Research Article |
Corresponding author: Elena V. Shaikevich ( elenashaikevich@mail.ru ) Academic editor: Vladimir Lukhtanov
© 2016 Elena V. Shaikevich, Ludmila S. Karan, Marina V. Fedorova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Shaikevich EV, Karan LS, Fyodorova MV (2016) Comparative analysis of the circadian rhythm genes period and timeless in Culex pipiens Linnaeus, 1758 (Diptera, Culicidae). Comparative Cytogenetics 10(4): 483-504. https://doi.org/10.3897/CompCytogen.v10i4.7582
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Nucleotide sequences of the circadian rhythm genes, period and timeless, were studied for the first time in mosquitoes Culex pipiens Linnaeus, 1758. In this work we evaluated variations of the studied genome fragments for the two forms of C. pipiens (forma “pipiens” – mosquitoes common for aboveground habitats, forma “molestus” – underground mosquitoes). We compared C. pipiens from Russia with transatlantic C. pipiens and subtropical Culex quinquefasciatus Say, 1823. Our results show that intraspecies variability is higher for the gene period than for the gene timeless. The revealed substitutions in nucleotide sequences and especially in amino acid sequences grouped the individuals of the two forms into distinct clusters with high significance. The detected fixed amino acid substitutions may appear essential for functioning of the circadian rhythm proteins in C. pipiens, and may be correlated with adaptations of the taxa within the group C. pipiens. Our results suggest that natural selection favors fixed mutations and the decrease in diversity of the genes period and timeless in mosquitoes of the C. pipiens f. “molestus” compared with the C. pipiens f. “pipiens”, is probably correlated with adaptive features of C. pipiens f. “molestus”. The studied genome regions may be considered as promising molecular-genetic markers for identification, population and phylogenetic analysis of similar species and forms of the Culex pipiens complex.
Culex pipiens , circadian rhythm genes, period , timeless , natural selection
The Culex pipiens Linnaeus, 1758 complex considered by some authors as a ‘polytypic species’ includes up to seven morphologically identical or very similar forms (
Only one species of the Culex pipiens complex, C. pipiens, has been found in Russia. This species includes two forms, C. pipiens f. “pipiens” and C. pipiens f. “molestus”, originally described as distinct species (
Among the specific behavioral/physiological traits which remained up to now the important criteria for defining populations of C. pipiens f. “pipiens” and C. pipiens f. “molestus”, differences in mating behavior are under the special interest. Mating activity of C. pipiens f. “pipiens” is restricted within the crepuscular period when males aggregate in swarms where they copulate with virgin females attracted to a swarm (
In insects the rhythms of mating activity are controlled by endogenous circadian clocks, which are under genetic control (
Clock genes, especially period and timeless, play an essential role in regulation of mating rhythms in insects. In Drosophila, null mutants of the clock gene period (per) lost the circadian rhythm in mating activity (
It may be suggested that differences in the rhythm of mating activity in two forms of C. pipiens resulted from the variations in circadian clocks genes. To test this hypothesis, we selected the genes per and tim. The aim of our work was to study variable nucleotide sequences in these genes, and to estimate the possible evolutionary significance of the detected variations.
