Molecular identification of blood meal sources in black flies (Diptera: Simuliidae) suspected as leucocytozoon vectors

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Molecular identification of blood meal sources in black flies (Diptera: Simuliidae) suspected as Leucocytozoon vectors Pairot Pramual , Jiraporn Thaijarern , Ubon Tangkawanit , Komgrit Wongpakam PII: DOI: Reference:

S0001-706X(20)30015-2 https://doi.org/10.1016/j.actatropica.2020.105383 ACTROP 105383

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Acta Tropica

Received date: Revised date: Accepted date:

8 January 2020 29 January 2020 29 January 2020

Please cite this article as: Pairot Pramual , Jiraporn Thaijarern , Ubon Tangkawanit , Komgrit Wongpakam , Molecular identification of blood meal sources in black flies (Diptera: Simuliidae) suspected as Leucocytozoon vectors, Acta Tropica (2020), doi: https://doi.org/10.1016/j.actatropica.2020.105383

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Highlights   

Domestic chickens are found to be preferred host of two black fly species. Leucocytozoon spp. is detected in black flies that feed on chickens. Two black fly species in Thailand are potentially natural vectors of Leucocytozoon.

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Molecular identification of blood meal sources in black flies (Diptera: Simuliidae) suspected as Leucocytozoon vectors Pairot Pramuala*, Jiraporn Thaijarerna, Ubon Tangkawanit b, Komgrit Wongpakamc,

a

Department of Biology, Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham 44150, Thailand

b

Department of Entomology and Plant Pathology, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002 Thailand

c

Walai Rukhavej Botanical Research Institute, Mahasarakham University, Kantharawichai District, Maha Sarakham, 44150 Thailand

*Corresponding author: Pairot Pramual, Ph.D. Department of Biology, Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham 44150 Thailand Phone: 66(0)43 754245 Fax: 66(0)43 754245 E-mail: [email protected]

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ABSTRACT Identification of the host blood meal of hematophagous insects can provide significant information regarding host preferences and the possibility of the transmission of disease agents. Currently, this knowledge is limited for black flies in the Oriental region. In this study, we used cytochrome b gene sequences for identification of blood meal sources of two black fly taxa, the Simulium asakoae complex and S. chumpornense Takaoka & Kuvangkadilok in Thailand. A total of 4,260 wild adult females were visually screened revealing 24 blood-engorged females from which cytochrome b was successfully amplified in 19 individuals. Comparisons in GenBank database revealed that all are closest to chicken (Gallus gallus) with sequence similarity of >98%. Therefore, these black fly species are feeding on chickens. We also molecularly investigated the hemosporidian blood protozoa genus Leucocytozoon in black flies and found 13 of 19 blood-engorged females positive for this protozoon. Sequence analysis revealed that this Leucocytozoon DNA could be assigned into two previously recognized groups, one with a Leucocytozoon reported from domestic chickens and black flies in Thailand, and another close to the L. schoutedeni. Our results indicate a high possibility that the S. asakoae complex and S. chumpornense are natural vectors of Leucocytozoon.

Keywords: blood parasite; Gomphostilbia; ornithophilic; Simulium

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1. Introduction Black flies (Diptera: Simuliidae) are important pests and are vectors of disease agents transmitted to humans and other animals (Adler et al., 2004; Adler and McCreadie, 2019). There are 2,310 living species of black flies reported globally (Adler, 2019). Females of more than 90% of these species require a blood meal to complete egg maturation (Adler and McCreadie, 2019). This implies that a large number of species feed on mammals and birds (the only two groups of animals from which female black flies take a blood meal), where they potentially transmit disease agents. Information regarding the host species of hematophagous insects is crucial for fully understanding disease epidemiology. However, only a fraction of host species are known, mostly human or economically significant livestock and knowledge is geographically biased towards North America and Europe. This knowledge lags far behind the advanced taxonomic understanding of the black flies in the Oriental region despite the region being globally ranked second in species richness (Takaoka, 2017). In Thailand, a total of 111 species of black flies have been reported (Takaoka et al., 2019; Thaijarern et al., 2019a). Seven species (Simulium chamlongi Takaoka & Suzuki, S. nigrogilvum Summers, S. nodosum Puri, S. monglaense Takaoka, Srisuka & Saeung, S. myanmarense Takaoka, Srisuka & Saeung, the S. tenebrosum complex and S. umphangense Takaoka, Srisuka & Saeung) and two species complex (S. asakoae complex, S. doipuiense complex) are known as human biters and three (the S. asakoae complex, S. nodosum and S. nigrogilvum) are potential vectors of Onchocerca spp. transmitted among cattle (Fukuda et al., 2003; Takaoka et al., 2003). Two species (S. nodosum and S. nakhonense Takaoka and Suzuki) feed on water buffalo (Takaoka et al., 2003). No information on black fly biting in other animals in Thailand has been reported. Leucocytozoonosis is a disease caused by blood protozoa of the genus Leucocytozoon and it is one of the most important diseases of poultry in which black flies are the principal

