Development of the muscular and nervous systems during the larval ontogeny of the stalked barnacle, Octolasmis angulata Aurivillius 1894 (Cirripedia: Thoracicalcerea: Poecilasmatidae).

The advancements in microscopic techniques have stimulated great interest in the muscular and neural architectures of invertebrates, specifically using muscle and neural structures to infer phylogenetic relationships. Here, we provide the data on the development of the muscular and nervous systems during the larval development of stalked barnacle, Octolasmis angulata using the phalloidin F-actin and immunohistochemical labelling (e.g. acetylated α-tubulin and serotonin) and confocal laser scanning microscopy analysis. All naupliar stages shared the same muscle and neural architectures with only the discrepancy in size. The nauplii have a complex muscle arrangement in their feeding apparatus and naupliar appendages. Most naupliar muscles undergo histolyse during the cyprid metamorphosis. The cyprid muscles form beneath the head shield at the end of nauplius VI. The naupliar and cyprid central nervous systems exhibit the typical tripartite brain comprising the protocerebrum, deutocerebrum and tritocerebrum. The serotonin-like immunoreactivity is mainly found in the naupliar brain, mandibular ganglia, cyprid brain and posterior ganglia. Our study revealed that numerous muscle and neural architectures in the naupliar and cyprids have phylogenetic significance, but future studies on the myoanatomy and neuroanatomy of other barnacle species are necessary to determine the homology of these structures.

Using DNA Barcodes to Aid the Identification of Larval Fishes in Tropical Estuarine Waters (Malacca Straits, Malaysia).

Larval descriptions of tropical marine and coastal fishes are very few, and this taxonomic problem is further exacerbated by the high diversity of fish species in these waters. Nonetheless, accurate larval identification in ecological and early life history studies of larval fishes is crucial for fishery management and habitat protection. The present study aimed to evaluate the usefulness of DNA barcodes to support larval fish identification since conventional dichotomous keys based on morphological traits are not efficient due to the lack of larval traits and the rapid morphological changes during ontogeny. Our molecular analysis uncovered a total of 48 taxa (21 families) from the larval samples collected from the Klang Strait waters encompassing both spawning and nursery grounds of marine and estuarine fishes. Thirty-two (67%) of the larval taxa were identified at the species level, two taxa (4%) at the genus level, and 14 taxa (29%) at family level. The relatively low rate of species-level identification is not necessarily due to the DNA barcoding method per se, but a general lack of reference sequences for speciose and non- commercial fish families such as Gobiidae, Blenniidae, and Callionymidae. Larval morphology remains important in species diagnoses when molecular matches are ambiguous. A lower ethanol percentage (50%) for larva preservation is also useful to keep the body of larvae intact for morphological identification, and to preserve DNA for subsequent molecular analyses. The 10% Chelex resin used to extract DNA is also cost- effective for long term monitoring of larval fishes. Hence, the DNA barcoding method is an effective and easy way to aid the identification of estuarine larval fishes at the species level.

Heat shock response and metabolic stress in the tropical estuarine copepod Pseudodiaptomus annandalei converge at its upper thermal optimum.

Heat shock response (HSR), in terms of transcription regulation of two heat shock proteins genes hsp70 and hsp90), was analysed in a widespread tropical copepod Pseudodiaptomus annandalei. The mRNA transcripts of both genes were quantified after copepods at a salinity of 20 underwent an acclimation process involving an initial acclimation temperature of 29 °C, followed by gradual thermal ramping to the target exposure temperature range of 24-36 °C. The respective cellular HSR and organismal metabolism, measured by respiratory activity at exposure temperatures, were compared. The fold change in mRNA expression for both hsp70 and hsp90 (8-9 fold) peaks at 32 °C, which is very close to 32.4 °C, the upper thermal optimum for respiration in the species. Unexpectedly, the modelled HSR curves peak at only 3 °C (hsp90) and 3.5 °C (hsp70) above the mean water temperature (29.32 °C) of the copepod in the field. We propose that copepods in tropical waters adopt a preparative HSR strategy, early at the upper limit of its thermal optimum, due to the narrow thermal range of its habitat thus precluding substantial energy demand at higher temperatures. However, the model suggests that the species could survive to at least 36 °C with short acclimation time. Nevertheless, the significant overlap between its thermal range of hsp synthesis and the narrow temperature range of its habitat also suggests that any unprecedented rise in sea temperature would have a detrimental effect on the species.

