The last hideout: Abundance patterns of the not-quite-yet extinct mayfly Prosopistoma pennigerum in the Albanian Vjosa River network

1. The mayfly Prosopistoma pennigerum (Müller, 1785) (Insecta: Ephemeroptera) once occurred in many European river networks. However, observations decreased in the last decades and the species can be considered largely extinct throughout Europe due to river alterations. 2. Only three extant populations are known from Cabriel (southern Spain), Volga (Russia) and Vjosa (Albania) rivers. 3. We recorded the species along a 150 km stretch in the Vjosa River in three sampling seasons (spring 2018, fall 2018 and fall 2019), counting up to 302 P. pennigerum per m2, the highest recorded abundance for the species to date. Moreover, we detected traces of environmental DNA in a newly designed targeted eDNA assay. 4. In our modelling approach we define the species’ niche in a theoretically available niche space given by the Vjosa River network and predict a high probability of presence (θ) in downstream located sections of this river. Expected abundances (λ) could be related to a set of environmental variables, importantly to higher discharge and increased sediment dynamics. 5. Simultaneous occurrence of larvae of different sizes at individual sites suggests an asynchronous life cycle, which may be advantageous to cope with the highly dynamic river hydrology. 6. The P. pennigerum population in the Vjosa is of key importance for the species’ global survival

Investigation of balloon dilation devices for treatment of Eustachian tube dysfunction

The presented three different balloon dilation devices for treating Eustachian tube dysfunction were compared regarding their geometries and designs. The balloon dilation behaviour was investigated by an in vitro test setup consisting of a test chamber with 37 °C water, a 2-axis laser scanner and a pressure controller. All balloons could be properly dilated up to the given nominal pressure (NP) or to rated burst pressure (RBP). The balloons reached their expected balloon diameter and length. The compliance data were 1.07 %/atm (TubaVent short), 1.16 %/atm (Acclarent Aera) and 1.28 %/atm (XprESS LoProfile). The measured profile and compliance data can be used for development of new devices for balloon dilation Eustachian tuboplasty

Polymeric stents for the Eustachian tube: development and human cadaver study

Impairment of Eustachian tube function with nonsufficient ventilation of the middle ear is a main cause for chronic otitis media. To provide an effective and safe therapy, the innovative concept of Eustachian tube stenting was established. Biodegradable polymeric stents are developed to restore impaired tube function and dissolve after fulfilling their supportive purpose. To evaluate the applicability of the stents in the Eustachian tube, prototypes in conjunction with corresponding implantation instruments were tested in human cadaver studies. Radiopaque markers and a diaphanoscopic approach were tested as additional features to prove correct positioning of catheter and stent in the tube. In the current study biodegradable polymeric stents were implanted in the Eustachian tube of human cadavers without difficulty. Correct positioning of the stents in the tube was proved by diaphanoscopy during intervention and postoperative tomographic and histological analyses. Once designs are optimized on the basis of cadaver studies, preclinical safety and efficacy studies using animal models will be initiated

Development of biodegradable stents for the treatment of Eustachian tube dysfunction

To provide an effective and safe therapy for chronic Eustachian tube dysfunction (ETD), biodegradable stents should be developed to restore important functions, e.g. middle ear ventilation and drainage. After defining general requirements specifications, stent designs of cardiovascular polymeric stents were modified according to dimensions and conditions of the Eustachian tube. Finite element simulations demonstrated the crimping capacity of the developed stent design and the ability of expansion in the specific geometry of the target location. Subsequent in vitro tests of stent prototypes showed satisfying properties concerning crimpability, expansion behavior and elastic recoil to demonstrate general feasibility. Further developments and additional testing will advance the implementation of a new treatment option of ETD

