Mitochondrial 12S Ribosomal RNA Gene Research Articles

An innovative molecular approach towards the cost-effective entomological authentication of honey

Honey authentication and traceability are crucial not only for economic purposes but also for ensuring safety. However, the widespread adoption of cutting-edge technologies in practical applications has been hampered by complex, time-consuming sample pre-treatment processes, the need for skilled personnel, and substantial associated expenses. This study aimed to develop a simple and cost-effective molecular technique to verify the entomological source of honey. By utilizing newly designed primers, we successfully amplified the mitochondrial 16S ribosomal RNA gene of honey bees from honey, confirming the high quality of the extracted DNA. Employing RFLP analysis with AseI endonuclease, species-specific restriction patterns were generated for honey derived from six closely related honey bees of the Apis genus. Remarkably, this method was proven equally effective in identifying heat-treated and aged honey by presenting the same RFLP profiles as raw honey. As far as we know, this is the initial research of the simultaneous differentiation of honey from closely related honey bee species using the restriction endonuclease AseI and mitochondrial 16S rRNA gene fragments. As a result, it holds tremendous potential as a standardized guideline for regulatory agencies to ascertain the insect origins of honey and achieve comprehensive traceability.

Development of molecular identification methods for Dryophytes suweonensis and D. japonicus, and their hybrids.

As hybridization can reduce biodiversity or cause extinction, it is important to identify both purebred parental species and their hybrids prior to conserving them. The Suwon tree frog, Dryophytes suweonensis, is an endangered wildlife species in Korea that shares its habitat and often hybridizes with the Japanese tree frog, D. japonicus. In particular, D. suweonensis, D. japonicus, and their hybrids often have abnormal ovaries and gonads, which are known causes that could threaten their existence. We collected 57 individuals from six localities where D. suweonensis is known to be present. High-resolution melting curve (HRM) analysis of the mitochondrial 12S ribosomal RNA gene was performed to determine the maternal species. Thereafter, the DNA sequences of five nuclear genes (SIAH, TYR, POMC, RAG1, and C-MYC) were analyzed to determine their parental species and hybrid status. The HRM analysis showed that the melting temperature of D. suweonensis was in the range of 79.0-79.3°C, and that of D. japonicus was 77.7-78.0°C, which clearly distinguished the two tree frog species. DNA sequencing of the five nuclear genes revealed 37 single-nucleotide polymorphism (SNP) sites, and STRUCTURE analysis showed a two-group structure as the most likely grouping solution. No heterozygous position in the purebred parental sequences with Q values ≥ 0.995 were found, which clearly distinguished the two treefrog species from their hybrids; 11 individuals were found to be D. suweonensis, eight were found to be D. japonicus, and the remaining 38 individuals were found to be hybrids. Thus, it was possible to unambiguously identify the parental species and their hybrids using HRM analysis and DNA sequencing methods. This study provided fundamental information for D. suweonensis conservation and restoration research.

Sympatric occurrence of Taenia saginata and Sarcocystis spp. in cattle from Narok County, Kenya: meat inspection findings with molecular validation.

The epidemiological picture of Taenia saginata infections in Kenya is fragmented with limited available data. Although Sarcocystis species are significant meat-borne parasites, few studies have explored their occurrence in Kenya. This study aimed to estimate the occurrence of bovine cysticercosis and screen for the presence of Sarcocystis spp. A meat inspection-based survey was conducted in ten abattoirs in Narok County, Kenya, and inspection for T. saginata cysticerci was limited to the Triceps brachii muscle. The apparent occurrence of the parasite was 5.4% (95% CI, 3.8, 7.6, n=573). Molecular confirmation of T. saginata was done via nested polymerase chain reaction targeting the mitochondrial 12S ribosomal RNA gene and restricted fragment length polymorphism. Sarcocystis species were identified using a multiplex polymerase chain reaction method targeting the 18S ribosomal RNA gene sequences and the mitochondrial cytochrome c oxidase subunit I gene. Of the 31 cystic lesions tested, 26/31 (83.9%) were confirmed to be T. saginata.Sarcocystis cruzi and S. hominis were detected in 8/31 (25.8%) and 1/31 (3.2%) of the cystic lesions, respectively. Co-infections of S. cruzi and T. saginata were found in 6/31 lesions (19.4%). The confirmation of bovine cysticercosis and S. hominis is suggestive of the presence of risky culinary and sanitation practices that facilitate transmission. This is the first report and molecular confirmation of Sarcocystis spp. in cattle in the country. The presence of both zoonotic S. hominis and pathogenic S. cruzi highlights an underexplored concern of veterinary and human health significance, warranting further epidemiological investigation.

