Sexual dimorphism in the proventriculus of the buff-tailed bumblebee Bombus terrestris (L. 1758) (Hymenoptera: Apidae).

Research on eusocial bee species like Bombus terrestris is primarily focused on the worker caste, which is why their morphology and anatomy are already well described. This includes the alimentary tract, which is adapted for feeding on nectar and pollen. Located at the transition between crop and ventriculus is a highly specialised compartment, the proventriculus. In female workers of B. terrestris, the proventriculus is surrounded by muscles and consists of four anterior lips. A detailed description, however, is only provided for B. terrestis worker bees while studies on the proventriculus of the male reproductive caste are absent. Here, we provide a detailed analysis of the differences between the proventriculus of the B. terrestris males and females through morphometrics, histology and scanning electron microscopy imaging, and unravel a distinct sexual dimorphism. The male proventriculus is wider resulting in a greater volume than the female proventriculus. Histological analysis revealed 4 distinctive chambers of the male proventriculus, which are completely covered with hairs on the inside. In contrast, those chambers in the proventriculus of female B. terrestris, are only rudimentarily present forming only small pouches with hairs in the junctions between the proventricular folds inside the proventriculus. The morphological differences in the proventriculus may be based on different modi vivendi, as males do not return to the colony and fly longer distances. This and the synthesis of sperm and mating plug might require higher energy reserves, leading to the necessity of higher food storage capacities.

The complexity of micro- and nanoplastic research in the genus Daphnia - A systematic review of study variability and a meta-analysis of immobilization rates.

In recent years, the number of publications on nano- and microplastic particles (NMPs) effects on freshwater organisms has increased rapidly. Freshwater crustaceans of the genus Daphnia are widely used in ecotoxicological research as model organisms for assessing the impact of NMPs. However, the diversity of experimental designs in these studies makes conclusions about the general impact of NMPs on Daphnia challenging. To approach this, we systematically reviewed the literature on NMP effects on Daphnia and summarized the diversity of test organisms, experimental conditions, NMP properties and measured endpoints to identify gaps in our knowledge of NMP effects on Daphnia. We use a meta-analysis on mortality and immobilization rates extracted from the compiled literature to illustrate how NMP properties, study parameters and the biology of Daphnia can impact outcomes in toxicity bioassays. In addition, we investigate the extent to which the available data can be used to predict the toxicity of untested NMPs based on the extracted parameters. Based on our results, we argue that focusing on a more diverse set of NMP properties combined with a more detailed characterization of the particles in future studies will help to fill current research gaps, improve predictive models and allow the identification of NMP properties linked to toxicity.

Localisation and identification of polystyrene particles in tissue sections using Raman spectroscopic imaging.

The uptake of microplastic particles (MPP) by organisms is frequently described and poses a potential risk for these organisms and ultimately for humans either through direct uptake or trophic transfer. Currently, the in-situ detection of MPP in organisms is typically based on histological examination of tissue sections after uptake of fluorescently-labelled MPP and is thus not feasible for environmental samples. The alternative approach is purification of MPP from whole organisms or organs by chemical digestion and subsequent spectroscopic detection (FT-IR or Raman). While this approach is feasible for un-labelled particles it goes along with loss of any spatial information related to the location in the tissue. In our study we aimed at providing a workflow for the localisation and identification of non-fluorescent and fluorescent polystyrene (PS) particles (fragments, size range 2-130 µm) in tissue sections of the model organism Eisenia fetida with Raman spectroscopic imaging (RSI). We provide methodological approaches for the preparation of the samples, technical parameters for the RSI measurements and data analysis for PS differentiation in tissue sections. The developed approaches were combined in a workflow for the in-situ analysis of MPP in tissue sections. The spectroscopic analysis requires differentiation of spectra of MPP and interfering compounds, which is challenging given the complexity of tissue. Therefore, a classification algorithm was developed to differentiate PS particles from haem, intestinal contents and surrounding tissue. It allows the differentiation of PS particles from protein in the tissue of E. fetida with an accuracy of 95%. The smallest PS particle detected in the tissue was 2 µm in diameter. We show that it is possible to localise and identify non-fluorescent and fluorescent ingested PS particles directly in tissue sections of E. fetida in the gut lumen and the adjacent tissue.

