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As a major component of freshwater ecosystems, insect species play an important role in nutrient cycling and are often used as bioindicators of water pollution. Although extensive studies have characterized insects from freshwater habitats, little is known about the distribution of these species along the Lower Sector of the Danube River. Therefore, this survey conducted in the Danube section within the Romanian territory aimed to identify insect larvae belonging to seven different species of Odonata, Trichoptera, Ephemeroptera, Lepidoptera and Megaloptera by DNA barcoding and to investigate their distribution, density and frequency. A total of 41 quantitative macrozoobenthic samples were collected during two consecutive years (2019 and 2020). Species showed large differences in the distribution and density along different sections, and an overall tendency to populate downstream areas, except for Sialis morio. On the other hand, only Hydropsyche bulgaromanorum, Triaenodes bicolor and S. morio larvae were identified in the upstream section (Sulina branch). These data provide baseline information on the larger range of some of the most common aquatic insects in the Romanian Danube section in relation to several environmental parameters based on the first molecular identification of these species using COI gene sequencing.

Despite the unique physiology and metabolic pathways of microbiomes from cold environments providing key evolutionary insights and promising leads for discovering new bioactive compounds, cultivable bacteria entrapped in perennial ice from caves remained a largely unexplored life system. In this context, we obtained and characterized bacterial strains from 13,000-years old ice core of Scarisoara Ice Cave, providing first isolates from perennial ice accumulated in caves since Late Glacial, and first culture-based evidences of bacterial resistome and antimicrobial compounds production. The 68 bacterial isolates belonged to 4 phyla, 34 genera and 56 species, with 17 strains representing putative new taxa. The Gram-negative cave bacteria (Proteobacteria and Bacteroidetes) were more resistant to the great majority of antibiotic classes than the Gram-positive ones (Actinobacteria, Firmicutes). More than 50% of the strains exhibited high resistance to 17 classes of antibiotics. Some of the isolates inhibited the growth of clinically important Gram-positive and Gram-negative resistant strains and revealed metabolic features with applicative potential. The current report on bacterial strains from millennia-old cave ice revealed promising candidates for studying the evolution of environmental resistome and for obtaining new active biomolecules for fighting the antibiotics crisis, and valuable cold-active biocatalysts.

Grapes’ infection with the fungi Botrytis cinerea is one of the major causes of economic loss in the winemaking sector worldwide. The laccase activity of grapes is considered an appropriate indicator of this type of fungal infection, and enzymatic activity higher than 3 U/mL indicates a high risk of irreversibly damaged grape must due to enzymatic browning. This work describes a fast test for the measurement of laccase activity based on a dual optical and electrochemical detection method. A paper sensor impregnated with the enzymatic substrate dye 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) provides a semi-quantitative optical measurement. While the paper sensor can be used independently, when combined with a screen-printed electrode and amperometry measurements, it enables the quantitative detection of laccase activities down to 0.4 U/mL in only 5 min. The method was applied for monitoring the artificial infection of white, rosé, and red grapes with different strains of Botrytis cinerea. The results were confirmed by parallel analysis using the spectrophotometric method of laccase activity determination based on syringaldazine. The influence of the fungal strain and type of grape on laccase activity levels is reported. The demonstrated robustness, simplicity, and versatility of the developed method make it ideal for application on-site in the vineyard or at grape processing points. Financial support provided by the Romanian Executive Agency for Higher Education, Research, Development and Innovation (UEFISCDI), ERANET-MANUNET-III-WINBIOTOOL-2, contract 151/9.03.2020 grant (for A.V), contract150/9.03.2020 (for P.E) and contract 152/9.03.2020 (for C.P. (Catalina Pantazi), E.B., and M.I.); the Romanian Academy grant RO1567-IBB05/2021 (for R.R. and C.P. (Cristina Purcarea)); the Basque Government and the European Union through the European regional development fund 2014–2020 (FEDER) (ZL-2020/00532 and ZL-2021/00340); and the Diputación Foral de Álava (ALAVA INNOVA program—INNOEM-2020/00045) for Bodegas de los Herederos del Marqués de Riscal; and the Diputación Foral de Álava (ALAVA INNOVA program— INNOEM-2020/00045) for Bodegas de los Herederos del Marqués de Risca is gratefully acknowledged.

