Mitochondrial Gene Regions Research Articles

Revealing the Genetic Differentiation of Rattus norvegicus (Berkenhout 1769) Populations by Analyzing Two Mitochondrial Markers

Rattus norvegicus (Brown rat) has a great importance for public health and economy because it lives in close association with human populations. However, molecular systematic studies on global populations of R. norvegicus are very few. In this study, sequences obtained from Europe, Asia, Africa, and America regions were analyzed using mitochondrial Cytochrome-b and Cytochrome oxidase-I gene regions and genetic differentiation levels between these populations were revealed. Accordingly, samples belonging to the studied populations did not split in Bayesian Inference trees and Median-joining networks; these samples also formed common haplotypes, and mean genetic distance and fixation index values were generally low. The results of the study showed that gene flow between these populations may be continuing due to the transportation activity by humans.

Expanded distribution and a new genus for rock-inhabiting sea pens (Cnidaria, Anthozoa, Octocorallia, Pennatuloidea)

Sea pens (Superfamily Pennatuloidea) are a specialized group of octocorals that evolved to live embedded in a soft-sedimented seafloor using their peduncles as anchors. Rock-inhabiting sea pens (“rock pens”) were first described in 2011; their peduncle is modified into a suction cup-like structure that allows them to attach onto the surface of hard substrates, an adaptation previously unknown in sea pens. There are currently four species that have been identified as rock pens based on their peduncular morphology: three of these are in the genus Anthoptilum (Anthoptilidae), and one in the genus Calibelemnon (Scleroptilidae). Herein, we explore the geographic distribution and depth ranges of rock pens using observations from remotely operated vehicles and investigate the evolutionary origins of the rock pens. We present a phylogenomic study of sea pens, based on DNA sequences from hundreds of ultraconserved elements (UCE) and compare these trees to those constructed using mitochondrial gene regions. Our results show that the ancestral sea pen had a typical elongated peduncle. The adaptation to attach onto hard, rocky substrates using the special suction cup-like peduncle evolved along a single derived lineage that is sister to a lineage comprising the genus Anthoptilum. We propose all known rock pen species be grouped into a single new genus, described herein, in the family Anthoptilidae, to reflect the phylogenetic history.

Genetic Diversity of Bluefish Population in Türkiye Coastal

Bluefish (Pomatomus saltatrix) is a commercially important, highly migratory predatory fish species distributed along the coasts of Türkiye. Conservation and sustainability of bluefish populations requires knowledge of the genetic structure of the population, but There are no genetic studies on bluefish in Türkiye. In this study, the population structure of Bluefish in the coastal regions of Türkiye was investigated by sequence analysis of mitochondrial DNA gene regions (Cytochrome b and Displacement-loop) and nuclear gene region (Rhodopsin). We tried to identify bluefish samples collected from 14 regions in the coastal region of Türkiye by analyzing two mtDNA gene sequences (Cyt b and D-loop) and nuclear gene region (Rhodopsin). While a total of 35 haplotypes were found in the Cytb gene region, a total of 132 haplotypes were found in the D-loop (control region) gene region. A single haplotype was detected in all stations for Rhodopsin. Also, the maximum likelihood trees based on Cyt b and D-loop sequences suggested relationship of Bluefish populations from Türkiye and the world. Results showed a low level of genetic differentiation between Bluefish populations in the study area, but Aegean and Mediterranean populations are more diverse than other populations due to migratory trait of this species.

Identification and Molecular Characterization of Clogmia (Telmatoscopus) albipunctatus (Drain Fly) Larva in a Rare Case of Urinary Myiasis in a Domestic Dog in India.

The study aimed to identify Clogmia (Telmatoscopus) albipunctata larva recovered from the urinary tract of a four-year-old female Siberian Husky dog in India using morphological assessment and molecular techniques. Larval specimen obtained from the dog urine underwent preliminary morphological assessments followed by polymerase chain reaction (PCR) to selectively amplify segments of the Cytochrome b (Cytb), NADH1 genes, and the tRNA-Ser gene within mitochondrial DNA. Species identification was achieved through sequence analysis of the amplified product and subsequent phylogenetic analysis. Larva recovered from dog urine underwent morphological examination, revealing notable features characteristic of Clogmia genus. The primers targeting the mitochondrial DNA gene region of fly larva, amplified a distinct product of approximately 788bp. Integration of morphological and molecular biology techniques confirmed the identification of the C. albipunctata larva in the dog urinary tract. This study elucidates the initial molecular evidence of C. albipunctata presence in a four-year-old female Siberian Husky dog exhibiting symptoms of haematuria and frequent micturition in India. It reveals an instance of a domestic dog in India contracting a C. albipunctata larva infection in its urinary tract, emphasizing the importance of clinical vigilance towards this infection.

