This study aimed to determine phylogenetic relationship between and among selected species of sea cucumbers (Echinodermata: Holothuroidea) using 16S mitochondrial ribosomal RNA (rRNA) gene. Phylogenetic analyses of 37 partial sequences of 16S mitochondrial rRNA gene using three main methods namely neighbour joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) showed the presence of five main genera of sea cucumbers: Molpadia from order Molpadiida and four genera of order Aspidochirotida namely Holothuria, Stichopus, Bohadschia and Actinopyga. All of the 17 species obtained from Malaysia distributed among the main genera except within Actinopyga. Interestingly, Holothuria excellens was out of Holothuria group causing Holothuria to be paraphyletic. High bootstrap value and consistent clustering made Molpadia, Stichopus, Bohadschia and Actinopyga monophyletic. The relationship of Actinopyga with the other genera was unclarified and Stichopus was sister to Molpadia. The latter finding caused the resolution at order level unclear. The pairwise genetic distance calculated using Kimura 2-parameter model further supported and verified findings from the phylogenetic trees. Further studies with more samples and different mitochondrial DNA genes need to be done to get a better view and verification on the molecular phylogeny of sea cucumbers.
Staphylococcus kloosii, an orange pigment-producing bacterium, was isolated from the respiratory tree of Holothuria (Mertensiothuria) leucospilota (Brandt 1835) from Teluk Nipah, Pangkor Island, Perak, Malaysia. This report is the first documentation of this Gram-positive strain, referred to as Strain 68 in Malaysia. A partial 16S ribosomal RNA gene sequence of the mesophilic strain has been registered with GenBank (National Center for Biotechnology Information, US National Library of Medicine) with accession number JX102547. Phylogenetic analysis using the neighbour-joining method further supported the identification of Strain 68 as S. kloosii. The circular strain produced orange pigments on tryptone glucose yeast extract agar (TGYEA) and in nutrient broth (NB) at approximately pH 7. The visible spectra of ethanolic and methanolic pigment extracts of the bacterial strain were considered identical with λmax at 426, 447 and 475 nm and λmax at 426, 445 and 473 nm, respectively. Both visible spectra resemble the visible spectra of lutein, which is a commercial carotenoid; however, further analyses are required to confirm the identity of this pigment. The methanolic extracts of the intracellular pigments comprised at least three pigment compounds: an orange pigment compound (major compound), a yellow pigment compound (the least polar) and a pink pigment compound (the most polar). These findings are the first documentation of the pigment composition of S. kloosii as no such record could be found to date.
This preliminary study aimed to isolate and identify microbes that inhabit the coelomic fluid of two local species of sea cucumbers collected from Malaysian waters - a ‘gamat’ species i.e. Stichopus chloronotus Brandt, 1835 and the most abundant ‘timun laut’ species in Malaysia i.e. Holothuria (Mertensiothuria) leucospilota (Brandt 1835). Phylogenetic analyses of partial 16S rRNA mtDNA gene sequences suggested the presence of at least eight microbial genera i.e. five bacterial genera - Bacillus, Exiguobacterium, Pseudomonas, Stenotrophomonas and Vibrio - isolated from the coelomic fluid of H. leucospilota and three genera of gram-positive bacteria from the Micrococcaceae family - Kytococcus, Micrococcus and either Kocuria or Rothia - isolated from the coelomic fluid of S. chloronotus. We speculate that less diverse microbial population in S. chloronotus as compared to H. leucospilota could be due to a number of environmental factors e.g. penetration of light surrounding the habitats of both species, the feeding behaviour of H. leucospilota and the higher level of antimicrobial properties of coelomic fluid in S. chloronotus. In terms of antimicrobial-resistance capability test, an isolate from genus Pseudomonas that is suspected to be P. alcaligenes exhibited high resistance towards streptomycin. Another isolate from genus Stenotrophomonas that was suspected to be S. maltophilia showed moderate resistance towards streptomycin and lower resistance towards kanamycin. Both isolates were from the coelomic fluid of H. leucospilota. Tetracycline inhibited the growth of all bacterial isolates tested. Further studies with more specimens of S. choronotus and H. leucospilota from broader geographical locations and the use of complete mtDNA genes along with morphological approaches for species identification may facilitate to provide better insights into the microbial population in the coelomic fluid of both local sea cucumber species.
The high popularity of a commercial sea cucumber species on Pangkor Island (i.e. Stichopus horrens) has resulted in a lack of studies on Holothuria (Mertensiothuria) leucospilota from Pangkor Island, Perak, Malaysia. S. horrens is the main ingredient of gamat-based traditional medicine on the resort island, while H. leucospilota is the most dominant species. For that reason, this study aimed to isolate ossicles from a specimen of morphospecies H. leucospilota collected from Teluk Nipah Beach, Pangkor Island; and to determine the shapes of the ossicles for species verification. A number of six ossicle shapes were observed. Beside buttons and tables as the common ossicle shapes; I-shaped rod, long I-shaped rod, branched rod, and spider-shaped rod were also documented. Nonetheless, the spider-shaped rod is an unusual ossicle shape in H. leucospilota as there was no previous record on it, thus it could be considered as a new finding. In summary, the results of this research indicate the uniqueness of the specimen of morphospecies H. leucospilota as a result of the presence of the spider-shaped rod. More studies with more samples, more morphological approaches as well as more molecular techniques need to be incorporated in future for a better insight.