The larvae of mosquitoes of both intraspecific forms were collected mostly in August 2006 in Volgograd City and nearby areas. The sampling sites, methods of larvae collection and rearing in lab, and methods of evaluating autogenity have been described earlier (
The DNA was isolated using the kit DIAtom™ DNA Prep (Isogen Russia). Each of the amplification reactions used 0.1 μg of the total DNA. The polymerase chain reaction (PCR) was run on the thermocycler GeneAmpR PCR System 2700 (Applied Biosystems USA), with amplification Encyclo PCR kit (Evrogen Russia), following the manufacturer’s instructions. For PCR and sequencing of amplification products, specific primers were constructed which were complementary to the conserved sequences of exons in the published sequences of the genes period and timeless from the total genome of a similar species C. quinquefasciatus (Vector Base Gene ID CPIJ007193 and CPIJ007082, respectively) (
primer | sequence | Tm (°C) | region |
---|---|---|---|
PerF2 | 5’-AGTTCCAAATCGCGCCACAG-3’ | 54 | per exon 2 |
PerR2 | 5’-TTGGGTTTGCTCGCTTCGTTC-3’ | 54 | per exon 2 |
PerF3 | 5’-ACAATGCATAGCCAACCGCAAG-3’ | 55 | per exon 3 |
PerR3 | 5’-GTTCGTCCCTTGACCATGATC-3’ | 54 | per exon 3 |
PerF4 | 5’-AACGGCTGTTATCTCGTACTG-3’ | 52 | per exon 4 |
PerR4 | 5’-GCATCGCGTGGTACATCATCG-3’ | 56 | per exon 4 |
TimF1 | 5’-AATGGTTGCTAGCGAATCCG-3’ | 52 | tim exon1 |
TimR1 | 5’-AGTAGAGTTCTCGACACCCG-3’ | 54 | tim exon1 |
TimF5 | 5’-GATTGGTCGGATTTGATTGAG-3’ | 50 | tim exon5 |
TimR5 | 5’-GTATGTCATCAACCGCCTTG-3’ | 52 | tim exon5 |
TimF5-1 | 5’-GGAAACCAGCAAAAGACTCG-3’ | 52 | tim intron5-6, exon6, intron6-7 |
TimR7 | 5’-TACGAGAGCACGTTGAACTG-3’ | 52 | tim intron5-6, exon6, intron6-7 |
TimF7 | 5’-ACATACTGTACAACATTGCCCTG-3’ | 53 | tim intron7-8 |
TimR8 | 5’-TCAGGTCGAACTTGATGATG-3’ | 50 | tim intron7-8 |
TimF9 | 5’-GCTGCGGCCGAAAGCGCCAG-3’ | 60 | tim intron9-10 |
TimR10 | 5’-ATTTCCATCGCTCGTGTGCTG-3’ | 54 | tim intron9-10 |
The DNA sequences were translated into amino acids sequences using ExPASy software (Swiss Institute of Bioinformatics), and compared with amino acids sequences of C. quinquefasciatus (
Phylogeny analysis was run in MEGA6. The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. All ambiguous positions were removed for each sequence pair.
Natural selection and the probability of rejecting the null hypothesis of strict-neutrality (dN = dS) was evaluated using MEGA6. For these purposes was used a codon-based Z-test (MEGA6). For a pair of sequences, this is done by first estimating the number of synonymous substitutions per synonymous site (dS) and the number of nonsynonymous substitutions per nonsynonymous site (dN), and their variances: Var(dS) and Var(dN), respectively. With this information, we tested the null hypothesis that there is no impact of selection (dN = dS) and the probability (P) of rejecting the null hypothesis of strict-neutrality. Also was tested an alternative hypothesis of purifying selection (dN < dS) and the probability of rejecting the null hypothesis of strict-neutrality in favor of the alternative hypothesis using a codon-based Z-test (MEGA6). Values of P determine statistical significance in a hypothesis test. A low P value suggests that sample provides enough evidence for the rejecting of the null hypothesis for the entire population. Values of P less than 0.05 are considered significant at the 5% level. The variance of the difference was computed using the analytical method (
The structure of the gene per was studied in three individual C. pipiens f. “molestus” and in three individual C. pipiens f. “pipiens”. Coding sequences of the three exons of the gene per were analysed: exon 2, 333 bp, exon 3, 738 bp, and exon 4, 1229 bp. In total, the 18 compared sequences spanned each 2300 bp (Suppl. material
In the exon 2 of the gene per (333 bp) 11 variable sites were found; six of these substitutions resulted in amino acid substitutions in both forms of C. pipiens (Fig.
Variable amino acid sites of the gene period in C. pipiens. C. pipiens from the USA (KM355980) and C. quinquefasciatus (CPIJ007193) are taken for the comparison. Exons 2 (sites 1-111), 3 (113-358), and 4 (360-768) are separated with blank columns. Positions of the variable sites relative to combined sequences as presented in Suppl. material
Estimates of Evolutionary Divergence over per and tim sequence pairs between Culex pipiens complex members.
