Feeding strategy and feed protein level affect the gut microbiota of common carp (Cyprinus carpio).

Common carp (Cyprinus carpio) were fed food with different protein concentrations following different feeding regimes, which were previously shown to affect growth, nitrogen excretion and amino acid catabolism. 16S rRNA gene amplicon sequencing was performed to investigate the gut microbiota of these fish. Lower dietary protein content increased microbial richness, while the combination of demand feeding and dietary protein content affected the composition of the gut microbiota. Hepatic glutamate dehydrogenase (GDH) activity was correlated to the composition of the gut microbiota in all dietary treatments. We found that demand-fed carp fed a diet containing 39% protein had a significantly higher abundance of Beijerinckiaceae compared to other dietary groups. Network analysis identified this family and two Rhizobiales families as hubs in the microbial association network. In demand-fed carp, the microbial association network had significantly fewer connections than in batch-fed carp. In contrast to the large effects of the feeding regime and protein content of the food on growth and nitrogen metabolism, it had only limited effects on gut microbiota composition. However, correlations between gut microbiota composition and liver GDH activity showed that host physiology and gut microbiota are connected, which warrants functional studies into the role of the gut microbiota in fish physiology.

Influence of tree cover on carcass detection and consumption by facultative vertebrate scavengers.

Scavenging mammals and vultures can exploit and deplete carcasses much faster than other birds and invertebrates. Vultures are strongly influenced by habitat type, e.g. tree cover, since they rely on their eyesight to detect carcasses. It remains unclear whether and how facultative scavengers - both other birds and mammals - are influenced by tree cover and how that affect carcass decomposition time, which in turn affects biodiversity and ecological processes, including the cycle of energy and nutrients. We studied whether the carcass detection and consumption, hence carcass decomposition speed, by facultative avian and mammalian scavengers varies with tree cover in areas without vultures. Fresh mammal carcasses were placed in different landscapes across the Netherlands at locations that widely varied in tree cover. Camera traps were used to record carcass exploitation by facultative avian and mammalian scavengers and to estimate carcass decomposition time. We found that carcass detection and consumption by birds, wild boar, and other mammals varied between locations. Carcass decomposition speed indeed increased with carcass detection and exploitation by mammals, especially by wild boar. However, this variation was not related to tree cover. We conclude that tree cover is not a major determinant of carcass exploitation by facultative scavengers in areas without obligate scavengers and large carnivores.

Ionomic Variation Among Tissues in Fallow Deer (Dama dama) by Sex and Age.

(1) In mammals, the mineral nutrient and trace elemental composition of the body - the ionome - differs among individuals. It has been hypothesized that these differences may be related to age and sex, both for ecotoxic and essential elements. (2) We investigated whether and how intraspecific ionomic variation is related to age and sex in Fallow deer (Dama dama). We tested the predictions that concentrations of ecotoxic elements increase with age, that ionomic variation is lower among young individuals than among older individuals, and that reproductive females (does) have the lowest concentrations of essential elements. (3) Culled animals of different sex and age were obtained from a single protected area. The animals were dissected to collect 13 tissues, and concentrations of 22 different elements were measured in a sample of each tissue. (4) We described substantial ionomic variation between individuals. Some of this variation was related to age and sex, as predicted. Based on the limited existing knowledge on chemical element allocation and metabolism in the body, sex-related differences were more difficult to interpret than age-related differences. Since reference values are absent, we could not judge about the consequences of the elemental values that we found. (5) More extensive ionomic surveys, based on a wide range of elements and tissues, are needed to enlarge the understanding of within-species ionomic variation and potential biological, ecological, and metabolic consequences.

Female pond bats hunt in other areas than males and consume lighter prey when pregnant.