Diversity, Distribution and DNA Barcoding Tribe of Anomalini beetles (Coleoptera: Scarabaeidae: Rutelinae) in Wheat Fields of Van, Türkiye

Anomalini beetles (Coleoptera: Scarabaeidae: Rutelinae) constitute a significant pest group responsible for substantial crop losses in wheat cultivation areas worldwide, including Turkey. This study aimed to comprehensively evaluate the phylogeny, diversity, abundance, and distribution of Anomalini beetles in wheat fields of Van province, Turkey. Surveys were conducted between April and August 2021, involving monthly sample collection at predetermined locations within six districts: Başkale, Çaldıran, Erciş, Gevaş, İpekyolu, and Tuşba. Standard traps and hand collection methods were employed to gather samples. In molecular studies, the mitochondrial COI gene region has been amplified and sequenced using universal primers. Anomalini beetles were detected in all sampling areas except Çatak district. Seven species were identified: Anisoplia austriaca, A. signata, A. lata, Brancoplia leucaspis, Blitopertha nigripennis, Chaetopteroplia segetum, and an unidentified Anisoplia sp. Chaetopteroplia segetum emerged as the most prevalent and abundant species across all districts. Notably, all identified Anomalini species represent the first records for Van province and its environs. While Anomalini beetles were present in the region, their population densities were not considered high enough to cause economic damage.

Investigation of Mitochondrial Cytb Gene Region of Both Echinococcus granulosus Eggs from Dogs and Cystic Echinococcosis Isolates Obtained from Sheep and Cattle by Molecular Methods.

We aimed to determine the common Echinococcus granulosus genotypes in Ağrı, Türkiye and to obtain information on the transmission of this parasite. Cystic echinococcosis samples from 100 slaughtered cattle and 100 slaughtered sheep and faecal samples from 200 stray dogs were included in 2021. Collected cyst fluid samples and faces were examined microscopically. DNA was isolated from the germinal membrane of the cysts and from the parasite eggs in the stool samples. The mitochondrial cytb gene region of the parasite was amplified by PCR. Genotypes were determined using the Basic Local Alignment Search Tool (BLAST) after sequence analysis of PCR amplicons. The highest percentage of cysts was found in the lungs of sheep and the liver of cattle. In addition, 75% of sheep cysts and 25.6% of cattle cysts were fertile. Taenia spp./Echinococcus spp. eggs were found in 6% of the faeces of 200 dogs examined microscopically. E. granulosus eggs were detected in 4 out of 50 stool samples analysed by PCR. All samples analysed by sequence analysis were identified as E. granulosus s.s. G1 genotype. Sequence comparison revealed revealed one or more-point mutations in different regions of the five samples. E. granulosus s.s. G1 genotype, known as sheep strain, is common in the Ağrı, Türkiye. The controlled slaughter of livestock, especially sheep, and the avoidance of feeding hydatid cyst organs to dogs, together with public education, were necessary to prevent the spread of the disease.

Assessing eDNA capture method from aquatic environment to optimise recovery of human mt-eDNA

