The new Internal Transcribed Spacer 2 diagnostic tool clarifies the taxonomic position and geographic distribution of the North American malaria vector Anopheles punctipennis

The malaria mosquito Anopheles punctipennis, a widely distributed species in North America, is capable of transmitting human malaria and is actively involved in the transmission of the ungulate malaria parasite Plasmodium odocoilei. However, molecular diagnostic tools based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA are lacking for this species. Anopheles punctipennis is a former member of the Anopheles maculipennis complex but its systematic position remains unclear. In this study, ITS2 sequences were obtained from 276 An. punctipennis specimens collected in the eastern and midwestern United States and a simple and robust Restriction Fragment Length Polymorphism approach for species identification was developed. The maximum-likelihood phylogenetic tree was constructed based on ITS2 sequences available through this study and from GenBank for 20 species of Anopheles. The analysis demonstrated a consistent ITS2 sequence length and showed no indications of intragenomic variation among the samples based on ITS2, suggesting that An. punctipennis represents a single species in the studied geographic locations. In this study, An. punctipennis was found in urban, rural, and forest settings, suggesting its potential broad role in pathogen transmission. Phylogeny based on ITS2 sequence comparison demonstrated the close relationship of this species with other members of the Maculipennis group. This study developed molecular tools based on ITS2 sequences for the malaria vector An. punctipennis and clarified the phylogenetic position of the species within the Maculipennis group.

been a significant problem in North America where it was introduced in the seventeenth century during the slave trade and, because of the presence of native competent malaria vectors, it rapidly spread through the entire continent [3]. In the USA, numerous outbreaks of malaria occurred from as far north as Massachusetts to subtropical southern states in the eighteenth and nineteenth centuries. Starting in the 1930s, intensive efforts to control malaria were undertaken and, in 1954, malaria was declared eliminated from the USA [3]. Nevertheless, each year, the Center for Disease Control and Prevention (CDC) reports between 1,000 and 1,500 cases of malaria in the USA [4], with 63 outbreaks of locally transmitted malaria occurring since 1957 [5]. Climate change, human migration, political instability, and the presence of competent malaria vectors increase the risk of malaria and other tropical diseases being imported and transmitted in regions where they were eradicated [5].
Three species are considered dominant malaria vectors in North America-Anopheles freeborni, Anopheles pseudopunctipennis, and Anopheles quadrimaculatus [6]. In addition to these species, Anopheles punctipennis, the most common and widely distributed anopheline mosquito species in North America [7], was an important contributor to malaria transmission in the USA in the past [8]. Similar to An. quadrimaculatus, this species is capable of transmitting two major human malaria parasites, Plasmodium vivax and Plasmodium falciparum [9]. Moreover, An. punctipennis can also transmit Plasmodium odocoilei, which infects wild hooved animals, such as deer. A recent study conducted in 17 states across the USA estimated a 25% P. odocoilei infection rate in whitetailed deer [10]. Malaria in wildlife populations may also have an impact on biodiversity, as infection reduces the deer population by killing fawns [11]. This can also present problems for humans due to the possibility of zoonotic transmission to livestock ungulates vectored from wild ungulates [11]. The ability of An. punctipennis to transmit arboviruses, such as Cache Valley [12] and eastern equine encephalomyelitis [13] viruses, as well as a nematode Dirofilaria immitis [14] has also been reported.
Continuous study of mosquitoes in the field requires rapid and robust identification of the species. The availability of PCR-based molecular technologies enable the use of ribosomal DNA (rDNA) for species diagnostics [15]. rDNA genes are organized in arrays of multiple copies in eukaryotic genomes and undergo "concerted evolution" which leads to unification of gene and intergenic sequences [16]. Unlike Intragenic Spacer and Intergenic Transcribed Spacer 1, Intergenic Transcribed Spacer 2 (ITS2) is a reliable tool for species diagnostics in the Anopheles genus [15]. ITS2 sequences have been utilized for species diagnostics in the Anopheles gambiae complex [17], the Anopheles funestus group [18], and the Anopheles bancroftii group [19]. One nucleotide difference in ITS2 led to the discovery of the M and S forms of An. gambiae, which have now been elevated to species status as An. gambiae (former S form) and Anopheles coluzzii (former M form) [20]. Three new Eurasian species-Anopheles artemievi [21], Anopheles persiensis [22], and Anopheles daciae [23]-were described based on differences in their ITS2 sequences. ITS2 sequences have been successfully used for the identification of North American mosquitoes, including a newly identified species, Anopheles hermsi [24], and four new species in the Quadrimaculatus complex [25]. More recently ITS2based diagnostic tools were developed for species from the Anopheles crucians complex [26]. Despite the wide distribution in North America and its obvious role in pathogen transmission, a molecular diagnostic tool using ITS2 of the rDNA from An. punctipennis has not been developed yet.
In the past, An. punctipennis was a member of the former Anopheles maculipennis complex, which included 7 Eurasian and 6 North American species [27]. Although, considered distance by the classical morphological approach, An. punctipennis was still placed within the An. maculipennis complex based on interspecies hybridization with other members of the complex and the structure of its polytene chromosomes [28]. In 2004, Harbach proposed a new classification for the genus Anopheles that was largely based on morphological characteristics and ITS2 sequence analyses [29]. According to this classification, the An. maculipennis complex was transformed to the Maculipennis group, which was subdivided into two North American subgroups, Freeborni and Quadrimaculatus, and one Eurasian Maculipennis subgroup. Anopheles punctipennis was excluded from the Maculipennis group and placed into a separate Punctipennis group that included An. punctipennis, Anopheles perplexens, and the An. crucians complex.
In this study, malaria mosquitoes were collected from 10 sites in eastern (Virginia and Florida) and midwestern states (Iowa and Minnesota) and 2 assays based on the sequencing of ITS2 of the rDNA and Restriction Fragment Length Polymorphism (RFLP) of ITS2 for molecular diagnostics of An. punctipennis were developed. In addition, ITS2 sequences were used to clarify the phylogenetic relationships of An. punctipennis.

