processing for DNA extraction, specimens were dried at room temperature to evaporate the ethanol. DNA was extracted from individual specimens using the GF-1 Nucleic Acid DNA Extraction Kit (Vivantis Technologies Sdn. Bhd., Shah Alam, Selangor, Malaysia), following the manufacturer’s protocol. The primers LCO1490 and HCO2198 [27] were used to amplify an approximately 650-bp fragment of the COI gene. The PCR reaction conditions followed those of Tangkawanit et al. [28]. PCR products were stained with Novel Juice (GeneDireX, Taoyuan, Taiwan, Republic of China) and were checked by 1% agarose gel electrophoresis. Successful amplifications were then purified using the PureDirex PCR CleanUp & Gel Extraction Kit (Bio-Helix, Taiwan, Republic of China), following the manufacturer’s protocol. The purified PCR products were sequenced at ATCG Company Limited (Thailand Science Park, Pathumthani, Thailand) using the same primers as for PCR.

Host blood-meal identification

Among the 11,718 specimens collected, only four were blood engorged and all were S. khelangense. These specimens were used for molecular identification of the host blood source. DNA was extracted from the whole individual using the same method as for PCR-based identification of black flies. The vertebrate host blood mitochondrial cyt b was amplified using primers L14841 and H15149 [29], with PCR reaction conditions as described by Malmqvist et al. [30]. PCR product checking, purification and sequencing were as for the DNA barcoding study but the primers for cyt b were used for sequencing.

Molecular detection of Leucocytozoon

A total of 1150 female black flies were randomly selected for molecular detection of Leucocytozoon. Of these, 300 females of the S. asakoae complex were used individually and 850 females of S. khelangense were pooled into groups of five individuals each for a total of 170 pools. The DNA extraction method was the same as that used in the PCR-based identification of black flies. The nested PCR method described by Hellgren et al. [31] was used to amplify Leucocytozoon DNA in black flies. Specific primers (HaemNFI and HaemNR3 for the first round and HaemFL and HaemR2L for the second round PCR) were used to amplify an approximately 500-bp fragment of cyt b of the Leucocytozoon mitochondrial DNA [31]. The PCR reaction conditions followed those of Jumpato et al. [10]. Purification and sequencing of the PCR products used the same method as described for the COI gene but with the specific primers for Leucocytozoon for sequencing.

Data analysis

Sequences of all genes were checked for quality using the “Edit/View sequencer file” option in MEGA X [32]. Three COI sequences (accession nos. PQ146532-PQ146534) were obtained from representative specimens identified morphologically as S. khelangense. These sequences were compared using the Basic Local Alignment Search Tool (BLAST); all had > 99% similarity with S. khelangense. The cyt b sequences (accession nos. PQ425370–PQ425372) of the host blood meals were successfully obtained from three of the four blood-engorged specimens. These sequences were compared with those of vertebrate cyt b in the National Center for Biotechnology Information (NCBI) GenBank, using BLAST. The vertebrate host was considered identified if sequence similarity was > 98%.

The cyt b sequences of Leucocytozoon parasites were checked and evaluated based on the sequence chromatogram. Only clear chromatograms, with no double peaks that might have indicated the possibility of co-infection, were used for further analysis. These sequences were submitted to NCBI GenBank (accession nos. PQ155266–PQ155417). Identification of Leucocytozoon lineages was performed using the BLAST method in the MalAvi database [14] (accessed 20 April 2024). A lineage was considered to be known if the sequence was a 100% match with any of those in the database; in this case, the lineage names reported in the MalAvi database were used. If the sequences obtained in the present study showed < 100% match (i.e., single-base differentiation), we considered it to be a new lineage. The new lineage sequences were then submitted to MalAvi and named following the database instructions by using the abbreviation of the host species name followed by a number [14]. Neighbor-joining (NJ) and maximum likelihood (ML) were used to examine genetic relationships between cyt b lineages of Leucocytozoon parasites found in the present study (54 lineages) plus those previously reported in black flies from Thailand (12 lineages) [10, 13, 21, 22]. Branch support was estimated using 1000 bootstrapping replications. The NJ and ML trees were inferred using MEGA X [32].

