A Silurian armoured aplacophoran and implications for molluscan phylogeny

The Mollusca is one of the most diverse, important and well-studied invertebrate phyla; however, relationships among major molluscan taxa have long been a subject of controversy(1-9). In particular, the position of the shell-less vermiform Aplacophora and its relationship to the better-known Polyplacophora (chitons) have been problematic: Aplacophora has been treated as a paraphyletic or monophyletic group at the base of the Mollusca(3,6,8), proximate to other derived clades such as Cephalopoda(2,3,10), or as sister group to the Polyplacophora, forming the clade Aculifera(1,5,7,11,12). Resolution of this debate is required to allow the evolutionary origins of Mollusca to be reconstructed with confidence. Recent fossil finds(13-16) support the Aculifera hypothesis, demonstrating that the Palaeozoic-era palaeoloricate 'chitons' included taxa combining certain polyplacophoran and aplacophoran characteristics(5). However, fossils combining an unambiguously aplacophoran-like body with chiton-like valves have remained elusive. Here we describe such a fossil, Kulindroplax perissokomos gen. et sp. nov., from the Herefordshire Lagerstatte(17,18) (about 425 million years BP), a Silurian deposit preserving a marine biota(18) in unusual three-dimensional detail. The specimen is reconstructed three-dimensionally through physical-optical tomography(19). Phylogenetic analysis indicates that this and many other palaeoloricate chitons are crown-group aplacophorans.

The effects of winter ice cover on the trophic ecology of whitefish (Coregonus lavaretus L.) in subarctic lakes

Lakes in Arctic and subarctic regions display extreme levels of seasonal variation in light, temperature and ice cover. Comparatively little is known regarding the effects of such seasonal variation on the diet and resource use of fish species inhabiting these systems. Variation in the diet of European whitefish Coregonus lavaretus (L.) during periods of ice cover in this region is often regarded as 'common knowledge'; however, this aspect of the species' ecology has not been examined empirically. Here, we outline the differences in invertebrate community structure, fish activity, and resource use of monomorphic whitefish populations between summer (August-September) and winter (February-March) in three subarctic lakes in Finnish Lapland. Benthic macroinvertebrate densities did not exhibit measurable differences between summer and winter. Zooplankton diversity and abundance, and activity levels of all fish species (measured as catch per unit effort) were lower in winter. The summer diet of C. lavaretus was typical of a generalist utilising a variety of prey sources. In winter, its dietary niche was significantly reduced, and the diet was dominated by chironomid larvae in all study sites. Pelagic productivity decreases during winter, and fish species inhabiting these systems are therefore restricted to feeding on benthic prey. Sampling time has strong effect on our understanding of resource utilisation by whitefish in subarctic lakes and should be taken into account in future studies of these systems. © 2012 John Wiley & Sons A/S.

Primary assembly of soil communities: disentangling the effect of dispersal and local environment

It has long been recognised that dispersal abilities and environmental factors are important in shaping invertebrate communities, but their relative importance for primary soil community assembly has not yet been disentangled. By studying soil communities along chronosequences on four recently emerged nunataks (ice-free land in glacial areas) in Iceland, we replicated environmental conditions spatially at various geographical distances. This allowed us to determine the underlying factors of primary community assembly with the help of metacommunity theories that predict different levels of dispersal constraints and effects of the local environment. Comparing community assembly of the nunataks with that of non-isolated deglaciated areas indicated that isolation of a few kilometres did not affect the colonisation of the soil invertebrates. When accounting for effects of geographical distances, soil age and plant richness explained a significant part of the variance observed in the distribution of the oribatid mites and collembola communities, respectively. Furthermore, null model analyses revealed less co-occurrence than expected by chance and also convergence in the body size ratio of co-occurring oribatids, which is consistent with species sorting. Geographical distances influenced species composition, indicating that the community is also assembled by dispersal, e.g. mass effect. When all the results are linked together, they demonstrate that local environmental factors are important in structuring the soil community assembly, but are accompanied with effects of dispersal that may "override" the visible effect of the local environment.

