Genetic diversity and population structure of Phlebotomus argentipes: Vector of Leishmania donovani in Sri Lanka

Dulani Ruwanika K Pathirage; Thilini C Weeraratne; Sanath C Senanayake; S H P Parakrama Karunaratne; Nadira D Karunaweera

doi:10.1371/journal.pone.0256819

. 2021 Sep 16;16(9):e0256819. doi: 10.1371/journal.pone.0256819

Genetic diversity and population structure of Phlebotomus argentipes: Vector of Leishmania donovani in Sri Lanka

Dulani Ruwanika K Pathirage ¹, Thilini C Weeraratne ², Sanath C Senanayake ¹, S H P Parakrama Karunaratne ^2,^‡, Nadira D Karunaweera ^1,^‡,^*

Editor: Maria Stefania Latrofa³

PMCID: PMC8445420 PMID: 34529694

Abstract

Phlebotomus argentipes is the vector of Leishmania donovani which causes the disease leishmaniasis, a neglected tropical disease and a growing health problem in Sri Lanka. A proper understanding of the population genetic structure of sand fly vectors is considered important prior to planning and implementation of a successful vector control program. Thus, the present study was conducted to determine the population genetic structure of sand fly vectors in Sri Lanka. Two mitochondrial genes namely Cytochrome c oxidase subunit 1 (Cox 1) and Cytochrome b (Cytb), and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization. Analyses included maximum likelihood method, network analysis and DNA polymorphisms. The outcome revealed unique sequences of all genomic regions studied except the cox 1 gene had a relationship with sand flies isolated previously from Sri Lanka, India and Israel and cytb gene of 4 sand flies that aligned with those isolated earlier from Sri Lanka and 3 from Madagascar. Furthermore, cox 1 gene and ITS 2 region analyses based on F_ST values indicated a possible gene flow between the study sites whereas cytb gene analysis favoured the existence of genetically distinct populations of P. argentipes in each of the study sites. Poor population differentiation of P. argentipes, a possible consequence of a gene flow, is indeed of concern due to the risk imposed by promoting the spread of functionally important phenotypes such as insecticide resistance across the country, making future vector control efforts challenging.

Introduction

Cutaneous leishmaniasis (CL), muco-cutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL) or “kala-azar” are the three main clinical forms of the disease leishmaniasis [1]. The disease is caused by an obligate intracellular flagellated protozoan belonging to the genus Leishmania. Phlebotomus argentipes (Diptera: Psycodidae) is the known vector of Leishmania donovani, the causal agent of the clinical form VL which is considered the second parasitic disease cause of a high number of deaths, in India, Nepal and Bangladesh [2–4]. Thus VL is recognized as the most virulent form among the three types of the disease [4]. However, the majority of L. donovani infections in Sri Lanka manifest as CL with only a few MCL and VL cases [5]. Although it was an exclusively imported disease prior to 1990s [6], now the disease is considered as widely prevalent over the country with case numbers increasing since 2001 and considered ‘notifiable’ in the health sector [1,5]. No national control programme is yet in place to contain the situation although leishmaniasis is a rapidly growing health threat in Sri Lanka [1].

Use of insecticides is the main strategy used for vector control in Sri Lanka, which may have promoted the development of insecticide resistance to indoor residual spraying of DDT and malathion [7]. This situation necessitated the use of organophosphates (e.g. fenitrothion), pyrethroids such as λ-cyhalothrin, cyfluthrin, and deltamethrin or the pseudo-pyrethroid etofenprox since 1994–2012 [8]. Rotation of insecticides every 3–5 years and sprayings restricted to targeted areas are used to overcome the development of resistance by the Sri Lankan malaria vector control programme. Sand flies were not directly targeted however, those flies in malaria endemic areas in the dry zone, which covers two-thirds of the country may have been regularly exposed to insecticides [9]. The presence of kdr mutation which is located at the position of 1014 with the amino acid changes from leucine to phenylalanine in a notable proportion of flies in Sri Lanka [10] will spread from one population to another by means of gene flow in sand flies similar to the case of mosquitoes [11–14].

The genetic structure for this sand fly species has been previously described [15,16]. However, the diversity of the phlebotomine sand flies is believed to be high in the country and the taxonomy based on morphology makes it hard to discriminate species and sibling species, which is more abundant among several species of phlebotomine sand flies.

DNA barcoding is considered as an efficient tool for genetic characterization of many species of insects including the New world sand flies and Old world sand flies by the use of Cytochrome oxidase subunit I (cox 1) sequences [16–19]. The 18s, 28s rDNA and cox 1, internal transcribed spacer 2 (ITS2) and Cytochrome b (cytb) sequences have been used to confirm the identification of species Phlebotomus argentipes complex in Sri Lanka. Cytochrome oxidase subunit I and cytb sequences were variable within the P. argentipes complex while the 18s and 28s sequences have not exhibited any variation within the sibling species [15].

The cox 1, cytb and ITS2 genes are useful genetic markers that are widely used in investigating the genetic characterization of Phlebotominae sand flies to determine the haplotypes, phylogeography and genetic structure [20,21].

The primary goal of this study was to use mitochondrial and ribosomal DNA sequence information to construct a DNA barcode to study the effect of evolutionary forces that may spread resistance genes from one population to another.

Materials and methods

Collection of sand flies

Adult sand flies were collected for two days at a time, every two months for a period of two years from November 2015 to November 2017 covering the sites Talawa (8°14’11.468"N, 80°21’2.782"E) in Anuradhapura district (North-Central Province), Pannala (7°19’43.608"N, 80°1’26.3316"E) in Kurunegala district (North-Western Province), Mamadala (6°07’16.80"N, 81°07’12.60"E) in Hambantota district (Southern Province), and Mirigama (7°13’30.72"N, 80°7’40.439"E) in Gampaha district (Western Province) in Sri Lanka (Fig 1). As the country is arbitrarily divided into dry, intermediate and wet zones based on the annual rainfall, Talawa and Mamadala are located within the dry zone, Pannala in the intermediate zone and Mirigama in the wet zone (Fig 1). The collection locations were determined based on climatic zones in the country, leishmaniasis case burden [22] and vector prevalence data [23]. The standard cattle-baited net traps and Center for Disease Control (CDC) light traps were used for overnight sand fly collections in each study site and flies were collected each morning at 6:00 h.

Fig 1 — The locations of the sand fly collection sites are indicated. Reprinted from authors’ previous work [Pathirage *et al*. 2020 [10]].

Morphological identification

Identification of individual sand flies prior to PCR amplification was carried out using morphometric characteristics, up to the species level by examination under a light microscope at a magnification of 400× using standard taxonomic keys [24,25]. Identified samples were preserved in −20°C [16] for PCR amplification.

PCR amplification and sequencing of mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA)

Full genomic DNA was isolated from individual sand flies (minimum of 10 individuals per population) collected from different study sites using DNeasy Blood and Tissue kit (QIAGEN-Germany).

PCR amplification of a variable part of the cytochrome oxidase subunit I gene was done using primers LCO 1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO 2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) [18] in a 50 μl PCR reaction which contained 0.4 μl of 10 μm of each primer, 10 μl of 5X Colourless GoTaq^® Flexi Reaction Buffer (Promega, USA), 2 μl of 25 mM MgCl_2, 1 μl of 10 mM of each dNTP and 5 units of GoTaq^® Flexi DNA polymerase (Promega, USA) with the following conditions: an initial denaturation at 95° C for 5 min; followed by 5 cycles at 94° C for 40 s, 45° C for 1 min and 72° C for 1 min; 35 cycles at 94° C for 40 s, 51° C for 1 min, 72° C for 1 min; and a final extension at 72° C for 7 min.