AA\NA | gene period | gene timeless | |||||||
---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | ||
1 | molestus | 0.010 | 0.010 | 0.027 | 0.012 | 0.025 | 0.028 | ||
2 | pipiens | 0.011 | 0.008 | 0.029 | 0,008 | 0.031 | 0.030 | ||
3 | pipiensUSA | 0.013 | 0.008 | 0.027 | 0.018 | 0.018 | 0.009 | ||
4 | quin | 0.036 | 0.036 | 0.041 | 0.018 | 0.020 | 0.004 |
The obtained sequences of the gene per of C. pipiens from Volgograd and C. pipiens f. “pipiens” from the USA (GenBank acc. number KM355980) using BLAST software were compared. The identity of nucleotide sequences of C. pipiens f. “pipiens” mosquitoes from different continents is 98-99%; 4-16 amino acid substitutions were detected. Pairwise comparison showed that C. pipiens f. “pipiens” from the USA is slightly different from the Volgograd C. pipiens f. “pipiens” (0.008) and from C. pipiens f. “molestus” (0.013); these values are comparable with the differences between the studied C. pipiens f. “pipiens” and C. pipiens f. “molestus” (Table
The identity of the nucleotide sequences of the gene per for the two species was 97%. Comparison of the DNA from both forms of C. pipiens and C. quinquefasciatus (CPIJ007193) revealed 113-116 (4.9–5.5%) variable nucleotide sites (Suppl. material
Using the DNA from the same three individual mosquitoes C. pipiens f. “molestus” and three individual mosquitoes C. pipiens f. “pipiens”, the three longest coding sequences of the gene tim were studied: exon 1 (1037 bp), exon 5 (376-379 bp), and exon 6 (145 bp). In total, the 18 compared sequences each spanned 1557-1560 bp. (Suppl. material
In exon 1 of the gene tim (1037 bp) 15 variable nucleotide sites and five variable amino acid sites were found, four of them showing variations only for the C. pipiens f. “pipiens” and were not found in C. pipiens f. “molestus” (Fig.
Variations of amino acid sites in the gene tim from C. pipiens. C. pipiens from the USA (KM355979), and C. quinquefasciatus (CPIJ007082) are taken for the comparison. Dashes show deletion in exon 1 in C. pipiens from the USA (KM355979). Exon 1 (sites 1-345) and exon 5 (sites 347-472) are separated by blank columns. Positions of the variable sites in combined tim sequences shown on the top.
Comparing the nucleotide sequences of the gene tim for the specimens of C. pipiens f. “molestus” one variable DNA site was found, the detected nucleotide substitution does not result in amino acid substitution. The aligned DNA sequences of the C. pipiens f. “pipiens” had 11 variable sites, one mutation resulting in amino acid substitution (Fig.
The obtained sequences of the gene tim for C. pipiens from Volgograd and C. pipiens f. “pipiens” from the USA (KM355979) were compared using BLAST software. Identity of nucleotide sequences for mosquitoes of C. pipiens f. “pipiens” from different continents is 96-97%. We found 7-12 amino acid substitutions. Unexpectedly, we found a 60-bp deletion within the coding sequence of exon 1 in C. pipiens f. “pipiens” from the USA (KM355979), positions 263-282 in Fig.
Comparison of DNA sequences of exons 1, 5 and 6 of the gene tim between representatives of the two species, C. pipiens and C. quinquefasciatus, revealed 50 variable sites (see Suppl. material
Our results showed that exon 1 of the gene tim in C. pipiens f. “molestus” differ from that of in C. pipiens f. “pipiens” (Fig.
The obtained nucleotide sequences showed overlapping peaks in one or more sites for 6 individuals. Four of them were identified as C. pipiens f. “molestus” and two as C. pipiens f. “pipiens”. Exon 1 of the gene tim of these six samples was studied by cloning and the DNA of the five clones for each specimen was sequenced. In total 79 sequences were obtained for comparative analysis (Suppl. material
49 variable nucleotide sites and 23 distinct haplotypes were found in exon 1 of the gene tim (Fig.