Previous studies have shown that environmental DNA (eDNA) from human sources can be recovered from natural bodies of water, and the generation of DNA profiles from such environmental samples may assist in forensic investigations. However, fundamental knowledge gaps exist around the factors influencing the probability of detecting human eDNA and the design of optimal sampling protocols. One of these is understanding the particle sizes eDNA signals are most strongly associated with and the most appropriate filter size needed for efficiently capturing eDNA particles. This study assessed the amount of mitochondrial eDNA associated with different particle sizes from human blood and skin cells recovered from freshwater samples. Samples (300 mL) were taken from experimental 10 L tanks of freshwater spiked with 50 µL of human blood or skin cells deposited by vigorously rubbing hands together for two minutes in freshwater. Subsamples were collected by passing 250 mL of experimental water sample through six different filter pore sizes (from 0.1 to 8 µm). This process was repeated at four time intervals after spiking over 72 hours to assess if the particle size of the amount of eDNA recovered changes as the eDNA degrades. Using a human-specific quantitative polymerase chain reaction (qPCR) assay targeting the HV1 mitochondrial gene region, the total amount of mitochondrial eDNA associated with different particle size fractions was determined. In the case of human blood, at 0 h, the 0.45 µm filter pore size captured the greatest amount of mitochondrial eDNA, capturing 42 % of the eDNA detected. The pattern then changed after 48 h, with the 5 µm filter pore size capturing the greatest amount of eDNA (67 %), and 81 % of eDNA at 72 h. Notably, a ten-fold dilution proved to be a valuable strategy for enhancing eDNA recovery from the 8 µm filter at all time points, primarily due to the PCR inhibition observed in hemoglobin. For human skin cells, the greatest amounts of eDNA were recovered from the 8 µm filter pore size and were consistent through time (capturing 37 %, 56 %, and 88 % of eDNA at 0 hours, 48 hours, and 72 hours respectively). There is a clear variation in the amount of eDNA recovered between different cell types, and in some forensic scenarios, there is likely to be a mix of cell types present. These results suggest it would be best to use a 5 µm filter pore size to capture human blood and an 8 µm filter pore size to capture human skin cells to maximize DNA recovery from freshwater samples. Depending on the cell type contributing to the eDNA, a combination of different filter pore sizes may be employed to optimize the recovery of human DNA from water samples. This study provides the groundwork for optimizing a strategy for the efficient recovery of human eDNA from aquatic environments, paving the way for its broader application in forensic and environmental sciences.

DETERMINATION OF ENTOMOPATHOGENIC NEMATODE FAUNA IN ŞIRNAK, TURKIYE

This study focuses on the isolation, identification, and characterization of entomopathogenic nematodes (EPNs) in Şırnak Province, Turkey. Between July and September 2023, we collected 256 soil samples, from which 11 EPN isolates were successfully recovered,representing a 4.3% of the samples. These isolates were identified using morphological examinations and molecular diagnostics, including mitochondrial COI and 18S rDNA gene regions. Our findings revealed a diverse population of EPNs, predominantly Heterorhabditis bacteriophora with five isolates, alongside four Steinernema species (one Steinernema carpocapsae, one Steinernema affine, and two Steinernema sp.). Phylogenetic analysis, employing the mitochondrial cytochrome oxidase subunit I (COXI) and the 18S rDNA gene regions, indicated low genetic variability within H. bacteriophora and greater diversity among Steinernema species. The study contributes significant insights into the EPN biodiversity in Şırnak Province and underscores the potential of these nematodes in biological pest control, offering a sustainable alternative to chemical pesticides. The results highlight the importance of local EPN isolates in the development of effective biological control strategies and the conservation of agricultural biodiversity.

Population genetic structure of bottlenose and whitespotted wedgefishes from the Southwest Indian Ocean using a dual marker approach

Wedgefishes (Rhinidae) are threatened by unsustainable fishing globally, and especially in the Southwest Indian Ocean (SWIO), due to their high-value fins in the shark trade. The whitespotted wedgefish Rhynchobatus djiddensis and the bottlenose wedgefish R. australiae are both classified as Critically Endangered on the IUCN Red List of Threatened Species, yet a lack of species-specific knowledge and taxonomic uncertainty still exists within this genus. Genetic approaches aid in taxonomic classification and identifying distinct populations for targeted conservation. Morphological specimen identification of samples (n = 189) collected across the SWIO was confirmed based on the cytochrome oxidase c subunit I (COI) and/or nicotinamide adenine dehydrogenase subunit 2 (ND2) gene regions. The genetic diversity and population structure within and between species and sampling locations were investigated using a dual marker approach: (1) 2 concatenated mitochondrial gene regions, namely COI and the control region (n = 117), and (2) 9 nuclear microsatellite markers (n = 146). The overall genetic diversity was moderate, with an indication that different evolutionary forces are at play on a mitochondrial versus nuclear level. The 2 species were delineated based on both marker types, and for R. djiddensis, the sampling locations of South Africa and Mozambique were genetically homogeneous. For R. australiae, significant differentiation was found between sampling locations, with Madagascar and Tanzania being genetically the most similar. This information provides critical insights into the distribution range and population structure of the whitespotted wedgefish species complex that can support the sustainable management of wedgefishes.

