Leptopoma is a species rich genus with approximately 100 species documented. Species-level identification in this group has been based on shell morphology and colouration, as well as some anatomical features based on small sample sizes. However, the implications of the inter- and intra-species variations in shell form to the taxonomy of Leptopoma species and the congruency of its current shell based taxonomy with its molecular phylogeny are still unclear. There are four Leptopoma species found in Sabah, Borneo, and their taxonomy status remains uncertain due to substantial variation in shell forms. This study focuses on the phylogenetic relationships and geographical variation in shell form of three Leptopoma species from Sabah. The phylogenetic relationship of these species was first estimated by performing Maximum Likelihood and Bayesian analysis based on mitochondrial genes (16S rDNA and COI) and nuclear gene (ITS-1). Then, a total of six quantitative shell characters (i.e., shell height, shell width, aperture height, aperture width, shell spire height, and ratio of shell height to width) and three qualitative shell characters (i.e., shell colour patterns, spiral ridges, and dark apertural band) of the specimens were mapped across the phylogenetic tree and tested for phylogenetic signals. Data on shell characters of Leptopoma sericatum and Leptopoma pellucidum from two different locations (i.e., Balambangan Island and Kinabatangan) where both species occurred sympatrically were then obtained to examine the geographical variations in shell form. The molecular phylogenetic analyses suggested that each of the three Leptopoma species was monophyletic and indicated congruence with only one of the shell characters (i.e., shell spiral ridges) in the current morphological-based classification. Although the geographical variation analyses suggested some of the shell characters indicating inter-species differences between the two Leptopoma species, these also pointed to intra-species differences between populations from different locations. This study on Leptopoma species is based on small sample size and the findings appear only applicable to Leptopoma species in Sabah. Nevertheless, we anticipate this study to be a starting point for more detailed investigations to include the other still little-known (ca. 100) Leptopoma species and highlights a need to assess variations in shell characters before they could be used in species classification.
A new species of limestone karst-adapted gecko of the Cyrtodactylus pulchellus complex, C. dayangbuntingensis sp. nov., is described from Dayang Bunting Island of the Langkawi Archipelago off the northwest coast of Peninsular Malaysia. It is the third species of the group to be described from the archipelago after C. langkawiensis and C. macrotuberculatus. The new species can be distinguished from all other species of Cyrtodactylus based on molecular evidence from the mitochondrial gene ND2 and its flanking tRNAs as well as having unique combinations of morphological and color pattern characteristics. This discovery underscores the need for continued surveys of the many islands in the archipelago to properly ascertain its true herpetological diversity.
A polymerase chain reaction (PCR) assay for the assessment of dog meat adulteration in meatballs was developed. The assay selectively amplified a 100-bp region of canine mitochondrial cytochrome b gene from pure, raw, processed and mixed backgrounds. The specificity of the assay was tested against 11 animals and 3 plants species, commonly available for meatball formulation. The stability of the assay was proven under extensively autoclaving conditions that breakdown target DNA. A blind test from ready to eat chicken and beef meatballs showed that the assay can repeatedly detect 0.2% canine meat tissues under complex matrices using 0.04 ng of dog DNA extracted from differentially treated meatballs. The simplicity, stability and sensitivity of the assay suggested that it could be used in halal food industry for the authentication of canine derivatives in processed foods.
Invasive snails in the genus Pomacea have spread across Southeast Asia including Peninsular Malaysia. Their effects on natural and agricultural wetlands are appreciable, but species-specific effects are less clear because of morphological similarity among the species. Our objective was to establish diagnostic characteristics of Pomacea species in Malaysia using genetic and morphological criteria. The mitochondrial COI gene of 52 adult snails from eight localities in Peninsular Malaysia was amplified, sequenced, and analysed to verify species and phylogenetic relationships. Shells were compared using geometric morphometric and covariance analyses. Two monophyletic taxa, P. canaliculata and P. maculata, occurred in our samples. The mean ratio of shell height: aperture height (P = 0.042) and shell height: shell width (P = 0.007) was smaller in P. maculata. P. maculata co-occurred with P. canaliculata in five localities, but samples from three localities contained only P. canaliculata. This study is the first to confirm the presence of two of the most invasive species of Pomacea in Peninsular Malaysia using a molecular technique. P. canaliculata appears to be the more widespread species. Despite statistical differences, both quantitative and qualitative morphological characteristics demonstrated much interspecific overlap and intraspecific variability; thus, shell morphology alone cannot reliably verify species identity. Molecular techniques for distinguishing between these two highly invasive Pomacea species are needed to understand their specific ecological niches and to develop effective protocols for their management.