In-depth characterization revealed polymer type and chemical content specific effects of microplastic on Dreissena bugensis.

In aquatic ecosystems, filter feeders like mussels are particularly vulnerable to microplastics (MP). However, little is known about how the polymer type and the associated properties (like additives or remaining monomers) of MP impact organisms, as the predominant type of MP used for effect studies on the organismic level are micron grade polystyrene spheres, without considering their chemical composition. Therefore, we exposed the freshwater mussel Dreissena bugensis (D. bugensis) to in-depth characterized fragments in the same concentration and size range (20-120 µm): recycled polyethylene terephthalate from drinking bottles, polyamide, polystyrene, polylactic acid, and mussel shell fragments as natural particle control. Real-time valvometry, used to study behavioral responses via the movement of the mussels' valves, showed that mussels cannot distinguish between natural and MP particles, and therefore do not cease their filtration, as when exposed to dissolved pollutants. This unintentional ingestion led to polymer type-dependent adverse effects (activity of antioxidant enzymes and proteomic alterations), related to chemicals and residual monomers found in MP. Overall, recycled PET elicited the strongest negative effects, likely caused by anthranilamide, anthranilonitrile and butylated hydroxytoluene, contained in the fragments, which are toxic to aquatic organisms. As PET is among the most abundant MP in the environment, sublethal effects may gradually manifest at the population level, leading to irreversible ecosystem changes.

MALDI mass spectrometry imaging workflow for the aquatic model organisms Danio rerio and Daphnia magna.

Lipids play various essential roles in the physiology of animals. They are also highly dependent on cellular metabolism or status. It is therefore crucial to understand to which extent animals can stabilize their lipid composition in the presence of external stressors, such as chemicals that are released into the environment. We developed a MALDI MS imaging workflow for two important aquatic model organisms, the zebrafish (Danio rerio) and water flea (Daphnia magna). Owing to the heterogeneous structure of these organisms, developing a suitable sample preparation workflow is a highly non-trivial but crucial part of this work and needs to be established first. Relevant parameters and practical considerations in order to preserve tissue structure and composition in tissue sections are discussed for each application. All measurements were based on high mass accuracy enabling reliable identification of imaged compounds. In zebrafish we demonstrate that a detailed mapping between histology and simultaneously determined lipid composition is possible at various scales, from extended structures such as the brain or gills down to subcellular structures such as a single axon in the central nervous system. For D. magna we present for the first time a MALDI MSI workflow, that demonstrably maintains tissue integrity during cryosectioning of non-preserved samples, and allows the mapping of lipids in the entire body and the brood chamber inside the carapace. In conclusion, the lipid signatures that we were able to detect with our method provide an ideal basis to analyze changes caused by pollutants in two key aquatic model organisms.

Taking advantage of transparency: A proof-of-principle for the analysis of the uptake of labeled microplastic particles by organisms of different functional feeding guilds using an adapted CUBIC protocol.

The analysis of the ingestion of microplastics (MP) by biota is frequently performed through invasive procedures such as chemical digestion protocols or by histological analysis of thin sections. Different, promising approaches for the observation of ingested MP particles pose so called tissue clearing methods. They are currently applied to organs, tissue samples, or whole organisms, rendering the sample transparent and enable to look inside an otherwise opaque environment. To date, there is a lack of methods to detect labeled MP inside an opaque organism's digestive tract without interfering with the sample's integrity. Therefore, our goal was to adapt the CUBIC tissue clearing protocol (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational Analysis) for aquatic and terrestrial organisms of various functional feeding groups for the analysis of the uptake of fluorescent labeled microplastic (MP) particles. We included the buff-tailed bumblebee Bombus terrestris, the compost worm Eisenia fetida, the woodlouse Porcellio scaber, the freshwater shrimp Gammarus roeselii, and the quagga mussel Dreissena bugensis in the analysis. The adapted CUBIC method has led to transparency in all normally opaque organisms. It further offers a simple way of locating fluorescent labeled MP inside the digestive system of the different organisms while leaving them intact.

