Functional morphology and ultrastructure of the mouth apparatus in the freshwater mite larvae Limnochares aquatica (L., 1758) (Acariformes, Limnocharidae).

The mouth apparatus in larvae of the freshwater mite Limnochares aquatica (L., 1758) (Acariformes, Limnocharidae), belonging to the lower Hydrachnidia, was studied with light microscopy, scanning (SEM) and transmission (TEM) electron microscopy, as well as with confocal laser scanning microscopy (CLSM) to reveal its main morphological and functional characteristics. The gnathosoma, functioning as a mouth organ takes a prognathous position, and inclines to the body axial line at the angle of around 30°. The gnathosoma is composed of segments of the two pairs of anterior appendages and their derivatives - two-segmented chelicerae lying above the infracapitulum - the compound medial portion of the palpal coxae, and five-segmented palps, articulated with the infracapitulum laterally. The palp tibia bears the bifid palpal claw facing downward nearly at right angle. The basal cheliceral segments are free, i.e., are not covered with the integumental fold posteriorly, and do not fuse with each other. Apically, they articulate with the relatively short movable digits composed of the basal portion (lever) and the distal portion (the cheliceral blades) curved upward by their ends. The flexible fixed digits protrude forward from the distal parts of the basal cheliceral segments. The ventral wall of the gnathosoma (mentum) transforms into a very specific papillary area (velum). A papillary area with long slim papillae is formed of a light cuticle and likely acts as a sucker but does not have contractile elements. The hypostome possesses the well developed lateral lips (galea) tightly embracing the distal portions of the chelicerae and hiding the movable digits. The roof of the infracapitulum (the cervix, or epistome) lying beneath the chelicerae, shows a compound structure. Anteriorly it connects with the inner hypostomal walls forming a particular pharyngeal ring framing the narrow mouth opening. We saw no evidence of a labrum. A characteristic valve projects forward from the dorsal surface of the cervix. A crescent-shaped pharynx runs along the bottom of the infracapitulum and is attached but not fused with the cuticle of the papillary area. The dorsal pharyngeal dilators originate on the cervix, sigmoid pieces and the lateral walls of the infracapitulum at their junction with the dorsal walls of the basal cheliceral segments. A pair of sigmoid pieces having small cavities inside serves for termination of the cheliceral elevators originated on the posterior portions of the dorsal walls of the basal cheliceral segments. The organizational pattern of the gnathosoma in larvae of L. aquatica is rather specific and is quite different from those of other aquatic or terrestrial parasitengonin larvae.

The structural analysis of secretion in the freshwater mite Limnesia maculata (Acariformes, Limnesiidae) supports the idea of a new form of arthropod silk.

The structural characteristics of silk secretion of the freshwater mite Limnesia maculata (O.F. Müller) (Acariformes, Limnesiidae) are described and analyzed for the first time based on light, atomic force and electron-microscopical approaches. The common dermal glands (14 pairs scattered over the body) produce silk mostly during the warm summer season. The process of silk secretion lasts from several hours to several days. The silk may appear like barely recognized clouds of a fine whitish substance. An individual silk thread is an indefinitely long uniform unbranched and non-stretchable tube, hollow or with a vesicular electron-dense residual content. In the silk bundle, threads may be freely interlaced, bent, curved or occasionally broken. The diameter of the tubes is in the range of 0.9-1.5 µm. The width of the tube walls varies greatly from 60 to 300 nm. Chaotically interlaced fine fibrils build the tube walls. On the external surface of the tube wall, these fibrils are loosely organized and frequently rising vertically, whereas on the internal side they are packed more tightly sometimes showing a mesh. The walls may reveal a layered structure or, contrary, are quite thin with through foramens. The revealed organization of silk in the freshwater mites is found to be the simplest among that of other arthropods. We propose a role of the silk in the capture of potential prey in the summer season. Silk in water mites significantly widens the wholesome area for the mites' life and gives them better chances in competition for potential resources.

A simple and low-cost method to visualize musculature and other aspects of anatomy by confocal microscopy.

