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Article

Novel Mitochondrial DNA Lineage Found among Ochlerotatus communis (De Geer, 1776) of the Nordic-Baltic Region

by
Heli Kirik
1,*,
Lea Tummeleht
2,
Tobias Lilja
3 and
Olavi Kurina
1
1
Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Friedrich Reinhold Kreutzwaldi 5D, 51006 Tartu, Estonia
2
Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Friedrich Reinhold Kreutzwaldi 62, 51006 Tartu, Estonia
3
Department of Microbiology, Swedish National Veterinary Institute, 751 89 Uppsala, Sweden
*
Author to whom correspondence should be addressed.
Insects 2020, 11(6), 397; https://doi.org/10.3390/insects11060397
Submission received: 14 May 2020 / Revised: 21 June 2020 / Accepted: 23 June 2020 / Published: 26 June 2020
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Abstract

:
The Ochlerotatus (Oc.) communis complex consist of three Northern American species as well as a common Holarctic mosquito (Diptera: Culicidae) Oc. communis (De Geer, 1776). These sister species exhibit important ecological differences and are capable of transmitting various pathogens, but cannot always be differentiated by morphological traits. To investigate the Oc. communis complex in Europe, we compared three molecular markers (COI, ND5 and ITS2) from 54 Estonian mosquitoes as well as two COI marker sequences from Sweden. These sequences were subjected to phylogenetic analysis and screened for Wolbachia Hertig and Wolbach symbionts. Within and between groups, distances were calculated for each marker to better understand the relationships among individuals. Results demonstrate that a group of samples, extracted from adult female mosquitoes matching the morphology of Oc. communis, show a marked difference from the main species when comparing the mitochondrial markers COI and ND5. However, there is no variance between the same specimens when considering the nuclear ITS2. We conclude that Oc. communis encompasses two distinct mitochondrial DNA lineages in the Nordic-Baltic region. Further research is needed to investigate the origin and extent of these genetic differences.

1. Introduction

Ochlerotatus (Oc.) communis complex includes four closely related mosquito species [1]: Oc. communis (De Geer, 1776), Oc. churchillensis (Ellis and Burst, 1973), Oc. nevadensis (Chapman and Barr, 1964) and Oc. tahoensis (Dyar, 1916). Morphology-based delimitation of these species is highly problematic due to a lack of reliable distinguishing traits, especially in adults [2]. Thus, researchers have employed both DNA sequencing, using mainly the mitochondrial cytochrome c oxidase subunit I (COI or COX1), and restriction fragment length polymorphism (RFLP) patterns to help with differentiation [3]. The namesake of the group, Oc. communis, is a common and often numerous Holarctic pest, whereas the other three species appear to be native to Northern America [1]. Due to the ubiquity of Oc. communis and because of its observed morphological variability, it is highly likely that this complex could have additional sister species in other parts of the world besides Northern America [2].
The phylogeography of the Oc. communis complex has received relatively little attention. At first these species were distinguished by morphologic as well as morphometric traits and the length differences of select loci, apparent in electrophoresis [1,4]. In 2014, the journal Canadian Entomologist published an article from H. H. Namin et al. describing barcoding (COI) results and designing a new diagnostic RFLP pattern for use with Oc. communis, Oc. churchillensis and Oc. tahoensis [3]. Since that, Oc. churchillensis, Oc. nevadensis and Oc. tahoensis have only been rarely sequenced, for example as part of vector disease investigations [5]. There are more studies on Oc. communis, but this species is still often diagnosed based on morphology alone, although genetic identification is also used [6,7,8,9,10]. Generally speaking, the Oc. communis complex does not currently appear to be under close study.
Mosquitoes from the Oc. communis complex have been associated with many pathogens. The Jamestown Canyon virus has been isolated from North American Oc. communis mosquitoes, which may be one of the species acting as an overwintering reservoir for the pathogen [5]. Oc. communis individuals, in some cases both adults and larva, have been found to carry Sindbis virus (known in Sweden as Ockelbo, in Finland as Pogosta and in Russia as Karelian virus) Batai virus, Francisella (F.) tularensis bacteria as well as different strains of the Inkoo virus in Scandinavian field studies [7,11,12]. Oc. communis could also be one of the main vectors of Dirofilaria repens, a filarial nematode which is currently expanding its area northward [8,13]. According to older studies, Oc. communis mosquitoes have also tested positive for Tahyna virus in Russia and six strains of California encephalitis virus in Canada [14,15].
Individuals within a phylogenetic group, even within isomorphic species, can often differ in their medical importance [16,17] and there is a noticeable lack of information regarding how the sister species within Oc. communis complex vary in their vector capacity and competence. Especially as some biological and ecological differences have been observed within the group. Firstly, Oc. churchillensis is the only autogenous species in the group and thought to be non-biting [1]. Both Oc. nevadensis and Oc. tahoensis seem to only be found in mountainous regions, the latter preferring higher elevations, while Oc. churchillensis inhabits forests near the North American tundra [4]. Because of these factors, it has been theorized that the sister species comprising Oc. communis complex may have derived from allopatric as well as sympatric speciation [3].
Maternally inherited Wolbachia Hertig and Wolbach, 1924 symbionts can also contribute to speciation within arthropods. Wolbachia is a genus of cytoplasmically transmitted bacteria that infect the tissues of many arthropods and some nematodes [18,19]. These endosymbionts have shown to cause cytoplasmic incompatibility, parthenogenesis and the death or feminization of biological males (reviewed by Correa and Ballard [20]). Because of this, Wolbachia infections have been seen as possible drivers of microevolution and even speciation [21,22,23]. Wolbachia strains have been detected in several different mosquito species, but infection rates vary [24,25]. At this time, no strains have been found in Oc. communis [8]. However, if detected, it could help explain some genetic results.
A larva with a COI sequence similar but not identical to Oc. tahoensis was recently found in Sweden [7], but the discovery was not further investigated. At the same time, similar cases were found with adult female mosquitoes in Estonia. Taking into account the possibility of additional Oc. communis complex species in Europe [2], a special attention was paid to the recently collected Estonian mosquitoes. The primary aim of this study was to search for a possible novel species within the Oc. communis complex. For this reason, both mitochondrial (mtDNA) and nuclear DNA (nDNA) markers from Nordic-Baltic mosquitoes were analyzed. These samples were also screened for infection with Wolbachia symbionts. Here we present the genetic information of 54 Estonian mosquitoes and compare those to two Swedish samples as well as to reference material from public nucleotide databases.

