Organic matter processing in a [simulated] offshore wind farm ecosystem in current and future climate and aquaculture scenarios.

The rapid development of blue economy and human use of offshore space triggered the concept of co-location of marine activities and is causing diverse local pressures on the environment. These pressures add to, and interact with, global challenges such as ocean acidification and warming. This study investigates the combined pressures of climate change and the planned co-location of offshore wind farm (OWF) and aquaculture zones on the carbon flow through epifaunal communities inhabiting wind turbines in the North Sea. A 13C-labelled phytoplankton pulse-chase experiment was performed in mesocosms (4 m3) holding undisturbed hard-substrate (HS) communities, natural sediment with infauna, and mobile invertebrate predators. Carbon assimilation was quantified under current and predicted future-climate conditions (+3 °C and -0.3 pH units), as well as a future-climate co-use scenario with blue mussel (Mytilus edulis) aquaculture. Climate change induced an increase in macrofaunal carbon assimilation as well as an organic enrichment of underlying sediments. Dynamic (non-)trophic links between M. edulis and other HS epifauna resulted in shifts among the species contributing most to the phytoplankton-derived carbon flow across climate scenarios. Increased inter- and intraspecific resource competition in the presence of M. edulis aquaculture prevented a large increase in the total assimilation of phytoplankton by HS fauna. Lower individual carbon assimilation rates by both mussels and other epifauna suggest that if filter capacity by HS epifauna would approach renewal by advection/mixing, M. edulis individuals would likely grow to a smaller-than-desired commercial size. In the same scenario, benthic organic carbon mineralisation was significantly boosted due to increased organic matter deposition by the aquaculture set-up. Combining these results with in situ OWF abundance data confirmed M. edulis as the most impactful OWF AHS species in terms of (total) carbon assimilation as well as the described stress responses due to climate change and the addition of bivalve aquaculture.

A cluster of Legionnaires' disease in Belgium linked to a cooling tower, August-September 2016: practical approach and challenges.

A cluster of Legionnaires' disease (LD) with 10 confirmed, three probable and four possible cases occurred in August and September 2016 in Dendermonde, Belgium. The incidence in the district was 7 cases/100 000 population, exceeding the maximum annual incidence in the previous 5 years of 1.5/100 000. Epidemiological, environmental and geographical investigations identified a cooling tower (CT) as the most likely source. The case risk around the tower decreased with increasing distance and was highest within 5 km. Legionella pneumophila serogroup 1, ST48, was identified in a human respiratory sample but could not be matched with the environmental results. Public health authorities imposed measures to control the contamination of the CT and organised follow-up sampling. We identified obstacles encountered during the cluster investigation and formulated recommendations for improved LD cluster management, including faster coordination of teams through the outbreak control team, improved communication about clinical and environmental sample analysis, more detailed documentation of potential exposures obtained through the case questionnaire and earlier use of a geographical information tool to compare potential sources and for hypothesis generation.

Physiological responses to ocean acidification and warming synergistically reduce condition of the common cockle Cerastoderma edule.

The combined effect of ocean acidification and warming on the common cockle Cerastoderma edule was investigated in a fully crossed laboratory experiment. Survival of the examined adult organisms remained high and was not affected by elevated temperature (+3 °C) or lowered pH (-0.3 units). However, the morphometric condition index of the cockles incubated under high pCO2 conditions (i.e. combined warming and acidification) was significantly reduced after six weeks of incubation. Respiration rates increased significantly under low pH, with highest rates measured under combined warm and low pH conditions. Calcification decreased significantly under low pH while clearance rates increased significantly under warm conditions and were generally lower in low pH treatments. The observed physiological responses suggest that the reduced food intake under hypercapnia is insufficient to support the higher energy requirements to compensate for the higher costs for basal maintenance and growth in future high pCO2 waters.

Cryptic diversity and ecosystem functioning: a complex tale of differential effects on decomposition.

