How to Read Cytochrome C Oxidase I

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Molecular evolution of cytochrome C oxidase-I protein of insects living in Saudi Arabia

• Samar Rabah,
• Haitham Yacoub,
• Nahid H. Hajrah,
• Ahmed Atef,
• Mohammed Al-Matary,
• Sherif Edris,
• Mona G. Alharbi,
• Magdah Ganash,
• Jazem Mahyoub,
• Rashad R. Al-Hindi,
• Khalid Chiliad. Al-Ghamdi,
• Neil Hall,
• Ahmed Bahieldin,
• Majid R. Kamli,
•  [ ... ],
• Irfan A. Rather
• [ view all ]
• [ view less ]

Molecular development of cytochrome C oxidase-I protein of insects living in Kingdom of saudi arabia

• Jamal South. Grand. Sabir,
• Samar Rabah,
• Haitham Yacoub,
• Nahid H. Hajrah,
• Ahmed Atef,
• Mohammed Al-Matary,
• Sherif Edris,
• Mona G. Alharbi,
• Magdah Ganash,
• Jazem Mahyoub

x

• Published: November 4, 2019
• https://doi.org/x.1371/journal.pone.0224336

Figures

Abstruse

The study underpins barcode characterization of insect species nerveless from Kingdom of saudi arabia and explored functional constraints during evolution at the DNA and protein levels to await the possible mechanisms of poly peptide development in insects. Codon structure designated AT-biased insect barcode of the cytochrome C oxidase I (COI). In add-on, the predicted 3D structure of COI protein indicated tyrosine in close proximity with the heme ligand, depicted commutation to phenylalanine in two Hymenopteran species. This change resulted in the loss of chemical bonding with the heme ligand. The estimated nucleotide commutation matrices in insect COI barcode more often than not showed a college probability of transversion compared with the transition. Computations of codon-past-codon nonsynonymous substitutions in Hymenopteran and Hemipteran species indicated that almost half of the codons are nether positive development. All the same, codons of COI barcode of Coleoptera, Lepidoptera and Diptera are more often than not under purifying selection. The results reinforce that codons in helices 2, 5 and 6 and those in loops ii–three and 5–half-dozen are by and large conserved and approach strong purifying selection. The overall results argue the possible evolutionary position of Hymenopteran species amongst those of other insects.

Citation: Sabir JSM, Rabah S, Yacoub H, Hajrah NH, Atef A, Al-Matary M, et al. (2019) Molecular evolution of cytochrome C oxidase-I poly peptide of insects living in Saudi Arabia. PLoS Ane 14(11): e0224336. https://doi.org/10.1371/periodical.pone.0224336

Editor: Vivek K. Bajpai, Dongguk Academy, Democracy OF KOREA

Received: August 21, 2019; Accustomed: Oct 10, 2019; Published: November 4, 2019

Copyright: © 2022 Sabir et al. This is an open admission commodity distributed nether the terms of the Artistic Eatables Attribution License, which permits unrestricted utilise, distribution, and reproduction in any medium, provided the original writer and source are credited.

Data Availability: All relevant data are inside the paper and its Supporting Information files.

Funding: This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, nether grant no. (99-130-35-HiCi) to JSMS. The authors, therefore, acknowledge with thanks DSR technical and financial support. The funders had no role in report design, data drove and analysis, determination to publish, or training of the manuscript.

Competing interests: The authors accept alleged that no competing interests exist.

Introduction

The fact that many insect species are difficult to be discriminated at the morphological level, as well as the huge number of cryptic species, makes the global species count uncertain [i]. The adoption of DNA-based molecular markers represents a satisfactory alternative. Since the proposal of Deoxyribonucleic acid barcoding in 2003, subunit I (658 bp) of the mitochondrial cytochrome C oxidase (COX) gene (namely COI) became the almost universal marking for species identification in the animal kingdom [two,3]. The recently developed Barcode Index Number (BIN) organisation [4] (Ratnasingham and Hebert, 2013) can human activity as a powerful culling to morphological species that easily distinguishes the occurrence of variety or possible speciation [5,6,7,8,9,10,xi,12]. Technically speaking, this arrangement complements the molecular-based approaches of species identification to strengthen and support the evolutionary analysis in insects.

