Cytochrome C Oxidase Subunit I Research Articles

Multi-local analysis supports the transfer of Omolicna fulva to the genus Agoo and establishment of a new species in the genus from Costa Rica

Recent survey work in Costa Rica has resulted in the discovery of a series of new species of derbid and cixiid planthoppers associated with palms. Here a new species of Agoo was collected by sweeping palms and is described as Agoo keili sp. nov. Sequence data are generated for the new species and Omolicna fulva for the cytochrome c oxidase subunit I (COI) gene, 18S rRNA gene and the D9 to D10 expansion region for the 28S rRNA gene to support the placement of the novel taxon in the genus Agoo. Omolicna fulva is transferred to Agoo, as A. fulvus comb. nov.

New Records of Phenacoccus solenopsis Natural Enemies in Europe and Taxonomic Additions on Anagyrus matritensis

The cotton mealybug Phenacoccus solenopsis (Hemiptera: Pseudococcidae) is a polyphagous invasive species native to America and considered one of the major cotton pests in Asia. It is currently threatening horticultural and ornamental protected crops in Mediterranean countries. Due to ecological and environmental concerns, the conventional chemical control of P. solenopsis in new areas of introduction is being replaced by exploring the potential of indigenous natural enemies as a sustainable biological control tool. After P. solenopsis introduction in Sicily (Italy), field surveys were conducted on native natural enemies attacking the mealybug to select promising biocontrol agents for field applications. For the first time, Aenasius arizonensis (Hymenoptera: Encyrtidae) was reported in Europe, and the native Anagyrus matritensis (Hymenoptera: Encyrtidae) was recorded in association with P. solenopsis. The two parasitoid species were identified by morphological features and molecularly using a portion of the mitochondrial cytochrome oxidase subunit I (mtCOI) gene. Because of missing information, additional morphological features were provided for the morphological identification of A. matritensis. In addition, the generalist predators Cryptolaemus montrouzieri, Hippodamia variegata and Parexochomus nigripennis (Coleoptera: Coccinellidae) were also recorded attacking the invasive mealybug.

Description of the morphology, morphometric, and molecular of Aphelenchoides fragariae (Aphelenchida: Aphelenchoididae) causing crimp disease of strawberry in Indonesia

Aphelenchoides fragariae, commonly known as strawberry crimp nematodes, primarily target the aerial parts of plants, affecting both internal and external structures. In Indonesia, where strawberries are predominantly cultivated in highland regions, the presence of strawberry crimp disease has been confirmed. Infected plants exhibit symptoms such as stunted growth, reddened foliage, crimped or curled leaves, and malformed buds and blooms. Aboveground damage caused by the nematodes includes contorted shoots, undersized leaves, and reddish petioles, often accompanied by discolored patches on the foliage. These symptoms significantly impair the growth and productivity of strawberry plants, highlighting the nematode’s potential as a serious pest in these regions. The identification of A. fragariae was achieved through a combination of morphological and molecular characterization methods. Species confirmation relied on PCR amplification of the nematode’s cytochrome oxidase subunit I (COI) gene, using primers (COI F and COI R) designed in the laboratory. The amplification yielded a specific fragment of approximately 550 base pairs, which was sequenced for further analysis. Sequence alignment revealed identity levels ranging from 82.8% to 99.7%, confirming the presence of A. fragariae. The resulting sequences were deposited in GenBank under the accession numbers LC804455 (A. fragariae isolate RB) and LC804456 (A. fragariae isolate LB), providing a valuable resource for future studies on this nematode species.

A new record of the genus Parabioxys (Hymenoptera, Braconidae, Aphidiinae) and a redescription of Bioxys japonicus from South Korea

The genera Bioxys Starý & Schlinger, 1967 and Parabioxys Shi & Chen, 2001 (Hymenoptera: Braconidae: Aphidiinae) are each known to contain only one species worldwide. Bioxys japonicus Starý & Schlinger, 1967 was recorded in South Korea in 1983, but this record should be considered doubtful. Parabioxys songbaiensis Shi & Chen, 2001 is now reported for the first time in South Korea. In this study, we provide detailed descriptions and photographic documentation of these two little-known species, B. japonicus and P. songbaiensis. Additionally, we present mitochondrial cytochrome c oxidase subunit I (COI) barcode region data and conduct a Bayesian tree analysis of closely related taxa.

