Cytochrome C Oxidase Subunit I Sequences Research Articles

The Partial Nucleotide Sequences of the Mitochondrial Genes, COI and 16s rRNA, of Fireflies in the Genera Pygoluciola, Trisinuata, and Medeopteryx (Coleoptera: Lampyridae)

Fireflies in the genera Pygoluciola, Medeopteryx, and Trisinuata distribute throughout lower northern Thailand. Five morphospecies of these fireflies were collected and nucleotide sequences were analyzed. We sequenced the partial of two mitochondrial genes; Cytochrome c oxidase subunit I (COI) and 16s rRNA, and the phylogenetic trees were constructed and compared with the firefly nucleotide sequences in the NCBI database. The COI gene was amplified using LCO1490 and HCO2198 primers while specific primers of LR-J-13020a and LR-N-13398a were used to analyze amplicons of 16s rRNA. The results showed the average GC-contents in the nucleotide of five firefly morphospecies were found to average 30.28% and 22.15% in (COI) and 16s rRNA, respectively. The variable site in COI nucleotide sequences was 399 of the total 520 nucleotides (76.73%) whereas 91 of the total 418 nucleotides (21.77%) were found in 16s rRNA nucleotide sequences. Construction of the phylogenetic trees of those five firefly morphospecies with reference accession samples indicated five clades when analyzed by COI nucleotide sequences and two clades were revealed in 16s rRNA gene. According to these results, COI nucleotides are one of the most effective methods for differentiating between and within firefly species. The addition of the 16s rRNA gene analysis showed an increase in the precision of firefly identification. Those five firefly morphospecies were separated using both the COI and 16s rRNA nucleotide sequences.

First Report of Kiwifruit Root Rot Caused by Globisporangium spinosum in China.

Kiwifruit is widely cultivated for its high vitamin C content and nutritional value. In January 2022, root rot symptoms were found in about 30% of Actinidia chinensis cv. Jinyan plants grafted on A. deliciosa rootstocks in an orchard located in Sanming (26.32°N, 117.23°E), Fujian Province of China. The affected plants appeared stunted, with brown and decaying roots, some of which were covered with white hyphae. To isolate the pathogen, the surfaces of typical symptomatic roots were sterilized for 30 s using 75% ethanol, followed by four rinses in sterile water, placing on potato dextrose agar (PDA), and incubating away from light at 25°C for 7 days. 16 Globisporangium-like isolates were obtained through hyphal tip isolation, displaying a milky-white appearance with irregular protuberances on the surface, and yellow-white backs with radial fold lines. The isolates were then cultured on corn meal agar for 5 days at 25°C in dark for morphological characteristics. Under microscope, the hyphae appeared as long strips without septa and 4.1 to 8.2 µm wide (average 6.7 µm), containing irregularly sized spherical droplets. Both terminal and intercalary hyphae swellings were observed; these appeared either spherical or subspherical, with some having projections. Their dimensions were 12.3 to 27.6 µm (average 17.3 µm). The oospores were mostly spherical, either plerotic or aplerotic, 11.8 to 22.3 µm wide (average 18.9 µm), with occasional projections. The antheridia were rod-shaped and curved, with one end attached to the oogonia. Amplification of the sequences of internal transcribed spacer (ITS) regions and cytochrome c oxidase subunit I (COI) were conducted using the primers ITS1/ITS4 (White et al. 1990) and OomCoxI-Levlo/OomCoxI-Levup (Robideau et al. 2011), respectively. The sequencing results revealed identical ITS and COI sequences in all 16 isolates. BLASTn analysis of the 969-bp ITS sequence ON202808 showed 99.38-99.59% similarity (965/971bp, 967/971bp) with the KJ162353 and AY598701 sequences from Globisporangium spinosum isolates, while the 700-bp COI sequence ON075783 showed 100% and 99.41% identity (680/680bp, 676/680bp) with the GenBank sequences HQ708835 and HQ708832, respectively, from G. spinosum. Phylogenetic analysis also showed that the obtained isolate (termed MA16) clustered with isolates from G. spinosum on the same evolutionary branch. For pathogenicity testing, four-month-old healthy Jinyan (A. chinensis) plants grown in sterilized media were transferred to sterile petri dishes covered with wet filter paper, and their roots were inoculated with a 5-mm-wide disk of MA16 when cultivated on PDA medium for 5 days. Miliang-1 (A. deliciosa) and Hongyang (A. chinensis) plants were treated similarly. The control groups each included three plants that were inoculated with non-colonized PDA. The plants were kept at 25 °C with a 12-/12-h light/dark cycle for 10 days when the inoculated plants exhibited root rot symptoms similar to those seen in the field, together with rotting and browning of the leaves. The control plants appeared healthy with no symptoms. After re-isolated from infected tissues, the pathogen was verified to be G. spinosum according to its ITS sequence, thus fulfilling the Koch's postulates. Recently, Pythium spinosum has been classified as G. spinosum according to whole-genome sequencing and phylogenomic analysis (Nguyen et al. 2022). Based on the morphological features and pathogenicity results, MA16 was identified as G. spinosum (van der Plaats-Niterink 1981; Huo et al. 2023). This report appears to be the first description of kiwifruit root rots caused by G. spinosum in China, and its identification will assist the development of strategies to counteract the disease.

