831. Looking beyond virus detection in RNA sequencing data : lessons learned from a community-based effort to detect cellular plant pathogens and pestsAnnelies Haegeman, Yoika Foucart, Kris De Jonghe, Thomas Goedefroit, Maher Al Rwahnih, Neil Boonham, Thierry Candresse, Yahya Gaafar, Oscar Hurtado-Gonzales, Zala Kogej Zwitter, Denis Kutnjak, Janja Lamovšek, Irena Mavrič Pleško, 2023, original scientific article Abstract: High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well. Keywords: plant viruses, plant virus detection, plant virology, high-throughput sequencing, RNA sequencing, plant tissues, plant pathogen, diagnostics, high-throughput sequencing, metagenomics, metatranscriptomics Published in DiRROS: 12.07.2024; Views: 144; Downloads: 115
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832. Leafhopper males compensate for unclear directional cues in vibration-mediated mate localizationJernej Polajnar, Ana Kuhelj, Rok Janža, Nada Žnidaršič, Tatjana Simčič, Meta Virant-Doberlet, 2023, original scientific article Abstract: Ambient noise and transmission properties of the substrate pose challenges in vibrational signal-mediated mating behavior of arthropods, because vibrational signal production is energetically demanding. We explored implications of these challenges in the leafhopper Aphrodes makarovi (Insecta: Hemiptera: Cicadellidae) by exposing males to various kinds of vibrational noise on a natural substrate and challenging them to find the source of the female playback. Contrary to expectations, males exposed to noise were at least as efficient as control males on account of similar searching success with less signaling effort, while playing back male–female duets allowed the males to switch to satellite behavior and locate the target without signaling, as expected. We found altered mitochondrial structure in males with high signaling effort that likely indicate early damaging processes at the cellular level in tymbal muscle, but no relation between biochemical markers of oxidative stress and signaling effort. Analysis of signal transmission revealed ambiguous amplitude gradients, which might explain relatively low searching success, but it also indicates the existence of behavioral adaptations to complex vibrational environments. We conclude that the observed searching tactic, emphasizing speed rather than thorough evaluation of directional cues, may compensate for unclear stimuli when the target is near. Keywords: biotremology, vibrational communication, sexual behavior of insects, impact of noise on behavior, metabolism of vibrational production Published in DiRROS: 12.07.2024; Views: 110; Downloads: 149
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833. Increasing information content and diagnosability in family-level classificationsMatjaž Kuntner, Klemen Čandek, Matjaž Gregorič, Eva Turk, A. Chris Hamilton, Lisa Chamberland, James Starrett, Ren-Chung Cheng, Jonathan A. Coddington, Ingi Agnarsson, Jason E. Bond, 2023, original scientific article Abstract: Higher-level classifications often must account for monotypic taxa representing depauperate evolutionary lineages and lacking synapomorphies of their better-known, well-defined sister clades. In a ranked (Linnean) or unranked (phylogenetic) classification system, discovering such a depauperate taxon does not necessarily invalidate the rank classification of sister clades. Named higher taxa must be monophyletic to be phylogenetically valid. Ranked taxa above the species level should also maximize information content, diagnosability, and utility (e.g., in biodiversity conservation). In spider classification, families are the highest rank that is systematically catalogued, and incertae sedis is not allowed. Consequently, it is important that family level taxa be well defined and informative. We revisit the classification problem of Orbipurae, an unranked suprafamilial clade containing the spider families Nephilidae, Phonognathidae, and Araneidae sensu stricto. We argue that, to maximize diagnosability, information content, conservation utility, and practical taxonomic considerations, this “splitting” scheme is superior to its recently proposed alternative, which lumps these families together as Araneidae sensu lato. We propose to redefine Araneidae and recognize a monogeneric spider family, Paraplectanoididae fam. nov. to accommodate the depauperate lineage Paraplectanoides. We present new subgenomic data to stabilize Orbipurae topology which also supports our proposed family-level classification. Our example from spiders demonstrates why classifications must be able to accommodate depauperate evolutionary lineages, e.g., Paraplectanoides. Finally, although clade age should not be a criterion to determine rank, other things being equal, comparable ages of similarly ranked taxa do benefit comparative biology. Keywords: classification, family rank, phylogenomics, systematics, monophyly, spider phylogeny, zoology Published in DiRROS: 12.07.2024; Views: 143; Downloads: 135
