c Principal Coordinate Analysis PCoA of samples according to the predictive functional profile. d Significantly expressed MetaCyc pathways predicted functional profile according to LEfSe. The bigger the LDA value obtained for a feature, the more significant.

Only significant features are plotted in the histogram. d Nonoxipent: Pentose phosphate pathway I non-oxidative branch , UMP syn I: UMP biosynthesis I, Pyr syn III: Pyrimidine deoxyribonucleotides de novo biosynthesis III, Glucurocat: β- d -glucuronosides degradation, L-Arg syn III: l -arginine biosynthesis III via N-acetyl- l -citrulline.

PCoAs were plotted using Vega editor v5. Overall, Enterobacteriaceae bacteria promoted the presence of other Enterobacteriaceae, especially between bacteria negatively correlated with Commensalibacter. In the predictive functional profile, natural and agricultural environments presented either the lowest or highest recruitment of features.

Similarity analysis via PCoA demonstrated clustering of environments along the PCo1 axis Fig. Natural samples indicated significant recruitment of several anabolic reactions for the generation of precursor metabolites, nucleosides and nucleotides, while Arginine biosynthesis and β-D-glucuronoside degradation pathways were more representative of agricultural samples Fig.

The semi-natural environment retained no significant pathways, even though it had intermediate abundances for every agricultural- and natural-significant pathway detected.

Environmental effects were clear, with only Sphingomonas, Bradyrhizobium and Methylobacterium abundantly present in all environments. Agricultural and semi-natural apiaries were overrun by Proteobacteria and more enriched than natural samples for Firmicutes Lactobacillus, Staphylococcus, Streptococcus, Paenibacillus.

Gammaproteobacteria mainly Arsenophonus, Stenotrophomonas and Pseudomonas were representative of agricultural samples, as was Lactococcus Firmicutes Fig.

Natural samples had a divergent microbial profile, with abundance of both Actinobacteria and Bacteroidia classes. Two genera significant for agriculture, Arsenophonus and Lactococcus , showed intermediate abundances in semi-natural samples Fig. Species wise, Paenibacillus larvae and Lactobacillus kunkeei both Bacilli plus Corynebacterium afermentans sub.

larvae present in natural samples and practically absent in agricultural hives, while L. kunkeei and C. afermentans were present in agricultural hives but absent in natural hives Supplementary Table S4. Characterization of the bacterial communities in hive entrance samples.

a Significantly enriched bacteria in each environment, according to LEfSe. Agricultural hives were rich in Gammaproteobacteria and Lactococcus. The classes Actinobacteria and Bacteroidia were prevalent in natural samples. c Principal Coordinate Analysis PCoA of samples according to the predictive functional profile MetaCyc pathways.

d Significantly recruited functions according to LEfSe. e Significantly enriched enzymes according to LEfSe. The enzymes Endo X3 EC 3. Median relative abundances of bacterial and functional biomarkers showing intermediate values in the semi-natural location for gut, hive entrance and bee bread samples.

The scales of the axes vary according to the relative abundance of the plotted feature. b Predicted functional biomarkers in the gut. d Predicted functional biomarkers in the hive entrance. e Bacterial biomarkers in bee bread.

Nonoxipent: Pentose phosphate pathway I non-oxidative branch , L-Arg syn III: l -arginine biosynthesis III via N-acetyl- l -citrulline , UMP syn I: UMP biosynthesis I, Pyr syn III: Pyrimidine deoxyribonucleotides de novo biosynthesis III, Glucurocat: β- d -glucuronosides degradation.

Plotting: Schematics were done in INKSCAPE v0. Concerning interactions among bacteria, correlations were mostly grouped by taxa. Exclusion, marked by negative correlation, was detected between several Actinobacteria or Bacteroidia versus genera such as Arsenophonus, Corynebacterium 1, Micrococcus and Gaiella Fig.

In the predictive functional profile, natural hives clustered together explaining None of the significantly recruited functions were exclusive to one environment. The only non-ubiquitous function was lipopolysaccharide LPS biosynthesis absent in natural colonies Fig.

Natural colonies exhibited increased frequency of the Bifidobacterium shunt, l -methionine biosynthesis mostly mediated by transsulfuration occurring from oxaloacetate , and a tricarboxylic acid cycle specific to acetate-producers TCA cycle VII Fig. Agricultural hives possessed enriched synthesis of nucleotides, cofactors, nicotinamide adenine dinucleotide NAD , membrane components Kdo2 lipid A, LPS, mycolate and fatty acids.

The tRNA processing pathway resulting in tRNA activation, and the stringent specific ppGpp metabolism were also significantly expressed Fig. Semi-natural samples had significant recruitment of the tryptophan 7-halogenase enzyme Fig. Contrasting environments shared similar microbiomes, with differences primarily found in abundances of scarce taxa.

Sphingomonas and Methylobacterium Alphaproteobacteria were overall the most abundant genera, followed by Acinetobacter in the natural environment and Bradyrhizobium in the other two Supplementary Table S2. Environmental effects were scarce for internal air, and only seen in the enrichment of Enterobacteriaceae mostly Arsenophonus , Curtobacterium and Massilia in agricultural samples Supplementary Fig.

Beta-Diversity results of gut bacterial communities and predicted functions showed that semi-natural samples clustered between natural and agricultural hives Figs. This effect was also apparent for predicted functions of hive entrance Figs.

In concordance, semi-natural hives showed intermediate relative abundances for several taxa and functional pathways, while natural and agricultural environments exhibited either lowest or highest relative abundances Fig. This trend was more evident at a functional Fig. Bacterial representatives showing intermediate abundances for gut samples included Comensalibacter , which was enriched in natural, scarce in semi-natural and mostly absent in agricultural samples.

An unknown Rhizobiaceae genus exhibited the opposite tendency enrichment in agriculture and absence or nearly absence in natural samples Fig. Same pattern was observed for Lactococcus in hive entrance Fig. A less pronounced transition was detected for Lactobacillus in gut samples and Pseudomonas in the hive entrance.

Both were present in all environments but augmented in natural and agricultural environments, respectively, while the semi-natural environment had intermediate abundances. Bee bread and gut samples also showed overall intermediate abundances for some non-environmentally relevant bacteria, such as Bradyrhizobium in bee bread and Gilliamella in gut Supplementary Table S2.

Besides the aforementioned bacteria showing intermediate abundances, all significantly recruited functions in gut and hive entrance samples had highest or lowest recruitment in natural and agricultural colonies, and intermediate values in the semi-natural environment Fig.

This behaviour was clear in the agricultural recruitment of NAD and Kdo2-lipid A synthesis, both displaying relative mean abundances under 0. The exceptions to the rule were the gondoate anaerobic synthesis enriched in semi-natural hive entrance and the Bifidobacterium shunt with equally low relative abundances in both anthropized locations Fig.

Several studies have revealed anthropization-induced bacterial shifts in the honey bee gut microbiota 14 , 16 , 17 , 19 , 30 , disturbing gut microbial abundances, composition and functions.

In this study, environmental anthropization resulted in the enrichment of potential pathogens and bacteria capable of surviving in contaminated landscapes, as well as recruitment of stress response-related functional pathways in bee gut and hive entrance samples.

Importantly, semi-natural hives, despite being genetically identical to the agricultural hives formed from same origin bees, queens and food reserves but unrelated to the natural ones, showed intermediate values at taxonomic and functional levels. This mixing of agricultural and natural traits likely stemmed from environmental factors e.

pollen diversity or pollution. Overall, bacterial communities associated with natural hives did not differ from the expected core profile for gut samples 13 , and were devoted to performing essential functions. Bacteria adapted to the natural environment were mainly found in the hive entrance.

These natural hives were heavier but less populated than the non-natural beehives. Reduced colony sizes have been associated with Varroa -surviving colonies Worker guts from the natural environment displayed bacterial profiles associated with good health, and were enriched in Acetobacteraceae and the gut core members Snodgrasella , Lactobacillus and Commensalibacter involved in nutrient acquisition and immune responses.

Some Lactobacilli are core members of the honey bee gut microbiome, ferment bee-diet byproducts 9 , 10 , 11 and can inhibit the pathogen Paenibacillus larvae in the gut of larvae 7.