Dithiocarbamate fungicides (DTFs) are widely used to control various fungal diseases in crops and ornamental plants. Maximum residual limits in the order of ppb-ppm are currently imposed by legislation to prevent toxicity problems associated with excessive use of DTFs. The specific analytical determination of DTFs is complicated by their low solubility in water and organic solvents. This review summarizes the current analytical procedures used for the analysis of DTF, including chromatography, spectroscopy, and sensor-based methods and discusses the challenges related to selectivity, sensitivity, and sample preparation. Biosensors based on enzymatic inhibition demonstrated potential as analytical tools for DTFs and warrant further research, considering novel enzymes from extremophilic sources. Meanwhile, Raman spectroscopy and various sensors appear very promising, provided the selectivity issues are solved.

Ice caves constitute the newly investigated frozen and secluded model habitats for evaluating the resilience of ice-entrapped microbiomes in response to climate changes. This survey identified the total and active prokaryotic and eukaryotic communities from millennium-old ice accumulated in Scarisoara cave (Romania) using Illumina shotgun sequencing of the ribosomal RNA (rRNA) and messenger RNA (mRNA)-based functional analysis of the metatranscriptome. Also, the response of active microbiome to heat shock treatment mimicking the environmental shift during ice melting was evaluated at both the taxonomic and metabolic levels. The putatively active microbial community was dominated by bacterial taxa belonging to Proteobacteria and Bacteroidetes, which are highly resilient to thermal variations, while the scarcely present archaea belonging to Methanomicrobia was majorly affected by heat shock. Among eukaryotes, the fungal rRNA community was shared between the resilient Chytridiomycota and Blastocladiomycota, and the more sensitive Ascomycota and Basidiomycota taxa. A complex microeukaryotic community highly represented by Tardigrada and Rotifera (Metazoa), Ciliophora and Cercozoa (Protozoa), and Chlorophyta (Plantae) was evidenced for the first time in this habitat. This community showed a quick reaction to heat shock, followed by a partial recovery after prolonged incubation at 4°C due to possible predation processes on the prokaryotic cluster. Analysis of mRNA differential gene expression revealed the presence of an active microbiome in the perennial ice from the Scarisoara cave and associated molecular mechanisms for coping with temperature variations by the upregulation of genes involved in enzyme recovery, energy storage, carbon and nitrogen regulation, and cell motility. This first report on the active microbiome embedded in perennial ice from caves and its response to temperature stress provided a glimpse into the impact of glaciers melting and the resilience mechanisms in this habitat, contributing to the knowledge on the functional role of active microbes in frozen environments and their response to climatic changes.

The microbial diversity and quantitative dynamics during the insect’s development stages constitute recently developed putative tools in forensic and medical studies. Meanwhile, little is known on the role of insects in spreading foodborne pathogenic bacteria and on the impact of these pathogens on the overall insects and feeding substrate microbiome composition. Here, we provide the first characterization of the bacterial communities harbored in adult and immature stages of Lucilia sericata, one of the first colonizers of decomposed human remains, in the presence of the foodborne pathogen Salmonella enterica using 16S rRNA Illumina sequencing and qPCR. The pathogen transmission from the wild adults to the second generation was observed, with a 101.25× quantitative increase. The microbial patterns from both insect and liver samples were not influenced by the artificial introduction of this pathogenic foodborne bacteria, being dominated by Firmicutes and Proteobacteria. Overall, our results provided a first detailed overview of the insect and decomposed substrate microbiome in the presence of a human pathogen, advancing the knowledge on the role of microbes as postmortem interval estimators and the transmission of pathogenic bacteria.

Here, we report the draft genome sequence of Flavobacterium sp. strain PL002, isolated from Antarctic Porphyra algae. The 4,299,965-bp genome sequence is assembled into 170 contigs, has 32.92% GC content, and 3,734 predicted genes.

Here, we report the draft genome sequence of Flavobacterium sp. strain PL002, isolated from Antarctic Porphyra algae. The 4,299,965-bp genome sequence is assembled into 170 contigs, has 32.92% GC content, and 3,734 predicted genes.

The draft genome sequence of Streptomyces fildesensis strain INACH3013, a psychrotrophic bacterium isolated from Northwest Antarctic soil, was reported. The genome sequence totaling 9,306,785 bp resulted from 122 contigs characterized by a GC content of 70.55%.