Exploring the Potential in Combining the Two Molecular Approaches, COI Barcoding and PCR-RFLP (COIBar-RFLP) in Identification of Selected Species of the Family Carangidae

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

Population structure, dispersion patterns and genetic diversity of two major invasive and commensal zoonotic disease hosts (Rattus norvegicus and Rattus tanezumi) from the southeastern coast of China.

Background: The invasive brownrat (Rattus norvegicus) and the Oriental rats (Rattus tanezumi) are common commensal murid that are important hosts for rodent-borne diseases in southeast Asia. Understanding their population structure and genetic diversity is essential to uncover their invasion biology and distribution dynamics that are essential for controlling rodent-borne diseases. Methods: TA total of 103 R. norvegicus and 85 R. tanezumi were collected from 13 to 9 coastal areas of six provincial monitoring sentinel sites, respectivelyto assess patterns in their microsatellite loci and their mitochondrial coxl gene region. Results: Eleven sampled populations of R. norvegicus were divided into two major clusters by region. The observed heterozygosity values of all regional populations were smaller than expected genetic diversity heterozygosity values and deviated from Hardy-Weinberg equilibrium Nine sample populations of R. tanezumi were divided into three clusters; two that included sample from Hainan and Fujian provinces, and one that included samples from the other provinces and cities. The genetic diversity of R. tanezumi was highest in samples from Jiangsu and Guangdong provinces. Conclusion: The data in this paper confirm the two invasive rodent species from the southeastern coastal region of China may have relied on maritime transport to spread from the southern region of China to the Yangtze River basin. R. tanezumi may then hanve migrated unidirectionally, along the southeastern provinces of China towards the north, while R. norvegicus spread in a complex and multidirectional manner in Hainan, Fujian, Zhejiang and Jiangsu Provinces of the country.

Genetic Diversity of Kırklareli Honey Bee (Apis mellifera L.) Populations in Thrace Region of Turkey: Identification of Mitochondrial COI and ND5 Gene Regions

The main goal of genetic resource conservation is to keep as much genetic diversity as possible within each species. In this respect, some difficulties in the protection of honey bee gene resources make it necessary to reveal the genetic structures of the subspecies and the genetic relationships between the subspecies. In this study, Kırklareli honey bee populations which were officially registered as an ecotype of Turkey’s honey bee (Apis mellifera L.) gene resources by the Republic of Turkey, Ministry of Agriculture and Forestry, were examined in the COI and ND5 genes of mitochondrial DNA. The restriction fragment length polymorphism (RFLP) together with the polymerase chain reaction (PCR) was used to define Apis mellifera populations. A total of 117 worker bee samples were used which were collected from mostly the Kırklareli province. A newly found single nucleotide polymorphism (SNP), G→A transition in the COI gene region formed a novel NcoI restriction site resulting in a new haplotype. This new haplotype has been abbreviated as haplotype C. As a result of the COI/SspI digestion, the previously reported C haplotype was determined. No restriction was found with the treatment of COI/StyI enzyme. On the other hand, as a result of ND5/AluI restriction, 2 restriction site and previously reported haplotype C was obtained in all of the studied samples. No restriction was screened with ND5/FokI and ND5/HincII enzymes in the whole samples, only a reported uncut B haplotype was observed. Within this study, novel genetic information has been revealed for the Kırklareli honey bee ecotype registered as the Thrace honey bee of Turkey’s honey bee gene resources. Moreover, detailed studies with larger sample sizes should be conducted to characterize the origin and the subspecies of Kırklareli honey bees in detail. It is thought that this study will be useful in the identification and registration of the Kırklareli honey bees to be carried out in the future, and also in the creation of a database.

Facilitating taxonomy and phylogenetics: An informative and cost-effective protocol integrating long amplicon PCRs and third-generation sequencing

Phylogenetic inference has become a standard technique in integrative taxonomy and systematics, as well as in biogeography and ecology. DNA barcodes are often used for phylogenetic inference, despite being strongly limited due to their low number of informative sites. Also, because current DNA barcodes are based on a fraction of a single, fast-evolving gene, they are highly unsuitable for resolving deeper phylogenetic relationships due to saturation. In recent years, methods that analyse hundreds and thousands of loci at once have improved the resolution of the Tree of Life, but these methods require resources, experience and molecular laboratories that most taxonomists do not have. This paper introduces a PCR-based protocol that produces long amplicons of both slow- and fast-evolving unlinked mitochondrial and nuclear gene regions, which can be sequenced by the affordable and portable ONT MinION platform with low infrastructure or funding requirements. As a proof of concept, we inferred a phylogeny of a sample of 63 spider species from 20 families using our proposed protocol. The results were overall consistent with the results from approaches based on hundreds and thousands of loci, while requiring just a fraction of the cost and labour of such approaches, making our protocol accessible to taxonomists worldwide.