Predator cue studies reveal strong trait-mediated effects in communities despite variation in experimental designs

Nonconsumptive or trait-mediated effects of predators on their prey often outweigh density-mediated interactions where predators consume prey. For instance, predator presence can alter prey behaviour, physiology, morphology and/or development. Despite a burgeoning literature, our ability to identify general patterns in prey behavioural responses may be influenced by the inconsistent methodologies of predator cue experiments used to assess trait-mediated effects. We therefore conducted a meta-analysis to highlight variables (e.g. water type, predator husbandry, exposure time) that may influence invertebrate prey's behavioural responses to fish predator cues. This revealed that changes in prey activity and refuge use were remarkably consistent overall, despite wide differences in experimental methodologies. Our meta-analysis shows that invertebrates altered their behaviour to predator cues of both fish that were fed the focal invertebrate and those that were fed other prey types, which suggests that invertebrates were not responding to specific diet information in the fish cues. Invertebrates also altered their behaviour regardless of predator cue addition regimes and fish satiation levels. Cue intensity and exposure time did not have significant effects on invertebrate behaviour. We also highlight that potentially confounding factors, such as parasitism, were rarely recorded in sufficient detail to assess the magnitude of their effects. By examining the likelihood of detecting trait-mediated effects under large variations in experimental design, our study demonstrates that trait-mediated effects are likely to have pervasive and powerful influences in nature.

Chiton phylogeny (Mollusca Polyplacophora) and the placement of the enigmatic species Choriplax grayi (H. Adams & Angas)

Shallow marine chitons (Mollusca:Polyplacophora:Chitonida) are widespread and well described from established morphoanatomical characters, yet key aspects of polyplacophoran phylogeny have remained unresolved. Several species, including Hemiarthrum setulosum Carpenter in Dall, 1876, and especially the rare and enigmatic Choriplax grayi (Adams & Angas, 1864), defy systematic placement. Choriplax is known from only a handful of specimens and its morphology is a mosaic of key taxonomic features from two different clades. Here, new molecular evidence provides robust support for its correct association with a third different clade: Choriplax is placed in the superfamily Mopalioidea. Hemiarthrum is included in Cryptoplacoidea, as predicted from morphological evidence. Our multigene analysis of standard nuclear and mitochondrial markers demonstrates that the topology of the order Chitonida is divided into four clades, which have also been recovered in previous studies: Mopalioidea is sister to Cryptoplacoidea, forming a clade Acanthochitonina. The family Callochitonidae is sister to Acanthochitonina. Chitonoidea is resolved as the earliest diverging group within Chitonida. Consideration of this unexpected result for Choriplax and our well-supported phylogeny has revealed differing patterns of shell reduction separating the two superfamilies within Acanthochitonina. As in many molluscs, shell reduction as well as the de novo development of key shell features has occurred using different mechanisms, in multiple lineages of chitons.

Predicting the predatory impacts of the “demon shrimp” Dikerogammarus haemobaphes, on native and previously introduced species

Biological invasions continue to exert pressure on ecosystems worldwide and we thus require methods that can help understand and predict the impacts of invasive species, on both native species and previously established invaders. Comparing laboratory derived functional responses among invasive and native predators has emerged as one such method, providing a robust proxy for field impacts. We used this method to examine the likely impacts of the Ponto–Caspian amphipod Dikerogammarus haemobaphes, known as the “demon shrimp”, a little investigated invader in European freshwaters that has recently established in the British Isles. We compared the functional responses on two prey species of D. haemobaphes with two other amphipod species: Dikerogammarus villosus, a congeneric invasive with well-documented impacts on macro-invertebrate communities and a native amphipod, Gammarus pulex. Prey species were native Chironomus sp. and the invasive Chelicorophium curvispinum, a tube-building amphipod also originating from the Ponto–Caspian region. D. villosus showed higher Type II functional responses towards both prey species than did D. haemobaphes and G. pulex, with the latter two predators exhibiting similar impacts on the native prey. However, D. haemobaphes had higher functional responses towards the invasive C. curvispinum than did G. pulex, both when prey individuals were tubeless and resident in their protective mud tubes. Thus, we demonstrate that functionally equivalent invasive congeners can show significantly different impacts on prey, regardless of shared evolutionary history. We also show that some predatory invaders can have impacts on native prey equivalent to native predator impacts, but that they can also exert significant impacts on previously introduced prey. We discuss the importance of invasion history and prey identity when attempting to understand and predict the impacts of new invaders.

