Mosaic or melting pot: The use of monogeneans as a biological tag and magnifying glass to discriminate introduced populations of Nile tilapia in sub-Saharan Africa.

The origin of introduced Nile tilapia stocks in sub-Saharan Africa is largely unknown. In this study, the potential of monogeneans as a biological tag and magnifying glass is tested to reveal their hosts' stocking history. The monogenean gill community of different Nile tilapia populations in sub-Saharan Africa was explored, and a phylogeographic analysis was performed based on the mitogenomes of four dactylogyrid species (Cichlidogyrus halli, C. sclerosus, C. thurstonae, and Scutogyrus longicornis). Our results encourage the use of dactylogyrids as biological tags. The magnifying glass hypothesis is only confirmed for C. thurstonae, highlighting the importance of the absence of other potential hosts as prerequisites for a parasite to act as a magnifying glass. With the data generated here, we are the first to extract mitogenomes from individual monogeneans and to perform an upscaled survey of the comparative phylogeography of several monogenean species with unprecedented diagnostic resolution.

Explosive networking: The role of adaptive host radiations and ecological opportunity in a species-rich host-parasite assembly.

Many species-rich ecological communities emerge from adaptive radiation events. Yet the effects of adaptive radiation on community assembly remain poorly understood. Here, we explore the well-documented radiations of African cichlid fishes and their interactions with the flatworm gill parasites Cichlidogyrus spp., including 10,529 reported infections and 477 different host-parasite combinations collected through a survey of peer-reviewed literature. We assess how evolutionary, ecological, and morphological parameters determine host-parasite meta-communities affected by adaptive radiation events through network metrics, host repertoire measures, and network link prediction. The hosts' evolutionary history mostly determined host repertoires of the parasites. Ecological and evolutionary parameters predicted host-parasite interactions. Generally, ecological opportunity and fitting have shaped cichlid-Cichlidogyrus meta-communities suggesting an invasive potential for hosts used in aquaculture. Meta-communities affected by adaptive radiations are increasingly specialised with higher environmental stability. These trends should be verified across other systems to infer generalities in the evolution of species-rich host-parasite networks.

Regenerative responses following DNA damage - ß-catenin mediates head regrowth in the planarian Schmidtea mediterranea.

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Schmidtea mediterranea Exposure to the genotoxic compound methyl methanesulfonate (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component ß-catenin-1 increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt signalling gradient along the body axis modulate stem cell responses to MMS.

Population genomics of introduced Nile tilapia Oreochromis niloticus (Linnaeus, 1758) in the Democratic Republic of the Congo: Repeated introductions since colonial times with multiple sources.

During colonial times, Nile tilapia Oreochromis niloticus (Linnaeus, 1758) was introduced into non-native parts of the Congo Basin (Democratic Republic of the Congo, DRC) for the first time. Currently, it is the most farmed cichlid in the DRC, and is present throughout the Congo Basin. Although Nile tilapia has been reported as an invasive species, documentation of historical introductions into this basin and its consequences are scant. Here, we study the genetic consequences of these introductions by genotyping 213 Nile tilapia from native and introduced regions, focusing on the Congo Basin. Additionally, 48 specimens from 16 other tilapia species were included to test for hybridization. Using RAD sequencing (27,611 single nucleotide polymorphisms), we discovered genetic admixture with other tilapia species in several morphologically identified Nile tilapia from the Congo Basin, reflecting their ability to interbreed and the potential threat they pose to the genetic integrity of native tilapias. Nile tilapia populations from the Upper Congo and those from the Middle-Lower Congo are strongly differentiated. The former show genetic similarity to Nile tilapia from the White Nile, while specimens from the Benue Basin and Lake Kariba are similar to Nile tilapia from the Middle-Lower Congo, suggesting independent introductions using different sources. We conclude that the presence of Nile tilapia in the Congo Basin results from independent introductions, reflecting the dynamic aquaculture history, and that their introduction probably leads to genetic interactions with native tilapias, which could lower their fitness. We therefore urge avoiding further introductions of Nile tilapia in non-native regions and to use native tilapias in future aquaculture efforts.