An assessment of South African small mammal barcode sequence libraries: Implications for future carnivore diet analyses by DNA

AbstractDNA metabarcoding requires reference libraries that link DNA sequences to species. Mitochondrial gene regions cytochrome c oxidase I (COI), 12S ribosomal RNA (12S rRNA), 16S ribosomal RNA (16S rRNA), cytochrome b (cyt b) and the hypervariable control region (D‐loop) are routinely used in studies that measure genetic diversity in animal species. This study aimed to review the state of DNA reference libraries for small South African mammals as small mammals constitute a large portion of small and medium carnivore diet. Analyses of DNA records revealed that of 193 small mammal species in South Africa, only 141 have DNA sequences available for one or more of the mitochondrial genes examined. Cyt b had the highest species coverage, with 59.1% of South African species represented in the reference libraries. COI only has 33.7%, 12S rRNA has 23.8%, D‐loop has 17.6%, and 16S rRNA has the lowest species coverage of 15%. This study supports the use of multiple gene regions when performing scat metabarcoding, particularly when wanting to determine the small mammal component of the diet. Additionally, it emphasises the need to build comprehensive DNA reference libraries linking sequences to taxonomically identified species.

Choice of primer pairs and PCR polymerase affect the detection of fish eDNA

Efficient biomonitoring is essential for fish protection and management. Environmental DNA (eDNA) has become a promising tool for fish surveys, and its accuracy and robustness are closely related to the primer pairs and DNA polymerases, especially for different environmental samples. However, there is still a lack of sufficient efforts to assess the effects of both two factors on fish biomonitoring. Here, we selected ten primer pairs in the mitochondrial 12S rRNA gene region and three commercial DNA polymerases and analyzed their effects on fish eDNA monitoring in surface water and sediment samples of Dianchi Lake. We found that primer pairs and DNA polymerases significantly affected fish biomonitoring in surface water and sediments of Dianchi Lake. First, there were significant variations in annotated fish eDNA sequences in different groups of primer pairs and DNA polymerases, the percentage of fish sequences amplified by the groups related to primers Riaz-12S and 12S-V5 was more than 90% of the total sequences. Second, the composition of different classification levels of fish taxa varied considerably across groups of primer pairs and DNA polymerases, and the groups related to primers Riaz-12S (i.e., Taq Master‒Riaz-12S, Rapid Taq‒Riaz-12S) and 12S-V5 (i.e., Taq Master‒12S-V5, Rapid Taq‒12S-V5) identified more taxa than other groups. Third, primer pairs had greater impacts on the structure of fish communities than DNA polymerases, and the interactions between two factors had more significant effects than any single one. This study highlights that primer pairs and DNA polymerases play critical roles in fish biomonitoring, and this work aimed to provide methodological guidance for assisting the design of the fish eDNA survey scheme.

New species of the genus Trichosetodes Ulmer, 1915 (Trichoptera, Leptoceridae) from Ratanakiri province, Cambodia, based on morphological and molecular data.

Three new species of Trichosetodes, namely T.carmelaesp. nov., T.katiengensissp. nov. and T.ratanakiriensissp. nov. are described and illustrated by male specimens. The male genitalia of T.carmelaesp. nov. can be distinguished from the other 16 species of the genus found in Southeast Asia by the shape of the phallicata. The phallicata of T.carmelaesp. nov. bears a tuft of long hairs in the middle of the dorsal edge. Trichosetodeskatiengensissp. nov. can be distinguished from the other species in Southeast Asia by the shape of the phallicata which is divided into dorsal and ventral branches in lateral view, and T.ratanakiriensissp. nov. by the characters of the left inferior appendage and the shape of segment IX. The posterior end of the left inferior appendage of T.ratanakiriensissp. nov. is not forked and the ventral and lateral views of the posteroventral lobes of segment IX are rounded. Illustrations of male genitalia of Trichosetodeskampongspeuensis Malicky & Kong, 2020 are provided for comparison. The molecular diversity of new Trichosetodes species was analyzed using the mitochondrial large subunit ribosomal rRNA gene region (16S rRNA). In terms of their genetic divergence, T.ratanakiriensissp. nov. and T.kampongspeuensis exhibited remarkable proximity, with only a 1.4% distance. On the contrary, T.carmelaesp. nov. displayed genetic disparity exceeding 6.3% when compared to both T.ratanakiriensissp. nov. and T.kampongspeuensis.