Limnonectes kuhlii and Limnonectes leporinus are two of the Bornean fanged frogs (without advertisement call) which are widely distributed, thus thought to exhibit different evolutionary lineages and the existence of genetically cryptic species. Yet, the two species are still under study especially at the molecular level. Hence, cytochrome c oxidase I (COI) of mitochondrial gene was used to investigate suitable parameters for DNA amplification using the Polymerase Chain Reaction (PCR) method. Three PCR programmes (varied in the temperatures and period of each PCR step) were employed to identify the most efficient parameters in amplifying PCR products for both species. From the three programmes, Programme B (Initial denaturation: 96°C for 5 min; denaturation: 95°C for 45 sec; annealing: 48-53°C for 1 min 30 sec; extension: 72°C for 1 min 30 sec; final extension: 72°C for 10 min, 30 cycles) showed the highest percentage (53%) of optimal PCR products. The other two programmes showed non-specific products or “primer-dimers”. The results also suggest that the annealing temperature of 52°C, 0.025-0.05 units/µl of 1.5mM Taq polymerase, 0.04 mM of
dNTPs mix and optimal concentrations of magnesium in 50 µl of reaction mixture were sufficient enough to amplify high quality PCR products for both species. However, using Programme B, the re-amplification of the PCR products yielded “primer-dimer”. In addition, a ‘Hot-Start’ PCR method was also applied and mostly yielded in an optimal PCR amplification. Nevertheless, further research on the second amplification of the two species should be conducted to determine the causes of the primer-dimer production.
Sooty blotch and flyspeck (SBFS) fungi infect the cuticle of fruit, including apple fruit, and produce pigmented colonies. A new member of this fungal complex in the genus Peltaster is described on the basis of molecular and morphological evidence. The SBFS complex is a diverse group of ectophytic fungi that reside primarily within the order Capnodiales. Sooty blotch and flyspeck isolates from apple orchards in the central United States were subjected to parsimony and Bayesian analyses based on the internal transcribed spacer regions of nuc rDNA, the partial translation elongation factor 1-α gene, and the partial mitochondrial small subunit rRNA gene. Phylogenetic analysis delineated a new species, Peltaster gemmifer, from P. cerophilus and P. fructicola. Peltaster gemmifer conidiophores bear primary conidia that produce secondary conidia either through budding or through microcyclic conidiation; these were not seen in cultures of P. cerophilus and P. fructicola. On cellulose membrane that was placed on water agar amended with apple juice, P. gemmifer produced brown to black pycnothyria in a superficial brownish mycelial mat, similar to the colonies produced on apple fruit. Findings from the present study add to the >80 named and putative SBFS species so far described worldwide.
A method of PCR-restriction fragment length polymorphism (RFLP) has been utilized to differentiate the mitochondrial genes of pork and wild boar meat (Sus scrofa). The amplification PCR products of 359 bp and 531 bp were successfully amplified from the cyt b gene of these two meats. The amplification product of pork and wild boar using mt-12S rRNA gene successfully produced a single band with molecular size of 456 bp. Three restriction endonucleases (AluI, HindIII and BsaJI) were used to restrict the amplification products of the mitochondrial genes. The restriction enzymes of AluI and BsaJI were identified as potential restriction endonucleases to differentiate those meats. HindIII enzyme was unable to restrict the PCR product of both meats. The genetic differences within the cyt b gene among the two meats were successfully confirmed by PCR-RFLP analysis.
Friedreich ataxia (FRDA) is the most frequent progressive autosomal recessive disorder associated with unstable expansion of GAA trinucleotide repeats in the first intron of the FXN gene, which encodes for the mitochondrial frataxin protein. The number of repeats correlates with disease severity, where impaired transcription of the FXN gene results in reduced expression of the frataxin protein. Gene expression studies provide insights into disease pathogenicity and identify potential biomarkers, an important goal of translational research in neurodegenerative diseases. Here, using real-time PCR (RT-PCR), the expression profiles of mitochondrial (mtDNA) and nuclear DNA (nDNA) genes that encode for the mitochondrial subunits of respiratory oxidative phosphorylation (OXPHOS) complex I in the blood panels of 21 FRDA patients and 24 healthy controls were investigated. Here, the expression pattern of mtDNA-encoded complex I subunits was distinctly different from the expression pattern of nDNA-encoded complex I subunits, where significant (p<0.05) down-regulation of the mitochondrial ND2, ND4L, and ND6 complex I genes, compared to controls, were observed. In addition, the expression pattern of one nDNA-encoded gene, NDUFA1, was significantly (p<0.05) down-regulated compared to control. These findings suggest, for the first time, that the regulation of complex I subunit expression in FRDA is complex, rather than merely being a reflection of global co-regulation, and may provide important clues toward novel therapeutic strategies for FRDA and mitochondrial complex I deficiency.