Somewhere I belong: phylogeny and morphological evolution in a species-rich lineage of ectoparasitic flatworms infecting cichlid fishes.

A substantial portion of biodiversity has evolved through adaptive radiation. However, the effects of explosive speciation on species interactions remain poorly understood. Metazoan parasites infecting radiating host lineages could improve our knowledge because of their intimate host relationships. Yet limited molecular, phenotypic and ecological data discourage multivariate analyses of evolutionary patterns and encourage the use of discrete characters. Here, we assemble new molecular, morphological and host range data widely inferred from a species-rich lineage of parasites (Cichlidogyrus, Platyhelminthes: Monogenea) infecting cichlid fishes to address data scarcity. We infer a multimarker (28S/18S rDNA, ITS1, COI mtDNA) phylogeny of 58 of 137 species and characterize major lineages through synapomorphies inferred from mapping morphological characters. We predict the phylogenetic position of species without DNA data through shared character states, a morphological phylogenetic analysis, and a classification analysis with support vector machines. Based on these predictions and a cluster analysis, we assess the systematic informativeness of continuous characters, search for continuous equivalents for discrete characters, and suggest new characters for morphological traits not analysed to date. We also model the attachment/reproductive organ and host range evolution using the data for 136 of 137 described species and multivariate phylogenetic comparative methods (PCMs). We show that discrete characters not only can mask phylogenetic signals, but also are key for characterizing species groups. Regarding the attachment organ morphology, a divergent evolutionary regime for at least one lineage was detected and a limited morphological variation indicates host and environmental parameters affecting its evolution. However, moderate success in predicting phylogenetic positions, and a low systematic informativeness and high multicollinearity of morphological characters call for a revaluation of characters included in species characterizations.

Wind-associated detours promote seasonal migratory connectivity in a flapping flying long-distance avian migrant.

It is essential to gain knowledge about the causes and extent of migratory connectivity between stationary periods of migrants to further the understanding of processes affecting populations, and to allow efficient implementation of conservation efforts throughout the annual cycle. Avian migrants likely use optimal routes with respect to mode of locomotion, orientation and migration strategy, influenced by external factors such as wind and topography. In self-powered flapping flying birds, any increases in fuel loads are associated with added flight costs. Energy-minimizing migrants are therefore predicted to trade-off extended detours against reduced travel across ecological barriers with no or limited foraging opportunities. Here, we quantify the extent of detours taken by different populations of European nightjars Caprimulgus europaeus, to test our predictions that they used routes beneficial according to energetic principles and evaluate the effect of route shape on seasonal migratory connectivity. We combined data on birds tracked from breeding sites along a longitudinal gradient from England to Sweden. We analysed the migratory connectivity between breeding and main non-breeding sites, and en route stopover sites just south of the Sahara desert. We quantified each track's route extension relative to the direct route between breeding and wintering sites, respectively, and contrasted it to the potential detour derived from the barrier reduction along the track while accounting for potential wind effects. Nightjars extended their tracks from the direct route between breeding and main non-breeding sites as they crossed the Mediterranean Sea-Sahara desert, the major ecological barrier in the Palaearctic-African migration system. These clockwise detours were small for birds from eastern sites but increased from east to west breeding longitude. Routes of the tracked birds were associated with partial reduction in the barrier crossing resulting in a trade-off between route extension and barrier reduction, as expected in an energy-minimizing migrant. This study demonstrates how the costs of barrier crossings in prevailing winds can disrupt migratory routes towards slightly different goals, and thereby promote migratory connectivity. This is an important link between individual migration strategies in association with an ecological barrier, and both spatially and demographic population patterns.

Do you have the nerves to regenerate? The importance of neural signalling in the regeneration process.