Animals with large energy requirements are forced to optimize their hunting strategy, which may result in differentiation of the diet between sexes and across seasons. Here, we examined spatiotemporal variation in the diet of both sexes of the Pond Bat Myotis dasycneme, a species known to have spatial segregation of sexes when the young are born and lactating. Fecal pellets were collected from live animals for a period of 15 years at various locations in the Netherlands. A total of 535 pellets were successfully analyzed by microscopy and an additional 160 pellets by DNA metabarcoding. Morphological and molecular analyses showed that the diet of pregnant and lactating pond bats differed significantly from the diet of females with no reproductive investment. Further analyses of the data showed that pregnant female pond bats are highly dependent on small prey and pupae, mainly nonbiting midges and mosquitoes (Diptera: Chironomidae and Culicidae). These insects can be found in large quantities in peatlands intersected with shallow waterways, the habitat type in which female pond bats were observed more often than males. Our results suggest that during pregnancy the spatial segregation of sexes coincides with sex-specific diets, which might reflect habitat selection based on energy requirements, in addition to lowered intraspecific competition.

Variability of the Ionome of Wild Boar (Sus scrofa) and Red Deer (Cervus elaphus) in a Dutch National Park, with Implications for Biomonitoring.

The ionome-an important expression of the physiological state of organisms-is poorly known for mammals. The focus on particular tissues-such as liver, kidney, and bones-in biomonitoring of environmental pollution and potential deficiencies is based on widely held assumptions rather than solid knowledge of full mammalian ionomes. We examined the full ionome of Red deer (Cervus elaphus) and Wild boar (Sus scrofa), two commonly used mammals for biomonitoring, in a Dutch protected nature reserve (Veluwezoom). We used four individuals per species. We dissected 13 tissues and organs from each individuals (eight in total) of each species and measured 22 elemental concentrations in each. We assessed, for each element, how concentrations varied across tissues within and between individuals. Based on existing literature, we put our findings in the context of their function in the mammalian body. We found that the ionome was highly variable between as well as within the two species. For most elements, tissues containing the highest and lowest concentration differed between individuals. No single tissue accurately represented the accumulation of toxic elements or potential deficiencies in the bodies. Our assessment of the element's biological roles revealed a serious lack of reference values. Our findings imply that analyses of commonly used tissues in biomonitoring do not necessarily capture bioaccumulation of toxins or potential deficiencies. We recommend establishing a centralized database of mammalian ionomes to derive reference values in future. To our knowledge, our study is one of the most complete assessments of mammalian ionomes to date.

Towards a mechanistic understanding of the impacts of nitrogen deposition on producer-consumer interactions.

Nitrogen (N) deposition has increased substantially since the second half of the 20th century due to human activities. This increase of reactive N into the biosphere has major implications for ecosystem functioning, including primary production, soil and water chemistry and producer community structure and diversity. Increased N deposition is also linked to the decline of insects observed over recent decades. However, we currently lack a mechanistic understanding of the effects of high N deposition on individual fitness, species richness and community structure of both invertebrate and vertebrate consumers. Here, we review the effects of N deposition on producer-consumer interactions, focusing on five existing ecological frameworks: C:N:P ecological stoichiometry, trace element ecological stoichiometry, nutritional geometry, essential micronutrients and allelochemicals. We link reported N deposition-mediated changes in producer quality to life-history strategies and traits of consumers, to gain a mechanistic understanding of the direction of response in consumers. We conclude that high N deposition influences producer quality via eutrophication and acidification pathways. This makes oligotrophic poorly buffered ecosystems most vulnerable to significant changes in producer quality. Changes in producer quality between the reviewed frameworks are often interlinked, complicating predictions of the effects of high N deposition on producer quality. The degree and direction of fitness responses of consumers to changes in producer quality varies among species but can be explained by differences in life-history traits and strategies, particularly those affecting species nutrient intake regulation, mobility, relative growth rate, host-plant specialisation, ontogeny and physiology. To increase our understanding of the effects of N deposition on these complex mechanisms, the inclusion of life-history traits of consumer species in future study designs is pivotal. Based on the reviewed literature, we formulate five hypotheses on the mechanisms underlying the effects of high N deposition on consumers, by linking effects of nutritional ecological frameworks to life-history strategies. Importantly, we expect that N-deposition-mediated changes in producer quality will result in a net decrease in consumer community as well as functional diversity. Moreover, we anticipate an increased risk of outbreak events of a small subset of generalist species, with concomitant declines in a multitude of specialist species. Overall, linking ecological frameworks with consumer life-history strategies provides a mechanistic understanding of the impacts of high N deposition on producer-consumer interactions, which can inform management towards more effective mitigation strategies.