Mosquito collection
A collection of approximately 500 Anopheles mosquitoes were sampled from multiple locations across four states during the summers of 2017 and 2018 as shown in Table 1. The collections from Iowa were provided by the colleagues from Iowa State University. Geographical positions of the mosquito collection sites are shown on a map using the program ArcGis Pro [30] in Fig. 1. Mosquitoes were collected from larval breeding sites, typically small ponds or bodies of water. These breeding sites were located near aquatic plants known as the filamentous algae. More specifically the plants among these breeding sites were identified as Cladophora sp., Spirogyra sp., and Pithophora sp. The pan dipping method was used to collect samples of Anopheles in their natural habitat. A pan was dipped under the water so that the surface water spilled into the pan. Once in the pan, a disposable pipette was implemented to draw the larvae from the water, which was then placed into a collection container. 50 ml centrifuge tubes were used for temporary storage while in the field. After that, the larvae were fixed in 70% ethanol. The mosquito larvae were individually labelled for identification and placed in 1.5 ml tubes and stored at − 20 °C.

DNA extraction
DNA extractions were conducted from mosquito larvae, except at the location in Iowa, where DNA were extracted from adults. DNA extractions were attempted for ~ 500 specimens. Each individual carcass was placed in a sterile 1.5 ml tube to prevent contamination. Extraction of genomic DNA from each specimen was conducted using the standard protocol for Qiagen DNeasy Blood and Tissue Kits (Qiagen, Germantown, MD, USA) with some modification. DNA elution was performed with 50 μl of molecular grade water (Research Products International, Mt. Prospect, IL, USA). Purified DNA was checked using a Nanodrop spectrometer (Thermo Fisher Scientific, Haverhill, MA, USA) to determine quantity and quality and then stored at − 20 °C.

