Species identification and molecular phylogenetics of processed sea cucumbers from Malaysian market based on 12s mitochondrial rRNA gene

Extensive processing of sea cucumber causes body deformation of the marine organism, hence causing difficulties in species identification of processed sea cucumbers. Due to the copresence of cases of unlabelled or mislabelled sea cucumber products in Malaysian markets and worldwide, a study was conducted to determine the species identities of processed sea cucumbers from selected Malaysian markets using non-protein-coding 12S mitochondrial rRNA gene. Phylogenetic analyses based on the distance-based Neighbour Joining method, and the character-based methods i.e. the Maximum Parsimony method, Maximum Likelihood method, and the Bayesian Analysis method of 81 ingroup sequences representing 63 processed sea cucumber specimens, 13 fresh and processed reference samples for species identification, and five fresh additional specimens from Teluk Nipah Beach, Pangkor Archipelago and Manukan Island, Sabah suggested the presence of three main clusters i.e. a gamat family cluster consisting of family Stichopodidae (Stichopus horrens, Stichopus vastus, and Thelenota anax) and two clusters of timun laut family comprising family Holothuriidae (Holothuria (Mertensiothuria) leucospilota, Holothuria (Metriatyla)scabra, Holothuria (Metriatyla) lessoni, Holothuria (Halodeima) atra, and Holothuria (Halodeima) edulis) and family Caudinidae (Acaudina molpadioides). The outcomes of this study also highlighted the availability of 40 new 12S mitochondrial rRNA gene sequences deposited in the GenBank that can be utilised by the enforcement agencies to monitor and overcome the issues of species substitution and product mislabelling of processed sea cucumber products in Malaysian markets

Species identification of processed sea cucumbers from Malaysian market based on concatenated gene sequences of mitochondrial rRNA genes

Species identification of sea cucumbers that have undergone body deformation due to extensive food processing e.g. beche-de-mer is difficult especially with the copresence of cases of unlabelled or mislabelled sea cucumber-based products in the markets. Therefore, a study was done to determine the species identities of processed sea cucumbers from selected Malaysian markets using concatenated gene sequences of non-protein-coding 12S and 16S mitochondrial rRNA genes. Phylogenetic analyses based on the distance-based Neighbour Joining method, and the character-based methods i.e. the Maximum Parsimony method, Maximum Likelihood method, and the Bayesian Analysis method of 47 ingroup sequences representing 37 processed sea cucumber specimens, 6 reference samples, and 4 additional specimens suggested the presence of 3 main clusters i.e. gamat family consisting of genus Stichopus and genus Thelenota; and timun laut family comprising family Holothuriidae. A number of 3 gamat species i.e. Stichopus horrens, Stichopus vastus, and Thelenota anax were recorded. Meanwhile, the specimens of Holothuria (Halodeima) atra, Holothuria (Halodeima) edulis, Holothuria (Metriatyla) lessoni, Holothuria (Mertensiothuria) leucospilota, and Holothuria (Metriatyla) scabra were the 5 timun laut species that grouped under the family Holothuriidae. The outcomes of this study can be utilised by the enforcement agencies to monitor and overcome the issues of species substitution and product mislabelling of processed sea cucumber products in Malaysian markets

Phylogenetic relationships within the genus holothuria inferred from 16s mitochondiral rRNA gene sequences

This study aimed to resolve the taxonomic status of a morphologically undetermined sea cucumber species of order Apodida from Malaysia (GenBank accession no.: FJ223867) using partial 16S mitochondrial rRNA gene sequences and subsequently to determine the validity of morphological taxonomy of Holothuria species into its current subgenera. The undetermined species clustered with all taxa of Holothuria in previous study. Phylogenetic analyses using maximum parsimony and Bayesian methods suggest that the undetermined species was genetically closer to Holothuria (Lessonothuria) pardalis and Holothuria (Acanthotrapeza) coluber; and its position in both phylogenetic trees further suggests its status as a Holothuria taxon. Subgenera of Holothuria, Merthensiothuria and Metriatyla are monophyletic with strong bootstrap supports and posterior probabilities of clades, thus strengthening their morphological taxonomies. Nonetheless, the non-monophyly of subgenera of Halodeima, Microthele and Platyperona suggests a requirement for their taxonomic revisions using integrative taxonomy. The status of Holothuria (Halodeima) edulis subgroups in the maximum parsimony and Bayesian trees is indistinct and further taxonomic revisions are necessary. In terms of sister relationship, both phylogenetic trees suggest that subgenus Holothuria is a sister taxon of subgenus Roweothuria while the other sister relationships were unclear due to the undetermined species, paraphyly and polyphyly of a number of subgenera. Further studies with more specimens of genus Holothuria from broader geographical locations and various mtDNA genes along with morphological approaches may facilitate to provide better insights into the molecular phylogeny of subgenera of Holothuria

