The generic assignment of the draconine lizard Gonocephalus robinsonii from the highlands of West-Malaysia has been uncertain since the original description. Here we present a study based on morphology, previously published karyotype data and molecular phylogenetics using 16S rRNA sequences to evaluate the systematic status of G. robinsonii. As a result we describe Malayodracon gen. nov. to accommodate the species.
The megophryid frogs Leptobrachella brevicrus, Leptolalax dringi and Megophrys dringi are species exclusively known from highly localised areas in isolated mountain ranges on Borneo. The tadpoles and adults in this study were collected at the shared type locality for the three species in Gunung Mulu National Park, Sarawak, Malaysia (Borneo). The species identities of larvae were determined via comparison to syntopic adults using DNA barcoding techniques based on partial 16S rRNA mitochondrial gene sequences. The genetic data supported the status of the three taxa as valid species. Descriptions of colouration in life and after preservation, external morphological features, morphometric measurements and ecological notes in comparison to congeneric species are supplied. The tadpoles of L. brevicrus and L. dringi show similar adaptations to a fossorial lifestyle. These include an elongated, vermiform body, a relatively long tail and small eyes. Both were found in the gravel beds of a small mountain stream. In contrast, the larvae of M. dringi are adapted to occupying and feeding at the surface of pools within the stream.
Eutropis rugifera has long been identified as a widespread species complex distributed in Nicobar, Peninsular Malaysia, Greater Sundaic Islands, Bali, Sulawesi and the Philippines. This skink was described by Stoliczka in 1870 from Nicobar Island based on a single specimen (holotype by monotypy). Later, Peters (1871), Bartlett (1895) and Werner (1896) described three more species which were morphologically similar to Euprepes percarinatus (from Java), Mabuia rubricollis (Borneo) and M. quinquecarinata (Sumatra) respectively, which are currently considered junior objective synonyms of Eutropis rugifera. We examined all the available synonym types and voucher specimens of Eutropis rugifera deposited at several museums. A morphological examination of the types of this species and mtDNA analysis (584 bp of 16S rRNA) of the samples from different biogeographic regions revealed that Eutropis rugifera from Nicobar Island, Bali Island, and Bawean Island are composed of a monophyletic species. However, the taxonomic status of the above population requires further clarification, and the population in Bawean Island may represent a cryptic species. Finally, we provide a complete redescription of E. rugifera based on its holotype.
A new species of Argiope Audouin 1826, A. hoiseni new species is described from Perak and Selangor, Peninsular Malaysia based on morphology and DNA information of the mitochondrial (16S rRNA, COI and COII) and nuclear-encoded (H3A, 18S rRNA) molecular markers. Epigynal structure suggested Argiope hoiseni to be similar to A. jinghongensis Yin, Peng Wang 1994, A. luzona (Walckenaer 1841), A. pulchella Thorell 1881 and A. taprobanica Thorell 1887. Molecular sequence data including the new species inferred that it is monophyletic with an intraspecific variation of 0.87-3.59 % based on the 16S+COI+COII+H3A dataset. Phylogenetic analyses also revealed insights into the evolutionary lineages of Argiope species in Southeast Asia as well as corroborated recent taxonomic changes and species synonymies associated with Argiope. Two new distribution records were also reported for A. chloreis Thorell,1877 and A. doleschalli Thorell, 1873 in Peninsular Malaysia.
Micryletta inornata (Boulenger 1890), the type species of the genus Micryletta, was originally described from the island of Sumatra in Indonesia. Subsequently, this species has been widely reported from Sundaland (Sumatra and Malay Peninsula), Indo-China, Northeast India and South Andaman, up to southern China and Taiwan. However, since the original description there has been no further report of this species from the type locality or the island. During a herpetofaunal survey in Sumatra, several specimens that are morphologically concordant with the original description and the syntypes of M. inornata were found, and thus the species was rediscovered after 125 years. Here, we provide a redescription of the species based on the freshly collected specimens, along with a detailed morphological and molecular comparison with known congeners. Further, using molecular data from the mitochondrial 16S rRNA gene, our study recovered the Sumatran M. inornata as a phylogenetically distinct lineage from all other populations previously referred to this species. This confirms that all known Micryletta 'inornata' populations from regions outside Sumatra constitute several other lineages representing either new species or previously available names currently considered as synonyms, consequently requiring taxonomic validation in the future.