First evidence for temporary and permanent adhesive systems in the stalked barnacle cyprid, Octolasmis angulata.

Although there have been extensive studies on the larval adhesion of acorn barnacles over the past few decades, little is known about stalked barnacles. For the first time, we describe the larval adhesive systems in the stalked barnacle, Octolasmis angulata and the findings differ from previous reports of the temporary (antennulary) and cement glands in thoracican barnacles. We have found that the temporary adhesives of cyprid are produced by the clustered temporary adhesive glands located within the mantle, instead of the specialised hypodermal glands in the second antennular segment as reported in the acorn barnacles. The temporary adhesive secretory vesicles (TASV) are released from the gland cells into the antennule via the neck extensions of the glands, and surrounded with microtubules in the attachment disc. Cement glands undergo a morphological transition as the cyprid grows. Synthesis of the permanent adhesives only occurs during the early cyprid stage, and is terminated once the cement glands reach maximum size. Evidence of the epithelial invaginations on the cement glands supports the involvement of exocytosis in the secretion of the permanent adhesives. This study provides new insight into the larval adhesives system of thoracican barnacles.

Response of marine copepods to a changing tropical environment: winners, losers and implications.

Background. Climate change concurrent with anthropogenic disturbances can initiate serial changes that reverberate up the food chain with repercussions for fisheries. To date, there is no information available concerning the combined effects of global warming and human impacts on tropical marine food webs. While temperate copepods respond differently to warming and environmental stressors, the extent to which tropical copepods can adapt to rising temperature of already warm waters remains unknown. We hypothesize that sea warming and other anthropogenic disturbances over the long term will have the greatest impact on the copepod community in nearshore waters where their effects are accentuated, and therefore vulnerable and resilient species could be identified. Methods. Zooplankton samples were collected during two time periods (1985-86 and 2014-15) interposed by marked anthropogenic disturbances, and at the same five stations located progressively from inshore to offshore in Klang Strait, Malaysia, following the asymmetrical before-after-control-impact (BACI) design. Copepods were identified to species, and results were interpreted by univariate (ANOVA) and multivariate (PERMANOVA, PCO) analyses of the computed species abundance and diversity measures. Results. Copepod total abundance was not significantly different among stations but higher after disturbance than before disturbance. However, changes in the abundance of particular species and the community structure between time periods were dramatic. Coastal large-bodied calanoid species (e.g., Acartia spinicauda, Calanopia thompsoni, Pseudodiaptomus bowmani and Tortanus forcipatus) were the most vulnerable group to disturbance. This however favored the opportunistic species (e.g., Oithona simplex, O. attenuata, Hemicyclops sp., Pseudomacrochiron sp. and Microsetella norvegica). Small-bodied copepods (e.g., Paracalanus sp., Parvocalanus crassirostris and Euterpina acutifrons) were unaffected. Centropages tenuiremis was likely an introduced species. There was no significant loss in species richness of copepods despite the dramatic changes in community structure. Discussion. Sea warming and other human-induced effects such as eutrophication, acidification and coastal habitat degradation are likely the main factors that have altered copepod community structure. The large-bodied estuarine and coastal calanoid copepods are surmised to be vulnerable to eutrophication and hypoxia, while both resilient and opportunistic species are largely unaffected by, or adaptable to, degraded coastal environments and observed sea surface temperature (SST) rise. It is forecasted that SST rise with unmitigated anthropogenic impacts will further reduce large-bodied copepod species the favoured food for fish larvae with dire consequences for coastal fish production.

