Diversity, Distribution and Evolution of the Planktonic Diatom Family Chaetocerotaceae

The number and abundance of diatom species in environmental samples are counted traditionally by means of light microscopy (LM). However, recognizing –let alone, counting- species is often challenging because of the existence of cryptic species and intraspecific phenotypic plasticity. Proper characterization requires isolation of cells, growing them into monoclonal cultures, and characterizing the cultures genetically and morphologically. However, not all species grow in culture, featureless ones are less likely to be isolated, and the procedure is laborious. High-throughput sequencing (HTS) metabarcoding bypasses morphology; DNA is collected from environmental samples, a particular marker sequenced, and the resulting sequences sorted into clusters or terminal clades assumed to represent species. Yet, reference barcodes of taxonomically validated species are needed to identify these clades. This exercise is the main aim of my thesis. Since it is impossible to do this for all the diversity within a PhD thesis project, we selected Chaetocerotaceae, an abundant and diverse family of marine planktonic diatoms, containing two genera: Chaetoceros and Bacteriastrum. Its members uniquely share setae; thin siliceous tubes emerging from the valve corners, facilitating detection in samples. Strains were obtained from the Gulf of Naples (GoN), from Central Chile and Roscoff – at sites for which LTER time series data are available. A total of 270 strains were obtained from these sites, and their 18S- and partial 28S rDNA sequences and morphological information gathered. The strains grouped into 60 genetically distinct species, thus providing a dataset of validated Chaetocerotacean 18S reference barcodes. Inferred molecular phylogenies showed monophyletic Chaetocerotaceae as well as monophyletic Bacteriastrum inside paraphyletic Chaetoceros, and the presence of cryptic diversity. To start with taxonomic updates, the species C. sporotruncatus and C. dichatoensis were described within the C. socialis species-complex based on spore morphology and sequence differences. Several rDNA sequences contained spliceosomal introns (ca. 100bp) and/or group-I introns (ca. 400bp). Phylogenies inferred from the introns did not corroborate rDNA phylogenies, suggesting horizontal gene transfer. Presence/absence of introns in conspecific strains sampled in different seasons suggests population differentiation between these seasons. A HTS dataset consisting of V4-sequences (part of 18S) from 48 seawater samples taken over the seasons in the GoN revealed 76 terminal clades of which 46 grouped with a reference barcode. Some of these species occur year-round whereas most others are seasonal. Surprisingly, of the 30 clades belonging to unknown Chaetocerotacean species, two appear to be among the most abundant in the GoN

Effect of hydrodynamic cavitation on zooplankton: a tool for disinfection

Application of hydrodynamic cavitation for disinfection of water is gaining momentum, as it provides environmentally and economically sound options. In this effort, the effect of cavitating conditions created by differential pump valve opening and that created by flowing through a cavitating element (orifice plates) on the microbes (zooplankton in sea water) is described. The experimental results are compared with modelling of cavitating conditions that includes cavity dynamics, turbulence generated by individual oscillating cavity, cell wall strength and geometrical and operating parameters of cavitation device. Theoretical model for quantifying the cavitationally generated turbulent shear and extent of microbial disinfection has been developed. Experimental results indicated that cavitation and/or turbulent fluid shear dominantly originating from cavitation are effective tools for sea water disinfection as more than 80% of the zooplankton present in the sea water were killed. It was also observed that shock waves generated due to cavitation is not the sole cause for zooplankton disruption. A correct physical mechanism accounting fluid turbulence and shear, generated from stable oscillation of cavity, significantly contribute towards the disruption. Further refinement of the model presented will serve as a basis for higher degree of disinfection and provide a practical tool for sea water disinfection

Annotated 18S and 28S rDNA reference sequences of taxa in the planktonic diatom family Chaetocerotaceae

The species-rich diatom family Chaetocerotaceae is common in the coastal marine phytoplankton worldwide where it is responsible for a substantial part of the primary production. Despite its relevance for the global cycling of carbon and silica, many species are still described only morphologically, and numerous specimens do not fit any described taxa. Nowadays, studies to assess plankton biodiversity deploy high throughput sequencing metabarcoding of the 18S rDNA V4 region, but to translate the gathered metabarcodes into biologically meaningful taxa, there is a need for reference barcodes. However, 18S reference barcodes for this important family are still relatively scarce. We provide 18S rDNA and partial 28S rDNA reference sequences of 443 morphologically characterized chaetocerotacean strains. We gathered 164 of the 216 18S sequences and 244 of the 413 28S sequences of strains from the Gulf of Naples, Atlantic France, and Chile. Inferred phylogenies showed 84 terminal taxa in seven principal clades. Two of these clades included terminal taxa whose rDNA sequences contained spliceosomal and Group IC1 introns. Regarding the commonly used metabarcode markers in planktonic diversity studies, all terminal taxa can be discriminated with the 18S V4 hypervariable region; its primers fit their targets in all but two species, and the V4-tree topology is similar to that of the 18S. Hence V4-metabarcodes of unknown Chaetocerotaceae are assignable to the family. Regarding the V9 hypervariable region, most terminal taxa can be discriminated, but several contain introns in their primer targets. Moreover, poor phylogenetic resolution of the V9 region affects placement of metabarcodes of putative but unknown chaetocerotacean taxa, and hence, uncertainty in taxonomic assignment, even of higher taxa.info:eu-repo/semantics/publishedVersio