Documentation of insect diversity in the Sahara-Arabian region including Saudi Arabia has recently taken place [13] as the biodiversity data available for this region is bereft, compared with those in Canada or the U.s.. Documentation based on the criteria of the International Union for Conservation of Nature allows recognizing invasive alien species or list threatened species. Malaise traps were successfully used in scoring richness of insect species and biodiversity surveillance in regions that are difficult-to-admission [xiv] and/or in the absence of formal taxonomic assignments [15].

COX enzyme functionally participates in the electron transport chain by reducing oxygen and pumping protons across the inner mitochondrial membrane. The encoded poly peptide of COI comprising 219 amino acids (AAs) consists of 6 polypeptide chains and a few metallic ligands. The latter includes 2 iron atoms bound in 2 heme groups, three coppers, one zinc and ane magnesium [16,17]. Changes in the AA sequence within the COI region likely reduce cellular free energy metabolism, especially when changes occur close to the active sites of the enzyme [18]. Purifying choice predominantly occurs for animate being mitochondrial poly peptide-coding genes, thus, results in the deficient of AA substitutions, especially in the COX genes [19,xx,21]. However, bear witness of nonsynonymous AA commutation supports the notion of positive selection mostly in fauna and specifically in course Insecta. Due to the functional constraints benchmark, changes should be biased to the nonfunctioning regions of the gene supporting the merits that evolution is non neutral [20,22]. Positive selection for changes in mitochondrial proteins during evolution could exist caused past lifestyle changes, and this has been observed in snakes changing their metabolic rate to complement lifestyle [21].

In this piece of work, we innovate a model study on the COI subunit of specimens of six insect orders nerveless from Kingdom of saudi arabia to generate DNA barcodes and detect variation among species and orders at the AA level. Based on the latter, changes in COI protein structure at the 3-dimensional model are projected. Through this model study, we gained new insights into the possible mechanisms by which COI poly peptide evolves in insects living in Saudi arabia.

Materials and methods

For sample collection, a Malaise trap [23,24] (Hutcheson and Jones, 1999; Loma et al., 2005) was installed at Hada Al-Sham station, King Abdulaziz University (KAU) located in the western region (near Makkah) of Saudi Arabia (21.795^oNorth, 39.711^oE). Samples were nerveless on a weekly footing for 4 weeks (May 1–28, 2017) into 95% ethanol and stored at -20°C for farther assay. Specimens were morphologically classified downwards to the species level, and so total DNAs were recovered from individual tissue samples according to Evans and Paulay [25]. PCR was performed to recover the COI gene fragment (650 bp) every bit described by Ashfaq et al. [13] and amplicons were shipped to Macrogen (South Korea) for Sanger sequencing. Recovered sequences were checked for quality and those meeting the standard criteria were further assigned to subsequent analysis. Good quality sequences were trimmed and algorithmically aligned with Clustal Omega with strict parameters and the resulting alignments were manually refined before translation. Deduced AA sequences were recovered consulting the invertebrate mitochondrial code. Samples with nucleotide deletions were excluded upon Deoxyribonucleic acid multi-sequence alignment, and but in-frame AA sequences were retained. A consensus sequence of Odonata (accretion no. JN294479) was used as a reference for comparison. This society is the near aboriginal in the hexapoda insect phenogram [26]. Binary data matrices were entered into TFPGA (version 1.three) and analyzed using qualitative routine to generate a similarity coefficient. Contrast coefficients were used to construct dendrograms using unweighted pair group method with arithmetic boilerplate (UPGMA) and sequential hierarchical and nested clustering (Neighbor-Joining or NJ) routine using NTSYSpc (version 2.10, Exeter software). Phylogeny tree generated by Bybee et al. [26], was taken every bit a reference ancestral order for testing insect lineage and evolution.

The AA sequence of cattle (Bos taurus) was used every bit a reference [27] for detecting changes of COI poly peptide of selected species at the 3D construction level. We recruited the bovine protein X-ray structure (Protein Data Banking concern ID 1OCC) as a homology model of the COI barcode region to build 3D structures of selected insect species using the I-TASSER Suite [28]. Distance measurements between AAs, on one side, and the two heme ligands referenced as 515 and 516, on the other side, were estimated using UCSF Chimera 1.13.one [29]. Electrostatic potential of the COI protein was estimated using DeepView—Swiss-PdbViewer (v4.1) (http://www.expasy.org/spdbv)

Nucleotide substitution patterns, e.thou., synonymous (S) and nonsynonymous (N), were generated using the Approximate Substitution Matrix feature in MEGA five. 6 and estimates of the numbers of Southward and Due north substitutions per site were made using the joint Maximum Likelihood reconstructions of ancestral states [30] of codon exchange and Felsenstein model [31] of nucleotide exchange. Changes in AA sequences were scored referring to the consensus sequence of Odonata.