A Case of Myiasis Caused by Cordylobia anthropophaga (Diptera: Calliphoridae) in an Italian Traveler Returning from Senegal.

Myiases are infestations of human and animal tissues by fly larvae. These conditions are widespread in tropical countries and travelers in those areas are at risk of becoming infested. Although Cordylobia anthropophaga (Blanchard & Berenger-Feraud, 1872) is one of the most common myiasis-causing species, few high-quality images and molecular sequences are available for this fly. We present a case of C. anthropophaga infestation in an Italian patient returning from Senegal, with the aim of increasing both visual and molecular data for this species. After removal, the larva was determined following standardized morphological keys and photographed under a digital microscope. Molecular characterization of the Cytochrome c oxidase subunit I (COI) was performed using universal primers. The general appearance, the structural organization of the cephalic region, of the cephaloskeleton, and of the posterior tracheal spiracles suggested that the causative agent of the myiasis was a third instar larva of C. anthropophaga. The morphological data are further supported by the molecular data: the COI sequence showed high levels of identity with the already published verified COI sequences of C. anthropophaga. We provide high-quality morphological and molecular data useful for the identification of larvae of C. anthropophaga. We highlight that myiasis might be common in Senegal and better data about its prevalence in travelers and in the endemic countries are needed to understand the burden of this condition.

The great gerbil (Rhombomys opimus) as a host for tick species in Gurbantunggut Desert

BackgroundRodents play an important role in the life cycle of ixodid and argasid ticks, particularly as hosts of larvae and nymphs. The great gerbil (Rhombomys opimus), the preferred prey item of several carnivores (e.g. the red fox and marbled polecat), is the dominant rodent species in the Gurbantunggut Desert in northwestern China. The aim of this study was to investigate tick species associated with different hosts in the habitat of great gerbils, including wildlife and livestock.MethodsDuring 2018–2023, ticks were removed from 326 great gerbils, two red foxes (Vulpes vulpes), three marbled polecats (Vormela peregusna), 35 pastured sheep (Ovis aries), and one long-eared desert hedgehog (Hemiechinus auritus) in the Gurbantunggut Desert. Ticks were identified according to standard morphological keys. Then, they were further analyzed by molecular and phylogenic methods based on two mitochondrial markers, 16S rDNA and cytochrome c oxidase subunit I (COI) genes.ResultsA total of 889 ticks were collected, representing five species. These included Hyalomma asiaticum (n = 425: 24 larvae, 79 nymphs and 322 adults), Rhipicephalus turanicus (n = 153: 2 nymphs and 151 adults), Haemaphysalis erinacei (n = 298: 4 larvae, 7 nymphs and 287 adults), Ixodes acuminatus (n = 7: 4 nymphs and 3 adults) and Ornithodoros tartakovskyi (6 adults). Based on COI sequences, molecular and phylogenetic analyses showed that (i) I. acuminatus from great gerbils and marbled polecats clustered with I. acuminatus reported from Europe; (ii) O. tartakovskyi found in northwestern China belonged to an independent clade; (iii) Hy. asiaticum, R. turanicus and Ha. erinacei had 100% sequence identities to conspecific ticks sampled previously in China.ConclusionsThe great gerbil is an important host for the developmental stages of I. acuminatus, O. tartakovskyi, Ha. erinacei, Hy. asiaticum and R. turanicus, thus supporting the life cycle of several tick species which, as adults, parasitize predators (red fox and marble polecat) as well as pastured sheep and hedgehogs in the Gurbantunggut Desert. Ixodes acuminatus and O. tartakovskyi were found for the first time on great gerbil and marbled polecat, respectively.Graphical