Wing pattern diversity in Eunica butterflies (Nymphalidae: Biblidinae): phylogenetic analysis implies decoupled adaptive trends in dorsal sexual dimorphism and ventral eyespot evolution.

Butterfly eyespots are wing patterns reminiscent of vertebrate eyes, formed by concentric rings of contrastingly coloured scales. Eyespots are usually located close to the wing margin and often regarded as the single most conspicuous pattern element of butterfly wing colour displays. Recent efforts to understand the processes involved in the formation of eyespots have been driven mainly by evo-devo approaches focused on model species. However, patterns of change implied by phylogenetic relationships can also inform hypotheses about the underlying developmental mechanisms associated with the formation or disappearance of eyespots, and the limits of phenotypic diversity occurring in nature. Here we present a combined evidence phylogenetic hypothesis for the genus Eunica, a prominent member of diverse Neotropical butterfly communities, that features notable variation among species in eyespot patterns on the ventral hind wing surface. The data matrix consists of one mitochondrial gene region (COI), four nuclear gene regions (GAPDH, RPS5, EF1a and Wingless) and 68 morphological characters. A combined cladistic analysis with all the characters concatenated produced a single most parsimonious tree that, although fully resolved, includes many nodes with modest branch support. The phylogenetic hypothesis presented corroborates a previously proposed morphological trend leading to the loss of eyespots, together with an increase in the size of the conserved eyespots, relative to outgroup taxa. Furthermore, wing colour pattern dimorphism and the presence of androconia suggest that the most remarkable instances of sexual dimorphism are present in the species of Eunica with the most derived eyespot patterns, and are in most cases accompanied by autapomorphic combinations of scent scales and "hair pencils". We discuss natural and sexual selection as potential adaptive explanations for dorsal and ventral wing patterns.

First detection of the potato cyst nematode, Globodera rostochiensis, infecting potato in the central region of Colombia.