2. Materials and Methods

2.1. Sampling and Morphological Identification

This study is based on 54 Estonian mosquitoes and two Swedish COI sequences (Table A1). Of the Estonian individuals, 26 were morphologically identified as Oc. communis, six as Oc. punctor (Kirby, 1837), eight as Oc. hexodontus (Dyar, 1916), 13 as Oc. cataphylla (Dyar, 1916) and one as Anopheles (An.) messeae (Falleroni, 1926). While Oc. communis is the main focus, Oc. punctor, Oc. hexodontus and Oc. cataphylla samples were included in the analysis to compare how intra- as well as interspecific genetic variation of the Oc. communis complex relates to other common species of the genus Ochlerotatus. An. messeae was used as an outgroup. All Estonian mosquitoes were collected from six different sites during 2015–2016, using automated Mosquito Magnet® Independence (Woodstream Corp., Lititz, PA, USA) machines. Four of these sampling sites were located on the Estonian mainland and two on the largest islands of the country—Saaremaa and Hiiumaa (Figure 1). Insects were stored at −20 °C until temporarily thawed and identified under a stereomicroscope Olympus SZ61 (Olympus Corporation, Shinjuku, Tokyo, Japan) to species level, using a standard taxonomic key [2]. Swedish COI sequences originate from Västerbotten County, first from a larva caught in 2014 [7] and the second from an adult female mosquito sent to the Swedish National Veterinary Institute (SVA) in 2017.

2.2. DNA Extraction

DNA was extracted from the Estonian mosquitoes using either DNeasy Blood and Tissue Kit (Qiagen, Hilden, North Rhine-Westphalia, Germany) or PrepMan® Ultra Sample Preparation Reagent (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). There were no qualitative differences between the used DNA extraction methods. Whole mosquitoes were used for the DNeasy Blood and Tissue Kit, while three legs from each specimen were taken for extracting DNA with the PrepMan® Ultra Sample Preparation Reagent. DNeasy Blood and Tissue Kit was used in accordance with the manufacturers manual and the DNA extractions made with PrepMan® Ultra Sample Preparation Reagent were conducted as specified in previous work [26].

2.3. DNA Markers

Two protein coding mitochondrial and two nuclear markers were amplified and sequenced from all Estonian mosquitoes used in this study. However, only three markers were used for further analysis as the D2 region of the large subunit 28S rDNA gene was too conserved between Ochlerotatus species to be of use, although it has been successfully utilized for species identification in other mosquito genera [27,28]. The 5’ region of the cytochrome c oxidase (COI) subunit I was chosen as one of the markers for its widespread use in mosquito identification and its generally good ability to differentiate between species, although it can at times either over- or underestimate the true number of well distinguished monophyletic groups [16,29,30]. The nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 5 (ND5) gene was used as an additional mitochondrial marker. ND5 is generally thought to have faster evolution rates compared to COI and thus has been used for inter- as well as intraspecies studies in mosquitoes [31,32,33]. Finally, the nuclear internal transcribed spacer 2 (ITS2), a region of the ribosomal RNA gene, was used as the most conserved marker. ITS2 sequences have been used for species identification in many animal groups and may be the most used nDNA marker for mosquitoes [16,34,35]. It has generally been proposed as a good marker to analyze alongside COI [16].
Each mosquito sample was also screened for Wolbachia symbionts by amplifying part of the Wolbachia surface protein (WSP) gene.