Results and word

COI barcode was analyzed at the Dna and protein levels for insects living in Kingdom of saudi arabia in guild to gain new insights as to how this gene and its encoded protein evolve among different insect orders. A number of 560 samples were collected by the Angst trap within a catamenia of four weeks. Photographs were taken for all specimens, while the number was somewhen narrowed to one photo per species as shown in S1 Fig. DNAs were purified from these samples and the recovered mitochondrial COI cistron fragments were sequenced. We projected to include only sequences with ≥ 634 nucleotides encoding all loops and helices of the COI protein. The number of deduced AAs is 211 starting at codon position 12 referring to the numbering made by Pentinsaari et al. [32]. Based on the rigid quality control criterion, the number of samples was narrowed to 175 for further analysis. The consensus DNA sequence of Odonata was included for comparison equally this order has an ancient phylogenetic position being the representative of the starting time ancient winged insects in the hexapoda phylogeny [26]. This social club has extensively been used every bit a reference in contemporary evolutionary genomic studies [33]. BLAST analysis indicated that a number of 30 species representing six insect orders were collected during the course of this study. Ii of which vest to Hemimetabolous, e.chiliad., Blattodea (one species) and Hemiptera (v species), while iv belong to Holometabolous, due east.yard., Hymenoptera (six species), Coleoptera (six species), Lepidoptera (v species) and Diptera (7 species). One of the Hymenopteran species was not precisely identified at both the morphological and molecular levels, while showed the closest relationship (98%) with Apoidea sp.

Multiple Deoxyribonucleic acid sequence alignment indicated that a number of 265 nucleotides out of 634 are common (conserved) in the recovered COI barcode, while the rest varied among orders and species as shown in S2 Fig. A phylogenetic tree constructed from the sequences indicated the close relationship between the species of Odonata and Blattodea as both orders are hemimetabolous (S3 Fig). Recent research indicated that Blattodea is the closest amongst the six orders of the nowadays report to Odonata (Fig 1) [26]. Interestingly, a shut relationship was shown between species of Hemiptera and Hymenoptera although the beginning is hemimetabolous, while the second is holometabolous. Species of Coleoptera were shown in close relationship with those of Diptera (S3 Fig).

Fig ane. Distribution of nucleotides within codons across six insect orders.

Nucleotide frequencies at each of the three codon positions in COI barcode sequence of species of half dozen insect orders in add-on to an Odonata sp. used for comparing.

https://doi.org/ten.1371/journal.pone.0224336.g001

As it is the case with animal mitochondrial Deoxyribonucleic acid [32], the DNA COI barcode sequences analyzed for the vi orders is AT-biased (eastward.g., AT content = ~68%) as shown in Fig 1. The AT content was lowest (~57%) for the nucleotides in the get-go position of all codons, while ~58% in the second position and as high equally ~90% in the third position. The G nucleotide has proven to be the to the lowest degree degenerate among the four nucleotides in the third position. Interestingly, A nucleotide in the second position was unexpectedly low (<15%), while C was high (~25%). No Thousand nucleotide was scored in the third position for four species namely Hymenoptera sp., Belenois aurota, Asyndetus sp. and Carpomya vesuviana. The commencement two species are Hymenoperan and Lepidopteran, respectively, while the other 2 are Dipteran. The lowest C nucleotide in the tertiary position was scored for the ii Hymenoptran species Tachytes crassus (<ii%) and Nomioides facilis (<1%) (Fig 1).

