An integrative description of a population of Mesobiotus radiatus (Pilato, Binda & Catanzaro, 1991) from Kenya

In a moss sample collected from Mount Kulal in Kenya, a new population of Mesobiotus radiatus was found. Given that the original description of M. radiatus was based solely on the morphology observed by light microscopy and measurements based mostly on a single individual, here we describe the new population by means of integrative taxonomy and a large sample size. We provide an integrative description comprising a comprehensive set of morphometric and morphological data from light and scanning microscopy as well as nucleotide sequences of three nuclear fragments (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial fragment (COI). Mesobiotus radiatus is most similar to M. binieki, M. coronatus, M. patiens, M. perfidus, M. philippinicus, M. pseudocoronatus, M. pseudopatiens, M. rigidus, M. simulans, and M. wuzhishanensis, but differs from them mainly by egg morphology and morphometry, and some characters of adult specimens

Observation of Live Ticks (Haemaphysalis flava) by Scanning Electron Microscopy under High Vacuum Pressure

Scanning electron microscopes (SEM), which image sample surfaces by scanning with an electron beam, are widely used for steric observations of resting samples in basic and applied biology. Various conventional methods exist for SEM sample preparation. However, conventional SEM is not a good tool to observe living organisms because of the associated exposure to high vacuum pressure and electron beam radiation. Here we attempted SEM observations of live ticks. During 1.5×10−3 Pa vacuum pressure and electron beam irradiation with accelerated voltages (2–5 kV), many ticks remained alive and moved their legs. After 30-min observation, we removed the ticks from the SEM stage; they could walk actively under atmospheric pressure. When we tested 20 ticks (8 female adults and 12 nymphs), they survived for two days after SEM observation. These results indicate the resistance of ticks against SEM observation. Our second survival test showed that the electron beam, not vacuum conditions, results in tick death. Moreover, we describe the reaction of their legs to electron beam exposure. These findings open the new possibility of SEM observation of living organisms and showed the resistance of living ticks to vacuum condition in SEM. These data also indicate, for the first time, the usefulness of tick as a model system for biology under extreme condition

New records of tardigrades from China, with zoogeographical remarks

Tardigrades are small (0.05-1.20 mm), hygrophilous micrometazoans. This paper reports on 18 species of tardigrades found in Sichuan, Yunnan, and Xizang Provinces in China, primarily in lichens and mosses. Eight species are new records for China: Echiniscus nepalensis Dastych, E. reticulatus Murray, E. spiniger Richters, Isohypsibius sattleri (Richters), Diphascon (D.) pingue (Marcus), Diphascon (A.) prorsirostre Thulin, Mesocrista spitsbergensis (Richters), and Platicrista angustata (Murray). Two species are new records for both Sichuan and Yunnan: Minibiotus intermedius (Plate) and Hypsibius pallidus Thulin; one species is new for Sichuan: Pseudechiniscus suillus (Ehrenberg); and three species are new for Yunnan: Cornechiniscus lobatus (Ramazzotti), Pseudechiniscus jiroveci Bartoš , and Doryphoribius flavus (Iharos). This and previous papers have reported on 86 species and subspecies of Tardigrada from China. This is only ca. 9% of the world limno-terrestrial and marine fauna of water bears. Among these 86 species only one is marine, three are found in freshwaters, and the others are terrestrial

Light-sheet fluorescent microscopy in tardigrade anoxybiosis

Tardigrades are invertebrates that are known for their tolerance to extreme conditions. This research focuses on anoxia, the total lack of oxygen, and the adaptation and survival of anoxiainduced experiments. Cryptobiosis is a form of dormancy that enables the survival of the animals, however, many forms of cryptobiosis are still poorly understood and the mechanisms and physiological responses are not entirely explained. This study aimed to create a protocol that utilizes fluorescent dyes to visualize the transition into anoxybiosis and morphometric changes at the cellular level involved in the phenomenon. Lightsheet fluorescent microscopy enabled fast 3D volumetric scanning of the transition and revealed what happened to the animal when anoxia was chemically applied. Results were aligned with the expectations: during anoxybiosis, tardigrades became immobilized and swollen leading to the relocation and reorganization of cells. Importantly it was observed that variation throughout the experiments was quite significant and in further studies, this should be outlined. In this research, two specimens of M. ripperi were used and obtained data were compared including cell number, volume, and displacement over time. The most fundamental issue is how to gain stable and reproducible results. Tardigrade cuticle and overall variation of body state of the animals create sources of error, particularly dyeing by soaking. By soaking, there are very few possibilities to control, how the dye is distributed and attached to the cellular compartments, therefore in this study we did not pay so close attention to the statistical significance of the results. Animals chosen for the experiments were random and therefore variables such as sex, age, and fasting were not included. Fluorescent microscopy is a widely used method in biological studies and this study showed that it can be used in live imaging of tardigrades. However, as a pilot experiment, this led to many open questions and features to improve, especially in the image analysis part. Large datasets need a lighter pipeline to gain a higher throughput method. In the future, the (light sheet) fluorescent imaging can be a beneficial tool for similar cellular studies; however, the individual sources of variation need to be minimized. Tardigrades can be a promising model organism for studies including cell survival, cancer research, and storage and storing solutions for various drug components when understanding the mechanisms that enable the animal stress tolerance and survival

