Expecting the unexpected: Random tissue barcoding reveals the presence of Pieridae in the diet of ground-dwelling Tenebrionidae (Insecta: Lepidoptera, Coleoptera)

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.

Dear Editor, Multiple infections with small liver flukes and minute intestinal flukes are the serious public health concern in the lower Mekong basin [1,2]. Although the epidemiological survey for those trematode infections are primarily carried out based on copro-parasitological examination, detection/identification of fecal eggs/worms is a tedious job and often problematic because of the morphological similarities of eggs/worms. Along with the popularization of PCR-sequencing methods, copro-DNA diagnosis and molecular phylogenetic identification/speciation have been introduced in epidemiological studies. Among various genes and non-coding lesions of nuclear and mitochondrial DNAs, mitochondrial cytochrome c oxidase subunit I (COXI) is one of the most widely used inter- and intra-species marker. Using COXI and some other markers, Lee and his colleagues performed molecular phylogenetic analyses on small liver flukes (Lee SU, Huh S. Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis. Korean J Parasitol 2004; 42: 145-148) and on minute intestinal flukes (Lee SU, Huh S, Sohn WM, Chai JY. Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai. Korean J Parasitol 2004; 42: 129-135). The COX1 gene sequences appeared in those articles are; Clonorchis sinensis ({type:entrez-nucleotide,attrs:{text:AF184619,term_id:296940320,term_text:AF184619}}AF184619, {type:entrez-nucleotide,attrs:{text:AF181889,term_id:285026682,term_text:AF181889}}AF181889, {type:entrez-nucleotide,attrs:{text:AF188122,term_id:285809842,term_text:AF188122}}AF188122), Metagonimus yokogawai ({type:entrez-nucleotide,attrs:{text:AF096230,term_id:297039733,term_text:AF096230}}AF096230), Metagonimus takahashii ({type:entrez-nucleotide,attrs:{text:AF096231,term_id:297039734,term_text:AF096231}}AF096231), Metagonimus miyatai ({type:entrez-nucleotide,attrs:{text:AF096232,term_id:297039735,term_text:AF096232}}AF096232), Pygidiopsis summa ({type:entrez-nucleotide,attrs:{text:AF181884,term_id:288563287,term_text:AF181884}}AF181884), and Stellantchasmus falcatus ({type:entrez-nucleotide,attrs:{text:AF181887,term_id:285804435,term_text:AF181887}}AF181887). In addition, Park [3] compared his COXI sequence of Opisthorchis viverrini Laotian isolate (AY055 382) to those of Gymnophalloides seoi ({type:entrez-nucleotide,attrs:{text:AF096234,term_id:297039736,term_text:AF096234}}AF096234) and Neodiplostomum seoulense ({type:entrez-nucleotide,attrs:{text:AF096233,term_id:285026845,term_text:AF096233}}AF096233) registered in the DNA database (Lee et al. unpublished). For the phylogenetic analyses of COXI gene of minute intestinal flukes of our own data, we have downloaded all those above mentioned COXI of Lee et al. and aligned them including our own COXI sequence of Haplorchis taichui ({type:entrez-nucleotide,attrs:{text:EF055885,term_id:119855482,term_text:EF055885}}EF055885) [4] and Paragonimus bangkokensis ({type:entrez-nucleotide,attrs:{text:AB354227,term_id:155369203,term_text:AB354227}}AB354227) [5]. Surprisingly, those sequence data were divided into 2 distinct groups without any similarities (Fig. 1). Eventually, we realized that this astonishing result is due to the reverse complementary sequences of COXI data deposited by Lee et al. (in the bottom half of the figure). We also noticed similar mixed-up deposition of the forward and reverse sequences of COXI gene of Fasciola spp., which were also included in Fig. 1 ({type:entrez-nucleotide,attrs:{text:AJ628024,term_id:88319716,term_text:AJ628024}}AJ628024, {type:entrez-nucleotide,attrs:{text:AJ628039,term_id:88319746,term_text:AJ628039}}AJ628039, {type:entrez-nucleotide,attrs:{text:FJ469984,term_id:238631966,term_text:FJ469984}}FJ469984; Zhu XQ et al. unpublished). Fig. 1 The DNA sequence alignment of the partial COXI gene of some trematodes obtained from the GenBank. Seven sequences from the top are the forward strands with JB3 primer sequence, and 2 in the middle are the forward strands without primer. Eight sequences ... For the determination of partial COX1 sequences of Platyhelminthes, the primer set of JB3 (5'-TTT TTT GGG CAT CCT GAG GTT TAT-3') and JB4.5 (5'-TAA AGA AAG AAC ATA ATG AAA ATG-3') [6] was widely used for investigating the inter- and intra-species variations of trematodes and cestodes. We noticed the mixed-up of the forward and reverse COXI sequences by Lee et al. as well as Zhu et al. because of the presence of the characteristic feature of this primer set (boxed in Fig. 1) in the sequences. The primer sequence should be deleted from the sequence data because it is not always identical with the real DNA sequence of the gene and the inclusion of the primer sequences sometimes causes the misreading in phylogenetic analyses [7]. In 3 reverse sequences, {type:entrez-nucleotide,attrs:{text:AF181884,term_id:288563287,term_text:AF181884}}AF181884, {type:entrez-nucleotide,attrs:{text:AY055380,term_id:22203992,term_text:AY055380}}AY055380, and {type:entrez-nucleotide,attrs:{text:AF096233,term_id:285026845,term_text:AF096233}}AF096233 seems to contain also the partial sequence of the cloning vector, which should be trimmed off before deposition. In general, raw data of forward and reverse sequences obtained from the sequencer should be aligned manually by cross-checking of the wave patterns because some 10-20 bases downstream from the forward primer and upstream from the reverse primer often contain erroneous base pairs [8]. Deposition of the reverse sequence means that those sequences were not aligned against forward sequence and not quite reliable. Since each sequence data in GenBank are opened for the public use, an accuracy of the sequence data is critically important for the mutual reliability of the scientists. The scientists should aware how to deposit accurate sequence data to the DNA data base. The reappraisal and correction of those sequences mentioned above is urgently necessary.