Using the long-term genetic monitoring network ARMS-MBON to detect marine non-indigenous species along the European coasts

The increasing prevalence of non-indigenous species (NIS) in marine ecosystems poses significant challenges for biodiversity conservation and ecosystem management. Advances in molecular techniques enable early detection and long-term monitoring of such taxa, especially when coupled with spatio-temporally wide sampling by networks such as the European ARMS Marine Biodiversity Observation Network (ARMS-MBON). This initiative performs standardised sampling campaigns using autonomous reef monitoring structures (ARMS) along European coasts and adjacent regions, providing open-access DNA metabarcoding data sets. We tested the potential of genetic observatory networks to detect and monitor marine NIS by analysing all publicly available ARMS-MBON cytochrome c oxidase subunit I (COI) and 18S rRNA amplicon sequencing data as of February 2024 using a customised bioinformatic pipeline. Screening against the World Register of Introduced Marine Species (WRiMS) and applying manual curation, we identified 63 marine taxa considered non-indigenous at one or more locations. This included widespread taxa and potential new introductions, such as Eucheilota menoni in the Adriatic Sea. We found no significantly higher number of NIS in samples from locations particularly impacted by maritime traffic compared to other areas. Our results suggest that the genetic observatory network approach is powerful for detecting and monitoring marine NIS, and that manual curation still is an essential step for obtaining reliable results. We recommend key improvements including more spatially intense sampling across diverse environments as well as enhancement of NIS reference checklists and genetic databases to ensure accurate identification of both known and unknown NIS across Europe.

A Long-Term Ecological Research Data Set From the Marine Genetic Monitoring Program ARMS-MBON 2018-2020.

Molecular methods such as DNA/eDNA metabarcoding have emerged as useful tools to document the biodiversity of complex communities over large spatio-temporal scales. We established an international Marine Biodiversity Observation Network (ARMS-MBON) combining standardised sampling using autonomous reef monitoring structures (ARMS) with metabarcoding for genetic monitoring of marine hard-bottom benthic communities. Here, we present the data of our first sampling campaign comprising 56 ARMS units deployed in 2018-2019 and retrieved in 2018-2020 across 15 observatories along the coasts of Europe and adjacent regions. We describe the open-access data set (image, genetic and metadata) and explore the genetic data to show its potential for marine biodiversity monitoring and ecological research. Our analysis shows that ARMS recovered more than 60 eukaryotic phyla capturing diversity of up to ~5500 amplicon sequence variants and ~1800 operational taxonomic units, and up to ~250 and ~50 species per observatory using the cytochrome c oxidase subunit I (COI) and 18S rRNA marker genes, respectively. Further, ARMS detected threatened, vulnerable and non-indigenous species often targeted in biological monitoring. We show that while deployment duration does not drive diversity estimates, sampling effort and sequencing depth across observatories do. We recommend that ARMS should be deployed for at least 3-6 months during the main growth season to use resources as efficiently as possible and that post-sequencing curation is applied to enable statistical comparison of spatio-temporal entities. We suggest that ARMS should be used in biological monitoring programs and long-term ecological research and encourage the adoption of our ARMS-MBON protocols.

Rejection of the concept of hemiurid genus Pulmovermis (Digenea: Hemiuridae) and other taxonomic propositions: new morphological and molecular data regarding Lecithochirium cyanovitellosum (Coil and Kuntz, 1960) Urabe and Sokolov, comb. nov. (formerly Pulmovermis cyanovitellosus).