We used regression analysis to test the relationship between Leucocytozoon diversity, prevalence and environmental factors (minimum and maximum temperature). In addition to data obtained in the present study, we also included information in the regression analysis on the habitats and Leucocytozoon diversity and prevalence from our previous studies of black flies [10, 13, 21, 22]. Leucocytozoon diversity was estimated using the Shannon H′ index, with each Leucocytozoon lineage (= cyt b haplotype) [14] equal to a species. The Shannon H′ index was calculated in PAST ver. 4.12b [33]. Because each collection was associated with a different number of collectors and durations of collection time, we standardized black fly abundance as the number of adult flies per collector per hour (number of adult flies/collector/hour). The prevalence of Leucocytozoon for pooled specimens was estimated in the Epitools Epidemiological Calculators (https://epitools.ausvet.com.au/ppfreqone; accessed 24 April 2024). Minimum and maximum air temperatures were obtained from the Thai National Hydroinformatics Data Center (https://www.thaiwater.net/weather; accessed 13 July 2024) for the closest weather station to the sampling sites. Because generation time of black flies in tropical regions, such as Thailand, is approximately 1 month [34], the minimum and maximum air temperatures were determined for a 1-month period dating from the collection date. Variables (Shannon H′ index, prevalence and minimum and maximum air temperature) were tested for normality and transformed using log10 or square root, if necessary, before regression analysis was performed.

Results

Host blood meal and black fly identification

Four blood-engorged females were identified morphologically as S. khelangense. The COI barcoding sequences of these blood-fed females corroborated the morphological identification with > 99% sequence similarity. Three of the four blood-engorged females were successfully sequenced for host source of DNA in the blood, and all were identical with chicken (Gallus gallus).

Prevalence of Leucocytozoon parasites in black flies

In total, 169 samples were positive for Leucocytozoon DNA from the 300 individual females of the S. asakoae complex and 170 pools of S. khelangense. However, only 152 samples (34 from the S. asakoae complex and 118 from S. khelangense) were successfully sequenced (Table 1). The overall prevalence of Leucocytozoon parasites in the S. asakoae complex and in S. khelangense was 11.3% and 21.1%, respectively (Table 1). In each collection, the prevalence of Leucocytozoon in the S. asakoae complex varied from 9% at the CM559 and CM556 sites in Chiang Mai province in the northern region to 13% at the CM613 site also in Chiangmai province. The prevalence rates for a location at Ban Pang Bong, Chiangmai province, where specimens were collected in 2019, 2020 and 2023 varied from 9% in 2019 to 13% in 2023 (Table 1).

For S. khelangense, prevalence rates varied from 13% in the collections from Beung Khong Long, Buengkan province (BK) in the northeastern region to 37% in BK644 in the same province. Leucocytozoon prevalence was also high (32%) at a sampling site in Loei province (LO648) in the northeastern region (Table 1). Regression analysis revealed that Leucocytozoon prevalence in black flies was not significantly related to black fly abundance or temperature. However, correlation analysis indicated that Leucocytozoon prevalence in black flies was significantly and positively related to black fly abundance (r = 0.551, P = 0.012).

Diversity of Leucocytozoon in black flies

A total of 54 lineages were identified from the 152 cyt b sequences obtained in the present study. Among these, 10 (GALLUS06, GALLUS07, GALLUS17, GALLUS18, GALLUS34, GALLUS35, GALLUS37, GALLUS41, GALLUS44, and GALLUS46) were lineages that were present in the MalAvi database, and the remaining 44 were novel lineages found in our study. Nine lineages were found in the S. asakoae complex in a total of 34 cyt b sequences, and 45 lineages were found in S. khelangense in 118 cyt b sequences. The most common lineage was GALLUS17, which was shared by 22 individuals, and GALLUS44, shared by 15 individuals. These common lineages were each specific to a black fly taxon; GALLUS17 was found only in S. khelangense, whereas GALLUS44 was found only in the S. asakoae complex. In addition, nine lineages were detected exclusively in the S. asakoae complex and 45 were found only in S. khelangense (Additional File 1: Table S1). Analysis of similarity (ANOSIM) of Leucocytozoon lineages in the S. asakoae complex and those of S. khelangense indicated that these were significantly different (R = 0.3556, P = 0.0216).

Diversity of Leucocytozoon, as measured with the Shannon H′ index, indicated that S. khelangense (H′ = 3.408) possessed much greater Leucocytozoon diversity than did the S. asakoae complex (H′ = 1.920). In each sampling location, the NK637 population of S. khelangense from Nongkhai province in the northeastern region showed the greatest diversity, with 21 Leucocytozoon lineages and H′ = 3.044 (Table 2). Multiple regression analysis revealed that Leucocytozoon lineage diversity based on the Shannon H′ index was positively related to black fly abundance and negatively related to maximum air temperature (H′ = 4.260 + 0.766Abundance – 0.115Maximum temperature; F = 10.135; P < 0.001; df = 2, 19; R2adj = 46.5%).