DNA haplotypes and variable amino acid positions in the exon 1 of the gene tim from C. pipiens f. “pipiens” and C. pipiens f. “molestus”. Haplotypes numbers and variable nucleotide sites are shown on the left. Variable amino acid sites are shown on the right. Only variable haplotypes are shown, all 79 sequences are presented in Suppl. material
The DNA polymorphism of the exon 1 of gene tim among specimens of the C. pipiens f. “pipiens” (0.007) was ten times higher than for the C. pipiens f. “molestus” (0.0006), and variability of the amino acid sequences was 0.0053 and 0.0001, respectively. Genetic distance between the two forms was 0.011 for DNA sequences and 0.009 for amino acid sequences. Genetic distance between C. pipiens of both forms and C. quinquefasciatus was 0.029 for DNA and 0.02 for amino acid sequences (Suppl. material
The sequences of some non-coding regions were analysed, expecting to find differences not only in coding DNA structure but also in intron size between C. pipiens f. “pipiens” and C. pipiens f. “molestus”. The primers were constructed for the conserved sites of the exons using the obtained sequences, and by homology with the gene tim from C. quinquefasciatus (CPIJ007082). The sequences of the introns 1-2 (7158 bp in length) and 10-11 (5189 bp), being too long for efficient PCR and sequencing and containing numerous repeats were not analysed. As for the other introns, sequencing of the PCR products showed no variability between two intraspecific forms in intron between exons 5 and 6 (59 bp). In intron 6-7 (61 bp) three variable sites and in intron 7-8 (160 bp) six variable sites were found. Studied introns showed no mutations common with either of the two C. pipiens forms. In the intron 9-10 (167 bp) seven variable sites were found, six of which differed between the two forms (Suppl. material
Phylogenetic dendrograms were constructed applying the Neighbor-Joining method to amino acid sequences of the three coding regions of genes per and tim, C. quinquefasciatus was used as an out-group. C. quinquefasciatus and C. pipiens form two well differentiated clusters. Basing on similarity of the gene per the individuals of the C. pipiens f. “pipiens” group together and form a joint cluster with a bootstrap coefficient of 97. The studied specimens of the C. pipiens f. “molestus” had more polymorphic amino acid sequences, but also are grouped into one cluster with bootstrap coefficient of 63 (Fig.
Evolutionary relationships of the studied taxa. Neighbor-joining trees of C. pipiens based on Aperiod and Btimeless inferred amino acid sequences with the C. quinquefasciatus (CPIJ007193) as the outgroup. Percent bootstrap support based on 1000 replicates. Seven amino acid sequences were analysed with a total of 766 positions of PERIOD (A) and 519 positions of TIMELESS (B) in the final datasets.
On the dendrogram for the exon 1 of the gene tim, constructed using the results of our extended study, most specimens of the C. pipiens f. “molestus” form separate clusters with a bootstrap coefficient of 96. A separate subcluster is formed by sequences of the hybrid V219 clones with haplotype H4. The studied specimens of the C. pipiens f. “pipiens” have polymorphic DNA sequences (Suppl. material
One way to test whether natural selection is operating on a gene is to compare the relative abundance of synonymous and nonsynonymous substitutions within the gene sequences (
Analysis of dN-dS between the 79 nucleotide sequences of exon 1 of the gene tim indicates that the probability of rejecting the null hypothesis of strict-neutrality between intraspecific forms ranges from 0.002 to 0.15 across the sequences with an overall average of 0.05. The number of synonymous substitutions per site (dS) was higher that the number of non-synonymous substitutions per site (dN), indicating Purifying Selection. The probability of rejecting the null hypothesis of strict-neutrality (dN = dS) in favor of the alternative hypothesis of Purifying Selection (dN < dS) ranges for the tim nucleotide sequences of C. pipiens f. “pipiens” and C. pipiens f. “molestus” from 0.001 to 0.10 with an overall average of 0.025 (Suppl. material
For the first time the genetic structure of the circadian rhythm genes (per and tim) were analysed for mosquitoes C. pipiens f. “molestus”. Our results have shown that DNA variation in individuals of C. pipiens f. “molestus” is smaller than in individuals of C. pipiens f. “pipiens”. Extended study of exon 1 of the gene tim revealed 4 DNA haplotypes in C. pipiens f. “molestus” and 19 haplotypes in C. pipiens f. “pipiens”. Decrease in DNA variability for the underground mosquitoes of C. pipiens f. “molestus” was also reported earlier in our study of mitochondrial DNA (
In coding sequences of both genes per and tim, variations between physiologically different forms of C. pipiens were found (Table
Comparison of exons and introns variability between C. pipiens f. “pipiens” and f. “molestus” from Russia.