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vector of the disease agents (Valkiũnas, 2005; Forrester and Greiner, 2008; Adler and McCreadie, 2019). Leucocytozoonosis can cause significant damage to the poultry industry through lethal effects or by decreasing productivity (Adler and McCreadie, 2019). There are at least 21 black fly species known as vectors of Leucocytozoon spp. (Adler and McCreadie, 2019). Leucocytozoon is a common haemosporidian blood parasite of domestic chickens found in Thailand with infection rates varying from 29 – 74% (Worasing et al., 2000, Singjam and Ruksachat, 2011; Jaijan et al., 2012; Takang et al., 2017). Leucocytozoon infections in domestic chickens can reduce egg productivity by 40% with a mortality rate of 1.5 – 40% (Worasing et al., 2000; Prasittirat et al., 2001). Recently, a molecular approach based on the cytochrome b sequence detected Leucocytozoon spp. In the S. asakoae complex and in S. chumpornense. DNA sequence analyses of the Leucocytozoon found in both black fly species revealed high similarity with those of these blood parasite protozoa found in domestic chickens (Jumpato et al., 2019) thus, suggesting that these black fly species are potential vectors of this disease agent. However, in order to demonstrate that suspected blood sucking insects are natural vectors of disease agents, at least two key criteria need to be met.(i) the suspected insect vector is demonstrated carrying an infective life stage of the disease agent and (ii) these insects are demonstrated to feed on the parasitic host. There is no information for the biting habit of S. chumpornense while the S. asakoae complex is known only to bite humans. In this study, a molecular approach based on specific amplification of the vertebrate mitochondrial cytochrome b (cyt b) gene was used to identify blood meal sources of these black fly species in Thailand. This method has been used successfully for identification of black fly vertebrate hosts in other geographic regions (Malmqvist et al., 2004; Hellgren et al., 2008; Imura et al., 2010). Information gathered from these studies has provided significant

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information on host preferences and also the possibility that black fly species might transmit disease agents (Hellgren et al., 2008; Imura et al., 2010). In addition to the identification of blood meal sources, we also use molecular approaches to detect Leucocytozoon in black fly specimens that are positive for vertebrate blood host amplifications.

2. Materials and methods 2.1 Specimen collection and identification Wild adult black fly specimens were collected from 11 sites (20 collections) in north and northeastern Thailand (Table 1 and Fig. 1) using a sweep net. Specimens were collected in the morning (07.00) or late afternoon (16.00), during time periods when black flies are actively searching for a host blood meal. For each collection event, sampling was conducted by four people with 1 hour sampling effort. Specimens were preserved in 80% ethanol and kept at -20° C until use. Identification of the black fly specimens followed the most recent keys to species of black flies in Thailand (Takaoka et al., 2019). 2.2 DNA extraction, amplification and sequencing DNA was extracted from whole body of the engorged female specimen using the GF1 Tissue DNA Extraction Kit (Vivantis, Malaysia). The mitochondrial cyt b of the vertebrate host blood in the blood meal was amplified using the primers L14841 and H15149 (Kocher et al., 1989). The temperature profile for the cyt b amplification followed the method described in Malmqvist et al. (2004). We also screened for Leucocytozoon in the blood engorged female black flies using primers specific for the cyt b gene of this protozoa (Hellgren et al., 2004). PCR reaction conditions and temperature profile followed Jumpato et al. (2019). PCR products of vertebrate and Leucocytozoon cyt b gene were sequenced using the same primers as in PCR. Sequencing was performed at 1st BASE (Malaysia) DNA sequencing service.