Site-specific PCR identification of animal species and formula particles of Scorpio

Scorpio, a commonly used animal medicine in China, is derived from Buthus martensii as recorded in the Chinese Pharmacopoeia. China harbors rich species of Scorpionida and adulterants exist in the raw medicinal material and deep-processed products of Scorpio. The microscopic characteristics of the deep-processed products may be incomplete or lost during processing, which makes the identification difficult. In this study, the maximum likelihood(ML) tree was constructed based on the morphology and cytochrome C oxidase subunit I(COⅠ) to identify the species of Scorpio products. The results showed that the main adulterant of Scorpio was Lychas mucronatus. According to the specific SNP sites in the COⅠ sequence of B. martensii, the stable primers were designed for the identification of the medicinal material and formula granules of Scorpio. The polymerase chain reaction(PCR) at the annealing temperature of 61 ℃ and 30 cycles produced bright specific bands at about 150 bp for both B. martensii and its formula particles and no band for adulterants. The adaptability of the method was investigated, which showed that the bands at about 150 bp were produced for Scorpio medicinal material, lyophilized powder, and formula granules, and commercially available formula granules. The results showed that the established method could be used to identify the adulterants of Scorpio and its formula granules, which could help to improve the quality control system and ensure the safe clinical application of Scorpio formula granules.

Genetic diversity and phylogeography of Phlebotomus argentipes (Diptera: Psychodidae, Phlebotominae), using COI and ND4 mitochondrial gene sequences.

Phlebotomus argentipes complex is the primary vector for cutaneous leishmaniasis, a burgeoning health concern in contemporary Sri Lanka, where effective vector control is important for proper disease management. Understanding the genetic diversity of the P. argentipes population in Sri Lanka is vital before implementing a successful vector control program. Various studies have indicated that genetic divergence, caused by genetic drift or selection, can significantly influence the vector capacity of arthropod species. To devise innovative control strategies for P. argentipes, exploring genetic diversity and phylogeography can offer valuable insights into vector competence, key genetic trait transfer, and impact on disease epidemiology. The primary objective is to analyze the genetic diversity and phylogeography of the P. argentipes complex in Sri Lanka, based on two mitochondrial genomic regions in modern representatives of P. argentipes populations. A total of 159 P. argentipes specimens were collected from five endemic areas of cutaneous leishmaniasis and identified morphologically. Two mitochondrial regions (Cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4) were amplified using the total DNA and subsequently sequenced. Partial sequences of those mitochondrial genes were utilized to analyze genetic diversity indices and to explore phylogenetic and phylogeographic relationships. Among five sampling locations, the highest genetic diversity for COI and ND4 was observed in Hambantota (Hd-0.749, π-0.00417) and Medirigiriya (Hd-0.977, π-0.01055), respectively. Phylogeographic analyses conducted using COI sequences and GenBank retrieved sequences demonstrated a significant divergence of P. argentipes haplotypes found in Sri Lanka. Results revealed that they have evolved from the Indian ancestral haplotype due to historical- geographical connections of the Indian subcontinent with Sri Lanka. Utilizing high-mutation-rate mitochondrial genes, such as ND4, can enhance the accuracy of genetic variability analysis in P. argentipes populations in Sri Lanka. The phylogeographical analysis of COI gene markers in this study provides insights into the historical geographical relationship between India and P. argentipes in Sri Lanka. Both COI and ND4 genes exhibited consistent genetic homogeneity in P. argentipes in Sri Lanka, suggesting minimal impact on gene flow. This homogeneity also implies the potential for horizontal gene transfer across populations, facilitating the transmission of genes associated with traits like insecticide resistance. This dynamic undermines disease control efforts reliant on vector control strategies.