2.4. Primers

For COI, which is by far the most commonly sequenced marker for mosquitoes, we used the universal primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTG G-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′), that consistently resulted in 710 bp long sequences [36]. A 450 bp segment of the ND5 region was amplified and sequenced using the primers 6500 (5′-TCCTTAGAATAAAATCCCGC-3′) and 7398 (5′-GTTTCTGCTTTAGTTCATTCTTC-3′) which were originally designed for Aedes (Ae.) albopictus [37]. Primer pair 5.8S (5′-TGTGAACTGCAGGACACATG-3′) and 28S (5′-ATGCTTAAATTTAGGGGGTA-3′) was used for ITS2, producing approximately 368 bp to 387 bp long sequences [38]. The ITS2 primers were initially developed to differentiate between cryptic Anopheline mosquitoes, but can be used for other mosquito genera as well. DNA from the Wolbachia symbionts WSP gene was amplified with the primers wsp 81F (5′-TGGTCCAATAAGTGATGAAGAAAC-3′) and wsp 691R (5′-AAAAATTAAACGCTACTCCA-3′) that would have resulted in 600 bp sequences [39].

2.5. Polymerase Chain Reaction (PCR) and Sequencing

While the composition of the polymerase chain reaction (PCR) mixes remained the same throughout, thermal cycler programs were adjusted for each primer pair to maximize the amplification yield of the respective marker regions. All PCR reactions contained 12.5 μL DreamTaq DNA Polymerase Master Mix (2X) (Thermo Fisher Scientific Inc., Waltham, MA, USA), 0.4 µM of each primer (0.04 µmol, TAG Copenhagen, Frederiksberg, Denmark), 10.5 μL nuclease-free water and 1 μL DNA template. The PCR program for amplifying the COI marker region was as follows: 95 °C for 2 min 15 s, followed by 35 cycles of 95 °C for 30 s, 57 °C for 45 s, 72 °C for 45 s and a final elongation step at 72 °C for 5 min. Although mostly identical, the PCR program for the ITS2 sequence introduced a much lower annealing temperature: 95 °C for 2 min 15 s, 35 cycles of 95 °C for 30 s, 45 °C for 45 s, 72 °C for 45 s and an elongation at 72 °C for 5 min. On the other hand, amplifying the mitochondrial ND5 region required a much longer PCR program, which contained 2 different sets of cycles on low annealing temperatures. The final program was: 94 °C for 3 min denaturation followed by 10 cycles of 94 °C for 30 s, 38 °C for 30 s, 65 °C for 45 s, then 50 cycles of 94 °C for 30 s, 38 °C for 30 s, 65 °C for 45 s and a last elongation of 65 °C for 3 min. The WSP region of the symbiont was amplified using a previously published PCR program [24] and a positive control sample from Culex pipiens was also added to the PCR. All samples were amplified with ESCO Swift Maxi Thermal Cycler (ESCO Micro Pte. Ltd., Changi South Street, Singapore, Singapore).
PCR products were checked for signals by electrophoresis using 1.6% agarose gel infused with ethidium bromide. Amplified samples were tinted with DNA Gel Loading Dye (6X) (Thermo Fisher Scientific Inc., Waltham, MA, USA) prior to electrophoresis. Positive signals were compared to GeneRuler 100 bp DNA Ladder, ready-to-use (Thermo Fisher Scientific Inc., Waltham, MA, USA) or, in the case of the WSP samples, to the GeneRuler 100 bp Plus DNA Ladder, ready-to-use (same company) to visually determine the approximate lengths of the replicated DNA strands. PCR products were cleaned and sequenced with Applied Biosystems 3130xl Genetic Analyzer by a two-directional procedure (Thermo Fisher Scientific Inc., Waltham, MA, USA).

