Region Of Cytochrome Oxidase Research Articles

First record of the genus Haritalodes Warren, 1890 and H. derogata (Fabricius, 1775) (Lepidoptera: Crambidae: Spilomelinae) from Türkiye and its morphological and molecular identification.

The genus Haritalodes Warren, 1890 is represented by 12 species worldwide except in the Nearctic and Neotropical regions. Haritalodes derogata (Fabricius, 1775), commonly known as the cotton leaf roller moth, is the most widely distributed species of the genus and is common in the West Palaearctic Region. This species is one of the most serious pests of Malvaceae. The larvae primarily feed on the leaves, and severe population levels may lead to significant yield loss in the primary host, cotton. In a survey in 2023, the shrubs of the Hibiscus sp. were found to be infested by a group of larvae with a very dense population feeding on the leaves in Trabzon Province, Türkiye. The larvae were reared for pupation and adult emergence. Morphological identification according to male and female genitalia as well as sequencing of the mitochondrial cytochrome oxidase region I (COI) from larvae and adults revealed that the species is H. derogata. This is the first report of the genus Haritalodes and Haritalodes derogata in Türkiye.

In support of morphology: Molecular analysis successfully delineates the Afrotropical genus Atylotus (Diptera: Tabanidae) into species

Previous studies of the horse fly genus Atylotus in the Afrotropics has shown little to no differentiation into species based on the barcoding region of cytochrome oxidase I (COI), largely due to morphological misidentifications. Using field caught specimens and a museum reference collection together with type comparisons, COI and 16S ribosomal RNA sequences were generated from two specimens of Atylotus agrestis, two A. albipalpus, four A fuscipes and one A. nigromaculatus. Phylogenetic analysis of these sequences produced four separate species clades with strong support. The results showed that COI does delineate the species of Afrotropical Atylotus and that misidentifications of specimens is a common problem. Additionally, Atylotus fuscipes is revived from synonymy and given full species status. Finally, a comprehensive review of the COI barcodes, publicly available on GenBank and BOLD is included that highlights some problems with using sequences from public databases.

Two new species and two new records of Onychiurinae (Collembola, Onychiuridae) from the Nilgiris, India.

Four species of Onychiurinae are reported from the Nilgiris, Western Ghats, southern India, including two new species, Thalassaphorura udagaiensis sp. nov. and Protaphorura sholai sp. nov., and two new records for India: Protaphorura fimata (Gisin, 1952) and Orthonychiurus folsomi (Schffer, 1900). Thalassaphorura udagaiensis sp. nov. can be separated easily from the similar species T. encarpata by the presence of 10/011/221110 dorsal S-chaetae and shorter anal spines. Protaphorura sholai sp. nov. is the most similar to the species P. microtica, P. subarctica and P. levantina as having the same number of ventral pseudocelli and parapseudocelli on abdomen; it can be easily distinguished by the position of head chaeta p2 in relation to p1 and p3, shorter M and Sp chaetae on Abd V and longer anal spines. We also sequenced the mitochondrial barcode region of cytochrome oxidase I (658 base pairs) to confirm the species status.

The first reported case of accidental intestinal myiasis in a domestic dog by the flesh fly, Sarcophaga africa (Wiedeman, 1824)

Myiasis occurs when fly larvae, or maggots, feed on the tissue, secretions or digestive content of a live vertebrate. Here, a rare case of accidental intestinal or enteric myiasis is reported in a domestic dog. The species of fly is molecularly identified as Sarcophaga africa (Wiedeman, 1824) using the barcoding region of cytochrome oxidase I (COI). A brief critique on the usage of the term “pseudomyiasis” is provided and the complex taxonomy of S. africa is briefly summarised in order to shed light on the erroneous use of S. cruenata and S. haemorrhoidalis with obvious downstream effects. Finally, a comparative assessment to the limited cases in the literature is provided. These few cases are however highly fragmented and our understanding of accidental intestinal myiasis and the clinical manifestations thereof remain incomplete.

Lurking in the water: testing eDNA metabarcoding as a tool for ecosystem-wide parasite detection.