Comparison of the gill and gut microbiomes of common carp (Cyprinus carpio) and zebrafish (Danio rerio) and their RAS environment.

Recirculating aquaculture systems (RAS) are increasingly being used to grow fish, as intensive water reuse reduces water consumption and environmental impact. RAS use biofilters containing nitrogen-cycling microorganisms that remove ammonia from the aquaculture water. Knowledge of how RAS microbial communities relate to the fish-associated microbiome is limited, as is knowledge of fish-associated microbiota in general. Recently, nitrogen-cycling bacteria have been discovered in zebrafish and carp gills and shown to detoxify ammonia in a manner similar to the RAS biofilter. Here, we compared RAS water and biofilter microbiomes with fish-associated gut and gill microbial communities in laboratory RAS housing either zebrafish (Danio rerio) or common carp (Cyprinus carpio) using 16S rRNA gene amplicon sequencing. The phylogeny of ammonia-oxidizing bacteria in the gills and the RAS environment was investigated in more detail by phylogenetic analysis of the ammonia monooxygenase subunit A (amoA). The location from which the microbiome was sampled (RAS compartments and gills or gut) had a stronger effect on community composition than the fish species, but species-specific differences were also observed. We found that carp- and zebrafish-associated microbiomes were highly distinct from their respective RAS microbiomes, characterized by lower overall diversity and a small core microbiome consisting of taxa specifically adapted to the respective organ. The gill microbiome was also defined by a high proportion of unique taxa. Finally, we found that amoA sequences from the gills were distinct from those from the RAS biofilter and water. Our results showed that the gut and gill microbiomes of carp and zebrafish share a common and species-specific core microbiome that is distinct from the microbially-rich RAS environment.

Effects of demand-feeding and dietary protein level on nitrogen metabolism and symbiont dinitrogen gas production of common carp (Cyprinus carpio, L.).

Ammonia accumulation is a major challenge in intensive aquaculture, where fish are fed protein-rich diets in large rations, resulting in increased ammonia production when amino acids are metabolized as energy source. Ammonia is primarily excreted via the gills, which have been found to harbor nitrogen-cycle bacteria that convert ammonia into dinitrogen gas (N2) and therefore present a potential in situ detoxifying mechanism. Here, we determined the impact of feeding strategies (demand-feeding and batch-feeding) with two dietary protein levels on growth, nitrogen excretion, and nitrogen metabolism in common carp (Cyprinus carpio, L.) in a 3-week feeding experiment. Demand-fed fish exhibited significantly higher growth rates, though with lower feed efficiency. When corrected for feed intake, nitrogen excretion was not impacted by feeding strategy or dietary protein, but demand-fed fish had significantly more nitrogen unaccounted for in the nitrogen balance and less retained nitrogen. N2 production of individual fish was measured in all experimental groups, and production rates were in the same order of magnitude as the amount of nitrogen unaccounted for, thus potentially explaining the missing nitrogen in the balance. N2 production by carp was also observed when groups of fish were kept in metabolic chambers. Demand feeding furthermore caused a significant increase in hepatic glutamate dehydrogenase activities, indicating elevated ammonia production. However, branchial ammonia transporter expression levels in these animals were stable or decreased. Together, our results suggest that feeding strategy impacts fish growth and nitrogen metabolism, and that conversion of ammonia to N2 by nitrogen cycle bacteria in the gills may explain the unaccounted nitrogen in the balance.

Influence of photoperiod on thermal responses in body size, growth and development in Lycaena phlaeas (Lepidoptera: Lycaenidae).