Addressing GHG emissions from land transport in a developing country

The number of motor vehicles in Malaysia is growing at a significant rate, from around 15 million in 2005 to around 25 million in 2014. Based on the National GHG Inventory as reported to UNFCC, as a whole the transport sector has consistently remained the second largest GHG emitting sector in the country, accounting for 20% of the country’s total GHG emissions in 2014 of which about 18% comes from road transportation. In this paper, a possible approach in addressing the GHG emissions from the land transport sector is presented. The avoid-shiftimprove strategy is employed to determine the possible measures to deal with mitigating the GHG emissions. Computation of GHG emissions for 2014 revealed that car is the vehicle with the biggest contribution, due to its large numbers and also VKT. Motorcycles, on the other hand, have relatively lower GHG contribution despite its huge numbers, while goods vehicle have significant GHG contribution despite its small number of vehicles. Public transport (both rail and bus) can significantly reduce GHG emission for each passenger-kilometer compared to other alternatives. Opting for smaller vehicle can also reduce GHG emissions. Another potential solution towards lower GHG emission is adoption of electric mobility and alternative fuel

Phylogenetic relationships within the Genus Holothuria inferred from 16S Mitochondiral rRNA Gene Sequences

This study aimed to resolve the taxonomic status of a morphologically undetermined sea cucumber species of order Apodida from Malaysia (GenBank accession no.: FJ223867) using partial 16S mitochondrial rRNA gene sequences and subsequently to determine the validity of morphological taxonomy of Holothuria species into its current subgenera. The undetermined species clustered with all taxa of Holothuria in previous study. Phylogenetic analyses using maximum parsimony and Bayesian methods suggest that the undetermined species was genetically closer to Holothuria (Lessonothuria) pardalis and Holothuria (Acanthotrapeza) coluber; and its position in both phylogenetic trees further suggests its status as a Holothuria taxon. Subgenera of Holothuria, Merthensiothuria and Metriatyla are monophyletic with strong bootstrap supports and posterior probabilities of clades, thus strengthening their morphological taxonomies. Nonetheless, the non-monophyly of subgenera of Halodeima, Microthele and Platyperona suggests a requirement for their taxonomic revisions using integrative taxonomy. The status of Holothuria (Halodeima) edulis subgroups in the maximum parsimony and Bayesian trees is indistinct and further taxonomic revisions are necessary. In terms of sister relationship, both phylogenetic trees suggest that subgenus Holothuria is a sister taxon of subgenus Roweothuria while the other sister relationships were unclear due to the undetermined species, paraphyly and polyphyly of a number of subgenera. Further studies with more specimens of genus Holothuria from broader geographical locations and various mtDNA genes along with morphological approaches may facilitate to provide better insights into the molecular phylogeny of subgenera of Holothuria

16S rRNA Barcoding technique for species identification of processed sea cucumbers from selected Malaysian markets

In food industry, wide-ranging processing of sea cucumbers (Phylum Echinodermata: Class Holothuroidea) including gutting, boiling, roasting, and subsequent preservation procedures are frequently needed prior to marketing. These processes cause body deformation of the sea cucumbers, thus leading to difficulties in species identification and confirmation of the processed sea cucumbers or beche-de-mer. Furthermore, beche-de-mer products in Malaysian markets are often unlabelled or mislabelled. Economic fraud, health hazards, and illegal trade of protected species are the potential major consequences of the issues. Therefore, a reliable, reproducible, and rapid technique for species identification is required. For that reason, this study was conducted to determine species identity of 25 beche-de-mer specimens that were not tagged with species details from four selected Malaysian markets. Five reference samples were also included in the analyses consisting of fresh samples that were morphologically identified as Stichopus horrens and Holothuria (Mertensiothuria) leucospilota from Pangkor Island, Perak, Malaysia. Phylogenetic analyses of 30 partial sequences of non-protein-coding 16S mitochondrial ribosomal RNA (rRNA) gene using five main methods i.e. Neighbour-Joining (NJ), Maximum Likelihood (ML), Minimum Evolution (ME), Unweighted Pair Group Method with Arithmetic Mean (UPGMA), and Maximum Parsimony (MP) showed the presence of two main clusters of the beche-demer specimens: Stichopodidae (gamat family) and Holothuriidae (timun laut family). The beche-de-mer specimens of S. horrens, Stichopus herrmanni, and Thelenota anax were the three gamat species that clustered under the family Stichopodidae. Meanwhile, the beche-de-mer specimens of H. leucospilota, Holothuria (Halodeima) edulis, Holothuria (Metriatyla) scabra, and H. scabra var. versicolor were the four timun laut species that clustered under the family Holothuriidae. In fact, the outcomes of this study suggested the potential of 16S mitochondrial rRNA gene sequencing technique to be used by the enforcement agencies in monitoring and overcoming the issues of species substitution and product mislabeling of beche-de-mer products in Malaysian markets