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2.3 Data analysis Sequences were aligned using the Clustal W option in BioEdit 7.2.6 (Hall, 1999) with final adjustment manually. The Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) in the National Centre of Biotechnology Information database was used to determine the sequence similarity of the vertebrate cyt b gene sequences. To examine genetic relationships between Leucoctyzoon cyt b sequences obtained in this study with those of a previous report in Thailand (Jumpato et al., 2019), phylogenetic analyses were conducted based on the neighbor-joining (NJ) method. The NJ tree analysis was implemented in MEGA X (Kumar et al., 2018) based on the Kimura 2-parameter (K2P) model. Branch support was calculated using the bootstrapping method with 1000 replicates. Sequences of Haemoproteus spp. (accession no. KT290922 – KT290924) were used as outgroups for phylogenetic analysis.

3. Results and Discussion A total of 24 blood engorged females were found among 4,620 adult specimens of the S. asakoae complex and S. chumpornense. Amplification of the vertebrate cyt b gene from blood meals was successful for 19 specimens; two from the S. asakoae complex and 17 from S. chumpornense (Table 1). NCBI BLAST results indicated that all of these sequences are closest to the cyt b gene of chicken (Gallus gallus) with >98% similarity (Table 2). Thus, the results suggested that S. chumpornense and the S. asakoae complex feed on chicken. This is consistent with the observation that all of the blood engorged specimens of both the S. asakoae complex and S. chumpornense were collected from or very close to village areas where domestic chickens were very common.

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Black fly biting of poultry has been reported in other geographic regions such as in Africa (El Bashir et al., 1976) and North America (Pinkovsky et al., 1981). In Asia, although there is good information regarding anthropophilic black fly species (Takaoka et al., 2003; Pramual et al., 2016; Jomkamsing et al., 2019), knowledge of the biting habit of black flies on other animals is very limited. There is just one report, of Japanese black flies, which is based on molecular identification of the blood meals and which indicated that four species were feeding on wild birds (Imura et al., 2010). This is the first report in Asia of black fly species of the subgenus Gomphostilbia feeding on domestic chickens. It is also the first report of the biting habit of S. chumpornense, a species that has previously been recognized as a potential vector of Leucocytozoon spp. (Jumpato et al., 2019) and Trypanosoma spp. (Thaijarern et al., 2019b). Like S. chumpornense, the S. asakoae complex is also a potential vector of these parasites (Jumpato et al., 2019; Thaijarern et al., 2019b). Both black fly taxa possess bifid claws which is a characteristic of ornithophilic species (Adler et al., 2004) although the S. asakoae complex can also feed on humans (Choochote et al., 2005). The results of the present study found that these black fly species can feed on chickens and thus provide further support for the view that the S. asakoae complex and S. chumpornense have high potential to be natural vectors of Leucocytozoon. All of the blood engorged female specimens of S. chumpornense were found to have fed on chickens. The association between this ornithophilic black fly species and avian host could be due to habitat sharing or host preference (Hellgren et al., 2008). We have collected large numbers (3,770 individuals) of wild adult females of this species in a village (i.e. Ban Nong Bua, Phu Ruea, Loei Province) (Table 1) where domestic chickens are very common. However, searches for the breeding site (i.e. running water) were unsuccessful, finding no suitable habitat for S. chumpornense close to this area. The nearest locality we have seen immature stages of this species was approximately 40 km away from the Ban Nong Bua

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collection site. Therefore, it is very likely that the large number of these black fly species found in the area where domestic chicken is common is an indication of host choice preference rather than sharing same habitat. In addition to the identification of sources of blood meals, we also detected Leucocytozoon DNA in 68% (13 from 19) of blood engorged black fly specimens. A much higher percentage of black fly specimens were found to be infected with Leucocytozoon in this study compared to Jumpato et al. (2019) who reported 29% for the S. asakoae complex and 7% for S. chumpornense. All black fly specimens screened for Leucocytozoon in this study contained chicken blood while those reported in Jumpato et al. (2019) were without host blood. Therefore, the high frequency of Leucocytozoon infection found in the present study tentatively indicates that this blood protozoan came from chickens. There were nine haplotypes of the Leucocytozoon DNA among 13 sequences (GenBank accession numbers: MN907103-MN907115). Phylogenetic analyses (data not shown) indicated that there was no evidence of the association between host cyt b haplotype and Leucocytozoon lineages. Phylogenetic analyses (Fig. 2) with cyt b sequences of the Leucocytozoon previously reported in black flies (Jumpato et al., 2019) and of domestic chicken in Thailand revealed that these Leucocytozoon could be assigned into two previously recognized groups (Jumpato et al., 2019). The majority (11 of 13) of Leucocytozoon DNA detected in blood engorged black flies were closely related to those of this hemosporidian parasite found in domestic chicken and black flies (Jumpato et al., 2019). Two additional sequences from S. chumpornense belonged to the clade that includes Leucocytozoon previously reported from black flies in Thailand (Jumpato et al., 2019) and are closely related to L. schoutedeni which was detected in red jungle fowl (Gallus gallus spadiceus) reported from Malaysia (GenBank accession number: KT290937). Experimental study in Africa has shown that L. schoutedeni can be transmitted to chickens by the black fly, S. adersi (Fallis et