In the light of global biodiversity change and emerging disease, there is an urgent need to establish efficient monitoring programmes of parasites in aquatic ecosystems. However, parasite identification is time-consuming, requires a high degree of taxonomic expertize and in general requires lethal sampling. The use of environmental DNA methodology to identify parasites has the potential to circumvent these limitations. This study evaluates the use of eDNA metabarcoding to detect the presence of all species of nematode and platyhelminth parasites in two New Zealand lakes. We developed two novel metabarcoding primer pairs targeting a region of cytochrome oxidase I gene (COI) specific to platyhelminths and nematodes. We successfully detected parasite DNA in both lakes. Platyhelminth DNA yield was in general greater than nematode DNA yield. This most likely results from the larger biomass of the former quantified using traditional methods, or the presence of free-swimming life stages in the life cycle of many platyhelminths. By using eDNA, we did not detect all expected parasite families revealed through traditional methods, likely due to a lack of sequencing data available from public databases such as GenBank. As such, genetic resources need to include full reference sequences if parasitology is to truly harness eDNA to characterize and monitor parasite biodiversity in natural systems.

Strategy for DNA extraction and detection from insect pests in stored home grain samples RESEARCH

Stored grains are subjected to infestations with more than 60 species of insects, that responsible for millions of dollars’ loss and cause several health problems including allergies and gastrointestinal disorders. Traditional detection techniques are laborious, expensive and not sensitive to detect insect contamination at the egg and larvae stages. Therefore, alternative methods are needed for rapid and sensitive detection. One obvious approach is to develop a molecular approach utilizing genetic information of the potential insect species that infest grains for amplification of specific target gene fragment utilizing polymerase chain reaction [PCR]. In the present study, a number of known infested grain samples were used in standardizing a method to isolate larvae and adult insects that were based on centrifugation washing method and a filtration washing method. The isolated insects were subjected to DNA extraction and PCR amplification of defined regions of cytochrome oxidase I (COI) gene followed by sequencing to identify the di"erent pest species. For PCR amplification new primers were designed and for this purpose the obtained COI sequences from di"erent insects were aligned to design two sets of primers (named: COI-PCR4 and COI-PCR5) specific for the indicated insect mitochondrial COI gene. The designed primers were tested for their specificity and sensitivity. The suitability of PCR primers and DNA extraction methods were evaluated on eleven samples of commercial grains utilizing each primer set with the two extraction methods.

Application of COI gene for identification of some economically and forensically important muscid flies of India (Diptera: Muscidae)

Muscid flies are of great medical and veterinary importance because of their ability to transmit diseases to humans and animals. Their larvae are found on dead bodies and are used as a forensic indicator for estimating Post-Mortem Interval (PMI). A drawback of morphological identification of these larvae is lack of an identification key which covers all the life stages of the taxa. To overcome the problem, entomologists now focus on the alternative methods, which can speed up the identification process. Barcoding region of Cytochrome oxidase I (COI)l is believed to be the best among other markers for precise identification and to mark out phylogeny. During the present study, total 63 specimens of 24 species belonging to 7 genera representing the various muscid subfamilies were used to check the efficacy of COI gene in identification. 11 species were collected from various regions (Punjab, Haryana, Himachal Pradesh, Uttarakhand) of North India during the years 2015–2019, and sequences of rest of the species were downloaded from the database of Genbank. The percentage of intraspecific divergences ranged from 0%-0.5% for species of India and 0%-3.0% for the sequences from other countries. The interspecific divergence ranged from 3.1% to 17.3% and 3.3% to 16.7% respectively for Indian species and species from the other part of the world. 1000 bootstrapped Neighbor-joining with Kimura-2 parameter phylogenetic tree constructed based on COI gene sequences, clearly separated 24 species of these Muscid flies. This is the first ever study on COI gene based identification of medico-legal, veterinary and forensically important Muscid flies from India.

Molecular Genetic Characters of Species of the Genus Jordanita Verity, 1946 (Lepidoptera: Zygaenidae, Procridinae): DNA Barcodes and Corresponding Amino Acid Sequences

The paper presents qualitative and quantitative composition of the nucleotide sequences of the 5'‑region of the first subunit of the mitochondrial cytochrome oxidase (COI) gene (decoded in the framework of the International DNA Barcoding Project) and the variability of the corresponding amino acid sequences in the genus Jordanita Verity, 1946. The number of variable positions in the COI region (219 amino acid residues) is 14.2% that significantly exceeds the data for other Lepidoptera. Thirty one amino acid variable positions were revealed, and two of them are localized near the active site of the enzyme.