Predicting invasive species impacts: a community module functional response approach reveals context dependencies

Summary
-Predatory functional responses play integral roles in predator–prey dynamics, and their assessment promises greater understanding and prediction of the predatory impacts of invasive species.
-Other interspecific interactions, however, such as parasitism and higher-order predation, have the potential to modify predator–prey interactions and thus the predictive capability of the comparative functional response approach.
-We used a four-species community module (higher-order predator; focal native or invasive predators; parasites of focal predators; native prey) to compare the predatory functional responses of native Gammarus duebeni celticus and invasive Gammarus pulex amphipods towards three invertebrate prey species (Asellus aquaticus, Simulium spp., Baetis rhodani), thus, quantifying the context dependencies of parasitism and a higher-order fish predator on these functional responses.
-Our functional response experiments demonstrated that the invasive amphipod had a higher predatory impact (lower handling time) on two of three prey species, which reflects patterns of impact observed in the field. The community module also revealed that parasitism had context-dependent influences, for one prey species, with the potential to further reduce the predatory impact of the invasive amphipod or increase the predatory impact of the native amphipod in the presence of a higher-order fish predator.
-Partial consumption of prey was similar for both predators and occurred increasingly in the order A. aquaticus, Simulium spp. and B. rhodani. This was associated with increasing prey densities, but showed no context dependencies with parasitism or higher-order fish predator.
-This study supports the applicability of comparative functional responses as a tool to predict and assess invasive species impacts incorporating multiple context dependencies.

Expression pattern of arenicins - the antimicrobial peptides of polychaete Arenicola marina

Immune responses of invertebrate animals are mediated through innate mechanisms, among which production of antimicrobial peptides play an important role. Although evolutionary Polychaetes represent an interesting group closely related to a putative common ancestor of other coelomates, their immune mechanisms still remain scarcely investigated. Previously our group has identified arenicins - new antimicrobial peptides of the lugworm Arenicola marina, since then these peptides were thoroughly characterized in terms of their structure and inhibitory potential. In the present study we addressed the question of the physiological functions of arenicins in the lugworm body. Using molecular and immunocytochemical methods we demonstrated that arencins are expressed in the wide range of the lugworm tissues - coelomocytes, body wall, extravasal tissue and the gut. The expression of arenicins is constitutive and does not depend on stimulation of various infectious stimuli. Most intensively arenicins are produced by mature coelomocytes where they function as killing agents inside the phagolysosome. In the gut and the body wall epithelia arenicins are released from producing cells via secretion as they are found both inside the epithelial cells and in the contents of the cuticle. Collectively our study showed that arenicins are found in different body compartments responsible for providing a first line of defence against infections, which implies their important role as key components of both epithelial and systemic branches of host defence.

Water and Temperature Relations of Growth of the Entomogenous Fungi Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus

The effects of temperature (5-50°C), water availability (0.998-0.88 water activity, aw), and aw × temperature interactions (15-45°C) on growth of three entomogenous fungi, Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus, were evaluated on a Sabouraud dextrose-based medium modified with the ionic solute KCl, the non-ionic solute glycerol, and an inert solute, polyethylene glycol (PEG) 600. The temperature ranges for growth of B. bassiana, M. anisopliae, and P. farinosus were 5-30, 5-40, and 5-30°C, and optimum growth temperatures were 25, 30, and 20°C, respectively. All three species grew over a similar aw range (0.90-0.998) at optimum temperatures for growth. However, there were significant interspecies variations in growth rates on media modified with each of the three aw-modifying solutes. Growth aw optima ranged between 0.99 and 0.97 on KCl-, glycerol-, and PEG 600-modified media for M. anisopliae and P. farinosus. B. bassiana grew optimally at 0.998 aw, regardless of aw. Comprehensive two-dimensional profiles of aw × temperature relations for growth of these three species were constructed for the first time. The results are discussed in relation to the environmental limits that determine efficacy of entomogenous fungi as biocontrol agents in nature. © 1999 Academic Press.