Marine ecosystems are experiencing accelerating population and species loss. Some ecosystem functions are decreasing and there is growing interest in the link between biodiversity and ecosystem functioning. The role of cryptic (morphologically identical but genetically distinct) species in this biodiversity-ecosystem functioning link is unclear and has not yet been formally tested. We tested if there is a differential effect of four cryptic species of the bacterivorous nematode Litoditis marina on the decomposition process of macroalgae. Bacterivorous nematodes can stimulate or slow down bacterial activity and modify the bacterial assemblage composition. Moreover, we tested if interspecific interactions among the four cryptic species influence the decomposition process. A laboratory experiment with both mono- and multispecific nematode cultures was conducted, and loss of organic matter and the activity of two key extracellular enzymes for the degradation of phytodetritus were assessed. L. marina mainly influenced qualitative aspects of the decomposition process rather than its overall rate: an effect of the nematodes on the enzymatic activities became manifest, although no clear nematode effect on bulk organic matter weight loss was found. We also demonstrated that species-specific effects on the decomposition process existed. Combining the four cryptic species resulted in high competition, with one dominant species, but without complete exclusion of other species. These interspecific interactions translated into different effects on the decomposition process. The species-specific differences indicated that each cryptic species may play an important and distinct role in ecosystem functioning. Functional differences may result in coexistence among very similar species.

Coexisting cryptic species of the Litoditis marina complex (Nematoda) show differential resource use and have distinct microbiomes with high intraspecific variability.

Differences in resource use or in tolerances to abiotic conditions are often invoked as potential mechanisms underlying the sympatric distribution of cryptic species. Additionally, the microbiome can provide physiological adaptations of the host to environmental conditions. We determined the intra- and interspecific variability of the microbiomes of three cryptic nematode species of the Litoditis marina species complex that co-occur, but show differences in abiotic tolerances. Roche 454 pyrosequencing of the microbial 16S rRNA gene revealed distinct bacterial communities characterized by a substantial diversity (85-513 OTUs) and many rare OTUs. The core microbiome of each species contained only very few OTUs (2-6), and four OTUs were identified as potentially generating tolerance to abiotic conditions. A controlled experiment in which nematodes from two cryptic species (Pm1 and Pm3) were fed with either an E. coli suspension or a bacterial mix was performed, and the 16S rRNA gene was sequenced using the MiSeq technology. OTU richness was 10-fold higher compared to the 454 data set and ranged between 1118 and 7864. This experiment confirmed the existence of species-specific microbiomes, a core microbiome with few OTUs, and high interindividual variability. The offered food source affected the bacterial community and illustrated different feeding behaviour between the cryptic species, with Pm3 exhibiting a higher degree of selective feeding than Pm1. Morphologically similar species belonging to the same feeding guild (bacterivores) can thus have substantial differences in their associated microbiomes and feeding strategy, which in turn may have important ramifications for biodiversity-ecosystem functioning relationships.

Learning to lie: effects of practice on the cognitive cost of lying.

Cognitive theories on deception posit that lying requires more cognitive resources than telling the truth. In line with this idea, it has been demonstrated that deceptive responses are typically associated with increased response times and higher error rates compared to truthful responses. Although the cognitive cost of lying has been assumed to be resistant to practice, it has recently been shown that people who are trained to lie can reduce this cost. In the present study (n = 42), we further explored the effects of practice on one's ability to lie by manipulating the proportions of lie and truth-trials in a Sheffield lie test across three phases: Baseline (50% lie, 50% truth), Training (frequent-lie group: 75% lie, 25% truth; control group: 50% lie, 50% truth; and frequent-truth group: 25% lie, 75% truth), and Test (50% lie, 50% truth). The results showed that lying became easier while participants were trained to lie more often and that lying became more difficult while participants were trained to tell the truth more often. Furthermore, these effects did carry over to the test phase, but only for the specific items that were used for the training manipulation. Hence, our study confirms that relatively little practice is enough to alter the cognitive cost of lying, although this effect does not persist over time for non-practiced items.

Anthelmintic tolerance in free-living and facultative parasitic isolates of Halicephalobus (Panagrolaimidae).

