Molekularnom analizom ribljih larvi utvrđena potencijalna prisutnost rijetke vrste: Buenia massutii Kovačić, Ordines i Schliewen, 2017. u Jadranskom moru

By introduction of DNA barcodes in Adriatic larval fish identification possible presence of new Gobiidae - Buenia massutii Kovačić, Ordines, and Schliewen, 2017, was noticed. Till now, occurrence of this species was restricted only to the Western Mediterranean and to the neighbouring part of the Atlantic Ocean.Larva nedavno opisane vrste Buenia massutii Kovačić, Ordines i Schliewen, 2017, s poznatom geografskom distribucijom ograničenom na zapadno Sredozemlje i susjedni dio Atlanskog oceana, je pronađena u Jadranskom moru. Naime, tijekom ihtioplanktonskog uzorkovanja je izolirana larva navedene vrste, čija je taksonomija utvrđena molekularnom analizom (DNA barcoding)

Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study

Reproduktivna biologija oslića, Merluccius merluccius (L. 1758), u Jadranskom moru

Within this paper, reproduction traits of the Adriatic population European hake, Merluccius merluccius (Linnaeus, 1758), were investigated. Specimens of the target species were monthly collected (N=1173) during 2019 along the eastern Adriatic Sea with commercial bottom trawlers. Overall, European hake total body length varied between 15.5 and 49.5 cm (mean±SD=27.5±5.0 cm) and domination of females was noted in larger length classes (TL>31.0 cm). The sexual ratio of all specimens was m/f=0.64; male prevalence was established over the year, except during the spawning peaks (December, June) when the sex ratio was in favour of the females. According to European hake’s maturity stages, gonad weights, gonadosomatic indexes and histological analysis of the gonad tissues, this species in Adriatic spawn twice in one year; the spawning period was from December to February, while second smaller signs of spawning were observed at the beginning of the summer, in June. 50% of the European hake male and female specimens sexually matured at 20.7 cm and 22.4 cm of total length, respectively.U okviru ovog rada je istražena reproduktivna biologija jadranske populacije oslića, Merluccius merluccius (Linnaeus, 1758). Uzorci istraživane vrste su prikupljeni iz uzoraka lovina ostvarenih komercijalnom pridnenom mrežom koćom s područja istočnog Jadrana (Hrvatsko ribolovno more) jednom mjesečno (N = 1173) tijekom 2019. godine. Raspon totalnih dužina tijela oslića se kretao između u rasponu od 15,5 cm do 49,5 cm (srednja vrijednost ± SD = 27,5 ± 5,0 cm) te je pri većim dužinskim razredima zabilježena dominacija ženki (TL> 31,0 cm). Omjer spolova (m/ž) je iznosio 0,64; prevalencija mužjaka je utvrđena tijekom čitave godine izuzev razdoblja mrijesta (prosinac, lipanj) kada je omjer spolova bio u korist ženki. Analizom stadija zrelosti, mase gonada, gonadosomatskog indeksa te histoloških preparata tkiva spolnih žlijezda utvrđeno je da se ova vrsta u Jadranu mrijesti dva puta u jednoj godini; utvrđeno je razdoblje mrijesta od prosinca do veljače, dok je drugo razdoblje mrijesta nešto slabijeg intenziteta utvrđeno početkom ljeta, točnije u lipnju. Prva spolna zrelost svih analiziranih jedinki oslića je nastupila pri totalnoj dužini tijela od 20,7 cm za mužjake odnosno 22,4 cm za ženke

Presentation_1_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.PDF

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p

Data_Sheet_2_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.XLSX

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p

Data_Sheet_5_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.XLSX

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p

Data_Sheet_1_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.DOCX

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p

Data_Sheet_4_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.XLSX

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p

Biometry, Distribution and Genetic Characterization of Blue Jack Mackerel Trachurus picturatus (Bowdich, 1825), a Rare Pelagic Fish Species in the Adriatic Sea

The blue jack mackerel Trachurus picturatus (Bowdich, 1825) specimens (N = 155) were collected during the MEDITS survey, done along the eastern side, precisely, of the Croatian fishing ground in July 2018. Biometrical analysis of ten morphometric and five meristic characters, as well as genetic analysis proved that the collected specimens were blue jack mackerel. The total length (TL) and weight (W) of all observed specimens ranged from 9.2 to 33.7 cm (12.15 &plusmn; 2.95 cm) and from 5.79 to 384.94 g (17.64 &plusmn; 39.42 g), respectively. All calculated length&ndash;length relationships were linear (r &gt; 0.923). Sex was determined only on two larger specimens (28 cm &lt; TL &lt; 32.8 cm), which were females. In the length&ndash;weight relationship, positive allometry was established (b = 3.1789). Based on 37 partial cytochrome b sequences, the overall haplotype diversity (h) of 0.812 &plusmn; 0.048 and nucleotide diversity (&pi;) of 0.0064 &plusmn; 0.0007 indicated high levels of haplotype and low nucleotide diversity. The obtained sequences were compared to previously published research within the Northeast Atlantic Ocean and the Mediterranean Sea, confirming the absence of genetic structure among these populations

Data_Sheet_3_Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.XLSX

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.</p