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Little phylogeographic structure is presumed for highly mobile species in pelagic zones. Lake Tanganyika is a unique ecosystem with a speciose and largely endemic fauna famous for its remarkable evolutionary history. In bathybatine cichlid fishes, the pattern of lake-wide population differentiation differs among species. We tested the magnifying glass hypothesis for their parasitic flatworm Cichlidogyrus casuarinus. Lake-wide population structure of C. casuarinus ex Hemibates stenosoma was assessed based on a portion of the mtCOI gene combined with morphological characterisation. Additionally, intraspecific mitogenomic variation among 80 individuals within one spatially constrained parasite metapopulation sample was assessed using shotgun NGS. While no clear geographic genetic structure was detected in parasites, both geographic and host-related phenotypic variation was apparent. The incongruence with the genetic north-south gradient observed in the host may be explained by the broad host range of this flatworm as some of its other host species previously showed no lake-wide restriction of gene flow. Our results are consistent with host driven morphological variation without genetic differentiation of the parasite, and highlight the importance of integratively approaching parasites` potential as “tags” for their hosts. We present the first parasite mitogenome from Lake Tanganyika and propose a methodological framework for studying intraspecific mitogenomic variation of dactylogyrid monogeneans.

Gastrotricha and Platyhelminthes form a clade called Rouphozoa. Representatives of both taxa are main components of meiofaunal communities, but their role in the trophic ecology of marine and freshwater communities is not sufficiently studied. Traditional collection methods for meiofauna are optimized for Ecdysozoa, and include the use of fixatives or flotation techniques that are unsuitable for the preservation and identification of soft-bodied meiofauna. As a result, rouphozoans are usually underestimated in conventional biodiversity surveys and ecological studies. Here, we give an updated outline of their diversity and taxonomy, with some phylogenetic considerations. We describe successfully tested techniques for their recovery and study, and emphasize current knowledge on the ecology, distribution, and dispersal of freshwater gastrotrichs and microturbellarians. We also discuss the opportunities and pitfalls of (meta)barcoding studies as a means of overcoming the taxonomic impediment. Finally, we discuss the importance of rouphozoans in aquatic ecosystems and provide future research directions to fill in crucial gaps in the biology of these organisms needed for understanding their basic role in the ecology of benthos and their place in the trophic networks linking micro-, meio-, and macrofauna of freshwater ecosystems. Balsamo, M (corresponding author), Univ Urbino, Dept Biomol Sci, Urbino, Italy. maria.balsamo@uniurb.it; tom.artois@uhasselt.be; smithj@winthrop.edu; antonio.todaro@unimore.it; loretta.guidi@uniurb.it; bleander@mail.ubc.ca; niels.vansteenkiste@botany.ubc.ca

Species interactions are a key aspect of evolutionary biology. Parasites, specifically, are drivers of the evolution of species communities and impact biosecurity and public health. However, when using interaction networks for evolutionary studies, interdependencies between distantly related species in these networks are shaped by ancient and complex processes. We propose using recent interacting host-parasite radiations, e.g. African cichlid fishes and cichlid gill parasites belonging toCichlidogyrus(Dactylogyridae, Monogenea), as macroevolutionary model of species interactions. The cichlid-Cichlidogyrusnetwork encompasses 138 parasite species and 416 interactions identified through morphological characteristics and genetic markers in 160 publications. We discuss the steps required to develop this model system based on data resolution, sampling bias, and reporting quality. In addition, we propose the following steps to guide efforts for a macroevolutionary model system for species interactions: first, evaluating and expanding model system outcome measures to increase data resolution; second, closing knowledge gaps to address underreporting and sampling bias arising from limited human and financial resources. Identifying phylogenetic and geographic targets, creating systematic overviews, enhancing scientific collaborations, and avoiding data loss through awareness of predatory journal publications can accelerate this process; and third, standardising data reporting to increase reporting quality and to facilitate data accessibility.