Table 2 Black fly abundance, number of Leucocytozoon lineages, Shannon H′ index and maximum and minimum air temperature for each collection of the Simulium asakoae complex and S. khelangense in Thailand
Full size table

Phylogenetic analysis of Leucocytozoon in black flies from Thailand

The NJ and ML analyses of Leucocytozoon lineages in black flies from Thailand revealed similar tree topologies; therefore, only the ML tree is presented here (Fig. 2). The ML tree indicated no major divergent clade. All lineages identified in the MalAvi database as Leucocytozoon schoutedeni (GALLUS06, GALLUS07) belonged to a clade that also included two lineages (SIMKHE08 and SIMKHE09) of unidentified Leucocytozoon. This is the only minor clade that received high (> 97%) bootstrap support. For Leucocytozoon sp., although there was no clear divergent clade, the lineages detected in the S. asakoae complex and S. khelangense were largely isolated. Only one lineage (SIMASA10) from the S. asakoae complex was genetically closer to those from S. khelangense.

Fig. 2
figure 2

Maximum likelihood tree based on the mitochondrial cytochrome b gene sequences of 66 Leucocytozoon lineages detected in the Simulium asakoae complex (blue text) and S. khelangense (black text) in Thailand. The lineage found in both black fly species is indicated in purple. Bold indicates new lineages reported in the present study. Bootstrap values based on 1000 replications for ML and NJ analyses are shown near the branch

Full size image

Discussion

We report sequences of 44 additional genetic lineages of Leucocytozoon in two taxa of black flies (S. asakoae complex and S. khelangense) in Thailand. All of these belong to the Leucocytozoon lineage reported from chickens, except for one that is closest to Leucocytozoon reported from owls. This latter lineage was originally reported from a brown hawk-owl (Ninox scutulata) in Japan [35] and has also been reported in this bird species in Thailand [19]. Because Leucocytozoon lineages are specific to vertebrate hosts [2, 8, 11, 36], identifying the genetic lineages of these parasites can provide a link to the host associations of the blood-sucking vectors [11]. Thus far, the most common blood hosts of the two abundant ornithophilic black fly taxa in Thailand, the S. asakoae complex and S. khelangense, are chickens, although both species also feed on humans [21, 37]. Another avian species that has been recorded as a blood host of S. khelangense (= S. chumpornense reported by Gomontean et al. [37]) is turkey (Meleagris gallopavo domesticus). Thus, finding this black fly carrying the Leucocytozoon lineage of owls indicates that S. khelangense also feeds on owls; this is the first record of this association for Southeast Asian black flies. The only record to date of wild birds fed upon by Asian black flies is the Japanese rock ptarmigan (Lagopus muta japonica). In Japan, at least three black fly species, Simulium japonicum, Prosimulium hirtipes and Stegopterna takeshii (as Cnephia mutata), feed on this ptarmigan [38]. Two of these black flies (S. japonicum and P. hirtipes) as well as S. uchidai are possible vectors of Leucocytozoon lovati to this endangered subspecies of bird [7].

The authors of a previous study reported a high prevalence (49%) of Leucocytozoon in the S. asakoae complex collected in January 2018 at Ban Pang Bong in Chiangmai province, Thailand, compared with other sampling sites [10]. We found that Leucocytozoon prevalence rates in the S. asakoae complex at this location from 2019 to 2023 (9% in 2019, 12% in 2020 and 13% in 2023) were much lower than those in 2018 reported by Jumpato et al. [10]. The high prevalence of Leucocytozoon in the S. asakoae complex collected in 2018 was possibly due to the majority of specimens used for Leucocytozoon detection being adult flies resting on vegetation [10], perhaps resting after taking a blood meal from the hosts (e.g. chickens). In this scenario, these specimens had a greater chance of being positive for Leucocytozoon than did those swept from the air, which were more likely to have been searching for a host at time of collection.