Parasite infection of specific host genotypes relates to changes in prevalence in two natural populations of bumblebees

The antagonistic relationship between parasites and their hosts is strongly influenced by genotype-by-genotype interactions. Defense against parasitism is commonly studied in the context of immune system-based mechanisms and, thus, the focus in the search for candidate genes in host-parasite interactions is often on immune genes. In this study, we investigated the association between prevalence of parasite infection and host mitochondrial DNA (mtDNA) haplotypes in two natural populations of bumblebees (Bombus terrestris). The two most common haplotypes of the host populations, termed A and B, differ by a single nonsynonymous nucleotide substitution within the coding region of cytochrome oxidase I, an important player in metabolic pathways. We screened infection by Nosema bombi, a common endoparasite of bumblebees, and the corresponding host mtDNA-haplotype frequencies in over 1400 bumblebees between 2000 and 2010. The island population of Gotland showed lower mtDNA diversity compared to the mainland population in Switzerland. Over time, we observed large fluctuations in infection prevalence, as well as variation in host haplotype frequencies in both populations. Our long-term observation revealed that N. bombi infection of specific host genotypes is transient: We found that with increasing infection prevalence, proportionally more individuals with haplotype B, but fewer individuals with haplotype A were infected. This suggests that the presence of N. bombi in specific host genotypes relates to infection prevalence. This may be a result of parasite competition, or differential resilience of host types to ward off infections. The findings highlight the important role of host mtDNA haplotypes in the interaction with parasites.

Enterobiasis epidemiology and molecular characterization of Enterobius vermicularis in healthy children in north-eastern Poland

Summary Enterobiasis is a human intestinal parasitic disease caused by human pinworm, Enterobius vermicularis. Despite being the most prevalent nematode infection in Europe and North America, predominantly among in school aged children, the data concerning infection rate and knowledge of genetic variability of pinworms are incomplete. The aim of the study was the estimation of prevalence and molecular typing of Enterobius vermicularis among healthy children in north-eastern Poland. In 2013 – 2015, 296 individuals (aged 2 – 18 years) from 12 kindergartens, schools and orphanages were examined by the adhesive cellophane tape method. Data on socio-demographic status were collected using a questionnaire. Molecular analysis was performed using the DNA of adult female pinworms and primers targeting the region of cytochrome oxidase I gene. The overall prevalence of enterobiasis was 10.1 %. Enterobius vermicularis infection rates were 3.9 % in children living in families and 32.8 % among the orphans (OR=0.08; 95 % CI: 0.04 – 0.19; p<0.001). There were no associations between distribution of enterobiasis and gender, pets possession and the season of examination. In 43.3 % of the infected children enterobiasis was asymptomatic. Based on a molecular marker three different haplotypes of pinworm were identified. All sequences clustered within type B, together with human E. vermicularis isolates from Denmark, Germany, Greece, and Japan. This paper provides complementary data on the occurrence and intraspecific variability of E. vermicularis in human population in Europe.

Uncovered Diversity of a Predominantly Andean Butterfly Clade in the Brazilian Atlantic Forest: a Revision of the Genus Praepedaliodes Forster (Lepidoptera: Nymphalidae, Satyrinae, Satyrini)