Gynura procumbens or longevity spinach plant is a type of medicinal plant that
distributes widely in Southeast Asia including Malaysia. G. procumbens is
commonly called as pokok Sambung Nyawa, Sabong Nyawa, Akar Sebiak, or
Kecam Akar by Malaysians; and often consumed as ulam or salad. Thus, there
might be several microorganisms particularly bacteria that still inhabiting the
leaves. Intensive studies on the therapeutic potential of G. procumbens have
indeed been carried out over time. However, studies focusing on the link between
medicinal plants and bacteria that inhabit the plant specifically epiphytic bacteria
on the leaf surface have yet to be fully appreciated. Besides, only one available
study on the diversity of bacteria inhabiting the phyllosphere was available
showing the lack of related studies. Thus, this study aimed to identify epiphytic
bacteria of G. procumbens leaves based on morphology through Gram-staining
and genetics using 16S ribosomal RNA gene sequencing; and to determine the
possible contribution of the bacteria to the medicinal properties of G. procumbens
by further readings. The findings from this research suggested the presence of
five species of bacteria i.e. Bacillus subtilis, Bacillus amyloliquefaciens, Pantoea
agglomerans, Sphingomonas melonis, and Burkholderia cepacia. From further
readings, it is known that some of the bacterial species have the potentials towards
medicinal applications. Also, it is hypothesised that the production of some
secondary metabolites in the plant might be due to the presence of the bacteria.
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.
Pigment-producing microorganisms have become a main focus due to the search
of food-grade microbial pigments for natural food colourant industry. Therefore,
this study aimed to isolate and genetically identify bacteria including pigmentproducing
bacterial strains associated with the tentacles of Holothuria
(Lessonothuria) pardalis (Selenka 1867) from Pulau Tinggi, Johor.
Morphological characteristics of bacteria based on Gram staining were observed
using a digital compound microscope. Partial 16S ribosomal RNA (rRNA) gene
sequencing was also incorporated to support the morphological approach. A
number of six partial gene sequences of non-protein coding 16S rRNA were
obtained and the Basic Local Alignment Search Tool (BLAST) analysis
suggested the presence of three known bacteria species from the genera of
Pseudomonas, Chryseobacterium and Klebsiella. The neighbourhood-joining
method and Maximum Likelihood method used in the phylogenetic analysis
supported the identification and classification of the bacteria species. Among the
bacterial isolates, only strain B14 identified as Chryseobacterium sp. produced
pigment i.e. orange pigment. The highest point of cell density of
Chryseobacterium sp. was recorded at 16 hours. However, further analysis is
needed to confirm the pigment characterisation and the potential of this pigment
to be exploited as natural colourant in the food industry.
Holothuria (Mertensiothuria) leucospilota (Brandt 1835), white threads fish or locally known as bat puntil is currently considered as the most abundant sea cucumber species in Malaysia. This study aimed to generate the genetic profile of H. leucospilota from Malaysia and then to determine the phylogenetic relationship between H. leucospilota and other members of genus Holothuria using partial sequences of cytochrome c oxidase I (COI) mitochondrial DNA (mtDNA) gene. In this study, specimens of H. leucospilota were collected from Intan Besar Island, Langkawi, Kedah Darul Aman in the west coast of Peninsular Malaysia. Three main methods namely neighbour joining, maximum parsimony and maximum likelihood were used for the phylogenetic tree reconstruction. Tree topologies showed that H. leucospilota has its own monophyletic clade clearly distinct from the other species. The pairwise genetic distance calculated further supported these findings. In addition, the results also should that the COI mtDNA gene is capable to unravel the phylogenetic relationship of H. leucospilota.
Nursyamfarhani Akashah, Siti Najihah Solehin, Marsheilla Lia Janing, Nur Aina Yusra Manap, Nor Shahida Ab Rahman, Muhammad Abdul Latiff Abu Bakar, et al.
Sea cucumber or locally known as gamat and timun laut is a well-known attraction
of Pulau Langkawi, Kedah, Malaysia. Not only limited to Pulau Langkawi, sea
cucumber can also be found in other coastal water areas of Malaysia. However,
the species richness and genetic diversity of sea cucumber in Pulau Tinggi, Johor
is still unknown to the marine scientific communities, especially for Holothuria
(Mertensiothuria) leucospilota, the most dominant species in Malaysia.
Therefore, this study aimed to generate DNA barcodes of H. leucospilota from
Pulau Tinggi. Protein-coding cytochrome c oxidase 1 (CO1) mtDNA gene was
used as it is commonly and widely used for molecular species identification via
DNA barcoding. Polymerase chain reaction (PCR) and DNA sequencing were
incorporated to generate the DNA barcodes of CO1 mtDNA gene. The BLAST
program confirmed the species status of the sea cucumber specimens as H.
leuscospilota or locally known as bat puntil or white threads fish. The application
for GenBank, NCBI, U.S. National Library of Medicine registration was
successfully accepted on October 17, 2019 with accession numbers of MN580537
and MN580538). The phylogenetic trees i.e neighbor joining tree (NJ, distancebased
method), maximum parsimony tree (MP, character-based method) and
maximum likehood tree (ML, character-based method) reconstructed using
MEGA X software version 10.0.5 (BETA) further supported the species status of
the sea cucumber specimens from Pulau Tinggi as H. leuscospilota. This is the
first record of CO1 mtDNA barcodes of H. leuscospilota from Pulau Tinggi,
Johor, Malaysia.