Amino acid sequences of COI barcode were sorted out in this written report based on their chemical properties into standard groups: nonpolar aliphatic (G, A, V, L, M and I), polar uncharged (S, T, C, P, N and Q), aromatic (F, Y and W), positively charged (K, R and H) and negatively charged (D and E). The barcode sequences of unlike species largely encode nonpolar AAs (Fig 2). In that location are differences in the AA composition of the barcode sequences amongst orders. Alanine (A) and valine (V) are lower in barcodes of Hymenopteran species likewise as of the Hemipteran species Batracomorphus angustatus compared with those of the other orders. On the other hand, lysine (G) is exclusively encoded by barcodes of the latter species although there are very few. Four species of the latter two orders) Hymenoptera and Hemiptera) also exclusively encode cysteine (C). The two AAs Chiliad and C do not exist in the sequence of cattle COI barcode [32]. We speculate that they appeared due to AA substitutions in several positions of the animate being AA sequence of the barcode of Batracomorphus angustatus. Three other AAs seem to exist encoded in very small amounts in few orders such as glutamine (Q), tyrosine (Y) and glutamic acid (E). Similar results were previously reached by Pentinsaari et al. [32] when studying Coleopteran and Lepidopteran species. The scarcity of these AAs in barcodes can perhaps be compensated by AAs of the same chemical groups at the same position in the polypeptide concatenation. For instance, tyrosine (Y) exists in iii positions, due east.one thousand., 38, 113 and 215. The position in the heart is conserved amidst the half dozen orders, while substituted to phenylalanine (F) in the other two positions. The 2 AAs exist in the aforementioned chemic group (aromatic). Interestingly, the five rare AAs (C, G, Q, Y, and East) are encoded by twofold degenerate codons. Cysteine in the COI region exists in four species of Hymenoptera and one species in Hemiptera although in very pocket-size amounts, while completely absent in other species of the six orders. Changes in cysteine in the barcode sequence can severely affect the secondary construction also as the stabilization of tertiary and quaternary structures of the COI protein.

Fig 2. Distribution of aminoacids within the barcodes across six insect orders.

Amino acid frequencies in barcode sequences of species of six insect orders in addition to an Odonata sp. used for comparing. AAs are grouped vertically based on their biochemical properties.

https://doi.org/10.1371/journal.pone.0224336.g002

Multiple AA sequence alignment of the COI barcode of the thirty species is shown in S4 Fig. The encoded AA sequences of the Dna barcode fragment across the six orders cover 211 AAs starting from position 12 to 222 based on the numbering fabricated by Pentinsaari et al. [32]. This portion of the barcode includes the enzymatically active part of COX mediating electron transfer from Cu to heme. Amino acid sequence variation in each position was scored in which nosotros referred to conserved AAs by asterisks while increasing variability by reducing the number from nine to 1 (S4 Fig). Based on the benchmark followed past Pentinsaari et al. (2016), the exchange of AAs with the same chemic property at any given position is not considered to significantly influence enzyme function. This also depicts the substitution of AAs from i chemical group to the other in a position far from the enzyme ligands. Nonetheless, substitutions that change the AA chemical group occur close to the enzyme ligands, may probable influence enzyme part. Equally indicated earlier, the secondary structure of the barcode region includes six α-helices connected past five loops (run across Fig 3 in [32]). The loops embrace 62 AAs of which all the eight-plus 11 AAs of loops 1–two and 3–iv, respectively, vary amidst the six orders. AAs in loop 2–3 vary in three out of the eight AAs, while 10 out of 23 and five of 12 for loops three–4 and 5–6, respectively. The loop 3–4 is of import for pointing towards the heme group at the active site of the COI protein. The higher number of conserved AAs in this study was expected due to the narrow genetic distances among the six insect orders compared with those shown by Pentinsaari et al. [32] across the Metazoan barcodes. The number of the conserved AAs of the barcode sequence amidst the six insect order is 102 including the known 23 conserved AAs among Metazoan [32]. Overall, AA variations occurring at positions in the helices iii, 4 and 5 probable have less influence on enzyme part compared with that in the helices 1 or 2. Too, AA variations are more pronounced in loops than in helices.