Many ectotherms species grow faster but attain a smaller body size when reared under warmer conditions, a phenomenon known as the Temperature-Size Rule (TSR). This rule appears to be stronger in aquatic ectotherms than in terrestrial ectotherms. The difference could be related to difficulties for oxygen uptake in water, whereas on land, adaptive responses in body size may relate to seasonal time constraints. To assess the role of seasonal time constraints in temperature size response of terrestrial ectotherms, we reared the small copper Lycaena phlaeas at three temperatures (18 ˚C, 23˚C and 28˚C) and two photoperiods (16L: 8D and 12L: 12D). We examined whether differences in body size across treatments was related to (1) differences in growth and development, (2) differences in breakpoints during growth trajectories, or (3) differences in ommatidia size (as a proxy for cell size). We found a weak inverse relationship between developmental temperature and the body size of adult butterflies; adult size decreased by approximately 1% for every degree warmer. Under warmer temperatures, caterpillars developed more quickly and had higher growth rates but reached a smaller body size. Under a short photoperiod, both growth and development were slower, especially at the two lower temperatures, but the body size resulting from slow growth over a longer developmental period did not vary with photoperiod. Breakpoints in growth trajectories occurred when larvae reached ∼40% of their maximum mass and these breakpoints were strongly correlated with the size of the resulting adults, suggesting that adult size is predetermined at an early stage. Temperature did not appear to cause reductions in body size through reductions in cell size. Butterflies were largely able to buffer their body size by modulating larval growth and development in tandem. They appear to use photoperiod as a cue to gauge the availability of time (with 12L: 12D indicating less time available) while temperature speeds up growth and development and as such governs the amount of time they need to complete a developmental cycle. Temperature and photoperiod thus induce changes in voltinism to fit a discrete number of generations into a growing season.

Shrinking body sizes in response to warming: explanations for the temperature-size rule with special emphasis on the role of oxygen.

Body size is central to ecology at levels ranging from organismal fecundity to the functioning of communities and ecosystems. Understanding temperature-induced variations in body size is therefore of fundamental and applied interest, yet thermal responses of body size remain poorly understood. Temperature-size (T-S) responses tend to be negative (e.g. smaller body size at maturity when reared under warmer conditions), which has been termed the temperature-size rule (TSR). Explanations emphasize either physiological mechanisms (e.g. limitation of oxygen or other resources and temperature-dependent resource allocation) or the adaptive value of either a large body size (e.g. to increase fecundity) or a short development time (e.g. in response to increased mortality in warm conditions). Oxygen limitation could act as a proximate factor, but we suggest it more likely constitutes a selective pressure to reduce body size in the warm: risks of oxygen limitation will be reduced as a consequence of evolution eliminating genotypes more prone to oxygen limitation. Thus, T-S responses can be explained by the 'Ghost of Oxygen-limitation Past', whereby the resulting (evolved) T-S responses safeguard sufficient oxygen provisioning under warmer conditions, reflecting the balance between oxygen supply and demands experienced by ancestors. T-S responses vary considerably across species, but some of this variation is predictable. Body-size reductions with warming are stronger in aquatic taxa than in terrestrial taxa. We discuss whether larger aquatic taxa may especially face greater risks of oxygen limitation as they grow, which may be manifested at the cellular level, the level of the gills and the whole-organism level. In contrast to aquatic species, terrestrial ectotherms may be less prone to oxygen limitation and prioritize early maturity over large size, likely because overwintering is more challenging, with concomitant stronger end-of season time constraints. Mechanisms related to time constraints and oxygen limitation are not mutually exclusive explanations for the TSR. Rather, these and other mechanisms may operate in tandem. But their relative importance may vary depending on the ecology and physiology of the species in question, explaining not only the general tendency of negative T-S responses but also variation in T-S responses among animals differing in mode of respiration (e.g. water breathers versus air breathers), genome size, voltinism and thermally associated behaviour (e.g. heliotherms).

Stoichiometric variation within and between a terrestrial herbivorous and a semi-aquatic carnivorous mammal.