Defining and assessing animal pain

The detection and assessment of pain in animals is crucial to improving their welfare in a variety of contexts in which humans are ethically or legally bound to do so. Thus clear standards to judge whether pain is likely to occur in any animal species is vital to inform whether to alleviate pain or to drive the refinement of procedures to reduce invasiveness, thereby minimizing pain. We define two key concepts that can be used to evaluate the potential for pain in both invertebrate and vertebrate taxa. First, responses to noxious, potentially painful events should affect neurobiology, physiology and behaviour in a different manner to innocuous stimuli and subsequent behaviour should be modified including avoidance learning and protective responses. Second, animals should show a change in motivational state after experiencing a painful event such that future behavioural decision making is altered and can be measured as a change in conditioned place preference, self-administration of analgesia, paying a cost to access analgesia or avoidance of painful stimuli and reduced performance in concurrent events. The extent to which vertebrate and selected invertebrate groups fulfil these criteria is discussed in light of the empirical evidence and where there are gaps in our knowledge we propose future studies are vital to improve our assessment of pain. This review highlights arguments regarding animal pain and defines criteria that demonstrate, beyond a reasonable doubt, whether animals of a given species experience pain.

Effects of Elodea nuttallii on temperate freshwater plants, microalgae and invertebrates: small differences between invaded and uninvaded areas

The invasive aquatic plant species Elodea nuttallii could pose a considerable risk to European freshwater ecosystems based on its current distribution, rate of spread and potential for high biomass. However, little research has been conducted on the impacts of this species on native biota. This study takes an ecosystem-wide approach and examines the impact of E. nuttallii on selected physicochemical parameters (dissolved oxygen and pH), algae, invertebrate and macrophyte communities. Elodea nuttallii had small but significant impacts on plant, invertebrate and algal species. The richness of algal periphyton was lower on E. nuttallii than on native macrophytes. The taxonomic composition of invertebrate communities associated with E. nuttallii differed from that associated with similar native plant species, but did not differ in terms of total biomass or species richness. Macrophyte species richness and total cover were positively correlated with percentage cover of E. nuttallii. Not all macrophyte species responded in the same way to E. nuttallii invasion; cover of the low-growing species, Elodea canadensis and charophytes were negatively correlated with E. nuttallii cover, whilst floating-rooted plants were positively correlated with E. nuttallii cover. All observed differences in the macrophyte community were small relative to other factors such as nutrient levels, inter-annual variation and differences between sites. Despite this, the observed negative association between E. nuttallii and charophytes is a key concern due to the rarity and endangered status of many charophyte species.

Detoxification of microcystins (cyanobacterial hepatotoxins) using TiO2 photocatalytic oxidation

TiO₂ photocatalysis has been used to destroy microcystin-LR in aqueous solution. The destruction of this toxin was monitored by HPLC, and the disappearance was accompanied by the appearance of seven UV detectable compounds. Spectral analysis revealed that some of these compounds retained spectra similar to the parent compound suggesting that the Adda moiety, thought to be responsible for the characteristic spectrum, remained intact whereas the spectra of some of the other products was more radically altered. Six of the seven observed reaction products did not appear to undergo further degradation during prolonged photocatalysis (100 min). The degree to which microcystin-LR was mineralized by photocatalytic oxidation was determined. Results indicated that less than 10% mineralization occurred. Mass spectral analysis of the photocatalyzed microcystin-LR allowed tentative characterization of the reaction process and products. Reduction in toxicity due to the photocatalytic oxidation was evaluated using an invertebrate bioassay, which demonstrated that the disappearance of microcystin-LR was paralleled by a reduction in toxicity. These findings suggest that photocatalytic destruction of microcystins may be a suitable method for the removal of these potentially hazardous compounds from drinking water.

Young, Old, and Weathered Carbon—Part 2: Using Radiocarbon and Stable Isotopes to Identify Terrestrial Carbon Support of the Food Web in an Alkaline, Humic Lake