The cytochrome oxidase b gene was amplified using primers CB3: 5 ′- CA [T/C] ATT CAA CC[A/T]GAATGATA-3 ′; N1N-PDR: 5 ′ - GGTA [C/T] [A/T] TTGC CTCGA [T/A] TTCG [T/A]TATGA– 3′ [26] in a 50 μl PCR reaction which contained 0.6 μl of 10 μm of each primer, 10 μl of 5X Colourless GoTaq^® Flexi Reaction Buffer (Promega, USA), 3 μl of 25 mM MgCl_2, 1 μl of 10 mM of each dNTP and 5 units of GoTaq^® Flexi DNA polymerase (Promega, USA). The cytb gene was amplified using the following conditions: an initial denaturation at 95° C for 5 min; followed by 10 cycles at 94° C for 30 s, 40° C for 30 s and 72° C for 1.5 min; 30 cycles at 94° C for 30 s, 45° C for 30 s, 72° C for 1.5 min; and a final extension at 72° C for 10 min.

Universal ITS2 primers forward primer (5.8S): 5’- ATC ACT CGC CTC ATG GAT CG 3’; reverse primer (28S): 5’- ATG CTT AAA TTT AGG GGG TAG TC 3’ [15] were used to amplify the ITS2 region. PCR amplification was done in a 25 μl PCR reaction which contained 0.820 μl of 10 μm of 5.8s and 0.730 μl of 2.8s each primer, 5 μl of 5X Colourless GoTaq^® Flexi Reaction Buffer (Promega, USA), 1.5 μl of 25 mM MgCl_2, 0.5 μl of 10 mM of each dNTP and 5 units of GoTaq^® Flexi DNA polymerase (Promega, USA) with cycling conditions of an initial denaturation at 95° C for 5 min; followed by 35 cycles at 95° C for 30 s, 55° C for 45 s and 72° C for 1 min; and a final extension at 72° C for 10 min.

PCR products were electrophoresed on a 1% agarose gel stained with ethidium bromide. Amplified PCR products were sent to Macrogen, Korea for purification and DNA sequencing using an Applied Biosystems 3730 DNA Analyzer. Sequences were received for both forward and reverse directions.

Sequence analysis

Trace files of DNA sequences were manually edited and aligned using ClustalW in BioEdit 7.2.5 software. The finalized sequences were subjected to a BLAST search, on the National Center for Biotechnology information (NCBI) database GenBank (http://www.ncbi.nlm.nih.gov) for species confirmation. Sequences obtained from each genetic marker was deposited in Genbank database (Accession numbers: cox 1- MW256437-MW256478, cytb- MW571044- MW571085 and ITS2- MW322926-MW322967). Phylogenetic analysis was conducted using MEGA version 6.06 software (http://www.megasoftware.net/) and the trees were constructed using the Kimura-2 parameter distance model in maximum likelihood method [27] for Phlebotomus argentipes sensu lato complex in Sri Lanka. Number of haplotypes (h), genetic diversity indices [Haplotype Diversity Index (Hd) and Nucleotide Diversity Index (Pi)] and, Neutrality tests (Tajima’s D and Fu’s Fs) were determined using DNA Sequences Polymorphism software (dnaSP) (version 5.1.10). Pairwise differences and population structures of each species were evaluated by analysis of molecular variance (AMOVA) in Arlequin software (version 3.11) (cmpg.unibe.ch/software/arlequin3/) and significance was evaluated based on 1000 permutations. Median joining (MJ) network analysis was conducted based on the number of nucleotide differences and haplotype networks of these three regions, and haplotype networks were constructed using Network software 5.0.0.1 (http://www.fluxus-engineering.com) to determine the interrelationship between haplotypes.

Results

Morphological identification

A total of 126 of adult sand flies were collected and were identified as Phlebotomus argentipes [15,24,25] and then subjected to PCR amplification followed by DNA sequencing.

Phylogenetic analysis

A minimum of 10 good quality sequences of mtDNA cox 1 (~650bp), cytb (~550bp) genes and rDNA ITS2 ( ~350bp) region of P. argentipes specimens collected from each locality were used in the phylogenetic analysis. Alignment of cox 1, cytb and ITS2 sequences with those available in public domain in Genbank confirmed their identification as P. argentipes (Nucleotide identity: 99.57% for cox 1 gene, 100.00% for cytb gene and 94.15% for ITS2 region) (S1–S3 Tables).

The evolutionary analysis by maximum likelihood method respectively for cox 1, cytb and ITS2 sequences of P. argentipes are shown in Fig 2. Representative haplotypes obtained for sand flies collected from different study sites were included in a single clade. There was no separation of clades based on the study sites suggesting the variations within this species complex and characteristics of individual sand flies are shared among studied populations.

The phylogenetic tree obtained for cox 1, cytb genes and ITS2 region were separated in to 2 different clades except SLITSI42 sample from Thalawa population (Fig 2A–2C). Each clade of cox 1 gene (A and B) comprised 21 individuals (29–85% bootstrap value) (Fig 2A) and the clade of cytb gene (X and Y) comprised 35 and 7 individuals (Fig 2B) in each clade. The two different clades (P and Q) of ITS2 region respectively comprised 34 and 7 individuals (48–88% bootstrap value) (Fig 2C).

Sand Flies originated from clade A (18 sand flies) of cox 1 gene clustered with P. argentipes previously reported in Sri Lanka and 2 individual sequences from Mirigama and Thalawa from the same clade A grouped with P. argentipes found in Kerala, India. Clade B of the cox 1 gene clustered with P. argentipes found in Israel (Fig 2A). Sequences (3 sand flies from Pannala and 1 sand fly from Thalawa) from clade Y of the cytb tree grouped with P. argentipes sibling sp. A and B previously reported in Sri Lanka and 3 individual sequences from Pannala from the same clade Y grouped with P. argentipes found in Madagaskar and nothing from clade X clustered with any publicly available sequence in NCBI (Fig 2B). None of the sequences from clade P and Q of ITS2 region grouped with publicly available sequences in NCBI (Fig 2C).

DNA sequence polymorphism

Respectively 16, 6 and 3 unique haplotypes were identified with a haplotype diversity (Hd) of 0.878, 0.513 and 0.324 for mtDNA cox 1 (~650bp), cytb (~550bp) genes and rDNA ITS2 ( ~350bp) region respectively. The total number of polymorphic sites were 263, 105 and 83 respectively for cox 1, cytb genes and ITS2 region. The haplotype diversities (Hd) and nucleotide diversities (Pi) were similarly high for all species in each study site for cox 1 gene. P. argentipes from Mamadala population had the lowest haplotype diversity (0.182) and nucleotide diversity (0.007) with haplotype diversities ranging from 0.182 to 0.733 and nucleotide diversities from 0.007 to 0.276 when compared with data from other populations for cytb gene. Similar haplotype diversities values were observed for cytb gene and ITS2 region in both Mamadala and Thalawa (Table 1).

Table 1. Genetic diversity indices, neutrality test values for Cox 1, Cytb and ITS2 regions of P. argentipes in each study site (n≤ 10 from each study site).