Pulmovermis cyanovitellosus Coil and Kuntz, 1960 is a species of hemiurid trematode that localizes in the lung of sea snakes, an unusual trait for this group of parasites. Recent molecular phylogenetic studies based on 28S rRNA gene sequences have shown that this species is closely related to members of the genus Lecithochirium Lühe, 1901. This finding is unexpected given that Pulmovermis Coil and Kuntz, 1960 and Lecithochirium are currently classified in different subfamilies of Hemiuridae (Pulmoverminae Sandars, 1961 vs. Lecithochiriinae Lühe, 1901). To investigate this discrepancy, we conducted a comparative morphological study on new specimens of P. cyanovitellosus collected from the same locality as those in our previous research. These new specimens were compared with the paragenophores from our earlier study and with existing descriptions in literatures. This study provides the following new morphological data regarding P. cyanovitellosus: body surface covered with minute scales; massive muscular pads present on both sides of body at level of terminal genitalia; small esophageal pouch and Drüsenmagen present; seminal vesicle connecting to pars prostatica via aglandular duct; small, cylindrical sinus-sac present; ejaculatory vesicle absent; Juel's organ present; metraterm separated from uterus by conspicuous sphincter and connecting to distal end of pars prostatica. Additionally, we assessed the phylogenetic relationships of P. cyanovitellosus using the 28S rRNA gene, the internal transcribed spacer 2 (ITS2), and the cytochrome c oxidase subunit I (COI) gene markers. Phylogenetic analyses based on these markers demonstrated that P. cyanovitellosus clustered with species of Lecithochirium. Based on this molecular evidence, we conclude that Pulmovermis is a junior synonym of the genus Lecithochirium, and propose L. cyanovitellosum (Coil and Kuntz, 1960) Urabe and Sokolov comb. nov. Given the morphological similarities between Pulmovermis and Hydrophitrema Sandars, 1960, we also propose synonymizing the latter with Lecithochirium, and L. giganticum (Sandars, 1960) Urabe and Sokolov comb. nov. and L. pingtanense (Wang, 1980) Urabe and Sokolov comb. nov. are proposed. As a result, Pulmoverminae should be considered a junior synonym of Lecithochiriinae Lühe, 1901.

Opoptera Aurivillius, 1882 (Lepidoptera: Nymphalidae, Satyrinae, Brassolini): Taxonomic revision, molecular phylogeny and description of a new species

Opoptera Aurivillius, 1882 (Lepidoptera: Nymphalidae, Brassolini) is a Neotropical genus, with species occurring from Mexico to Argentina. Although previous phylogenetic hypotheses involving Opoptera species have been proposed, an investigation of the species relationships based on molecular data, supported by a systematic review, has not been carried out to date. This study presents a taxonomic revision of Opoptera, which currently comprises eight species and two subspecies, proposing a hypothesis of relationships among the taxa based on DNA, morphology, geographic distribution, and natural history data. To carry out this study, 530 specimens of all species of the genus were examined, including type material of all proposed taxa. For the phylogenic hypothesis, the maximum likelihood method was used, and five molecular markers: one mitochondrial, cytochrome c oxidase subunit I (COI), and four nuclear, elongation factor 1-alpha (EF-1α), ribosomal protein S5 (RpS5), wingless (wg), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Opoptera was recovered as monophyletic and the relationships between species corroborate the previous hypotheses. From the taxonomic review, two synonyms are proposed: Opoptera hilaris colombiana (Rothschild, 1916) syn. nov. and Opoptera hilaris fuscata Stichel, 1908 syn. nov. of Opoptera hilaris Stichel, 1901. A new status is proposed: Opoptera mexicana J. Maza & R. G. Maza, 1989 stat. nov. A new species is described: Opoptera iracildae sp. nov. In order to stabilize the nomenclature of the group, three neotypes are designated for Caligo syme Hübner, [1821], Opsiphanes arsippe Hopffer, 1874, and Opoptera aorsa hilaris Stichel, 1901; and ten lectotypes for Morpho aorsa Godart, [1824], Opoptera aorsa litura Fruhstorfer, 1907, Opoptera arsippe bracteolata Stichel, 1901, Opoptera aorsa fuscata Stichel, 1908, Opsiphanes sulcius Staudinger, 1887, Opsiphanes sulcius var. sodalis Röber, 1906, Papilio alcimedon Dalman, 1823, Morpho acadina Godart, [1824], Opoptera syme fumosa Stichel, 1902, and Opsiphanes staudingeri Godman & Salvin, 1894. This study recognizes ten valid species and eight synonyms.