Potato cyst nematodes (PCNs; Globodera spp.) cause significant losses in worldwide cultivated potato (Solanum tuberosum) crops. In Colombia, PCN was first reported in 1970 (Baeza 1972), although this report lacked a comprehensive species description and diagnosis. After that, G. pallida has been the only PCN species reported affecting potatoes in the main producing regions of Colombia (Evans et al. 1975; Nieto et al. 1983; Vallejo et al. 2021). However, in the survey conducted by Vallejo et al. (2021), a single sample from Chocontá, Cundinamarca in the central region of the country (N 5,22396046668291, W -73,6571338400244) showed molecular characters similar to G. rostochiensis. As correct identification is essential for effective pest management, the location was re-sampled in September 2022. From the soil samples collected, PCN cysts and second-stage juveniles (J2s) were retrieved from soil using Fenwick and centrifugation methods, respectively. Morphometric characters of cysts (n = 53) were consistent with G. rostochiensis, with a length without neck (L) ranging from 451 to 614 μm (X̅ = 546.9 ± 20.3 μm), width (W) from 424 to 658 μm (X̅ = 546.9 ± 25.5 μm) and L/W ratio was 1.00 ± 0.02. Distance from anus to vulva varied from 41 to 109 μm (X̅ =75.67 ± 13.8 μm), Granek's ratio from 2.3 to 5.5 μm (X̅ = 3.89 ± 0.7 μm), and the number of cuticular ridges between the vulva and the anus were 14 to 20 (X̅ = 16.19 ± 1.7). The second-stage juvenile (n = 90) length ranged from 394 to 547 μm (X̅ = 495.62 ± 31.0 μm), the stylet length varied from 18 to 24 μm (X̅ = 21.21 ± 0.9 μm) with rounded knobs. The length of the hyaline tail ranged from 20 - 31 μm (X̅ = 24.09 ± 1.92) and the true tail from 31- 56 μm (X̅ = 48.30 ± 5.71 μm). Molecular analyses confirmed morphological identification. DNA was extracted from cysts and J2s. PCR was performed for the 28S rDNA D2-D3 segment using primers D2A and D3B (Subbotin et al. 2006), and for the mitochondrial COI gene region using primers JB3 and JB5 (Derycke et al. 2005). BLAST analyses of target 28S rDNA D2-D3 sequences (OP293373-OP293380) showed 100% identity of the 658 bp to other sequences on Genbank, including isolates from Turkey, United Kingdom, and Iran (MK311329.1, MG994942.1, KU297659.1, and KU297658.1). Similarly, the target COI region sequences (OP297993-OP298001) were 100% identical to the 407 bp of G. rostochiensis POT01 isolate from Germany, and 99.75% identical to voucher NRM67 from Indonesia, and isolate CD2200 from USA (MF773722.1, MT240262.1, and MN095979.1). Phylogenetic analysis of both gene regions strongly supported G. rostochiensis, with the Colombian sequences clustering with MH399815.1, and KU297654.1 isolates for the COI and 28S regions, respectively (Fig. 1S). In addition, a pathogenicity test was conducted in the greenhouse. For this, ten cysts were inoculated to potato plants of Criolla variety grown in 5 pots of 15 cm diameter with sterile soil and sand (1:1). Noninoculated plants served as controls (three replicates each). After three months, 54 ± 23 cysts/100 g of soil were isolated from inoculated plants (Fig. 2S), resulting in a reproduction factor (R=Pf/Pi) of 4.54 ± 0.86, while no yellow females or cysts were observed on the control plants. To our knowledge, this is the first report of G. rostochiensis in Colombia. This is an important pest that causes serious yield losses of potatoes and is a quarantine nematode in many countries (EPPO 2017). Further studies are necessary to prevent the spread of this PCN species in the main producing potato regions of Colombia.

Integrated morphological and molecular characterization of the fish parasitic nematode Rhabdochona (Rhabdochona) gendrei Campana-Rouget, 1961 infecting Labeobarbus altianalis (Boulenger, 1900) in Kenya.

During a parasitological survey carried out between May and August 2022 in the River Nyando, Lake Victoria Basin, a single species of Rhabdochona Railliet, 1916 (Nematoda: Rhabdochonidae) was recorded from the intestine of the Rippon barbel, Labeobarbus altianalis (Boulenger, 1900) (Cyprinidae). Based on light microscopy (LM), scanning electron microscopy (SEM) and DNA analyses the parasite was identified as Rhabdochona (Rhabdochona) gendrei Campana-Rouget, 1961. Light microscopy, SEM and DNA studies on this rhabdochonid resulted in a detailed redescription of the adult male and female. The following additional taxonomic features are described in the male: 14 anterior prostomal teeth; 12 pairs of preanal papillae: 11 subventral and one lateral; six pairs of postanal papillae: five subventral and one lateral, with the latter pair at the level of first subventral pairs when counted from the cloacal aperture. For the female: 14 anterior prostomal teeth and the size and absence of superficial structures on fully mature (larvated) eggs dissected out of the nematode body. Specimens of R. gendrei were genetically distinct in the 28S rRNA and cytochrome c oxidase subunit 1 (cox1) mitochondrial gene regions from known species of Rhabdochona. This is the first study that provides genetic data for a species of Rhabdochona from Africa, the first SEM of R. gendrei, and the first report of this parasite from Kenya. The molecular and SEM data reported herein provide a useful point of reference for future studies on Rhadochona in Africa.

A New, Non-Invasive Methodology for the Molecular Identification of Adult Sarcophagidae from Collections.

Correct species identification is the cornerstone of all scientific studies that involve insects. Alongside traditional morphological identification techniques, molecular identification based on the characterization and analysis of specific mitochondrial or nuclear gene regions is becoming commonplace. Despite the good results that can be achieved, DNA extraction usually involves invasive techniques that lead to the partial or total destruction of specimens. In this work, a non-invasive DNA extraction technique is described. The technique was tested on the abdomens of dry-preserved Sarcophagidae (Diptera) specimens collected between 1889 and 2015. This allowed for the correct identification of species without impairing diagnostic morphological structures useful for further studies.