International audience; Carbon nanofibers (CNF) are efficient electrode modifiers in electrochemical biosensors that enhance the electrochemical active area, induce electrocatalytic effect toward the oxidation of the enzymatic cofactor nicotinamide adenine dinucleotide (reduced form, NADH), and enable the quantitative immobilization of enzymes. Combining CNF with efficient and stable mediators radically augments the speed of electron transfer between NADH and solid electrodes and leads to electrochemical sensors characterized by high sensitivity and stability. The main aim of this work was to investigate the performance of a novel mediator for NADH with advantageously low solubility in an electrochemical detector based on a screen-printed CNF electrode as well as its potential in biosensing. Using a mediator, prepared from Meldola Blue and Ni hexamine chloride, a stable and sensitive electrochemical NADH sensor is provided with a detection limit of 0.5 μmol L−1. Further on, covalent immobilization of a recently described aldehyde dehydrogenase from the Antarctic Flavobacterium PL002 strain on the surface of the mediator-modified electrode produced a stable biosensor for the detection of aldehydes. When integrated in a flow injection analysis (FIA) setup with amperometric detection at 0.1 V vs. Ag/AgCl, the measurement of benzaldehyde with a detection limit of 10 μmol L−1 over a linear range of 30–300 μmol L−1 is possible. Determination of trace benzaldehyde impurities in a pharmaceutical excipient was also demonstrated and results compared with a chromatographic method.

For the last decades, forensic microbiology became an emerging complementary tool in criminalistics. Although the insect-microbe interactions regarding pathogen transmission were extensively studied, only scarce information is available on bacterial transfer from necrophagous insects to host tissues. Our data provides the first report on the occurrence of Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica in Lucilia illustris Meigen, 1826 (Diptera: Calliphoridae), and the quantitative dynamics of the two bacterial species along the insect life-stages and transfer to beef and pork host tissues using qPCR gyrase b specific primers. The content of both bacterial species increased along the insect life stages. W. chitiniclastica was detected in all developmental stages independent of the feeding substrate. I. indica was measurable with 102 gene copies ng−1 DNA threshold starting from the third instar larvae when feeding on beef, and from the egg stage with a 102× higher representation when using the pork substrate. The transfer of bacterial species to both tissues occurred after 3 colonization days except for I. indica that was visible in beef liver only during day 5. Considering the utilization of pork tissues as human analogues, these quantitative microbial dynamics data provides first insect-specific bacterial candidates as potential colonization biomarkers in forensic investigations.

Ice entrenched microcosm represents a vast reservoir of novel species and a proxy for past climate reconstitution. Among glacial ecosystems, ice caves represent one of the scarcely investigated frozen habitats. To characterize the microbial diversity of perennial ice from karst ecosystems, Roche 454 sequencing of 16S rRNA gene amplicons from the underground ice block of Scarisoara Ice Cave (Romania) was applied. The temporal distribution of bacterial and archaeal community structures from newly formed, 400, and 900 years old ice layers was surveyed and analyzed in relation with the age and geochemical composition of the ice substrate. The microbial content of cave ice layers varied from 3.3 104 up to 7.5 105 cells mL−1, with 59–78% viability. Pyrosequencing generated 273,102 reads for the five triplicate ice samples, which corresponded to 3,464 operational taxonomic units (OTUs). The distribution of the bacterial phyla in the perennial cave ice varied with age, organic content, and light exposure. Proteobacteria dominated the 1 and 900 years old organic rich ice deposits, while Actinobacteria was mostly found in 900 years old ice strata, and Firmicutes was best represented in 400 years old ice. Cyanobacteria and Chlorobi representatives were identified mainly from the ice block surface samples exposed to sunlight. Archaea was observed only in older ice strata, with a high incidence of Crenarchaeota and Thaumarchaeaota in the 400 years old ice, while Euryarchaeota dominated the 900 years old ice layers, with Methanomicrobia representing the predominant taxa. A large percentage (55.7%) of 16S rRNA gene amplicons corresponded to unidentified OTUs at genus or higher taxa levels, suggesting a greater undiscovered bacterial diversity in this glacial underground habitat. The prokaryotes distribution across the cave ice block revealed the presence of 99 phylotypes specific for different ice layers, in addition to the shared microbial community. Ice geochemistry represented an important factor that explained the microbial taxa distribution in the cave ice block, while the total organic carbon content had a direct impact on the cell density of the ice microcosm. Both bacterial and archaeal community structures appeared to be affected by climate variations during the ice formation, highlighting the cave ice microbiome as a source of putative paleoclimatic biomarkers. This report constitutes the first high-throughput sequencing study of the cave ice microbiome and its distribution across the perennial underground glacier of an alpine ice cave.