16S rRNA barcoding technique for species identification of processed sea cucumbers from selected Malaysian markets

In food industry, wide-ranging processing of sea cucumbers (Phylum Echinodermata: Class Holothuroidea) including gutting, boiling, roasting, and subsequent preservation procedures are frequently needed prior to marketing. These processes cause body deformation of the sea cucumbers, thus leading to difficulties in species identification and confirmation of the processed sea cucumbers or beche-de-mer. Furthermore, beche-de-mer products in Malaysian markets are often unlabelled or mislabelled. Economic fraud, health hazards, and illegal trade of protected species are the potential major consequences of the issues. Therefore, a reliable, reproducible, and rapid technique for species identification is required. For that reason, this study was conducted to determine species identity of 25 beche-de-mer specimens that were not tagged with species details from four selected Malaysian markets. Five reference samples were also included in the analyses consisting of fresh samples that were morphologically identified as Stichopus horrens and Holothuria (Mertensiothuria) leucospilota from Pangkor Island, Perak, Malaysia. Phylogenetic analyses of 30 partial sequences of non-protein-coding 16S mitochondrial ribosomal RNA (rRNA) gene using five main methods i.e. Neighbour-Joining (NJ), Maximum Likelihood (ML), Minimum Evolution (ME), Unweighted Pair Group Method with Arithmetic Mean (UPGMA), and Maximum Parsimony (MP) showed the presence of two main clusters of the beche-demer specimens: Stichopodidae (gamat family) and Holothuriidae (timun laut family). The beche-de-mer specimens of S. horrens, Stichopus herrmanni, and Thelenota anax were the three gamat species that clustered under the family Stichopodidae. Meanwhile, the beche-de-mer specimens of H. leucospilota, Holothuria (Halodeima) edulis, Holothuria (Metriatyla) scabra, and H. scabra var. versicolor were the four timun laut species that clustered under the family Holothuriidae. In fact, the outcomes of this study suggested the potential of 16S mitochondrial rRNA gene sequencing technique to be used by the enforcement agencies in monitoring and overcoming the issues of species substitution and product mislabeling of beche-de-mer products in Malaysian markets

Morphological and molecular identification of Holothuria (Merthensiothuria) leucospilota and Stichopus horrens from Pangkor Island, Malaysia

Kajian awal ini bertujuan untuk mengenalpasti satu spesies komersil gamat iaitu Stichopus horrens Selenka, 1867 dan satu spesies timun laut iaitu Holothuria (Mertensiothuria) leucospilota (Brandt, 1835) dari Pulau Pangkor, Perak, Malaysia dengan mengaplikasikan teknik-teknik morfologi berdasarkan bentuk-bentuk osikel dan teknik-teknik molekul menggunakan gen sitokrom c osidasi I (COI) mitokondria DNA (mtDNA). Di Malaysia, gamat didefinisikan sebagai spesies timun laut daripada famili Stichopodidae yang memiliki nilai-nilai perubatan manakala timun laut dirujukkan kepada spesies bukan gamat. Spesies S. horrens adalah amat popular di Pulau Pangkor sebagai bahan utama dalam penghasilan air gamat dan minyak gamat secara tradisional manakala H. leucospilota merupakan spesies paling dominan di Malaysia. Berbeza dengan kajian-kajian sebelum ini, bahagian dalaman tubuh iaitu pohon respirasi dan gastrousus telah disertakan dalam kajian ini untuk mendapatkan kesimpulan yang lebih baik berdasarkan morfologi. Keputusan-keputusan menunjukkan tiada osikel telah hadir di dalam gastrousus H. leucospilota dan ciri tersebut dicadangkan sebagai penanda diagnostik yang unik untuk spesies timun laut tersebut. Di samping itu, kehadiran rod berbentuk Y di dalam pohon respirasi S. horrens seterusnya menyokong potensi bahagian dalaman tubuh untuk mengenalpasti spesies gamat tersebut. Selanjutnya, analisis-analisis filogenetik gen COI mtDNA spesimen-spesimen timun laut tersebut menggunakan kaedah hubungkait jiran dan kaedah persamaan maksimum seterusnya mengesahkan status spesies H. leucospilota dan S. horrens dari Pulau Pangkor, Perak, Malaysia. Jujukan-jujukan gen COI mtDNA tersebut telah didaftarkan dengan GenBank, National Center for Biotechnology Information (NCBI), US National Library of Medicine (no. akses GenBank: KC405565-KC405568). Walaupun lebih banyak spesimen dari pelbagai lokasi diperlukan untuk menghasilkan keputusan-keputusan muktamad yang lebih baik, penemuan-penemuan semasa telah memberi gambaran yang lebih baik tentang kepentingan pendekatan-pendekatan yang saling lengkap-melengkapi iaitu teknik-teknik morfologi dan molekul dalam pengenalpastian kedua-dua spesies timun laut Malaysia tersebut