Interestingly, Paenibacillus appeared in the natural apiary at low abundances across all the apibiome, except for beebread where its abundances equalled the ones found in agricultural samples.

larvae was also present in natural hive entrance samples, yet no clinical symptoms of American Foulbrood were seen, suggesting spore inactivation or suppression of the pathogen by the bee gut bacterial community. Lactobacillus impoverishment as detected in our agricultural hives has been detected following antibiotic application Snodgrasella further contributes to improved honey bee gut equilibrium by facilitating biofilm formation in the gut 34 , maintaining anaerobiosis for gut symbionts 9 , and expressing immune genes after Escherichia coli infection Protozoan inhibition in natural honey bee gut through a combined enrichment of Lactobacillus , Commensalibacter and Snodgrasella was previously hypothesised as likely 30 , and known to occur in bumblebees Consistently, Commensalibater and Lactobacillus were positively correlated in our study and were dominant in the natural environment.

All in all, the consortium of genera found in guts of natural colonies maintained gut environment homeostasis anaerobiosis and biofilms and possibly hindered infectious or opportunistic colonizations, supporting honey bee welfare by both suppressing pathogen infections or allowing its tolerance e.

larvae , and by favouring the intake of essential molecules and nutrients. Taken together, this natural taxonomic profile represented a balanced honey bee microbiome. In contrast, agricultural gut bacterial communities were enriched in non-core bacteria, similar to the findings of Muñoz-Colmenero et al.

The agricultural guts were rich in Enterobacteriaceae and Rhizobiaceae, both anthropization-related, with Rhizobiaceae having been linked to sugar syrup feeding 15 and Enterobacteriaceae to Coumaphos and Chlorothalonil pesticide usage 18 as well as to anthropization Several Enterobacteriaceae species are also opportunistic environmental bacteria and their presence in the honey bee gut has been associated with colony health weakening 37 and illness Various Enterobacteriaceae within our samples were negatively correlated with Commensalibacter and Bartonella.

Augmentation of Bartonella was detected in winter bees 32 and in newly emerged bees after nutritional stress and Nosema ceranae infection Thus, whilst Commensalibacter maintained homeostasis of natural bacterial communities, Bartonella might have performed a similar function in semi-natural colonies.

In summary, enrichment of opportunistic bacteria and reduction of beneficial taxa was evident in agricultural colonies, confirming the detrimental impact of environmental anthropization.

In between both extremes, semi-natural profiles were shifting towards natural communities despite genetic similarities and geographic proximity to agricultural colonies. Honey bee gut bacteria show a diminished metabolism 39 specialised in the usage of recalcitrant compounds sugars, flavonoid glycosides, etc.

derived from the bee diet In this sense, most gut organisms conduct anaerobic carbohydrate fermentation 11 , 39 while a few take part in biofilm formation or cell adhesion, such as Gilliamella apicola, Snodgrasella alvi, Lactobacillus and Bifidobacterium 12 , In concordance, the natural environment displayed an inherent metabolism supporting the synthesis of indispensable metabolites UMP and pyrimidine deoxyribonucleotides and the recruitment of ubiquitous anabolic reactions non-oxidative branch of the pentose phosphate pathway, PPP.

These inherent pathways, depleted in agricultural guts, showed intermediated recruitment in semi-natural samples, suggesting a weakened metabolism in both anthropized locations.

Pyrimidine synthesis was especially low in agriculture, which could reflect the diminished Lactobacillus abundances in that location as Lactobacillus synthesise pyrimidine exclusively In combination with a less active anabolism, agricultural samples exhibited stress-related pathways.

They had increased arginine Arg biosynthesis, linked to the aftermath of cold stress in the diptera Bactrocera dorsalis 40 , and increased β- d -glucuronoside degradation. Glucuronosides are formed in mammals as byproducts of hepatic glucuronidation, enabling detoxification of unwanted and toxic compounds 41 , and are later excreted into the gut.

Recruitment of β- d -glucuronoside degradation within bee guts suggests glucuronoside presence following glucuronidation of toxic molecules by honey bees.

The presence of xenobiotics might force the gut bacterial microbiome to invest most of its energy into defence mechanisms, thus neglecting pathways needed for the community to thrive i. functions seen in natural guts. Agricultural honey bees endure under these conditions, but are unable to achieve a sound health state.

Aside from the honey bee gut, other niches within the apibiome also showed a strong environmental impact.

The hive entrance, being directly in contact with the hive external area, appeared as a valuable indicator of the landscape. The coastal location of natural colonies in Unije island promoted enrichment of mostly aquatic and salt-tolerant bacteria.

Bacteroidetes , for instance, are often enhanced in haloalkaline habitats 42 such as our natural shoreline i. high salinity and humidity. More relevant to our study was the decreased presence of the contamination-resistant Sphingomonas 43 , and of the potential pathogen Arsenophonus 37 , The bacteria enriched in agricultural and semi-natural landscapes are common in environments associated with plants e.

soils and roots. Resistance to environmental contamination has been reported or suspected for some of these bacteria. Gammaproteobacteria, enriched in agricultural colonies, are highly adaptable chemotrophs suggested to resist unfavourable conditions 42 , while the Sphingomonas and Gemmatimonas genera in semi-natural hives enriched and present respectively are key bacteria in cadmium-contaminated and saline-alkaline stressed soils Increased Gemmatimonas abundance has also been linked to Pyraclostrobin fungicide application 44 and to long-term use of organic and inorganic fertilisers Sphingomonas are often associated with plant microbiomes, capable of degrading several recalcitrant substances and common helpers of fungi and plants during metal-degradation of soils 43 , Sphingomonas proliferation in leaf microbiomes has been reported after anti-pathogen treatment of plants A similar Sphingomonas enrichment to the one observed here in semi-natural hives was reported for petroleum-contaminated soils Indeed, semi-natural and agricultural apiaries were situated near traditional oil and natural gas exploitations Enrichment of potential pathogens was another common trait of semi-natural and agricultural hives.

Agricultural samples were particularly enriched in Pseudomonas 50 and Arsenophonus. In bees, Arsenophonus has been linked to increased death rates 26 and weakened colony health 37 , whilst enrichment of one Arsenophonus candidate was associated with increased incidence of Colony Collapse Disorder Interestingly, one study showed that both environment and social interactions play an important role in honey bee Arsenophonus acquisition 26 , and Arsenophonus was enriched in all agricultural hive niches.

Arsenophonus could be a biomarker of anthropization, transmitted through social activities. However, the same study 26 demonstrated that Arsenophonus abundances within honey bee guts were very location-dependent, with nearby hives sharing similar abundances.

Thus, we cannot discard the influence of apiary in the differences detected for Arsenophonus abundances. Semi-natural hives also revealed several potential honey bee pathogens, including the human-affecting Streptococcus 51 , Anaerococcus 52 and Paenibacillus.

The latter genus posed a great risk to honey bees, since Paenibacillus larvae is the causative agent of American Foulbrood AFB If transmitted from entrance to bees, Paenibacillus could infect the brood and threaten semi-natural colonies.

Likewise, the Lactobacillus kunkeei present in these semi-natural beehives, if transmitted to the brood, could protect semi-natural colonies by increasing brood resistance against both P. larvae and Nosema ceranae 7 , Further studies would be needed to determine P.

larvae transmission within beehives. On the contrary, the hive entrances of natural hives exhibited an adapted and overall more pathogen-poor bacterial community, with the exception of P. larvae more abundant than in agriculture.

Agricultural hive entrances, compared to semi-natural, had less positive reinforcements against pathogens and contamination i. less abundance of Lactobacillus kunkeei and bioremediators.

Whilst natural hive entrances recruited functions prevalent among balanced metabolisms e. methionine synthesis , agricultural hive entrances had more active stress-related pathways. They recruited Gram negative bacterial pathways for synthesis of outer membrane components e. Kdo2-lipid A, LPS as well as the stringent response-inducing ppGpp metabolism, which enables bacterial persistence and pathogenicity 55 , The semi-natural profiles shifted towards natural abundances.

Hive internal air and bee bread were the least influenced niches in the apibiome. Internal air, formed by floating abiotic and biotic particles found within hives, was expected to act as an indicator of beehive fitness. Indeed, shifts in airborne microbiome composition have been linked to soil, flora and possibly pollution 57 , as well as urbanisation Consequently, we expected differences between anthropic and natural landscapes, but the internal hive air microbiota turned out to be mostly stable and largely unaffected by environmental factors.