Molecular phylogenetic analyses of the sister species Sphenomorphus stellatus and S. praesignis based on the mitochondrial genes 12S and 16S rRNA recover the former as paraphyletic with respect to the latter in that a specimen of S. stellatus from the type locality in Peninsular Malaysia is more closely related to S. praesignis than to Indochinese populations of S. stellatus. Furthermore, the phylogeny indicates that the Indochinese populations represent two species, thus resulting in four major lineages within this clade. These relationships are consistent with multivariate and univariate analyses of morphological and discrete color pattern data which statistically define and diagnose the four lineages and together with the molecular data, provide the foundation for robust, testable, species-level hypotheses. As such, S. stellatus is herein restricted to Peninsular Malaysia; S. annamiticus is resurrected for the circum-continental populations ranging through southeastern Thailand, southern Cambodia, and southern Vietnam; a new species-S. preylangensis sp. nov.-is described from an isolated mountain, Phnom Chi, from the Prey Lang Wildlife Sanctuary in central Cambodia; and the taxonomy of S. praesignis remains unchanged. The description of S. preylangensis sp. nov. underscores the necessity to conserve this remnant of lowland evergreen rainforest in the Prey Lang Wildlife Sanctuary.
A new species of Ansonia is described from the Shan Plateau of Myanmar based on an integrative taxonomic analysis that differentiates it from all other congeners. Molecular phylogenetic analyses based on the mitochondrial genes 12S and 16S rRNA and tRNA-val recover A. kyaiktiyoensis sp. nov. as the sister species to A. inthanon from Thailand but differs from it and other congeners by at least a 5.0% sequence divergence. It is further differentiated by the following combination of morphological characters: (1) maximum SVL 24 mm in males and females; (2) first finger shorter than second; (3) absence of interorbital and tarsal ridges; (4) presence of light-coloured interscapular spot; (5) presence of yellow rictal tubercle; (6) absence of wide, light-coloured patch below eye; (7) presence of large, discrete, bright-yellow submandibular spots along the underside of lower jaw; (8) iris yellow-gold; (9) presence of markings on the snout consisting of streaks below the eye to the lip, and on the canthus rostralis to the nostril; (10) dorsum grey-brown with orange-beige spots, a dark-brown X-shaped marking on the back surrounding the interscapular spot, and dark-coloured markings on rump; (11) fore- and hind limbs with orange-beige cross-bars; and (12) venter light-gray with yellow spotting, especially near flanks and underside of hind limbs. Ansonia kyaiktiyoensis sp. nov. is the westernmost known record for the genus and the only species west of the Salween Basin. Its discovery echoes the increasing number of herpetological discoveries being made in upland regions fringing the Ayeyarwady and Salween Basins.
Examination of types and recently collected specimens revealed that Ansonia anotis Inger, Tan, and Yambun, 2001 and Pedostibes maculatus (Mocquard, 1890), both described from Kinabalu, Sabah, Malaysia, are hardly differentiated morphologically. Analyses of a total of 2,427 bp of the 12S rRNA, tRNA(val), and 16S mitochondrial rRNA genes revealed that the two species are very close genetically. Thus A. anotis is regarded as conspecific and is synonymized with P. maculatus. Genetically, this species proved to form a lineage distinct from other bufonids from Southeast Asia, including species of Ansonia and Pedostibes. Because the species has also some unique morphological traits different from known bufonid genera, we propose to establish a new genus for Nectophryne maculata Mocquard, 1890.