The importance of nerve-derived signalling for correct regeneration has been the topic of research for more than a hundred years, but we are just beginning to identify the underlying molecular pathways of this process. Within the current review, we attempt to provide an extensive overview of the neural influences during early and late phases of both vertebrate and invertebrate regeneration. In general, denervation impairs limb regeneration, but the presence of nerves is not essential for the regeneration of aneurogenic extremities. This observation led to the "neurotrophic factor(s) hypothesis", which states that certain trophic factors produced by the nerves are necessary for proper regeneration. Possible neuron-derived factors which regulate regeneration as well as the denervation-affected processes are discussed.

Toxic effects of cadmium on flatworm stem cell dynamics: A transcriptomic and ultrastructural elucidation of underlying mechanisms.

Stem cells or undifferentiated cells can cope more easily with external stresses. To evaluate the impact of toxic compounds on stem cell dynamics in vivo, in relation to other biological responses, we use the carcinogenic element cadmium and the regenerating model organism Macrostomum lignano. Through both BrdU and anti-histone H3 immunostainings, cadmium-induced effects were investigated at different stages of the stem cell cycle. A 24-h exposure to 100 and 250 µM CdCl2 significantly decreased the number of stem cells (neoblasts) in mitosis, whereas the number of cells in the S phase remained unchanged. After this short-term exposure, the ultrastructure of the neoblasts was minimally affected in contrast to the epidermal tissues. These results were supported by gene expression data: transcripts of cdc2 and pig3 were significantly upregulated during all treatments. Both genes are involved in the cell cycle progression and are transcribed in the gonadal region, where stem cells are highly represented. Based on a substantial increase in gene expression of heat shock proteins (HSP) and their high activity in the gonadal region, we hypothesize that these proteins are key players in the protection of stem cells against external stresses. Apart from the strong HSP induction, other protective processes including cell division, apoptosis and anti-oxidative defence, were also activated. We, therefore, conclude that the protection of stem cells against external stressors may be based on the interplay between stem cell maintenance, i.e. repair and recovery through division, on one hand and apoptosis on the other hand. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1217-1228, 2016.

Toxicity profiles and solvent-toxicant interference in the planarian Schmidtea mediterranea after dimethylsulfoxide (DMSO) exposure.

To investigate hydrophobic test compounds in toxicological studies, solvents like dimethylsulfoxide (DMSO) are inevitable. However, using these solvents, the interpretation of test compound-induced responses can be biased. DMSO concentration guidelines are available, but are mostly based on acute exposures involving one specific toxicity endpoint. Hence, to avoid solvent-toxicant interference, we use multiple chronic test endpoints for additional interpretation of DMSO concentrations and propose a statistical model to assess possible synergistic, antagonistic or additive effects of test compounds and their solvents. In this study, the effects of both short- (1 day) and long-term (2 weeks) exposures to low DMSO concentrations (up to 1000 µl l(-1) ) were studied in the planarian Schmidtea mediterranea. We measured different biological levels in both fully developed and developing animals. In a long-term exposure set-up, a concentration of 500 µl l(-1) DMSO interfered with processes on different biological levels, e.g. behaviour, stem cell proliferation and gene expression profiles. After short exposure times, 500 µl l(-1) DMSO only affected motility, whereas the most significant changes on different parameters were observed at a concentration of 1000 µl l(-1) DMSO. As small sensitivity differences exist between biological levels and developmental stages, we advise the use of this solvent in concentrations below 500 µl l(-1) in this organism. In the second part of our study, we propose a statistical approach to account for solvent-toxicant interactions and discuss full-scale solvent toxicity studies. In conclusion, we reassessed DMSO concentration limits for different experimental endpoints in the planarian S. mediterranea.

Revision of Acrochordonoposthia Reisinger, 1924 (Rhabditophora, Typhloplanidae, Protoplanellinae) with the description of one new species.

An overview of the morphology, taxonomy and distribution of all known species of the rhabdocoel taxon Acrochordonoposthia is provided. One new species A. vandeputae n. sp. is described from Graz (Austria). This new species can easily be distinguished from its congeners by the morphology of its copulatory organ, which contains a straight cirrus lined with spines. All species of Acrochordonoposthia are compared with one another, and an updated identification key is provided.