Cracking the glass‐perchlet code: Integrative taxonomy uncovers high species‐level diversity within the glass‐perchlet genus Ambassis (Ambassidae) in tropical Asia

AbstractGlass‐perchlets of the genus Ambassis (Teleostei; Ambassidae) form an important component of the brackish and marine coastal fish communities of tropical Asia. However, their species‐level diversity is still poorly documented because of the absence of recent taxonomic revisions in this region and the limited availability of specimens for research. In addition, long‐standing taxonomic and nomenclatural issues complicate the studies of this genus. Herein, we examine the diversity of Ambassis in Peninsular Malaysia using an integrative taxonomic approach and a large set of recently collected specimens from this region. Our initial morphological observations of 260 specimens revealed the presence of eight species, identified as Ambassis dussumieri, Ambassis interrupta, Ambassis kopsii, Ambassis macracanthus, Ambassis nalua, Ambassis octava, Ambassis urotaenia and Ambassis vachellii. We then sequenced the barcode fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene for 122 of our specimens, representing all eight morpho‐species. Automatic species delimitation methods recovered nine Molecular Operational Taxonomic Units (MOTUs) because A. interrupta is made of two MOTUs. Morphological re‐examination within A. interrupta detected variation at one character, congruent with molecular delimitation. Overall, our integrative approach unveiled rich species‐level diversity within the genus Ambassis in Peninsular Malaysia, with the presence of nine species. Further comparisons between our COI dataset and the COI sequences archived in the Barcode of Life Data System (BOLD) from specimens of Ambassis broadly collected in tropical Asian regions, indicated regional‐scale hidden diversity and identification conflicts, triggering the need for a complete taxonomic revision of this genus.

Cryptic Diversity in Sympatric Migonemyia migonei (Diptera: Psychodidae), Eventual Meaning for Leishmaniasis Transmission.

Migonemyia migonei (Franҫa, 1920) (Diptera: Psychodidae) belongs to the subfamily Phlebotominae, of epidemiological importance due to its role as a vector in leishmaniasis transmission cycles and its broad geographic distribution in South America. Few morphometric and genetic studies have demonstrated the existence of variability among geographically distant populations in Brazil. The aim of the study was to estimate the genetic distance within the morphospecies Mg. migonei through the analysis of cytochrome C oxidase subunit I (COI) sequences of specimens captured in Argentina and those available in online databases. The COI sequences from specimens collected in different localities of Argentina and sequences available in online databases were utilized. Genetic distances were analyzed and a median-joining haplotype network was constructed. Finally, phylogenetic reconstruction was performed according to Bayesian inference. The analyses led to the identification of at least two haplogroups: haplogroup I with sequences of specimens from Colombia, Brazil and Argentina, and haplogroup II with sequences of specimens from Argentina. Interestingly, specimens from Argentina whose haplotypes corresponded to both haplogroups, were collected in sympatry. The results suggest that Mg. migonei could be a species complex with at least two distinct members. This hypothesis could explain the known characteristics of adaptability and vector permissiveness of the species, as the putative cryptic species of the complex could differ in traits of epidemiological importance.

Molecular evidence for multiple origins and high genetic differentiation of non-native winter crane fly, Trichocera maculipennis (Diptera: Trichoceridae), in the maritime Antarctic

Native biodiversity and ecosystems of Antarctica safeguarded from biological invasion face recent threats from non-native species, accelerated by increasing human activities and climate changes. Over two decades ago, the winter crane fly, Trichocera maculipennis, was first detected on King George Island. It has now successfully colonized several research stations across King George Island. To understand the origin, genetic diversity, and population structure of this Holarctic species, we conducted mitochondrial DNA cytochrome c oxidase subunit I (COI) sequence analysis across both its native and invasive ranges. In parallel, we performed microsatellite loci analysis within the invasive ranges, utilizing 12 polymorphic microsatellite markers. Furthermore, we compared body sizes among adult males and females collected from three different locations of King George Island. Our COI sequence analysis exhibited two different lineages present on King George Island. Lineage I was linked to Arctic Svalbard and Polish cave populations and Lineage II was related to Canadian Terra Nova National Park populations, implying multiple origins. Microsatellite analysis further exhibited high levels of genetic diversity and significant levels of genetic differentiation among invasive populations. Body sizes of adult T. maculipennis were significantly different among invasive populations but were not attributed to genetics. This significant genetic diversity likely facilitated the rapid colonization and establishment of T. maculipennis on King George Island, contributing to their successful invasion. Molecular analysis results revealed a substantial amount of genetic variation within invasive populations, which can serve as management units for invasive species control. Furthermore, the genetic markers we developed in the study will be invaluable tools for tracking impending invasion events and the travel routes of new individuals. Taken together, these findings illustrate the highly invasive and adaptable characteristics of T. maculipennis. Therefore, immediate action is necessary to mitigate their ongoing invasion and facilitate their eradication.