In order to elucidate the taxonomic status of the Fejervarya limnocharis complex relative to Malaysia and Japan populations, morphological observations and molecular phylogenetic analysis were carried out using three populations from Indonesia (type locality), Malaysia, and Japan. In addition, we conducted histological and spermatogenic observations using hybrids among these populations. Principal component and cluster analyses demonstrated that these populations could be clearly separated from one another. Abnormal testes were found in the hybrids between the Japan and Indonesia populations and between the Japan and Malaysia populations, but testes of the controls and hybrids between the Malaysia and Indonesia populations were quite normal. The mean number of univalents per cell was 5.42, 4.58, and 0.20 in hybrids between the Indonesia and Japan populations, Malaysia and Japan populations, and Indonesia and Malaysia populations, respectively. Sequence divergences in 16S rRNA and Cyt b genes were 0-0.4% (xbar=0.2%) and 0.3-1.5% (xbar=1.0%), respectively, between the Malaysia and Indonesia populations, and 2.4-2.6% (xbar=2.5%) and 11.0-12.0% (xbar=11.5%) between the Japan population and F. limnocharis complex, including the Malaysia and Indonesia populations and F. multistriata from China. This study indicated that the Malaysia population and F. multistriata from China should be designated as a subspecies of topotypic F. limnocharis, and that the Japan population should be regarded as a distinct species.
A new scleractinian coral species, Pachyseris inattesa sp. n., is described from the Red Sea. Despite a superficial resemblance with some species in the agariciid genus Leptoseris with which it has been previously confused, P. inattesa sp. n. has micro-morphological characters typical of the genus Pachyseris. This genus, once part of the Agariciidae, is comprised of five extant species and is widely distributed throughout the tropical Indo-Pacific. It is currently incertae sedis as a result of recent molecular analysis and appears to be closely related to the Euphylliidae. A molecular phylogenetic reconstruction including P. inattesa sp. n., the genus type species P. rugosa, and P. speciosa, all present in the Red Sea, was performed using the mitochondrial intergenic spacer between COI and 16S-rRNA. The results confirm that P. inattesa sp. n. is a monophyletic lineage closely related to the other Pachyseris species examined.
A wild-type, Gram-positive, rod-shaped, endospore-forming and motile bacteria has been isolated from palm oil mill sludge in Malaysia. Molecular identification using 16S rRNA gene sequence analysis indicated that the bacteria belonged to genus Paenibacillus. With 97 % similarity to P. alvei (AUG6), the isolate was designated as P. alvei AN5. An antimicrobial compound was extracted from P. alvei AN5-pelleted cells using 95 % methanol and was then lyophilized. Precipitates were re-suspended in phosphate buffered saline (PBS), producing an antimicrobial crude extract (ACE). The ACE showed antimicrobial activity against Salmonella enteritidis ATCC 13076, Escherichia coli ATCC 29522, Bacillus cereus ATCC 14579 and Lactobacillus plantarum ATCC 8014. By using SP-Sepharose cation exchange chromatography, Sephadex G-25 gel filtration and Tricine SDS-PAGE, the ACE was purified, which produced a ~2-kDa active band. SDS-PAGE and infrared (IR) spectroscopy indicated the proteinaceous nature of the antimicrobial compound in the ACE, and liquid chromatography electrospray ionization mass spectroscopy and de novo sequencing using an automatic, Q-TOF premier system detected a peptide with the amino acid sequence F-C-K-S-L-P-L-P-L-S-V-K (1,330.7789 Da). This novel peptide was designated as AN5-2. The antimicrobial peptide exhibited stability from pH 3 to 12 and maintained its activity after being heated to 90 °C. It also remained active after incubation with denaturants (urea, SDS and EDTA).
Disused tin-mining ponds make up a significant amount of water bodies in Malaysia particularly at the Kinta Valley in the state of Perak where tin-mining activities were the most extensive, and these abundantly available water sources are widely used in the field of aquaculture and agriculture. However, the natural ecology and physicochemical conditions of these ponds, many of which have been altered due to secondary post-mining activities, remains to be explored. As ammonia-oxidizing bacteria (AOB) are directly related to the nutrient cycles of aquatic environments and are useful bioindicators of environmental variations, the focus of this study was to identify AOBs associated with disused tin-mining ponds that have a history of different secondary activities in comparison to ponds which were left untouched and remained as part of the landscape. The 16S rDNA gene was used to detect AOBs in the sediment and water sampled from the three types of disused mining ponds, namely ponds without secondary activity, ponds that were used for lotus cultivation and post-aquaculture ponds. When the varying pond types were compared with the sequence and phylogenetic analysis of the AOB clone libraries, both Nitrosomonas and Nitrosospira-like AOB were detected though Nitrosospira spp. was seen to be the most ubiquitous AOB as it was present in all ponds types. However, AOBs were not detected in the sediments of idle ponds. Based on rarefaction analysis and diversity indices, the disused mining pond with lotus culture indicated the highest richness of AOBs. Canonical correspondence analysis indicated that among the physicochemical properties of the pond sites, TAN and nitrite were shown to be the main factors that influenced the community structure of AOBs in these disused tin-mining ponds.