This could be because free floating particles within hives, as happens with pollen granules, might stick to bee bodies and reduce the pool of available bacteria that can be sampled from the in-hive air. Anthropization had a meagre effect on bee bread, but differences among environments were consistent with the changes detected in gut and hive entrance.

Bee bread sample composition resembled previous studies 8 , 23 , 30 , and overall natural hives had slight enrichment of acidic and sugar-tolerant bacteria while agricultural hives were enriched for Arsenophonus , previously described in bee bread obtained from multiple habitats Herein, Arsenophonus was most likely transmitted from agricultural bees to their food stores or vice versa, as agricultural bee guts possessed slight Arsenophonus enrichment.

Contamination of the food reserves by potential pathogens such as Arsenophonus might negatively impact honey bee health at the agricultural environment, as consumption of said food could result in gut microbiome dysbiosis or affect the whole apibiome.

One of the main findings of this study was the intermediated relative abundances observed in semi-natural hives for some bacteria e. Comensalibacter , Lactobacillus , Arsenophonus and several predicted pathways e. The intermediate state was more widespread for pathway recruitment than for community composition, indicating an early functional response of the beehive bacterial community.

Importantly, 16 days of exposure to an anthropization gradient were sufficient to shift the bacterial fraction of the apibiome of hives. Agricultural hives stayed anthropized while semi-natural bacterial apibiomes became more balanced, resembling the profiles found in a non-anthropized apiary.

As a parallel, quick adaptability of the gut microbiome when under pressure has been reported in humans 59 , 60 and other mammals Similarly, honey bee colony-wide analysis of molecular biomarkers also demonstrated an overall increase in the expression of vitellogenin regulatory protein within bees , antioxidant enzymes and immune proteins in hives situated near areas under wildlife recuperation for the US Conservation Reserve Program Our results show that placing beehives in less anthropized areas more natural with less agricultural pressure would lead to recruitment of beneficial bacteria e.

Lactobacillus and Commensalibacter in honeybee guts and induce functional reorganisation. Indeed, habitat restoration in agricultural areas by planting native flora can favour recovery of pollinator populations 3 , including wild bees Decreased anthropization of hives increased the relative abundances of beneficial bacteria in all of the sampled hive niches of the apibiome, albeit at different rates, and induced shifts in predicted functional profiles of guts and hive entrances.

These results highlight the quick adaptability of honey bee-associated microbiomes. They offer straightforward management strategies to strengthen bee colonies by reducing the impact of anthropization by planting of indigenous flora around crops, or relocating hives to more natural areas whilst maintaining current agricultural production.

These results also highlight the relevance of the hive as the unit of study for microbial research, as opposed to bees, in order to understand the contribution of each niche to colony health and resilience, as well as the importance of their interactions. Larger longitudinal and long-term analyses considering seasonal changes would enable the identification of global patterns of anthropization and of core microbes within hive niches, and contribute towards the identification of: 1 beneficial profiles that could be targeted to strengthen honey bee health at any time-period, 2 bacteria that weaken colonies, and 3 biomarkers, as Arsenophonus appears in this work, indicating the risk status of hives under anthropization.

Thus, a checklist of safety and hazard markers could be developed as a management tool to employ as a bioindicator of beehive health. Samples were obtained from 3 apiaries within Croatia. On 20 May , 33 hives were formed in the agricultural region of Marijančaci Hives contained one super each.

All frames were standard Langstroth, and sister queens and same-origin worker bees were utilised to avoid genetic variation. Hives were moved the next day.

Twenty-two hives were relocated 24 aerial km away to the agricultural region of Kozarac The remaining 11 colonies were moved to Vardarac These 11 colonies were designated as the semi-natural apiary. Ten additional hives were located in Unije Seven hives were already established on this natural location beforehand.

All 7 had 1 lacked management since including pesticide treatments , 2 survived Varroa destructor infestations and 3 been previously studied by Muñoz-Colmenero et al. Two supers were added to all 10 hives located in this natural landscape.

All three apiaries remained untreated during this experiment. Agricultural exploitations and commercial beekeeping practices are regular in Osječko-Baranjska encompassing both Kozarac and Vardarac apiaries. Grasslands, fruit trees apple and plum , and intensive commercial crops such as rapeseed, wheat, sunflower, corn, soybeans and barley surrounded the agricultural apiary 30 , 64 , In contrast, the natural location of Unije had pastureland, tufted hair grass, maquis olive groves , coniferous woodland, and mixed broad-leaved trees holm oaks Arable land was limited to small grassland and shrub plantations around the village Colony strength parameters were assessed on the 6th of June following the Liebefeld method 67 , Portable electronic scales were used to weigh entire hives.

Both sides of all frames were checked for in-field measurement of adult bees, brood and pollen areas dm 2 , as well as frame walls and bottom board for measurement of adults.

Total adult bee, brood and pollen loads were calculated by multiplying the area by adult bees or brood and pollen as required for standard LR Langstroth frames Varroa load was measured simultaneously by the Powdered Sugar method Statistical analyses for colony strength differences among environments were performed in R Rv3.

Samples were collected in June 6, , 17 days after colony formation. When possible, 4 sample types were collected per hive: young worker bees collected from brood frames most likely nurses for gut dissection G , 8 cm 2 of bee bread comb randomly selected from a single frame PB , microorganisms stuck to the hive entrance and collected by swab scrubbing the entire entrance S , and filters containing vacuum filtered internal air F from the hive.

Older workers could not be sampled due to the short period elapsed since hive formation. The entrance was swabbed by scrubbing left and right around 6 times per swab tip 3—4 swabs per hive. Sampling of air was done by placing, on top of the honey super, a plastic dome Lekliško cupola, produced by Dubravko Leskovic with a perforated side attached to a vacuum hoover Hf, J.

Holdings with filters. The vacuum was left running for 10 min. Air samples were not collected in the semi-natural environment due to material limitations.

In total, samples were collected from the 43 hives comprising this study. The supernatant was collected and placed in a clean 1. This process was repeated once more by adding µl 1xPBS to recover the maximum liquid sample size.

Cell lysis and DNA extraction was performed following the protocol established by Muñoz-Colmenero et al. Then the tubes were vortexed and incubated at 56 °C and rpm for 90 min. The resulting supernatant was collected and placed in a clean 2 mL tube. This step was performed twice in order to recover as many microorganisms as possible, after which both supernatants were combined and μL of ethanol were added.

A 2 mL PowerBead Tube with 0. Samples were homogenised using a Precellys 24 tissue homogenizer Bertin Technologies for 4 min. Then the tubes were incubated at 65 °C for 15 min and centrifuged at 10,× g for 30 s. The supernatant was collected and combined with μL of solution C2, after which tubes were again incubated at 4 °C for 5 min and centrifuged at 10,× g for 1 min.

Approximately μL of supernatant were then transferred to a 2 ml collection tube, where μL of pre-shaken solution C4 were added. These primers contained Illumina sequencing adaptors and a 12 bp barcode sequence bound to the forward primers, allowing sample identification. PCR products were examined on a 1.

The DNA purification of the PCR products, the preparation of the libraries and the paired-end sequencing were performed at the Sequencing and Genotyping Unit of the University of the Basque Country SGIKER. Quality of raw sequences was checked with FastQC High Throughput Sequence QC Report v0.

Demultiplexing of the sequences without Golay error correction , in-depth sequence quality control by the denoise-paired DADA2 method 72 , and taxonomic assignment were performed in Qiime2 v2. Amplicon sequence variants ASVs present in a single sample were removed. The original feature table was split by sample type to create sample type specific datasets gut, bee bread, hive entrance and internal air datasets.

Phylogenetic trees were generated from these datasets using mafft and fasttree alignment in Qiime2. Taxonomic analysis was preceded by mitochondrial and chloroplast sequence removal.

Relative abundances of bacteria were represented for phyla and genera via qiime taxa barplot. A common sequencing depth for all sample types was determined through alpha rarefaction curves, and utilised to calculate and compare Alpha diversity values among sample types.