Functional annotation of the reference transcriptome of Teleaulax amphioxeia

Raw reads were pre-processed by removing the adaptors and low-quality reads using BBMap. The filtered reads were normalized for depth based on kmer counts using BBNorm function. De novo transcriptomes were generated using both Trinity and velvet-oases. CD-HIT-EST was used to merge the two de novo transcriptomes and reduce the transcript redundancy to 98% similarity and generate unique genes. Transcriptome assembly completeness was evaluated with BUSCO (Benchmarking Universal Single Copy Orthologs) database. Functional annotation was done using blastp function of ncbi-blast using the nr database with evalue 1E-20 and num_alignments 3.Funded by NOAA/ECOHAB: NA19NOS4780182-722372-71268

De novo transcriptome assembly and gene annotation for the toxic dinoflagellate Dinophysis

Abstract Species within the dinoflagellate genus Dinophysis can produce okadiac acid and dinophysistoxins leading to diarrhetic shellfish poisoning. Since the first report of D. ovum from the Gulf of Mexico in 2008, reports of other Dinophysis species across US have increased. Members of the D. cf. acuminata complex (D. acuminata, D. acuta, D. ovum, D. sacculus) are difficult to differentiate due to their morphological similarities. Dinophysis feeds on and steals the chloroplasts from the ciliate, Mesodinium rubrum, which in turn has fed on and captured the chloroplasts of its prey, the cryptophyte Teleaulax amphioxeia. The objective of this study was to generate de novo transcriptomes for new isolates of these mixotrophic organisms. The transcriptomes obtained will serve as a reference for future experiments to assess the effect of different abiotic and biotic conditions and will also provide a useful resource for screening potential marker genes to differentiate among the closely related species within the D. cf. acuminata-complex. The complete comprehensive detailed workflow and links to obtain the transcriptome data are provided

Gut fluorescence analysis of barnacle larvae: an approach to quantify the ingested food

Gut fluorescence analysis can provide a snapshot of ingested food and has been employed in feeding studies of various organisms. In this study we standardised the gut fluorescence method using laboratory-reared barnacle larvae (Balanus amphitrite) fed with mono-algal diet Chaetoceros calcitrans, a unicellular diatom at a cell concentration of 2 × 105 cells ml-1. The gut fluorescence of IV–VI instar nauplii was found to be 370(±12) ng chlorophyll a larva-1 and in faecal pellets it was 224(±63) ng chlorophyll a larva-1. A phaeopigment concentration in larval gut was found to be 311(±13) ng larva-1 and in faecal pellets it was 172(±61) ng larva-1. The study also analysed larval samples collected from the field during different seasons from a tropical environment influenced by monsoons (Dona Paula bay, Goa, west coast of India), with characteristic temporal variations in phytoplankton abundance and diversity. Gut fluorescence of larvae obtained during the post-monsoon season was consistently higher when compared to the pre-monsoon season, suggesting the predominance of autotrophic forms in the larval gut during the post-monsoon season. Whereas, the low gut fluorescence obtained during the pre-monsoon season indicated the ingestion of food sources other than autotrophs. Such differences observed in the feeding behaviour of larvae could be due to differential availability of food for the larvae during different seasons and indicate the capability of larvae to feed on wide range of food sources. This study shows the value of the fluorescence method in feeding studies of planktotrophic organisms and in the evaluation of ecosystem dynamics

Stable isotopic analysis of Barnacle larvae and their faecal pellets to evaluate the ingested food