DNA amplification
For rDNA ITS2 amplification, the direct primer from Djadid et al. [31] with some modification 5′-ATC ACT CGG CTC TCG TGG ATCG-3′ and the reverse primer design by Proft et al. 5′-ATG CTT AAA TTT AGG GGG TA-3′ were used [32]. For the PCR reaction, Hot Start ImmoMix ™ (Bioline, Taunton, MA, USA) was used. PCR reaction mixtures were conducted using 3 μl of genomic DNA, 15 μl 2 × ImmoMix, 1 μl of both forward and reverse primers at 10 µM concentration, and 10 μl of molecular grade water. PCR amplification was conducted using a thermal cycler (Eppendorf, Hauppauge, NY, USA) with the following parameters: 95 °C for 10 min, followed by 30 cycles of 94 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s, and the final step at 72 °C for 5 min.
The mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified using the following primers that were designed using Primer 3 software: Forward 5′-TGG AGC CTT TAT TGG AGA CG-3′ and Reverse 5′-ATA GTA GAA AAT GGG GCC GG-3′. PCR amplification was conducted with the following parameters for COI: 95 °C for 10 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final step at 72 °C for 7 min. PCR products were analysed by electrophoresis in 1% agarose gel. GelRed ® Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA) was used to visualize the bands. For DNA sequencing, amplicons were purified with a Wizard ™ PCR Clean Up kit (Promega, Fitchburg, WI, USA). The product was stored at − 20 °C until processed for sequencing.

DNA sequencing and species diagnostics
10 µl of PCR products were mixed with 3 μl forward or reverse primers at 3.2 μM concentration and followed be a standard Sanger sequencing procedure [33]. The raw sequences obtained from local collections were uploaded to Sequencher 5.3 software (Gene Codes Corp. Ann Arbor, MI) for further analysis. Species were identified by comparing the obtained sequences with sequences available in GenBank using NCBI BLAST [34]. Because ITS2 sequences were unavailable for An. punctipennis at the time when the research was conducted this species was identified using additional sequencing of the COI gene; samples were identified as An. punctipennis if they had ≥ 99% identity with An. punctipennis COI sequences from GenBank [30]. This comparison allowed to match the ITS2 sequence with the COI sequence from the same individual. In addition, samples from Iowa were analysed morphologically as An. punctipennis at the adult stage [35].
Sequences were analysed for quality and for 114 specimens sequenced from both ends the forward and reverse sequences were assembled to obtain the complete sequences of ITS2. Resulting ITS2 nucleotide sequence of An. punctipennis (MW581372-MW581485) and a consensus sequence (LR877261) were submitted to the Gen-Bank [30].

DNA restriction
To obtain DNA fragments with different lengths for An. punctipennis, 4 μl of the ITS2 PCR product, 2 μl of the 10 × CutSmart ® Buffer, 5 units (0.5 µl) of NaeI restriction enzyme (New England BioLabs, Inc., Ipswich, MA, USA), and 18 μl of ddH 2 O were added to a sterile PCR tube. The tubes were then placed in a thermocycler at 37 °C for 60 min. PCR products were analysed by electrophoresis in 3% agarose gel. GelRed ® Nucleic Acid Gel Stain was used to visualize the bands.

Phylogenetic analysis
ITS2 sequences representing 20 Anopheline species were downloaded from the GenBank [34]. The sequences were aligned using the webPRANK server [36] and then the model of nucleotide substitutions was estimated using jModelTest 2 software [37] and Bayesian information criterion. The maximum-likelihood tree was constructed using MEGA X [38] software with the HKY-G model and tested using 500 bootstrap-replications. All sites with gaps or missing data were excluded from the alignment during tree construction.