A new genus with six new species of Typhlopolycystidinae Evdonin, 1977 (Platyhelminthes, Kalyptorhynchia, Polycystididae).

Five new species of the new taxon Brunetorhynchus n. gen. are described: B. deconincki n. sp., B. microstylis n. sp. and B. complicatus n. sp. are from the Mediterranean, B. canariensis n. sp. is from the Canary Island Lanzarote, B. cannoni n. sp. is from the Australian East coast and one species from the Galapagos, formerly described as Limipolycystis spec., is transferred to the new genus as B. dubius n. sp.. As in Limipolycystis, these species have a single stylet, (accessory stylet type II), an accessory secretion vesicle (type II) and a prostate vesicle (type III) in the male atrium, although the latter vesicle is absent in some species. Unlike the species of Limipolycystis, where the seminal receptacle is a sclerotized tubule, the species of the new taxon have a pear-shaped seminal receptacle on the oviduct. 

First record of a proseriate flatworm predating on a rhabdocoel (Platyhelminthes: Proseriata and Rhabdocoela).

Microturbellarian flatworms comprise a diverse assemblage amongst meiofauna. These animals primarily exhibit carnivorous feeding habits, preying on various organisms, such as crustaceans, annelids and even other microturbellarians. However, details of their diet are poorly known. This study represents the first documentation of a proseriate preying upon a rhabdocoel. The proseriate was extracted from the sediment and studied alive. Within its digestive tract, structures of the reproductive systems of its prey were observed and identified as belonging to Phonorhynchopsishaegheni, a predatory turbellarian as well. No remains of any other organisms were detected. This finding underscores the relevance of Proseriata as top-level predators within the meiofaunal trophic web, a role that warrants further consideration beyond what has been previously acknowledged.

Brachyrhynchus n. gen. n. sp., a new genus of Polycystididae Graff, 1905 (Rhabdocoela: Kalyptorhynchia), with the description of three new species from the Mediterranean and the Indian Ocean.

Three new species of Polycystididae are described: Brachyrhynchus triplostylis n. gen. n. sp., B. acutus n. sp. and B. oosterlyncki n. sp. They are the first species of Polycystididae to be described with a second, single-walled accessory stylet connected to a small glandular vesicle in the male system. The three species can be distinguished from each other by the detailed morphology and the relative lengths of the three stylets in the male system. The relationships of these species with other representatives of Polycystididae are discussed.

Green roofs and pollinators, useful green spots for some wild bee species (Hymenoptera: Anthophila), but not so much for hoverflies (Diptera: Syrphidae).

Urbanisation has become one of the major anthropogenic drivers behind insect decline in abundance, biomass and species richness over the past decades. As a result, bees and other pollinators' natural habitats are reduced and degraded. Green roofs are frequently recommended as ways to counter the negative impacts of urbanisation on nature and enhance the amount of green space in cities. In this study we evaluated the pollinator (more specifically wild bees and hoverflies) diversity, abundance and species richness on twenty green roofs in Antwerp, Belgium. We analysed the influence of roof characteristics (age, surface area, height, percent cover of green space surrounding each site) on species richness or abundance of pollinators. In total we found 40 different wild bee species on the green roofs. None of the physical roof characteristics appear to explain differences in wild bees species richness and abundance. Neither could we attribute the difference in roof vegetation cover, i.e. roofs build-up with only Sedum species and roofs with a combined cover of Sedum, herbs and grasses, to differences in diversity, abundance, or species richness. We found a positive trend, although not significant, in community weighted mean body size for wild bees with an increase in green roof surface area. Roof wild bee communities were identified as social polylectic individuals, with a preference for ground nesting. Only eleven individuals from eight different hoverfly species were found. Our results show that green roofs can be a suitable habitat for wild bee species living in urban areas regardless of the roofs' characteristics, but hoverflies have more difficulties conquering these urban green spaces.

