Musculature of two bdelloid rotifers, Adineta ricciae and Macrotrachela quadricornifera: organization in a functional and evolutionary perspective

Organization of muscles in microinvertebrates has often been studied to answer functional questions and understand phylogenetic relationships among taxa. In this study, the musculature of two bdelloid species, Adineta ricciae and Macrotrachela quadricornifera, was illustrated, and their organization was compared with other rotifer taxa to generate possible hypotheses of evolutionary relationships among Rotifera. The two species share a common habitat but differ from each other in feeding and locomotion. A. ricciae feeds on the biofilm by scraping it, is unable to swim, and slides on the head cilia using the foot to propel over the substratum. M. quadricornifera feeds by filtration, can swim, and advances by looping in a leech-like motion. Their musculature, stained with TRITC-phalloidin, was observed using confocal laser scanning microscopy. Major differences between the two species were observed in the muscles of head and foot, possibly reflecting differences in their life style. Muscles of the trunk were similarly arranged: circular muscles surrounded longitudinal bands, which were inserted at different points on the body wall. In both bdelloids, circular muscles of the trunk were incomplete ventrally, a condition also present in Seison and in soft-bodied monogononts from benthos. Within rotifers, circular muscles in the form of complete rings are present in acanthocephalans and in soft-bodied planktonic monogononts but are absent in loricate monogononts, which generally possess dorsoventral bands. The diversity of muscle organization among rotifers was interpreted and discussed. Riassunto Lo studio dell'organizzazione muscolare e stato condotto in numerosi microinvertebrati nel tentativo di comprenderne la funzione ma anche i rapporti evolutivi tra i vari taxa. Seguendo la medesima linea investigativa, abbiamo osservato la muscolatura di due rotiferi bdelloidei, Adineta ricciae e Macrotrachela quadricornifera, quindi confrontato i risultati con i dati di letteratura presenti per altri rotiferi. Le due specie investigate condividono lo stesso habitat ma differiscono per modalita di alimentazione e locomozione. Adineta ricciae si nutre grattando il fondo, e incapace di nuotare e si muove strisciando sul substrato utilizzando la forza propulsiva del piede; M. quadricorniferae filtratrice, e in grado di nuotare e di spostarsi sul fondo con caratteristico movimento a sanguisuga. La loro muscolatura, evidenziata con falloidina rodaminata, e stata osservata con microscopio confocale. I risultati mostrano differenze nella regione del capo e del piede, differenze che verosimilmente riflettono lo stile di vita delle specie, la muscolatura del tronco invece mostra un'organizzazione simile: muscoli circolari incompleti esterni e muscoli longitudinali interni inseriti in diversi punti del corpo. I muscoli circolari del tronco sono interrotti ventralmente in entrambe le specie investigate, condizione condivisa con i Seison e con i monogononti bentonici a corpo molle. Estendendo il confronto ad altre specie di rotiferi, si evince che muscoli circolari completi sono presenti in ploimidi planctonici a corpo molle e negli acantocefali, mentre mancano nei monogononti loricati, i quali possiedono muscoli dorsoventrali. Differenze nell'organizzazione muscolare vengono interpretate e discusse sulla base dell'attuale visione filogenetica dei rotiferi

Confocal Laser Scanning Microscopy Applied to a New Species Helps Understand the Functioning of the Reproductive Apparatus in Stylet-Bearing Urodasys (Gastrotricha: Macrodasyida)

Gastrotrichs are highly diverse and abundant in all aquatic ecosystems; however, they are often overlooked. During a biodiversity survey in Sardinia (Italy), a new species of gastrotrich herein described was discovered. Specimens of Urodasys bifidostylis sp. nov. were found in sandy sediments from two submarine caves. Using an integrative approach of traditional light (DIC) and high-resolution (CLSM) microscopies, we herein reveal, for the first time, the fine structure and function of the reproductive organ in an Urodasys representative. This is particularly relevant considering the complex reproductive organs and strategies of this group. Results allow comparisons between the reproductive apparatus and sperm transfer modalities in Urodasys and the closely related genus Macrodasys. One similarity is that both groups transfer male gametes in packets, suggesting the production of spermatophores to be a common phenomenon in Gastrotricha. Unique to Urodasys is the ability of multiple and consecutive copulations and sperm transfers and, differently than Macrodasys, the transfer of sperms unlikely occurs simultaneously between the two hermaphroditic partners. These findings provide new insights into the reproductive strategies of Urodasys and are expected to advance future studies on the evolution of reproductive strategies and the rise of interspecific reproductive barriers in interstitial meiofauna