The present study aimed to isolate actinobacteria from soil samples and characterized them using molecular tools and screened their secondary metabolites for antimicrobial activities. Thirty-nine strains from four different location of Barrientos Island, Antarctica using 12 types of isolation media was isolated. The isolates were preceded to screening of secondary metabolites for antimicrobial and antifungal activities. Using high-throughput screening methods, 38% (15/39) of isolates produced bioactive metabolites. Approximately 18% (7/39), 18% (7/39), 10% (4/39) and 2.5% (1/39) of isolates inhibited growth of Candida albicans ATCC 10231(T), Staphylococcus aurues ATCC 51650(T), methicillin-resistant Staphylococcus aurues (MRSA) ATCC BAA-44(T) and Pseudomonas aeruginosa ATCC 10145(T), respectively. Molecular characterization techniques like 16S rRNA analysis, Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), Random amplified polymorphic DNA (RAPD) and composite analyses were used to characterize the actinobacteria strains. Analysis of 16S rRNA sequences is still one of the most powerful methods to determine higher taxonomic relationships of Actinobacteria. Both RAPD and ERIC-PCR fingerprinting have shown good discriminatory capability but RAPD proved to be better in discriminatory power than ERIC-PCR. Our results demonstrated that composite analysis of both fingerprinting generally increased the discrimination ability and generated best clustering for actinobacteria strains in this study.
Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterial isolate codenamed π9 was selected for the cloning of the gene encoding triose phosphate isomerase (TIM), an enzyme in the glycolytic pathway. Based on 16S rRNA gene sequence analysis, this isolate was identified as a species of the genus Pseudomonas under the P. fluorescens group. The cloning of a 816 bp fragment of TIM gene which covers the 756 bp open reading frame was achieved by a combination of degenerate and splinkerette PCRs. The partial sequence of this gene was first PCR amplified by using degenerate primers and the flanking sequences were subsequently amplified by splinkerette PCR technique. Amino acid sequence of the cloned TIM was 97% identical to TIM from Pseudomonas fluorescens and shared 51% identity with the TIM from psychrophilic Vibrio sp. This work demonstrated the use of multiple PCR techniques to clone a gene without prior knowledge of its sequence. The cloning of the TIM gene by PCR was more rapid and cost effective compared to the traditional genomic library construction and screening method. Homology model of the TIM protein in this study was generated based on Escherichia coli TIM crystal structure. The model could serve as a hypothetical TIM structure from a psychrophilic microorganism for further investigation into areas that showed deviations from the known mesophilic TIM structures.
Banana is one of the most important fruits cultivated in Malaysia, and it provides many health benefits. However, bacterial wilt disease, which attacks bananas, inflicts major losses on the banana industry in Malaysia. To understand the complex interactions of the microbiota of bacterial wilt-diseased banana plants, we first determined the bacterial communities residing in the pseudostems of infected (symptomatic) and diseased-free (non-symptomatic) banana plants. We characterized the associated microorganisms using the targeted 16S rRNA metagenomics sequencing on the Illumina MiSeq platform. Taxonomic classifications revealed 17 and nine known bacterial phyla in the tissues of non-symptomatic and symptomatic plants, respectively. Cyanobacteria and Proteobacteria (accounted for more than 99% of the 16S rRNA gene fragments) were the two most abundant phyla in both plants. The five major genera found in both plant samples were Ralstonia, Sphingomonas, Methylobacterium, Flavobacterium, and Pseudomonas. Ralstonia was more abundant in symptomatic plant (59% out of the entire genera) as compared to those in the non-symptomatic plant (only 36%). Our data revealed that 102 bacterial genera were only assigned to the non-symptomatic plant. Overall, this study indicated that more diverse and abundant microbiota were associated with the non-symptomatic bacterial wilt-diseased banana plant as compared to the symptomatic plant. The higher diversity of endophytic microbiota in the non-symptomatic banana plant could be an indication of pathogen suppression which delayed or prevented the disease expression. This comparative study of the microbiota in the two plant conditions might provide caveats for potential biological control strategies.