Extremophilic biocatalysts represent an enhanced solution in various industrial applications. Integrating enzymes with high catalytic potential at low temperatures into production schemes such as cold-pressed silymarin processing not only brings value to the silymarin recovery from biomass residues, but also improves its solubility properties for biocatalytic modification. Therefore, a cold-active lipase-mediated biocatalytic system has been developed for silybin acylation with methyl fatty acid esters based on the extracellular protein fractions produced by the psychrophilic bacterial strain Psychrobacter SC65A.3 isolated from Scarisoara Ice Cave (Romania). The extracellular production of the lipase fraction was enhanced by 1% olive-oil-enriched culture media. Through multiple immobilization approaches of the cold-active putative lipases (using carbodiimide, aldehyde-hydrazine, or glutaraldehyde coupling), bio-composites (S1–5) with similar or even higher catalytic activity under cold-active conditions (25 °C) have been synthesized by covalent attachment to nano-/micro-sized magnetic or polymeric resin beads. Characterization methods (e.g., FTIR DRIFT, SEM, enzyme activity) strengthen the biocatalysts’ settlement and potential. Thus, the developed immobilized biocatalysts exhibited between 80 and 128% recovery of the catalytic activity for protein loading in the range 90–99% and this led to an immobilization yield up to 89%. The biocatalytic acylation performance reached a maximum of 67% silybin conversion with methyl decanoate acylating agent and nano-support immobilized lipase biocatalyst.

Considering that most industrial processes are carried out under harsh physicochemical conditions, which would inactivate enzymes from commonly isolated mesophilic organisms, current studies are geared toward the identification of extremophilic microorganisms producing enzymes resistant to extreme salt concentrations, temperature and pH. Among the extremophiles, halophilic microorganisms are an important source of salt-tolerant enzymes that can be used in varying biotechnological applications. In this context, the aim of the present work was to isolate and identify halophiles producing hydrolases from the Atacama Desert, one of the harshest environments on Earth. Isolates were recovered from halite samples and screened for the presence of seven different hydrolase activities (amylase, caseinase, gelatinase, lipase, pectinase, cellulase and inulinase) using agar plate-based assays. From a total of 23 halophilic bacterial isolates, most showed lipolytic (19 strains) and pectinolytic (11 strains) activities. The molecular identification of eight selected isolates showed a strong similarity to members of the Halomonas and Idiomarina genera. Therefore, the present study represents a preliminary, but essential, step to identify novel biological sources of extremozymes in an environment once thought to be devoid of life.

This experimental procedure was designed to isolate total the RNA aiming to obtain intact and high-quality integrity (RIN>7) for further next generation sequencing analysis. The protocol was developed to analyse the environmental microbial active community providing a clear overview of the microbial transcriptional activity in a specific time or after a different condition exposition sets, such as the investigation of thermal adaptation after heat-shock. The procedure is specifically designed to extract total-RNA from water samples although it can be used for various environmental samples as done in Schostag et al. and Bang-Andreasen et al. 2019 in soil with similar accuracy results. The total-RNA yield was >1000 ng/mL with a RIN ranging from 7 to 8.5. The final Illumina library preparation yield ranged between 15 and 31 ng/µL. This protocol isa compiled by using the Qiagen RNeasy® PowerSoil® Total RNA Kit with additional steps according the DNeasy Max®Kit (Qiagen), NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, Inc), KAPA Library Quantification Kit for Illumina® Platforms (Kapa Biosystems), Qubit 2.0 (Thermo Fisher Scientific, Hvidovre, Denmark), Bioanalyzer® (Agilent Technologies, Inc.) sample Purification Beads (New England Biolabs) and Liquid G2 DNA/RNA Enhancer (Ampliqon) manufacturer protocols.