Carbon (C) and nitrogen (N) stable isotope analysis (SIA) has been used to identify the terrestrial subsidy of freshwater food webs. However, SIA fails to differentiate between the contributions of old and recently fixed terrestrial C and consequently cannot fully determine the source, age, and biochemical quality of terrestrial carbon. Natural abundance radiocarbon (∆14C) was used to examine the age and origin of carbon in Lower Lough Erne, Northern Ireland. 14C and stable isotope values were obtained from invertebrate, algae, and fish samples, and the results indicate that terrestrial organic C is evident at all trophic levels. High winter δ15N values in calanoid zooplankton (δ15N = 24‰) relative to phytoplankton and particulate organic matter (δ15N = 6‰ and 12‰, respectively) may reflect several microbial trophic levels between terrestrial C and calanoid invertebrates. Winter and summer calanoid ∆14C values show a seasonal switch between autochthonous and terrestrial carbon sources. Fish ∆14C values indicate terrestrial support at the highest trophic levels in littoral and pelagic food webs. 14C therefore is useful in attributing the source of carbon in freshwater in addition to tracing the pathway of terrestrial carbon through the food web.

Fulfilling the 3Rs: Galleria mellonella as an In Vivo Model to Assess the Antimicrobial Efficacy of Self-assembling Peptide Hydrogels

The concept of ‘The Three Rs’ (The 3Rs: reduction, refinement and replacement) is an important consideration in the development of alternatives to animal testing in medical research. Invertebrate models such as Galleria mellonella are advantageous both economically and ethically.1 Galleria have proven to be effective alternatives to assess the antimicrobial activity of novel therapeutics.2
In this study Galleria mellonella are validated and used as an in vivo infection model to determine the antimicrobial activity of a novel self-assembling antimicrobial peptide NapFFKK.3 The peptide was considered as being non-toxic to the Galleria with 100% survival 120 hours post inoculation with NapFFKK. Following inoculation with Pseudomonas aeruginosa PAO1, Escherichia coli ATCC 11303, Staphylococcus epidermidis ATCC 35984 and Staphylococcus aureus ATCC 6538, the highest concentration allowing survival was selected and used as the test inoculum. Haemolymph was extracted from inoculated and peptide treated Galleria at either 24 or 72 hours post-treatment. Reduction in bacterial load was determined in comparison to a positive control. Bacterial load was decreased in all treated Galleria with decreasing antimicrobial activity demonstrated with a decreased concentration of peptide (2- log cycle reduction achieved in Escherichia coli inoculated Galleria treated with 2% NapFFKK). The results are promising regarding the use of Galleria mellonella as an infection model and NapFFKK as an effective novel antimicrobial.

The use of biomarkers to diagnose the ecological impact of pollutants in Mediterranean rivers

Um assunto que requer atenção é a avaliação ecológica da qualidade da água de ecossistemas de água doce. Uma abordagem que surge como promissora é a biomonitorização baseada em biomarcadores, porque pode avaliar a saúde dos organismos e obter sinais de alerta precoce acerca dos riscos ambientais. Até agora, porém, o uso de biomarcadores em espécies de invertebrados, para diagnosticar danos ecológicos nos rios, é escasso. Por essa razão, existe uma necessidade urgente de desenvolver biomarcadores nas principais espécies de macroinvertebrados dos ecossistemas fluviais que são alvo de estudo. Esta tese tem como objectivo averiguar se as respostas in situ, aliadas aos biomarcadores, podem ser um método viável para avaliar os danos ecológicos de contaminantes em ecossistemas de água doce. Numa primeira fase, os biomarcadores foram usados para averiguar os mecanismos fisiológicos de adaptação genética de clones de Daphnia magna ao pesticida organofosforado fenitrothion. Numa segunda fase, os biomarcadores foram usados como ferramentas de diagnóstico de poluição em zonas ribeirinhas. Estes estudos foram realizados com três espécies-chave de macroinvertebrados: Daphnia magna, Corbicula fluminea e Hydropsyche exocellata, nos rios Besós e Llobregat e no Delta do rio Ebro (NE Espanha). Além disso, foram realizados com animais capturados nos rios, ou com ensaios de transplantes, e foram complementados com índices biológicos de macroinvertebrados e análises químicas da água e dos animais. Como os contaminantes químicos têm vários modos toxicológicos de acção e, portanto, afectam várias respostas bioquímicas dos organismos, foram analisados nas três espécies um conjunto de biomarcadores pertencentes a diferentes vias metabólicas. A abordagem experimental indica que o uso combinado de biomarcadores e outras medidas, tais como índices biológicos e testes in situ, contribui para diagnosticar os efeitos prejudiciais de contaminantes nas comunidades ribeirinhas.