2.6. Sequence Analysis

Forward and reverse DNA strands were aligned and assembled into consensus sequences in BioEdit Sequence Alignment Editor version 7.2.5 [40]. Primers and low-quality areas were trimmed to produce the final sequences. MEGA X version 10.0.5 [41] was used for sequence alignment and data analysis. All original DNA sequences were uploaded to the online database GenBank. Reference sequences were added to the analysis via the Blast Search tool in MEGA X (Table A2). This was effective for COI and ITS2 markers. However, the mtDNA marker ND5 has so far received little attention in mosquitoes from the Ochlerotatus genus and, therefore, no previous sequences could be found from the database.
Protein-coding gene sequences were aligned based on codons, whereas the DNA strands for ITS2 were aligned by nucleotides alone and allowed gaps for indels. In all cases, the Multiple Sequence Comparison by Log-Expectation (MUSCEL) tool with default options was used for aligning sequences. The Find Best-Fit Substitution Model feature was used for all of the analyzed markers to determine the most appropriate model and Rates among Sites variable. These results were then used to calculate mean within and between group genetic distances measuring the proportion of nucleotide sites with differences between each sequence pair (p-distances). This was done using the Compute within Group Mean Distance and Compute between Group Mean Distance functions in MEGAX. Gaps/Missing Data Treatment was set to complete deletion, which ensured that all sequences of the same marker were trimmed to identical lengths: 441 bp for COI, 321 bp for ND5 and 251 bp for the ITS2 marker region.
Phylogenetic trees were constructed with the maximum likelihood method, while also using the analysis model and Rates among Sites recommended by the Find Best-Fit Substitution Model feature. The Gamma Parameter (+G) was set to 6, Gaps/Missing Data Treatment was set to complete deletion and Bootstrap with 1000 replications was employed each time. All trees were annotated and rooted using An. messeae as an outgroup. Trees were then modified to only display bootstrap values > 75% and distance values ≥ 0.01. Differences between population sizes were not accounted for. While Oc. communis, Oc. punctor and Oc. cataphylla are all common in Estonia, there is very little information about their exact effective population sizes. However, mosquitoes of the Oc. sp. group likely have a much smaller effective population size than the normal type Oc. communis.

3. Results

Of the 54 Estonian mosquitoes used in this study, 26 were identified as Oc. communis by morphological evaluation, but only 14 of these were grouped together by all three DNA markers. The remaining 12 individuals formed a separate monophyletic group within the Oc. communis complex based to their mitochondrial markers, similarly to two COI sequences received from Sweden. However, this pattern was not apparent when examining the nDNA results.
According to the phylogenetic tree based on the COI marker (Figure 2), 12 sequences from mosquitoes caught at three different sites (sites 1, 2 and 5) in Estonia (Figure 1) and 2 sequences from Sweden cluster together (hereafter referred to as Oc. sp.), distinct from Oc. communis and closer to the North American species Oc. tahoensis and Oc. churchillensis. In fact, there is on average about 0.063 (standard error (S.E) 0.018) substitutions per base difference between Oc. communis specimens and the Oc. sp. group. Meanwhile, the p-distances between Oc. sp. individuals and Oc. tahoensis as well as Oc. churchillensis are smaller, 0.046 (S.E. 0.013) and 0.054 (S.E. 0.015) respectively. It should also be noted, that the Oc. sp. COI sequences show a high similarity to each other, being almost genetically identical. This is in contrast to the larger genetic diversity within other groups. These results show that there is a previously unknown group of genetically distinct individuals belonging to the Oc. communis complex found in Europe.
The ND5 marker region also suggests that mosquitoes forming the Oc. sp. group are a distinct genetic unit, separate from traditional Oc. communis mtDNA sequences (Figure 3). Compared to the COI region, the ND5 marker sequences are even more variable between groups. Between Oc. communis and the Oc. sp. cluster, there is a difference of on average 0.083 (S.E 0.031) base substitutions per site. Yet, within group average evolutionary distances remain small. In the Oc. sp. group, there are only on average 0.001 (S.E 0.001) base substitutions per site over all of the sequence. The p-distance is once again larger among the traditional Oc. communis samples, averaging 0.008 (S.E 0.009) base substitutions. ND5 marker sequences associated with Oc. punctor, Oc. hexodontus and Oc. cataphylla clusters are even more variable. Unfortunately, there are no ND5 marker sequences from Oc. tahoensis, Oc. churchillensis or Oc. nevadensis currently available in GenBank. Likewise, because the ND5 region is a less popular marker than COI and ITS2, there were also no reference sequences for Oc. communis, Oc. punctor, Oc. hexodontus or Oc. cataphylla. However, the ND5 sequences included in this study support the conclusions drawn from the COI marker.
While mitochondrial markers outlined Oc. sp. group as a separate entity, this is not the case for the nuclear marker ITS2. In fact, there are no differences between the ITS2 sequences from Oc. communis and the Oc. sp. samples (Figure 4). On the other hand, Oc. churchillensis reference sequences downloaded from GenBank maintain their genetic distance from Oc. communis, being on average 0.025 (S.E. 0.009) substitutions per base apart. The same is true for Oc. punctor, Oc. hexodontus and Oc. cataphylla clusters, which differ on average from the Oc. communis group by 0.035 (S.E 0.011) and 0.079 (S.E 0.017) base substitutions per site, respectively. It should also be noted that the ITS2 sequences of the Oc. communis cluster have no notable within-group genetic variance. Within group average genetic variance is also relatively low in other groups. From these results we can see that the ITS2 region of Estonian Oc. communis individuals is quite conserved and does not echo the variance shown by the mtDNA markers.
Estonian mosquito samples were screened for Wolbachia surface protein as it could offer an explanation for the divergent mtDNA lineage within the analyzed Oc. communis individuals. However, there were no positive electrophoresis signals for any of the samples except for the positive control. Therefore, there is no evidence of Wolbachia symbionts in the Oc. communis individuals used in this study.