This survey presents the first high-throughput characterisation of fungal distribution based on ITS2 Illumina sequencing of uncultured microbiome from a 1500 years old perennial ice deposit in Scărișoara Ice Cave, Romania. Of the total of 1 751 957 ITS2 sequences, 64% corresponded to 182 fungal operational taxonomic units, showing a low diversity, particularly in older ice strata, and a distinct temporal distribution pattern. Ascomycota was the major phylum in all ice samples, dominating the 400 and 1500 years old ice strata deposited during the cold Little Ice Age (LIA) and Dark Ages Cold Period, while Basidiomycota was mostly present in 900-years old ice formed during the Medieval Warm Period (MWP). Chytridiomycota and Mucoromycota phyla were present in recently formed and 400-years old ice, respectively. Among the 80 identified genera, Cryptococcus victoriae, commonly found in glacial habitats, was identified in all strata. A positive correlation between fungal distribution and ice conductivity, Ca, Na and Sr concentrations was observed across the ice block, with pH values trailing climate variations during LIA and MWP, respectively. Our record highlighted the presence of a complex climate and environmental-driven fungal community in perennial ice strata accumulated during the last 1500 years in Scărișoara Ice Cave.

Extremophilic microorganisms are equipped with enzymes able to catalyze various reactions under extreme conditions of temperature, salinity, pH, hydrostatic pressure, radiation, etc. The particular characteristics of these enzymes, also known as extremozymes, represent valuable assets for advanced industrial applications. Most recently, extremophiles were used for the “green” and cost-effective production of high-quality nanomaterials on a large scale. The eco-friendly production of metallic nanoparticles and reduced graphene oxide using extremozymes, discussed here, represents an important breakthrough for various applications. Further benefits of extremozymes immobilized on a variety of nanomaterials, resulting in highly effective biohybrids including light-harvesting devices, biofuel cells, and biosensors, are also discussed. For biosensor development, the dual strategy for improving their stability and sensitivity is based on utilization of extremozymes as biocatalyst and of nanomaterials as high-loading support with role also in signal transduction. While a low assortment of such biosensors was developed so far, a largely unexploited applicative potential of combined use of extremozymes and nanomaterials in biosensing is anticipated due to their already demonstrated industrial benefits.

Cave ice ecosystems represent a poorly investigated glacial environment. Diversity of cave ice bacteria and their distribution in perennial ice deposits of this underground glacial habitat could constitute a proxy for microbial response to climatic and environmental changes. Scarisoara Ice Cave (Romania) hosts one of the oldest and largest cave ice blocks worldwide. Here we report on cultured microbial diversity of recent, 400, and 900 years-old perennial ice from this cave, representing the first characterization of a chronological distribution of cave-ice bacteria. Total cell density measured by SYBR Green I epifluorescence microscopy varied in the 2.4 x 104 – 2.9 x 105 cells mL-1 range. The abundance of cultured bacteria (5 x 102 – 8 x 104 CFU mL-1) representing 0.3-52% of the total cell number decreased exponentially with the ice age, and was higher in organic rich ice sediments. Cultivation at 4˚C and 15˚C using BIOLOG EcoPlates revealed a higher functional diversity of cold-active bacteria, dependent on the age, sediment content and physicochemical properties of the ice. The composition dissimilarity of ice microbiota across the ice block was confirmed by growth parameter variations when cultivated in different liquid media at low and high temperatures. PCR-DGGE and sequencing of bacterial 16S rRNA gene fragments from the cultured ice samples led to the identification of 77 bacterial amplicons belonging to Gammaproteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, showing variation in distribution across the ice layers. Several identified OTUs were homologous to those identified in other glacial and karst environments and showed partial conservation across the ice block. Moreover, our survey provided a glimpse on the cave-ice hosted bacteria as putative biomarkers for past climate and environmental changes.

During violent criminal actions in which the perpetrator disposes of the victim’s remains by burial, the analysis of insects and bacterial colonization patterns could be necessary for postmortem interval (PMI) estimation. Our research aimed to assess the decomposition process of buried rat carcasses from shallow graves (40 cm), the diversity and dynamics of insects and bacteria throughout the decomposition stages, and the environmental parameters’ influence on these variations. The results provide further insight on decomposition in soil and contribute to a broader understanding of the factors involved in decomposition by qualitatively and quantitatively analysing the decomposer community (bacteria and insects). Additionally, two bacterial taxa, Enterococcus faecalis and Clostridium paraputrificum that were investigated for the first time as PMI indicators using quantitative polymerase chain reaction (qPCR) showed differential abundance over time, promising data for PMI estimation. The current study on the decomposition of buried rat carcasses in a natural environment will strengthen the current knowledge on decomposed remains from shallow graves and represents an effort to quantify insect and bacterial taxa as PMI estimators.