Population		n	S	H	Hd (±SD)	Pi	DT	DD	DF
Whole population	Cox 1	42	263	16	0.878±0.037	0.427	-3.150	-0.526	-0.464
	Cytb	42	105	6	0.513±0.089	0.154	0.392	1.913	1.626
	ITS2	42	83	3	0.324±0.081	0.074	0.583	0.656	0.750
Mamadala	Cox 1	11	252	7	0.818±0.119	0.304	2.659	1.524	2.064
	Cytb	11	8	2	0.182±0.144	0.007	-1.934	-2.320	-2.511
	ITS2	11	76	2	0.182±0.144	0.042	-2.230	-2.745	-2.967
Mirigama	Cox 1	11	251	6	0.836±0.089	0.290	2.262	1.609	2.020
	Cytb	11	15	4	0.600±0.154	0.022	-1.331	-0.737	-1.007
	ITS2	11	76	2	0.436±0.133	0.101	1.326	1.651	1.780
Pannala	Cox 1	10	253	3	0.689±0.104	0.202	2.019	1.672	1.430
	Cytb	10	100	3	0.733±0.076	0.276	2.430	1.656	2.091
	ITS2	10	76	2	0.356±0.159	0.082	0.029	1.647	1.403
Thalawa	Cox 1	10	262	7	0.867±0.107	0.304	2.013	1.241	1.622
	Cytb	10	103	3	0.378±0.181	0.109	-2.141	-2.522	-2.743
	ITS2	10	83	3	0.378±0.181	0.087	0.510	0.636	0.395

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n, number of DNA sequences for each gene and location; S, number of polymorphic sites; H, number of haplotypes; Hd, haplotype diversity; Pi, nucleotide diversity; DT, Tajima’s D; DD, Fu & Li’s D*; DF, Fu & Li’s F*; SD, Standard deviation.

According to neutrality test results of cox 1 gene, both Tajima’s D and Fu’s Fs values were significant in Mamadala (P<0.02), Thalawa (P<0.05) and Mirigama (P<0.05) populations whereas these values were not significant in Pannala (P>0.1) population (Table 1).

Tajima’s D index was positive (+) for all study sites of cox 1 gene that may indicates a bottleneck situation except in Pannala population. Both Tajima’s D and Fu’s Fs values in cytb gene were not significant (P>0.1) in Mirigama population whereas significant in other populations (P<0.05) where the population may have experienced a bottleneck event in the past (Table 1). When consider neutrality results of ITS2 region, both Tajima’s D and Fu’s Fs values were significant only in Mamadala population (P<0.02) where the population might have experienced a bottleneck event in the past (Table 1).

Pairwise F_ST values and population structure

The pairwise F_ST values obtained for three different genes of P. argentipes in this study are presented in Table 2. The pairwise F_ST values of P. argentipes between different study sites were not significant (P>0.1) with an absence of genetic differentiation between populations. Interestingly, the pairwise F_ST values between Mirigama and Pannala (P = 0.01) and between Mamadala and Pannala (P = 0.00) for cytb gene were significant favouring existence of genetic differentiation between populations (S4 Table).

Table 2. Pairwise F_ST values and P values obtained for Cox 1, Cytb and ITS2 regions of P. argentipes collected in 4 different localities.

Study site	Mirigama	Mamadala	Thalawa	Pannala
Cox 1 region
Mirigama	0.00000
Mamadala	0.08011	0.00000
Thalawa	0.09859	0.09670	0.00000
Pannala	0.24943	0.13873	0.19531	0.00000
Cytb region
Mirigama	0.00000
Mamadala	0.03186	0.00000
Thalawa	0.00144	0.00711	0.00000
Pannala	0.53985^*	0.55809^*	0.35835	0.00000
ITS2 region
Mirigama	0.00000
Mamadala	0.01579	0.00000
Thalawa	0.08035	0.05162	0.00000
Pannala	0.08952	0.05307	0.10333	0.00000

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* Significantly different pairwise F_ST values (p<0.02).

The effects of evolutionary forces on population

F_ST (Fixation index) values were 0.0579, 0.4221 and 0.0607 for cox 1, cytb genes and ITS2 region respectively for P. argentipes in this study (Table 3). According to the AMOVA analysis, variations of population genetic structure was not observed among or between populations for cox 1 gene and ITS2 region whereas significant for (P<0.05) cytb gene among studied localities (S4 Table).

Table 3. F_ST values obtained for AMOVA analysis of Cox 1, Cytb and ITS2 regions of P. argentipes collected in each study site.

Region	F_ST values	P value
Cox 1	0.0579	0.1964±0.0121
Cytb	0.4221^*	0.0020±0.0014
ITS2	0.0607	0.7801±0.0142

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* Significantly different F_ST values (p = 0.02).

Haplotypes interrelationship

The lowest number of haplotypes (n = 3) were shown by ITS2 region when compared to the cytb (6 haplotypes) and cox 1 (8 haplotypes) genes of P. argentipes. The most dominant haplotypes were haplotype 5 and 6 for cox 1 and cytb genes respectively (42.86% and 69.05% of the total population in cox 1 and cytb genes respectively), and haplotype 2 (80.95% of the total population) in ITS2 region (Fig 3A–3C). The lowest haplotype sharing was shown by cox 1 gene where only 36.36% (haplotypes 1, 3, 4 and 5) were observed while 66.67% haplotypes sharing were shown by both cytb gene (haplotypes 3, 4, 5 and 6) and ITS2 (haplotypes 1 and 2). Out of shared haplotypes, only haplotype 5 and 6 respectively for cox 1 gene and cytb gene shared among 4 localities while other haplotypes shared among 2 or 3 study localities (Fig 3A(i) and 3B(i)). However, both haplotypes 1 and 2 in ITS2 region shared among 4 localities in this study (Fig 3C(i)).

Fig 3 — Haplotype networks generated using Network 5.0.0.1 for A)*Cox 1* B)*Cytb* C)ITS2 region of P. *argentipes* collected from four geographical locations in Sri Lanka. i) for total population ii) Comparison with NCBI references. Each haplotype is represented by a circle and the size of the circle is proportional to the number of individuals with each haplotype. Geographical localities are colour coded. Mirigama; red, Pannala; yellow, Mamadala; black, Thalawa; green. Reference sequences are indicated in different colours.

With reference to the cox 1 gene of P. argentipes, haplotype 6, 7 and 8 respectively had a relationship with sand flies isolated previously from Sri Lanka, India and Israel (Fig 3A(ii)). Haplotype 1 (sand flies from Pannala) and 2 (sand flies from Pannala and Thalawa) in Cytb region of P. argentipes were related to sand flies originated from Madagascar and from Sri Lanka respectively (Fig 3B(ii)). None of the sequences of sand flies shared haplotypes with other reference sand fly sequences in NCBI and therefore ITS2 region of sand flies were considered as unique (Fig 3C(ii)).

Discussion

Leishmaniasis transmission may be curtailed by the introduction of vector control strategies in Sri Lanka. Previous work by authors demonstrated the existence of insecticide tolerance in local sand fly populations and also the presence of a known genetic mutation associated with insecticide resistance, with direct implications on future sand fly control [10]. Thus, the present study was conducted to investigate the genetic diversity and population genetic structure of leishmaniasis vectors, which are applicable for effective planning of future vector control programmes in Sri Lanka.

This is the first attempt, as far as it is known, to study the population genetic structure of P. argentipes using multiple markers to understand the possible evolutionary forces that may operate in local sand fly populations.

The phylogenetic trees generated in the study demonstrated two clades. Interestingly, most of the haplotypes were shared among four locations, which indicate the close genetic relatedness between sand fly populations in the four regions studied.

Phylogenetic tree analysis and network analysis of cox 1 sequences of this study also indicated that the P. argentipes were closely related to specimens of the same species collected in Sri Lanka, India and Israel. However, the cytb sequences of three sand flies originated from Pannala were closely related to P. argentipes found in Madagaskar, which may need further confirmation with a larger sample. The ITS2 region analysis indicated unique sequences that did not show any resemblance to existing sequences available in the NCBI database.

This is the first attempt to obtained sequences and population genetic analysis for cox 1, cytb genes and ITS2 region for P. argentipes in Sri Lanka.