Studies on anthelmintic resistance in equine parasites do not include facultative parasites. Halicephalobus gingivalis is a free-living bacterivorous nematode and a known facultative parasite of horses with a strong indication of some form of tolerance to common anthelmintic drugs. This research presents the results of an in vitro study on the anthelmintic tolerance of several isolates of Halicephalobus to thiabendazole and ivermectin using an adaptation of the Micro-Agar Larval Development Test hereby focusing on egg hatching and larval development. Panagrellus redivivus and Panagrolaimus superbus were included as a positive control. The results generally show that the anthelmintic tolerance of Halicephalobus to both thiabendazole and ivermectin was considerably higher than that of the closely related Panagrolaimidae and, compared to other studies, than that of obligatory equine parasites. Our results further reveal a remarkable trend of increasing tolerance from fully free-living isolates towards horse-associated isolates. In vitro anthelmintic testing with free-living and facultative parasitic nematodes offers the advantage of observing drug effect on the complete life cycle as opposed to obligatory parasites that can only be followed until the third larval stage. We therefore propose Halicephalobus gingivalis as an experimental tool to deepen our understanding of the biology of anthelmintic tolerance.

Investigating the ecology and evolution of cryptic marine nematode species through quantitative real-time PCR of the ribosomal ITS region.

The presence of morphologically similar but genetically distinct species has impacted biogeographical and ecological paradigms. In marine sediments, free-living nematodes form one of the most abundant and diverse faunal groups. Inferring the importance of nematode diversity for ecosystem functioning requires species-level identification, which is hampered by the lack of easily observable diagnostic characters and the presence of cryptic species. New techniques are urgently needed to adequately study the ecology and evolution of cryptic species. The aim of the present study was to evaluate the potential of a quantitative real-time PCR (qPCR) assay using the internal transcribed spacer (ITS) region of the ribosomal DNA to detect and quantify cryptic species of the R. (P.) marina complex. All primer pairs proved to be highly specific, and each primer pair was able to detect a single juvenile in a pool of 100 nematodes. C(t) values were significantly different between developmental stages for all species except for PmIII. Despite differences between developmental stages, a strong correlation was observed between the amount of extracted DNA and the number of nematodes present. Relative and absolute quantification estimates were comparable and resulted in strong positive correlations between the qPCR estimate and the actual number of nematodes present in the samples. The qPCR assay developed here provides the ability to quickly identify and quantify cryptic nematode species and will facilitate their study in laboratory and field settings.

Effects of barium and cadmium on the population development of the marine nematode Rhabditis (Pellioditis) marina.

Offshore oil and gas drilling often involves the use of fluids containing barium and traces of other heavy metals. These may affect the environment, but information on their toxicity to benthic biota remains scant. Here, we present results of a 10-day bioassay with the marine nematode Rhabditis (Pellioditis) marina at different loads of barium (0-10 ,000 ppm nominal concentrations) and cadmium (0-12 ppm) in the range of concentrations reported from drilling-impacted sediments. Barium did not affect the fitness and population development of R. (P.) marina at concentrations up to 300 ppm, but did cause a decrease in population abundance and an increase in development time from concentrations of 400-2000 ppm onwards. Increased mortality occurred at 4800 ppm Ba. For cadmium, LOEC and EC50 values for total population abundance were 2.95 and 8.82 ppm, respectively. Cd concentrations as low as 2.40 to 2.68 caused a decrease in the abundance of adult nematodes, indicating that assays covering more generations would likely demonstrate yet more pronounced population-level effects. Our results indicate that oil and gas drilling activities may potentially have important implications for the meiobenthos through the toxicity of barium and associated metals like cadmium.

Phylogeography of the Rhabditis (Pellioditis) marina species complex: evidence for long-distance dispersal, and for range expansions and restricted gene flow in the northeast Atlantic.

Pinpointing processes that structure the geographical distribution of genetic diversity of marine species and lead to speciation is challenging because of the lack of obvious dispersal barriers and the likelihood of substantial (passive) dispersal in oceans. In addition, cryptic radiations with sympatric distributions abound in marine species, challenging the allopatric speciation mechanism. Here, we present a phylogeographical study of the marine nematode species complex Rhabditis (Pellioditis) marina to investigate processes shaping genetic structure and speciation. Rhabditis (P.) marina lives on decaying macroalgae in the intertidal, and may therefore disperse over considerable distances. Rhabditis (P.) marina consists of several cryptic species sympatrically distributed at a local scale. Genetic variation in the COI gene was screened in 1362 specimens from 45 locations around the world. Two nuclear DNA genes (ITS and D2D3) were sequenced to infer phylogenetic species. We found evidence for ten sympatrically distributed cryptic species, seven of which show a strong genetic structuring. A historical signature showed evidence for restricted gene flow with occasional long-distance dispersal and range expansions pre-dating the last glacial maximum. Our data also point to a genetic break around the British Isles and a contact zone in the Southern Bight of the North Sea. We provide evidence for the transoceanic distribution of at least one cryptic species (PmIII) and discuss the dispersal capacity of marine nematodes. The allopatric distribution of some intraspecific phylogroups and of closely related cryptic species points to the potential for allopatric speciation in R. (P.) marina.