4. Discussion

There is a discrepancy between the mtDNA and nDNA markers among mosquitoes morphologically identified as Oc. communis in the Nordic-Baltic region. The mitochondrial markers COI and ND5 clearly distinguish two clusters of individuals within the Oc. communis complex: One identical to Oc. communis reference sequences and the other representing a new group. However, this difference is not reflected in the ITS2 sequences of the same mosquitoes. Based on the nuclear marker, all examined Oc. communis-like individuals have identical ITS2 marker sequences, whereas reference sequences for Oc. churchillensis, which also belongs to the Oc. communis complex, remain distinct from Oc. communis. There appears to be a previously unreported mtDNA lineage within the Nordic-Baltic Oc. communis populations, but no variance within the nDNA to point to a distinct species.
Both COI and ND5 marker regions have high support for similar monophyletic clades and both trees show some amount of nucleotide changes between traditional Oc. communis individuals, while Oc. sp. sequences are largely identical. This shows, that the Oc. sp. clade is evolutionarily younger than the traditional Oc. communis and its members probably less numerous, as the mtDNA of this group has yet to accrue many mutations [44]. However, the nuclear marker ITS2 shows noticeably less divergence between all of the analyzed Ochlerotatus species. Moreover, the D2 region of the large subunit 28S, also originally sequenced for this study, was unable to provide any notable differences within Ochlerotatus, although it has been shown to work for anopheline mosquitoes [28]. It seems that the species analyzed for this study are in general more closely related to each other than those most often sequenced in other mosquito genera. Therefore, it is currently difficult to say if any of the markers used in this work might underestimate the true diversity of the Ochlerotatus species. This matter would be greatly improved by finding and analyzing nDNA markers with better resolution for the Ochlerotatus genus. It would also be useful to sequence a larger number of Oc. communis, in order to obtain a better overview of its natural intraspecific variance.
The reason for mitochondrial differences between coexisting groups can be hard to pinpoint. As the common type Oc. communis and the individuals with differing mtDNA coexist in the same communities and have identical ITS2 sequences, it is probable that they are intermixing. MtDNA variation in arthropod populations can be influenced by symbiotic bacteria like the maternally inherited Wolbachia [45]. However, none of the samples used in this study were positive for Wolbachia DNA and this coincides with previous observations [8]. There could be other speciation-driving factors in play, but that is impossible to determine at this juncture. Importantly, the fauna of the Nordic-Baltic region is relatively young, only emerging after the Last Glacial Period more than 9000 years ago when it was recolonized by organisms from different glacial refugiums [46,47,48]. It is possible that Eurasian Oc. communis complex individuals with the differing mtDNA used to be geographically separated, but not genetically different enough as to have evolved reproductive barriers.
Uncertainties in mtDNA sequencing results can also be caused by pseudogenes or heteroplasmy. Many organisms are known to have nuclear insertions of mitochondrial sequences (NuMts) as well as multiple types of functional mtDNA in the same individual (heteroplasmy)—both of these can interfere with the amplification and sequencing of mitochondrial markers, resulting in erroneous conclusions if not recognized [49]. Sequences from COI and ND5 genes have been known to be incorporated into nDNA as NuMts [50]. Furthermore, starting with Aedes aegypti (Linnaeus, 1762) and Culex quinquefasciatus (Say, 1823), NuMts have now been found in many mosquito species [51,52]. However, these insertions in nDNA are non-functional and, therefore, not under the same constraints or mutation rates as the real mtDNA [53,54]. These pseudogenes tend to contain inappropriate stop-codons and indels as well as more point mutation than normal [49,55]. Such problems are not evident in the sequences analyzed in this study. Heteroplasmy can also result in ambiguous reads in Sanger sequencing, but can be harder to identify compared to NuMts [16]. It cannot be completely ruled out that the less common Oc. communis mtDNA lineage reported in this study is a sign of heteroplasmy.
While Oc. communis is not currently considered an important disease vector, it has been indicated as a possible carrier of several viral and bacterial pathogens. This species is known for its wide distribution and can be numerous at times [2]. Because of this, genetic deviations within Oc. communis populations may be important from future vector and pest control standpoints. Estonian and Swedish faunas are relatively young and it is reasonable to assume that Oc. communis individuals carrying the different mtDNA variant are not limited to the Nordic-Baltic region, but can be found within a much wider area. However, there has thus far been little indication of notable genetic discrepancies within this species in Central and Western Europe. Because of this, more sampling efforts should be directed towards Eastern Eurasia. Also, there is more work to be done in regards to sequencing additional nDNA markers from Oc. communis complex species and finding more informative nDNA markers to use with genus Ochlerotatus in general.