The Effects of Dissolved and Suspended Solids on Freshwater Meiofauna

Meiofauna include small-sized animals (\u3c 1mm) distributed in all aquatic ecosystems on Earth, where they play fundamental trophic and ecological roles. The biodiversity of marine meiofauna and its links with anthropogenic activities is routinely investigated, however, freshwater communities are less known. This is particularly true for the Southeastern United States, which is greatly investigated and elected a hotspot of biodiversity for larger species but very little is known about the meiofauna. The purpose of this research is to reveal the biodiversity of meiofauna from the Tennessee River and test for potential correlations with anthropogenic activities. As a proxy for pollution, dissolved and suspended solids were considered in this study. The research hypothesis is that meiofaunal biodiversity would be affected by possible changes of dissolved and suspended solids in the water column. Possible mechanisms causing biodiversity shifts could be ascribed to osmotic stresses of animals to cope with variation in dissolved solids or, more indirectly, because different sunlight penetration caused by suspended solids would affect primary production. To test the hypothesis, water samples were collected from nine stations located along the Tennessee River in Hamilton County. Each station was visited three times, and, during each visit, environmental parameters (including dissolved and suspended solids) were measured. Meiofauna biodiversity (estimated as richness, community composition, and phylogenetic diversity) was revealed using a metagenomic approach. Statistical analyses were applied to test for possible correlations between the biodiversity estimates and the measured environmental parameters. Results show a high biodiversity of meiofauna with more than 200 amplicon sequence variants distributed across 10 metazoan phyla. Environmental conditions are highly variable among stations and statistical analyses show that while both dissolved solids (TDS) and turbidity (suspended solids, NTU) did not significantly affect meiofauna biodiversity in the collected samples, various other water and sediment metrics were found to be significant predictors of meiofauna biodiversity. In conclusion, the results of this project not only reveal for the first time the meiofauna biodiversity from the Tennessee River, but also suggest that meiofauna could be used as a bioindicator for several anthropogenic activities in freshwater ecosystems

First report of meiofauna biodiversity from the Tennessee River

Small-sized (\u3c 1mm) invertebrates, commonly known as meiofauna, are abundant and ubiquitous in all aquatic ecosystems, performing key functions such as nutrient cycling and sediment stability. Yet, their unexplored diversity and response to disturbances limit our capacity to understand, mitigate, and remediate the consequences of pollution and environmental changes. The biodiversity of meiofauna is largely overlooked especially in freshwater habitats. For example, the Tennessee River hosts more aquatic species than any other region in North America and contains one of the most diverse aquatic ecosystems in the world, however, little is known about its meiofaunal biodiversity. This work includes results obtained between 2019 and 2021 thanks to the contribution of research students working in Leasi’s lab and students enrolled in the Meiofauna Biodiversity class. An integrated morphological and molecular approach revealed the presence of over 200 meiofaunal species distributed across 10 phyla. Results support that the Tennessee River could be a hotspot of biodiversity for small-sized animals as well. Future works will focus on understanding how anthropogenic inputs affect meiofauna biodiversity in this important ecosystem

A complete three-dimensional reconstruction of the myoanatomy of Loricifera:comparative morphology of an adult and a Higgins larva stage

INTRODUCTION: Loricifera is a group of small, marine animals, with undetermined phylogenetic relationships within Ecdysozoa (molting protostome animals). Despite their well-known external morphology, data on the internal anatomy of loriciferans are still incomplete. Aiming to increase the knowledge of this enigmatic phylum, we reconstruct for the first time the three-dimensional myoanatomy of loriciferans. Adult Nanaloricus sp. and the Higgins larva of Armorloricus elegans were investigated with cytochemical labeling techniques and CLSM. We discuss our findings with reference to other loriciferan species and recently established phylogenies. RESULTS: The somatic musculature of both adult and larval stages is very complex and includes several muscles arranged in three orientations: circular, transverse and longitudinal. In adult Nanaloricus sp., the introvert is characterized by a net-like muscular arrangement, which is composed of five thin circular fibers crossed by several (up to 30) thin longitudinal fibers with bifurcated anterior ends. Two sets of muscles surround the pre-pharyngeal armature: 6 buccal tube retractors arranged 3 × 2 in a conical shaped structure, and 8 mouth cone retractors. Additionally, a thick, circular muscle marks the neck region and a putative anal sphincter is the posteriormost myoanatomical feature. In the Higgins larva of A. elegans, two circular muscles are distinguished anteriorly in the introvert: a dorsal semicircular fiber and a thin ring muscle. The posteriormost region of the body is characterized by an anal sphincter and a triangular muscle. CONCLUSIONS: Based on the currently available knowledge, the myoanatomical bodyplan of adult loriciferans includes: (i) 8 mouth cone retractors, (ii) a pharynx bulb composed of transversal fibers arranged radially, (iii) circular muscles of the head and neck, (iv) internal muscles of the spinoscalids, (v) longitudinal muscles spanning all body regions, and (vi) transverse (circular) muscles in the abdomen. Concerning the Higgins larva, the muscle subsets assigned to its myoanatomical ground pattern are the (i) longitudinal retractors of the mouth cone, introvert, and abdomen, (ii) abdominal transverse muscles, and (iii) a pharynx bulb composed of transverse, radial fibers. In a comparison with phyla traditionally regarded as phylogenetically close, our data show that the overall myoanatomy of Loricifera is more similar to Kinorhyncha and Nematomorpha than to Priapulida. However, the head musculature of all these groups is very similar, which supports homology of their introverts and head morphology