Functional constraints during the evolution of the insect COI barcode was checked in the predicted three-dimensional structure in terms of the distances betwixt AAs and the two heme ligands referred to every bit hemes 515 and 516 (Fig 3). The analysis indicated a number of 16 AAs mostly in close proximity with heme 515 (Fig 4A). These AAs exist in helices 2 (10) and 1 (four) and loop 3–4 (2). They are Thr (T), Ser (South), Ile (I), Arg (R), Thy (Y), Ile (I), Val (V), His (H), Ala (A), Met (M), Ile (I), Met (Yard), Val (V), Ile (I), Gly (G) and Trp (W). They exist at codon positions 15, eighteen, 21, 22, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 109 and 110, respectively. Six of them at positions 15, 21, 38, 41, 42 and 57 vary amidst the six insect orders, while the residue are conserved. We selected Batrachedra amydraula as a model barcode of the insect conserved sequences for the sixteen AAs in the heme 515 vicinity in which the predicted 3D structure was generated (Fig 3). We mainly focused on 4 AAs that direct share six bonds with hemes 515 and 516. These AAs are R (position 22), Y (position 38), H (position 45) and W (position 110). 2 bonds exist between H at position 45 and heme 515 (Fig 4B) and two other bonds exist between W and the two heme ligands 515 and 516 (Fig 3C). Chemic bonds occur between NH1 molecule of R (position 22) and OMA molecule of heme 515, while OH molecule of Y (position 38) and O1A molecule of heme 515. Two chemic bonds occur between NE2 molecule of H (position 45) side chain and both NA and NC molecules of heme 515. W (position 110) shares N atom of its backbone with O1D molecule of heme 515 and NE1 molecule of its side chain with O2D molecule of heme 516. Distances between the iv selected AAs and heme ligands of COI barcode are shown in S6 Fig. All the 4 AAs are < 4 Å apart from the respective heme ligand.

Fig 3. Predicted iii-dimensional COI poly peptide structure of Batrachedra amydraula.

A model barcode of the insect consensus AA sequence in the heme 515 vicinity. A displays the xvi AAs that probably affect functions of the two hemes 515 and 516. These AAs are threonine (T), sulfur (S), isoleucine (I), arginine (R), tyrosine (Y), isoleucine (I), valine (V), histidine (H), alanine (A), methionine (M), isoleucine (I), methionine (M), valine (V), isoleucine (I), glycine (Thousand) and tryptophan (Westward). These AAs exist at positions 15, 18, 21, 22, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 109 and 110 of the COI polypeptide chain, respectively, following AA numbering of Pentinsaari et al. (2016). Four AAs sharing six bonds with hemes 515 and 516 are shown inside red circles. One actress bail as well exists between Due south at position 18 and R at position 22. B indicates the occurrence of two bonds between H (position 45) and heme 515. C indicates the occurrence of two bonds of W (position 110); one with heme 515 and the other with heme 516. D indicates the absence of hydrogen bail with heme 515 at position 38 in the Camponotus maculatus barcode sequence due to substitution of tyrosine (Y) for phenyl alanine (F). As indicated in C, default position of W during 3D structure prediction is 126, while position 110 following poly peptide model of Pentinsaari et al. (2016). Different types of atoms in the construction are indicated by different color.

https://doi.org/10.1371/journal.pone.0224336.g003

Fig 4. Alignment of the predicted iii-dimensional structures of COI proteins of Batrachedra amydraula and Camponotus maculatus.

A indicates the occurrence of a hydrogen bond between tyrosine of the first insect at position 38 and Heme 515, while lack of bonding betwixt phenyl alanine of the second insect at the same position. B indicates the side chain structures of the two AAs facing Heme 515. C indicates the alignments of the half dozen helices and five loops of the two 3D structures.