5. Conclusions

The current study presents evidence for an additional discrete mtDNA lineage within Oc. communis in Europe. This common Holarctic pest is the namesake of the Oc. communis complex, which it shares with three closely related Northern American species. While these sister species have sometimes been regarded as subspecies to Oc. communis, there are clear differences in their ecology, genetic material and in some cases morphology. It has been long theorized that there could be more species belonging to the Oc. communis complex in other parts of the world besides Northern America. In this study we show, based on the COI and ND5 markers, that there is a group of Oc. sp. individuals with a distinct mtDNA lineage within morphologically identified Oc. communis mosquitoes in Estonia and Sweden. We also show that these differences are not apparent in the nDNA of the same individuals. It was also determined that the analyzed mosquitoes had no detectable Wolbachia infection, ruling these maternally inherited symbionts out as a possible explanation for the mitochondrial differences.

Author Contributions

Conceptualization, H.K., L.T. and O.K.; Data curation, H.K.; Formal analysis, H.K. and T.L.; Funding acquisition, L.T. and O.K.; Investigation, H.K. and T.L.; Methodology, H.K., L.T., T.L. and O.K.; Project administration, L.T. and O.K.; Supervision, L.T. and O.K.; Validation, H.K.; Visualization, H.K.; Writing—original draft, H.K.; Writing—review and editing, H.K., L.T., T.L. and O.K. All authors have made notable contributions to this study as well as have read and agreed to the published version of the manuscript.

Funding

This study was supported by institutional research funding IUT21-1 of the Estonian Research Council and by Estonian University of Life Sciences research and development project 8P160014VLVP.

Acknowledgments

The authors would like to especially thank Magnus Evander (Umeå University, Sweden) for providing the COI sequence H2_GB1960-1936R from Västerbotten County, Sweden and Erkki Õunap (Tartu University, Estonia) for guidance in phylogenetic analyses. We are also grateful to everyone involved in maintaining and operating insect traps.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Appendix A