Three new species of Australian miracine parasitoid wasps collected by regional schools as part of the Insect Investigators citizen science project (Hymenoptera, Braconidae, Miracinae)

Miracinae is a poorly known and rarely collected subfamily of parasitoid wasps belonging to the family Braconidae. Here, three new species are described from material collected by Australian regional schools as part of the Insect Investigators citizen science project and named in collaboration with students: Mirax supremus Slater-Baker, sp. nov., Mirax ceduna Slater-Baker, sp. nov. and Mirax kaatijan Slater-Baker, sp. nov. The barcoding region of the cytochrome c oxidase subunit I (COI) gene was obtained for each new species and analysed alongside all publicly available COI DNA barcodes for Miracinae on the Barcode of Life Database, to produce a molecular framework to guide species delimitation and efficient morphological examination. Full morphological descriptions are provided along with high quality images for each new species. A key to described Australian Miracinae is provided.

Pleistocene Refugia Inferred from Molecular Evidence in a Forest-Dwelling Harvestman (Arachnida, Opiliones, Gonyleptidae) Support a Biogeographic Split in Subtropical Argentina.

This paper addresses the population genetic structure of the forest-dwelling gonyleptid Geraeocormobius sylvarum (Arachnida, Opiliones). Phylogeographic analyses using cytochrome oxidase subunit I (COI) were conducted on 186 individuals from 43 localities in Argentina and Paraguay, arranged into nine operational sectors and defined upon geographic and vegetation features. Given the current environmental uniformity, it was aimed to assess whether molecular fingerprints of G. sylvarum correlate with Pleistocene fragmentation events, inferred through forest contraction/expansion cycles. The network of 87 haplotypes displayed an unstructured pattern; 75 were found in unique localities (54 on single individuals), with most haplotypes restricted to a single operational sector. The calibrated phylogenetic tree revealed significant admixture, with each clade mainly related to one operational sector. Results suggest multiple fragmentation events; most COI diversity arose in the Late Pleistocene/Early Holocene; recent and relict lineages coexist at a few sites. Banks of major rivers (Paraná and Uruguay) in Misiones Province may have served as main refuge areas, with dispersal within each basin being frequent but sporadic across the watershed divide, keeping basins separated for long periods. The split of the Misiones opiliogeographical area into two sectors corresponding to the major basins is proposed.

Life in the dead shell: utilization of dead Pinna shells by the blenny fish Petroscirtes variabilis Cantor, 1849 (Blennidae).

The seagrass ecosystem supports a diverse range of marine life, including fish, crabs, and seahorses. It plays a vital role in providing essential services such as habitat creation, nutrient cycling, and shoreline stabilization. In the present study, we conducted line intercept transects to assess the abundance of Pinna shells in seagrass meadows of the Sangumal regionin the Palk Bay,India. Among the 30 transects surveyed, we observed a total of 33 Pinna shells. Ten shells were found to be empty, while 6 hosted blenny fish. The fish were identified as saber-tooth blenny Petroscirtes variabilis Cantor, 1849, via integrative taxonomy (morphology + cytochrome oxidase subunit I gene phylogeny). The phylogenetic tree based on the maximum likelihood inference method indicated that P. variabilis clustered together with other sequences of P. variabilis available in the NCBI GenBank. We also revealed the importance of empty Pinna shells acting as refugia for blenny fish, which was evident from the presence of eggs attached to the shells.

Molecular evidence of Macracanthorhynchus hirudinaceus (Pallas, 1781) in cockchafers in rural areas of Elazig, Türkiye.