Morphological and molecular characterisation of ghost jellyfish Cyanea nozakii (Cnidaria: Scyphozoa) forming a swarm in the Bay of Bengal, Indian Ocean

The swarm of Scyphozoa, Cyanea nozakii is considered as a nuisance since it causes ecological catastrophe and dangerous stings to humans. This species is distributed in the Pacific and Indian Ocean. The present study reports the first swarm of C. nozakii Kishinouye, 1891 from the coastal waters of Northwestern Bay of Bengal during winter monsoon, 2021. The species was identified by using integrated tools of morphometry and molecular taxonomy. DNA barcoding of mitochondrial Cytochrome c Oxidase subunit I (COI) was used to confirm the species identity after taxonomic identification. Our sample clustered closely with previously published sequences of C. nozakii from the coastal waters of China. The COI sequence alignment translated into 242 amino acid residues, comprising of 36 variable sites, 199 conserved sites and 15 parsimony informative sites. C. nozakii was present with a mean abundance of 270 ± 50 ind. m−3 in the study area. This study was undertaken to monitor the geospatial spread of jellyfish species diversity and to document the recent increase in jellyfish swarms from Indian waters.

Taxonomic Contributions on Macrogynoplax Enderlein and Enderleina Jewett (Plecoptera: Perlidae) from Brazil, Including a New Species Supported by Molecular and Morphological Data.

Stoneflies (Plecoptera) are aquatic insects widely used in ecological and monitoring studies as indicators of environmental quality due to their high sensitivity to pollution. Among Brazilian Plecoptera, Perlidae is the most diverse family, with four genera (Anacroneuria Klapálek 1909, Enderleina Jewett 1960, Kempnyia Klapálek 1914, and Macrogynoplax Enderlein 1909) and about 150 species. Herein, based on data from the barcode region of the mitochondrial cytochrome c oxidase subunit I (COI) gene and morphological characters, we studied specimens of Enderleina and Macrogynoplax, mainly from the Brazilian Savanna. Our alignment with a total length of 608 base pairs included 23 COI sequences, of which 10 are new and 13 were obtained from GenBank. The sequences and congruence of the obtained species clusters were analyzed using neighbor joining. As a result, we describe a new species of Enderleina, the first of this genus in the Brazilian Savanna, and update the geographic distribution of some Macrogynoplax species. A new record was included for Macrogynoplax veneranda Froehlich 1984 in São Paulo state and the first record of M. yupanqui Stark 1996 in Brazil was also included. First records were made for Macrogynoplax matogrossensis Bispo and Neves 2005 and a Macrogynoplax specimen in Goiás and Bahia states, respectively. Consequently, we expanded the distribution of the genus in the Brazilian Savanna and Atlantic Forest. In addition, we provide observations on the morphological differences found among these species, discuss about the distribution of the genera, present molecular data discussing their contributions to the knowledge of the genus, and comment on the recommendations for future research considering a scenario of integrative approaches. Lastly, we present a key to identify the adult males of Enderleina.

Genetic variation of the lymnaeid Radix rubiginosa (Michelin, 1831) (Mollusca, Gastropoda) and other freshwater snails in Thailand examined using mitochondrial COI sequences

ABSTRACT Freshwater lymnaeid snails, for example Radix rubiginosa, may serve a crucial role as the sole intermediate hosts of several trematodes of medical or veterinary importance causing diseases in livestock and humans including fascioliasis, schistosomiasis and echinostomiasis. In Thailand, there is little information on the genetic variation of lymnaeid snails. Thus, this study explores the mitochondrial cytochrome c oxidase subunit I (COI) sequence variation of lymnaeid and physid snails collected from different geographical areas in Thailand, with the main focus on R. rubiginosa. In all, 175 R. rubiginosa, 7 Orientogalba sp., 9 Racesina sp., and 61 Physella acuta were collected from 36 localities in Thailand. A high level of genetic variation was detected in R. rubiginosa, with 40 haplotypes being recorded. Phylogenetic analyses revealed two clades (possibly species) in the material identified as R. rubiginosa. One clade contained all samples examined in this study, together with a sequence from Indonesia, while the second clade comprised specimens found in Singapore, Malaysia, and Vietnam. Seven haplotypes of P. acuta were clustered into clade A, the globally invasive lineage of this species.