Environmental DNA compliments traditional sampling for monitoring fish communities in a Texas estuary

AbstractObjectiveEnvironmental DNA (eDNA) metabarcoding has become an important method for inventorying and monitoring biota in aquatic systems. The Texas Parks and Wildlife Department conducts regular fishery‐independent sampling of biotic communities using traditional sampling gears, such as gill nets and bag seines, in all of the major estuaries of adjacent to the Gulf of Mexico in Texas. Previous studies have shown that eDNA approaches can complement traditional sampling methods.MethodsWe compared fish community structure data in the Cedar Lakes estuary system obtained with traditional sampling gears with data obtained using eDNA sampling using a small sequence of mitochondrial 12S ribosomal RNA gene and a validated taxonomic reference file.ResultFor spring and fall of 2022, eDNA metabarcoding detected a larger number of species than either bag seines or gill nets. Species richness detected via eDNA in two seasons in a single year was comparable with the species richness of agency’s historical record based on traditional gears for Cedar Lakes.ConclusionSeasonal and spatial variation in species richness was similar between traditional and eDNA sampling; however, eDNA metabarcoding allowed detection of several species that would be difficult or impossible to capture with either bag seines or gill nets. We observed two limitations of eDNA metabarcoding. Read depth was not a good index of relative abundance, which limits our ability to infer relative biomass using single samples. Secondly, we observed detection bias in our eDNA results. Specifically, eDNA failed to detect two species of elasmobranchs present when water sampling was performed and eDNA also performed poorly compared to traditional sampling gears for some species of bony fishes. Despite these limitations, eDNA metabarcoding proved to be an efficient and cost‐effective alternative and compliment to traditional fisheries sampling gears for fishery‐independent monitoring of community structure and composition in estuaries of the Gulf of Mexico.

The smallest of its kind: Description of a new cryptic Amnirana species (Amphibia, Anura, Ranidae) from West African rainforests

The distribution of the White-Lipped Frog Amnirana albolabris was long assumed to extend from eastern-central to western African rainforests. However, recent molecular phylogenetic analyses revealed that this taxon likely represents several undescribed species. Because the name-bearing types were collected in Gabon, the distantly related West African populations clearly represent an undescribed species that partly occurs in sympatry with Amnirana fonensis. Based on an integrative taxonomic approach, including molecular, morphological, and acoustic data, we describe the 'albolabris' populations from the Upper Guinean Forest Zone as a new species, and redescribe the morphologically similar A. fonensis on the bases of a larger series of genotyped individuals, including the first known females. We also provide new biological information for A. fonensis, including their advertisement call, habitat, and reproductive data. The new species is sister to A. fonensis and the two species differ by 5.8% in the mitochondrial 16S ribosomal RNA gene. Morphologically, males of the new species are smaller and have relatively smaller humeral glands. Females of the new species seem to have shorter legs than A. fonensis. In comparison to A. fonensis, the advertisement call of the new species has a higher dominant frequency and more pronounced frequency modulation. The two species differ in their distribution and habitat preferences, as revealed by environmental niche modelling. Whereas the new species is restricted to the Upper Guinean forests and thus is a true lowland-rainforest inhabitant, A. fonensis lives predominantly in Guinean montane forests in the forest-savanna mosaic zone.

First detection of Echinococcus multilocularis in Bosnia and Herzegovina.