Screening of 1,000-years old ice layers from the perennial ice block of Scărișoara Ice Cave (NW Romania) revealed the presence of fungal communities. Using culture-dependent methods and molecular techniques based on DGGE fingerprinting of 18S rRNA gene fragments and sequencing, we identified 50 cultured and 14 uncultured fungi in presently-forming, 400 and 900 years old ice layers, corresponding to 28 distinct operational taxonomic units (OTUs). The dominant ice-contained fungal OTUs were related to Ascomycota, Basidiomycota and Cryptomycota phyla. Representatives of Mucoromycota and Chytridiomycota were also isolated from recent and 400 years old ice samples. The cryophilic Mrakia stokesii was the most abundant fungal species found in the cave ice samples of all prospected ages, alongside other cryophilic fungi also identified in various glacial environments. Ice deposits formed during the Little Ice Age (dated between AD 1,250 and 1,850) appeared to have a higher fungal diversity than the ice layer formed during the Medieval Warm Period (prior to AD 1,250). A more complex fungal community adapted to low temperatures was obtained from all analyzed ice layers when cultivated at 4 °C as compared to 15 °C, suggesting the dominance of cold-adapted fungi in this glacial habitat. The fungal distribution in the analyzed cave ice layers revealed the presence of unique OTUs in different aged-formed ice deposits, as a first hint for putative further identification of fungal biomarkers for climate variations in this icy habitat. This is the first report on fungi from a rock-hosted cave ice block.

The decomposition process of human (and other mammalian) remains is influenced by numerous factors such as the environmental temperature, relative humidity, precipitation, geographical location of the remains, as well as the medical conditions of and any injuries sustained by the deceased person. The decomposition process generally follows a consistent pattern (fresh, bloat, active decomposition, advanced decomposition, and dry stage). Understanding the time-span of each stage of decomposition and the influence of the biotic and abiotic factors involved is imperative when trying to estimate the time elapsed since death (postmortem interval [PMI]). Over the course of decomposition, the tissues are gradually consumed by necrophagous insects and bacteria. The environmental temperature and its variations influence how insects colonize the remains, having a significant impact on their presence and developmental cycle. Additionally, the bacterial community colonizing decomposing tissues is also greatly affected by variations in environmental temperature. Because both the rate of decomposition of human remains and the relative abundance of certain insects and bacterial species are all temperature-dependent, insect and bacterial colonization data are useful as key points for the PMI estimation. This article reviews the current literature documenting the data collected on the occurrence and development cycles of predominant necrophagous Diptera (Calliphoridae, Muscidae, Sarcophagidae) and bacterial taxa involved in the decomposition of various carcasses at different temperatures. This review will impact the forensic community by providing an overview on the temperature, insect and bacterial records for the PMI estimation, seeking to aid forensic entomologists, microbiologists, pathologists, and the legal community.

The identification of necrophagous insect diversity and dynamics has forensic significance for postmortem interval estimation specific to burial. Few studies regarding the necrophagous entomofauna from buried remains have been performed to date. In contrast to the exposed carcasses, the accessibility of soil to insects is limited due to burial depth and is dependent on soil type. This study highlights the colonization behavior of Conicera similis (Haliday 1833) (Diptera: Phoridae) during carcass decomposition, a previously unobserved taxon in Romania. Adult and larvae specimens were collected from rat (Rattus norvegicus) carcasses buried at 40 cm depth in an urban park environment in June 2016 during active decomposition and their presence and activity period was correlated with the environmental parameters variation. Bacterial diversity from C. similis female adult and larvae specimens was determined via 16S rRNA gene Illumina sequencing to further characterize these commonly encountered and forensically important necrophagous insects. This report signals the easternmost geographical location in Europe (Bucharest, Romania) of C. similis to date.