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834. Coastal fish fauna in the Cystoseira s.l. algal belts : experiences from the Northern Adriatic SeaLovrenc Lipej, Danijel Ivajnšič, Valentina Pitacco, Domen Trkov, Borut Mavrič, Martina Orlando-Bonaca, 2023, original scientific article Abstract: Cystoseira s.l. forests are recognised as important habitats which host diverse coastal fish assemblages. Many fish species use such habitats as feeding, breeding, and nursery grounds. Since the coastal fish community depends on the availability of dense macroalgal belts, the decline of these habitats in the Mediterranean Sea also affects the density of coastal fish species. We studied the coastal fish assemblage in Cystoseira s.l. forests in three consecutive years 2019–2021 in the Gulf of Trieste (Adriatic Sea). Data on coastal fish fauna were collected by visual counts conducted by SCUBA diving. Data on algal cover and habitat types were obtained by recording with a video camera. Similarities and differences in the fish community were analysed in terms of habitat and substrate preferences. A total of 34 species were recorded in Cystoseira forests. The results of the present study show that the different algal cover and associated depth gradient have different effects on the fish assemblage in coastal waters, affecting species composition and abundance. For many species, particularly labrids and sea breams, there is a decreasing temporal trend in frequency of occurrence and density. However, fish community trends can be used as a good “proxy” to evaluate the algal belt status. Our results indicate that rapid conservation and restoration actions are needed to stem the decline of Cystoseira s.l. forests. Keywords: Cystoseira s.l. forests, fish assemblage, fish biodiversity, habitat preference, macroalgal belt, Mediterranean Sea Published in DiRROS: 12.07.2024; Views: 126; Downloads: 110
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835. New records of introduced species in the Mediterranean Sea (April 2023)Ana Fortič, Reem Al-Sheikh Rasheed, Zouhair Almajid, Ali Badreddine, José Carlos Báez, Ángel Belmonte-Gallegos, Nicola Bettoso, Diego Borme, Federica Camisa, Martina Orlando-Bonaca, 2023, original scientific article Abstract: This Collective article reports 17 introduced species and 22 new locations for these species in the Mediterranean Sea. The reports are from three different Marine Strategy Framework Directive (MSFD) subregions (Aegean-Levantine Sea, Adriatic Sea and Western Mediterranean Sea) and the Sea of Marmara and cover ten different countries. The goal of consistent and detailed reporting of introduced species is to complement the existing species inventories and serve as a basis for establishing monitoring strategies and other conservation measures. Some of the reports from this article are the first species records for the Mediterranean Sea, namely the green alga Udotea flabellum from the Aegean Sea (Turkey) and the deepbody boarfish Antigonia capros from the Balearic Sea (Spain). In addition, new records of introduced species are included for different seas, namely the moon crab Matuta victor for the Aegean Sea (Greece), the whale shark Rhincodon typus and the lionfish Pterois miles for the Alboran Sea (Spain), the almaco jack Seriola rivoliana for the Tyrrhenian Sea (Italy), and the hound needlefish Tylosurus crocodilus for the Adriatic Sea (Italy). Furthermore, reports on first country records are included: the red alga Colaconema codicola from Slovenia, the nudibranch Melibe viridis from Bosnia and Herzegovina, the lionfish Pterois miles from Montenegro, and the goldstripe sardinella Sardinella gibbosa from Syria, which also represents a second record for the Mediterranean Sea. Furthermore, the occurrence of the scleractinian coral Oculina patagonica was noted in Gulf of Lion (France). Four polychaete species, namely Leodice antennata, Timarete punctata and Branchiomma bairdi, are reported from the vermetid reef habitat and two of them (L. antennata and B. bairdi) are also recorded for the first time in Lebanon. Evidence for established populations of the Asian date mussel Arcuatula senhousia in the Sea of Marmara (Turkey) and the rayed pearl oyster Pinctada radiata around the island of Sardinia (Italy) is provided. Published in DiRROS: 12.07.2024; Views: 157; Downloads: 121