Echinococcus multilocularis has been spreading through Central Eastern Europe but has not yet been reported in Bosnia and Herzegovina (B&H). Recently, this parasite is confirmed in Croatia suggesting the movement of the parasite's distribution limit further south. Given that there is no surveillance or monitoring system for echinococcosis in B&H, our study was designed as a pilot study of E. multilocularis. A total of 57 red foxes originating from 24 localities all over the country were collected during the routine rabies monitoring, autopsied and examined for the presence of echinococcosis. Based on intestinal scraping technique and microscopy, E. multilocularis adult worms have been detected in one (1/57, 1.75%) red fox. To verify this finding and to differentiate Echinococcus spp., DNA extracted from adult worms was subjected to species-specific PCR targeting part of the mitochondrial 12S ribosomal RNA gene. E. multilocularis PCR-positive samples were further confirmed by NGS sequencing of a 203 bp amplified fragment of 12S rRNA, which has been deposited in GenBank (Accession no.: OP047920). This finding represents the first detection of E. multilocularis in B&H, strongly suggesting its presence in the country. The confirmation of the parasite in the same locality where migrants/refugees temporarily stay on their route to Western Europe highlights the need for a One Health approach in addressing all future questions. Moreover, the first detection of E. multilocularis in B&H warrants the need for the implementation of an appropriate state surveillance program.

Molecular phylogenetic investigation of toads based on sequencing of the mitochondrial 16S ribosomal RNA gene

Amphibians inhabit a wide variety of habitats and environments, such as arboreal, terrestrial and freshwater aquatic ecosystems, and are considered among the vertebrate classes to exhibit one of the most diverse distributions. Their life histories, behaviour and diversity play vital roles in assessing environmental and evolutionary theories. The mitochondrial 16S rRNA gene sequence analysis has been widely used in molecular taxonomy and is frequently used in systematic analyses of families and genera. This study examines taxonomic relationships among 18 specimens of the Arabian toad (Sclerophrys arabica) and the Dhofar toad (Bufo dhufarensis) from Al Madinah Province, Saudi Arabia by estimating phylogenetic relationships using the mitochondrial 16S ribosomal RNA gene (16S rRNA). Results indicate the presence of four new lineages of toads among the samples in this study. The generated phylogenetic tree grouped all samples of Arabian toad in one clade, while the Dhofar toad samples were divided into three subclades. This is the first study to be reported the genetic diversity of Al Madinah Province toads.

Discovery of a new limestone karst-restricted odorous frog from northern Guangdong, China (Anura, Ranidae, Odorrana).

Karstic landscapes play an important role in biodiversity formation and often contain high levels of endemism. However, site-endemic taxa in karstic landscapes are being threatened by exploitation and weak legal protection. In this study, we describe Odorranaconcelata Wang, Zeng, & Lin, sp. nov., a limestone karst-restricted odorous frog from northern Guangdong, China. This new species shows distinctive genetic divergence and morphological differences from its congeners. Phylogenetic results suggest that the new species represents an independent lineage that is grouped with O.lipuensis and O.liboensis based on the mitochondrial 16S and 12S ribosomal RNA genes. We recommend the new species be listed as Vulnerable (VU) in the IUCN categorization as it is only known from the type locality with limited microhabitats and is threatened by habitat degradation.

Robust environmental DNA assay development and validation: A case study with two vulnerable Australian fish

Abstract Analysis of environmental (e)DNA can facilitate an understanding of the presence and distribution of aquatic species. However, eDNA detection using quantitative PCR requires validated and standardized species‐specific assay designs. This study presents two eDNA assays to detect Murray cod, Maccullochella peelii, and mulloway, Argyrosomus japonicus (two ecologically vulnerable Australian species), based on small fragments of the mitochondrial 12S ribosomal RNA gene. A comprehensive description of species‐specific assay development, from assay design to testing in silico, in vitro and in situ, has been included to guide effective assay design and validation in future studies. The results indicate that the assay was species specific for M. peelii within its natural distribution. However, the assay also amplified genomic DNA from two allopatric and endangered congeners (Maccullochella ikei and Maccullochella mariensis), thus potentially facilitating their eDNA detection elsewhere. In contrast, the A. japonicus assay was highly species specific with no amplification among close relatives. Both target‐species assays are highly sensitive to as few as four and 10 copies per PCR reaction, respectively. This study has demonstrated that the assays assessed are effective tools for detecting the targeted species in situ from environmental DNA samples, which will assist efforts to conserve and manage their stocks.