Live Imaging in Planarians: Immobilization and Real-Time Visualization of Reactive Oxygen Species.

Imaging of living animals allows the study of metabolic processes in relation to cellular structures or larger functional entities. To enable in vivo imaging during long-term time-lapses in planarians, we combined and optimized existing protocols, resulting in an easily reproducible and inexpensive procedure. Immobilization with low-melting-point agarose eliminates the use of anesthetics, avoids interfering with the animal during imaging-functionally or physically-and allows recovering the organisms after the imaging procedure. As an example, we used the immobilization workflow to image the highly dynamic and fast-changing reactive oxygen species (ROS) in living animals. These reactive signaling molecules can only be studied in vivo and mapping their location and dynamics during different physiological conditions is crucial to understand their role in developmental processes and regeneration. In the current protocol, we describe both the immobilization and ROS detection procedure. We used the intensity of the signals together with pharmacological inhibitors to validate the signal specificity and to distinguish it from the autofluorescent nature of the planarian.

All quiet on the western front? The evolutionary history of monogeneans (Dactylogyridae: Cichlidogyrus, Onchobdella) infecting a West and Central African tribe of cichlid fishes (Chromidotilapiini).

Owing to the largely unexplored diversity of metazoan parasites, their speciation mechanisms and the circumstances under which such speciation occurs - in allopatry or sympatry - remain vastly understudied. Cichlids and their monogenean flatworm parasites have previously served as a study system for macroevolutionary processes, e.g., for the role of East African host radiations on parasite communities. Here, we investigate the diversity and evolution of the poorly explored monogeneans infecting a West and Central African lineage of cichlid fishes: Chromidotilapiini, which is the most species-rich tribe of cichlids in this region. We screened gills of 149 host specimens (27 species) from natural history collections and measured systematically informative characters of the sclerotised attachment and reproductive organs of the parasites. Ten monogenean species (Dactylogyridae: Cichlidogyrus and Onchobdella) were found, eight of which are newly described and one redescribed herein. The phylogenetic positions of chromidotilapiines-infecting species of Cichlidogyrus were inferred through a parsimony analysis of the morphological characters. Furthermore, we employed machine learning algorithms to detect morphological features associated with the main lineages of Cichlidogyrus. Although the results of these experimental algorithms remain inconclusive, the parsimony analysis indicates that West and Central African lineages of Cichlidogyrus and Onchobdella are monophyletic, unlike the paraphyletic host lineages. Several instances of host sharing suggest occurrences of intra-host speciation (sympatry) and host switching (allopatry). Some morphological variation was recorded that may also indicate the presence of species complexes. We conclude that collection material can provide important insights on parasite evolution despite the lack of well-preserved DNA material.