Isolation and Characterization of Aquatic-Borne Klebsiella pneumoniae from Tropical Estuaries in Malaysia.

Klebsiella pneumoniae is an opportunistic pathogen that is responsible for causing nosocomial and community-acquired infections. Despite its common presence in soil and aquatic environments, the virulence potential of K. pneumoniae isolates of environmental origin is largely unknown. Hence, in this study, K. pneumoniae isolated from the estuarine waters and sediments of the Matang mangrove estuary were screened for potential virulence characteristics: antibiotic susceptibility, morphotype on Congo red agar, biofilm formation, presence of exopolysaccharide and capsule, possession of virulence genes (fimH, magA, ugE, wabG and rmpA) and their genomic fingerprints. A total of 55 strains of K. pneumoniae were isolated from both human-distributed sites (located along Sangga Besar River) and control sites (located along Selinsing River) where less human activity was observed, indicated that K. pneumoniae is ubiquitous in the environment. However, the detection of potentially virulent strains at the downstream of Kuala Sepetang village has suggested an anthropogenic contamination source. In conclusion, the findings from this study indicate that the Matang mangrove estuary could harbor potentially pathogenic K. pneumoniae with risk to public health. More studies are required to compare the environmental K. pneumoniae strains with the community-acquired K. pneumoniae strains.

Fully-automated identification of fish species based on otolith contour: using short-time Fourier transform and discriminant analysis (STFT-DA).

Background. Fish species may be identified based on their unique otolith shape or contour. Several pattern recognition methods have been proposed to classify fish species through morphological features of the otolith contours. However, there has been no fully-automated species identification model with the accuracy higher than 80%. The purpose of the current study is to develop a fully-automated model, based on the otolith contours, to identify the fish species with the high classification accuracy. Methods. Images of the right sagittal otoliths of 14 fish species from three families namely Sciaenidae, Ariidae, and Engraulidae were used to develop the proposed identification model. Short-time Fourier transform (STFT) was used, for the first time in the area of otolith shape analysis, to extract important features of the otolith contours. Discriminant Analysis (DA), as a classification technique, was used to train and test the model based on the extracted features. Results. Performance of the model was demonstrated using species from three families separately, as well as all species combined. Overall classification accuracy of the model was greater than 90% for all cases. In addition, effects of STFT variables on the performance of the identification model were explored in this study. Conclusions. Short-time Fourier transform could determine important features of the otolith outlines. The fully-automated model proposed in this study (STFT-DA) could predict species of an unknown specimen with acceptable identification accuracy. The model codes can be accessed at http://mybiodiversityontologies.um.edu.my/Otolith/ and https://peerj.com/preprints/1517/. The current model has flexibility to be used for more species and families in future studies.

Preliminary Taxonomic Survey and Molecular Documentation of Jellyfish Species (Cnidaria: Scyphozoa and Cubozoa) in Malaysia.