The prevalence of Leucocytozoon found in the present study (11.3% for S. asakoae complex and 21.1% for S. khelangense) was much higher than rates reported from black flies in Japan (1.6%) [7] but lower than those from Sweden (62%) [36], the USA (46.2% in S. silvestre) [8] and Germany (29.4%) [11]. Vectors acquire haemosporidian parasites from vertebrate hosts only via blood-feeding. Therefore, the prevalence of Leucocytozoon in black flies depends on its prevalence in the vertebrate host and the probability that the vector and host encounter each other. Prevalence rates of Leucocytozoon in Thailand are low in wild birds (2% in raptors [19] and 8.1% in 12 wild bird species from Chiangmai province [20]) but much higher in domestic chickens (18.0–89.5%) [18, 22, 39, 40]. The relatively high prevalence of Leucocytozoon in the S. asakoae complex and S. khelangense compared with its prevalence in wild birds corresponds with the host blood source, primarily domestic chickens [21, 37]. We found a positive relationship between prevalence rates and black fly abundance (r = 0.551, P = 0.012). This finding agrees with previous reports that the prevalence of Leucocytozoon in black flies is associated with environmental factors that promote the production of large populations of these insects [9, 16, 17]. Given the high prevalence (up to 89%) of Leucocytozoon in domestic chickens in many areas of Thailand [18], a high abundance of black flies should increase the chance of uptake of the parasite and an increase in its prevalence in the vectors.

At least 48 black fly species are known as vectors of Leucocytozoon, and most of these can transmit diverse lineages of Leucocytozoon [3, 6,7,8,9,10,11,12,13]. However, some black fly species are specific to a particular parasite lineage. For example, Simulium annulus is specific to the Leucocytozoon IGRYS1 lineage, perhaps because of a possible host preference, as all analyzed S. annulus in northern Sweden fed only on the Eurasian crane (Grus grus) despite being collected from geographically distant (200 km) locations [36]. We found variations in the degree of specific vector–parasite associations. Among 66 Leucocytozoon lineages in black flies in Thailand, only one was shared between the S. asakoae complex and S. khelangense. Both taxa also possessed the dominant Leucocytozoon lineages that were found in geographically widespread populations but were exclusive to either the S. asakoae complex or to S. khelangense. Lineages GALLUS17, GALUUS35 and GALLUS37 were the dominant lineages in S. khelangense and occurred across a geographically widespread area covering all sampling sites of S. khelangense, encompassing > 500 km transects. Similarly, the GALLUS06, GALLUS44 and SIMASA01 lineages were predominant in the S. asakoae complex. The latter lineage is also found in a location (Song Khon Waterfall, Phu Ruea, Loei province) [10]) in close geographical proximity (< 12 km) to the lineages of S. khelangense (Ban Nong Bua, Phu Ruea, Loei province) [21]). Furthermore, L. schoutedeni was also significantly more frequently in the S. asakoae complex than in S. khelangense (χ2 = 31.72, df = 1, P < 0.0001). Because both species feed predominately on chickens, different lineage assemblages in these black flies are unlikely to be a result of different host preferences [36, 41]; rather, different Leucocytozoon lineages in the S. asakoae complex and S. khelangense are possibly related to different ecologies and phenologies of these black flies. The S. asakoae complex attains peak abundance in the rainy season [23, 42], whereas S. khelangense (= S. chumpornense in Pramual et al. [22]) predominates during the hot season [22]. In addition, the S. asakoae complex is dominant at higher elevations (> 600 m a.s.l.) compared with S. khelangense (mostly < 600 m a.s.l.) [37]. Season and elevation are strongly related to temperature, one of the main environmental factors related to haemosporidian parasite prevalence [16, 17]. Accordingly, we found a significant relationship between diversity of Leucocytozoon lineages and temperature. Thus, differences in Leucocytozoon lineages in the S. asakoae complex and S. khelangense might be a result of co-adaptation between vector and parasite in response to the temperature in different seasons and at different elevations. To gain further insight into vector–parasite co-evolution, studies are needed to examine the relationship between black fly haplotypes and those of Leucocytozoon lineages [43].

Conclusions

We found a high diversity of Leucocytozoon in black flies of two ornithophilic taxa, the S. asakoae complex and S. khelangense, in Thailand. Our results indicate an association between Leucocytozoon lineages and these black flies even though they feed on the same hosts, possibly indicating vector–parasite co-adaptation in response to the environmental conditions of their respective habitats. Further investigation based on the individual haplotypes of the insect vector and the Leucocytozoon lineage will be helpful in testing the hypothesis of the vector–parasite co-evolution system [43].