Epithelial growth factor receptor (EGFR) is a cell surface transmembrane receptor that mediates the tyrosine signaling pathway to carry the extracellular messages inside the cell and thereby alter the function of nucleus. This leads to the generation of various protein products to up or downregulate the cellular function. It is encoded by cell erythroblastosis virus oncogene B1, so called C-erb B1/ERBB2/HER-2 gene that acts as a proto-oncogene. It belongs to the HER-2 receptor-family in breast cancer and responds best with anti-Herceptin therapy (anti-tyrosine kinase monoclonal antibody). HER-2 positive breast cancer patient exhibits worse prognosis without Herceptin therapy. Similar incidence and prognosis are reported in other epithelial neoplasms like EGFR + lung non-small cell carcinoma and glioblastoma (grade IV brain glial tumor). Present study highlights the role and connectivity of EGF with various cancers via signaling pathways, cell surface receptors mechanism, macromolecules, mitochondrial genes and neoplasm. Present study describes the EGFR associated gene expression profiling (in breast cancer and NSCLC), relation between mitrochondrial genes and carcinoma, and several in vitro and in vivo models to screen the synergistic effect of various combination treatments. According to this study, although clinical studies including targeted treatments, immunotherapies, radiotherapy, TKi-EGFR combined targeted therapy have been carried out to investigate the synergism of combination therapy; however still there is a gap to apply the scenarios of experimental and clinical studies for further developments. This review will give an idea about the transition from experimental to most advanced clinical studies with different combination drug strategies to treat cancer.
Certain species of Phlebotomine sandflies (Diptera: Psychodidae) are vectors of the protozoa which causes leishmaniasis. Sandflies are found breeding in enclosed places like caves. Thailand is a popular tourist destination, including for ecotourism activities like caving, which increases the risk of contact between tourists and sandflies. Surveillance of sandflies is important for monitoring this risk but identification of species based on morphology is challenged by phenotypic plasticity and cryptic diversity. DNA barcodes have been used for the identification of sandflies in Thailand. We collected sandflies using CDC light trap from four tourist caves in Northern Thailand. Female sandflies were provisionally sorted into 13 morphospecies and 19 unidentified specimens. DNA was extracted from the thorax and legs of sandflies and the DNA barcode region of cytochrome c oxidase I mtDNA amplified and sequenced. The specimens were sorted into 22 molecular operational taxonomic units (MOTU) based on the 145 DNA barcodes, which is significantly more than the morphospecies. Several of the taxa thought to be present in multiple caves, based on morphospecies sorting, split into cave-specific MOTU which likely represent cryptic species. Several MOTU reported in an earlier study from Wihan Cave, Thailand, were also found in these caves. This supports the use of DNA barcodes to investigate species diversity of sandflies and their useful role in surveillance of sandflies in Thailand.
Gymnothorax minor is a moray eel of the family Muraenidae found in the Western Pacific Ocean. We report here
its complete mitogenome as determined by Illumina next-generation sequencing and the phylogenetic relationship
with its congeners and other taxa of the family Muraenidae. The whole mitogenome of G. minor had a total length
of 16,574 bp, comprising 37 genes - 13 protein-coding genes (PCGs), two ribosomal ribonucleic acid (rRNA) and 22
transfer ribonucleic acid (tRNA) genes - and a control region. Excepting cox1 with GTG, the other 12 PCGs had ATG
start codon. Seven of its PCGs had incomplete stop codon - five (nad2; cox1; cox2; nad3 and nad4) with T and two
(atp6 and cox3) with TA. Molecular phylogeny based on 13 PCGs was concordant with 15 mitochondrial genes (13 PCGs
and 2 rRNA genes). The subfamily Muraeninae as well as the subfamily Uropterygiinae were monophyletic. However,
the genus Gymnothorax was paraphyletic, with G. minor forming a sister group with Rhinomuraena quaesita in the
lineage containing also G. kidako and G. formosus forming a sister group with Enchelynassa canina. The phylogenetic
relationship of the genus Gymnothorax and related taxa of the family Muraenidae, based on the mitochondrial cob
gene, was in general similar to that based on 15 mt-genes. The mitogenome is useful for future studies on phylogenetics
and systematics of eels of the family Muraenidae and other taxa of the order Anguilliformes.