The primary definitive host of the giant acanthocephalan, also known as the giant thorny-headed worm Macracanthorhynchus hirudinaceus (Pallas, 1781), is Sus scrofa. The definitive host ingests the parasite by consuming infected scarabaeoid or hydrophilid beetles. This study aimed to ascertain the presence of M. hirudinaceus in the intermediate hosts through molecular analysis. The cockchafers were collected from Elazig province of Türkiye. A total of 30 pools, comprising 10 pools for each of three districts were obtained from cockchafers collected from 10 areas. The gDNA was isolated and PCR was conducted using specific primers which amplify the mitochondrial cytochrome c oxidase subunit 1 (mt-CO1) gene of M. hirudinaceus. Then, the PCR-positive samples were sequenced, and phylogenetic and haplotype analyses were performed. A total of 300 cockchafer adults and/or larvae were collected for this study from different regions of three districts (Sivrice, Baskil, and Keban) in Elazig province of Türkiye. No PCR band was observed in any of the samples in Sivrice (0%). However, a total of 16 samples (5.3%), 10 from the Baskil (10%) and 6 from Keban (6%), showed a PCR band of 491 bp. All sequences were confirmed as M. hirudinaceus. Two distinct haplotypes were detected at two points. Of the total number of sequences, twelve were found to consist of a single haplotype. One of the two haplotypes was comprised of 10 isolates, while the other included six isolates. This study is one of the limited studies on the molecular identification and haplotyping of M. hirudinaceus in cockchafers.

Diversity in Thrips palmi: exploration of genetic and ecological heterogeneity.

Thrips palmi Karny (Thysanoptera: Thripidae), an impactful pest in Southeast and East Asia, spread to Africa, Oceania, and the Americas in the past decades. Besides being a principal pest of vegetables, legumes, fibre, and ornamental crops, T. palmi serves as the vector for several plant viruses that cause substantial economic losses. The impact of intraspecies heterogeneity in the evolution of pesticide-resistant, host-specific, and geo-ecological strains is known for several thrips species. The present study aims to explore the genetic basis of the intraspecies heterogeneity of T. palmi populations if any. The genetic diversity within T. palmi has been recognized by 35 microsatellite markers, sequencing mitochondrial cytochrome oxidase subunit I (mtCOI), and internal transcribed spacer 2 (ITS2) markers. A total of 93 populations were collected from diverse ecological niches of India. Out of which 20 populations were identified as T. palmi. A total of 286 mtCOI and 66 ITS2 sequences of T. palmi originating from different parts of the world including the sequences generated in this study were considered to ascertain the genetic diversity. All the collected Indian populations are clustered into two distinct ancestries in Structure analysis, with a predominant inclination towards Ancestry II. A high intraspecific diversity of global populations of T. palmi up to 19% based on mtCOI sequences and 15% in ITS2 sequences is indicative of the presence of cryptic species. Phylogenetic analyses and haplotype networking clustered the global population of T. palmi into five lineages. Restricted gene flow was anticipated among the major lineages of T. palmi. The results suggest the existence of reproductively isolated populations due to potential geographical barriers. Excess of rare alleles suggested a recent selective sweep or population expansion after a recent bottleneck that seeks a further evolutionary understanding of T. palmi population globally, and its introduction and ecesis in new areas. The study proposes that T. palmi populations across the globe represent a species complex with broader genetic boundaries and reproductive isolation that suggest further taxonomical appraisal.

First Report of Phytopythium vexans Causing Root Rot on Tulip Poplar in Tennessee and the United States.