The Lower Congo Basin is characterized by a mangrove-lined estuary at its mouth and, further upstream, by many hydrogeographical barriers such as rapids and narrow gorges. Five localities in the mangroves and four from (upstream) left bank tributaries or pools were sampled. On the gills of Coptodon tholloni, Coptodon rendalli, Hemichromis elongatus, Hemichromis stellifer and Tylochromis praecox, 17 species of parasites (Dactylogyridae & Gyrodactylidae, Monogenea) were found, eight of which are new to science. Six of these are herein described: Cichlidogyrus bixlerzavalai n. sp. and Cichlidogyrus omari n. sp. from T praecox, Cichlidogyrus calycinus n. sp. and Cichlidogyrus polyenso n. sp. from H. elongatus, Cichlidogyrus kmentovae n. sp. from H. stellifer and Onchob-della ximenae n. sp. from both species of Hemichromis. On Cichlidogyrus reversati a ridge on the accessory piece was discovered that connects to the basal bulb of the penis. We report a putative spillback effect of the native parasites Cichlidogyrus berradae, Cichlidogyrus cubitus and Cichlidogyrus flexicolpos from C. tholloni to the introduced C. rendalli. From our results, we note that the parasite fauna of Lower Congo has a higher affinity with the fauna of West African and nearby freshwater ecoregions than it has with fauna of other regions of the Congo Basin and Central Africa.

Accurate assessments of biodiversity are crucial to advising ecosystem-monitoring programs and understanding ecosystem function. Nevertheless, a standard operating procedure to assess biodiversity accurately and consistently has not been established. This is especially true for meiofauna, a diverse community (>20 phyla) of small benthic invertebrates that have fundamental ecological roles. Recent studies show that metabarcoding is a cost-effective and time-effective method to estimate meiofauna biodiversity, in contrast to morphological-based taxonomy. Here, we compare biodiversity assessments of a diverse meiofaunal community derived by applying multiple taxonomic methods based on comparative morphology, molecular phylogenetic analysis, DNA barcoding of individual specimens, and metabarcoding of environmental DNA. We show that biodiversity estimates are strongly biased across taxonomic methods and phyla. Such biases affect understanding of community structures and ecological interpretations. This study supports the urgency of improving aspects of environmental high-throughput sequencing and the value of taxonomists in correctly understanding biodiversity estimates.

Even at the start of the 21st century, the actual biodiversity of the world's marine ecosystems is still largely unknown. Not surprisingly, especially the smallest creatures are much understudied. Among the least known taxonomical groups are the microturbellarians, small free-living flatworms that inhabit sandy beaches or live epiphytically on algae. With a rough conservative estimation of about 15.000 species still to be described, an enormous task still lays ahead for classical morphology-based taxonomy. And not only is the relative number of species already described limited, but on top of that most of them are described from a few marine ecoregions only, mostly from the Western and Northern Europe, and the Mediterranean. The major part of the world's coasts and deep sea habitats never were investigated. This lack of knowledge greatly hampers further research regarding biogeography, ecology or even phylogeny. In recent years we have tried to shed light on several questions regarding the evolution and ecology of these animals, including the origin of a symbiotic life style, the history of major habitat shifts and the "Everything small is everywhere"-hypothesis, research that is still ongoing. These studies were only possible because of a big taxonomical effort and trustable databases, and the results will briefly be presented.

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.

Five new species of Limipolycystis Schilke, 1970 are described and their morphology is compared to that of the only species known, L. curvitubo Schilke, 1970. L. sicilicula n. sp., L. falx n. sp., L. friedae n. sp., L. libra n. sp. and L. wallbergi n. sp. all largely resemble L. curvitubo as to the internal anatomy. They all have a caudally-situated testis and, at least in those species for which good material is available, a seminal receptacle could be seen. This seminal receptacle is tubiform, connecting the bursa with the most proximal part of the oviduct, which is filled with sperm. All these species are mainly distinguishable from each other by details in the shape of the stylet. Diagnoses are provided for all new species and the diagnosis of the genus is emended to reflect the new finds.