Internal Transcribed Spacer 2 sequencing
The primary goal of this study was to develop a molecular tool based on ITS2 for the identification of An. punctipennis. To accomplish this goal, mosquitoes were collected from multiple locations in several eastern and midwestern states in the USA (  (Fig. 2). Based on the restriction enzyme map of ITS2 sequences, a NaeI restriction enzyme was chosen that cleaves the PCR product of An. punctipennis, resulting in the appearance of two fragments of 168 bp and 283 bp. There are no sites for this restriction enzyme in the ITS2 sequences in any of the other species that were found together in all mosquito collections (Table 1). This approach was tested on 50 samples from the various sample locations: 9 samples each from the Defuniak Springs location in Florida and in the Des Moines location in Iowa, and 3 samples from the Minnetrista location in Minnesota. The overall analysis of the 50 samples of An. punctipennis evaluated by the RFLP-ITS2 assay indicated that 100% of the samples displayed a double band. The assays with all the collected anopheline species determined that An. punctipennis was the only species that displays a double band result, indicating high reliability of this approach the identification of An. punctipennis (Fig. 2). In addition to the species collected in this study, the ITS2 sequences from the other 704 species from genus Anopheles, were also analysed using data from GenBank [34]. Of 55 species with NaeI site, only six species were from North America: Anopheles aquasalis, An. crucians B, Anopheles darlingi, Anopheles neivai, Anopheles punctimacula, and An. pseudopunctipennis. However, most of these species can be easily distinguished from An. punctipennis by the size of the PCR products and restriction fragment patterns with the only exception of An. punctimacula (Table 2). However, this species has no geographic overlap with An. punctipennis in the USA. Thus, NaeI-based RFLP approach should be applicable for the identification of An. punctipennis in different populations throughout the USA.

Anopheles species distribution in the eastern and midwestern USA
In addition to the development of a tool for molecular diagnosis of An. punctipennis, the species composition was analysed in all locations from the eastern and midwestern United States (Table 1, Fig. 1). In the Midwest, An. punctipennis was found in Minnetrista, Minnesota where ~ 8% of the specimens were represented by An. punctipennis and nearly 92% were An. quadrimaculatus. This location is indicated on the map as KW (Fig. 1a). Additional samples from the Midwest were collected in Iowa near Des Moines (IO). In contrast, (See figure on next page.) Fig. 1 Species composition in mosquito collections in eastern and midwestern United States. Species distribution [35] is shown on the map by different shades (a). The map was developed using the program ArcGis Pro [30]. Species abundance is shown by different colors in pie charts. Details of the site locations and species compositions in Virginia and Florida are shown in panels B and C, respectively. Another collection set was made in the Appalachian Mountains in Virginia, with elevations ranging from 600 m to 1.2 km (Fig. 1b). Collections in Virginia were made in 3 different environments: town, village, and forest. The town collection was made at the Duck Pond (DP) on the Virginia Tech campus, which is in the center of a small college town of Blacksburg, Virginia. In this setting, 94% of the mosquitoes were An. punctipennis with a high population density. The village collection was made in a rural setting in nearby Pandapas Pond (P, PP) in Montgomery County, Virginia. This location contained farms and wildlife. The proportion of An. punctipennis in this location reached 65% of the samples collected. The rest was represented by 22% of An. quadrimaculatus, 3.7% of An. crucians C, and 5.6% of each of An. crucians D and An. crucians E. The third collection was made in a forest location at the high elevation of 1.2 km in Giles County, Virginia near Mountain Lake (MM). In this location, 100% of the mosquitoes were An. punctipennis, again with a higher population density than in rural location.
The next set of collections were made in Florida along the coast. Mosquitoes were collected in Western Florida near Panama City Beach and in Eastern Florida near Orlando in urban, rural, and forest locations. An. punctipennis was found in only one urban location in Defuniak Springs, Florida in Bruce Creak (BC). The collection consisted of approximately 73% of An. punctipennis, 23% of An. crucians A, 2% of An. crucians C, and 2% of An. crucians E. In the coastal areas, the prevalent mosquitoes were from the An. crucians complex. In the mineral springs in Panacea, Florida (PF) the sample collection included 92% Anopheles bradleyi and 8% An. quadrimaculatus. In the more urban setting along the coast, Carrebelle, Florida (CF), approximately 39% An. crucians C and approximately 61% An. crucians D were found. Finally, in the remote forest settings of Apalachicola National Forest in Apalachicola, Florida (SR), 100% of the collection was An. crucians D.