Classification of target tissues of Eisenia fetida using sequential multimodal chemical analysis and machine learning.

Acquiring comprehensive knowledge about the uptake of pollutants, impact on tissue integrity and the effects at the molecular level in organisms is of increasing interest due to the environmental exposure to numerous contaminants. The analysis of tissues can be performed by histological examination, which is still time-consuming and restricted to target-specific staining methods. The histological approaches can be complemented with chemical imaging analysis. Chemical imaging of tissue sections is typically performed using a single imaging approach. However, for toxicological testing of environmental pollutants, a multimodal approach combined with improved data acquisition and evaluation is desirable, since it may allow for more rapid tissue characterization and give further information on ecotoxicological effects at the tissue level. Therefore, using the soil model organism Eisenia fetida as a model, we developed a sequential workflow combining Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) for chemical analysis of the same tissue sections. Data analysis of the FTIR spectra via random decision forest (RDF) classification enabled the rapid identification of target tissues (e.g., digestive tissue), which are relevant from an ecotoxicological point of view. MALDI imaging analysis provided specific lipid species which are sensitive to metabolic changes and environmental stressors. Taken together, our approach provides a fast and reproducible workflow for label-free histochemical tissue analyses in E. fetida, which can be applied to other model organisms as well.

Uncovering the chemistry behind inducible morphological defences in the crustacean Daphnia magna via micro-Raman spectroscopy.

The widespread distribution of Crustacea across every aquatic ecological niche on Earth is enabled due to their exoskeleton's versatile properties. Especially mineralization of the exoskeleton provides protection against diverse environmental threats. Thereby, the exoskeleton of some entomostracans is extremely phenotypically plastic, especially in response to predators. For instance, the freshwater zooplankton Daphnia forms conspicuous inducible morphological defenses, such as helmets, and can increase the stability of its exoskeleton, which renders them less vulnerable to predation. In this study, we reveal for the first time the chemical composition of the exoskeleton of Daphnia magna, using Raman spectroscopy, to be composed of α-chitin and proteins with embedded amorphous calcium carbonate (ACC). Furthermore, we reveal the exoskeleton's chemical changes associated with inducible defense mechanisms in the form of more substantial mineralization, which is probably correlated with enhanced carapace stability. We, therefore, highlight the importance of calcium-biominerals for inducible morphological defenses in Daphnia.

Predator-specific inducible morphological defenses of a water flea against two freshwater predators.

The expression of inducible morphological defenses in Daphnia in response to a single predator is a well-known phenomenon. However, predator-specific modifications of the same defensive traits as an adaption to different predator regimes is so far only described for Daphnia barbata. It is unknown if this accounts only for this species or if it is a more widespread, general adaptive response in the genus Daphnia. In the present study, we therefore investigated whether a clone of the pond-dwelling species Daphnia similis responds to different predatory invertebrates (Triops cancriformis; Notonecta maculata) with the expression of predator-specific modifications of the same defensive traits. We showed that Triops-exposed individuals express a significantly longer tail-spine, while body width decreased in comparison to control individuals. Additionally, they also expressed inconspicuous defenses, that is, significantly longer spinules on the dorsal ridge. The Notonecta-exposed D. similis showed a significantly longer tail-spine, longer spinules and a larger spinules bearing area on the dorsal ridge than control individuals as well. However, a geometric morphometric analysis of the head shape revealed significant, predator-specific changes. Triops-exposed individuals expressed a flattened head shape with a pronounced dorsal edge, while Notonecta-exposed individuals developed a high and strongly rounded head. Our study describes so far unrecognized inducible defenses of D. similis against two predators in temporary waters. Furthermore, the predator-dependent change in head shape is in concordance with the 'concept of modality', which highlights the qualitative aspect of natural selection caused by predators.