Title: À l'Ouest, rien de nouveau ? L'histoire évolutive des monogènes (Dactylogyridae : Cichlidogyrus, Onchobdella) infectant une tribu de poissons cichlidés (Chromidotilapiini) d'Afrique occidentale et centrale. Abstract: En raison de la nature largement inexplorée de la diversité des parasites métazoaires, leurs mécanismes de spéciation et les circonstances dans lesquelles cette spéciation se produit­allopatrie ou sympatrie­restent très peu étudiés. Les cichlidés et leurs parasites Plathelminthes monogènes ont déjà servi de modèle pour l'étude des processus macro-évolutifs, par exemple pour le rôle des radiations d'hôtes de l'Afrique de l'Est sur les communautés de parasites. Ici, nous étudions la diversité et l'évolution des monogènes peu étudiées qui infestent une lignée de poissons cichlidés d'Afrique occidentale et centrale : les Chromidotilapiini, qui est la tribu de cichlidés la plus riche en espèces dans cette région. Nous avons examiné les branchies de 149 spécimens hôtes (27 espèces) provenant de musées d'histoire naturelle et mesuré systématiquement les caractères informatifs des pièces sclérifiées du hapteur et des organes copulateurs des parasites. Dix espèces de monogènes (Dactylogyridae : Cichlidogyrus et Onchobdella) ont été trouvées ; huit sont nouvelles pour la science et une est redécrite. Les positions phylogénétiques des espèces de Cichlidogyrus infectant les chromidotilapiines ont été déduites par une analyse de parcimonie des caractères morphologiques. En outre, nous avons utilisé des algorithmes d'apprentissage automatique pour détecter les caractéristiques morphologiques associées aux principales lignées de Cichlidogyrus. Bien que les résultats de ces algorithmes expérimentaux restent peu concluants, l'analyse de parcimonie indique que les lignées de Cichlidogyrus et d'Onchobdella de l'Afrique de l'Ouest et Central sont monophylétiques, contrairement aux lignées d'hôtes qui sont paraphylétiques. Plusieurs cas de partage d'hôtes suggèrent des occurrences de spéciation synxénique (sympatrie) et de changement d'hôte (allopatrie). Certaines variations morphologiques ont été enregistrées et peuvent également indiquer la présence de complexes d'espèces. Nous concluons donc que le matériel de collection peut fournir des informations importantes sur l'évolution des parasites malgré le manque d'ADN exploitable.

Can avian flyways reflect dispersal barriers of clinostomid parasites? First evidence from the mitogenome of Clinostomum complanatum.

Clinostomum complanatum (Rudolphi, 1814) is an economically important parasitic flatworm (Trematoda, Digenea), yet little is known on the population structure of these animals. We characterise a new mitochondrial genome for C. complanatum, derived from an Iranian specimen. The newly obtained sequence is used to position the species in the digenean tree of life. The first-ever intraspecific comparison at mitogenome scale within C. complanatum revealed a high degree of similarity to the previously sequenced mitogenome of a distant (Italian) population. Avian migratory routes mirror phylogenetic clustering, and hence we suggest that infection of a flying host enables genetic exchange between parasites across large geographic distances. Comparative mitogenomic work in Clinostomum spp. at both the intra- (C. complanatum) and interspecific (C. complanatum-C. sinensis) level further shows that usage of new and/or additional mitochondrial markers is preferred over single-gene methods for high-resolution diagnostics and population biology.

Sharing is caring? Barcoding suggests co-introduction of dactylogyrid monogeneans with Nile tilapia and transfer towards native tilapias in sub-Saharan Africa.

Invasive Nile tilapias negatively impact native tilapia species through hybridisation and competition. However, the co-introduction of parasites with Nile tilapia, and subsequent changes in parasite communities, are scarcely documented. Monogeneans are known pathogens of cultured Nile tilapia, although little is known about their fate once Nile tilapias establish in new ecosystems. We investigate the parasitological consequences of Nile tilapia introduction on native tilapias in basins in Cameroon, the Democratic Republic of the Congo (DRC), and Zimbabwe, focusing on ectoparasitic dactylogyrids (Monogenea). Using the mitochondrial cytochrome oxidase c subunit I (COI) and nuclear 18S-internal transcribed spacer 1 (18S-ITS1) rDNA region of 128 and 166 worms, respectively, we evaluated transmission of several dactylogyrid species. Parasite spillover from Nile tilapia was detected for Cichlidogyrus tilapiae to Coptodon guineensis in Cameroon, Cichlidogyrus thurstonae to Oreochromis macrochir in the DRC, and Cichlidogyrus halli and C. tilapiae to Coptodon rendalli in Zimbabwe. Parasite spillback to Nile tilapia was detected for Cichlidogyrus papernastrema and Scutogyrus gravivaginus from Tilapia sparrmanii and Cichlidogyrus dossoui from C. rendalli or T. sparrmanii in the DRC, and Cichlidogyrus chloeae from Oreochromis cf. mortimeri and S. gravivaginus from O. macrochir in Zimbabwe. 'Hidden' transmissions (i.e. transmission of certain parasite lineages of species that are naturally present on both alien and native hosts) were detected for C. tilapiae and Scutogyrus longicornis between Nile tilapia and Oreochromis aureus and C. tilapiae between Nile tilapia and Oreochromis mweruensis in the DRC, and Cichlidogyrus sclerosus and C. tilapiae between Nile tilapia and O. cf. mortimeri in Zimbabwe. A high density of Nile tilapia occurring together with native tilapias, and the broad host range and/or environmental tolerance of the transmitted parasites, are proposed as factors behind parasite transmission through ecological fitting. However, continuous monitoring and the inclusion of environmental variables are necessary to understand the long-term consequences of these transmissions on native tilapias and to elucidate other underlying factors influencing these transmissions.