Seasonal dynamics of population genetic structure in cryptic taxa of the Pellioditis marina complex (Nematoda: Rhabditida).

The distribution patterns and genetic structure of the Pellioditis marina species complex in Belgium and The Netherlands were compared between four consecutive seasons. Different types of habitats (coast, estuary, semi-estuary and lake) with different degrees of connectivity were sampled. In addition, each habitat type was characterised by either temporal or permanent algal deposits. We screened 426 bp of the mitochondrial cytochrome oxidase c (COI) gene with the single-strand conformation polymorphism (SSCP) method in 1615 individuals of Pellioditis marina. The 51 haplotypes were divided into four (sympatric) lineages, with divergences ranging from 0.25 to 10.6%. Our results show that the lineages have different temporal dynamics, which may be linked to abiotic factors. Analysis of Molecular Variance (AMOVA) indicated a significant structuring in the PmI lineage, which correlated with habitat characteristics and which changed over time (Mantel, r = 0.51; p = 0.126). Intrapopulational diversity was similar in all locations, and temporal changes in haplotype frequencies were not higher in temporary than in permanent algal deposits. Instead, the results of the temporal survey indicated that (some) P. marina populations are characterised by a metapopulation structure. It is emphasized that a complete and correct interpretation of processes causing genetic structuring within species and of the genetic structure itself can only be done when analyses are performed at several time points.

Overproduction of Arabidopsis thaliana FeSOD confers oxidative stress tolerance to transgenic maize.

Transgenic maize (Zea mays L.) plants have been generated by particle gun bombardment that overproduce an Arabidopsis thaliana iron superoxide dismutase (FeSOD). To target this enzyme into chloroplasts, the mature Fesod coding sequence was fused to a chloroplast transit peptide from a pea ribulose-1,5-bisphosphate carboxylase gene. Expression of the chimeric gene was driven by the CaMV 35S promoter. Growth characteristics and in vitro oxidative stress tolerance of transgenic lines grown in control and chilling temperatures were evaluated. The transgenic line with the highest transgenic FeSOD activities had enhanced tolerance toward methyl viologen and had increased growth rates.

Spontaneous acquisition of tumorigenicity and invasiveness by mouse lens explant cells during culture in vitro.

The lens of the eye is one of the rare organs in which tumors do not occur spontaneously. It therefore appeared to us that lens cells would not present the background of spontaneous transformation toward malignancy found with many other cell cultures. We have cultured C3H/HeA mouse lens explant (MLE) cells for 70 wk and analyzed changes in malignancy-related phenotypes in function of the number of passages. In vitro, we studied morphology, colony forming efficiency on tissue culture plastic substrate (CFEtc) and in soft agar, population doubling time, saturation density, and invasiveness into precultured chick heart fragments. In vivo, tumorigenicity, invasion, and metastasis were analyzed after injection of cell suspensions subcutaneously and intraperitoneally, after implantation of cells aggregated to collagen sponges under the renal capsule and after implantation of cell aggregates subcutaneously into the tail and into the pinna. The CFEtc, population doubling time, and saturation density increased as the number of passages of culture in vitro increased, but colony formation in soft agar was never observed. MLE cells till passage 16 were not invasive in vitro, but hereafter consistently were found to be invasive. After about 17 passages, corresponding to 25 wk of culture, MLE cells acquired the capacity to form tumors in syngeneic mice. These tumors were invasive but metastases were not observed. We concluded that MLE cells acquired in an apparently spontaneous way a number of malignancy-related phenotypes, without, however, reaching the stage of metastasis.