The genus Praepedaliodes Forster, 1964, the only representative of the mega-diverse mostly Andean Pedaliodes complex lineage in the Brazilian Atlantic Forest, is revised. Prior to this study, four species were known, P. phanias (Hewitson, 1862), P. granulata (Butler, 1868), P. amussis (Thieme, 1905) and P. exul (Thieme, 1905). Here, a further six are described, all from SE Brazil, expanding to 10 the number of species in this genus. Lectotypes are designated for P. phanias, P. granulata and P. amussis. The genus is most diverse in the Serra da Mantiqueira (São Paulo, Rio de Janeiro, Minas Gerais) and in the Serra Geral (Paraná, Santa Catarina) with seven species occurring in both ranges. Praepedaliodes phanias is the most widespread species and the only one found in the western part of the Atlantic Forest; only this species and P. duartei Dias, Dolibaina & Pyrcz n. sp. occurring to near sea level. Other species, P. zaccae Dolibaina, Dias & Pyrcz n. sp., P. francinii Freitas & Pyrcz n. sp., P. sequeirae Pyrcz, Dias & Dolbaina n. sp., P. landryi Pyrcz & Freitas n. sp. and P. pawlaki Pyrcz & Boyer n. sp. are strictly montane and the highest species richness is reached at 1400–1800 m. One species, P. sequeirae n. sp., is a narrow endemic found only at timberline in the Agulhas Negras massif above 2300 m. Immature stages are described for two species, P. phanias and P. landryi n. sp. Molecular data (barcode region of cytochrome oxidase, subunit I) and adult morphology, including male and female genitalia, support the genus as monophyletic, belonging to a predominantly Andean clade of the Pedaliodes Butler, 1867 complex. Morphological evidences, in particular female genitalia comparative analysis, indicate the genera Physcopedaliodes Forster, 1964 and Panyapedaliodes Forster, 1964 as possibly the closest relatives to Praepedaliodes. Molecular data are inconclusive in this respect.

Molecular diversity of thrips on rabi/summer groundnut in North Karnataka

Molecular Diversity of Thrips on Rabi/Summer Groundnut in North Karnataka Thrips population was collected on Rabi/summer groundnut from six geographical regions viz., Kerur (Bagalkot; Badami), Korti (Bagalkot; Bilgi), Devapur (Vijayapur) and Handigona (Uttar Kannada; Kumta), Narendra (Dharwad) and Naregal (Gadag). Genomic DNA was isolated and subjected to molecular analysis of COI gene and the results of COI gene sequences of all the six geographical locations indicated as Scirtothrips dorsalis cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial. The sequence comparison revealed that there is more than 94 per cent similarity with NCBI published sequence of S. dorsalis COI gene (KM355471.1) between the thrips samples of all the six locations. The sequences of all the six population were compared using alignment editor ‘Bioedit’. There was no sequence variation among the six population(isolates) for the 190 bp region of cytochrome oxidase I (COI) gene, indicating that the populations were similar with respect to this region of cytochrome oxidase I (COI) gene.....

Development of species-specific primers with potential for amplifying eDNA from imperilled freshwater unionid mussels.

Environmental DNA (eDNA) is emerging as a potentially powerful tool for inferring species' presence, and hence occupancy, from DNA that is shed into environmental samples such as water. Although eDNA screening has been used to detect DNA from a variety of taxonomic groups, it has not yet been used to identify DNA from species with numerous potentially sympatric confamilial species, a situation that may preclude the development of species-specific markers. There are 41 native freshwater mussel species (Unionidae) in Ontario, Canada. Many of these are potentially sympatric, and 14 species have been formally assessed as endangered, threatened, or special concern. We investigated whether there was sufficient variation within the cytochrome oxidase region (COI) to develop species-specific eDNA markers for at-risk unionids. We developed 32 COI markers for eight unionid species, and tested each of these on the target species plus 29 potentially sympatric unionid taxa. Six of these markers amplified DNA only from the intended target species. We then extracted and amplified mussel eDNA from rearing-tank water samples. We conclude that despite high species diversity, it should be possible to develop eDNA COI markers and screen water samples for habitat occupancy by unionid mussels.

Congruence between cytochrome oxidase I (COI) and morphological data in Anuraphis spp. (Hemiptera, Aphididae) with a comparison between the utility of the 5' barcode and 3' COI regions.

The discrimination of species in the genus Anuraphis is particularly difficult due to the overlap of morphological characters. In this study, we used the 5’ (barcode) and 3’ regions of cytochrome oxidase I (COI) to test their utility in the identification of species in this genus as well as closely related species. Both regions were useful to discriminate all the species tested. However the non-barcode 3’ region resulted in higher resolution and support for species relationships when the data were analyzed using both Maximum Likelihood and MrBayes. We propose the development of an integrated database that encompasses morphological, molecular, life-cycle, host plant and bibliographic information to facilitate and increase the accuracy of aphid identification.