The microbial life thriving in a wide variety of frozen environments raised a high interest in understanding their adaptation mechanisms to temperatures below 0°C and for using their advantageous applicative characteristics. Recent findings revealed the presence of complex microbial communities, comprising bacteria, archaea, and fungi in perennial ice blocks located in ice caves. The diversity of these relatively secluded glacial ecosystems responds to their particular sunlight regime, substrate geochemistry and age of the subterranean ice block deposits. This current synopsis of total and culturable microorganisms reported so far in ice caves in relation to the geochemical characteristics of the ice substrate and their spatial and temporal distribution highlights the viability and structural/functional diversity of this particular microcosm, and argues for the putative role of key microbial species from such ice habitats as proxies for palaeoclimate dating.

The present study represents the first report on the presence of Meroplius fukuharai (Diptera: Sepsidae) in Romania. The research area was located in Bucharest. Meroplius fukuharai was recorded during an experiment for investigating necrophagous insect species dynamics. Adult specimens were sampled during the summer (August 2013) from swine carcasses at the beginning of the advanced decay stage. The species had a sporadic occurrence, only four male specimens being sampled and identified both morphologically and genetically during the four-month survey. The recorded environmental parameters during the sampling period showed an air temperature of 28-33°C and a relative humidity of 53-57%. This report on the presence of M. fukuharai in Romania leads to the expansion of its known distribution range in the South Eastern part of Europe.

The estimation of postmortem interval (PMI) is affected by several factors including the cause of death, the place where the body lay after death, and the weather conditions during decomposition. Given the climatic differences among biogeographic locations, the understanding of necrophagous insect species biology and ecology is required when estimating PMI. The current experimental model was developed in Romania during the warm season in an outdoor location. The aim of the study was to identify the necrophagous insect species diversity and dynamics, and to detect the bacterial species present during decomposition in order to determine if their presence or incidence timing could be useful to estimate PMI. The decomposition process of domestic swine carcasses was monitored throughout a 14-wk period (10 July-10 October 2013), along with a daily record of meteorological parameters. The chronological succession of necrophagous entomofauna comprised nine Diptera species, with the dominant presence of Chrysomya albiceps (Wiedemann 1819) (Calliphoridae), while only two Coleoptera species were identified, Dermestes undulatus (L. 1758) and Creophilus maxillosus Brahm 1970. The bacterial diversity and dynamics from the mouth and rectum tissues, and third-instar dipteran larvae were identified using denaturing gradient gel electrophoresis analysis and sequencing of bacterial 16S rRNA gene fragments. Throughout the decomposition process, two main bacterial chronological groups were differentiated, represented by Firmicutes and Gammaproteobacteria. Twenty-six taxa from the rectal cavity and 22 from the mouth cavity were identified, with the dominant phylum in both these cavities corresponding to Firmicutes. The present data strengthen the postmortem entomological and microbial information for the warm season in this temperate-continental area, as well as the role of microbes in carcass decomposition.

This report on the occurrence of Themira nigricornis (Meigen, 1826) (Sepsidae: Diptera) on Romanian territory represents the first record of this species within south-eastern Europe. Adults of T. nigricornis were collected in spring, in the second half of April, from an experimental setting placed in an urban location (Bucharest) in southern Romania. The bait consisted of domestic pig carcass in advanced decomposition stage, placed on soil ground and surrounded by vegetation. The visiting specimens were located around the posterior segment of the pig carcass. The registered abundance of adult individuals was relatively low, with 10 specimens distinguished during two weeks interval. The species was taxonomically characterized and identified. This study, documenting the presence of T. nigricornis in the southern part of Romania, expanded the Fauna Europaea distribution range record of this species, assigning the southernmost location of T. nigricornis in Europe.

We examined the potential for natural attenuation of ten hydrokarst systems (HKS) in three mountain units in the Carpathian Mountains. We sampled in places where water enters below ground and where water emerges back to the surface in springs and is used as drinking water by the local communities. Water samples were used to assess the degree of chemical and microbiological pollution. Although the water in the ten HKS was rather clean, a general decrease in the concentration of most chemical compounds was observed along the flow path, regardless of the number of tributaries the underground stream receives. Dilution caused by tributaries could not account for the decrease in the concentration of most compounds. The contribution of other chemical immobilization processes, such as retention of pollution in the subsurface or sorption to sediment particles was suggested, in combination with the activity of microorganisms. The bacteria diversity is complex and decreases from upstream to downstream locations due to dilution with water provided by tributaries or retention of bacteria in the subsurface by adhesion to substrates. We suggest that karst can have a significant potential for natural attenuation by retaining the pollution underground, in combination with biodegradation performed by microorganisms.