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al., 1973). However, to conclude that Leucocytozoon found in S. chumpornense is L. schoutedeni would require additional information such as comparison of the morphology of the protozoa. Our results presented in this study further support the previous finding that ornithophilic black fly species in Thailand; the S. asakoae complex and S. chumpornense, are potential natural vectors of heamosporidian parasites of the genus Leucocytozoon transmitted among domestic chickens. We also report for the first time on the chicken-biting habits of S. chumpornense and S. asakoae. Our results provided a good association between host, vector, and parasite although further study is required to confirm that these two species are natural vectors of the Leucocytozoon.

Acknowledgements This research was financially supported by Mahasarakham University (Grant year 2020). We would like to thank Dr. Adrian Plant for valuable comments on an earlier version of the manuscript.

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Wilson, A. C., 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl Acad. Sci. USA 86, 6196–6200. Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K., 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549. Malmqvist, B., Strasevicius, D., Hellgren, O., Adler, P.H., Bensch, S., 2004. Vertebrate host specificity of wild-caught blackflies revealed by mitochondrial DNA in blood. Philos. Trans. R. Soc. B Biol. Sci. 271, S152–S155 Pinkovsky, D. D., Forrester, D. J., Butler, J. F., 1981. Investigations on black fly vectors (Diptera: Simuliidae) of Leucocytozoon smithi (Sporozoa: Leucocytozoidae) in Florida. J. Med. Entomol. 18(2), 153–157. Pramual, P., Thaijarern, J., Wongpakam, K., 2016. DNA barcoding of human-biting black flies (Diptera: Simuliidae) in Thailand. Acta Trop. 164, 33–40. Prasittirat1, M., Vongpakorn, M., Chompoochan, T., Intarachote, U., Moekaew, K., Anucha Sookruen, A. 2001. Treatment of Leucocytozoonosis in native chickens by sulfadimethoxine and mosquito nets. Proceedings of the 39th Kasetsart University Annual Conference, 387 – 393. (in Thai). Singjam, S., Ruksachat, N., 2011. Case Report: Outbreak of Leucocytozoonosis in Captive Wild Birds, Khao Kor Wildlife Captive Breeding Center, Veterinary Research and Development Center, Lower Northern Region News. (accessed 14 October 2018. http://region6.dld.go.th/webnew/pdf/academic/4/7.pdf. Takaoka, H. 2017. Speciation, faunal affinities and geographical dispersal of black flies (Diptera: Simuliidae) in the Oriental Region. Acta Trop. 166, 234–240. Takaoka, H., Choochote, W., Aoki, C., Fukuda, M., Bain, O., 2003. Black flies (Diptera:

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Table 1 Sampling locations, number of adult female black fly specimens, number of positive PCR of cytochrome b gene of vertebrate and Leucocytozoon. Black fly species

Location (code)

Latitude /

Collection date

n

Longitude

No. positive host

No. positive

blood gene

Leucocytozoon

S. asakoae

Song Khon Waterfall, Phu Ruea,

17°21′13″ N/

22 May 2017

40

0

0

complex

Loei (LO1)

101°24′23″ E

17 Mar 2019

9

0

0

Ban Pang Bong 2, Doi Saket,

18°59′03″ N/

22 Jan 2018

222

0

0

Chiang Mai (CM1)

99°20′07″ E

Phu Ruea Highland Agricultural

17°17′53″ N/

12 Feb 2019

16

1

0

Experiment Station, Loei (LO2)

101°24′37′′ E

19 Jan 2019

27

0

0

Suan Hom Waterfall, Nong Hin,

17°02′49″ N/

16 Mar 2019

10

0

0

Loei (LO3)

101°45′42″ E

Mork Fah waterfall, Chiang Mai

19°01′37″ N/

5 Apr 2019

1

1

1

(CM2)