Assessment of four DNA fragments (COI, 16S rDNA, ITS2, 12S rDNA) for species identification of the Ixodida (Acari: Ixodida)

BackgroundThe 5’ region of cytochrome oxidase I (COI) is the standard marker for DNA barcoding. However, COI has proved to be of limited use in identifying some species, and for some taxa, the coding sequence is not efficiently amplified by PCR. These deficiencies lead to uncertainty as to whether COI is the most suitable barcoding fragment for species identification of ticks.MethodsIn this study, we directly compared the relative effectiveness of COI, 16S ribosomal DNA (rDNA), nuclear ribosomal internal transcribed spacer 2 (ITS2) and 12S rDNA for tick species identification. A total of 307 sequences from 84 specimens representing eight tick species were acquired by PCR. Besides the 1,834 published sequences of 189 tick species from GenBank and the Barcode of Life Database, 430 unpublished sequences representing 59 tick species were also successfully screened by Bayesian analyses. Thereafter, the performance of the four DNA markers to identify tick species was evaluated by identification success rates given by these markers using nearest neighbour (NN), BLASTn, liberal tree-based or liberal tree-based (+threshold) methods.ResultsGenetic divergence analyses showed that the intra-specific divergence of each marker was much lower than the inter-specific divergence. Our results indicated that the rates of correct sequence identification for all four markers (COI, 16S rDNA, ITS2, 12S rDNA) were very high (> 96%) when using the NN methodology. We also found that COI was not significantly better than the other markers in terms of its rate of correct sequence identification. Overall, BLASTn and NN methods produced higher rates of correct species identification than that produced by the liberal tree-based methods (+threshold or otherwise).ConclusionsAs the standard DNA barcode, COI should be the first choice for tick species identification, while 16S rDNA, ITS2, and 12S rDNA could be used when COI does not produce reliable results. Besides, NN and BLASTn are efficient methods for species identification of ticks.

Taxonomy of the Crematogaster degeeri-species-assemblage in the Malagasy region (Hymenoptera: Formicidae)

We revise the species-level taxonomy of the Crematogaster (Crematogaster) degeeri-species-assemblage, a group of related ants occuring in Madagascar and the wider Malagasy region, and further provide an identification key to all species-groups of the genus Crematogaster in this region. Within the C. degeeri-assemblage, we recognize twelve species based upon morphological data from worker, queen and male ants, as well as genetic data from the barcode region of cytochrome oxidase I. Seven new species are described: Crematogaster alafara Blaimer sp. nov., C. bara Blaimer sp. nov., C. mafybe Blaimer sp. nov., C. maina Blaimer sp. nov., C. malahelo Blaimer sp. nov., C. masokely Blaimer sp. nov., C. ramamy Blaimer sp. nov. Crematogaster tricolor Gerstäcker, 1859 (stat. rev.) and C. dentata Dalla Torre, 1893 (stat. nov.) are raised to species level, and the following new synonymies are proposed: Crematogaster degeeri lunaris Santschi, 1928 as a synonym of C. degeeri Forel, 1886; Crematogaster sewelli improba Forel, 1907 and C. sewelli mauritiana Forel, 1907 as synonyms of C. dentata Dalla Torre, 1893, and C. pacifica Santschi, 1919 as a synonym of C. lobata Emery, 1895. Species descriptions, images, and distribution maps and identification keys based on worker ants, as well as on queen ants where available, are presented for all twelve species. In addition, we present a molecular gene tree for cytochrome oxidase I and summarize levels of sequence divergence within and between species of the C. degeeri-species-assemblage. Our findings are discussed in the light of previous work on Malagasy Crematogaster ants.

Linking Larvae and Adults of Apharyngostrigea cornu, Hysteromorpha triloba, and Alaria mustelae (Diplostomoidea: Digenea) Using Molecular Data

Because the taxonomy of trematodes is based on adults, the larval stages of most digeneans cannot be identified to species based on morphology alone. Molecular data provide a means of linking larval stages to known adults. We obtained sequences from the barcode region of cytochrome oxidase I (CO1) from adult and larval parasites of fish, frogs, birds, and mammals across North America. Sequences from adult Apharyngostrigea cornu, Hysteromorpha triloba, and Alaria mustelae (Diplostomoidea: Digenea) from definitive hosts matched those of meta- and mesocercariae from fish and frogs. These data provided new information on the distributions of all 3 parasite species. Metacercariae of A. cornu, which have not been previously reported in North American hosts, were found in Notemigonus crysoleucas, Pimephales notatus, and Catostomus commersonii in the St. Lawrence River. Metacercariae of H. triloba are reported in Canadian waters and in N. hudsonius for the first time. Alaria mustelae is reported for the first time in frogs from Quebec, Canada, and an additional species of Alaria was detected in California. Sequences of internal transcribed spacer rDNA from a subset of specimens support the same species boundaries indicated by CO1 divergence. There was little divergence in CO1 sequences from an unidentified diplostomid species sampled at a large spatial scale.