Phylogenetic analysis based on Internal Transcribed Spacer 2
Phylogenetic analyses based on ITS2 sequences using the maximum-likelihood approach [38] allowed us to construct a reliable tree of the major phylogenetic subgroups of the studied species (Fig. 3). The analysis included 20 species: An.punctipennis; 8 North American species from the Maculipennis group (Anophelesfreeborni, Anopheles hermsi, Anopheles occidentalis, and 5 species from An.quadrimaculatus complex); 5 Eurasian species (Anopheles beklemishevi, Anophelesmartinius, Anopheles messeae, Anopheles persiensis, and Anopheles sacharovi), and an outgroup Asian species, Anopheles sinensis (Table 3). Overall, the tree separated all species into two large groups corresponding to species from the Maculipennis group together with An. punctipennis and species from the An. crucians complex with strong bootstrap support. Within the Maculipennis clade sequences from An. punctipennis formed a separate sister clade to An. quadrimaculatus with high bootstrap support (79%). Thus, the phylogenetic placement of An. punctipennis is unambiguously inside the Maculipennis group according to the analysis of ITS2 sequences.

Discussion
Among other malaria mosquito species, An. punctipennis has an extensive geographic distribution throughout North America [35] and can transmit two important species of human malaria, P. falciparum and P. vivax, the ungulate malaria parasite P. odocoilei, and viruses [10]. Despite these facts, An. punctipennis represents one of the most understudied and neglected vectors of malaria in North America, in particular because in the USA malaria was declared eradicated and the major research projects from the last two decades have focused on African malaria mosquito species [2]. Moreover, a molecular diagnostic tool based on ITS2 sequences, the most reliable species identification tool for malaria mosquitoes [15], had never been developed for An. punctipennis prior to current investigation. In this study, ITS2 fragments of rDNA were sequenced from 494 field collected mosquito samples in the eastern and midwestern USA.
Among them, 276 samples were identified as An. punctipennis based on morphological characteristics or COI sequences available at NCBI [34]. All of the An. punctipennis samples had a consistent ITS2 sequence length and there was no indication of interindividual variations. Thus, this study determined that An. punctipennis represents a single species in the eastern and midwestern states in the USA. However, samples from California and other western states were not included in this study, which requires further attention in the future. In addition, the RFLP approach based on ITS2 sequences was optimized for 7 species of malaria mosquitoes that were available through current mosquito collections ( Table 1). The presence of a unique site in An. punctipennis ITS2 sequences for the restriction enzyme NaeI allowed to cleave the PCR product of the ITS2 into two fragments that could be easily identified by gel-electrophoresis. Based on ITS2 sequence  (Table 2). However, among these species only two An. crucians B, An. pseudopunctipennis have a geographical overlap with An. punctipennis in the USA [39]. Moreover, most of these six species have different sizes of PCR products and RFLP fragment patterns. Thus, this method represents a simple and robust technique for identifying An. punctipennis among other local species of Anopheles in the USA. The approach also reduces the time and cost of molecular identification and represents a robust method for species identification in field collected mosquitoes.
In current survey, Anopheles mosquito collections were generated from samples collected at 11 locations within 4 states (Florida, Iowa, Minnesota, and Virginia) from urban, rural, and forest settings. An. punctipennis was found in 6 locations; only the coastal collection sites in Florida had no An. punctipennis samples (Fig. 1). Thus, the geographical distribution analysis showed that An. punctipennis is a very abundant species of Anopheles in the eastern and midwestern USA. Despite the fact that An. punctipennis was considered as a woodland mosquito [35], this species was detected in all of the settings that were examined (i.e., urban, rural, or forest), suggesting a possible broad role of this mosquito in transmission of pathogens. Interestingly, the