Spermatogenesis and the structure of the testes were studied ultrastructurally in Isodiametra pulchra (Smith and Bush, Transactions of the American Microscopical Society 1991; 110: 12; Hooge and Tyler, Journal of zoological systematics and evolutionary research 2005; 43: 100). The testes are paired, compact, non-follicular and lie dorsally and dorso-laterally to the paired ovaries, partially enfolding them. All stages of spermatogenesis, including spermiogenesis, are described at the ultrastructural level and their spatial organization within the testes is discussed. The cells at the early stages of spermatogenesis (spermatogonia and spermatocytes) are located on the dorsal and dorso-lateral sides of the testes, while the late stages (spermatids and filiform spermatozoa with 9+2 axonemes) lie at the ventral and inner periphery of the testes, adjacent to ovaries. All the cell types can be found both at the anterior and the posterior end of the testes. The value of the structure of the testes as a phylogenetic marker is addressed.

The neoblast stem cell system of flatworms is considered to be unique within the animal kingdom. How this stem cell system arises during embryonic development is intriguing. Therefore we performed bromodeoxyuridine labelling on late stage embryos of Macrostomum lignano to assess when the pattern of proliferating cells within the embryo is comparable to that of hatchlings. This pattern can be found in late embryonic stages (stage 8). We also used the freeze cracking method to perform macvasa embryonic labelling. Macvasa is a somatic and germ line stem cell marker. We showed macvasa protein distribution during the whole embryonic development. In the macvasa-positive blastomeres the protein is localized around the nucleus in the putative chromatoid bodies. However, at a specific embryonic stage, it is also ubiquitously present in the cytoplasm of some blastomeres. We compare our data with what is known from Schmidtea polychroa of the expression of the vasa-like gene SpolvlgA and the protein distribution of the chromatoid body component Spoltud-1. The embryonic origin of the somatic stem cell system and the germ line is discussed.

The development of macrostomid flatworms is of interest for evolutionary developmental biology research because these taxa combine characteristics of the canonical spiral cleavage pattern with significant deviations from this pattern. One such deviation is the formation of hull cells, which surround the remaining embryonic primordium during early development. Using live observations with a 4D microscope system, histology, and 3D reconstructions, we analyzed the ontogeny of these hull cells in the macrostomid model organism Macrostomum lignano. Our cell lineage analysis allowed us to find the precursors of the hull cells in this species. We discuss the relation between macrostomid development and the development of other spiralians and the question of whether hull cells are homologous within rhabditophoran flatworms.

Morphological and karyological studies have suggested the existence of many cryptic species within Gyratrix hermaphroditus Ehrenberg, 1831, a cosmopolitan, euryhaline ‘species’ of free living flatworms. Molecular data, however, are completely lacking. In this study we have investigated the genetic relationships between morphologically identical, European populations (both freshwater and marine). The analysis is performed using a part of the nuclear ITS1 gene sequence. These analyses revealed the presence of 28 haplotypes. The Neighbour-joining tree shows a freshwater and a monophyletic marine clade. This result raises some very interesting questions concerning the evolutionary history and the biogeography of this species complex. Furthermore, very different haplotypes, belonging to different deep monophyletic clades, occur at the same location. This is an indication that a high number of species can occur sympatrically.

A new species of polyclad flatworm from Papua New Guinea is described. It is found symbiotic in the ophiuroid Ophiothrix purpurea von Martens, 1867 (Echinodermata: Ophiuroidea). Apparently it belongs to the taxon Discoplana Bock, 1913 and can be distinguished from the six previously described Discoplana species by its very short ejaculatory duct and a penial papilla covered with a penial sheath, but without any true sclerotised structures such as a stylet or spines. The cladistic analysis of the Discoplana/Euplana species, based on morphological features and including two outgroups, reveals that all species of Discoplana, except D. pacificola, form a monophyletic taxon, that is not a synonym of Euplana Girard, 1893. Therefore the name Discoplana is conserved and the new species will be described as Discoplana malagasensis sp. nov. A key for the Discoplana/Euplana group is provided. In this key the biogeographical distribution and possible synonyms are given.