Sphaeroma terebrans, a wood-boring isopoda, is distributed worldwide in tropical and subtropical mangroves. The taxonomy of S. terebrans is usually based on morphological characteristics, with its molecular identification still poorly understood. The number of teeth on the uropodal exopod and the length of the propodus of the seventh pereopod are considered as the major morphological characteristics in S. terebrans, which can cause difficulty in regards to accurate identification. In this study, we identified S. terebrans via molecular and morphological data. Furthermore, the validity of the mitochondrial cytochrome c oxidase subunit I (COI) gene as a DNA barcode for the identification of genus Sphaeroma, including species S. terebrans, S. retrolaeve, and S. serratum, was examined. The mitochondrial COI gene sequences of all specimens were sequenced and analysed. The interspecific Kimura 2-parameter distances were higher than intraspecific distances and no intraspecific-interspecific distance overlaps were observed. In addition, genetic distance and nucleotide diversity (π) exhibited no differences within S. terebrans. Our results revealed that the mitochondrial COI gene can serve as a valid DNA barcode for the identification of S. terebrans. Furthermore, the number of teeth on the uropodal exopod and the length of the propodus of the seventh pereopod were found to be unreliable taxonomic characteristics for S. terebrans.

基于线粒体细胞色素c氧化酶亚基I基因序列的帘蛤科贝类分子系统发育研究

Metabarcoding extra‐organismal DNA from environmental samples is now a key technique in aquatic biomonitoring and ecosystem health assessment. Of critical consideration when designing experiments, and especially so when developing community standards and legislative frameworks, is the choice of genetic marker and primer set. Mitochondrial cytochrome c oxidase subunit I (COI), the standard DNA barcode marker for animals, with its extensive reference library, taxonomic discriminatory power and predictable sequence variation, is the natural choice for many metabarcoding applications. However, for targeting specific taxonomic groups in environmental samples, the utility of COI has yet to be fully scrutinized. Here, by using a case study of marine and freshwater fishes from the British Isles, we quantify the in silico performance of twelve primer pairs from four mitochondrial loci – COI, cytochrome b, 12S and 16S – in terms of reference library coverage, taxonomic discriminatory power and primer universality. We subsequently test in vitro four primer pairs – three COI and one 12S – for their specificity, reproducibility, and congruence with independent datasets derived from traditional survey methods at five estuarine and coastal sites around the English Channel and North Sea. Our results show that for aqueous extra‐organismal DNA at low template concentrations, both metazoan‐targeted and fish‐targeted COI primers perform poorly in comparison to 12S, exhibiting low levels of reproducibility due to non‐specific amplification of prokaryotic and non‐target eukaryotic DNAs. An ideal metabarcode would have an extensive reference library upon which custom primers could be designed, either for broad assessments of biodiversity, or taxon specific surveys. Such a database is available for COI, but low primer specificity hinders practical application, while conversely, 12S primers offer high specificity, but lack adequate references. The latter, however, can be mitigated by expanding the concept of DNA barcodes to include whole mitochondrial genomes generated by genome‐skimming existing tissue collections.