Tajima’s D is the difference between observed average pairwise difference (π) and expected average pairwise difference (ϴ) [28]. Tajima’s D index of cox 1 gene was positive (+) in all localities i.e. Thalawa, Mirigama and Mamadala, (except in Pannala population) that demonstrates the presence of high variations that exist within study sites with observed average pairwise differences (π) being higher than the expected average pairwise differences (ϴ). Thus the natural selection may have led to a low variation, which might result in a bottleneck event. This effect was further confirmed using ITS2 region analysis, which resulted in a positive (+) Tajima’s D value in Pannala.

Tajima’s D of cytb gene was minus (-) for Thalawa and Mamadala indicating a low genetic variation within the study sites and therefore, the π is lower than ϴ. This might indicate a bottleneck event in the past in these sites. Such an effect could be confirmed in Mamadala also based on ITS2 region analysis.

High F_ST value of 0.4221 with cytb gene analysis in this study suggest genetically distinct populations between the sites (P<0.02). However, that assumption does not hold true when F_ST values from cox 1 gene and ITS2 region were considered (P>0.05). Interestingly, the pairwise F_ST values between Mirigama and Pannala and between Mamadala and Pannala for cytb gene were different (P<0.02), which may demonstrate genetic differentiation between populations.

The lack of population differentiation and haplotype sharing between the sites that was revealed in the population structure analysis (both pairwise comparisons and F_ST values derived through the analysis of molecular variance) using cox 1 and ITS2 markers may indicate a possible gene flow between regions (though it’s not supported by the outcome of cytb region analysis in Mamadala, Mirigama and Pannala). Sri Lanka is an island with a relatively small land mass and there are no major geographical barriers between the studied localities. Hence, regardless of the geographic distance the likelihood of a gene flow between the study sites could be considerably high. The presence of a known genetic mutation associated with insecticide resistance in a notable proportion of flies in these study locations was previously reported [10]. A possible gene flow therefore, would pose a risk of dispersal of genes of significance, such as insecticide resistance genes, between populations with the risk of spread of insecticide resistance that will have implications for future vector control. The findings related to gene flow could be further strengthened through future studies involving a larger sample size.

The molecular markers have been extensively used for genotyping and study of evolutionary history of mosquitoes. The analysis using cytb gene has demonstrated to be useful to detect the lack of population differentiation and the presence of gene flow in some species of dipterans in South-east Asia [29]. The gene flow between species of insect vectors may lead to alterations in the disease patterns [30]. Low genetic structure variation in some sand fly species as a result of geographical isolation and restricted gene flow may have led to reduced flight abilities and formation of cryptic species that ultimately influence the capacity to transmit parasites such as Leishmania in Latin America [31]. Moreover, cytb investigations have demonstrated the high genetic differentiation and restricted gene flow among populations of Lutzomyia cruciata in Mexico [32]. The low intraspecific genetic divergence between specimens from Mexico and those from north-western Colombia of Lu. shannoni population may be interpreted as a northward expansion of the species from South America through Central America via the Isthmus of Panama. Interestingly, a recent population expansion of Lu. shannoni in the U.S coincides with the conclusion of the most recent great glaciation period at the end of the Pleistocene Epoch [unpublished work].

Population genetic structure studies of sand fly vectors is important to investigate the presence of possible gene flow among populations. The possible gene flow might act as a carrier and transfer genes from one population to another even in geographically distinct populations (devoid of geographical barriers). If the genes are responsible for insecticide resistance, the gene flow might enhance the number of species having insecticide resistance genes with adverse consequences on vector control programs. Thus it is vital to investigate the population genetic structure of sand flies in Sri Lanka to enable effective sand fly control programs in the future.

Conclusions

Populations of P. argentipes in Sri Lanka seem to be distinct from sand flies that are found elsewhere except for their sequences related to cox 1 gene (100%) and cytb gene (16.7%) that were found to be shared between study populations. Furthermore, the findings related to the analyses of both cox 1 and ITS2 indicated a possible gene flow with lack of population differentiation between geographically distant populations of P. argentipes in Sri Lanka, perhaps due to the absence of geographic barriers and the continuity of habitats. Therefore, there is a high risk of transferring genes of functional significance, such as those that confer insecticide resistance from one population to another with the risk of eventually spreading across the country. However, confirmation of such an assumption would require further investigations and supportive evidence probably with the use of more neutral genotyping markers such as microsatellites, which have been used in other Phlebotomus sp. for similar investigations including for those related to the influence of geographic distance and altitude [33].

The study also confirms the possible use of cox 1, cytb genes and ITS2 region as genotyping markers for the determination of the population genetic structure of Phlebotominae sand flies in Sri Lanka. Thus, the findings will pave the way for more extensive investigations, probably with the use of more genotyping markers, to aid a vector control strategy that may need to be adopted within a future national leishmaniasis control programme.

Supporting information

S1 Table. Nucleotide identities for cox 1 gene.

Nucleotide identities obtained through NCBI blast results for cox 1 gene.

(PDF)

Click here for additional data file.^{(305.3KB, pdf)}

S2 Table. Nucleotide identities for cytb gene.

Nucleotide identities obtained through NCBI blast results for cytb gene.

(PDF)

Click here for additional data file.^{(307.2KB, pdf)}

S3 Table. Nucleotide identities for ITS2 region.

Nucleotide identities obtained through NCBI blast results for ITS2 region.

(PDF)

Click here for additional data file.^{(306.2KB, pdf)}

S4 Table. Analysis of Molecular Variance (AMOVA) of cox 1, cytb genes and ITS2 region.

Analysis of Molecular Variance (AMOVA) of cox 1, cytb genes and ITS2 region using Arlequin software (version 3.11) (cmpg.unibe.ch/software/arlequin3/).

(PDF)

Click here for additional data file.^{(100.9KB, pdf)}

Acknowledgments

We acknowledge Mr. Sunil Shantha and Mr. M. P. Ariyapala for field assistance to collect sand flies, Mr. Wasantha Senadeera for providing a map of study locations, the Head and staff of the Parasitic Diseases Research Unit, Department of Parasitology, Faculty of Medicine, University of Colombo for logistical help.

Data Availability

All relevant relevant data have been uploaded to GenBank: cox 1- MW256437-MW256478 cytb- MW571044- MW571085 ITS2- MW322926-MW322967.

Funding Statement

This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, USA, under award number U01AI136033. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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PLoS One. doi: 10.1371/journal.pone.0256819.r001

Decision Letter 0

Maria Stefania Latrofa

22 Jun 2021

PONE-D-21-16188

Genetic diversity and population structure of Phlebotomus argentipes: vector of leishmaniasis in Sri Lanka.

PLOS ONE

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Additional Editor Comments:

Dear Authors, thought the study is of interest for the readers, it is pivotal to modify the ms. according to comments of both reviewers and focusing the population genetic analysis on microsatellite loci; otherwise, you have to limit the study only on the molecular typing of sand fly species examined.

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Reviewer #1: No

Reviewer #2: Partly

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Revision of the paper “Genetic diversity and population structure of Phlebotomus argentipes: vector of leishmaniasis in Sri Lanka” by Pathirage et al.

Leishmaniasis is a significant vector-borne disease. Understanding the degree of gene flow among vector populations is essential for planning efficient control actions and avoiding resistant allele spread. At this aim, studying the genetic structure of populations is an important and well-recognized approach.

In this paper the authors aimed to determine the population genetic structure of sand fly vectors in Sri Lanka. Two mitochondrial genes (Cox 1 and Cytb) and the internal transcribed spacer (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization.