Samples presenting lower sequence depths were thus filtered out. Visualisation was conducted in R with ggplot2 and dplyr. Sequencing depth for each sample type was then determined through alpha rarefaction curves, and rarefied data sets were obtained per sample type for comparison of environments through Alpha and beta Diversity analyses.

For each hive niche, Alpha diversity analyses were performed again and for Beta diversity analyses Bray—Curtis distance community composition dissimilarity was computed using Qiime2 and visualised as Principal Coordinate Analysis PCoA via Vega editor v5.

Permutational multivariate analysis of variance PERMANOVA was calculated in Qiime2 based on rarefied Bray—Curtis matrices and with pairwise BH-FDR correction, to determine whether the bacterial communities between environments differed significantly. Considering that data dispersion can confound PERMANOVA results, homogeneity of group dispersion PERMDISP 77 for environments was calculated with betadisper on the same matrices.

Spearman correlation-based circular UPGMA trees unweighted pair group method with arithmetic mean were obtained in Qiime2 and displayed via iTOL Interactive Tree of Life tool, v6.

Colors indicating anthropization level within UPGMA trees were added via INKSCAPE v0. The feature frequency tables of each sample type were collapsed at genus level and transformed to relative abundances. Tables were uploaded to the Galaxy web application 79 where Linear Discrimination Analysis LDA size Effect LEfSe 80 was used to identify the bacteria driving the differences among environments.

LEfSe uses a non-parametric factorial Kruskal—Wallis sum rank test 81 to identify differentially abundant taxa, followed by a canonical method to calculate which taxa combinations contribute more to environmental differences. Histograms and cladograms of results were plotted within the Galaxy web application, and taxa names within graphs cleaned using INKSCAPE v0.

test from the Hmisc package, applying BH-FDR correction, and visualized using corrplot package. Non-linear sample distribution was checked before Spearman correlation analysis, using shapiro.

test normality test in R 82 and BH-FDR correction. Mean relative abundances of all significant genera were represented in tables using percentages. To determine if environmental changes could impact honey bee apibiome functionality, functional prediction of E.

enzymes 83 and MetaCyc pathways 84 were performed for gut and hive entrance samples using the PICRUSt2 v2. The resulting E. and pathway tables were rarefied for diversity analyses. BH-FDR correction was applied to p-values of pairwise analysis. Bray—Curtis distances were visualised via PCoA to determine environmental dissimilarities.

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In this case, nutrient loads are low compared with the environments discussed above and the ecosystem is far from being eutrophicated. However, as in the more complex environments Zhang et al. At times when perturbation was higher i.

summer , the composition of bacterial communities deviated clearly from that of the reference sites: a high proportion of sequences from the Gammaproteobacteria and Bacteroidetes , as well as a reduction of Alphaproteobacteria , was observed; typical oligotrophic groups such as SAR11 or SAR86 decreased or disappeared, and the main alphaproteobacterial species belonged to the Roseobacter clade Nogales et al.

These patterns are the same as those found in VH and the Venice lagoon Zhang et al. Therefore, independently of the particular phylotypes observed in each site which appeared to be different after sequence comparison , there seems to be a general response of microbial communities to nutrient enrichment, at least in seawater, which seems to be independent of the absolute magnitude of the nutrient load.

Thus, there seems to be a counter-selection against typical oligotrophic microorganisms and the proliferation of copiotrophic bacteria able to grow and respond to increased nutrient concentrations, such as certain groups of Gammaproteobacteria i.

Alteromonadales , Pseudoalteromonas , Pseudomonadales , OM60 group , members of the division Bacteroidetes and representatives of the Roseobacter clade in the Alphaproteobacteria.

All these groups have been associated with experimental nutrient enrichment or algal blooms in marine environments Eilers et al. These microorganisms probably react directly to nutrient enrichment by increasing their growth rate and their biomass, following the typical strategy of fast growers.

None of the studies conducted so far have explored at the same time the diversity of phytoplankton and bacterioplankton in nutrient-enriched environments, and, therefore, this aspect remains unknown.

A good model for studying more directly the effect of nutrient enrichment in marine ecosystems without the presence of additional stressors is aquaculture.

Fish production in aquaculture farms is a growing economical sector worldwide but has the drawback of releasing high amounts of organic matter resulting from uneaten fish food and faecal material to the seawater and the sediments below the cages.

Because aquaculture has been shown to have an effect on environmental quality and health, there are numerous studies addressing this issue.

Meta-analysis of published ecological data from the water column, as well as studies conducted in particular locations, have shown that fish farms have a significant local effect on the pools of particulate and dissolved organic and inorganic nutrients, such as particulate organic phosphorus, particulate organic nitrogen, dissolved organic nitrogen, dissolved organic carbon, ammonium and nitrite La Rosa et al.

Usually, no effects are observed on the concentration of phosphate and silicate. As in most complex environments nutrient enrichment from fish cages causes a significant increase in bacterioplankton abundance and heterotrophic production Sakami et al.

Although increases in phytoplankton abundance is not always evident from chlorophyll data, a stimulatory effect of fish cages on autotrophic eukaryotic phytoplankton has been observed as well, although this does not lead to the development of phytoplankton blooms because there seem to be a tight control by grazing Navarro et al.

Hydrology is probably also playing a role because fish farms are usually located in areas with good water exchange. Despite all these important perturbations of the marine microbial food web, few studies have reported which are the changes in microbial communities that occur under fish cages in comparison with control areas.

In a study performed in farms of milkfish Chanos chanos in the Philippines, Garren et al. There were few phylogenetic groups represented in libraries from the fish pens and the proportion of clones affiliated to the Cyanobacteria , the Proteobacteria and the Bacteroidetes were high.

The results of this study would indicate a reduction in the bacterioplankton diversity near fish cages in comparison with surrounding areas, contradicting the findings reported above about microbial diversity in nutrient-enriched coastal areas. This apparent reduction in bacterial diversity due to aquaculture should be confirmed in other systems because it might not be a general finding.

For example, Wei et al. However, it should be taken into account that fish pens and ponds are completely different aquaculture systems and therefore the effect on microbial communities might also be different. On the other hand, if we take into account the fact that the perturbation caused by a fish farm is more constant in amount, composition and periodicity of additions i.

fish food and fish faeces , we can hypothesize that this might lead to the development of microbial communities highly specialized in processing this particular type of organic load, maybe dominated by a few very efficient microorganisms, and therefore less diverse. The most dramatic effect of the nutrient load from fish farms occurs in sediments below the cages.

There, the high organic load, mainly in the form of particulate organic matter, increases oxygen demand and alters the biogeochemistry of the sediments. The benthic biomass in environments affected by fish farms becomes dominated by microbial components, and bacterial abundance increases significantly compared with control areas Mirto et al.

In contrast, fish farms cause a decline of some groups of benthic fauna and seagrasses Mirto et al. With respect to functionality, the organic load of fish farming stimulates microbial anaerobic respiration processes in sediments below fish farms, such as sulphate, iron and manganese reduction, denitrification, methanogenesis and fermentation Christensen et al.

By far the most important anaerobic process in fish-farm sediments, as in other nutrient-enriched environments, is sulphate reduction because sulphate is usually present in nonlimiting concentrations in marine sediments Holmer et al.

The production of high amounts of hydrogen sulphide by sulphate reduction, concomitant with a decrease in oxygen, might lead to the inhibition of important microbial processes in the nitrogen cycle such as nitrification oxidation of ammonium to nitrate , and hence, to lower the rates of coupled denitrification reduction of nitrate to nitrogen gas , which requires oxidized forms of nitrogen.

Coupled nitrification—denitrification is important for reducing the high nitrogen load of fish-farm sediments and for controlling the efflux of ammonium from the sediments to the water column McCaig et al. However, in reduced fish-farm sediments, the process of dissimilatory nitrate reduction to ammonium, which also needs oxidized forms of nitrogen, can be quantitatively more important than denitrification.

As a result of reduced nitrification, there is less nitrogen removed from the sediment as nitrogen gas ultimately released to the atmosphere and a higher efflux of ammonium to the water column, which stimulates primary production and maintains the nitrogen in the system Christensen et al.