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S4 Fig for the AA multiple sequence alignment indicated that the four AAs are conserved amidst insect orders except for tyrosine (position 38) that unexpectedly showed exchange to phenylalanine in ii Hymenopteran species, east.g, Camponotus maculatus and Monomorium junodi. These two AAs differ in the occurrence of an OH molecule at the side concatenation of tyrosine, which is lacking in phenylalanine. The predicted 3D structure of Camponotus maculatus was investigated at this position in gild to observe the possible severity accompanying this exchange (Fig 3D). Tyrosine and phenylalanine share the same chemic grouping, however, the structure indicates no chemical bonding between the AA and heme 515 albeit the possible occurrence of Van der Waals interaction. This is the event of polarity change due to substitution that increases the chance of splitting heme from the protein. Nosotros speculate that this change in the 3D construction of COI and its event can impair the process of electron transfer from Cu to heme, thus, potentially affect free energy metabolism in the mitochondria. Fig three depicts the alignment of the predicted three-dimensional structures of COI proteins of Batrachedra amydraula and Camponotus maculatus. The figure indicates the occurrence of a hydrogen bond betwixt tyrosine of the first insect at position 38 and Heme 515 while lacking bond between phenylalanine of the 2nd insect at the same position (Fig 4A and 4B). The figure as well underpins poor alignment of loop 1–2 or four–five of the two insect species (Fig 3C), which reflects the high rate of varied AA in these 2 regions in line with the results of Pentinsaari et al. [32] and those of the present study (S4 Fig). The three-dimensional structure of the protein too indicates that the change from tyrosine to phenylalanine resulted in the loss of bonding with heme, thus likely impact energy metabolism. The phenylalanine appears to form a proper geometric complementarity; however, lacks electrostatic complementarity due to the absenteeism of the hydroxyl group that contributes to the electrostatic interaction with the heme (Fig 4A), resulting in an increase in hydrophobicity. This suggests that the protein retains its structure, only non all of its functions. Moreover, the absence of a single hydrogen bond, computing the electrostatic potential showed no clear departure when tyrosine is substituted with phenylalanine (S5 Fig).

Phylogenetic tree based on multiple AA sequence alignment of COI barcode indicated that Hymenoptera is the most genetically distant of all the 6 insect orders under study (Fig five). The tree construction complements that of the tree generated based on multiple Dna sequence alignment (S2 Fig) in which Blattodea was shown to be closest to Odonata. The same benchmark applies to the relatedness between the two orders Hemiptera and Hymenoptera and the two other orders Coleoptera and Diptera. We further analyzed the six varied AAs at positions fifteen, 21, 38, 41, 42 and 57, existing within the distance of < 4Å from heme 515, in terms of transitions from one biochemical group to the other (Fig five). At position 15 of the COI protein, threonine is changed to either serine with the same chemic grouping (polar uncharged) or methionine (two-codon AA) with different chemic groups (nonpolar) in all species of Hymenoptera and Hemiptera, respectively. At position 21, the substitution of isoleucine to valine in the aforementioned chemic group (nonpolar) has taken identify in only Melanotus villosus species. At position 38, the substitution of tyrosine to phenylalanine in the same chemical grouping has taken identify in the two Hymenopteran species Monomorium junodi and Camponotus maculatus. At position 41, the commutation of isoleucine (two-codon AA) to either leucine (half dozen-codon AA) or methionine (ii-codon AA) in the same chemical group (nonpolar) has taken place in Hymenopteran species except for Camponotus maculatus. An actress substitution at the same position has taken place to valine too in the same chemical group (nonpolar). At position 42, the substitution of valine to isoleucine in the same chemical group has taken place in Hymenopteran species, which was not fully detected at the species level. Interestingly, commutation at position 57 from isoleucine (nonpolar) to phenylalanine in a different chemical group (aromatic) has taken identify in all Hymenopteran species. The AA transitions from one chemic group to the other might event in impaired energy metabolism. The study indicated a small distance betwixt Hymenoptera and Hemiptera in the phylogenetic tree than betwixt Coleoptera and Hemiptera. The data of AA substitution back up our speculation that Hymenoptera is the most genetically distant of all the other insect orders nether written report. As Hymenopteran species are shown closely related to Hemipteran species and the fact that Hymenopteran species showed AA substitutions in five out of the six varied AAs in close proximity with the heme ligand indicate that Hymenoptera might be among the oldest insect orders.

Fig five. Phylogenetic tree of species of the six insect orders based on multiple AA sequence alignment in COI barcode.

The tree also indicates substitutions of the six varied AAs (at positions 15, 21, 38, 41, 42 and 57) within the altitude of < 4Å from heme 515. Different colors are given to substitutions at different positions, while same colors for substitutions at the same position. AA sequence of Odonata was used for comparison.

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The estimated nucleotide substitution matrices in the COI barcode of the six orders generally showed a slightly college probability of transversion compared with transition (Fig vi). The probability of transversion ranged between ~40% for the Coleopteran species Melanotus villosus and ~65% for the Hymenopteran species Tachytes crassus. Substitutions of A followed by T to whatever other nucleotide represents the lowest probability compared with the rest. Changes from C to T followed by G to A showed the highest transition frequency compared with the others. These ii high transition frequencies justify the high TA ratio in insect orders and the bias against G or C during evolution. Both Coleoptera and Diptera showed the well-nigh notable bias confronting G.