Mohammed Rizman-Idid, Abu Bakar Farrah-Azwa, and Ving Ching Chong (2016) Scientific enquiries into jellyfish blooms and associated problems are often deterred by the lack of taxonomical and ecological studies worldwide. Taxonomic difficulty is attributed to the high degree of morphological variations among and within species. To date, only two scyphozoan jellyfish species have been documented from field surveys in Malaysian waters, whereas another four Malaysian scyphozoan and two cubozoan jellyfish species have been mentioned in toxicological studies. None of these species have; however, been verified. This study thus aimed to document and resolves the uncertainty of earlier identified species in the region using morphology and molecular DNA sequencing. Jellyfish specimens were collected from Malaysian waters in the Straits of Malacca, South-China Sea and the Sulu-Sulawesi Sea over two years (June 2008 to October 2010), and their DNA sequences were compared with those from the Atlantic and Pacific regions. Ten scyphozoan and two cubozoan species were recorded in Malaysian waters (South China Sea and Straits of Malacca). These jellyfish included eight species from the order Rhizostomeae (Rhizostomatidae, Lobonematidae, Mastigiidae, Catostylidae and Cepheidae), two species from Semaestomeae (Pelagiidae and Cyaneidae) and two species from class Cubozoa; one from order Carybdeida (family Carukiidae) and another from order Chirodropida (family Chiropsalmidae). Molecular identification of species using phylogenetic approaches was based on DNA sequences of partial cytochrome oxidase I (COI), 16S and internal transcribed spacer (ITS1) regions. The COI phylogenetic tree of Cubozoa and Scyphozoa species from the Atlantic and Pacific regions showed distinct clustering of six Malaysian jellyfish species. However, most of the deeper divergences and relationships between the families were unresolved, which were also observed in the 16S and ITS1 phylogenetic trees. The Malaysian edible species Lobonemoides robustus, Rhopilema hispidum and Rhopilema esculentum were grouped within Rhizostomeae, whereas other scyphozoans showed phylogenetic affinities to Semaestomeae and Kolpophorae. Chrysaora and Cyanea appeared non-monophyletic; however their paraphyly was not confirmed. This study has provided the much needed baseline information on the taxonomy of Malaysian jellyfish species which have been substantiated by partial COI, 16S and ITS1 sequences. A total of 12 putative species of jellyfish were identified, which encompassed 12 genera.

Next generation sequencing yields the complete mitochondrial genome of the Zebra moray, Gymnomuraena zebra (Anguilliformes: Muraenidae).

In this study, the complete mitogenome sequence of the Zebra moray, Gymnomuraena zebra (Anguilliformes: Muraenidae) has been sequenced by the next-generation sequencing method. The assembled mitogenome consisting of 16,576 bp includes 13 protein coding genes, 22 transfer RNAs, and two ribosomal RNAs genes. The overall base composition of Zebra moray is 30.2% for A, 26.8% for C, 17.2% for G, and 25.8% for T and show 80% identities to Kidako moray, Gymnothorax kidako. The complete mitogenome of the Zebra moray provides an essential and important DNA molecular data for further phylogeography and evolutionary analysis for moray eel phylogeny.

Complete mitogenome of two moray eels of Gymnothorax formosus and Scuticaria tigrina (Anguilliformes: Muraenidae).

In this study, the complete mitogenome sequence of two moray eels of Gymnothorax formosus and Scuticaria tigrina (Anguilliformes: Muraenidae) has been sequenced by the next-generation sequencing method. The assembled mitogenome, with the length of 16,558 bp for G. formosus and 16,521 bp for S. tigrina, shows 78% identity to each other. Both mitogenomes follow the typical vertebrate arrangement, including 13 protein coding genes, 22 transfer RNAs, two ribosomal RNAs genes, and a non-coding control region of D-loop. The length of D-loop is 927 bp (G. formosus) and 850 bp (S. tigrina), which is located between tRNA-Pro and tRNA-Phe. The overall GC content is 45.5% for G. formosus and 47.9% for S. tigrina. Complete mitogenomes of G. formosus and S. tigrina provide essential and important DNA molecular data for further phylogenetic and evolutionary analysis for moray eel.

Next-generation sequencing yields the complete mitochondrial genome of the longfang moray, Enchelynassa canina (Anguilliformes: Muraenidae).

In this study, the complete mitogenome sequence of the longfang moray, Enchelynassa canina (Anguilliformes: Muraenidae) has been sequenced by the next-generation sequencing method. The length of the assembled mitogenome is 16,592 bp, which includes 13 protein coding genes, 22 transfer RNAs, and 2 ribosomal RNAs genes. The overall base composition of longfang moray is 28.4% for A, 28.0% for C, 18.4% for G, 25.1% for T, and show 82% identities to Kidako moray, Gymnothorax kidako. The complete mitogenome of the longfang moray provides an essential and important DNA molecular data for further phylogeography and evolutionary analysis for moray eel phylogeny.

Automated identification of copepods using digital image processing and artificial neural network.

