Simulium reptans (Linnaeus, 1758)

Vaulin, Oleg V., Petrozhitskaya, Liudmila V. & Novgorodova, Tatiana A., 2024, Genetic diversity of populations of Simulium reptans s. l. (Diptera: Simuliidae) in the territory of Russia and Northern Kazakhstan, Zootaxa 5543 (3), pp. 329-342 : 334-336

Testing of the allele-specific PCR (AS-PCR) method for identification of Simulium reptans and S. reptantoides

From examined 122 specimens, the vast majority (121) had a PCR product predicted for S. reptans . One specimen had an additional band expected for S. reptantoides . Subsequent sequencing of the COI region revealed a nucleotide substitution near the 3’ end of the corresponding primer annealing site ( Figure 5 ). Despite the absence of S. reptantoides among the studied specimens, the spectrum of PCR products of the individual with an additional substitution characteristic of S. reptantoides indicates the possibility of differentiating these species using the proposed approach.

Sequencing of nuclear rRNA genes

Sequencing of a fragment of the COI gene and two regions of rRNA genes–ITS2 and the D 2 28S variable region –was performed for several individuals of each sample. Minimal polymorphism was detected for both rDNA sequences. In a number of specimens, polymorphism in ITS2 is expressed in the presence of several variants that differ in insertion/deletion, which makes it difficult to obtain complete reads.

In addition to insertions/deletions, polymorphism was noted for the A/T substitution near the 3’ end of the sequence, as well as the overlap of these nucleotides ( Figure 6 ). All three sequence variants occur in Asian populations; the variant with A/T overlap was noted in all 4 studied ITS2 sequences from the samples collected in the Murmansk Region.

Most of the studied specimens were identical for the D 2 28S region; two sequences originating from Ust`-Kut (Irkutsk Region) and the Novosibirsk Region had a superposition of two nucleotides at the same position. The D 2 28S region sequences also differ in three positions from the sequence EF417075, presented in the DNA database and referred to S. reptans . This result may be associated either with the peculiarities of species identification or with the presence of cryptic species.

Phylogenetic analysis by COI gene region

Despite the contradiction of data on restriction analysis and the results of species diagnostic PCR, all the studied sequences belonged to S. reptans ( Figure 7 ). Several groups of COI sequences uniting samples by geographical origin can be distinguished. Specimens of S. reptans A include a basal group of British specimens, as well as a group including specimens from Sweden and from the Murmansk Region of Russia. Form B of S. reptans includes the East European branch ( Slovakia, Slovenia, Latvia, Lithuania); the Eurasian branch, with a very wide distribution from Northern Europe to Eastern Siberia ( Great Britain, Sweden, Norway, part of the Siberian samples) and the Asian branch (Novosibirsk Region, Krasnoyarsk Krai, Irkutsk Region, Northern Kazakhstan).

The Eurasian branch corresponds to the BstF5 I restriction spectrum of 100+250+350 bp. and Alu I restriction spectrum–with one detectable fragment of 230 bp in size.

The Asian branch corresponds to the BstF5 I restriction spectrum of 600+100 bp. and Alu I restriction spectrum with fragments of 180+230 bp in size.

Thus, the Asian branch predominates in the Ob-Irtysh basin (Novosibirsk Region, Krasnoyarsk Krai and Northern Kazakhstan), while in the Irkutsk Region the proportions of the Asian and Eurasian branches were comparable (Table S3, Figure 3 ).

Figures

FIGURE 3. Proportions of two restriction spectra of Simulium reptans s.l. among the studied material collected in different localities in the territory of Ob-Irtysh basin. Si16, Si19, Si28–sample numbers; ns–non-significant differences (Fisher’s exact test, two-tailed, with Bonferroni correction, p> 0.017).

FIGURE 5. Primer annealing sites and multiplex AS-PCR results for samples of Simulium reptans s.l. from the Si16 sample (Novosibirsk). A–Annealing site of a primer specific for S. reptantoides; consensus sequences of molecular forms are given, as well as the sequence of a unique individual that we studied. B–Annealing site of a primer specific for S. reptans. C–results of electrophoresis of AS-PCR products from the Si16 sample, lane No. 4 corresponds to the unique sample Si16-4, which gives two PCR products, for the remaining specimens only the PCR product with the size predicted for S. reptans is visible (579 bp). Lane on the right (No. 11)–molecular weight marker 100bp+1.5+3. For A and B, vertical lines indicate complementary pairs, and the arrow indicates the order of nucleotides in the primer.

FIGURE 6. Typical results of ITS2 sequencing of Simulium reptans s.l. A–with nucleotide A in the position in question, B –with A/T (W) superposition, C–with nucleotide T.

FIGURE 7. Maximum likelihood tree constructed from the DNA fragment of the COI gene of S. reptans. A branch of S. reptantoides was used for rooting. The geographical origin of the individuals is indicated. Sequences taken from the NCBI database are designated by corresponding numbers. Bayesian posterior probabilities (above the branches) and bootstrap values (below the branches) are given.