Tulip poplar (Liriodendron tulipifera) is a member of the Magnolia family, is a large, fast-growing, long-lived, deciduous tree native to eastern North America. One-year-old tulip poplar seedlings grown under field conditions in a commercial nursery in Warren County, Tennessee, exhibited severe root rot in May 2024. Dark brown to black lesions were observed on the affected roots. Disease severity was 60% of the affected root area, and disease incidence was 45% of 300 plants. Symptomatic root tissues were surface-disinfected with 70% ethanol and washed twice with distilled water. Small sections of root tissue were placed in Petri dishes containing V8-PARPH (V8 juice agar amended with pimaricin, ampicillin, rifampicin, pentachloronitrobenzene, and hymexazol) agar and incubated at 24°C in an 8-hour photoperiod cycle. After 3 days of incubation, whitish cottony mycelia with radiate and chrysanthemum flower-like growth patterns were observed (Supplementary Fig. 1a). The hyphae were coenocytic and 5.5 μm wide. Sporangia were subglobose with papillate, filamentous to globose smooth oogonia (18.35 to 23.86 μm; n = 50), bell-shaped antheridia, and spherical zoospores that are characteristic of Phytopythium vexans (de Cock et al. 2015) (Supplementary Fig. 1b and c). Total DNA was extracted (DNeasy PowerLyzer Microbial Kit) from 7-day-old isolates FBG7781-1 and FBG7781-2 and amplified using the ITS1/ITS4 (White et al. 1990), NL1/NL4 (Baten et al. 2014), Levup/Fm85mod (Robideau et al. 2011) and Cox2F/Cox2RC4 (Hudspeth et al. 2000) primer pairs. Isolates were sequenced using for genetic markers, including the ribosomal internal transcribed spacer (ITS), the 28S large subunit (LSU) of ribosomal RNA, the mitochondrial cytochrome c oxidase subunit I (COXI) and cytochrome c oxidase subunit II (COXII), respectively. The sequences (GenBank accession nos. PQ555199 and PQ555200 for ITS, PQ555205 and PQ555206 for LSU, PQ562067 and PQ562068 for COXI, and PQ562065 and PQ562066 for COXII) were 100% identical to the ITS, LSU, COX1, and COXII genetic markers of P. vexans isolates in GenBank (ITS: PQ050141, LSU: AB468723, COXI: GU133478, COXII: AB468910). Pathogenicity tests were performed on 1-year-old tulip poplar seedlings grown in 1-gal containers (3.8 L) to fulfill Koch's postulates. Tulip poplar seedlings were drenched and inoculated (150 ml/plant) with a pathogen slurry (two plates of 7-day-old culture/liter) as described in Panth et al. 2021, using isolates FBG7781-1 and FBG7781-2 (five plants per isolate). Five plants were drenched with V8-PARPH agar slurry without the pathogen and served as controls. The study was conducted in a greenhouse maintained at 21 to 23°C and 70% relative humidity and watered twice daily for 2 min using an overhead irrigation system. Fifteen days after inoculation, dark brown lesions developed in the roots of all inoculated plants (Supplementary Fig. 2a). No symptoms were observed in the control plants (Supplementary Fig. 2b). Isolates resembling the morphological characteristics of P. vexans were recovered from inoculated plants using the method described above, and their identity as P. vexans was confirmed by DNA sequencing with the same four primer pairs previously described. Phytopythium vexans has been reported to cause root rot in flowering cherry, ginkgo, red maple, and redbud in Tennessee (Baysal-Gurel et al. 2021, Liyanapathiranage et al. 2023, Panth et al. 2021). To our knowledge, this is the first report of P. vexans causing root rot of tulip poplar in Tennessee, the United States and worldwide. This finding is significantly important for the development of a successful disease management strategy for P. vexans in tulip poplar.

The green lacewing genus Kuwayamachrysa Tsukaguchi & Tago, 2018 from China, with description of two new species based on morphological and molecular evidence

Previously, Kuwayamachrysa Tsukaguchi & Tago, 2018 (Neuroptera: Chrysopidae: Chrysopinae) was known as a monotypic green lacewing genus from the Palearctic region. Here we described two new species of Kuwayamachrysa from China: Kuwayamachrysa wujiaoensis sp. nov. and Kuwayamachrysa neptuna sp. nov. Also, Mallada qinlingensis Yang, 1989, currently named as Apertochrysa qinlingensis (Yang, 1989), is recognized as a junior synonym of Kuwayamachrysa kichijoi (Kuwayama, 1936). A key to the Kuwayamachrysa species is provided. The standard DNA barcoding region of cytochrome c oxidase subunit I (COI) of these species was sequenced for the verification of the new species.