The markers used in this paper are not suitable for assessing population genetic structure and gene flow among populations. At this aim, microsatellite or SNPs markers should be used. Consequently the inferred conclusions are not supported by genetic data.

The genetic analyses carried out in this paper revealed unique sequences of “all genomic regions studied except the cox 1 gene in 21 flies that aligned with those from Kerala, India and cytb gene of 4 flies that aligned with those isolated earlier from Sri Lanka and 3 from Madagascar”.

The authors say that Cox 1 gene and ITS 2 region analyses revealed gene flow between the study sites”. However, the lack of differentiation among populations is due to incomplete lineage sorting and the lack of power of these markers for intra-specific studies.

Reviewer #2: Genetic diversity and population structure of Phlebotomus argentipes: vector of leishmaniasis in Sri Lanka

General comment:

Kalawila et al. present a very interesting article about the genetic variability of the species Phlebotomus argentipes collected in four areas of Sri Lanka. This species is of great relevance for its role in the transmission of visceral leishmaniasis. They analyzed three genes, two mitochondrial and one ribosomal, with which they observed the presence of two well-differentiated communities in two different clades. Additionally, they analyzed the population structure, finding the gene flow between the analyzed populations. They increase the number of genetic sequences for three genes, but mainly provide the first ITS2 gene sequences for the region and the species.

In general, I consider that it is an article that contains interesting findings, but I suggest some changes that I consider necessary to favor the content of the article. I also suggest that the article be reviewed by an English language consultant.

I wish you much success and I hope that my suggestions will be useful to you. Congratulations for your article.

Particular comments

Abstract

Line 30: Two mitochondrial genes viz. Please correct this sentence, I consider the word “viz.” it is misused.

Line 31: please delete oxidase, just leave Cytochrome b (CytB)

Line 30-31: parentheses are not italicized

Line 33: Change Network to network

Line 35: change flies to sand flies

Line 38-42: Please restructure the sentences, because it is a bit confusing.

Introduction

Line 51-54: I suggest change the paragraph this way:

Phlebotomus argentipes (Diptera: Psycodidae) is the known vector of Leishmania donovani, the causal agent of the clinical form VL which is considered the second parasitic disease cause of a high number of deaths, in India, Nepal and Bangladesh [4].

Line 70: change weren´t to were not

Line 78, 83, 103: delete this space

Line 86: Colombia is not an Old world country, please make the correction. Additionally there are more barcode studies for species from the new world, such as USA, Mexico, Brazil, please keep it in mind

Line 99: change phylogeograpy to phylogeography

Line 61-77: Although the information is relevant is very long, please be more specific.

Line 93-102: I suggest that only provide the information regarding the relevance of the use of the cox1, cytb and ITS2 genes for the genetic characterization of Phlebotomus argentipes. And do not include information that is not relevant in your research. For example: Line 94-96:In South America the phylogenetic analysis of cox 1 sequences of Verrucarum species of New World sand flies of the genus Lutzomyia was investigated [20].Line:97-98: Mitochondrial introgression in the Lutzomyia townsendi in Colombia was studied using cytb gene [12].

Line 48-107: In general, the introduction is very long, and contains information that is not very relevance. I kindly suggest that it be summarized a bit to make it more clear and precise.

Line106-107: “to study the effect of evolutionary forces that may spread resistance genes from one population to another”.

I have a question. The genes to be amplified are not necessarily genes that are related to insecticide resistance. How do you plan to evaluate and/or justify that the genetic variability that you may find are related to resistance to insecticides?

It is not very clear to me. Please in the part of your introduction, where you talk about insecticides could explain what genes are useful for do this objective.

Material and Method

Line 111-116: A total of 126 of adult sand flies were collected, this sentences is a result not is part of the method. Please move this information to the next section. Also specify how many days you collected in each of those locations.

Results

Line:189-190: A total of 126 of adult sand flies were collected and were identified as Phlebotomus argentipes [15, 25, 26].

Please, also specify how many specimens you collected in each one of the four locations that you studied. Why you did not collect other sand fly species and only obtain specimens of Phlebotomus argentipes, this is very common in other studies? There are not other sand fly species in your country? How many males and females did you collect? Did you analysis 126 specimens by PCR or how many?

Line193: how did you select those 10 specimens of the best quality?

Line 214, 217,305,306,308: Change flies or fly to sand flies or sand fly according to the case in the entire document.

Line 217: correct P. argentipes

Line 237: In the table, please include the number of specimens that you analyzed, for each gene and locations.

Discussion

Line 322: two clades

Line 325: network analysis

Line 326: P. argentipes were closely related to specimens of the same species collected in Sri Lanka, India and Israel.

Line 327: of three sand flies

Line 327-328: cytb is a marker that shows variability at the intraespecific level, different from cox1 that shows differences at the interespecific level. So, why is relevant that some specimens are similar to specimens from Madagascar? Explain more about it, although you have a small sample, it is interesting that you have haplotypes similar to a locality that is considerably distant from your collection point.

Line 329: did not

Line 330: Add a sentence highlighting that your work is one of the first sequences for this gene and for this species. I think it is very important that you highlight this contribution of your work.

Line 345: does not.

Line 352-353: put the name of the area of study not numbers, because you did not listed before.

Line 354-360: Although it is an interesting hypothesis, I think it is very risky to assert something of this style, given that the number of specimens that you analyzed is very small. I do not consider that ten specimens are enough to think that there is gene flow in all communities of sand flies and those possible resistance genes are being inherited. Additionally, you did not analyze genetic markers for resistance.

Line 363-373: This information is not informative for this article, I suggest that you focus on the group of sand flies and compare your results with those of other researchers. I suggest you read the article by:

1. Pech-May et al. 2016. Genetic structure and divergence in populations of Lutzomyia cruciata, a phlebotomine sand fly (Diptera: Psychodidae) vector of Leishmania mexicana in southeastern Mexico https://doi.org/10.1016/j.meegid.2013.02.004

2. Wolkoff, 2018. Population structure, demographic history, and environmental niche of the sand fly disease vector Lutzomyia shannoni (Dyar) (Diptera: Psychodidae) in the U.S., Mexico, and Colombia https://scholarworks.uttyler.edu/biology_grad/53/

Line 362-382: I suggest that you change these paragraphs as follows and fill them in with additional information, so that you can keep comparing your results.

The molecular markers have been extensively used for genotyping and study of evolutionary history of mosquitoes [30, 31]. The analysis using cytb gene has demonstrated been useful to detect the lack of population differentiation and the presence of gene flow in some species of dipterans in South-east Asia [32]. The gene flow between species of insect vectors may lead to alterations in the disease patterns [35]. Low genetic structure variation in some sand fly species as a result of geographical isolation and restricted gene flow may have led to reduced flight abilities and formation of cryptic species that ultimately influence the capacity to transmit parasites such as Leishmania in Latin America [36].

Line 384-391: again, I do not agree with these statements, I suggest that you approach it in another way for greater support, or that you only suggest it as a possible hypothesis in a more subtle way.

References

Please check the structure of the citations again, to make it homogeneous. Scientific and gene names are also missing in italics, and there are spelling mistakes (e.g. Line 4,5,12, 16, 17, 26, 27, 38, 39)

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2021 Sep 16;16(9):e0256819. doi: 10.1371/journal.pone.0256819.r002

Author response to Decision Letter 0

3 Jul 2021

Author responses to editor’s and reviewers comments:

Additional Editor’s Comments:

Q: it is pivotal to modify the ms. according to comments of both reviewers and focusing the population genetic analysis on microsatellite loci; otherwise, you have to limit the study only on the molecular typing of sand fly species examined.