BACKGROUND: Copepods are planktonic organisms that play a major role in the marine food chain. Studying the community structure and abundance of copepods in relation to the environment is essential to evaluate their contribution to mangrove trophodynamics and coastal fisheries. The routine identification of copepods can be very technical, requiring taxonomic expertise, experience and much effort which can be very time-consuming. Hence, there is an urgent need to introduce novel methods and approaches to automate identification and classification of copepod specimens. This study aims to apply digital image processing and machine learning methods to build an automated identification and classification technique. RESULTS: We developed an automated technique to extract morphological features of copepods' specimen from captured images using digital image processing techniques. An Artificial Neural Network (ANN) was used to classify the copepod specimens from species Acartia spinicauda, Bestiolina similis, Oithona aruensis, Oithona dissimilis, Oithona simplex, Parvocalanus crassirostris, Tortanus barbatus and Tortanus forcipatus based on the extracted features. 60% of the dataset was used for a two-layer feed-forward network training and the remaining 40% was used as testing dataset for system evaluation. Our approach demonstrated an overall classification accuracy of 93.13% (100% for A. spinicauda, B. similis and O. aruensis, 95% for T. barbatus, 90% for O. dissimilis and P. crassirostris, 85% for O. similis and T. forcipatus). CONCLUSIONS: The methods presented in this study enable fast classification of copepods to the species level. Future studies should include more classes in the model, improving the selection of features, and reducing the time to capture the copepod images.

Diverse and abundant multi-drug resistant E. coli in Matang mangrove estuaries, Malaysia.

E.coli, an important vector distributing antimicrobial resistance in the environment, was found to be multi-drug resistant, abundant, and genetically diverse in the Matang mangrove estuaries, Malaysia. One-third (34%) of the estuarine E. coli was multi-drug resistant. The highest antibiotic resistance prevalence was observed for aminoglycosides (83%) and beta-lactams (37%). Phylogenetic groups A and B1, being the most predominant E. coli, demonstrated the highest antibiotic resistant level and prevalence of integrons (integron I, 21%; integron II, 3%). Detection of phylogenetic group B23 downstream of fishing villages indicates human fecal contamination as a source of E. coli pollution. Enteroaggregative E. coli (1%) were also detected immediately downstream of the fishing village. The results indicated multi-drug resistance among E. coli circulating in Matang estuaries, which could be reflective of anthropogenic activities and aggravated by bacterial and antibiotic discharges from village lack of a sewerage system, aquaculture farms and upstream animal husbandry.

Molecular and morphological analyses reveal phylogenetic relationships of stingrays focusing on the family Dasyatidae (Myliobatiformes).

Elucidating the phylogenetic relationships of the current but problematic Dasyatidae (Order Myliobatiformes) was the first priority of the current study. Here, we studied three molecular gene markers of 43 species (COI gene), 33 species (ND2 gene) and 34 species (RAG1 gene) of stingrays to draft out the phylogenetic tree of the order. Nine character states were identified and used to confirm the molecularly constructed phylogenetic trees. Eight or more clades (at different hierarchical level) were identified for COI, ND2 and RAG1 genes in the Myliobatiformes including four clades containing members of the present Dasyatidae, thus rendering the latter non-monophyletic. The uncorrected p-distance between these four 'Dasytidae' clades when compared to the distance between formally known families confirmed that these four clades should be elevated to four separate families. We suggest a revision of the present classification, retaining the Dasyatidae (Dasyatis and Taeniurops species) but adding three new families namely, Neotrygonidae (Neotrygon and Taeniura species), Himanturidae (Himantura species) and Pastinachidae (Pastinachus species). Our result indicated the need to further review the classification of Dasyatis microps. By resolving the non-monophyletic problem, the suite of nine character states enables the natural classification of the Myliobatiformes into at least thirteen families based on morphology.

Sound signatures and production mechanisms of three species of pipefishes (Family: Syngnathidae).