A comparative lightmicroscopical study of the male atrial system of the Polycystididae is presented. Firstly, the different glandular structures associated with the male atrial system are compared. This has lead to the recognition of eight different types of glandular vesicles, four of which are considered prostate, the other four accessory. Secondly, the different hard structures that can be found in the male atrium are discussed and compared. These hard structures can consist of numerous hard spines (armed cirrus), but mostly the hard structure is a single- or double-walled tubiform stylet, or a plate-shaped stylet. Different types of stylets can be recognised, some of which are associated with a prostate vesicle and therefore considered prostate stylets. Using the criteria of position and conjunction, conjectures of homologies are put forward. These primary homologies are compared with homology assessments found in earlier literature.

Two new species of Polycystididae are described: Duplacrorhynchus megalophallus sp. nov. from the bay of Marseille (France) and D. heyleni sp. nov. from Zanzibar (Tanzania). The female system of both is characterized by the presence of a muscular bulb at the proximal end of the bursal stalk, with three strong teeth in D. megalophallus, and in D. heyleni the muscle bulb is highly asymmetric. In D. megalophallus the copulatory bulb is divided by a septum, and the cirrus has small spines and a proximal umbrella-like expansion. In D. heyleni only the cirrus, which has strong teeth, is enclosed in the cirrus sack: the seminal and prostate vesicles are not. Relationships with the two known species of the genus (D. minor Schockaert & Karling, 1970 and D. major Schockaert & Karling, 1970) are discussed. The subfamily Duplacrorhynchinae Schockaert & Karlinger, 1970 may constitute a monophyletic taxon, characterised by the 3+2 configuration of the proboscis prostate vesicle. The taxon Duplacrorhynchus may be the sister taxon of all these genera.

Cadmium komt voor in mariene sedimenten dewelke de natuurlijke habitat zijn van de platworm Macrostomum lignano. Deze worm werd recent ontdekt als een nieuw modelorganisme binnen het fylum van de Platyhelminthes. Macrostomum lignano vertoont, net als vele andere platwormen, een enorme regeneratiecapaciteit. Deze regeneratiecapaciteit danken zij aan de aanwezigheid van neoblasten. De cellen die het unieke stamcelsysteem van de platworm vormen. Neoblasten zijn namelijk de enige cellen in adulte wormen die in staat zijn tot deling. De neoblast is dus een fundamenteel en essentieel celtype, dat instaat voor regeneratie en celvernieuwing. Omwille van deze reden is het interessant om het effect van cadmium op de neoblasten te onderzoeken. In deze studie werden de effecten van cadmium op de distributie, de migratie en differentiatie van delende neoblasten onderzocht. Dit werd mogelijk gemaakt met behulp van bromodeoxy-uridine (BrdU), een thymidine-analoog dat wordt geïncorporeerd in het DNA gedurende de S-fase van de celcyclus. Voor het nagaan van distributiepatronen en aantal delende neoblasten werden de wormen kortstondig blootgesteld aan BrdU, terwijl voor het nagaan van migratie- en differentiatiepatronen de wormen continu werden blootgesteld aan BrdU. Voor beide experimenten werden er naast een blancogroep, twee groepen blootgesteld aan respectievelijk 2,5 HM CdCl2 en 5 HM CdCl2 (sublethale concentraties). Het distributiepatroon van neoblasten bleek ongewijzigd te zijn onder invloed van cadmium. Voor het kwantificeren van het aantal delende neoblasten, al dan niet blootgesteld aan cadmium, werd er eerst een methode ontwikkeld om ze te kunnen tellen met ImageJ, gebaseerd op de verdeling van de celvolumes. Hierbij werd er een geautomatiseerde methode uitgewerkt die snel uitgevoerd kon worden en waarbij de foutenmarge zo klein mogelijk gehouden werd. Na deze beeldverwerking bleek dat bij een blootstelling aan 2,5 HM CdCl2 het aantal delende neoblasten na 2 dagen toeneemt. Na 4 dagen daalde het aantal delende neoblasten echter terug tot normale waarden en na 6 dagen daalde het aantal delingen nog meer. Hieruit kan men vaststellen dat een blootstelling aan 2,5 HM op korte termijn (2 dagen) het neoblastsysteem gaat reageren doordat het aantal delingen toeneemt. Op lange termijn (6 dagen) lijkt het echter alsof het neoblastsysteem aangetast wordt ten gevolge van cadmium waardoor de mitotische activiteit afneemt. Bij een blootstelling aan 5 HM werden er geen significante wijzigingen in het aantal delende neoblasten terug gevonden. Dit kan erop wijzen dat het effect van cadmium voor 2 dagen of na 6 dagen zal optreden, verder zou het kunnen dat cadmium bij deze concentratie andere pathways gaat aanspreken die geen invloed hebben op het neoblastsysteem. Een laatste hypothese hieromtrent is dat de algen bij 5 HM aangetast worden waardoor de wormen onvoldoende voedsel tot zich hebben kunnen nemen. Hierdoor gaat de stress van uithongering ervoor zorgen dat het aantal delende neoblasten afneemt wat een antagonistisch effect zou kunnen hebben ten opzicht van een eventuele stijging die 5 HM cadmium veroorzaakt. De effecten van 2,5 HM en 5 HM CdCl2 bleken op de migratie en differentiatie een toename in het aantal gemigreerde en gedifferentieerde cellen te veroorzaken na één en drie dagen blootstelling. Een kwantitatieve analyse was hier echter niet mogelijk omdat de continue blootstelling aan BrdUresulteerde in een aankleuring van teveel cellen.