Multiple copies of cytochrome oxidase 1 in species of the fungal genus Fusarium

Using data from published mitochondrial or complete genomes, we developed and tested primers for amplification and sequencing of the barcode region of cytochrome oxidase 1 (COX1) of the fungal genus Fusarium, related genera of the order Hypocreales, and degenerate primers for fungi in the subdivision Pezizomycotina. The primers were successful for amplifying and sequencing COX1 barcodes from 13 genera of Hypocreales (Acremonium, Beauveria, Clonostachys, Emericellopsis, Fusarium, Gliocladium, Hypocrea, Lanatonectria, Lecanicillium, Metarhizium, Monocillium, Neonectria and Stilbella), 22 taxa of Fusarium, and two genera in other orders (Arthrosporium, Monilochaetes). Parologous copies of COX1 occurred in several strains of Fusarium. In some, copies of the same length were detected either by heterozygous bases in otherwise clean sequences or in different replicates of amplification and sequencing events; this may indicate multiple transcribed copies. Other strains included one or two introns. Two intron insertion sites had at least two nonhomologous intron sequences among Fusarium species. Irrespective of whether the multiple copy issue could be resolved by sequencing RNA transcripts, developing a precise COX1-based barcoding system for Fusarium may not be feasible. The overall divergence among homologous COX1 sequences obtained so far is rather low, with many species sharing identical sequences.

DNA‐based discrimination between the sibling species Aphis gossypii Glover and Aphis frangulae Kaltenbach

Abstract Morphologically similar species occur in various groups of insects, including aphid pests. In Europe, Aphis frangulae Kaltenbach and Aphis gossypii Glover (sometimes considered as subspecies) are differentiated usually on the basis of life cycle and host plant. We used a sexual population of A. frangulae collected on the primary host and samples of A. gossypii collected on cucurbits or cotton for the development of molecular markers. DNA sequence data for the gene encoding cytochrome b and for the barcode region of cytochrome oxidase I, as well as a length polymorphism for an intron in the sodium channel para‐type gene discriminated unambiguously between the two taxa. These markers were also used as identification keys for aphids collected on crops belonging to the Solanaceae. The cytochrome b marker differentiates host‐related Aphis gossypii haplotypes, and the para‐type gene intron might be suitable for the resolution of taxonomic problems in other aphid species complexes.

Polymerase chain reaction-restriction fragment length polymorphism assays to distinguish Liriomyza huidobrensis (Diptera: Agromyzidae) from associated species on lettuce cropping systems in Italy.

The pea leafminer, Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae), is a serious insect pest infesting open field lettuce plantings in northern Italy. In these cropping systems, it coexists with several other agromyzid species that have negligible economic importance on open field vegetables. The rapid detection of L. huidobrensis is crucial for effective management strategies, but the identification of agromyzids to species can be very difficult at adult as well at immature stages. In this study, a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay is proposed to separate L. huidobrensis from Liriomyza bryoniae (Kaltenbach), Liriomyza trifolii (Burgess), and Chromatomyia horticola (Goureau), which usually occur in the same lettuce plantings. An approximately 1,031-bp region of the mitochondrial genome encompassing the 3' region of cytochrome oxidase I, the whole leucine tRNA, and all of the cytochrome oxidase II was amplified by PCR and digested using the enzymes PvuII and SnaBI separately. Both endonucleases cut the amplicons of L. huidobrensis in two fragments, whereas the original band was not cleaved in the other analyzed species. The presence of Dacnusa spp. DNA does not bias the assay, because the PCR conditions and the primer set here described do not amplify any tract of this endoparasitic wasp genome.