From bites to barcodes: uncovering the hidden diversity of black flies (Diptera: Simuliidae) in Vietnam

BackgroundPrompt and precise identification of black flies (Simuliidae) is crucial, given their biting behaviour and significant impact on human and animal health. To address the challenges presented by morphology and chromosomes in black fly taxonomy, along with the limited availability of molecular data pertaining to the black fly fauna in Vietnam, this study employed DNA-based approaches. Specifically, we used mitochondrial and nuclear-encoded genes to distinguish nominal species of black flies in Vietnam.MethodsIn this study, 135 mitochondrial cytochrome c oxidase subunit I (COI) sequences were established for 45 species in the genus Simulium in Vietnam, encompassing three subgenera (Gomphostilbia, Nevermannia, and Simulium), with 64 paratypes of 27 species and 16 topotypes of six species. Of these COI sequences, 71, representing 27 species, are reported for the first time.ResultsCombined with GenBank sequences of specimens from Malaysia, Myanmar, Thailand, and Vietnam, a total of 234 DNA barcodes of 53 nominal species resulted in a 71% success rate for species identification. Species from the non-monophyletic Simulium asakoae, S. feuerborni, S. multistriatum, S. striatum, S. tuberosum, and S. variegatum species groups were associated with ambiguous or incorrect identifications. Pairwise distances, phylogenetics, and species delimitation analyses revealed a high level of cryptic diversity, with discovery of 15 cryptic taxa. The current study also revealed the limited utility of a fast-evolving nuclear gene, big zinc finger (BZF), in discriminating closely related, morphologically similar nominal species of the S. asakoae species group.ConclusionThis study represents the first comprehensive molecular genetic analysis of the black fly fauna in Vietnam to our knowledge, providing a foundation for future research. DNA barcoding exhibits varying levels of differentiating efficiency across species groups but is valuable in the discovery of cryptic diversity.Graphical abstract

Molecular characterization and genetic diversity of three Stomoxys flies S. bengalensis, S. calcitrans, and S. sitiens (Diptera: Muscidae) in Central Thailand.

Stomoxys flies (Diptera: Muscidae) are hematophagous ectoparasites of medical and veterinary importance. In this study, three Stomoxys species, i.e. S. bengalensis, S. calcitrans, and S. sitiens, were collected from three provinces in Central Thailand with the aim of estimating the genetic divergence between species, for species identification, as well as within species, for a genetic diversity study based on the cytochrome c oxidase subunit I (COI) gene. Our results showed that the average intraspecific genetic divergences of Stomoxys flies ranged from 0.11% in S. sitiens to 0.98% in S. calcitrans, whereas the average interspecific genetic divergences ranged from 5.24% between S. sitiens and S. bengalensis to 6.69% between S. calcitrans and S. bengalensis. In addition, there was no overlap between the intraspecific and interspecific genetic divergences. The COI sequence analysis revealed a high haplotype diversity and low nucleotide diversity, reflecting a rapid population expansion after past bottlenecks. Moreover, there was no significant difference (P>0.05) in the pairwise population differentiation (Fst) among Stomoxys flies in Central Thailand, because of the lack of natural barriers, thus allowing genetic exchange between them. The monitoring of the haplotype network revealed that two lineages of S. calcitrans in Central Thailand were distributed in all study areas, including the Nakhon Pathom, Pathum Thani, and Saraburi Provinces. These findings may improve our understanding of the genetic patterns of these three Stomoxys flies, as well as the underlying biological mechanisms, which is knowledge that can be used for further effective control of these flies.