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836. Evaluation of DNA extraction methods for reliable quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosaAlexandra Bogožalec Košir, Dane Lužnik, Viktorija Tomič, Mojca Milavec, 2023, original scientific article Abstract: Detection and quantification of DNA biomarkers relies heavily on the yield and quality of DNA obtained by extraction from different matrices. Although a large number of studies have compared the yields of different extraction methods, the repeatability and intermediate precision of these methods have been largely overlooked. In the present study, five extraction methods were evaluated, using digital PCR, to determine their efficiency in extracting DNA from three different Gram-negative bacteria in sputum samples. The performance of two automated methods (GXT NA and QuickPick genomic DNA extraction kit, using Arrow and KingFisher Duo automated systems, respectively), two manual kit-based methods (QIAamp DNA mini kit; DNeasy UltraClean microbial kit), and one manual non-kit method (CTAB), was assessed. While GXT NA extraction kit and the CTAB method have the highest DNA yield, they did not meet the strict criteria for repeatability, intermediate precision, and measurement uncertainty for all three studied bacteria. However, due to limited clinical samples, a compromise is necessary, and the GXT NA extraction kit was found to be the method of choice. The study also showed that dPCR allowed for accurate determination of extraction method repeatability, which can help standardize molecular diagnostic approaches. Additionally, the determination of absolute copy numbers facilitated the calculation of measurement uncertainty, which was found to be influenced by the DNA extraction method used. Keywords: nucleic acid, dPCR, DNA extraction methods, Gram-negative bacteria Published in DiRROS: 12.07.2024; Views: 141; Downloads: 150
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837. Combined toxic effects of BPA and its two analogues BPAP and BPC in a 3D HepG2 cell modelMartina Štampar, Tim Ravnjak, Ana-Marija Domijan, Bojana Žegura, 2023, original scientific article Abstract: Bisphenol A (BPA) is one of the most commonly used substances in the manufacture ofvarious everyday products. Growing concerns about its hazardous properties, including endocrinedisruption and genotoxicity, have led to its gradual replacement by presumably safer analogues inmanufacturing plastics. The widespread use of BPA and, more recently, its analogues has increasedtheir residues in the environment. However, our knowledge of their toxicological profiles is limitedand their combined effects are unknown. In the present study, we investigated the toxic effectscaused by single bisphenols and by the combined exposure of BPA and its two analogues, BPAP andBPC, after short (24-h) and prolonged (96-h) exposure in HepG2 spheroids. The results showed thatBPA did not reduce cell viability in HepG2 spheroids after 24-h exposure. In contrast, BPAP andBPC affected cell viability in HepG2 spheroids. Both binary mixtures (BPA/BPAP and BPA/BPC)decreased cell viability in a dose-dependent manner, but the significant difference was only observedfor the combination of BPA/BPC (both at 40μM). After 96-h exposure, none of the BPs studiedaffected cell viability in HepG2 spheroids. Only the combination of BPA/BPAP decreased cellviability in a dose-dependent manner that was significant for the combination of 4μM BPA and 4μMBPAP. None of the BPs and their binary mixtures studied affected the surface area and growth ofspheroids as measured by planimetry. In addition, all BPs and their binary mixtures studied triggeredoxidative stress, as measured by the production of reactive oxygen species and malondialdehyde,at both exposure times. Overall, the results suggest that it is important to study the effects of BPsas single compounds. It is even more important to study the effects of combined exposures, as thecombined effects may differ from those induced by single compounds. Keywords: BP analogues, hepatic in vitro 3D cell model, combined exposure, viability, oxidative stress, toxicology Published in DiRROS: 12.07.2024; Views: 494; Downloads: 137
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838. Plant pathogen detection on a lab-on-a-disc using solid-phase extraction and isothermal nucleic acid amplification enabled by digital pulse-actuated dissolvable film valvesRohit Mishra, Lourdes AN. Julius, Jack Condon, Patricija Pavelskopfa, Philip L. Early, Matthew Dorrian, Katarina Mrvova, Grace Henihan, Faith Mangwanya, Tanja Dreo, 2023, original scientific article Published in DiRROS: 12.07.2024; Views: 128; Downloads: 94