The mitochondrial cytochrome c oxidase subunit 1 (COI) gene of ciliates was first successfully sequenced in species of the genera Tetrahymena and Paramecium (Class Oligohymenophorea). The sequence of the COI gene is extremely divergent from other eukaryotes and includes an insert, which is over 300 nucleotides long. In this study, we designed a primer pair that successfully amplified the COI gene of ciliates from five different classes: Heterotrichea, Spirotrichea, Oligohymenophorea, Nassophorea and Colpodea. These classes represent the diversity of the phylum Ciliophora very well, since they are widely distributed on the ciliate small subunit rRNA tree. The amplified region is approximately 850 nucleotides long and corresponds to the general barcoding region; it also includes the insert region. In this study, 58 new COI sequences from over 38 species in 13 orders are analysed and compared, and distance trees are constructed. While the COI gene shows high divergence within ciliates, the insert region, which is present in all classes, is even more divergent. Genetic distances calculated with and without the insert region remain in the same range at the intraspecific level, but they differ considerably at or above genus level. This suggests that the entire barcoding region is under similar selective constraints and that the evolutionary rate of the ciliate COI is extremely high and shows unequal rate variation. Although many problems still remain regarding standardization of barcoding methods in ciliates, the development of a universal or almost universal primer combination for the Phylum Ciliophora represents important progress. As shown in four examples, the resolution of COI at the intraspecific level is much greater than that of any nuclear genes and shows great potential to (1) identify species based on molecular data if a reliable database exists, and (2) resolve the relationships of closely related ciliate taxa and uncover cryptic species.

Among the Ixodid ticks, Hyalomma anatolicum is a well-known vector that transmits various pathogens to terrestrial and semi-terrestrial vertebrates including humans, and its population differ in ecology and vector competence. Expansion of this tick to new areas changes the genetic structure, and lead to affect the vector-pathogen interaction and disease outcomes. The present study was designed to infer the haplotype diversity, demographic dynamics, gene flow and genetic differentiation, and phylogeny of H. anatolicum from different countries based on the cytochrome oxidase I (COI) and 16S rDNA sequences. A total of 320 ticks were collected from cattle, buffaloes, and sheep in five districts of Khyber Pakhtunkhwa, Pakistan, morphologically identified as H. anatolicum, and subjected to genetic analysis. A total 85 and 138 sequences for COI and 16S rDNA, including 11 and 2 sequences generated in this study, respectively, were analyzed to assess haplotype network, population structure and divergence, demographic changes, and phylogenetic analysis. Analysis based on COI sequences yielded 29 haplotypes in which haplotype 1 and 15 were the predominant consisting of 35 and 20 sequences, respectively, from Pakistan, India, China, Bangladesh, Iraq, Saudi Arabia, Kazakhstan and Egypt. The 16S rRNA yielded 30 haplotypes in which haplotype 1 was predominant consisting of total 86 sequences from Pakistan, India, China, United Arab Emirates, Tajikistan, Kazakhstan, Turkey, Egypt, and Iraq. Complete haplotype network based on COI and 16S rRNA confirmed stellate structure, together with high haplotype diversity (COI 0.77899, 16S rRNA 0.60774) and low nucleotide diversity (COI 0.00445, 16S rRNA 0.00431), which support recent population expansion. Similarly, neutrality indices for the whole dataset, Tajima’s D (COI − 2.36363**, 16S rRNA − 2.54127***), Fu and Li’s D (COI − 5.72992, 16S rRNA − 6.31313*), and Fu and Li’s F (COI − 5.04435*, 16S rRNA − 5.56085*) were negative, indicating deviation from neutrality and recent population dispersal. In the phylogenetic tree based on the COI and 16S rDNA sequences, with exception of one sequence for a single haplotypes, which appeared independently, there is a single main clade that includes the largest number of sequences for all other haplotype. Based on COI and 16S rDNA sequences, the present study provided first detail information about the population genetics and haplotype networks of H. anatolicum.