A 16S Next Generation Sequencing Based Molecular and Bioinformatics Pipeline to Identify Processed Meat Products Contamination and Mislabelling.

Simple SummaryMeat adulteration and fraud encompasses the deliberate fraudulent addition or substitution of proteins of animal or plant origin in edible products primarily for economic gain. The mitochondrial 16S ribosomal (rRNA) gene was used to identify species that are present in pure and processed meat samples. The meat samples were sequenced using an Illumina sequencing platform, and bioinformatics analysis was carried out for species identification. The results indicated that pork was the major contaminant in most of the meat samples. The bioinformatics pipeline demonstrated its specificity through identification of species specific and quantification of the contamination levels across all samples. Food business operators and regulatory sectors can validate this method for food fraud checks and manage any form of mislabeling in the animal or plant protein food ecosystem.Processed meat is a target in meat adulteration for economic gain. This study demonstrates a molecular and bioinformatics diagnostic pipeline, utilizing the mitochondrial 16S ribosomal RNA (rRNA) gene, to determine processed meat product mislabeling through Next-Generation Sequencing. Nine pure meat samples were collected and artificially mixed at different ratios to verify the specificity and sensitivity of the pipeline. Processed meat products (n = 155), namely, minced meat, biltong, burger patties, and sausages, were collected across South Africa. Sequencing was performed using the Illumina MiSeq sequencing platform. Each sample had paired-end reads with a length of ±300 bp. Quality control and filtering was performed using BBDuk (version 37.90a). Each sample had an average of 134,000 reads aligned to the mitochondrial genomes using BBMap v37.90. All species in the artificial DNA mixtures were detected. Processed meat samples had reads that mapped to the Bos (90% and above) genus, with traces of reads mapping to Sus and Ovis (2–5%) genus. Sausage samples showed the highest level of contamination with 46% of the samples having mixtures of beef, pork, or mutton in one sample. This method can be used to authenticate meat products, investigate, and manage any form of mislabeling.

EDNA profiling of mammals, birds, and fish of surface waters by mitochondrial metagenomics: application for source tracking of fecal contamination in surface waters.

Knowing the composition of animals present in aquatic ecosystems can tell us about the anthropic pressures on these environments. One of these pressures is the occurrence of fecal contamination. However, this contamination can originate from more than one animal species in areas where urban and agricultural activities overlap. Mitochondrial DNA (mtDNA) has become the standard barcoding tool to identify the presence of animal species in environment. Amplicon-sequencing metagenomics is a powerful approach to derive the animal profile in an environment. However, PCR primers targeting mtDNA of a broad range of animals are highly degenerate or generate short DNA fragments that could cause ambiguous affiliation. Here we report the development of a new set of primers targeting the mitochondrial 16S ribosomal RNA genes of a broad range of terrestrial and aquatic animals, which include mammals, birds, and fishes. These primers successfully amplified mtDNA from environmental DNA (eDNA) extracted from surface waters. Sequencing the resulting amplicons revealed the presence of mammals and birds that may contribute in fecal contamination of surface water. In one of the river samples high in fecal indicator bacteria, human and bovine mtDNA accounted for 40.5% and 4.1% of the sequences, respectively, suggesting fecal contamination by these two animals. These findings indicate that our PCR primers coupled with amplicon-sequencing metagenomics contribute in profiling the animal diversity in the surface waters and its surrounding. This approach could be a valuable tool to identify simultaneously the potential contribution of various animals as sources of fecal contamination in surface waters.