R: The current study was designed to determine the population genetic structure of sand flies using two mitochondrial markers cox 1, cytb and a ribosomal region ITS2. However, no microsatellite loci were used for genotyping due their unavailability of such markers in P. argentipes. While we as a group, continue our efforts in developing such robust markers for P. argentipes, the authors believe the selected antigenic loci of P. argentipes are good enough for the said purpose due to the following reasons:

Previous studies have successfully used cox 1and cyb as the genetic marker in analysing the population genetic structure of insects including sand flies.

Ebrahimi, et al. Genetic dynamics in the sand fly (Diptera: Psychodidae) nuclear and mitochondrial genotypes: evidence for vector adaptation at the border of Iran with Iraq. Parasites Vectors 9, 319 (2016). https://doi.org/10.1186/s13071-016-1603-5.

Flanley, et al. Population genetics analysis of Phlebotomus papatasi sand flies from Egypt and Jordan based on mitochondrial cytochrome b haplotypes. Parasites Vectors. 2018; 11, 214, doi:10.1186/s13071-018-2785-9.

Guernaoui, et al. Population Genetics of Phlebotomus papatasi from Endemic and Nonendemic Areas for Zoonotic Cutaneous Leishmaniasis in Morocco, as Revealed by Cytochrome Oxidase Gene Subunit I Sequencing. Microorganisms. 2020; 8(7): 1010.

Arrivillaga et al. Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences. Infect Genet Evol. 2002; 2: 83-95.

Latrofaa et al. Multilocus molecular and phylogenetic analysis of phlebotomine sand flies ( Diptera : Psychodidae ) from southern Italy. Acta trop. 2011;119:91–8. doi:10.1016/j.actatropica.2011.04.013.

Cohnstaedt et al. Phylogenetics of the phlebotomine sand fly group Verrucarum (Diptera: Psychodidae: Lutzomyia). Am J Trop Med Hyg. 2011;84(6):913–22.

Weeraratne et al. Genetic diversity and population structure of malaria vector mosquitoes Anopheles subpictus, Anopheles peditaeniatus, and Anopheles vagus in five districts of Sri Lanka. Malar. J. 2018; 17: 271. https://doi.org/10.1186/s12936-018-2419-x.

Gene flow also successfully determined using these markers in following studies.

Marcondes et al. Introgression between Lutzomyia intermedia and both Lu. neivai and Lu. whitmani, and their roles as vectors of Leishmania braziliensis. Trans R Soc Trop Med Hyg. 1997; 91(6): 725-6.

Belen et al. Genetic structures of sand fly (Diptera: Psychodidae) populations in a leishmaniasis endemic region of Turkey. Journal of vector biology. 2011; 36 (Supplement 1): 32-48. https://doi.org/10.1111/j.1948-7134.2011.00110.x.

Studies appear below have proven that ITS regions could successfully determine the population genetic structure of many organisms including parasites and mosquitoes. Further, Manni et al (2015) have used microsatellites and ITS2 as the genetic markers and have proven that both markers are equally successful in determining the population genetic structure.

Manni, M., Gomulski, L.M., Aketarawong, N. et al. Molecular markers for analyses of intraspecific genetic diversity in the Asian Tiger mosquito, Aedes albopictus . Parasites Vectors 8, 188 (2015). https://doi.org/10.1186/s13071-015-0794-5

Villalobos, G., Orozco-Mosqueda, G.E., Lopez-Perez, M. et al. Suitability of internal transcribed spacers (ITS) as markers for the population genetic structure of Blastocystis spp. Parasites Vectors 7, 461 (2014). https://doi.org/10.1186/s13071-014-0461-2.

Latrofaa MS, Dantas-Torresa F, Weigla S, Taralloa VD, Parisib A, Traversac D, et al. Multilocus molecular and phylogenetic analysis of phlebotomine sand flies ( Diptera : Psychodidae ) from southern Italy. Acta trop. 2011;119:91–8. doi:10.1016/j.actatropica.2011.04.013.

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Reviewer Comments:

Reviewer 1:

Q1: Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Partly

R1: A minimum of 10 sequences of each marker from each study site was used in the analysis. Previous published work on population genetic structure analysis have used sample numbers between 6-10 from a single study location and have obtained successful results. I suggest adding one statement to the discussion in line 342-343 and changing the following sentence in the conclusion in line 370-373, 375-377–

‘Gene flow was evident with lack of population differentiation even between geographically distant populations of P. argentipes perhaps due to absence of geographic barriers and the continuity of habitats.’

‘Results also validated the use of cox 1, cytb genes and ITS2 region as a tool in understanding the population genetic structure of Phlebotominae sand flies in Sri Lanka.’

As,

Both cox 1 and ITS2 indicated a gene flow with lack of population differentiation even between geographically distant populations of P. argentipes perhaps due to absence of geographic barriers and the continuity of habitats.

Results showed the possible use of cox 1, cytb genes and ITS2 region as markers in determining the population genetic structure of Phlebotominae sand flies in Sri Lanka.

Q2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

R2: Standard statistical software and analysis were used in the study by referring to previous published work.

Q3: The markers used in this paper are not suitable for assessing population genetic structure and gene flow among populations. At this aim, microsatellite or SNPs markers should be used. Consequently the inferred conclusions are not supported by genetic data.

R3: The current study was designed to determine the population genetic structure of sand flies using two mitochondrial markers cox 1, cytb and a ribosomal region ITS2. However, no microsatellite loci were used for genotyping due their unavailability of such markers in P. argentipes. While we as a group, continue our efforts in developing such robust markers for P. argentipes, the authors believe the selected antigenic loci of P. argentipes are good enough for the said purpose due to the following reasons:

Most of the studies have been conducted to investigate population genetic analysis using these markers (Ebrahimi et al. 2016, Flanley et al, 2018, Guernaoui et al. 2020, Arrivillaga et al. 2002, Cohnstaedt et al. 2011, Testa et al. 2002, Latrofaa et al. 2011, Weeraratne et al. 2018).

Gene flow also successfully determined using these markers in following studies.

(Marcondes et al. 1997, Belen et al. 2011).

Further, several studies have conducted using one or two of the markers used in the present study and microsattelites and have proved that both could be used sucessfully in population genetic structure analysis (Manni et al. 2015, Villalobos et al. 2014).

we suggest changing the following sentence in the conclusion in line 370-373, 375-377–

‘Results also validated the use of cox 1, cytb genes and ITS2 region as a tool in understanding the population genetic structure of Phlebotominae sand flies in Sri Lanka.’

As,

Results showed the possible use of cox 1, cytb genes and ITS2 region as markers in determining the population genetic structure of Phlebotominae sand flies in Sri Lanka.

Reviewer 2:

Q1: Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Partly

R1: A minimum of 10 sequences of each marker from each study site was used in the analysis. Previous published work on population genetic structure analysis have used sample numbers between 6-10 from a single study location and have obtained successful results. I suggest changing the following sentence in the discussion and conclusion in line 342-343, 370-373, 375-377–

‘Results also validated the use of cox 1, cytb genes and ITS2 region as a tool in understanding the population genetic structure of Phlebotominae sand flies in Sri Lanka.’

As,

Results showed the possible use of cox 1, cytb genes and ITS2 region as markers in determining the population genetic structure of Phlebotominae sand flies in Sri Lanka.

Q2: Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

R2: The typographical and grammatical errors suggested by the reviewers were corrected and changes are highlighted in the manuscript.

Abstract

Q3: Line 30: Two mitochondrial genes viz. Please correct this sentence, I consider the word “viz.” it is misused.

R3: Agreed and changed the sentence in line 30.

Q4: Line 31: please delete oxidase, just leave Cytochrome b (CytB)

R4: Agreed and deleted it in line 31.