Background: Traditionally, morphological features of Rhipicephalus (Boophilus) annulatus from closely-related ticks have been considered for their identification and differentiation. However, it is difficult and requires expertise in order to accurately identify and differentiate engorged female ticks and some developmental stages such as larva and nymph from other similar ticks. Hence, molecular markers may be a suitable alternative. OBJECTIVES: Mitochondrial cytochrome c oxidase subunit I (COI) gene and the second internal transcribed spacer (ITS2) fragments of Rh. (Bo.) annulatus were sequenced to assess the use of molecular techniques for identifications and phylogenetic studies of these ticks. METHODS: Polymerase chain reaction (PCR) technique was performed based on the analyses of COI and ITS2 sequences of ticks collected from two different regions in Iran (Golestan and Mazandaran). RESULTS: The length of COI and ITS2 sequences were 1539 and 1158bp, respectively. The nucleotide similarity of COI gene was 91.3% between the ticks examined from the two different regions. The deduced amino acid sequences from COI showed 98.6% similarity between the ticks studied and showed 98.2 and 99.6% similarity with the only complete sequence of Rh. (Bo.) annulatus (AGH19677) registered in GenBank. The obtained complete nucleotide sequences of ITS2 from Rh. (Bo.) annulatus from Golestan and Mazandaran revealed 99.9% similarity, while the other ticks registered in GenBank 95 to 99% similarity (KC503267, AF271270, AF271272, JQ412126). CONCLUSIONS: It seems that COI and ITS2 sequences could provide suitable genetic markers for discrimination and genetic characterization of Rhipicephalus (Boophilus) annulatus.

The origin of the Tibetan Mastiff and species identification of Canis based on mitochondrial cytochrome c oxidase subunit I (COI) gene and COI barcoding

Phylogeny of scale-worms (Aphroditiformia, Annelida), assessed from 18SrRNA, 28SrRNA, 16SrRNA, mitochondrial cytochrome c oxidase subunit I (COI), and morphology

한국산 붉바리 집단에서 유전적 구조와 계통 유연관계를 mtDNA COI 유전자 서열의 다형성을 이용하여 조사하였다. COI 유전자 서열을 결정하였고 기존에 보고된 서열들과 비교하였다. 본 연구를 통해 결정된 COI 서열들은 기존에 보고된 EF607565에 대하여 99.1-99.8%의 동일성을 나타내었다. 전체 20가지의 haplotype들이 발견되었고, 한국산 붉바리 집단은 19가지의 haplotype을 나타내었다. 이들 중 Hap_03과 Hap_08은 각각 제주도와 중국-특이적인 COI 서열들을 보였다. 반면, Hap_07은 한국에서 채집된 시료들과 홍콩과 대만에서 보고된 기록 등 여러 COI 서열들을 포함하였다. COI haplotype들의 다형성에 근거한 계통 유전학적 분석을 통해 작성된 NJ tree는 Epinephelus 속 내에서 단계통적인 분지양상을 나타내었고, 이는 붉바리 집단들이 공통의 모계 선조에서 진화한 것임을 나타내었다. 또한 중국해에서 보고된 COI 서열만을 포함하였던 Hap_08은 NJ tree의 중앙부에서 위치하였고, Hap_07의 서열들과도 근연의 관계임을 보여주었다. 이 결과는 중국산 붉바리 역시 동아시아의 다른 집단들과 모계적으로 연관되어있음을 보여주었다. 결과적으로, 동아시아 붉바리 집단들은 모계적으로 연관되어있을 뿐만 아니라 공통의 진화 역사를 공유하고 있으며 여전히 동아시아 해류(Kuroshio 해류)에 의해 영향을 받는 집단이라고 할 수 있다. 본 연구는 붉바리의 유전적 구조와 계통 유연관계를 이해하는 데 도움을 줄 수 있으며, 인공증식과 산업화에 관련된 연구에 있어 중요한 역할을 담당할 것으로 기대된다. The genetic structure and phylogenetic relationship were investigated in Korean red spotted grouper populations using the nucleotide sequence polymorphisms of the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) gene. The COI gene was sequenced showed 99.1-99.8% identity with the EF607565 sequence previously reported. A total of twenty haplotypes were found, and the Korean population showed nineteen haplotypes. Among those, Hap_03 and Hap_08 showed Jeju-do and China-specific COI sequences, respectively. However, Hap_07 had twelve COI sequences from South Korea and records from Hong Kong and Taiwan. Neighbor-joining (NJ) trees constructed from the phylogenetic analyses based on the polymorphisms of the COI haplotypes showed a monophyletic branching pattern within the genus Epinephelus. This indicated that the red spotted grouper populations had evolved from common maternal ancestors. In addition, the Hap_08, which had the COI sequence recorded only from China Sea, was found in the middle of the NJ tree nearby Hap_07 and showed a close relationship with Hap_07. This indicates that Chinese red spotted grouper is also maternally related to other populations in East Asia. Consequently, East Asian red spotted grouper populations are maternally related, as well as sharing the same evolutionary history, and are still affected by the East Asian ocean current (Kuroshio). These findings help to explain the genetic structure and phylogenetic relationship of red spotted grouper and also contribute to research on artificial breeding and industrialization.