But even if nitrification is not inhibited, it might be insufficient to oxidize all the ammonium resulting from the organic load to nitrate, and consequently there is a net flux of ammonium to the water column Bissett et al. The bacterial composition in sediments under fish farms along gradients of organic pollution, and hence sulphide and oxygen concentrations, have been analysed by 16S rRNA gene-based approaches in Japan Asami et al.

Significant differences in composition of clone libraries from fish-farm and reference sediments are usually observed, both at a broad and at a fine phylogenetic resolution Bissett et al. These studies have also shown that bacterial community composition changes significantly after cessation of production, such as in periods of fallowing, but the communities do not revert to the composition before the impact, i.

they are not resilient Bissett et al. A distinctive characteristic composition of bacterial communities in fish-farm sediments cannot be easily drawn due to the high microbial diversity found in marine sediments Asami et al.

However, even when these farm sediments were located in different areas and they were dedicated to the production of different fish species, there were some common findings that allow the formulation of certain general trends. For example, an increase in the abundance of Deltaproteobacteria which includes most of the sulphate-reducing genera is generally observed.

This has been proven by a higher proportion of deltaproteobacterial 16S rRNA gene clones in fish-farm libraries Castine et al. Besides, in agreement with a high production of sulphide by sulphate reduction, a characteristically high proportion of clone sequences of putative sulphur-oxidizing Gammaproteobacteria Asami et al.

Sequences of the Epsilonproteobacteria are also abundant Bissett et al. This group is also related to the metabolism of reduced sulphur compounds and it is usually found in sulphide-rich sediments Campbell et al. The proportion of clones affiliated to the division Bacteroidetes Flavobacteria in particular , which are supposed to play a role in biopolymer degradation, tends to be high in fish-farm sediments as well, and the phylotypes recovered are different from those in control areas Asami et al.

Finally, clone sequences related to betaproteobacterial nitrifiers are usually not recovered in libraries from fish-farm sediments targeting total bacterial diversity Bissett et al.

However, using molecular tools targeting selectively this bacterial group, several authors have demonstrated a different composition of the betaproteobacterial nitrifiers in fish-farm sediments compared with control sites McCaig et al.

These changes in the composition of microorganisms participating in the degradation of the organic load and in the sulphur and nitrogen cycles, together with the changes in process rates i.

sulphate reduction , provide good evidence of an altered functionality of the microbial communities in sediments below fish farms. The perturbation is likely to have an effect on archaeal groups too, but the composition of archaeal communities or their role in biogeochemical processes such as ammonia oxidation or methanogenesis in these environments has not been explored so far.

Taking together the results of the studies conducted in environments receiving point and diffuse nutrient inputs, some general effects of nutrient enrichment on microbial community composition and function can be proposed Fig.

Thus, despite the composition of the microbial assemblage developing at a particular site, it seems clear that the structure of the microbial food web will be affected as well as important biogeochemical cycles, such as the nitrogen, carbon and sulphur cycles.

General effects of two examples of human-derived stressors on marine microbial communities. Persistent organic pollutants, such as herbicides aldrin, DDT, hexachlorobenzene, etc. They reach the marine environment by direct discharges from coastal areas or dumping into the sea , runoff from land, river discharges or atmospheric deposition.

They also adsorb to floating and stranded plastics, which contributes to their transport and persistence in the environment Rios et al. Among the different pollutants entering the marine environment, the effect of hydrocarbon pollution on marine microbial communities has been the subject of numerous studies, mainly driven by the concerns caused by tanker accidents such as that of the Exxon Valdez in Alaska or the more recent accident of the Prestige tanker in Spain.

Most of these studies have focused on analysing the effect of crude oil spills on microbial diversity, the changes in response to bioremediation trials usually involving the addition of nutrients and the extent of hydrocarbon biodegradation in polluted environments.

These topics are covered in excellent recent review articles Head et al. The results obtained show that the microbial communities developing in response to events of hydrocarbon contamination differ in composition although the efficacy in hydrocarbon removal is similar Head et al.

Events of acute contamination cause a reduction of microbial diversity in the short term. This is due to two main reasons: the disappearance of certain groups of microorganisms i.

archaea and cyanobacteria and the strong selection for specialist hydrocarbon-degrading marine bacteria i. Alcanivorax , Cycloclasticus , which become predominant particularly when nutrients are added to stimulate hydrocarbon degradation Head et al.

In contrast, microbial diversity is usually high in chronically or long-term hydrocarbon-polluted marine environments Hernandez-Raquet et al. This behaviour is exactly the same as that observed in environments polluted chronically with nutrients Zhang et al.

Typically, 16S rRNA gene sequences related to known hydrocarbon degraders [i. Alcanivorax , Cycloclasticus , etc. Yakimov et al. This might indicate that those hydrocarbon degraders are minor components of these communities, and therefore are missed because of method limitation, although they become predominant in bioremediation trials.

For example, sequences affiliated to Alcanivorax were not observed in mesocosms prepared with seawater from Messina Harbour but they became predominant 15 days after the addition of oil and nutrients Cappello et al. Also, most-probable-number counts of hydrocarbon degraders in two marinas in the United States were shown to be low Piehler et al.

Recent studies conducted in areas polluted after the Prestige tanker oil spill sampled one year after the accident revealed the importance of members of the suborder Corynebacterineae i. Rhodococcus and the family Sphingomonadaceae in the degradation of the alkane and aromatic fraction of this heavy oil, respectively Alonso-Gutiérrez et al.

These results show the wide diversity of bacterial hydrocarbon degraders in environmental samples and the importance of certain groups i. Actinobacteria as hydrocarbon degraders in rough environments i.

rocks polluted with heavy oil. Alternatively, there might be novel, uncharacterized degraders in these polluted environments Paissé et al. We have experimental evidence supporting this hypothesis coming from an analysis of aromatic ring-hydroxylating dioxygenase genes ARHD in polluted coastal sediments from Patagonia in Argentina Lozada et al.

Sequences representative of the already known phnAc -like genes of Alcaligenes faecalis AFK2, phnAI -like genes of Cycloclasticus spp. and nahAc -like genes of Pseudomonas spp. These novel ARHDs contained the conserved residues of bacterial ring-hydroxylating dioxygenase α-subunits Lozada et al.

We have a biased perception of the risk associated to hydrocarbon pollution towards contamination caused by tanker accidents and this has motivated most of the research on hydrocarbon pollution and bioremediation studies conducted so far.

However, the contribution of tanker accidents to total hydrocarbon contamination in the sea is very low. For example, in the Mediterranean Sea, 80 tonnes of oil were spilled in accidents between and [ Environmental European Agency EEA , ].

In contrast, tonnes are discharged per year due to shipping operations i. discharges of ballast water, tank washing, oil sludge, bilge water or engine room wastes and about tonnes per year from oil terminals and routine land-based operations EEA, Therefore, it is important to take into account this type of pollution and to assess properly its effects on coastal microbial communities.

An example of a study of the effect of land-based sources of hydrocarbon pollution on microbial communities is the one done in a chronically polluted coastal retention basin in the Mediterranean Sea Etang de Berre, France.

This coastal lagoon receives hydrocarbons from refineries, petrochemical plants and transportation systems. Samples were taken along the hydrocarbon pollution gradient Hernandez-Raquet et al. This means that, although other environmental factors were determining microbial composition in the area, hydrocarbons appeared as a key factor for diversification of communities.

When the composition of bacterial communities was analysed in detail, a predominance of sequences related to Delta - and Gammaproteobacteria , as usually observed in marine sediments, was obtained, although variability in different sampling years was high as happens also in nutrient-enriched environments Hernandez-Raquet et al.

As usual, only a few sequences could be related to hydrocarbon-degrading bacteria and these included sequences related to Marinobacter spp. Therefore, anaerobic metabolism of hydrocarbons by SRB seems to be common in polluted environments.

As mentioned above, activities derived from shipping are important sources of hydrocarbon contamination in the marine environment EEA, ; Halpern et al. A good proportion of the pollution caused by ships is due to illegal discharges. In the Mediterranean, for example, the density of spills can be correlated with major shipping routes Ferraro et al.

The effect of this type of diffuse hydrocarbon pollution in the composition of seawater microbial communities has not been addressed but it might be a factor to consider for explaining the described cosmopolitan distribution of specialist hydrocarbon degraders in the marine environment Yakimov et al.