BACKGROUND: The elemental composition of the mammalian body is widely believed to be more or less constant within and among species, yet reliable comparisons of elemental content are lacking. Here, we examine the elemental composition of two mammal species with different diet and provenance: terrestrial herbivorous Fallow deer (Dama dama) - collected from a single area - and semi-aquatic carnivorous Eurasian otter (Lutra lutra) - collected from different areas. METHODS: We compared twelve elemental contents for twelve different body tissues and organs, for four tissue samples per species. Homogeneous samples were tested for twelve elemental contents using ICP-OES. RESULTS: We found evidence for differences in elemental composition between species, between tissues, and between individuals. Herbivorous Fallow deer seemed more variable in its elemental composition compared to carnivorous Eurasian otter. The absolute concentration of some elements, e.g. Mn and Cu, showed differences between the species as well. CONCLUSION: Since we found stoichiometric variation among the species, these findings question the widely held assumption that mammals are under relative tight stoichiometrically homeostatic control.

Male long-distance migrant turned sedentary; The West European pond bat (Myotis dasycneme) alters their migration and hibernation behaviour.

During autumn in the temperate zone, insectivorous male bats face a profound energetic challenge, as in the same period they have to make energy choices related to hibernation, mating and migration. To investigate these energetic trade-offs, we compared the body mass of male and female pond bats (Myotis dasycneme) through the summer season, characterized the known hibernacula in terms of male or female bias, and subsequently compared their population trend during two study periods, between 1930-1980 and 1980-2015. Towards the end of summer, males began losing weight whilst females were simultaneously accumulating fat, suggesting that males were pre-occupied with mating. We also found evidence for a recent adaptation to this energetic trade-off, males have colonised winter roosts in formerly unoccupied areas, which has consequently led to a change in the migration patterns for the male population of this species. As male bats do not assist in raising offspring, males have ample time to restore their energy balance after hibernation. Our results suggest that choosing a hibernacula closer to the summer range not only decreases energy cost needed for migration, it also lengthens the mating season of the individual male. Our findings have important conservation implications, as male and female biased hibernation assemblages may differ critically in terms of microclimate preferences.

Hatching failure and accumulation of organic pollutants through the terrestrial food web of a declining songbird in Western Europe.

Population growth in passerine birds is largely driven by fecundity. If fecundity is affected, for instance by hatching failure, populations may decline. We noted high hatching failure of up to 27% per year in relict populations of the Northern wheatear (Oenanthe oenanthe) in The Netherlands, a strongly declining, migratory passerine in Europe. This hatching failure itself can cause population decline, irrespective of other adverse factors. Additionally, we investigated the cause of hatching failure. Unhatched eggs showed egg yolk infections or embryonic malformations, part of which is associated with the actions of dioxin-like compounds (DLCs). Indeed, DLCs appear to bioaccumulate in the local foodweb, where the soil contained only background concentrations, similar to those found at many other locations. DLC concentrations in Dutch eggs were six-fold higher than those in a reference population in Sweden, where egg failure was only 6%. However, Northern wheatears appear to be only moderately sensitive to the actions of DLCs, because of their specific Ah-receptor type which may moderate the receptor mediated effects of DLCs. This indicates that the concentrations of DLCs, although elevated, may not have caused the embryo malformations or the low hatching rates. We discuss whether other toxins may be important or imbalances in the nutrition and if inbreeding may play a larger role than expected.

More than 75 percent decline over 27 years in total flying insect biomass in protected areas.

Global declines in insects have sparked wide interest among scientists, politicians, and the general public. Loss of insect diversity and abundance is expected to provoke cascading effects on food webs and to jeopardize ecosystem services. Our understanding of the extent and underlying causes of this decline is based on the abundance of single species or taxonomic groups only, rather than changes in insect biomass which is more relevant for ecological functioning. Here, we used a standardized protocol to measure total insect biomass using Malaise traps, deployed over 27 years in 63 nature protection areas in Germany (96 unique location-year combinations) to infer on the status and trend of local entomofauna. Our analysis estimates a seasonal decline of 76%, and mid-summer decline of 82% in flying insect biomass over the 27 years of study. We show that this decline is apparent regardless of habitat type, while changes in weather, land use, and habitat characteristics cannot explain this overall decline. This yet unrecognized loss of insect biomass must be taken into account in evaluating declines in abundance of species depending on insects as a food source, and ecosystem functioning in the European landscape.

Species-area relationships are modulated by trophic rank, habitat affinity, and dispersal ability.