A new species of black fly, Simulium (Gomphostilbia) isanense, is described based on females, males, pupae and mature larvae from Thailand. This new species is placed in the Simulium epistum species-group of the subgenus Gomphostilbia Enderlein. It is characterized by the pupal gill with eight filaments arranged as 3+3+2 from dorsal to ventral, of which an inner filament of the ventral pair is slightly longer than its counter filament. Taxonomic notes are provided to distinguish this new species from S. (G.) angulistylum Takaoka & Davies from Peninsular Malaysia, and three other related species. The difference between this new species and S. (G.) angulistylum is supported by genetic distances using the mitochondrial COI gene.
Five new species of black flies, Simulium (Gomphostilbia) chiangraiense, S. (G.) huaikaeoense, S. (G.) khaokhoense, S. (G.) maeklangense, and S. (G.) pamiangense, are described based on adults, pupae and mature larvae from Thailand. These five species are similar to one another and are placed in the S. decuplum subgroup of the S. batoense species-group by having the male fore coxae darkened, ventral plate flat and transverse; pupal gill with 10 short filaments; and larval postgenal cleft deep approaching or reaching the posterior margin of the hypostoma. Simulium (G.) chiangraiense sp. nov. and S. (G.) pamiangense sp. nov. differ from the three other new species by the much longer pupal gill filaments and darkened dorsal surface of abdominal segments 1 and 2. Taxonomic notes are provided to distinguish these five new species from five other related species. The phylogenetic positions of these new species in the S. decuplum subgroup are presented based on the mitochondrial COI gene. Simulium (G.) pamiangense sp. nov. and S. (G.) huaikaeoense sp. nov. are highly similar to each other genetically, showing a sister relationship, though they are clearly different morphologically. On the contrary, S. (G.) khaokhoense sp. nov. is distantly positioned from S. (G.) huaikaeoense nov., though it is almost indistinguishable morphologically from the latter.
There are two morphotypes of Penaeus semisulcatus described hitherto in the Persian Gulf, namely the banded and non-banded antennae morphotypes. In this study, we used morphometric measurements and two mitochondrial genes (16S rRNA and cytochrome oxidase subunit I-COI) to assess relationships between the two morphotypes of P. semisulcatus. Out of 25 morphological characters examined, 10 characters were found significantly different between the two morphotypes when tested against separate sexes or both sexes combined. Results from the 16S rRNA and COI sequence analysis of two morphotypes of P. semisulcatus morphotype showed up to 6% and 17% sequence divergence, respectively. The 16S rDNA and COI sequences of the non-banding morphotype were not only very different to those of the banding morphotype but was also very different to all other Penaeus species (i.e., P. monodon, P. merguiensis, and P. indicus) included in the study. Both parsimony and Neighbor-Joining trees based on 16S rDNA and COI sequences provide similar tree topology that clearly separated the two morphotypes into two distinct groups. Based on these findings, we propose the two morphotypes of P. semisulcatus to be relegated as two sympatric species.
The mitochondrial genome sequence of the purple mottled shore crab, Cyclograpsus granulosus, is documented (GenBank accession number: LN624373), which makes it the third for genera of the superfamily Grapsoidea. Cyclograpsus granulosus has a mitogenome of 16,300 bp consisting of 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The base composition of the C. granulosus mitogenome is 36.15% for T, 19.54% for C, 33.14% for A and 11.17% for G, with an AT bias of 69.29%. The mitogenome gene order is atypical for the brachyuran crabs, but is identical to species of the genus Eriocheir from the same family.
The mitochondrial genome sequence of the porcellanid crab, Petrolisthes haswelli is provided, making it the second for the family Porcellanidae and the third for the superfamily Galatheoidea. Petrolisthes haswelli has a mitogenome of 15,348 bp consisting of 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The base composition of the P. haswelli mitogenome is 35.66% for T, 18.65% for C, 34.35% for A and 11.34% for G, with an AT bias of 70.01%. The mitogenome gene order is identical to the mitogenome of Neopetrolisthes maculatus, the only other species of the family with a sequenced mitogenome.