DNA barcoding of black flies (Diptera: Simuliidae) in Indonesia

BackgroundDNA barcoding is a valuable taxonomic tool for rapid and accurate species identification and cryptic species discovery in black flies. Indonesia has 143 nominal species of black flies, but information on their biological aspects, including vectorial capacity and biting habits, remains underreported, in part because of identification problems. The current study represents the first comprehensive DNA barcoding of Indonesian black flies using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences.MethodsGenomic DNA of Indonesian black fly samples were extracted and sequenced, producing 86 COI sequences in total. Two hundred four COI sequences, including 118 GenBank sequences, were analysed. Maximum likelihood (ML) and Bayesian inference (BI) trees were constructed and species delimitation analyses, including ASAP, GMYC and single PTP, were performed to determine whether the species of Indonesian black flies could be delineated. Intra- and interspecific genetic distances were also calculated and the efficacy of COI sequences for species identification was tested.ResultsThe DNA barcodes successfully distinguished most morphologically distinct species (> 80% of sampled taxa). Nonetheless, high maximum intraspecific distances (3.32–13.94%) in 11 species suggested cryptic diversity. Notably, populations of the common taxa Simulium (Gomphostilbia) cheongi, S. (Gomphostilbia) sheilae, S. (Nevermannia) feuerborni and S. (Simulium) tani in the islands of Indonesia were genetically distinct from those on the Southeast Asian mainland (Malaysia and Thailand). Integrated morphological, cytogenetic and nuclear DNA studies are warranted to clarify the taxonomic status of these more complex taxa.ConclusionsThe findings showed that COI barcoding is a promising taxonomic tool for Indonesian black flies. The DNA barcodes will aid in correct identification and genetic study of Indonesian black flies, which will be helpful in the control and management of potential vector species.Graphical abstract

Morpho-Molecular Characterization and Seasonality analysis of Greenidea psidii Van Der Goot on Guava in Terai Zone of West Bengal, India

Greenidea psidii, Guava aphid comes under the subfamily Greenideinae within the Aphididae and was first discovered in the World in 1916 on guava. In terai region of West Bengal these species are regarded as potential pest of guava (Psidium guajava). Therefore, quick and precise species identification is immediately needed for early detection and risk analysis followed by integrated management of the aphid pests. During morpho- taxonomy study we found that apterous aphids have long sized siphunculi, alongwith long setae present on the body. Moreover, the identity of the aphid species was confirmed through DNA barcoding of the mitochondrial cytochrome c oxidase sub unit I (COX-I) gene, another analytical tool for quick and precise identification. The COX-I sequence was successfully blasted for similarity search and uploaded in NCBI software (Accession No. OR085959). Maximum likelihood method for phylogenetic analysis reveals that all the G. psidii sequences were clustered in the same clade and confirmed the identity of the species of the present study. The seasonal fluctuation studies reveals that the maximum population of 21.85 aphids/ twigs and 17.45 aphids/ twigs were observed on 10th SW during 2021 and on 11th SW during 2022, respectively.

Genetic Diversity and Phylogeography of Taenioides cirratus in Five Geographical Populations Based on Mitochondrial COI and Cytb Gene Sequences

The genetic diversity of Taenioides cirratus was investigated on the basis of mitochondrial cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) gene sequences. A total of 159 specimens collected from the Chaohu Lake (CL), Nansihu Lake (NL), Taihu Lake (TL), Pearl River (PR), and Nandu River (NR) were sequenced. The total length of the sequence was 2485 bp with 412 polymorphic sites. A total of 73 haplotypes were identified, with Hap1 being the most widely distributed. The PR and NR populations showed high genetic diversity, while the CL population showed low genetic diversity. TL and NL showed high haplotype diversity but low nucleotide diversity. The analysis of molecular variance demonstrated that the sequence variations were mainly occurred among populations. T. cirratus populations are declining, and rare alleles are present at low frequencies, as analysed using a neutral test and a mismatched distribution analysis. There was a relatively high level of genetic differentiation among the populations of the Yangtze River basin (including NL), PR, and NR (Fst > 0.15). The two similar phylogenetic trees constructed by the maximum likelihood (ML) and Bayesian inference (BI) methods presented three major lineages, of which lineage II contains haplotypes from PR and NR, lineage III contains haplotypes from CL, NL, TL, and PR, whereas lineage I contains only a portion of haplotypes from NR. Based on the neutral test, mismatch analysis, and Bayesian Skyline Plot (BSP), geological and climatic events were inferred to have played an important role in the historical dynamics of T. cirratus population. Hap1, Hap25, and Hap58 were inferred as possible ancestral haplotypes by network analysis. Our study offers an essential foundation for resource preservation and additional taxonomic clarification of T. cirratus.