Availability of data and materials

No datasets were generated or analysed during the current study.

References

  1. Forrester DJ, Greiner EC. Leucocytozoonosis. In: Atkinson CT, Thomas NJ, Hunter DB, editors. Parasitic diseases of wild birds. Ames: Blackwell; 2008. p. 54–107.

    Chapter  Google Scholar 

  2. Valkiūnas G, Iezhova TA. Insights into the biology of Leucocytozoon species (Haemosporida, Leucocytozoidae): why is there slow research progress on agents of leucocytozoonosis? Microorganisms. 2023;11:1251.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Adler PH, McCreadie JW. Black flies (Simuliidae). In: Mullen GR, Durden LA, editors. Medical and veterinary entomology. San Diego: Elsevier; 2019. p. 237–59.

    Chapter  Google Scholar 

  4. Ševčík R, Mahlerová K, Riera FA, Zárybnická M. Leucocytozoon infection does not influence the survival of Boreal Owl Aegolius funereus nestlings. Avian Dis. 2024;68:134–40.

    Article  PubMed  Google Scholar 

  5. Akiba K. Studies on Leucocytozoon disease of chickens. II. On the transmission of L. caulleryi by Culicoides arakawae. Jpn J Vet Sci. 1960;22:309–17 (in Japanese).

    Article  Google Scholar 

  6. Santiago-Alarcon D, Palinauskas V, Schaefer HM. Diptera vectors of avian Haemosporidian parasites: untangling parasite life cycles and their taxonomy. Biol Rev. 2012;87:928–64.

    Article  PubMed  Google Scholar 

  7. Sato Y, Tamada A, Mochizuki Y, Nakamura S, Okano E, Yoshida C, et al. Molecular detection of Leucocytozoon lovati from probable vectors, black flies (Simuliudae [sic]) collected in the alpine regions of Japan. Parasit Res. 2009;104:251–5.

    Article  Google Scholar 

  8. Murdock CC, Adler PH, Frank J, Perkins SL. Molecular analyses on host-seeking black flies (Diptera: Simuliidae) reveal a diverse assemblage of Leucocytozoon (Apicomplexa: Haemospororida) parasites in an alpine ecosystem. Parasit Vectors 2015;8:1–7.

    Article  Google Scholar 

  9. Lotta IA, Pacheco MA, Escalante AA, González AD, Mantilla JS, Moncada LI, et al. Leucocytozoon diversity and possible vectors in the Neotropical highlands of Colombia. Protist. 2016;167:185–204.

    Article  PubMed  Google Scholar 

  10. Jumpato W, Tangkawanit U, Wongpakam K, Pramual P. Molecular detection of Leucocytozoon (Apicomplexa: Haemosporida) in black flies (Diptera: Simuliidae) from Thailand. Acta Trop. 2019;190:228–34.

    Article  PubMed  Google Scholar 

  11. Chakarov N, Kampen H, Wiegmann A, Werner D, Bensch S. Blood parasites in vectors reveal a united blackfly community in the upper canopy. Parasit Vectors 2020;13:1–8.

    Article  Google Scholar 

  12. Žiegytė R, Bernotienė R. Contribution to the knowledge on black flies (Diptera: Simuliidae) as vectors of Leucocytozoon (Haemosporida) parasites in Lithuania. Parasit Int. 2022;87:102515.

    Article  Google Scholar 

  13. Stangarm J, Mintara R, Jumpato W, Gomontean B, Thanee I, Wongpakam K, et al. Molecular detection of blood protozoa and identification of black flies of the Simulium varicorne species group (Diptera: Simuliidae) in Thailand. Acta Trop. 2024;254:107207.

    Article  PubMed  CAS  Google Scholar 

  14. Bensch S, Hellgren O, Pérez-Tris J. MalAvi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages. Mol Ecol Resour. 2009;9:1353–8.

    Article  PubMed  Google Scholar 

  15. Adler PH. World blackflies (Diptera: Simuliidae): a comprehensive revision of the taxonomic and geographical inventory. 2024. https://biomia.sites.clemson.edu/pdfs/blackflyinventory.pdf. Accessed 6 Aug 2024.