Fig half dozen. Nucleotide exchange matrix derived based on the COI barcode.

Maximum composite likelihood estimates of the pattern of nucleotide substitution to detect transition and transversion frequencies based on Deoxyribonucleic acid sequence alignment in COI barcode of species in the six insect orders.

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Maximum Likelihood computations of codon-by-codon synonymous (s) and nonsynonymous (n) substitutions in COI protein were conducted using the HyPhy software package in which Odonata was used as the reference ancestral society (Fig vii). The full number of nonsynonymous codons is 109, out of 211, of which 102 of them showed positive values at different rates across the half dozen orders. The other vii codons showed negative dN-dS values, hence, listed nether purifying selection. Hymenoperan followed past Hemipteran species are severely under positive selection as the values of dN-dS were positive in 90 and 61 out of the 102 codons, respectively. Twenty-two extremely high positive dN-dS values (> 1.0) in 13 codons were scored for species simply of these two orders; the most frequent are codons 98 (A, a four-codon AA) for Hymenopteran species and 126 (S, an eight-codon AA) for Hemipteran species. On the other hand, the only species (east.g., Balta vilis) of Blattodea showed positive dN-dS values for as low as 17 codons. This low number reflects the close relatedness of this order with Odonata in line with the results of the phylogenetic analysis. The other three orders, e.g., Lepidoptera, Coleoptera, and Diptera showed a total of 30, 38 and 34 positive dN-dS values across species, respectively. This indicates that virtually codons of COI barcode in these three orders seem to exist mostly under purifying pick. There are 16 unique codons to a given species of either Hymenopteran (13 codons) or Hemipteran (iii codons) that are nether positive selection. These Hymenopteran species are Camponotus maculatus (one codon), Hymenoptera sp. (4 codons), Nomioides facilis (3 codons) and Tachytes crassus (four codons), while Hemipteran species is Batracomorphus angustatus (four codons). On the other hand, 10 codons oft showed positive selection across most species of the six orders; 5 of them are located in the helix regions and five are located in the loop regions. Positions of these codons are sixteen, 23, 27, 48, 81, 118, 119, 123, 162 and 199. As for the dN-dS values in terms of domain structure, the results indicated that percentages of codons under positive selection are ~ 80, 31, 61, 52, 40 and ix% in helices ane to 6, while 100, 38, 52, 100 and 33% in loops one–two to 5–vi, respectively. These results point that codons in helices ii, 5 and 6 and in loops 2–3 and 5–half dozen are mostly conserved and are under strong purifying pick.

Fig 7. Maximum Likelihood computations of codon-by-codon synonymous (s) and nonsynonymous (due north) substitutions in COI protein.

Clarification of COI codons that are under positive selection based on the values of dN-dS in thirty species of six insect orders live in Saudi arabia. Sequence of COI in guild Odonata was used every bit a reference.

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The study indicates that COI barcodes tin can provide insights by which the encoded protein evolve and function in insect at unlike taxonomic scales. The COI or Folmer region is basically used for species identification based on variations at the DNA level [34]. The region is sufficiently conserved within species, while variable among species. This region is located in the enzymatically agile part of COI, thus involved in the respiratory chain by transferring electrons from Cu to heme. Accordingly, this region is nether functional constraints and mutations in close proximity with heme ought to exist lethal. There is less evolutionary pressure confronting variation in positions within the extra-membranous loop structures (e.grand., loops i–2 at AA positions 27–34, three–four at AA positions 102–124 and four–v at AA positions 156–166). This conclusion was previously reached by Panchenko et al. [35]. Amino acids in the two highly conserved loops (eastward.k., loops 2–3 and 5–6) seem of import in holding the two sides of the enzyme and avoid astringent conformational changes. However, tryptophan within the variable loop 3–4 at position 99 rigidly evolves as it is the only AA of COI protein that bonds with the two hemes. Another AA in positions shut to the active site is rigid membrane-embedded α-helices undergo functional constraints and restricted evolution (negative evolution). A similar blueprint of variation was observed in the protein encoded by mitochondrial ribosomal RNA gene, where the loops freely evolve [36].