Gene | Locus | size (bp) | Variable DNA sites | Differentiating DNA sites | Variable AA sites |
Differentiating AA sites |
---|---|---|---|---|---|---|
Per | exon2 | 333 | 11 | 6 | 6 | 4 |
exon3 | 738 | 12 | 2 | 3 | 1 | |
exon4 | 1229 | 27 | 9 | 4 | 1 | |
Tim | exon1 | 1037 | 49 | 2 | 10 | 1 |
exon5 | 376–379 | 5 | 1 | 0 | 0 | |
exon6 | 145 | 3 | 0 | 0 | 0 | |
intron5-6 | 59 | 0 | 0 | - | - | |
intron6-7 | 61 | 3 | 0 | - | - | |
intron7-8 | 160 | 6 | 0 | - | - | |
intron9-10 | 167 | 7 | 6 | - | - |
Codon-based Test of Neutrality between C. pipiens f. “pipiens” and C. pipiens f. “molestus”.
specimen | gene period | gene timeless | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 1 | 2 | 3 | 4 | 5 | 6 | ||
1 | pipiens1 | -1.950 | -2.829 | -3.130 | -3.682 | -3.309 | -2.307 | -2.009 | -2.830 | -2.779 | -2.679 | ||
2 | pipiens2 | 0.108 | -2.904 | -3.326 | -3.865 | -3.349 | 0.034 | -1.856 | -2.906 | -2.859 | -2.766 | ||
3 | pipiens3 | 0.010 | 0.008 | -3.775 | -4.284 | -3.788 | 0.089 | 0.0663 | -2.945 | -2.899 | -2.805 | ||
4 | molestus1 | 0.001 | 0.003 | 0.000 | -1.534 | -0.586 | 0.0047 | 0.0023 | 0.005 | -0.333 | -0.998 | ||
5 | molestus2 | 0.000 | 0.000 | 0.000 | 0.154 | -1.427 | 0.0056 | 0.0027 | 0.0058 | 0.774 | -0.665 | ||
6 | molestus3 | 0.002 | 0.003 | 0.000 | 0.502 | 0.15 | 0.0073 | 0.0034 | 0.0075 | 0.320 | 0.547 |
C. pipiens f. “pipiens” from N America clusters with C. pipiens f. “pipiens” from Volgograd basing on comparison of the gene per and with C. quinquefasciatus based on comparison of the gene tim. It remains unknown whether this is common for all American C. pipiens f. “pipiens”, shown using microsatellite analysis to differ from the European C. pipiens f. “pipiens” (
Genetic structure of the studied genes is polymorphic. However, the revealed substitutions in nucleotide sequences and especially in protein sequences grouped the individuals of the two forms into distinct clusters with high significance, a longer genetic distance separating the cluster of C. pipiens from C. quinquefasciatus. Although the two studied genes differed in variability, the results of analysis of the gene per, as well as the gene tim, show that the difference between C. pipiens and C. quinquefasciatus are 2.5–3 times higher than the difference between the forms of C. pipiens. The genetic distances again confirm the order of evolutionary events in the C. pipiens complex: the divergence of the form C. pipiens f. “molestus” from C. pipiens occurred considerably later than the divergence of C. pipiens and C. quinquefasciatus (
The non-coding genome sequences are considered to be highly variable. These sequences are often used to search for the markers to differentiate closely related organisms by size of the PCR products. For example, variation in spacers of the ribosomal genes cluster is a base for identification of some mosquito species of the genus Anopheles (
The Test of Neutrality rejects the null hypothesis of strict-neutrality at P < 5% level and imply that both per and tim loci evolve under strong selective constraint during the divergence of intraspecific forms. Our results suggest that natural selection favored the fixed mutations and the decreased diversity of the genes per and tim in mosquitoes C. pipiens f. “molestus” compared with the C. pipiens f. “pipiens”, probably preserving adaptive features of the form “molestus”. Well-documented data have been reported showing that new native mutations sometimes are rapidly spreading in a population and that polymorphism in one locus may provide adaptive variations in behavioral and morphological phenotypes of the insects in nature (