Q5: Line 30-31: parentheses are not italicized

R5: Agreed and done.

Q6: Line 33: Change Network to network

R6: Done it in line 33.

Q7: Line 35: change flies to sand flies

R7: Done it in line 35.

Q8: Line 38-42: Please restructure the sentences, because it is a bit confusing.

R8: Changed the sentence in line 38-42.

Introduction

Q9: Line 51-54: I suggest change the paragraph this way:

R9: Agreed and done the changes to line 50-52.

Q10: Line 70: change weren´t to were not

R10: Agreed and done in line 65.

Q11: Line 78, 83, 103: delete this space

R11: Deleted spaces.

Q12: Line 86: Colombia is not an Old world country, please make the correction. Additionally there are more barcode studies for species from the new world, such as USA, Mexico, Brazil, please keep it in mind.

Q12: Done the changes to the sentence in line 75-77.

Q13: Line 99: change phylogeograpy to phylogeography

R13: Changed as ‘phylogeography’ in line 85.

Q14: Line 61-77: Although the information is relevant is very long, please be more specific.

R14: Agreed and done in line 59-70.

Q15: Line 93-102: I suggest that only provide the information regarding the relevance of the use of the cox1, cytb and ITS2 genes for the genetic characterization of Phlebotomus argentipes. And do not include information that is not relevant in your research. For example: Line 94-96:In South America the phylogenetic analysis of cox 1 sequences of Verrucarum species of New World sand flies of the genus Lutzomyia was investigated [20].Line:97-98: Mitochondrial introgression in the Lutzomyia townsendi in Colombia was studied using cytb gene [12].

R15: Due to restricted studies in P. argentipes corrected statement in line 83-85.

Q16: Line 48-107: In general, the introduction is very long, and contains information that is not very relevance. I kindly suggest that it be summarized a bit to make it more clear and precise.

R16: The changes were made according to the comments suggested by the reviewers to the introduction to be summarized.

Q17: Line106-107: “to study the effect of evolutionary forces that may spread resistance genes from one population to another”.

R17: There is no link between the genes used in the study and the insecticide resistance genes. This statement was made as there is a high chance of entering such genes from one populations to another population in the absence of population genetic structure variation.

Q18: It is not very clear to me. Please in the part of your introduction, where you talk about insecticides could explain what genes are useful for do this objective.

R18: Explained in line 67-69.

Material and Method

Q19: Line 111-116: A total of 126 of adult sand flies were collected, this sentences is a result not is part of the method. Please move this information to the next section. Also specify how many days you collected in each of those locations.

R19: Moved it to the result section and added information to the material and method in line 92-97.

Results

Q20: Line:189-190: A total of 126 of adult sand flies were collected and were identified as Phlebotomus argentipes [15, 25, 26].

R20: Added it to line 171.

Q21: Please, also specify how many specimens you collected in each one of the four locations that you studied. Why you did not collect other sand fly species and only obtain specimens of Phlebotomus argentipes, this is very common in other studies? There are not other sand fly species in your country? How many males and females did you collect? Did you analysis 126 specimens by PCR or how many?

R21: About 42 specimens were collected in each one of the four locations with a total of 52 females and 74 males. There are Sergentomyia spp. in the country. However, trapping method was basically effective for cattle-baited net trap. Thus, Phlebotomus argetipes were found apart from one or two Sergentomyia spp. specimens collected from light traps in some days, not routinely. Due to absence of sufficient numbers of samples, Sergentomyia spp. was not included in the study. Due to the study were based on the vector species, Phlebotomus argentipes, a total of 126 species were analysed by PCR followed by the analysis and interpretation were based on vector control strategies.

Q22: Line193: how did you select those 10 specimens of the best quality?

R22: The chromatograms of each and every sequence was checked manually and only the good quality sequences were used in the study.

Q23: Line 214, 217,305,306,308: Change flies or fly to sand flies or sand fly according to the : case in the entire document.

R23: Agreed and corrected.

Q24: Line 217: correct P. argentipes

R24: Corrected it line 199.

Q25: Line 237: In the table, please include the number of specimens that you analyzed, for each gene and locations.

R25: Added to the Table 1.

Discussion

Q26: Line 322: two clades

R26: Agreed and done it in line 302.

Q27: Line 325: network analysis

R27: Done it in line 305.

Q28: Line 326: P. argentipes were closely related to specimens of the same species collected in Sri Lanka, India and Israel.

R28: Corrected it in line 306-307.

Q29: Line 327: of three sand flies

R29: Corrected it in line 307.

Q30: Line 327-328: cytb is a marker that shows variability at the intraespecific level, different from cox1 that shows differences at the interespecific level. So, why is relevant that some specimens are similar to specimens from Madagascar? Explain more about it, although you have a small sample, it is interesting that you have haplotypes similar to a locality that is considerably distant from your collection point.

R30: The larger sample will be used for further studies to confirm this specimens exactly from Madagascar or not. If so, there is a genetic exchange may apply in between those specimens.

Q31: Line 329: did not

R31: Corrected it in line 309.

Q32: Line 330: Add a sentence highlighting that your work is one of the first sequences for this gene and for this species. I think it is very important that you highlight this contribution of your work.

R32: Done it in line 311-312.

Q33: Line 345: does not.

R33: Corrected it in line 327.

Q34: Line 352-353: put the name of the area of study not numbers, because you did not listed before.

R34: Agreed it and corrected in line 334-335.

Q35: Line 354-360: Although it is an interesting hypothesis, I think it is very risky to assert something of this style, given that the number of specimens that you analyzed is very small. I do not consider that ten specimens are enough to think that there is gene flow in all communities of sand flies and those possible resistance genes are being inherited. Additionally, you did not analyze genetic markers for resistance.

R35: Previously published work on population genetic structure analysis have used sample numbers between 6-10 from a single study location and have obtained successful results. This study used a minimum of 10 sequences from each marker from each locality. However, as the sample size is not very high it was stated as “possible” gene flow.

Insecticide resistance genes was taken as an example gene that could be introduced into other populations due to gene flow.

The sentence, “The possible gene flow revealed in the current study demonstrate the risk of dispersal of such resistance genes between populations with the potential for the spread of insecticide resistance and implications for future vector control” could be changed as “The possible gene flow revealed in the current study demonstrate the risk of dispersal of genes such as insecticide resistance genes between populations with the potential for the spread of insecticide resistance and implications for future vector control” in line 339-342 in the discussion.

Q36: Line 363-373: This information is not informative for this article, I suggest that you focus on the group of sand flies and compare your results with those of other researchers. I suggest you read the article by:

R36: Added information to line 351-358.

Q37: Line 362-382: I suggest that you change these paragraphs as follows and fill them in with additional information, so that you can keep comparing your results.

R37: Added information to line 344-351.

Q38: Line 384-391: again, I do not agree with these statements, I suggest that you approach it in another way for greater support, or that you only suggest it as a possible hypothesis in a more subtle way.

R38: Corrected it in line 360-366.

References

Q39: Please check the structure of the citations again, to make it homogeneous. Scientific and gene names are also missing in italics, and there are spelling mistakes (e.g. Line 4,5,12, 16, 17, 26, 27, 38, 39)

R39: Agreed and done it.

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PLoS One. doi: 10.1371/journal.pone.0256819.r003

Decision Letter 1

Maria Stefania Latrofa

4 Aug 2021

PONE-D-21-16188R1

Genetic diversity and population structure of Phlebotomus argentipes: vector of leishmaniasis in Sri Lanka.