Background. Syngnathid fishes produce three kinds of sounds, named click, growl and purr. These sounds are generated by different mechanisms to give a consistent signal pattern or signature which is believed to play a role in intraspecific and interspecific communication. Commonly known sounds are produced when the fish feeds (click, purr) or is under duress (growl). While there are more acoustic studies on seahorses, pipefishes have not received much attention. Here we document the differences in feeding click signals between three species of pipefishes and relate them to cranial morphology and kinesis, or the sound-producing mechanism. Methods. The feeding clicks of two species of freshwater pipefishes, Doryichthys martensii and Doryichthys deokhathoides and one species of estuarine pipefish, Syngnathoides biaculeatus, were recorded by a hydrophone in acoustic dampened tanks. The acoustic signals were analysed using time-scale distribution (or scalogram) based on wavelet transform. A detailed time-varying analysis of the spectral contents of the localized acoustic signal was obtained by jointly interpreting the oscillogram, scalogram and power spectrum. The heads of both Doryichthys species were prepared for microtomographical scans which were analysed using a 3D imaging software. Additionally, the cranial bones of all three species were examined using a clearing and double-staining method for histological studies. Results. The sound characteristics of the feeding click of the pipefish is species-specific, appearing to be dependent on three bones: the supraoccipital, 1st postcranial plate and 2nd postcranial plate. The sounds are generated when the head of the Dorichthyes pipefishes flexes backward during the feeding strike, as the supraoccipital slides backwards, striking and pushing the 1st postcranial plate against (and striking) the 2nd postcranial plate. In the Syngnathoides pipefish, in the absence of the 1st postcranial plate, the supraoccipital rubs against the 2nd postcranial plate twice as it is pulled backward and released on the return. Cranial morphology and kinesis produce acoustic signals consistent with the bone strikes that produce sharp energy spikes (discrete or merged), or stridulations between bones that produce repeated or multimodal sinusoidal waveforms. Discussion. The variable structure of the sound-producing mechanism explains the unique acoustic signatures of the three species of pipefish. The differences in cranial bone morphology, cranial kinesis and acoustic signatures among pipefishes (and seahorses) could be attributed to independent evolution within the Syngnathidae, which warrants further investigation.

Worldwide genetic differentiation in the common fouling barnacle, Amphibalanus amphitrite.

Amphibalanus amphitrite is a common fouling barnacle distributed globally in tropical and subtropical waters. In the present study, the genetic (mitochondrial cytochrome oxidase subunit I) and morphological differentiation in A. amphitrite from 25 localities around the world were investigated. The results revealed three clades within A. amphitrite with a genetic divergence of ~ 4% among clades, whereas there were no diagnostic morphological differences among clades. Clade 1 is widely distributed in both temperate and tropical waters, whereas Clade 3 is currently restricted to the tropical region. The deep divergence among clades suggests historical isolation within A. amphitrite; thus, the present geographical overlaps are possibly a result of the combined effects of rising sea level and human-mediated dispersals. This study highlights the genetic differentiation that exists in a common, widely distributed fouling organism with great dispersal potential; future antifouling research should take into account the choice of lineages.

Detection of multiple potentially pathogenic bacteria in Matang mangrove estuaries, Malaysia.

The deltaic estuarine system of the Matang Mangrove Forest Reserve of Malaysia is a site where several human settlements and brackish water aquaculture have been established. Here, we evaluated the level of fecal indicator bacteria (FIB) and the presence of potentially pathogenic bacteria in the surface water and sediments. Higher levels of FIB were detected at downstream sampling sites from the fishing village, indicating it as a possible source of anthropogenic pollution to the estuary. Enterococci levels in the estuarine sediments were higher than in the surface water, while total coliforms and E. coli in the estuarine sediments were not detected in all samples. Also, various types of potentially pathogenic bacteria, including Klebsiella pneumoniae, Serratia marcescens and Enterobacter cloacae were isolated. The results indicate that the Matang estuarine system is contaminated with various types of potential human bacterial pathogens which might pose a health risk to the public.