Ganoderma boninense, the main causal agent of oil palm (Elaeis guineensis) basal stem rot (BSR), severely reduces oil palm yields around the world. To reduce reliance on fungicide applications to control BSR, we are investigating the efficacy of alternative control methods, such as the application of biological control agents. In this study, we used four Streptomyces-like actinomycetes (isolates AGA43, AGA48, AGA347 and AGA506) that had been isolated from the oil palm rhizosphere and screened for antagonism towards G. boninense in a previous study. The aim of this study was to characterize these four isolates and then to assess their ability to suppress BSR in oil palm seedlings when applied individually to the soil in a vermiculite powder formulation. Analysis of partial 16S rRNA gene sequences (512 bp) revealed that the isolates exhibited a very high level of sequence similarity (> 98%) with GenBank reference sequences. Isolates AGA347 and AGA506 showed 99% similarity with Streptomyces hygroscopicus subsp. hygroscopicus and Streptomyces ahygroscopicus, respectively. Isolates AGA43 and AGA48 also belonged to the Streptomyces genus. The most effective formulation, AGA347, reduced BSR in seedlings by 73.1%. Formulations using the known antifungal producer Streptomyces noursei, AGA043, AGA048 or AGA506 reduced BSR by 47.4, 30.1, 54.8 and 44.1%, respectively. This glasshouse trial indicates that these Streptomyces spp. show promise as potential biological control agents against Ganoderma in oil palm. Further investigations are needed to determine the mechanism of antagonism and to increase the shelf life of Streptomyces formulations.
Widespread and inadequate use of Monocrotophos has led to several environmental issues. Biodegradation is an ecofriendly method used for detoxification of toxic monocrotophos. In the present study, Msd2 bacterial strain was isolated from the cotton plant growing in contaminated sites of Sahiwal, Pakistan. Msd2 is capable of utilizing the monocrotophos (MCP) organophosphate pesticide as its sole carbon source for growth. Msd2 was identified as Brucella intermedia on the basis of morphology, biochemical characterization and 16S rRNA sequencing. B. intermedia showed tolerance of MCP up to 100 ppm. The presence of opd candidate gene for pesticide degradation, gives credence to B. intermedia as an effective bacterium to degrade MCP. Screening of the B. intermedia strain Msd2 for plant growth promoting activities revealed its ability to produce ammonia, exopolysaccharides, catalase, amylase and ACC-deaminase, and phosphorus, zinc and potassium solubilization. The optimization of the growth parameters (temperatures, shaking rpm, and pH level) of the MCP-degrading isolate was carried out in minimal salt broth supplemented with MCP. The optimal pH, temperature, and rpm for Msd2 growth were observed as pH 6, 35 °C, and 120 rpm, respectively. Based on optimization results, batch degradation experiment was performed. Biodegradation of MCP by B. intermedia was monitored using HPLC and recorded 78% degradation of MCP at 100 ppm concentration within 7 days of incubation. Degradation of MCP by Msd2 followed the first order reaction kinetics. Plant growth promoting and multi-stress tolerance ability of Msd2 was confirmed by molecular analysis. It is concluded that Brucella intermedia strain Msd2 could be beneficial as potential biological agent for an effective bioremediation for polluted environments.