presence of An. quadrimaculatus was determined at high altitude in the Appalachian Mountains (Fig. 1), where the presence of this species had not previously been determined [35]. Anopheles bradleyi, which was considered a brackish water mosquito [35], in current study, was found only in Mineral Springs in coastal area of Florida supporting the previous observations. Finally, the present investigation sheds some light on the phylogenetic relationships of An. punctipennis and its systematic position within other species from the genus Anopheles. Anopheles puctipennis was a former member of the An. maculipennis complex [27], but later was excluded from the complex based on morphological characteristics. According to Harbach [29], An. punctipennis belongs to a separate Punctipennis group that includes An. punctipennis, An. perplexens, and the An. crucians complex. Because of the absence of ITS2 sequences, An. punctipennis was not included in the phylogenetic analysis that was conducted for other species from the Maculipennis group [31,40,41]. The only phylogeny that included An. punctipennis was based on sequencing the D2 region of rDNA [42]. This analysis clearly separated the An. quadrimaculatus clade from the An. freeborni-An. occidentalis-An. earlei-An. punctipennis clade and suggested Anopheles walkeri as the most basal species for the entire group. However, the branches within these two clades remained poorly resolved.
For the better understanding the systematics position of An. punctipennis, phylogenetic analysis was performed for 20 species based on ITS2 sequences available from this work and from the GenBank [34]. In addition to An. punctipennis, the study included 13 species from the Maculipennis group, 5 species from the An. crucians complex, and an outgroup species, An. sinensis. The Maculipennis species were selected based on their systematics positions from two North American subgroups, Freeborni (An. freeborni, An hermsi and An. occidentalis) and Quadrimaculatus (Anopheles diluvialis, Anopheles smaragdius, Anopheles inundatus, Anopheles maverlius and An. quadimaculatus), and one Eurasian Maculipennis subgroup (An. beklemishevi, An. martinius, An. messeae, An. persiensis and An. sacharovi). The systematics species statuses of An. hermsi [24], species from Quadrimaculatus complex [25,43], and An. persiensis [31] were previously determined based on ITS2 sequencing. The species were also chosen based on their geographic distributions: in western (Freeborni subgroup) and eastern (Quadrimaculatus subgroup) parts in North America [6]; and in northern (An. beklemishevi, and An. messeae) and southern (An. persiensis, An, martinius, and An. sacharovi) parts of Eurasia [44][45][46]. Some of the species from An. crucians complex (An. crucians C, D, and E) included in this study were also identified based on ITS2 sequences [26]. These species are mostly found in eastern parts of North America. An outgroup species An. sinensis is a dominant malaria vector in Asia [44]. The maximum-likelihood tree, which was developed here, clearly separated the Maculipennis species from the An. crucians complex species and placed An. punctipennis as a sister taxon to An. quadrimaculatus. Thus, the phylogenetic analysis based on ITS2 sequences allowed us to reliably identify that An. punctipennis represents a species within the Maculipennis group and is a close relative to An. quadrimaculatus. Further analysis based on multiple genes or the entire genome will provide more details about the phylogenetic relationships of An. punctipennis and clarify its systematic position within the Maculipennis group of malaria mosquitoes.

Conclusions
This study reports the development of a new molecular diagnostic tool for An. punctipennis, one of the most widely spread species of malaria mosquitoes in North America. Two versions of the tool, based on either sequence analysis or a cost-effective RFLP approach, were optimized based on field collected material from the eastern and midwestern US. Sequence analysis demonstrated no variations in length or nucleotide polymorphism within the studied samples suggesting that An. punctipennis represents a single species in the area of study. The phylogenetic analysis based on ITS2 sequences placed An. punctipennis within the Maculipennis group species and separate from species within the An. crucians complex.