The study mostly indicated that virtually half the positions of the AA of the barcode region are variable, however, they are generally beyond the diminutive interaction distance of the heme groups as previously reported [27,16,37]. Six of these AAs exist within a distance of >4Å with four of them changed to AAs of the same chemical groups. However, the three-dimensional structure of the protein indicates that the change from tyrosine to phenylalanine resulted in the loss of bonding with heme, thus likely affect energy metabolism.

Estimates of dN-dS were used for detecting codons under either positive or purifying selection. dS is the number of synonymous substitutions per site (southward/S) and dN is the number of nonsynonymous substitutions per site (northward/Due north). Positive values indicate the glut of nonsynonymous substitutions in these positions. During evolution, positive (Darwinian) selection promotes the sweeps of new benign alleles, and negative (or purifying) option impedes the spread of harmful alleles [38]. The results indicated that 102 codons are under positive option and tend to evolve more rapidly.

Phenotypic plasticity or polyphenism is another potential role player of evolutionary changes and shaping of ecosystems that allows species to phenotypically adapt to different ecology conditions during evolution [39,xl,41,42]. This type of evolutionary force is not genome structure-based, rather, it relies on the differential factor expression (gene expression-biased) and is particularly prominent in insects. Cistron expression bias is predominant in molecular development when selection becomes less constructive at removing deleterious alleles. Studying polyphenism provides insight into the molecular basis of phenotypic differentiation during evolution [43,44,45,46, 47,48,49,50,51]. This approach is ideal especially for cases where low genetic differences be amidst species [52,53].

Hymenopteran species have the highest AA variation in line with the presumed age of the order that is likely among the oldest, thus had a long time to molecularly evolve past irresolute AA sequence. Previous reports speculated that differences in patterns of variation, substitution, and choice among insect orders are due to the number of metabolism-related factors that are high in actively flying insect species (like Hymenoptera) than in non-flying species [54]. Pick is biased towards the college active metabolic rate and the increase of the resting metabolic rate. Pentinsaari et al. [32] indicated a higher relative rate of CT/GA transitions in the insect is the consequence of higher oxidative damage to Deoxyribonucleic acid. This high variation is the result of a weak purifying selection in Hymenopteran species. We strongly recommend making wet laboratory assay to support whether the changes in AAs consequently affect metabolism in the target species or non. The overall results argue the possible evolutionary position of Hymenopteran species among those of other insect orders living in Saudi Arabia, especially Hemiptera and Coleoptera.

Supporting information

S3 Fig. Phylogenetic tree analysis of the insect orders.

Phylogenetic tree describing the genetic relatedness among species of the six orders based on Deoxyribonucleic acid sequences of the COI gene fragment. Consensus DNA sequence of Odonata was used for comparison.

https://doi.org/10.1371/journal.pone.0224336.s003

(TIF)

S4 Fig. Multiple sequence alignment of the COI protein sequences.

A comparative aminoacid sequence alignment of poly peptide fragment spanning 211 AA of the 30 insect species along with a reference sequence from Odonata for comparing. AA sequences starting time at position 12 and ends at position 222 following the numbering of Pentinsaari et al. (2016).

https://doi.org/10.1371/journal.pone.0224336.s004

(TIF)

S6 Fig. Description of the predicted three-dimensional model pf the COI poly peptide structure.

Predicted iii-dimensional model of the COI protein structure of Batrachedra amydraula indicating the bond distances (indicated by black arrows) between the 2 heme structures 515 and 516 in one hand and AAs arginine (R), tyrosine (Y), histidine (H) and tryptophan (W) on the other manus. The four AAs exist in positions 11, 27, 34 and 99 of the COI polypeptide chain, respectively. A displays bonds of heme/R (one bond), heme/Y (one bail) and heme/H (two bonds). The latter iii AAs bond with heme 515. B displays bonds of heme/West (two bonds) to point that Westward is the simply AA of the COI that bonds (once) with heme 516. C displays the unabridged six bonds of the 4 AAs and the two hemes. The distance between whatever given AA and heme 515 or between Westward and heme 516 is <4Å. Different types of atoms in the construction are indicated by a different colour.

https://doi.org/10.1371/periodical.pone.0224336.s006

(TIF)

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