Novel Mitochondrial DNA Markers for Scleractinian Corals and Generic-Level Environmental DNA Metabarcoding

Coral reefs, the most biodiverse habitats in the ocean, are formed by anthozoan cnidarians, the scleractinian corals. Recently, however, ongoing climate change has imperiled scleractinian corals and coral reef environments are changing drastically. Thus, convenient, high-density monitoring of scleractinian corals is essential to understand changes in coral reef communities. Environmental DNA (eDNA) metabarcoding is potentially one of the most effective means of achieving it. Using publicly available scleractinian mitochondrial genomes, we developed high-specificity primers to amplify mitochondrial 12S ribosomal RNA (12S) and cytochrome oxidase-1 (CO1) genes of diverse scleractinian corals, which could be used for genus-level metabarcoding analyses, using next-generation sequencing technologies. To confirm the effectiveness of these primers, PCR amplicon sequencing was performed using eDNA isolated along the seashore of Okinawa, Japan. We successfully amplified all eDNA samples using PCR. Approximately 93 and 72% of PCR amplicon sequences of 12S and CO1 primers originated from scleractinian 12S and CO1 genes, respectively, confirming higher specificities for coral mitochondrial genes than primers previously used for coral eDNA metabarcoding. We also found that hierarchical clustering, based on the percentage of mapped reads to each scleractinian genus, discriminates between sampling locations, suggesting that eDNA surveys are sufficiently powerful to reveal differences between coral communities separated by <1 km. We conclude that the method reported here is a powerful tool for conducting efficient eDNA surveys targeting scleractinian corals.

Detecting aquatic and terrestrial biodiversity in a tropical estuary using environmental DNA

AbstractEstuaries are characterized by a tidal regime and are strongly influenced by hydrodynamics and host diverse and highly dynamic habitats, from fresh, brackish, or saltwater to terrestrial, whose biodiversity is especially difficult to monitor. Here, we investigated the potential of environmental DNA (eDNA) metabarcoding, with three primer sets targeting different regions of the mitochondrial DNA 12S ribosomal RNA gene, to detect vertebrate diversity in the estuary of the Don Diego River in Colombia. With eDNA, we detected not only aquatic organisms, including fishes, amphibians, and reptiles, but also a large diversity of terrestrial, arboreal, and flying vertebrates, including mammals and birds, living in the estuary surroundings. Further, the eDNA signal remained relatively localized along the watercourse. A transect from the deep outer section of the estuary, across the river mouth toward the inner section of the river, showed marked taxonomic turnover from typical marine to freshwater fishes, while eDNA of terrestrial and arboreal species was mainly found in the inner section of the estuary. Our results indicate that eDNA enables the detection of a large diversity of vertebrates and could become an important tool for biodiversity monitoring in estuaries, where water integrates information across the ecosystem.Abstract in Spanish is available with online material.