The fine structure of the midgut epithelium in Xerobiotus pseudohufelandi (Iharos, 1966) (Tardigrada, Eutardigrada, Macrobiotidae)

The aims of our studies were to describe the ultrastructure of the midgut epithelial cells of the eutardigrade Xerobiotus pseudohufelandi and to determine if there are any differences in the ultrastructure of midgut epithelial cells between males and females. The analysis was performed with the use of the light and transmission electron microscopes. In X. pseudohufelandi the midgut epithelium is composed of digestive cells, but in the anterior portion of the midgut a group of cells with different ultrastructure has been observed. Histochemical staining showed the accumulation of reserve material in the cytoplasm of digestive cells. We suggest that some of them fulfil the role of regenerative cells (crescent-like cells, midgut stem cells), whereas others are differentiating cells which form new digestive cells. No differences in the ultrastructure of the midgut epithelium between males and females were distinguished except in the amount of multivesicular bodies

Microsco-pi: a novel and inexpensive way of merging biology and IT

It is well known that schools and colleges often have budget limitations that can hamper the effectiveness of practical education. This article looks at how cheap, off-the-shelf components can be used to produce a simple DIY digital microscope, and how this provides novel opportunities to integrate biology, physics, design technology and computer science in a fun and hands-on way

Using open-source hardware to investigate biological behaviours: a DIY approach aimed at educational institutions

This MPhil thesis is the result of some time working with the Raspberry Pi, a cheap, credit-card sized computer. This has been used in this project to offer teachers new opportunities and methods to study a curious microscopic organism, the tardigrade, and a more common animal, found everywhere- the ant. Through these short, education based experiments, I have attempted to identify how this hardware could be used in schools to boost cross-curricular teaching and to enhance the work that can be carried out in a school setting, while sticking to a very tight budget. The entire project has been accomplished for less than £200 and has incorporated a wide range of external sensors that provide real, useful data for studying the effect of environment on the behaviour of animals. This thesis takes the reader through a history of the technology and gives an introduction to the equipment and programmes used throughout, before applying this to two very practical studies which attempt to utilise the hardware to advance our knowledge of animal behaviour. It is, to my knowledge, the first time a Raspberry Pi has been used in ant behavioural studies, and provides schools and colleges ideas on how to utilise this equipment to look at animals in new ways

Selective neuronal staining in tardigrades and onychophorans provides insights into the evolution of segmental ganglia in panarthropods

Background: Although molecular analyses have contributed to a better resolution of the animal tree of life, the phylogenetic position of tardigrades (water bears) is still controversial, as they have been united alternatively with nematodes, arthropods, onychophorans (velvet worms), or onychophorans plus arthropods. Depending on the hypothesis favoured, segmental ganglia in tardigrades and arthropods might either have evolved independently, or they might well be homologous, suggesting that they were either lost in onychophorans or are a synapomorphy of tardigrades and arthropods. To evaluate these alternatives, we analysed the organisation of the nervous system in three tardigrade species using antisera directed against tyrosinated and acetylated tubulin, the amine transmitter serotonin, and the invertebrate neuropeptides FMRFamide, allatostatin and perisulfakinin. In addition, we performed retrograde staining of nerves in the onychophoran Euperipatoides rowelli in order to compare the serial locations of motor neurons within the nervous system relative to the appendages they serve in arthropods, tardigrades and onychophorans. Results: Contrary to a previous report from a Macrobiotus species, our immunocytochemical and electron microscopic data revealed contralateral fibres and bundles of neurites in each trunk ganglion of three tardigrade species, including Macrobiotus cf. harmsworthi, Paramacrobiotus richtersi and Hypsibius dujardini. Moreover, we identified additional, extra-ganglionic commissures in the interpedal regions bridging the paired longitudinal connectives. Within the ganglia we found serially repeated sets of serotonin- and RFamid-like immunoreactive neurons. Furthermore, our data show that the trunk ganglia of tardigrades, which include the somata of motor neurons, are shifted anteriorly with respect to each corresponding leg pair, whereas no such shift is evident in the arrangement of motor neurons in the onychophoran nerve cords. Conclusions: Taken together, these data reveal three major correspondences between the segmental ganglia of tardigrades and arthropods, including (i) contralateral projections and commissures in each ganglion, (ii) segmentally repeated sets of immunoreactive neurons, and (iii) an anteriorly shifted (parasegmental) position of ganglia. These correspondences support the homology of segmental ganglia in tardigrades and arthropods, suggesting that these structures were either lost in Onychophora or, alternatively, evolved in the tardigrade/arthropod lineage