  16. Sehgal RN. Manifold habitat effects on the prevalence and diversity of avian blood parasites. Int J Parasitol Parasites Wildl. 2015;4:421–30.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Murdock CC, Foufopoulos J. Simon CP A transmission model for the ecology of an avian blood parasite in a temperate ecosystem. PLoS ONE. 2013;8:e76126.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Boonchuay K, Thomrongsuwannakij T, Chagas CRF, Pornpanom P. Prevalence and diversity of blood parasites (Plasmodium, Leucocytozoon and Trypanosoma) in backyard chickens (Gallus gallus domesticus) raised in Southern Thailand. Animals. 2023;13:2798.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Lertwatcharasarakul P, Salakij C, Prasopsom P, Kasorndorkbua C, Jakthong P, Santavakul M. Molecular and morphological analyses of Leucocytozoon parasites (Haemosporida: Leucocytozoidae) in raptors from Thailand. Acta Parasitol. 2021;66:1406–16.

    Article  PubMed  Google Scholar 

  20. Buranapim N, Chaiwisit P, Wangkawan A, Tiwananthagorn S. A survey on blood parasites of birds in Chiang Mai province. Vet Integr Sci. 2019;17:65–73.

    Google Scholar 

  21. Pramual P, Thaijarern J, Tangkawanit U, Wongpakam K. Molecular identification of blood meal sources in black flies (Diptera: Simuliidae) suspected as Leucocytozoon vectors. Acta Trop. 2020;205:105383.

    Article  PubMed  CAS  Google Scholar 

  22. Pramual P, Tangkawanit U, Kunprom C, Vaisusuk K, Chatan W, Wongpakam K, et al. Seasonal population dynamics and a role as natural vector of Leucocytozoon of black fly, Simulium chumpornense Takaoka & Kuvangkadilok. Acta Trop. 2020;211:105617.

    Article  PubMed  CAS  Google Scholar 

  23. Choochote W, Takaoka H, Fukuda M, Otsuka Y, Aoki C, Eshima N. Seasonal abundance and daily flying activity of black flies (Diptera: Simuliidae) attracted to human baits in Doi Inthanon National Park, northern Thailand. Med Entomol Zool. 2005;56:335–48.

    Article  Google Scholar 

  24. Ishii Y, Choochote W, Bain O, Fukuda M, Otsuka Y, Takaoka H. Seasonal and diurnal biting activities and zoonotic filarial infections of two Simulium species (Diptera: Simuliidae) in northern Thailand. Parasite. 2008;15:121–9.

    Article  PubMed  CAS  Google Scholar 

  25. Takaoka H, Srisuka W, Saeung A. Checklist and keys for the black flies (Diptera: Simuliidae) of Thailand. Med Entomol Zool. 2019;70:53–77.

    Article  Google Scholar 

  26. Srisuka W, Aupalee K, Otsuka Y, Fukuda M, Takaoka H, Saeung A. A new species of Simulium (Gomphostilbia) (Diptera, Simuliidae) from Thailand, with a key to identify females of 14 species of the Simulium varicorne species-group. ZooKeys. 2022;1083:1.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Folmer O, Hoeh WR, Black MB, Vrijenhoek RC. Conserved primers for PCR amplification of mitochondrial DNA from different invertebrate phyla. Mol Mar Biol Biotechnol. 1994;3:294–9.

    PubMed  CAS  Google Scholar 

  28. Tangkawanit U, Wongpakam K, Pramual P. A new black fly (Diptera: Simuliidae) species of the subgenus Asiosimulium Takaoka and Choochote from Thailand. Zootaxa. 2018;4388:111–22.

    Article  PubMed  Google Scholar 

  29. Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, et al. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc Natl Acad Sci USA. 1989;86:6196–200.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Malmqvist B, Strasevicius D, Hellgren O, Adler PH, Bensch S. Vertebrate host specificity of wild–caught blackflies revealed by mitochondrial DNA in blood. Proc R Soc Lond B Biol Sci. 2004;271:S152–5.

    Article  Google Scholar 

  31. Hellgren O, Waldenstrom J, Bensch S. A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. J Parasitol. 2004;90:797–802.

    Article  PubMed  CAS  Google Scholar 

  32. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Hammer Ø, Harper DA, Ryan PD. Past: paleontological statistics software package for education and data analysis. Palaeontol Electron. 2021;4:1.

    Google Scholar 

  34. Pramual P, Kuvangkadilok C, Baimai V, Walton C. Phylogeography of the black fly Simulium tani (Diptera: Simuliidae) from Thailand as inferred from mtDNA sequences. Mol Ecol. 2005;14:3989–4001.