The true picture of environmental DNA, a case study on harvested fishponds

The applications of environmental DNA (eDNA) metabarcoding are constantly increasing. Many validation studies have been performed in laboratories, however, field experiments are necessary to verify the robustness of eDNA based monitoring. In summer and autumn 2019 water samples in 39 sites and an inflow across three ponds of sizes of approximately 3 (A), 10 (B) and 29 (C) hectares in the Czech Republic were collected. 500 ml from each sample was filtered through 0.45 µl membrane filters and extracted using the Mu-DNA water protocol. Uniquely indexed vertebrate primers aligning mitochondrial 12S ribosomal RNA gene were utilised for eDNA amplification. PCR negative and positive controls were included to each sublibrary. Sequence reads were analysed using metaBEAT pipeline. The sequences were assigned to species level with exception Perca fluviatilis and Sander lucioperca which could not be differentiated, further referred as Perca+Sander and used as a single species. At the end of autumn, ponds were harvested and 319,833 fish of 27,054 kg of 22 species were captured. The most abundant species was Gymnocephalus cernua (on average 34.8%) followed by Cyprinus carpio and Pseudorasbora parva. Cyprinus carpio was also the dominant species in biomass (on average 90.7%), followed by the Ctenopharyngodon idella and Esox Lucius. The total number of sequences across 240 eDNA samples was 35,627,310. 21,540,396 sequences remained after bioinformatic filtering with an average read count per sample of 68,111. All samples detected 1–6 species, except for summer pond B where 64% were negative. In total, more species were detected in autumn than summer in pond A 12/10 and pond B 9/2 and the same in pond C 11/11. The only species detected in all campaigns was Cyprinus carpio with the highest average reads count of 28,961. Ctenopharyngodon idella, Rutilus rutilus, and Scardinius erythrophthalmus were detected in all campaigns except the summer pond B samples. The site occupancy values were variable between ponds and seasons with exception of ubiquitous Cyprinus carpio. The numbers of detected species in inflows were higher in autumn compared to summer in pond A (7/5) and B (9/4) and identical in pond C (6/6). The detected species in inflows were mostly the same as species detected in ponds. Gobio gobio was the only detected species in all campaigns. Lota lota was detected in eDNA in summer and autumn only in inflow to pond A. In pond B Perca+Sander and in pond C Pseudorasbora parva were detected in eDNA inflow in both periods and in harvest, but not in eDNA in ponds. The percentage of shared species detected by eDNA and at pond harvests was >50% in all ponds. During the pond harvest 1, 5 and 4 species were captured not detected in ponds´ eDNA compared to 3, 0, and 2 species detected only in ponds´ eDNA in ponds A, B and C, respectively. Significant positive relationships were observed between fish read counts/species site occupancy and fish abundance/biomass with exception of data from pond B in autumn. This study highlights the importance of field experiments and methods validation. Dominant species detection is relatively straightforward; however, detection of rare species is more challenging depending on species behaviour, habitat complexity. It further supports that eDNA should be applied in optimal conditions to achieve highest overall detection, which has important implications for applying this method to aid management and policy initiatives. This study was supported by projects MSM200961901, “The true picture of eDNA”, QK1920011 ”Methodology of predatory fish quantification in drinking-water reservoirs to optimize the management of aquatic ecosystems”, and by CAS within the program of the Strategy AV 21.

Genetic variation and phylogenetic analysis of Indonesian indigenous catfish (baung fish) based on mitochondrial 12S rRNA gene.

Background and Aim:Baung fish is an essential commodity in Indonesia; however, few studies have explored the genetic diversity of Indonesian catfish. Thus, this study aimed to analyze the genetic variation and phylogenetic relationships among Indonesian catfish based on the mitochondrial 12S ribosomal RNA (rRNA) gene.Materials and Methods:In total, 28 catfish were collected from nine rivers in seven provinces and from the Indian Ocean. Catfish genomes were obtained from epaxial and hepaxial muscle samples. The mitochondrial 12S rRNA gene was amplified by polymerase chain reaction using a pair of primers (Baung12SF and Baung12SR). The 12S rRNA sequences were analyzed using MEGA X to determine genetic variation and phylogenetic relationships.Results:In total, 178 variation sites in the 12S rRNA gene were substituted among Indonesian catfish. The genetic distance between all Indonesian catfish samples was 0.1-16.0%. The closest genetic distance was between MP and PM catfish, whereas the farthest genetic distances were between BF and EM and PD and EM. For the entire population, based on mean diversity calculations, the number of base substitutions per site was 0.08.Conclusion:Indonesian catfish were divided into four clades based on the 12S rRNA gene. The catfish MP, KR, PM, MS, BB, and KS were grouped with Hemibagrus nemurus, the catfish EM was grouped with Mystus vittatus, the catfish BSBJ was grouped with Pangasius pangasius, and the catfish PD and BF were grouped with Netuma thalassina.

Rock island melody: A revision of the Afroedura bogerti Loveridge, 1944 group, with descriptions of four new endemic species from Angola

Four new species of flat geckos in the Afroedura bogerti Loveridge, 1944 group are described from south-western and west-central Angola. The description of these new species significantly restricts the distribution range of typical A. bogerti, a morphologically very similar species, from which they differ genetically by 5.9–12% divergence for the mitochondrial 16S ribosomal RNA gene. Morphologically and genetically, Angolan Afroedura are divided into two main groups: a mostly south-western coastal group and a west-central inland/highland group. These two groups are further divisible into three and two subgroups respectively, all geographically isolated, differing by a combination of the following features: colouration, average adult size, number of mid-body scale rows, number of scale rows on dorsal and ventral surface of each tail verticil and if nostril scales are in contact or not. All five Angolan species are morphologically distinguishable and in agreement with the molecular results. An updated dichotomous key to the Afroedura transvaalica group is provided. The new discovery adds to a growing number of endemic Pro-Namib reptiles described from Angola in recent years.