Confocal microscopy study of musculature and other anatomical structures in whole-mount preparations of arthropods and some other cuticle-bearing animals often presents a significant difficulty because the cuticle poses a barrier to fluorescent dyes and their pigmented tissues can cause attenuation of fluorescent signal. This paper describes a simple and inexpensive procedure based on the use of clove oil as a tissue-clearing, staining, and mounting medium that helps overcome the problem of slow dye penetration and tissue opaqueness and allows muscles and several other organ systems to be visualized by confocal or epifluorescent microscopy. This clove oil-induced fluorescence (COIF) method relies on the ability of clove oil to accumulate in muscles and some other tissues and become steadily fluorescent if irradiated at 488 nm. For this method, animals were fixed in 70% ethanol or 4% formaldehyde, then dehydrated and mounted in clove oil. Heavily pigmented animals were additionally bleached in hydrogen peroxide prior to the dehydration step. The COIF method showed excellent results in all major groups of arthropods and some mollusks and annelids revealing the three-dimensional arrangement of muscles, gonads, glands, cellular nuclei and some parts of the nervous and digestive systems. In the other animal groups tested, clove oil stained all tissues making it difficult to observe the anatomical details. The COIF method is advantageous in some respects over other methods such as phalloidin staining because of its tissue penetration and clearing abilities, low cost of the reagents, resistance to photodamage and the possibility of staining museum specimens.

Occurrences of Threatened Species included in the Third Edition of the Red Data Book of the Komi Republic (Russia).

BACKGROUND: The purpose of the data paper was to introduce into scientific literature the results of scientific work carried out for the third edition of the 'Red Data Book of the Komi Republic'. The article reflects methodological approaches to the formation of a list of rare and in need of protection species and describes the corresponding datasets published in GBIF. NEW INFORMATION: Information about 7,187 occurrences of 438 rare species and infraspecies included in the third edition of the 'Red Data Book of the Komi Republic' have been published.

Muscles of the male and female copulatory organs of Bursaphelenchus mucronatus and Chiloplacus sp. (Nematoda: Rhabditida).

Male and female copulatory organs figure prominently in nematode taxonomy, but the associated musculature remains insufficiently explored. The aim of this study was therefore to further our knowledge of the musculature of the vulva and male copulatory organs in nematodes by using phalloidin staining and confocal microscopy to examine two nematode species, Bursaphelenchus mucronatus and Chiloplacus sp. The musculature of the round vulva in Chiloplacus sp. comprises three pairs of radial vulval dilators and another pair of dilators of the anterior inner vulval plate. This arrangement is similar to that of the Rhabditida, but in Chiloplacus the anterior pair appears to have been transformed into the vulval plate muscles. The musculature of the slit-like vulva in B. mucronatus includes dilators of the vulval lips and external vulval flap, constrictors of the vulval slit and posterior transverse muscle bands. The opposing pairs of vulval dilators show quadrilateral symmetry as observed in the Rhabditida, but the constrictors running along the rim of the vulva have no counterparts in other species. The musculature of the male copulatory organ in Chiloplacus sp. comprises two pairs of spicule protractors and retractors and three pairs of gubernacular muscles. In B. mucronatus, as in the other Aphelenchoididae, the gubernaculum is absent and there is one pair of spicule protractors and two pairs of muscles inserted on the saddle (angular bend) of the spicules. The arrangement of the spicule saddle muscles resembles those of the gubernaculum, which may indicate that in this family the gubernaculum has become fused to the spicules. The literature review of muscles of nematode copulatory organs are given in a table for 15 muscle groups; it can be used for phylogenetic reconstruction and classification of the order Rhabditida.

Description of Bradyetes paramatthei sp. nov. (Copepoda: Calanoida), a new aetideid species from the deep Pacific Ocean with notes on the genus Bradyetes.

A new and rare aetideid species of the benthopelagic genus Bradyetes Farran, 1905 is described from female specimens collected near the seafloor from the abyss of the Pacific, Atlantic and Southern Oceans between 2000 and 2014. The new species, Bradyetes paramatthei sp. nov., is described from the Kurile-Kamchatka Trench (Pacific Ocean) and is additionally reported from the Angola and Guinea basins and from the Meteor Seamount of the Atlantic Ocean. Bradyetes paramatthei sp. nov. is related to the species B. matthei Johannessen, 1976, but differs in the shape of the prosome posterior corners, which are oval-rounded, and in the proximal segment of the antennal exopod, which is supplied with one seta. These two species are shown to constitute a separate species group within the genus Bradyetes. The other species group contains the remaining congeners including Bradyetes inermis Farran, 1905, for which morphological variability is discussed. Specimens of this species show diverse morphology and comprise 3 morphotypes distinguished by the P1, with a developed or absent lateral lobe , the P1 basal medial seta, which is either nude or supplied with setules, and by the different number of setae of the maxillule praecoxal arthrite.