The gut microbiome refers to the microorganism community living within the digestive tract. The environment plays a crucial role in shaping the gut microbiome composition of animals. The gut microbiome influences the health and behavior of animals, including the critically endangered Malayan tiger (Panthera tigris jacksoni). However, the gut microbiome composition of Malayan tigers, especially those living in their natural habitats, remains poorly understood. To address this knowledge gap, we used next-generation sequencing DNA metabarcoding techniques to analyze the gut microbiome of wild Malayan tigers using fecal samples collected from their natural habitats and in captivity. Our aim was to determine the gut microbiota composition of the Malayan tiger, considering the different types of habitat environments. The results revealed a diverse microbial community within the gut microbiome of Malayan tigers. The prominent phyla that were observed included Firmicutes, Proteobacteria, Actinobacteriota, Fusobacteriota and Bacteroidota. Beta diversity analysis revealed significant differences in gut microbiome composition of Malayan tigers that inhabited oil palm plantations, in villages and protected areas. Diversity analysis also revealed significant difference in the gut microbiome between wild and captive Malayan tigers. However, the distinctions of gut microbiome between wild and captive alpha diversity did not yield significant differences. The differences in microbiome diversity resulted from the interplay of dietary intake and environmental factors. This information will facilitate the establishment of focused conservation approaches and enhance our understanding of the effect of microbiome composition on Malayan tiger health.
Intensive aeration for nitrification is a major energy consumer in sewage treatment plants (STPs). Low-dissolved-oxygen (low-DO) nitrification has the potential to lower the aeration demand. However, the applicability of low-DO nitrification in the tropical climate is not well-understood. In this study, the potential of low-DO nitrification in tropical setting was first examined using batch kinetic experiments. Subsequently, the performance of low-DO nitrification was investigated in a laboratory-scale sequential batch reactor (SBR) for 42 days using real tropical sewage. The batch kinetic experiments showed that the seed sludge has a relatively high oxygen affinity. Thus, the rate of nitrification was not significantly reduced at low DO concentrations (0.5 mg/L). During the operation of the low-DO nitrification SBR, 90% of NH4-N was removed. The active low-DO nitrification was mainly attributed to the limited biodegradable organics in the sewage. Fluorescence in-situ hybridisation and 16S rRNA amplicon sequencing revealed the nitrifiers were related to Nitrospira genus and Nitrosomonadaceae family. Phylogenetic analysis suggests 47% of the operational taxonomic units in Nitrospira genus are closely related to a comammox bacteria. This study has demonstrated active low-DO nitrification in tropical setting, which is a more sustainable process that could significantly reduce the energy footprint of STPs.
The applicability of the enhanced biological phosphorus removal (EBPR) process for the removal of phosphorus in warm climates is uncertain due to frequent reports of EBPR deterioration at temperature higher than 25 °C. Nevertheless, a recent report on a stable and efficient EBPR process at 28 °C has inspired the present study to examine the performance of EBPR at 24 °C-32 °C, as well as the PAOs and GAOs involved, in greater detail. Two sequencing batch reactors (SBRs) were operated for EBPR in parallel at different temperatures, i.e., SBR-1 at 28 °C and SBR-2 first at 24 °C and subsequently at 32 °C. Both SBRs exhibited high phosphorus removal efficiencies at all three temperatures and produced effluents with phosphorus concentrations less than 1.0 mg/L during the steady state of reactor operation. Real-time quantitative polymerase chain reaction (qPCR) revealed Accumulibacter-PAOs comprised 64% of the total bacterial population at 24 °C, 43% at 28 °C and 19% at 32 °C. Based on fluorescent in situ hybridisation (FISH), the abundance of Competibacter-GAOs at both 24 °C and 28 °C was rather low (<10%), while it accounted for 40% of the total bacterial population at 32 °C. However, the smaller Accumulibacter population and larger population of Competibacter at 32 °C did not deteriorate the phosphorus removal performance. A polyphosphate kinase 1 (ppk1)-based qPCR analysis on all studied EBPR processes detected only Accumulibacter clade IIF. The Accumulibacter population shown by 16S rRNA and ppk1 was not significantly different. This finding confirmed the existence of single clade IIF in the processes and the specificity of the clade IIF primer sets designed in this study. Habitat filtering related to temperature could have contributed to the presence of a unique clade. The clade IIF was hypothesised to be able to perform the EBPR activity at high temperatures. The clade's robustness most likely helps it to fit the high-temperature EBPR sludge best and allows it not only to outcompete other Accumulibacter clades but coexist with GAOs without compromising EBPR activity.