Stable isotopes of carbon and nitrogen are useful in the evaluation of food web dynamics and have been used extensively in the feeding studies. Barnacles are dominant component of the intertidal and bio-fouling communities. Life cycle of barnacles includes planktonic naupliar stages followed by a non-feeding pre-settling cyprid stage. Development success of the nauplii depends on the food available for them in the natural environment. However, the food preference of the nauplii in the natural environment is still not well understood. In this study experiments were carried out initially to assess the 13C and 15N fractionation between the food and the Balanus amphitrite nauplii that were fed with Chaetoceros calcitrans. Laboratory reared larvae fed with C. calcitrans diet pointed out an enrichment in 13C by 0.95‰ and 15N by 1.42‰. The enrichment level in the faecal pellets was comparatively higher than that obtained from the larvae either fed or starved indicating the role of heterotrophic organisms. Expanding on these observations, efforts were made to evaluate the food of the acorn barnacle larvae collected from the wild during different seasons. The δ13C ratios of the larvae and their faecal pellets obtained during the post-monsoon season were heavier compared to pre-monsoon season indicating the feeding of larvae on isotopically heavier food sources during the post-monsoon season. However, the δ13C and δ15N ratios of the larvae and their faecal pellets did not differ significantly between the seasons and this could possibly be due to the broad range of isotopic signatures observed at the base of the food web

Settlement and recruitment of the barnacle Balanus amphitrite from a tropical environment influenced by monsoons

Studies on the settlement and the subsequent recruitment of intertidal organisms are crucial steps in understanding their population structure in a particular bioregion. However, studying the recruitment of intertidal organisms such as barnacles, with two-phase life cycle, and understanding the determinants of recruitment is a complex problem. Many processes which operate during the pelagic pre-settlement phase and benthic post-settlement phase at different spatial and temporal scales can determine the fate of barnacle populations. In this study, observations were carried out on settlement and recruitment of the intertidal barnacle Balanus amphitrite from a tropical environment influenced by monsoons at spatial and temporal scales. Settlement and recruitment both showed significant temporal variations. In general, settlement and recruitment was lower during the monsoon season except during monsoon breaks. Consistency in settlement and recruitment was mostly observed during the pre-monsoon season, and it coincided with peaks in larval abundance observed during the same season. In general, settlement remained more or less similar on different types of substrata, whereas recruitment differed significantly. Temporal variations observed in settlement and recruitment of barnacles could be due to the influence of monsoons in this region, which leads to seasonal fluctuations in physical factors such as local hydrodynamics, that influence the retention and dispersal of larvae in the adult habitats, and could also be due to variations in the availability of food for the larvae and the settled populations

What do barnacle larvae feed on? Implications in biofouling ecology

Barnacles are one of the dominant macrofouling organisms found in the intertidal region throughout the world. Among the different species of barnacles Balanus amphitrite (=Amphibalanus amphitrite) is a favoured candidate organism used in experimental studies. Larval development in this barnacle includes planktotrophic naupliar stages followed by pre-settling cyprid instar. Studies have shown that availability of food during naupliar development is of critical importance to successful metamorphosis of the cypris larva. Traditionally barnacle larvae are raised in the laboratory providing mono-algal cultures of diatoms as food organisms. Such a luxury is not a reality in the wild. Observations to quantify the food available for the nauplii deliberated by monitoring the faecal pellets egested by freshly captured larvae from a tropical estuarine environment (Dona Paula bay, Goa, west coast of India) influenced by monsoon and characteristic temporal variations in the phytoplankton abundance and diversity indicated that the percentage of defaecating larvae (an indicator of food consumed) was comparatively higher during the pre-monsoon season. Generally this season is characterized by lower chlorophyll-a concentration. However, the average number of faecal pellets defaecated by a larva remained constant irrespective of the season. Earlier work in the study area depicts temporal changes in phytoplankton community structure; diatoms dominate during the post-monsoon season whereas dinoflagellates dominate during the pre-monsoon season. These observations indicate a possible shift in the food available for the larvae. As the faecal pellets did not always have remnants of diatom frustules, it is possible to say that the larvae survived on food material other than diatoms. Settlement of barnacles on panels of aluminium in the vicinity was monitored throughout the year and peaked during the pre-monsoon season. It is thus possible to infer successful larval development and metamorphosis in this barnacle species on varying forms of food

Changes in the abundance and composition of zooplankton from the ports of Mumbai, India

Zooplankton abundance, biomass, and composition from the ports of Mumbai, India, were studied by selecting 14 stations in and around the area during three different periods between 2001 and 2002 (Nov 01, Apr 02, and Oct 02). The results are compared with the records available since the 1940s. Copepod species such as Canthocalanus sp., Paracalanus arabiensis, Cosmocalanus sp., Euterpina acutifrons, Nannocalanus minor, and Tortanus sp. which were not reported in the earlier studies were observed during the present investigation. Purely herbivorous forms like Nannocalanus minor, Paracalanus sp., and Temora discaudata were in reduced abundance during Apr 02 sampling which was coupled with reduction in the diatom population. Whereas increased abundance of some carnivorous and omnivorous forms during Apr 02 sampling can be related to the changes in the food web dynamics