In areas exploited for tourism, there is a consistent increase in the number of hydrocarbon spill detections during the high season, correlating with the increase in boating activity Ferraro et al. The pollution caused by maritime transport and recreational boats is likely to be a source of fuels i.

diesel and synthetic lubricants to seawater and sediments. There are few studies addressing the effect of this type of pollution. One experiment was performed in in situ mesocosms with water and sediments from a tropical estuary in Singapore, impacted by boating-derived activities.

The mesocosms were treated with diesel oil at concentrations reproducing the mean and the highest hydrocarbon concentrations measured in the area Nayar et al.

The response observed was negative for eukaryotic phytoplankton and picocyanobacteria Synechococcus at higher hydrocarbon concentrations, although picocyanobacteria were stimulated at the lower concentrations.

In contrast, the number of heterotrophic bacteria and production rates increased in response to the treatment, particularly at the higher concentrations Nayar et al. These results show that there is a rapid response of microbial communities to diesel pollution in the marine environment, as demonstrated for more complex hydrocarbon mixtures such as crude oil Head et al.

A second study tested the short-term effect of diesel oil, a biodegradable lubricant and a synthetic lubricant clean and used in a field experiment in pristine Antarctic sediments Powell et al. Although the bacterial communities differed from the control in all treatments, the most significant variations were due to treatment with diesel, followed by synthetic lubricants, but not with the biodegradable lubricant Powell et al.

These results show ways of reducing the impact of lubricants in the marine environment, particularly in sensitive areas such as the poles. Maritime activities require the building of ports and recreational marinas. Because of the constant increase of maritime transport United Nations Conference on Trade and Development, and the interest in increasing tourism-derived activities European Commission, , there is a constant pressure for the development of harbours and recreational marinas, and therefore, we can expect an increase in the environmental stress posed by them.

Some of the pollutants that are commonly found in harbours are hydrocarbons i. as result of boat traffic, accidental spills or discharge of bilge oil and ballast water , detergents, surfactants or antifouling compounds i. heavy metals, biocides , even though ports have facilities for the collection of wastes and for pollution control.

Additionally, they receive nutrient inputs, either directly due to harbour activities or through discharges of rivers and sewage effluents such as VH in Hong Kong Zhang et al.

Harbour seawater and sediments have been shown to hold particular bacterial communities, different from those in adjacent areas, highly diverse and highly variable at the temporal scale Schauer et al. Despite chemical pollution, the effect of nutrient enrichment seems to be the most important factor driving the composition of bacterioplankton in harbours, and for example nutrient enrichment might explain the high abundance of sequences related to Clostridium and Vibrio in the sediments of Milazzo Harbour, subjected to high organic load Yakimov et al.

Relationships between bacterioplankton community composition and hydrocarbon pollution were only found in the study conducted in Messina Harbour, where the relative abundance of putative hydrocarbon-degrading bacteria correlated with an increase in hydrocarbons Denaro et al.

In sediments, stimulation of hydrocarbon degraders has been observed after hydrocarbon addition, indicating that there is a primed microbial community able to use these pollutants, both aerobically and anaerobically Hayes et al. As in other hydrocarbon-polluted sediments, SRB seem to be involved in hydrocarbon degradation in harbour sediments.

For example, a recent study on the composition of the active sulphate-reducing assemblages in sediments of Boston Harbour by analysing transcripts mRNAs of dsrAB genes Chin et al.

Several studies have addressed the composition of bacterial communities in marine sediments polluted with heavy metals. As in the case of other pollutants, bacterial communities in sediments with different levels of heavy-metal pollution are shown to differ Gillan et al.

The same result has been observed for archaeal and photosynthetic communities Toes et al. Different from the cases of nutrient or hydrocarbon pollution, which seem to stimulate bacterial growth, total prokaryotic cell numbers appear to be negatively correlated with heavy metals such as cadmium, copper, zinc and lead Gillan et al.

However, when the calculations were done for particular phylogenetic groups, few statistically significant correlations could be established, and they seemed to be dependent on the sample. Therefore, it is difficult to determine the effect of heavy metal on particular groups of microorganisms with the data available so far.

In highly polluted sediments of a Norwegian fjord, the abundance of Gammaproteobacteria determined by FISH counts was negatively correlated with copper and zinc, and that of Bacteroidetes with copper, zinc and cadmium. In contrast, in Belgian sediments with lower pollution levels, the only significant correlation was found between Bacteroidetes and cadmium Gillan et al.

In these two sediments, bacterial community composition was also analysed by a cloning and sequencing approach. The results evidenced the presence of the main groups typically found in marine sediments: Gamma - and Deltaproteobacteria and Bacteroidetes. But some of the clones in the Norwegian sediment grouped with clones from an Antarctic sediment polluted with heavy metals and hydrocarbons, and a group of gammaproteobacterial sequences was found in both the Norwegian and the Belgian sediments Gillan et al.

This might indicate that certain bacterial populations might be favoured in heavy-metal-polluted sediments, although these relationships are even more difficult to draw than in the case of hydrocarbon pollution. A laboratory experiment was performed in microcosms to simulate the effect of sediment disturbance dredging in a sediment chronically polluted with heavy metals.

In addition, the effect of deposition of a 3 mm layer of polluted sediment on the surface of unpolluted sandy sediments was also analysed Toes et al. These two practices dredging and deposition onto a different location are common in the marine environment. Homogenization of the polluted sediment simulating dredging caused minor changes in the composition of microbial communities.

However, overlying of sandy sediments with polluted sediment caused significant changes in the composition in the three microbial components analysed Bacteria , Archaea and Cyanobacteria in comparison with the unpolluted control, although the effect was lower for Archaea Toes et al.

After one year of incubation, some common bacterial groups were observed in clone libraries from the artificially polluted sandy sediments in comparison with those from the chronically polluted sediment: representatives of the Roseobacter clade, the genus Vibrio and a member of the Flavobacteriaceae whose sequence was related to a clone from a heavy-metal-polluted Antarctic sediment Toes et al.

These sequences might represent microorganisms favoured by the conditions imposed by heavy-metal pollution, although to confirm this, it will be necessary to corroborate their absence or lower abundance in control, unpolluted sediments.

In the environment, removal of sediments polluted with mercury, PCBs and PAHs in a location in the Baltic Sea resulted in the development of significantly different bacterial communities.

Summarizing the studies presented on chemical and heavy-metal pollution, we can conclude that, as in the case of nutrient enrichment, this type of perturbation again alters the composition of microbial communities Fig. But because this type of pollution selects for those microorganisms that are capable of degrading or chemically transforming the pollutants, and sometimes these organisms belong to particular phylogenetic groups, the studies conducted so far can point to particular types of bacteria i.

oligotrophic marine hydrocarbon-degrading Gammaproteobacteria , Actinobacteria , Sphingomonaceae or certain SRB phylotypes likely to be contributing to alleviate the problem of chemical pollution in the environment.

However, in many cases, these is only indirect evidence that does not fill the gap in knowledge on fundamental aspects of the functionality of microbial communities in chemically polluted environments.

For example, novel or unrecognized microbial genera might be involved in the degradation of contaminants, most likely through metabolic cooperation with other microorganisms using degradation intermediates. Microorganisms as such, i. forming part of degradation networks, will not be isolated as typical pollutant degraders and therefore escape our knowledge, although they are probably key players in the environment.

Other fundamental aspects, such as how pollution affects bacterial production rates, respiration rates or other microbial processes have not been analysed in chemically polluted environments, in contrast to what has been done in nutrient-enriched environments.

The broad use of high amounts of antibiotics in intensive farming to prevent or treat animal infections is also causing environmental concern see review Cabello et al. The bactericidal action of antibiotics can cause changes in the composition of natural microbial communities by selectively inhibiting susceptible bacteria.

Besides, exposure of natural communities to antibiotics might lead to the selection of resistant bacteria, which can then transfer their resistance determinants to opportunistic pathogenic bacteria in the environment. In relation to the marine environment, there are numerous data coming from aquaculture, where administration of high amounts of a variety of antibiotics in fish farms for prophylactic or therapeutic reasons is routinely done Cabello, ; Sapkota et al.