Table
Table A1. Mosquitoes used in this study with their GenBank sequence numbers and upload year where possible as well as the country of origin.
Table A1. Mosquitoes used in this study with their GenBank sequence numbers and upload year where possible as well as the country of origin.
IDSite No.Morphologically Determined SpeciesCountry of OriginGenBank COI Acc. Num.GenBank ND5 Acc. Num.GenBank ITS2 Acc. Num.Upload Year
150040121Oc. hexodontusEstoniaMT149916MT150197MT1502472020
150040151Oc. cataphyllaEstoniaMT149946MT150204MT1502522020
150050021Oc. punctorEstoniaMT149917MT150196-2020
150050051Oc. punctorEstoniaMT149918--2020
150050071Oc. hexodontusEstoniaMT149919MT150198-2020
150050081Oc. cataphyllaEstoniaMT149920MT150205MT1502532020
150050101Oc. cataphyllaEstoniaMT149921MT150206MT1502542020
150050121Oc. communisEstoniaMT149922MT150192MT1502302020
150090252Oc. cataphyllaEstoniaMT149923MT150207MT1502552020
150090262Oc. cataphyllaEstoniaMT149924MT150208-2020
150090272Oc. communisEstoniaMT149925MT150184MT1502212020
150090282Oc. punctorEstoniaMT149926MT150195MT1502432020
150090302Oc. sp.EstoniaMT149927MT150171MT1502342020
150090312Oc. sp.EstoniaMT149928MT150172MT1502352020
150090362Oc. hexodontusEstoniaMT149929MT150199MT1502482020
150100042Oc. communisEstoniaMT149930MT150185MT1502222020
150130011Oc. cataphyllaEstoniaMT149931MT150209MT1502562020
150130031Oc. cataphyllaEstoniaMT149932MT150210MT1502572020
150130041Oc. communisEstoniaMT149933MT150183MT1502202020
150130051Oc. cataphyllaEstoniaMT149934MT150211MT1502582020
150130061Oc. punctorEstoniaMT149935MT150194-2020
150130151Oc. cataphyllaEstoniaMT149936MT150212MT1502592020
150160243Oc. communisEstoniaMT149937MT150182MT1502192020
150160303Oc. communisEstoniaMT149938MT150181MT1502182020
150160413Oc. hexodontusEstoniaMT149939MT150200MT1502492020
150180014Oc. hexodontusEstoniaMT149940-MT1502452020
150180074Oc. communisEstoniaMT149941MT150186MT1502232020
150180084Oc. hexodontusEstoniaMT149942-MT1502462020
150180114Oc. punctorEstoniaMT149943-MT1502442020
150200023Oc. cataphyllaEstoniaMT149944MT150213MT1502602020
150320024Oc. cataphyllaEstoniaMT149945MT150214MT1502612020
160080016Oc. cataphyllaEstoniaMT149947MT150215MT1502622020
160790955Oc. sp.EstoniaMT149948MT150173MT1502362020
160790975Oc. sp.EstoniaMT149949MT150169MT1502322020
160791025Oc. communisEstoniaMT149950-MT1502242020
160791105Oc. sp.EstoniaMT149951MT150170MT1502332020
160791125Oc. sp.EstoniaMT149952MT150174MT1502372020
160791205Oc. sp.EstoniaMT149953MT150175MT1502382020
161420015Oc. sp.EstoniaMT149954MT150176MT1502392020
161420025Oc. sp.EstoniaMT149955MT150177MT1502402020
161420055Oc. cataphyllaEstoniaMT149969MT150216MT1502632020
161420065Oc. communisEstoniaMT149956MT150187MT1502252020
161420075Oc. communisEstoniaMT149957MT150188MT1502262020
161420105Oc. sp.EstoniaMT149958MT150178MT1502412020
161420135Oc. communisEstoniaMT149959MT150189MT1502272020
161420165Oc. communisEstoniaMT149960MT150193MT1502312020
161420235Oc. sp.EstoniaMT149961MT150179MT1502422020
161420405An. messeaeEstoniaMT149962MT150217MT1502642020
161490011Oc. communisEstoniaMT149963MT150190MT1502282020
161490031Oc. hexodontusEstoniaMT149964MT150202MT1502502020
161490041Oc. punctorEstoniaMT149965MT150203-2020
161490061Oc. communisEstoniaMT149966MT150191MT1502292020
161490071Oc. hexodontusEstoniaMT149967MT150201MT1502512020
161490091Oc. sp.EstoniaMT149968--2020
H2_GB1960-1936R-N/ASweden----
hitta_myggan_14-N/ASweden----
Table
Table A2. GenBank reference sequences used in the phylogenetic analysis, selected based on their similarity to the Swedish and Estonian mosquito sequences.
Table A2. GenBank reference sequences used in the phylogenetic analysis, selected based on their similarity to the Swedish and Estonian mosquito sequences.
SpeciesCountry of OriginGenBank COI Acc. Num.GenBank ND5 Acc. Num.GenBank ITS2 Acc. Num.Upload Year
Oc. tahoensisUSAJX259677--2012
Oc. churchillensisUSAMG242480--2018
Oc. tahoensisCanadaJF868962--2018
Oc. communisCanadaJF868933--2018
Oc. punctorBelgiumKM258280--2015
Oc. hexodontusCanadaKR697054--2018
Oc. cataphyllaSwedenKP942759--2018
Oc. tahoensisCanadaKM648357--2019
Oc. tahoensisCanadaKM628572--2019
Oc. churchillensisCanadaKC713604--2013
Oc. tahoensisCanadaMF825642--2018
Oc. tahoensisCanadaKM645852--2019
Oc. tahoensisCanadaKM639864--2019
Oc. churchillensisCanadaKC713602--2013
Oc. churchillensisCanadaKC713603--2013
Oc. churchillensisCanadaKC713601--2013
Oc. communisCanada--KF5350222013
Oc. churchillensisCanada--KF5350132013
Oc. churchillensisUSA--MG2326132018
Oc. punctorCanada--KF5350722013
Oc. hexodontusCanada--KF5350392013
Oc. abserratusCanada--KF5350262013