In the face of ongoing habitat fragmentation, species-area relationships (SARs) have gained renewed interest and are increasingly used to set conservation priorities. An important question is how large habitat areas need to be to optimize biodiversity conservation. The relationship between area and species richness is explained by colonization-extinction dynamics, whereby smaller sites harbor smaller populations, which are more prone to extinction than the larger populations sustained by larger sites. These colonization-extinction dynamics are predicted to vary with trophic rank, habitat affinity, and dispersal ability of the species. However, empirical evidence for the effect of these species characteristics on SARs remains inconclusive. In this study we used carabid beetle data from 58 calcareous grassland sites to investigate how calcareous grassland area affects species richness and activity density for species differing in trophic rank, habitat affinity, and dispersal ability. In addition, we investigated how SARs are affected by the availability of additional calcareous grassland in the surrounding landscape. Beetle species richness and activity density increased with calcareous grassland area for zoophagous species that are specialists for dry grasslands and, to a lesser extent, for zoophagous habitat generalists. Phytophagous species and zoophagous forest and wet-grassland specialists were not affected by calcareous grassland area. The dependence of species on large single sites increased with decreasing dispersal ability for species already vulnerable to calcareous grassland area. Additional calcareous grassland in the landscape had a positive effect on local species richness of both dry-grassland specialists and generalists, but this effect was restricted to a few hundred meters. Our results demonstrate that SARs are affected by trophic rank, habitat affinity, and dispersal ability. These species characteristics do not operate independently, but should be viewed in concert. In addition, species' responses depend on the landscape context. Our study suggests that the impact of habitat area on trophic interactions may be larger than previously anticipated. In small habitat fragments surrounded by a hostile matrix, food chains may be strongly disrupted. This highlights the need to conserve continuous calcareous grassland patches of at least several hectares in size.

Museum specimens reveal loss of pollen host plants as key factor driving wild bee decline in The Netherlands.

Evidence for declining populations of both wild and managed bees has raised concern about a potential global pollination crisis. Strategies to mitigate bee loss generally aim to enhance floral resources. However, we do not really know whether loss of preferred floral resources is the key driver of bee decline because accurate assessment of host plant preferences is difficult, particularly for species that have become rare. Here we examine whether population trends of wild bees in The Netherlands can be explained by trends in host plants, and how this relates to other factors such as climate change. We determined host plant preference of bee species using pollen loads on specimens in entomological collections that were collected before the onset of their decline, and used atlas data to quantify population trends of bee species and their host plants. We show that decline of preferred host plant species was one of two main factors associated with bee decline. Bee body size, the other main factor, was negatively related to population trend, which, because larger bee species have larger pollen requirements than smaller species, may also point toward food limitation as a key factor driving wild bee loss. Diet breadth and other potential factors such as length of flight period or climate change sensitivity were not important in explaining twentieth century bee population trends. These results highlight the species-specific nature of wild bee decline and indicate that mitigation strategies will only be effective if they target the specific host plants of declining species.

Avian population consequences of climate change are most severe for long-distance migrants in seasonal habitats.

One consequence of climate change is an increasing mismatch between timing of food requirements and food availability. Such a mismatch is primarily expected in avian long-distance migrants because of their complex annual cycle, and in habitats with a seasonal food peak. Here we show that insectivorous long-distance migrant species in The Netherlands declined strongly (1984-2004) in forests, a habitat characterized by a short spring food peak, but that they did not decline in less seasonal marshes. Also, within generalist long-distance migrant species, populations declined more strongly in forests than in marshes. Forest-inhabiting migrant species arriving latest in spring declined most sharply, probably because their mismatch with the peak in food supply is greatest. Residents and short-distance migrants had non-declining populations in both habitats, suggesting that habitat quality did not deteriorate. Habitat-related differences in trends were most probably caused by climate change because at a European scale, long-distance migrants in forests declined more severely in western Europe, where springs have become considerably warmer, when compared with northern Europe, where temperatures during spring arrival and breeding have increased less. Our results suggest that trophic mismatches may have become a major cause for population declines in long-distance migrants in highly seasonal habitats.