Cadmium is een toxisch metaal dat bij zowel mens als dier ernstige schade kan veroorzaken aan ondermeer de longen, de nier, de lever en het botweefsel. Deze schade is het gevolg van de effecten op cellulair niveau, waar het metaal oxidatieve stress induceert. Oxidatieve stress is een onevenwicht tussen de pro- en anti-oxidanten in de cel, waardoor een overschot aan pro-oxidanten ontstaat. Dit leidt tot de oxidatie van proteïnen, lipiden en DNA. Een cel beschikt over verschillende beschermingsmechanismen, waaronder het anti-oxidatief systeem met enzymen, zoals catalase en superoxidedismutase, en metabolieten, zoals glutathion en vitamine E. Verder kunnen er ook heat-shock proteïnen geïnduceerd worden om de cel te beschermen, en herstellen indien nodig. In deze scriptie werd onderzoek verricht naar de effecten van blootstelling aan Cd op het cellulaire niveau bij het modelorganisme Macrostomum lignano. Momenteel wordt M. lignano reeds gebruikt als modelorganisme in ontogenetische studies, in onderzoek naar regeneratie en stamceldynamiek en in evolutionaire studies. Dankzij een EST-databank, die pas recent publiek beschikbaar werd gesteld, kan het onderzoek naar achterliggende, moleculaire effecten gestart worden. Het onderzoek dat in deze scriptie wordt voorgesteld vormt een eerste verkennende studie van moleculaire effecten veroorzaakt door Cd, een type onderzoek dat nog bij geen enkele platwormsoort werd uitgevoerd. In eerste instantie was het noodzakelijk de lysis van het weefsel en de RNA extractie te optimaliseren. Hieruit kon besloten worden dat het mogelijk is om RNA te isoleren uit kleine hoeveelheden materiaal door de stalen te malen en vervolgens gebruik te maken van de ‘RNeasy mini kit’ (Qiagen, Venlo). Als tweede werd de LC50-waarde van Cd bij M. lignano bepaald over 1 dag en 5 dagen. Vervolgens werd er gezocht naar genen die stabiel blijven tijdens de behandeling met Cd met geNormTM en NormFinder, en dus het meest aangewezen zijn om gebruikt te worden als referentiegenen in dit experiment. Hieruit blijkt dat -tubuline, caseïne kinase 2 en cytochroom c oxidase subeenheid IV de meest geschikte referentiegenen zijn. Bij analyse van de genexpressie van catalase, calmoduline, heat-shock proteïne 60 en 90 werden er geen significante verschillen gevonden na Cd blootstelling. Tenslotte werden er reactieve zuurstofvormen in vivo gedetecteerd bij M. lignano. H2O2 komt voornamelijk aan de meest frontale lichaamswand en in de vesicula seminalis voor, O2 -° is meer verspreid over het hele lichaam, maar ontbreekt in de gonaden.