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Insects 11 00397 g001 550
Figure 1. Map showing the six Estonian collection sites, indicated with green circles. Pie charts demonstrate the amount of Oc. sp. (yellow background) and Oc. communis (orange background) specimen caught from each site. In total this paper includes 20 mosquitoes from site 1 “Undi” (58°29′ N, 26°54′ E), eight from site 2 “Muraste” (59°28′ N, 24°27′ E), four from site 3 “Mändjala” (58°13′ N, 22°20′ E), five from site 4 “Vanajõe” (58°53′ N, 22°26′ E), 16 specimens from site 5 “Metsaküla” and one mosquito from site 6 “Tartu” (58°04′ N, 25°31′ E). 31 of these mosquitoes were caught in 2015 and 23 during 2016. Base map curtesy of ©OpenStreetMap contributors (https://www.openstreetmap.org/copyright) and ©MapTiler (https://www.maptiler.com/copyright/).
Figure 1. Map showing the six Estonian collection sites, indicated with green circles. Pie charts demonstrate the amount of Oc. sp. (yellow background) and Oc. communis (orange background) specimen caught from each site. In total this paper includes 20 mosquitoes from site 1 “Undi” (58°29′ N, 26°54′ E), eight from site 2 “Muraste” (59°28′ N, 24°27′ E), four from site 3 “Mändjala” (58°13′ N, 22°20′ E), five from site 4 “Vanajõe” (58°53′ N, 22°26′ E), 16 specimens from site 5 “Metsaküla” and one mosquito from site 6 “Tartu” (58°04′ N, 25°31′ E). 31 of these mosquitoes were caught in 2015 and 23 during 2016. Base map curtesy of ©OpenStreetMap contributors (https://www.openstreetmap.org/copyright) and ©MapTiler (https://www.maptiler.com/copyright/).
Insects 11 00397 g001
Insects 11 00397 g002 550
Figure 2. Phylogenetic tree based on 72 partial cytochrome c oxidase (COI) sequences (441). Calculated with the Maximum Likelihood method, using Tamura 3-parameter [42] model, with discrete gamma distribution (6 categories (+G, parameter = 0.1790)) Branch lengths are shown to scale and measured as the number of substitutions per site. An. messeae was used as an outgroup for rooting the tree. Oc. sp. sequences are genetically closer to North American species Oc. tahoensis and Oc. churchillensis than the widespread Oc. communis.
Figure 2. Phylogenetic tree based on 72 partial cytochrome c oxidase (COI) sequences (441). Calculated with the Maximum Likelihood method, using Tamura 3-parameter [42] model, with discrete gamma distribution (6 categories (+G, parameter = 0.1790)) Branch lengths are shown to scale and measured as the number of substitutions per site. An. messeae was used as an outgroup for rooting the tree. Oc. sp. sequences are genetically closer to North American species Oc. tahoensis and Oc. churchillensis than the widespread Oc. communis.
Insects 11 00397 g002
Insects 11 00397 g003 550
Figure 3. Phylogenetic tree representing the information of 48 dehydrogenase subunit 5 (ND5) marker region sequences (321 bp). The tree is calculated with the maximum likelihood method, using the Tamura 3-parameter [42] model and a discrete gamma distribution (6 categories (+G, parameter = 0.1516)). Branch lengths are shown to scale and measured based on the average number of substitutions per site between sequence pairs. An. messeae was used the outgroup in order to root the tree. Oc. sp. sequences cluster together, away from the Oc. communis group.
Figure 3. Phylogenetic tree representing the information of 48 dehydrogenase subunit 5 (ND5) marker region sequences (321 bp). The tree is calculated with the maximum likelihood method, using the Tamura 3-parameter [42] model and a discrete gamma distribution (6 categories (+G, parameter = 0.1516)). Branch lengths are shown to scale and measured based on the average number of substitutions per site between sequence pairs. An. messeae was used the outgroup in order to root the tree. Oc. sp. sequences cluster together, away from the Oc. communis group.
Insects 11 00397 g003
Insects 11 00397 g004 550
Figure 4. Phylogenetic tree based on the 53 internal transcribed spacer 2 (ITS2) marker sequences (251 bp). Calculated with the maximum likelihood method, using the Kimura 2-parameter model [43] and discrete Gamma distribution (6 categories (+G, parameter = 0.8737)). Branch lengths are shown to scale and measured based on the average number of substitutions per site between sequence pairs. An. messeae was used as the outgroup for rooting the tree. There appears to be no variation amid the ITS2 sequences Oc. sp. sequences and Oc. communis. However, Oc. churchillensis sequences obtained from GenBank remain as a separate group.
Figure 4. Phylogenetic tree based on the 53 internal transcribed spacer 2 (ITS2) marker sequences (251 bp). Calculated with the maximum likelihood method, using the Kimura 2-parameter model [43] and discrete Gamma distribution (6 categories (+G, parameter = 0.8737)). Branch lengths are shown to scale and measured based on the average number of substitutions per site between sequence pairs. An. messeae was used as the outgroup for rooting the tree. There appears to be no variation amid the ITS2 sequences Oc. sp. sequences and Oc. communis. However, Oc. churchillensis sequences obtained from GenBank remain as a separate group.
Insects 11 00397 g004

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Kirik, H.; Tummeleht, L.; Lilja, T.; Kurina, O. Novel Mitochondrial DNA Lineage Found among Ochlerotatus communis (De Geer, 1776) of the Nordic-Baltic Region. Insects 2020, 11, 397. https://doi.org/10.3390/insects11060397

AMA Style

Kirik H, Tummeleht L, Lilja T, Kurina O. Novel Mitochondrial DNA Lineage Found among Ochlerotatus communis (De Geer, 1776) of the Nordic-Baltic Region. Insects. 2020; 11(6):397. https://doi.org/10.3390/insects11060397

Chicago/Turabian Style

Kirik, Heli, Lea Tummeleht, Tobias Lilja, and Olavi Kurina. 2020. "Novel Mitochondrial DNA Lineage Found among Ochlerotatus communis (De Geer, 1776) of the Nordic-Baltic Region" Insects 11, no. 6: 397. https://doi.org/10.3390/insects11060397

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