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839. Halimeda tuna (Bryopsidales, Ulvophyceae) calcification on the depth transect in the northern Adriatic Sea; carbonate production on the microscale of individual segmentsYvonne Nemcova, Martina Orlando-Bonaca, Jiri Neustupa, 2023, original scientific article Abstract: Halimeda tuna (J. Ellis & Solander) J.V. Lamouroux is the only Halimeda species found in the Mediterranean Sea, and it is an important habitat former. In the northern Adriatic, H. tuna is among the ten most abundant seaweeds in the upper-infralittoral belt in spring and autumn. The modular thalli consist of serially arranged calcified segments. Calcification is closely related to photosynthesis, which causes alkalinization of the inter-utricular space and triggers aragonite formation. Understanding of the complex patterns of segment shape plasticity in relation to CaCO3content at different depth levels is still incomplete. Geometric morphometrics was used to investigate H. tuna segment shape variation on the depth transect at Cape Madona Nature Monument in the northern Adriatic Sea. The position on the thallus and the CaCO3 content of each studied segment were recorded, allowing slight changes in mineral content to be detected at the microscale of the segments. Our results showed that shape, size, or asymmetry of H. tuna segments were not significantly affected by depth. On the other hand, plants that grew deeper were generally more calcified. The apical and subapical segments contributed to the increase in CaCO3 content at the deeper sites, whereas the basal segments did not. This indicates that reniform or oval segments positioned apically or subapically play a key role in calcification of H. tuna in Mediterranean ecosystems. Keywords: calcification, depth transect, geometric morphometrics, green algae, Halimeda tuna, phenotypic plasticity, Ulvophyceae Published in DiRROS: 12.07.2024; Views: 120; Downloads: 98
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840. Evaluation of the rbcL marker for metabarcoding of marine diatoms and inference of population structure of selected generaTimotej Turk Dermastia, Ivano Vascotto, Janja Francé, David Stanković, Patricija Mozetič, 2023, original scientific article Abstract: Diatoms are one of the most important phytoplankton groups in the world’s oceans. There are responsible for up to 40% of the photosynthetic activity in the Ocean, and they play an important role in the silicon and carbon cycles by decoupling carbon from atmospheric interactions through sinking and export. These processes are strongly influenced by the taxonomic composition of diatom assemblages. Traditionally, these have been assessed using microscopy, which in some cases is not reliable or reproducible. Next-generation sequencing enabled us to study diversity in a high-throughput manner and uncover new distribution patterns and diversity. However, phylogenetic markers used for this purpose, such as various 18S rDNA regions, are often insufficient because they cannot distinguish between some taxa. In this work, we demonstrate the performance of the chloroplast-encoded rbcL marker for metabarcoding marine diatoms compared to microscopy and 18S-V9 metabarcoding using a series of monthly samples from the Gulf of Trieste (GoT), northern Adriatic Sea. We demonstrate that rbcL is able to detect more taxa compared to 18S-V9 metabarcoding or microscopy, while the overall structure of the diatom assemblage was comparable to the other two methods with some variations, that were taxon dependent. In total, 6 new genera and 22 new diatom species for the study region were identified. We were able to spot misidentification of genera obtained with microscopy such as Pseudo-nitzschia galaxiae, which was mistaken for Cylindrotheca closterium, as well as genera that were completely overlooked, such as Minidiscus and several genera from the Cymatosiraceae family. Furthermore, on the example of two well-studied genera in the region, namely Chaetoceros and particularly Pseudo-nitzschia, we show how the rbcL method can be used to infer even deeper phylogenetic and ecologically significant differences at the species population level. Despite a very thorough community analysis obtained by rbcL the incompleteness of reference databases was still evident, and we shed light on possible improvements. Our work has further implications for studies dealing with taxa distribution and population structure, as well as carbon and silica flux models and networks. Keywords: rbcL, metabarcoding, monitoring, diatoms, population genetics, Pseudo-nitzschia, Adriatic Published in DiRROS: 12.07.2024; Views: 131; Downloads: 98
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