Nucleotide sequences of the circadian rhythm genes were studied for the first time in mosquitoes C. pipiens f. “molestus” and compared with those for C. pipiens f. “pipiens” and C. quinquefasciatus. These results show that intraspecies variability is higher for the gene per than for the gene tim. Revealed substitutions in nucleotide sequences and especially in protein sequences grouped the individuals of the two ecological forms of C. pipiens into distinct clusters with high significance. The results suggest that natural selection favored the fixed mutations and the decreased diversity of the genes per and tim in mosquitoes of the C. pipiens f. “molestus” compared with the C. pipiens f. “pipiens”. The detected fixed amino acid substitutions may appear essential for functioning of the circadian rhythm proteins in C. pipiens, and may be related with adaptations of the taxa within the group C. pipiens. Moreover, under natural selection mutations in the key genes of circadian pattern may provide some advantage to the underground C. pipiens f. “molestus”. The studied genome regions may be considered as promising molecular-genetic markers for identification, population and phylogenetic analysis of similar species and forms of the C. pipiens complex.
This work was supported by the Russian Foundation of Fundamental Research, grants N 14-04-0112914, N 16-04-00091 and the European Commission in the framework of FP7-261391 EuroWestNile research project.
Aligned nucleotide sequences of per gene.
Data type: primary data
Explanation note: DNA sequences of three clones of each individual C. pipiens are presented and compared with sequences of C. quinquefasciatus (CPIJ007193) and C. pipiens from the USA (KM355980).
Aligned nucleotide sequences of tim gene.
Data type: primary data
Explanation note: DNA sequences of three clones of each individual C. pipiens are presented and compared with sequences of C. quinquefasciatus (CPIJ007193) and C. pipiens from the USA (KM355980).
Analysis of the divergent between two forms of C. pipiens based on comparison of the exon 1 of the gene tim sequences.
Data type: primary data
Explanation note: Nucleotide and amino acid sequences of the tim gene exon1 in compare with sequence of C. quinquefasciatus.
Aligned tim nucleotide non-coding sequences.
Data type: primary data
Explanation note: Intron’s DNA sequences of individual C. pipiens f. “pipiens” and C. pipiens f. “molestus” are presented.
Codon-based Test of Neutrality for analysis between per gene sequences of C. pipiens both forms.
Data type: measurement of Z-test value
Explanation note: The test statistic (dN - dS) and the probability of rejecting the null hypothesis of strict-neutrality (dN = dS) are shown base on the differences between per gene sequences of C. pipiens both forms.
Codon-based Test of Neutrality for analysis between tim gene sequences of C. pipiens both forms.
Data type: measurement of Z-test value
Explanation note: The test statistic (dN - dS) and the probability of rejecting the null hypothesis of strict-neutrality (dN = dS) are shown base on the differences between tim gene sequences of C. pipiens both forms.
Codon-based Test of of Purifying Selection for analysis between the exon1 of the gene tim sequences of C. pipiens both forms.
Data type: measurement of Z-test value
Explanation note: The test statistic (dN - dS) and the probability of rejecting the null hypothesis of strict-neutrality (dN = dS) are shown base on the differences between the exon1 of the gene tim sequences of C. pipiens both forms.