98°57′17″ E 325

2

1

Total for S. asakoae complex S. chumpornense

Ban Kok Bok, Wang Saphung,

17°23′34″ N/

22 Mar 2017

186

0

0

Loei (LO4)

101°34′18″ E

16 Mar 2019

20

0

0

20 Apr 2019

40

3

0

22 Mar 2017

28

0

0

Ban Huai Lad, Phu Ruea, Loei

17°27′01″ N/

(LO5)

101°25′49′′ E

16

Black fly species

Location (code)

Latitude /

Collection date

n

Longitude Suan Hom waterfall, Nong Hin,

17°02′49″ N/

Loei (LO3)

101°45′42″ E

Chaisathan, Nan (NN1)

18°46′08″ N/

No. positive host

No. positive

blood gene

Leucocytozoon

23 Mar 2017

125

0

0

5 Oct 2018

86

0

0

6 Oct 2018

5

0

0

6 Oct 2018

8

1

1

100°43′57′′ E Ban Nam Muab, Wiang Sa, Nan

18°29′04″ N/

(NN2)

100°55′54′′ E

Ban Tan Tong, Wiang Sa, Nan

18°29′47′′ N/

(NN3)

100°55′24′′ E

Ban Nong Bua, Phu Ruea, Loei

17°26′54″ N/

16 Mar 2019

1,171 1

0

(LO6)

101°20′38″ E

20 Apr 2019

89

2

1

17 Mar 2019

877

2

2

26 May 2019

369

2

2

16 Jun 2019

1,264 6

6

4,295 17

12

4,620 19

13

Total for S. chumpornense Total n, number of specimens.

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Table 2 Results of NCBI BLAST of vertebrate cytochrome b gene sequences obtained from adult female black fly species in Thailand. Black fly species S. askoae complex

Specimens ID

Closest species (% similarity)

AKBIl Gallus gallus (99%) AKBl5182 Gallus gallus (99%) S. chumpornense CPFM5401 Gallus gallus (99%) CPFM5402 Gallus gallus (99%) CPFM5221 Gallus gallus (100%) CPFM4891 Gallus gallus (100%) CPFM5231 Gallus gallus (99%) CPFM5232 Gallus gallus (100%) CPFM5271 Gallus gallus (99%) CPFM5272 Gallus gallus (99%) CPFM5273 Gallus gallus (99%) CPBI5312 Gallus gallus (100%) CPFM5312 Gallus gallus (100%) CPFM5466 Gallus gallus (100%) CPFM5465 Gallus gallus (100%) CPFM5464 Gallus gallus (98%) CPFM5462 Gallus gallus (100%) CPFM5461 Gallus gallus (100%) CPFM5467 Gallus gallus (100%) * +, PCR positive for Leucocytozoon; - PCR negative for Leucocytozoon.

GenBank accession number DQ512917, AY509649, FM205718, FM205716 DQ512917 , AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716 DQ512917, AY509649, FM205718, FM205716

Leucocytozoon detection* + + + + + + + + + + + +

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Figure legends

Fig. 1. Collection sites in Thailand for wild adult black flies used in this study. Details of collection sites are given in Table1.

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Fig. 2. Neighbor-joining tree of Leucocytozoon cyt b sequences obtained from blood engorged female black flies and sequences of these protozoa in Thailand reported in NCBI GenBank database. Bootstrap values are shown above or near the branch. The name of each sequence containing the GenBank accession number is followed by the name of the Leucocytozoon species and host species. Bold characters indicate specimens obtained in this study.

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GRAPHICAL ABSTRACT

22 Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

23 AUTHORSHIP STATEMENT All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in the Acta Tropica.

Authorship contributions Please indicate the specific contributions made by each author (list the authors’ initials followed by their surnames, e.g., Y.L. Cheung). The name of each author must appear at least once in each of the three categories below.

Category 1 Conception and design of study: P. Pramual, acquisition of data: J. Thaijarern, U. Tangkawanit, K. Wongpakam, P. Pramual; analysis and/or interpretation of data: J. Thaijarern, P. Pramual, Category 2 Drafting the manuscript: P. Pramual, J. Thaijarern, U. Tangkawanit; revising the manuscript critically for important intellectual content: P. Pramual. Category 3 Approval of the version of the manuscript to be published (the names of all authors must be listed): P. Pramual, J. Thaijarern, U. Tangkawanit, K. Wongpakam.