Musculature of the penial complex: A new criterion in unravelling the phylogeny of Hygrophila (Gastropoda: Pulmonata).

The taxonomy of freshwater pulmonates (Hygrophila) has been in a fluid state warranting the search for new morphological criteria that may show congruence with molecular phylogenetic data. We examined the muscle arrangement in the penial complex (penis and penis sheath) of most major groups of freshwater pulmonates to explore to which extent the copulatory musculature can serve as a source of phylogenetic information for Hygrophila. The penises of Acroloxus lacustris (Acroloxidae), Radix auricularia (Lymnaeidae), and Physella acuta (Physidae) posses inner and outer layers of circular muscles and an intermediate layer of longitudinal muscles. The inner and outer muscle layers in the penis of Biomphalaria glabrata consist of circular muscles, but this species has two intermediate longitudinal layers separated by a lacunar space, which is crossed by radial and transverse fibers. The muscular wall of the penis of Planorbella duryi is composed of transverse and longitudinal fibers, with circular muscles as the outer layer. In Planorbidae, the penial musculature consists of inner and outer layers of longitudinal muscles and an intermediate layer of radial muscles. The penis sheath shows more variation in muscle patterns: its muscular wall has two layers in A. lacustris, P. acuta, and P. duryi, three layers in R. auricularia and Planorbinae and four layers in B. glabrata. To trace the evolution of the penial musculature, we mapped the muscle characters on a molecular phylogeny constructed from the concatenated 18S and mtCOI data set. The most convincing synapomorphies were found for Planorbinae (inner and outer penis layers of longitudinal muscles, three-layered wall of the penis sheath). A larger clade coinciding with Planorbidae is defined by the presence of radial muscles and two longitudinal layers in the penis. The comparative analysis of the penial musculature appears to be a promising tool in unraveling the phylogeny of Hygrophila.

Architecture of the nervous system in metacercariae of Diplostomum pseudospathaceum Niewiadomska, 1984 (Digenea).

The development of metacercariae of Diplostomum pseudospathaceum Niewiadomska, 1984 is accompanied by profound morphological transformations often characterized as metamorphosis, which makes these metacercariae an interesting case for studying the morphogenesis of the digenean nervous system. Although the nervous system of D. pseudospathaceum is one of the most extensively studied among digeneans, there are still gaps in our knowledge regarding the distribution patterns of some neuroactive substances, most notably neuropeptides. The present study addresses these gaps by studying pre-infective metacercariae of D. pseudospathaceum using immunochemical staining and confocal microscopy to characterize the distribution patterns of serotonin (5-HT) and two major groups of flatworm neuropeptides, FMRFamide-related (FaRPs) and substance P-related (SP) peptides. The general morphology of the nervous system was examined with antibodies to alpha-tubulin. The nervous system of the metacercariae was shown to conform to the most common morphology of the nervous system in the hermaphroditic generation, with three pairs of posterior nerve cords and four pairs of anterior nerves. The patterns of FaRP- and 5-HT immunoreactivity (IR) were similar to those revealed in earlier studies by cholinesterase activity, which is in accordance with the known role of these neurotransmitters in controlling muscle activity in flatworms. The SP-IR nervous system was significantly different and consisted of mostly bipolar cells presumably acting as mechanoreceptors. The architecture of the nervous system in D. pseudospathaceum metacercariae is discussed in comparison to that in cercariae of D. pseudospathaceum and metacercariae of related digenean species.

Muscular Arrangement and Sclerite Morphology in the Haptor of Tetraonchus monenteron (Monogenea, Dactylogyridea).