The identification and phylogeny of muricids have been in a state of confusion for a long time due to the morphological convergence and plasticity. DNA-based identification and phylogeny methods often offer an analytically powerful addition or even an alternative. In this study, we employ a DNA barcoding method to identify 17 known and easily confused muricid species (120 individuals) from the whole China coast based on mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA sequences, and nuclear ITS-1 and 28S rRNA sequences. The phylogeny of muricid subfamilies is also analysed based on all mitochondrial and nuclear sequences. The universal COI and 16S rRNA primers did not work broadly across the study group, necessitating the redesign of muricid specific COI and 16S rRNA primers in this paper. Our study demonstrates that COI gene is a suitable marker for barcoding muricids, which can distinguish all muricid species studied. Phylogenetic analysis of 16S rRNA, ITS-1 and 28S rRNA data also provide good support for the species resolution observed in COI data. The relationships of muricid subfamilies are resolved based on the separate and combined gene data that showed the monophyly of each the subfamilies Ergalataxinae, Rapaninae, Ocenebrinae and Muricinae, especially that Ergalataxinae did not fall within Rapaninae.

DNA barcoding has become a promising tool for species identification and phylogeny in a wide range of animal taxa using mitochondrial cytochrome c oxidase subunit I (COI). The Corvidae (Aves: Passeriformes) is a species rich and morphologically diverse family. In the present study, we analyzed the COI barcodes of 39 species from 12 genera of Corvidae. COI gene was also used to examine phylogenetic relationships of Corvidae. Every species possessed a barcode distinct from that of other species. Kimura two-parameter distances were calculated between species barcodes. The average genetic distance between the species was 22 times higher compared to the average genetic distance within species. Maximum likelihood method was used to construct a phylogenetic tree. All the species could be discriminated by their distinct clades in the phylogenetic tree. COI gene data provided good evidence for the monophyly of the Corvidae. Members of Cyanopica and Pyrrhocorax were the first to split from the Corvidae lineage. Analysis of COI genes supported the others genera fell into two clades. DNA barcoding is an effective molecular tool for Corvidae species identification and phylogenetic inference.

Identification of hydrozoan species is challenging, even for taxonomic experts, due to the scarcity of distinct morphological characters and phenotypic plasticity. DNA barcoding provides an efficient method for species identification, however, the choice between mitochondrial cytochrome c oxidase subunit I (COI) and large subunit ribosomal RNA gene (16S) as a standard barcode for hydrozoans is subject to debate. Herein, we directly compared the barcode potential of COI and 16S in hydrozoans using 339 sequences from 47 pelagic hydrozoan species. Analysis of Kimura 2-parameter genetic distances (K2P) documented the mean intraspecific/interspecific variation for COI and 16S to be 0.004/0.204 and 0.003/0.223, respectively. An obvious “barcoding gap” was detected for all species in both markers and all individuals of a species clustered together in both the COI and 16S trees. These results suggested that the species within the studied taxa can be efficiently and accurately identified by COI and 16S. Furthermore, our results confirmed that 16S was a better phylogenetic marker for hydrozoans at the genus level, and in some cases at the family level. Considering the resolution and effectiveness for barcoding and phylogenetic analyses of Hydrozoa, we strongly recommend 16S as the standard barcode for hydrozoans.

The family Carangidae is considered a markedly diverse, widespread taxon. Due to the characteristic “cryptic diversity” and “hybrid speciation” within the family, there is an exigency for taxonomic approaches that go beyond traditional phenotypic modus in identifying species. Mitochondrial cytochrome c oxidase subunit - I (COI) barcoding gene region plays a significant role over phenotypic characters in identifying species. This study evaluates the combination of the two molecular approaches: the COI DNA barcoding and PCR-RFLP for judicious species discrimination. The partial mitochondrial COI gene region of the selected Carangid fish species was amplified, sequenced and confirmed sequences with a mean length of 655 bp were submitted to the main databases; NCBI and BOLD. Intraspecific and interspecific nucleotide divergences were computed by the Kimura-2-Parameter (K2P) and they ranged between (0.00–2.96 %) and (6.46–21.83 %), respectively. The possibility of acquiring the same RFLP profiles given by the restriction enzymes HaeIII and MbOII was observed by the theoretical cleavage of 250 reference sequences of the corresponding gene. Hence, major and minority composite haplotypes of each species were obtained based on the fragment types derived by both HaeIII and MbOII. The resulting divergence values were compatible with the previously reported values for marine fish species. All the species were clearly differentiated by both RFLP banding patterns and the highest probability assumption of getting the same RFLP profile was compatible with the most abundant composite haplotype of each species. This reveals the practicability of the combination of two consolidated molecular approaches, COI barcoding and PCR-RFLP (COIBar-RFLP).