The mitochondrial genome sequence of the ghost crab, Ocypode ceratophthalmus, is documented (GenBank accession number: LN611669) in this article. This is the first mitogenome for the family Ocypodidae and the second for the order Ocypodoidea. Ocypode ceratophthalmus has a mitogenome of 15,564 base pairs consisting of 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The base composition of the O. ceratophthalmus mitogenome is 35.78% for T, 19.36% for C, 33.73% for A and 11.13% for G, with an AT bias of 69.51% and the gene order is the typical arrangement for brachyuran crabs.
The Mictyris longicarpus (soldier crab) complete mitochondrial genome sequence is reported making it the first for the family Mictyridae and the second for the superfamily Ocypodoidea. The mitogenome is 15,548 base pairs made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The soldier crab mitogenome gene order is characteristic of brachyuran crabs with a base composition of 36.58% for T, 19.15% for C, 32.43% for A and 11.83% for G, with an AT bias of 69.01%.
The infraorder Anomura consists of a morphologically and ecologically heterogeneous group of decapod crustaceans, and has attracted interest from taxonomists for decades attempting to find some order out of the seemingly chaotic diversity within the group. Species-level diversity within the Anomura runs the gamut from the "hairy" spindly-legged yeti crab found in deep-sea hydrothermal vent environments to the largest known terrestrial invertebrate, the robust coconut or robber crab. Owing to a well-developed capacity for parallel evolution, as evidenced by the occurrence of multiple independent carcinization events, Anomura has long tested the patience and skill of both taxonomists attempting to find order, and phylogeneticists trying to establish stable hypotheses of evolutionary inter-relationships. In this study, we performed genome skimming to recover the mitogenome sequences of 12 anomuran species including the world's largest extant invertebrate, the robber crab (Birgus latro), thereby over doubling these resources for this group, together with 8 new brachyuran mitogenomes. Maximum-likelihood (ML) and Bayesian-inferred (BI) phylogenetic reconstructions based on amino acid sequences from mitogenome protein-coding genes provided strong support for the monophyly of the Anomura and Brachyura and their sister relationship, consistent with previous studies. The majority of relationships within families were supported and were largely consistent with current taxonomic classifications, whereas many relationships at higher taxonomic levels were unresolved. Nevertheless, we have strong support for a polyphyletic Paguroidea and recovered a well-supported clade of a subset of paguroids (Diogenidae + Coenobitidae) basal to all other anomurans, though this requires further testing with greater taxonomic sampling. We also introduce a new feature to the MitoPhAST bioinformatics pipeline (https://github.com/mht85/MitoPhAST) that enables the extraction of mitochondrial gene order (MGO) information directly from GenBank files and clusters groups based on common MGOs. Using this tool, we compared MGOs across the Anomura and Brachyura, identifying Anomura as a taxonomic "hot spot" with high variability in MGOs among congeneric species from multiple families while noting the broad association of highly-rearranged MGOs with several anomuran lineages inhabiting extreme niches. We also demonstrate the value of MGOs as a source of novel synapomorphies for independently reinforcing tree-based relationships and for shedding light on relationships among challenging groups such as the Aegloidea and Lomisoidea that were unresolved in phylogenetic reconstructions. Overall, this study contributes a substantial amount of new genetic material for Anomura and attempts to further resolve anomuran evolutionary relationships where possible based on a combination of sequence and MGO information. The new feature in MitoPhAST adds to the relatively limited number of bioinformatics tools available for MGO analyses, which can be utilized widely across animal groups.
Minnows of the genus Phoxinus are common and an often highly abundant fish species in Palearctic freshwater habitats. Phoxinus species have a complex evolutionary history, phylogenetic relationships are not well understood and there are a number of unresolved taxonomic problems. There are currently 23 different mitochondrial genetic lineages identified in the genus Phoxinus, 13 of which are recognized as valid species. The taxonomic status of these lineages requires resolution, including the degree to which they can interbreed. Suitable nuclear molecular markers for studies of population divergence and interbreeding between morphotypes and mitochondrial lineages are lacking for Phoxinus species. Therefore, the authors developed a set of microsatellite markers using genomic information from Phoxinus lumaireul and tested their suitability for this and two related species, Phoxinus krkae and Phoxinus marsilii. Out of 16 microsatellite candidate loci isolated, 12 were found to be in Hardy-Weinberg equilibrium when tested on two P. lumaireul senso lato populations. Seven loci amplified across the three species, enabling the study of intraspecific genetic diversity and population structure within P. marsilii and P. krkae. The markers were able to clearly resolve differences among the three tested species, including the recently described P. krkae, and are therefore suitable for the detection of introgression and hybridization among populations consisting of mixtures of two or more of P. lumaireul s. l., P. marsilii and P. krkae.