Genomic Profiling for Piroplasms in Feeding Ixodid Ticks in the Eastern Cape, South Africa.

Importation of tick-infected animals and the uncontrollable migration of birds and wild animals across borders can lead to geographical expansion and redistribution of ticks and pathogen vectors, thus leading to the emergence and re-emergence of tick-borne diseases in humans and animals. Comparatively, little is known about the occurrence of piroplasms in ixodid ticks in the Eastern Cape, South Africa, thus necessitating this study, which is aimed at detecting piroplasms (Theileria and Babesia) from feeding tick samples collected from cattle, sheep, and goats in selected sites in the Eastern Cape, South Africa. A total of 1200 feeding ixodid ticks collected from farm animals at selected homesteads were first subjected to molecular identification using mitochondrial 12S ribosomal RNA (rRNA) gene by PCR and were further tested for the presence of piroplasms through amplification of the 18S rRNA gene via nested-PCR followed by sequencing of the PCR products. The results indicated that 853 (71.1%) corresponded to the genus Rhipicephalus, 335 (27.9%) corresponded to genus Amblyomma, and 12 (1%) corresponded to genus Haemaphysalis. Amblyomma hebraeum and Rhipicephalus appendiculatus were the most common identified ticks from this study. The 18S rRNA nested-PCR revealed that 44 (3.7%) samples were confirmed positive for Theileria. A homology search for the generated sequences revealed a high percentage identity of 98–98.9% similarity to T. buffeli, T. orientalis, and T. sergenti in the GenBank. Based on the results obtained herein, we conclude that there is a big diversity of Theileria species; therefore, we suggest that this research should cover more geographical areas in order to reveal the true prevalence of this pathogen in the studied area because this will be a great step in the possible prevention of an outbreak that could have devastating effects on livestock production and human health in both the studied areas and South Africa at large.

Morphological and genetic identification of formalin-fixed gobioid larvae and description of postflexion larvae of Paragunnellichthys sp. and Ctenogobiops feroculus

Gobioid larvae collected from the coast of Okinawa Island, southern Japan, were identified on the basis of a combination of morphological characters and a sequence of a hypervariable region of mitochondrial 12S ribosomal RNA (12S rRNA) gene (138–145 base pairs). A short-term formalin fixation technique enabled identification using both morphological and genetic methods. Thirteen of the 21 types of gobioid larvae assessed were identified to the species level. Additionally, we described the morphology of the postflexion larvae of wormfish Paragunnellichthys sp. and shrimp-associated goby Ctenogobiops feroculus, identified for the first time in their respective genera.

Case Report: Periorbital Filariasis Caused by Brugia malayi

Brugia malayi is a lymphatic nematode that accounts for approximately 10% of lymphatic filariasis cases worldwide. It is endemic in several countries in South and Southeast Asia. In Thailand, B. malayi is endemic in the southern region. The extralymphatic presentation of B. malayi is rare. Here, we report the case of a woman residing in the central region of Thailand who presented with an erythematous periorbital nodule at the left medial canthal area caused by lymphatic filaria. A viable sexually mature filarial adult was removed from the lesion. The nematode species was identified as B. malayi by histology staining and DNA sequencing of the partial mitochondrial 12S ribosomal RNA (rRNA) gene. As far as we know, this is the first case report of B. malayi presenting with a periorbital nodule that has occurred in a disease non-endemic area of Thailand with possibly a zoonotic origin.

Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes

BackgroundMolecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics.MethodsTo study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms.ResultsThe phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers.ConclusionsThis study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.