Antibiotic treatment in fish farms leads to increased concentrations of these compounds in water and sediments below the cages as well as in the fish stock produced. As a result, there is a positive selection for antibiotic-resistant bacteria in water and sediments from fish cages, surrounding areas and among fish-associated bacteria Chelossi et al.

Often, the isolation of multiresistant bacteria i. bacteria resistant to several antibiotics is reported. This is in agreement with the simultaneous use of several types of antibiotics in fish farms Dang et al.

Antibiotic-resistant bacteria isolated from fish-farm water and sediments are diverse. Although most of them belong to the Gammaproteobacteria genera Vibrio , Photobacterium , Pseudomonas , Pseudoalteromonas , Alteromonas , Citrobacter , Salmonella , isolates from the Alphaproteobacteria , Firmicutes , Actinobacteria and Bacteroidetes have also been reported Furushita et al.

The presence of antibiotic-resistance genes in these isolates, as well as the ability of some of the isolates for transferring the resistance genes by conjugation to Escherichia coli recipient strains, have been demonstrated Rhodes et al.

Besides, closely related plasmids of the IncU group, carrying oxytetracycline-resistance determinants, have been found in isolates from hospitals in the UK and Germany and from fish-farm environments, demonstrating that plasmid transfer might occur between natural bacteria and potential human pathogens Rhodes et al.

Instead of isolating antibiotic-resistant bacteria, Hargrave et al. They measured profiles of resistance to the antibiotic oxytetracycline, widely used in aquaculture, in mixed bacterial communities from sediments around salmon aquaculture farms and in feed pellets which were proposed as the source of antibiotics.

Usually, numbers were higher in surface sediments but resistance levels were still high at intermediate depths, which mean that sediments around fish farms can constitute reservoirs for antibiotic-resistant bacteria in the marine environment Hargrave et al.

It is important to recognize that fish farms are not the only source of antibiotics in the marine environment. Thus, oxytetracycline-resistant bacteria are present in surface seawater and sediments receiving the input of sewage treatment plants, as demonstrated in Jiaozhou Bay in China and Halifax Harbour in Canada Dang et al.

The study conducted in the area of VH, where discharges of Anthropogenic use of coastal areas i. beaches also seems to have an effect on the natural abundance of antibiotic-resistant bacteria. Studies performed in sand from beaches of the Southern Baltic Sea coast showed that bacteria isolated from a recreational beach had higher resistance to antibiotics from different chemical families than those in nonrecreational areas Mudryk, ; Mudryk et al.

The level of multiresistance resistance to several antibiotics was also higher among isolates from the recreational beach. The examples presented here provide evidence of the potential health risk derived from antibiotic contamination in the marine environment because they demonstrate the selection and enrichment of multiresistant bacteria.

Some of them might be potential human pathogens i. Vibrio , Pseudomonas , Salmonella or might potentially transfer genetic determinants of resistance to pathogenic bacteria. But none of these studies have addressed how antibiotics alter the composition and functionality of microbial communities in marine environments.

Given the magnitude and worldwide extension of the problem of antibiotic pollution, it is imperative to design carefully environmental and laboratory studies to address this issue, to determine which microbial groups are affected by the presence of antibiotics in the water and how antibiotics alter the functionality of microbial communities, both in water and in sediments.

Several of the uses of coastal resources have the risk of introducing potential human pathogenic bacteria, as shown in Table 1. The highest risk is derived from direct sewage discharges, which are a source of human faecal bacteria.

Therefore, sewage treatment and routine controls for faecal indicators coliforms, E. coli , enterococci in seawater in areas dedicated to bathing are usual procedures in developed countries Stewart et al.

However, sewage discharge is not the only source of faecal bacteria in marine environments. Other human-derived sources of potential pathogens are runoff from land urban and agricultural areas , leaking septic tanks, sewer overflows, discharges from boats, etc.

Stewart et al. Sources of faecal bacteria other than human i. birds, pets, wildlife, etc. should not be disregarded Choi et al.

A recent publication has presented a novel and interesting approach to assess faecal pollution in coastal watersheds using community-based indicators Wu et al.

Using PhyloChip, a phylogenetic microarray, these authors identified operational taxonomic units OTUs characteristic of faecal samples mainly belonging to the phyla Firmicutes , Proteobacteria , Bacteroidetes and Actinobacteria which were designated as faecal subsample associated OTUs FSAO. By analysing the similarity of bacterial communities in several environmental samples from two watersheds in Santa Barbara CA to FSAO, they could identify the samples exposed to faecal sources.

The results obtained agree with traditional culture methods for the detection of faecal bacteria. In addition, they proposed a new community-based indicator to assess ecosystem health, based in the ratio of relative richness of three bacterial classes: Bacilli , Bacteroidetes and Clostridia to that of the Alphaproteobacteria BBC: A.

This ratio is higher in faecal and sewage samples and lower in samples not impacted by faecal material Wu et al. Approaches like this represent an important step forward in the analysis of faecal contamination in coastal areas because it is based in powerful molecular methods, and in the assessment of whole communities instead of using a few two or three indicator bacteria.

Faecal material is also a source of human pathogenic enteric viruses. Viruses from faecal origin belong to the families Adenoviridae adenovirus , Caliciviridae i.

Norwalk virus , Picornaviridae i. poliovirus, hepatitis A and Reoviridae reoviruses and rotaviruses. These viruses cause a variety of diseases in humans, either by direct exposure to water or after ingestion of contaminated seafood Griffin et al.

Viruses are shown to persist better in the environment than the bacterial indicators used for water quality monitoring Griffin et al. Apart from causing diseases in human and other marine mammals, epidemic viral infections can cause significant economic losses in aquaculture plants for commercial production of fish and shellfish Lang et al.

In addition to discharges of faecal material, other human activities such as bathing constitute a source of potential microbial contamination. For example, enterococci and Staphylococcus aureus are transferred directly from the skin of bathers in high loads into seawater 3—6 × 10 5 and 6.

These results highlight the importance of nonenteric bacteria as potential pathogens in the environment Stewart et al. Besides the health risk posed by allochthonous microorganisms from faecal or other origin, there are several potential human pathogenic species that are indigenous to marine and estuarine environments, such as Vibrio cholerae , Vibrio vulnificus and Vibrio parahaemolyticus Thompson et al.

The first two species, V. chlolerae and V. vulnificus , can cause severe, fatal diseases. In contrast, V. parahaemolyticus infections are usually not life-threatening but they are very common worldwide, mainly due to the consumption of contaminated seafood Collins et al.

These opportunistic pathogenic bacteria have a variety of virulence factors such as haemolysins V. vulnificus and V. parahaemolyticus , toxins cholera toxin , colonization factors, etc. Thompson et al. The presence of pathogenicity-associated genes from V.

cholerae and V. parahameolyticus has been proven in environmental isolates from different Vibrio spp. In the marine environment, vibrios are usually associated to surfaces and establish symbiotic relationships with phyto- and zooplankton i.

copepods , which might constitute vectors for disease transmission and for the development of disease outbreaks Lipp et al. Therefore, conditions favouring the development of phytoplankton blooms, such as in eutrophicated environments, might favour the development of vibrios.

In fact, as mentioned previously in this review, Vibrionaceae have been found in nutrient-rich environments Yakimov et al. In addition, vibrios proliferate at higher water temperatures and have a wide tolerance range to salinity, being able to survive well in low-salinity brackish water.

Therefore, their incidence is expected to increase in a scenario of global climate change. Different marine dinoflagellates Dinophysis spp. brevis and Gambierdiscus toxicus and a diatom Pseudonitzschia produce toxins with a variety of neurologic, gastrointestinal, respiratory and irritating effects.

Disease is usually caused by consumption of contaminated fish or shellfish, although aerosol inhalation and direct eye or skin exposure can also cause problems. Environmental perturbations derived from human activities that might have an effect in the increased incidence of harmful algal blooms are multiple and include: nutrient enrichment eutrophication , particularly in coastal waters; destruction of coastline due to coastal exploitation i.

Potential pathogens might be disseminated in the marine environment due to long-distance transport and discharge of ship ballast water used for ship stability and trim. For example, ballast water has been shown to contain epidemic-causing serotypes of V.

coli including strain O , Enterococcus spp. Concerns of the danger of ballast water discharges, not only for the spread of microbial pathogens but also of invasive species, have resulted in the establishment of guidelines for ballast water management and the promulgation of the International Convention for the Control and Management of Ship's Ballast Water and Sediments in by the International Maritime Organization.