Genetic variations suggests that Takifugu rubripes, T. chinensis, and T. pseudommus are the same species with a shared gene pool

IntroductionPufferfish are valuable ingredients and important fishery resources in Asia. Among them, approximately 25 Takifugu species have undergone explosive speciation in the marine environments of East Asia. Specifically, T. rubripes, T. pseudommus, and T. chinensis are remarkably similar in appearance and genetics, and their classification remains controversial. Here, we provide comprehensive genetic and genomic evidence regarding their speciation and classification.MethodsWhole genome sequencing was performed on T. pseudommus, leading to the identification and development of 15 novel tandem simple sequence repeats (SSRs) from its genomic data. Genetic diversity, differentiation, and population structure among T. rubripes, T. pseudommus, T. chinensis, and T. xanthopterus were subsequently analyzed using a combination of these 15 SSR loci and mitochondrial cytochrome oxidase subunit I (CoI) and cytochrome b (CytB) gene sequences. Furthermore, genomic variations, including single nucleotide polymorphisms, insertions, and deletions, were identified by comparing the genome of T. pseudommus with the reference T. rubripes genome retrieved from the National Center for Biotechnology Information. These variations were screened using ENSEMBL annotation and gene ontology analysis, and their potential association with morphological differences, such as dorsal spot patterns, was evaluated using reference Takifugu specimens.Results and discussionPopulation genetic analysis of T. rubripes, T. pseudommus, and T. chinensis using the CoI and CytB genes, combined with the 15 SSR loci, revealed clustering into a single genetic group characterized by remarkably low genetic diversity (four haplotypes, with diversity values ranging from 0.0000 to 0.00065) and minimal pairwise differentiation (microsatellite-based FST values ranging from -0.0021 to 0.0075). Additionally, comparative genomic analysis between T. pseudommus and the reference T. rubripes genome did not identify genetic variations that could directly explain the observed morphological differences among the species. These findings strongly suggest that T. rubripes, T. pseudommus, and T. chinensis represent a single species sharing a common genetic pool.

DNA barcoding of invasive Gambusia holbrooki Girard, 1859 and Atherina boyeri Risso, 1810 inhabiting Upper Euphrates River Basin, Türkiye

The main contributor to Türkiye’s abundant freshwater fish biodiversity is its geographic location. This fauna consists of endemic, native, and non-native fish species. The introduction of Gambusia holbrooki Girard, 1859 to Lake Amik in the 1920s for the biological control of malaria was the first introduction of nonnative species to Türkiye. Atherina boyeri Risso, 1810 and other nonnative fish species have recently been introduced to Türkiye’s freshwaters. In this research, the first records of invasive Gambusia holbrooki (Keban Dam Lake, in Elazığ Province) and Atherina boyeri (Karakaya Dam Lake, in Elazığ Province) are cited from the Upper Euphrates River Basin in the Eastern Anatolia Region of Türkiye. In situ electrofishing equipment was used to gather the specimens. Fish muscle samples were used to extract genomic DNA, which was then used to barcode the mitochondrial cytochrome c oxidase subunit I (COI) gene to identify different species of fish. The identification of invasive fish species using DNA barcoding is an effective technique, as evidenced by the comparison of amplified COI sequences to the BLAST database.

Development of a qPCR assay for early detection and quantification of Phytopythium vexans in kiwifruit plant and soil affected by Vine Decline Syndrome.