Investigation of Stent Prototypes for the Eustachian Tube in Human Donor Bodies

Chronic otitis media is often connected to Eustachian tube dysfunction. As successful treatment cannot be guaranteed with the currently available options, the aim is to develop a stent for the Eustachian tube (ET). Over the course of this development, different prototypes were generated and tested in ex vivo experiments. Four different prototypes of an ET stent and one commercially available coronary stent were implanted in the ET of seven human donor bodies. The position of the stents was verified by cone beam CT. The implanted ETs were harvested, embedded in resin and ground at 200 µm steps. Resulting images of the single steps were used to generate 3D models. The 3D models were then evaluated regarding position of the stent in the ET, its diameters, amount of squeezing, orientation of the axes and other parameters. Virtual reconstruction of the implanted ET was successful in all cases and revealed one incorrect stent placement. The cross-section increased for all metal stents in direction from the isthmus towards the pharyngeal orifice of the ET. Depending on the individual design of the metal stents (open or closed design), the shape varied also between different positions along a single stent. In contrast, the cross-section area and shape remained constant along the polymeric prototype. With the current investigation, insight into the behavior of different prototypes of ET stents was gained, which can help in defining the specifications for the intended ET stent

Tracing the almost extinct mayfly Prosopistoma pennigerum (Müller, 1785) – an eDNA approach

The rare mayfly Prosopistoma pennigerum was once widely distributed across Europe and occurred virtually in every large river. Today, it holds fast against the ever-growing destruction of its habitat with a few relic populations remaining. Preliminary data and information on its congeners suggest that free-flowing rivers with near-natural hydrodynamics are its primary habitat. Rivers where the species currently occurs should be primary targets for large-scale landscape conservation and protection. According to the Water Framework Directive, achieving longitudinal and lateral connectivity is a priority target, and occurrence and viability of Prosopistoma pennigerum populations could be an indicator of restoration success.We developed and validated a targeted qPCR protocol to detect this mayfly and applied it to standardized water samples filtered for eDNA analysis from the Vjosë (Albania) and Volga (Russia), two rivers with extant populations of P. pennigerum. In the Vjosë river, 45 sampling sites were sampled three times in 2018 and 2019. In the Volga river, we focused on a site with >15 years of continuous records of P. pennigerum at Rzhev and two downstream locations, where eDNA samples were collected in 2017. At each sampling site in the Vjosë, eDNA samples were collected by filtering 0.5 L of stream water through each of two 0.45 µm Sterivex filters. In the Volga, 2 L of stream water were filtered through a total of eight 0.7 µm glass fibre filters. Filters were stored and shipped at -20°C until further processing. Environmental DNA extraction was performed using the DNeasy® PowerWater® Sterivex™ Kit following the Experienced User protocol for Vjosë samples and via a Phenol-Chloroform Isoamyl extraction for the Volga samples. A Taqman qPCR assay was developed using a newly designed primer and probe set. Standard curves obtained from an amplicon dilution series yielded a reaction efficiency of 88% and an R2-value of 0.995, with a calculated limit of quantification of 1801 copies/µL and a limit of detection of 59 copies/µL. Each eDNA sampling replicate was tested using five qPCR replicates, yielding ten qPCR reactions per sampling site in the Vjosë river and 40 qPCR reactions in the Volga river. With each batch of eDNA samples, four negative and two positive controls were analysed. At each site, we also collected benthic MHS samples with a 25x25 cm 500 µm net where 20 samples were taken to reflect microhabitat distribution and dominance. Benthos samples were subsampled in the lab and all zoobenthos hand-picked. Prosopistoma pennigerum occurrence was assessed as areal density (number of specimens per m2).While P. pennigerum occured in high frequencies and abundances in benthic samples along the Vjosë river main stem in all sampling seasons, qPCR of eDNA samples suggested slightly different occurrence patterns. Conversely, P. pennigerum was detected with high consistency at two sites in the Volga river (at Rzhev and a site 99 km downstream), where its frequency is much lower than in the Vjosë river. The success of detecting a benthic species in eDNA samples depends on a variety of factors that may have affected DNA quality and prevented better detection of P. pennigerum in the Vjosë river eDNA samples.We demonstrate the principal applicability of molecular methods to search for rare species in hot-spots of biodiversity in Central Europe. The remnant populations of P. pennigerum in the hydrodynamically minimally impaired Vjosë and the Volga highlight the conservation and protection needs in Eastern and Southern Europe. At the European scale, restoration efforts should be geared towards creating viable habitat conditions for large-river species such as P. pennigerum. Here, our qPCR assay can deliver crucial data for better management of Europe’s large rivers