    Article  PubMed  CAS  Google Scholar 

  35. Inumaru M, Murata K, Sato Y. Prevalence of avian haemosporidia among injured wild birds in Tokyo and environs, Japan. Int J Parasitol Parasit Wildl. 2017;6:299–309.

    Article  Google Scholar 

  36. Hellgren O, Bensch S, Malmqvist B. Bird hosts, blood parasites and their vectors—associations uncovered by molecular analyses of blackfly blood meals. Mol Ecol. 2008;17:1605–13.

    Article  PubMed  CAS  Google Scholar 

  37. Gomontean B, Jumpato W, Wongpakam K, Tangkawanit U, Wannasingha W, Thanee I, et al. Diversity, distribution and host blood meal analysis of adult black flies (Diptera: Simuliidae) from Thailand. Insects. 2024;15:74.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Imura T, Sato Y, Ejiri H, Tamada A, Isawa H, Sawabe K, et al. Molecular identification of blood source animals from black flies (Diptera: Simuliidae) collected in the alpine regions of Japan. Parasit Res. 2010;106:543–7.

    Article  Google Scholar 

  39. Piratae S, Vaisusuk K, Chatan W. Prevalence and molecular identification of Leucocytozoon spp. in fighting cocks (Gallus gallus) in Thailand. Parasit Res. 2021;120:2149–55.

    Article  Google Scholar 

  40. Khumpim P, Chawengkirttikul R, Junsiri W, Watthanadirek A, Poolsawat N, Minsakorn S, et al. Molecular detection and genetic diversity of Leucocytozoon sabrazesi in chickens in Thailand. Sci Rep. 2021;11:16686.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Gutiérrez-López R, Bourret V, Loiseau C. Is host selection by mosquitoes driving vector specificity of parasites? A review on the avian malaria model. Front Ecol Evol. 2020;8:569230.

    Article  Google Scholar 

  42. Srisuka W, Sulin C, Aupalee K, Phankaen T, Taai K, Thongsahuan S, et al. Community structure, biodiversity and spatiotemporal distribution of the black flies (Diptera: Simuliidae) using malaise traps on the highest mountain in Thailand. Insects. 2021;12:504.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Martínez-de la Puente J, Martinez J, Rivero-de Aguilar J, Herrero J, Merino S. On the specificity of avian blood parasites: revealing specific and generalist relationships between haemosporidians and biting midges. Mol Ecol. 2011;20:3275–87.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank Adrian Plant for valuable comments and the initial English language revision of the manuscript.

Funding

This research project was funded by Mahasarakham University, Grant number 6720001/2567.

Author information

Authors and Affiliations

  1. Department of Biology, Faculty of Science, Mahasarakham University, Kantharawichai District, Mahasarakham, 44150, Thailand

    Waraporn Jumpato, Ronnalit Mintara & Pairot Pramual

  2. Center of Excellence in Biodiversity Research, Mahasarakham University, Mahasarakham, 44150, Thailand

    Wannachai Wannasingha, Chavanut Jaroenchaiwattanachote & Pairot Pramual

  3. Walai Rukhavej Botanical Research Institute, Mahasarakham University, Kantharawichai District, Mahasarakham, 44150, Thailand

    Komgrit Wongpakam

  4. Department of Plant and Environmental Sciences, Clemson University, Clemson, SC, 29634-0310, USA

    Peter H. Adler

Authors
  1. Waraporn Jumpato
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Wannachai Wannasingha
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Chavanut Jaroenchaiwattanachote
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Ronnalit Mintara
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Komgrit Wongpakam
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Peter H. Adler
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Pairot Pramual
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

WJ: Conceptualization, formal analysis, resources, data curation, writing—original draft preparation. WW: Data curation, resources, visualization. CJ: Investigation, data curation, visualization. RM: Investigation, data curation, resources. KW: Investigation, data curation, writing—review and editing. PHA: Conceptualization, writing—review and editing. PP: Funding acquisition, formal analysis, writing—original draft preparation, writing—review and editing. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Pairot Pramual.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors read and approved the final manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Table S1.

Number of each Leucocytozoon lineage detected in black flies from each collection site.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jumpato, W., Wannasingha, W., Jaroenchaiwattanachote, C. et al. Diversity and prevalence of Leucocytozoon in black flies (Diptera: Simuliidae) of Thailand. Parasites Vectors 17, 475 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13071-024-06567-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13071-024-06567-0

Keywords

Parasites & Vectors

ISSN: 1756-3305

Contact us