Reference genes for qPCR assays in toxic metal and salinity stress in two flatworm model organisms.

The flatworm species Schmidtea mediterranea and Macrostomum lignano have become new and innovative model organisms in stem cell, regeneration and tissue homeostasis research. Because of their unique stem cell system, (lab) technical advantages and their phylogenetic position within the Metazoa, they are also ideal candidate model organisms for toxicity assays. As stress and biomarker screenings are often performed at the transcriptional level, the aim of this study was to establish a set of reference genes for qPCR experiments for these two model organisms in different stress situations. We examined the transcriptional stability of nine potential reference genes (actb, tubb, ck2, cox4, cys, rpl13, gapdh, gm2ap, plscr1) to assess those that are most stable during altered stress conditions (exposure to carcinogenic metals and salinity stress). The gene expression stability was evaluated by means of geNorm and NormFinder algorithms. Sets of best reference genes in these analyses varied between different stress situations, although gm2ap and actb were stably transcribed during all tested combinations. In order to demonstrate the impact of bad normalisation, the stress-specific gene hsp90 was normalised to different sets of reference genes. In contrast to the normalisation according to GeNorm and NormFinder, normalisation of hsp90 in Macrostomum lignano during cadmium stress did not show a significant difference when normalised to only gapdh. On the other hand an increase of variability was noticed when normalised to all nine tested reference genes together. Testing appropriate reference genes is therefore strongly advisable in every new experimental condition.

Dactylogyridae 2022: a meta-analysis of phylogenetic studies and generic diagnoses of parasitic flatworms using published genetic and morphological data.

Dactylogyridae is one of the most studied families of parasitic flatworms with more than 1000 species and 166 genera described to date including ecto- and endoparasites. Dactylogyrid monogeneans were suggested as model organisms for host-parasite macroevolutionary and biogeographical studies due to the scientific and economic importance of some of their host lineages. Consequently, an array of phylogenetic research into different dactylogyrid lineages has been produced over the past years but the last family-wide study was published 16 years ago. Here, we provide a meta-analysis of the phylogenetic relationships of Dactylogyridae including representatives of all genera with available molecular data (n = 67). First, we investigate the systematic informativeness of morphological characters widely used to diagnose dactylogyrid genera through a parsimony analysis of the characters, character mapping, and phylogenetic comparative methods. Second, we provide an overview of the current state of the systematics of the family and its subfamilies, and summarise potentially poly- and paraphyletic genera. Third, we elaborate on the implications of taxonomic, citation, and confirmation bias in past studies. Fourth, we discuss host range, biogeographical, and freshwater-marine patterns. We found two well-supported macroclades which we assigned to the subfamilies Dactylogyrinae and Ancyrocephalinae. These subfamilies further include 16 well-supported clades with only a few synapomorphies that could be deduced from generic diagnoses in the literature. Furthermore, few morphological characters considered systematically informative at the genus level display a strong phylogenetic signal. However, the parsimony analysis suggests that these characters provide little information on the relationships between genera. We conclude that a strong taxonomic bias and low coverage of DNA sequences and regions limit knowledge on morphological and biogeographical evolutionary patterns that can be inferred from these results. We propose addressing potential citation and confirmation biases through a 'level playing field' multiple sequence alignment as provided by this study.