Kiwifruit Vine Decline Syndrome (KVDS) is a soilborne disease affecting Actinidia fruit trees in perennial cropping systems. Since its emergence in 2012, studies have increasingly identified the oomycete Phytopythium vexans as a major causative agent of the disease. P. vexans is also implicated in complex soilborne disease systems of woody perennial crops, including replant disease in apple and pear. To date, most molecular assays for the detection of P. vexans target the nuclear ribosomal internal transcribed spacer (ITS), a region which is ill-suited for distinguishing between closely related oomycete species. The cytochrome oxidase subunit I (COI) mitochondrial gene was targeted for the design of new primers because it was previously identified as a better marker for differentiating oomycete species. The FOR2/REV4RCA primer pair gave the best results regarding PCR specificity and was selected for use in a SYBR Green-based qPCR assay. The specificity of the qPCR assay was evaluated using 29 P. vexans strains (including different phylogenetic groups) as well as a wide variety of closely related off-target species associated with pathogenic soil communities of fruit trees. P. vexans strains were successfully quantified down to 20 fg in water and in DNA extracted from kiwifruit roots. P. vexans was also detected in artificially inoculated Actinidia plant roots as well as in a variety of naturally infected field samples of both kiwi and apple trees. These results suggest that the qPCR assay developed in this study is highly sensitive and specific to target pathogen, regardless of sample matrix.

Morphological and Molecular Identification of Obligatory Myiasis-Causing Species in Wild Cervids in Croatia.

The infestation of ruminants by fly larvae of the Oestridae family can lead to nasopharyngeal or subcutaneous myiasis, which can harm animal welfare and productivity on sheep and cattle farms. The infestation of wild ruminants allows them to serve as reservoirs for subsequent transfer to domestic animals. The fly species most commonly responsible for myiasis have been well studied in many parts of the world, but less so in southeastern Europe. Here, we identified three botfly species among 197 subcutaneous larvae and 138 nasopharyngeal larvae from 36 wild roe deer (Capreolus capreolus) and 9 wild red deer (Cervus elaphus) at 30 locations across Croatia. Instars were stored in 75% ethanol and their species and developmental stage were identified based on morphological and morphometric keys. Subcutaneous larvae in both cervid species were identified as Hypoderma diana, while nasopharyngeal larvae were identified as Cephenemyia simulator in roe deer and Pharyngomyia picta in red deer. Partial sequencing of the gene-encoding mitochondrial cytochrome oxidase subunit I confirmed the taxonomic classification of C. stimulator and P. picta but was unable to confirm that of H. diana for lack of previously published sequences. This study appears to be the first to combine the morphological and molecular characterization of myiasis-causing botflies in wild cervids in southeastern Europe.

Target site mutations and metabolic detoxification of insecticides in continental populations of Cimex lectularius and Cimex hemipterus (Hemiptera: Cimicidae).

In recent decades, the common and the tropical bed bugs have experienced a resurgence in many parts of the world. The evolution of insecticide resistance in bed bug populations is considered a significant factor contributing to this resurgence. We analyzed samples of Cimex lectularius L. and Cimex hemipterus (F.) from Europe (Spain 41, Switzerland 2, the Czech Republic 1), Asia (Hong Kong 34), North America (USA 14, Mexico 3), and South America (Colombia 3) to assess the prevalence and mechanisms of insecticide resistance. We identified specimens morphologically and barcoded them by sequencing the mitochondrial Cytochrome c oxidase subunit I (COI) and the 16S ribosomal RNA (16S rRNA) genes. Additionally, we screened segments of the voltage-gated sodium channel (VGSC) and the nicotinic acetylcholine receptor (nAChR) genes for point mutations associated with insecticide resistance and measured the activity of detoxifying enzymes. All samples from North America and Europe were identified as C. lectularius, whereas specimens from Hong Kong were C. hemipterus. Out of 64 C. lectularius samples tested for knockdown resistance (kdr) mutations, 90.6% contained at least 1 known mutation. All 35 C. hemipterus samples exhibited kdr mutations. A new mutation was identified in the pyrethroid target site in both common (F1524C) and tropical (F1450C) bed bugs. No resistance-associated mutations in the nAChR gene were found. Several populations that exhibited kdr mutations also showed elevated activity of detoxifying enzymes. The high frequency of kdr-associated mutations in bed bug populations from Spain and Hong Kong limits the efficacy of pyrethroids for their control.