Biodiversity estimates and ecological interpretations of meiofaunal communities are biased by the taxonomic approach

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 timeeffective 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

Enhancing metabarcoding efficiency and ecological insights through integrated taxonomy and DNA reference barcoding: A case study on beach meiofauna

Molecular techniques like metabarcoding, while promising for exploring diversity of communities, are often impeded by the lack of reference DNA sequences available for taxonomic annotation. Our study explores the benefits of combining targeted DNA barcoding and morphological taxonomy to improve metabarcoding efficiency, using beach meiofauna as a case study. Beaches are globally important ecosystems and are inhabited by meiofauna, microscopic animals living in the interstitial space between the sand grains, which play a key role in coastal biodiversity and ecosystem dynamics. However, research on meiofauna faces challenges due to limited taxonomic expertise and sparse sampling. We generated 775 new cytochrome c oxidase I DNA barcodes from meiofauna specimens collected along the Netherlands' west coast and combined them with the NCBI GenBank database. We analysed alpha and beta diversity in 561 metabarcoding samples from 24 North Sea beaches, a region extensively studied for meiofauna, using both the enriched reference database and the NCBI database without the additional reference barcodes. Our results show a 2.5-fold increase in sequence annotation and a doubling of species-level Operational Taxonomic Units (OTUs) identification when annotating the metabarcoding data with the enhanced database. Additionally, our analyses revealed a bell-shaped curve of OTU richness across the intertidal zone, aligning more closely with morphological analysis patterns, and more defined community dissimilarity patterns between supralittoral and intertidal sites. Our research highlights the importance of expanding molecular reference databases and combining morphological taxonomy with molecular techniques for biodiversity assessments, ultimately improving our understanding of coastal ecosystems

Fifteen species in one: deciphering the Brachionus plicatilis species complex (Rotifera, Monogononta) through DNA taxonomy

Understanding patterns and processes in biological diversity is a critical task given current and rapid environmental change. Such knowledge is even more essential when the taxa under consideration are important ecological and evolutionary models. One of these cases is the monogonont rotifer cryptic species complex Brachionus plicatilis, which is by far the most extensively studied group of rotifers, is widely used in aquaculture, and is known to host a large amount of unresolved diversity. Here we collate a dataset of previously available and newly generated sequences of COI and ITS1 for 1273 isolates of the B. plicatilis complex and apply three approaches in DNA taxonomy (i.e. ABGD, PTP, and GMYC) to identify and provide support for the existence of 15 species within the complex. We used these results to explore phylogenetic signal in morphometric and ecological traits, and to understand correlation among the traits using phylogenetic comparative models. Our results support niche conservatism for some traits (e.g. body length) and phylogenetic plasticity for others (e.g. genome size)

Patterns of Diversity in Soft-Bodied Meiofauna: Dispersal Ability and Body Size Matter

Background: Biogeographical and macroecological principles are derived from patterns of distribution in large organisms, whereas microscopic ones have often been considered uninteresting, because of their supposed wide distribution. Here, after reporting the results of an intensive faunistic survey of marine microscopic animals (meiofauna) in Northern Sardinia, we test for the effect of body size, dispersal ability, and habitat features on the patterns of distribution of several groups.Methodology/Principal Findings: As a dataset we use the results of a workshop held at La Maddalena (Sardinia, Italy) in September 2010, aimed at studying selected taxa of soft-bodied meiofauna (Acoela, Annelida, Gastrotricha, Nemertodermatida, Platyhelminthes and Rotifera), in conjunction with data on the same taxa obtained during a previous workshop hosted at Tjärnö (Western Sweden) in September 2007. Using linear mixed effects models and model averaging while accounting for sampling bias and potential pseudoreplication, we found evidence that: (1) meiofaunal groups with more restricted distribution are the ones with low dispersal potential; (2) meiofaunal groups with higher probability of finding new species for science are the ones with low dispersal potential; (3) the proportion of the global species pool of each meiofaunal group present in each area at the regional scale is negatively related to body size, and positively related to their occurrence in the endobenthic habitat.Conclusion/Significance: Our macroecological analysis of meiofauna, in the framework of the ubiquity hypothesis for microscopic organisms, indicates that not only body size but mostly dispersal ability and also occurrence in the endobenthic habitat are important correlates of diversity for these understudied animals, with different importance at different spatial scales. Furthermore, since the Western Mediterranean is one of the best-studied areas in the world, the large number of undescribed species (37%) highlights that the census of marine meiofauna is still very far from being complete

Supplemental Table 6

Values of Jaccard dissimilarity based on incidence datasets measured within and between communities. Identical = 0; completely dissimilar = 1. Abbreviations: MT, morphotypes; EE, evolutionary independent entities obtained with GMYC; OTU, operational taxonomic units obtained with single individuals; eOTU, operational taxonomic units obtained with environmental samples; SV, sequence variants. L, littoral; SL, sublittoral; O, offshore. Dissimilarity values were estimated using both the focal phyla (SV; eOTU) and whole meiofauna dataset (SV2; eOTU2)