These numbers are highly variable and depend on many factors such as the source region of the boat, season of the year and ballast-water management, i. whether or not there had been open-ocean exchange of water Drake et al.

The changes in the microbial component of ballast water during a transoceanic voyage in tanks with and without open-ocean exchange have been studied by quantifying bacterial and viral numbers Drake et al.

The results showed that containment of ballast water in boat tanks led to a decrease in the bacterial and viral load in comparison with the initial water, irrespective of performing open-ocean exchange or not Drake et al.

Bacterial communities in ballast water initially resemble those of the source seawater, which is usually from a coastal location Tomaru et al. This ballast water is replaced in the open ocean at some point during the voyage to reduce the risk of discharging invasive species in the reception port.

The open-ocean water filling the tanks has a significantly different microbial composition Tomaru et al. This means that allochthonous microorganisms from coastal regions are routinely discharged into the open ocean, and vice versa, open-ocean microorganisms are discharged into coastal regions.

However, the microbial assemblages discharged in each case are different from those in the source water because there is a change in composition during the voyage within the time frame of days Tomaru et al. A recent study analysed bacterial diversity in ballast water in a ship anchored in Xiamen Port, and compared it with that of the receiving harbour seawater Ma et al.

The source of the ballast water was Singapore and it had been partially replaced with water from the South China Sea. The analysis of the corresponding 16S rRNA gene clone libraries revealed that ballast water contained a less diverse bacterial community, with representatives of only two classes, Alpha - and Gammaproteobacteria.

There was no evidence of the presence of pathogenic bacteria. Within these two groups, the phylotypes retrieved in ballast water were different from those in Xiamen Harbour seawater Ma et al.

Thus, wherever it is done, the discharge of ballast water might potentially alter, at least transiently, the autochthonous microbial composition of the discharge site.

Whether this is significant or not has not been determined. If an open-ocean exchange is performed, we can expect that most of the coastal microorganisms will not be able to compete with the autochthonous bacteria in the more oligotrophic open ocean, and the opposite, i.

oligotrophic bacteria from the open ocean will not survive in coastal regions. Results from metagenomic studies show that microbial communities with dissimilar genomic composition i. genetic repertoire develop in different marine locations or at different depths DeLong et al.

Therefore, the success of an invasive microorganism, potentially dangerous such as a pathogen, would depend on the size of the inoculum, its capability to survive in the new environment and its competitive ability in facing a microbial community adapted to the conditions at the site of discharge.

As reported by Halpern et al. Thus, the interest in studying the effect of processes such as increase of seawater temperature and ocean acidification is increasing rapidly in the last years. No particular studies have been done targeting the microbial compartment but the information gathered so far provides clues on how microbial communities could be affected.

Anthropogenic activities such as the burning of fossil fuels, deforestation, industrialization and cement production are causing increased levels of atmospheric carbon dioxide CO 2 and, consequently, an increase in dissolved CO 2 in the oceans. The acidification of the oceans will be detrimental for calcifying organisms and will cause changes in species distribution and abundance, as well as in food-web dynamics and structure.

The increase in dissolved CO 2 has an effect in the carbon cycle of the oceans but also in the cycles of the major nutrient elements: nitrogen, phosphorus, silicon and iron for a review, see Hutchings et al. The information available indicates that three important processes in the nitrogen cycle might be affected.

For example, nitrogen fixation is predicted to increase in a high-CO 2 ocean, based on the results of experiments done with nitrogen-fixing Trichodesmium and unicellular Cyanobacteria. In contrast, a decrease in pH seems to reduce nitrification rates and the abundance of bacterial and archaeal nitrifiers in seawater Hutchings et al.

Consequently, the fluxes of oxidized nitrogen species, such as nitrate, will be affected. This in turn would have a negative effect on denitrification rates, although there can also be positive effects over this process due to the expansion of suboxic zones in the oceans.

An increase in the relative proportions of ammonium to nitrate due to decreased nitrification will cause a shift in the composition of primary producers, by favouring microbial components such as picocyanobacteria and nanoflagellates Hutchings et al. On the other hand, global warming will lead to an increase in seawater temperatures and cause a stronger stratification of the oceans.

This will limit the fluxes of nutrients from sediments and the deep sea i. phosphorus, iron , and potentially intensify problems of hypoxia and anoxia. Changes in the climate system can also cause alterations in the abundance and global distribution i.

spread from tropical areas to lower latitudes of pathogens such as Vibrio spp. The abundance of V. cholerae , the causative agent of cholera epidemics, is already shown to follow climatic patterns Lipp et al. Higher water temperatures, together with increased nutrient levels, would also increase the frequency and severity of harmful algal blooms Moore et al.

Environmental changes due to anthropogenic activities seawater warming or increased CO 2 and nutrient supply are also suspected of being involved in the expansion of coral diseases worldwide, diseases in which microorganisms are important Sokolov et al. In addition, strong rainfall events and flooding due to sea-level changes, both expected under a global change scenario, would result in an increase of estuarine and brackish low-salinity environments, which again would favour the growth of Vibrio spp.

Lipp et al. Moreover, floods may also cause problems of inefficient water sanitation and increase runoff from land, with the consequent increase in the risk of faecal bacterial and viral , nutrient and chemical contamination of the marine environment Kite-Powell et al.

The possibility of generating phytoplankton blooms by ocean fertilization with the aim of increasing the flux of organic matter towards the deep ocean causing carbon burial and alleviating problems of increasing CO 2 concentrations is catching the attention of companies for commercial exploitation.

This represents a new anthropogenic use of the marine environment. Several scientific experiments involving iron and phosphorous fertilization have been performed in order to test the premise that these nutrients were limiting primary production in different oceanic regions Thingstad et al.

However, based on the experience gathered on the functioning of marine ecosystems and the interplay of biogeochemical cycles, there is strong scientific criticism of the usefulness of ocean fertilization to sequester carbon, as well as concerns about the risks associated Secretariat of the Convention on Biological Diversity, In particular, the plans of the Australian company Ocean Nourishing Corporation to discharge urea in nitrogen-deficient areas of the ocean have been criticized on scientific grounds Glibert et al.

In this scenario, there will not be an increase in CO 2 sequestration and carbon burial, and in addition, undesired gases will be released into the atmosphere, aggravating the problem instead of alleviating it. Worldwide marine ecosystems suffer from the impact caused by human activities.

In view of the development of our societies and the increase in human population, the stress posed by humans to the marine environment will continue to increase. The microbial component of marine ecosystems has been neglected in many studies of anthropogenic impact.

Therefore, our knowledge on microbial communities of anthropogenically impacted environments lags far behind those involving higher organisms. Many studies report changes in prokaryotic numbers and a few have included measurements of microbial activity.

Changes in diversity have also been analysed, although in many cases, this has been done only by electrophoretic profiling methods. As a result, we have a partial view on the composition of microbial communities in stressed environments. Therefore, there is a need for a better cataloguing of microbial diversity in anthropogenically stressed environments.

New technologies such as remote ocean-observing systems, metagenomics, metatranscriptomics and massive tag sequencing can help us to gain more information on these communities. Thanks to the use of comparative studies of impacted and reference sites, we know that human impact causes significant changes in microbial community composition.

However, we are still unable to make predictions on how important this could be for the functioning of the ecosystems, particularly in the long term, especially if the stressor does not disappear or it is likely to increase.

For example, we know already that certain microbial groups are favoured in conditions of nutrient enrichment or chemical pollution, but most of this information is provided for broad phylogenetic groups i.

division, class and not for particular species or ecotypes. phylum, class, genera, species, etc. There is also a need for relating the changes in diversity to the metabolic processes, which are important for the functioning of the ecosystem.

Because of the complexity of marine ecosystems, these goals would need the combination of environmental observations as well as carefully designed microcosms or mesocosms experiments. In the context of global climate change, for which human activities can be blamed, microbial communities of human-stressed environments are of paramount importance, and therefore the study of their composition, dynamics and functioning is of the utmost relevance for the development of less destructive human practices or for alleviating problems already present in our environment.

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