First record on the occurrence of long fingered gurnard Lepidotrigla longimana (Scorpaeniformes: Triglidae) from Bangladesh

This study confirmed the presence of triglid fish Lepidotrigla longimana Li, (1981) for the first time in Bangladesh waters. Four individuals of this species were collected from Cox's Bazar (21.77 N 91.62 E) on 5 October 2018 as bycatch. The species was taxonomically identified through the traditional morphometric method which was further validated by a molecular approach based on partial cytochrome c oxidase subunit I (COI) sequences (DNA barcodes). The body length is three times of the head length and 4.1 times of the body height. Head length is 2.5 times larger than the snout length and 3.2 times of the eye diameter. The number of scales on the lateral line is 60. A smaller blue spot is present on the inner side of the pectoral fin. The average K2P distances of the COI sequence within species were 0.53. In the Maximum likelihood (ML) tree, the sequences clustered together under a single clade. Morphometric, meristic and molecular data thus confirmed the species as L. longimana. J. Bangladesh Acad. Sci. 47(1); 29-32: June 2023

Hitting the Road: Haplotype Diversity of Fire Ants Nesting on Disturbed Atlantic Forest Habitats.

Ants of the genus Solenopsis are globally distributed, presenting high diversity and many generalist species. In South America, the dominant species is Solenopsis saevissima (Smith, 1855), commonly found nesting in grassy fields surrounding humanized areas. In spite of being so common, there has been no research evaluating the effect of human disturbances on the mitochondrial DNA (mtDNA) haplotype diversity in this species. In this context, we here characterized the mtDNA haplotype diversity in S. saevissima nests by highway roadsides, dust roads, and forest borders of Atlantic Forest, based on partial sequences of cytochrome c oxidase subunit I (COI). Based on the facts that the species is a rapid colonizer of disturbed habitats, we specifically probed how the genetic diversity of native S. saevissima is impacted by highways and roads infrastructure expanding around the rainforest. Species diagnosis was established both by morphological characters and obtained mtDNA COI sequences. Overall, the species exhibited high haplotypes and nucleotide diversity, particularly around forest borders; though all haplotypes seemed closely related across the different habitats. We identified seven mitochondrial haplotypes (H1 to H7), where haplotype H1 was exclusively found in highway roadside nests, and H7 on dust roads; the remaining haplotypes were recorded from all habitats. Haplotype H1 was geographically isolated to the south of the Atlantic Forest, supporting previous suggestions that it acts as a biogeographical barrier. The pattern is suggestive of a recent species expansion, probably resulting from extensive habitat fragmentation. Taken together, our data demonstrates fire ant haplotypes prevailing in some anthropized habitats, characterizing how a native species lining the remnants of the Brazilian Atlantic Forest might be a concern for environmental conservation.

Molecular identification of hymenopteran parasitoids and their endosymbionts from agromyzids.

Three polyphagous pest Liriomyza spp. (Diptera: Agromyzidae) have recently invaded Australia and are damaging horticultural crops. Parasitic wasps are recognized as effective natural enemies of leafmining species globally and are expected to become important biocontrol agents in Australia. However, the hymenopteran parasitoid complex of agromyzids in Australia is poorly known and its use hindered due to taxonomic challenges when based on morphological characters. Here, we identified 14 parasitoid species of leafminers based on molecular and morphological data. We linked DNA barcodes (5' end cytochrome c oxidase subunit I (COI) sequences) to five adventive eulophid wasp species (Chrysocharis pubicornis (Zetterstedt), Diglyphus isaea (Walker), Hemiptarsenus varicornis (Girault), Neochrysocharis formosa (Westwood), and Neochrysocharis okazakii Kamijo) and two braconid species (Dacnusa areolaris (Nees) and Opius cinerariae Fischer). We also provide the first DNA barcodes (5' end COI sequences) with linked morphological characters for seven wasp species, with three identified to species level (Closterocerus mirabilis Edwards & La Salle, Trigonogastrella parasitica (Girault), and Zagrammosoma latilineatum Ubaidillah) and four identified to genus (Aprostocetus sp., Asecodes sp., Opius sp. 1, and Opius sp. 2). Phylogenetic analyses suggest C. pubicornis, D. isaea, H. varicornis, and O. cinerariae are likely cryptic species complexes. Neochrysocharis formosa and Aprostocetus sp. specimens were infected with Rickettsia. Five other species (Cl. mirabilis, D. isaea, H. varicornis, Opius sp. 1, and Opius sp. 2) were infected with Wolbachia, while two endosymbionts (Rickettsia and Wolbachia) co-infected N. okazakii. These findings provide background information about the parasitoid fauna expected to help control the leafminers.