PLOS ONE

Dear Dr. Karunaweera,

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PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

I agree with the authors on that the target genes analyzed are good for the specimen’s identification and for the analyses of the genetic divergence within and among phlebotomine sand fly populations, but not on the gene flow. Indeed, Weeraratne and colleagues only hypothesized the presence of the gene flow using the cox1, whilst Manni et al., define that SSR markers are, as expected, more informative than ITS2 in revealing the slight genetic diversity between native and derived populations both in terms of variability and differentiation. The authors have to be more cautious when talk on the gene flow. I suggest, for example, putting your considerations as a hypothesis of the “existence of a potential gene flow” based on the results obtained of the genes analyzed, and that the gene flow have to be further investigated or supported by the analysis of microsatellites.

See also the article Prudhomme et al., “Altitude and hillside orientation shapes the population structure of the Leishmania infantum vector Phlebotomus ariasi.” Scientific Reports, 2020 DOI: 10.1038/s41598-020-71319-w.

Change the title: since sand flies are vector of pathogen and not of diseases

Lines 36-37: I suggest changing “revealed gene flow” into “may indicate a potential gene flow”, due to the motivation above, and throughout the ms.

PLoS One. 2021 Sep 16;16(9):e0256819. doi: 10.1371/journal.pone.0256819.r004

Author response to Decision Letter 1

16 Aug 2021

Author responses to journal requirements and additional editor’s comments:

Journal Requirements:

Q: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

R: Reviewed the reference list and it was completed and corrected. Following changes were made in the reference list.

• Journal name abbreviation was corrected in line 428, 443.

• Page numbers were added in line 467.

• Journal name and page numbers were added in line 473.

• Journal name, issue, page numbers and DOI number were added in line 475-476.

Following reference was removed from the reference list as it contains unpublished work. Thus, the statement in line 353-358 referred to as “unpublished work” in line 358.

Wolkoff. Population structure, demographic history, and environmental niche of the sand fly disease vector Lutzomyia shannoni (Dyar) (Diptera: Psychodidae) in the U.S., Mexico, and Colombia. University of Texas at Tyler; 2018.

Additional Editor’s Comments:

Q1: I agree with the authors on that the target genes analyzed are good for the specimen’s identification and for the analyses of the genetic divergence within and among phlebotomine sand fly populations, but not on the gene flow. Indeed, Weeraratne and colleagues only hypothesized the presence of the gene flow using the cox1, whilst Manni et al., define that SSR markers are, as expected, more informative than ITS2 in revealing the slight genetic diversity between native and derived populations both in terms of variability and differentiation. The authors have to be more cautious when talk on the gene flow. I suggest, for example, putting your considerations as a hypothesis of the “existence of a potential gene flow” based on the results obtained of the genes analyzed, and that the gene flow have to be further investigated or supported by the analysis of microsatellites.

R1: Authors agreed and suggested changes have been added in the abstract section in line 32-42 and in the conclusion section in line 369-373 and 374-385.

“Therefore, there is a high risk of transferring genes of functional significance, such as those that confer insecticide resistance from one population to another with the risk of eventually spreading across the country. However, confirmation of such an assumption would require further investigations and supportive evidence probably with the use of more neutral genotyping markers such as microsatellites, which have been used in other Phlebotomus sp. for similar investigations including for those related to the influence of geographic distance and altitude (Prudhomme et al., 2020). The study also confirms the possible use of cox 1, cytb genes and ITS2 region as genotyping markers for the determination of the population genetic structure of Phlebotominae sand flies in Sri Lanka. Thus, the findings will pave the way for more extensive investigations, probably with the use of more genotyping markers, to aid a vector control strategy that may need to be adopted within a future national leishmaniasis control programme.”

Q2: Change the title: since sand flies are vector of pathogen and not of diseases

R2: The title was changed into ‘Genetic diversity and population structure of Phlebotomus argentipes: vector of Leishmania donovani in Sri Lanka.’

Q3. Lines 36-37: I suggest changing “revealed gene flow” into “may indicate a potential gene flow”, due to the motivation above, and throughout the ms.

R3: Changed as ‘may indicate a possible gene flow’ in line 37, 39-40, 333-334, 337, 339-343, 363-364, 372.

Authors suggested to acknowledge funding source in the Acknowledgements section in line 391-394.

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PLoS One. doi: 10.1371/journal.pone.0256819.r005

Decision Letter 2

Maria Stefania Latrofa

17 Aug 2021

Genetic diversity and population structure of Phlebotomus argentipes: vector of Leishmania donovani in Sri Lanka.

PONE-D-21-16188R2

Dear Dr. Karunaweera,

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PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0256819.r006

Acceptance letter

Maria Stefania Latrofa

3 Sep 2021

PONE-D-21-16188R2

Genetic diversity and population structure of Phlebotomus argentipes: vector of Leishmania donovani in Sri Lanka.

Dear Dr. Karunaweera:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Nucleotide identities for cox 1 gene.

Nucleotide identities obtained through NCBI blast results for cox 1 gene.

(PDF)

Click here for additional data file.^{(305.3KB, pdf)}

S2 Table. Nucleotide identities for cytb gene.

Nucleotide identities obtained through NCBI blast results for cytb gene.

(PDF)

Click here for additional data file.^{(307.2KB, pdf)}

S3 Table. Nucleotide identities for ITS2 region.

Nucleotide identities obtained through NCBI blast results for ITS2 region.

(PDF)

Click here for additional data file.^{(306.2KB, pdf)}

S4 Table. Analysis of Molecular Variance (AMOVA) of cox 1, cytb genes and ITS2 region.

Analysis of Molecular Variance (AMOVA) of cox 1, cytb genes and ITS2 region using Arlequin software (version 3.11) (cmpg.unibe.ch/software/arlequin3/).

(PDF)

Click here for additional data file.^{(100.9KB, pdf)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(37KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(18.9KB, docx)}

Data Availability Statement

All relevant relevant data have been uploaded to GenBank: cox 1- MW256437-MW256478 cytb- MW571044- MW571085 ITS2- MW322926-MW322967.

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Genetic diversity and population structure of Phlebotomus argentipes: Vector of Leishmania donovani in Sri Lanka

Dulani Ruwanika K Pathirage

Thilini C Weeraratne

Sanath C Senanayake

S H P Parakrama Karunaratne

Nadira D Karunaweera

Roles

Abstract

Introduction

Materials and methods

Collection of sand flies

Fig 1. Map of Sri Lanka.

Morphological identification

PCR amplification and sequencing of mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA)

Sequence analysis

Results

Morphological identification

Phylogenetic analysis

Fig 2. Evolutionary analysis.

DNA sequence polymorphism

Table 1. Genetic diversity indices, neutrality test values for Cox 1, Cytb and ITS2 regions of P. argentipes in each study site (n≤ 10 from each study site).

Pairwise FST values and population structure

Table 2. Pairwise FST values and P values obtained for Cox 1, Cytb and ITS2 regions of P. argentipes collected in 4 different localities.

The effects of evolutionary forces on population

Table 3. FST values obtained for AMOVA analysis of Cox 1, Cytb and ITS2 regions of P. argentipes collected in each study site.

Haplotypes interrelationship

Fig 3. Haplotypes interrelationship.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Maria Stefania Latrofa

Roles

Author response to Decision Letter 0

Decision Letter 1

Maria Stefania Latrofa

Roles

Author response to Decision Letter 1

Decision Letter 2

Maria Stefania Latrofa

Roles

Acceptance letter

Maria Stefania Latrofa

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Pairwise F_ST values and population structure

Table 2. Pairwise F_ST values and P values obtained for Cox 1, Cytb and ITS2 regions of P. argentipes collected in 4 different localities.

Table 3. F_ST values obtained for AMOVA analysis of Cox 1, Cytb and ITS2 regions of P. argentipes collected in each study site.