BACKGROUND : Tetraonchidae is a small and relatively monomorphic family of dactylogyroid monogeneans. Since the morphology of sclerites of the attachment organ (haptor) is one of the primary criteria in tetraonchid taxonomy, the confocal study of sclerites and associated musculature may provide valuable taxonomic information. PURPOSE : The aim of this study was to examine the haptoral sclerites and musculature in Tetraonchus monenteron (Wagener, 1857), a common parasite of pike. METHODS: Haptoral musculature of T. monenteron was studied using phalloidin staining and confocal microscopy, with sclerites visualized in the reflection confocal mode. RESULTS: Haptoral armature of T. monenteron consists of ventral and dorsal pairs of anchors, a ventral bar, eight pairs of marginal hooks and at least three pairs of accessory sclerites. Anchors are operated by 14 muscles, of which the most prominent are extrinsic muscles, the transverse muscle interconnecting the ventral anchors, three muscles connecting the ventral anchor to the ventral bar, and four muscles of the dorsal and ventral anchors inserting on the haptoral wall. The extrinsic muscles are attached to the braceshaped sclerites, which in turn are connected to the dorsal anchors via muscle bundles. CONCLUSION: The gaffing action of the dorsal anchors is likely to be achieved by the extrinsic muscles and the transverse muscle that clamps the extrinsic muscles against the body wall. The ventral anchors are probably held in attached position by the transverse muscle and four muscles inserting on the ventral bar and haptoral wall. The haptoral musculature may have potential utility for tetraonchid taxonomy.

Musculoskeletal and nervous systems of the attachment organ in three species of Diplectanum (Monogenea: Dactylogyroidea).

The taxonomy of Diplectanum Diesing, 1858, a genus of monopisthocotylean monogeneans, remains unsettled and needs to be revised based on new morphological criteria. Recent studies in monopisthocotyleans have shown that the muscle arrangement in the posterior attachment organ (haptor) differs between congeneric species and can be used as an additional criterion in genus-level taxonomy. To explore the possibility of using the haptoral musculature and nervous system in the taxonomy of Diplectanum, we conducted a detailed confocal-microscopy study of three species of Diplectanum (D. aculeatum Parona et Perugia, 1889, D. sciaenae van Beneden et Hesse, 1863 and D. similis Bychowsky, 1957) with phalloidin staining for muscle and indirect immunostaining for 5HT and FMRFamide. A further goal was to clarify the functional mechanics of the haptor and the role of its essential components (squamodiscs and anchors) in attachment to the host. The system of connecting bars and gaffing anchors was found to have a complex musculature consisting of 23 muscles in D. aculeatum and D. sciaenae, and 21 muscles in D. similis. The squamodiscs were shown to be operated by several groups of muscles attached primarily to the area termed the squamodisc fulcrum. Most of the haptoral musculature is identical in D. aculeatum and D. sciaenae and these species differ only in the presence of a muscle sheath around the tissue strand between the squamodiscs in D. sciaenae and in the different patterns of superficial squamodisc muscles. Diplectanum similis shows more significant differences from the other two species: besides lacking two of the haptoral muscles, it also differs in the shapes and arrangement of several other muscles. The nervous system of all three species conforms to the general pattern typical for the Dactylogyroidea and shows little variation between species.

Haptoral neuromusculature in two species of Dactylogyrus Diesing, 1850 (Monogenea: Dactylogyridae).

The taxonomy of Dactylogyrus Diesing, 1850 (Monogenea: Dactylogyridae), like that of most monopisthocotyleans, relies heavily on the morphology of sclerites of the posterior attachment organ (haptor). However, the associated neuromusculature is essentially unknown and, therefore, the aim of this study was to use confocal microscopy to examine the haptoral neuromusculature in two species of Dactylogyrus: D. amphibothrium Wagener, 1857 and D. crucifer Wagener, 1857. The monogeneans were stained with phalloidin for muscle and with antibodies for FMRFamide and 5HT and confocal reflectance microscopy was used to visualise the sclerites (i.e. anchors, marginal hooks and bars). Both species had a similar architecture of the anchoral musculature, with a pair of extrinsic muscles, two interconnecting muscles and muscles attached to the haptoral wall, anchoral openings and a connecting bar. The musculature of most marginal hooks consisted of retractors and protractors inserted on the proximal ends of the hooks. The two species differed significantly in the musculature of the accessory ventral bar: D. crucifer had a four-rayed bar with an elaborate musculature associated with the marginal hooks and D. amphibothrium had a rod-shaped bar with simple musculature. Patterns of neurotransmitter immunoreactivity (IR) in the haptor were similar in both species: RFamide-IR cells were clustered in a pair of pre-anchoral ganglia interconnected by a neurite bundle, 5HT-IR cells formed a ventral loop projecting neurites to the marginal hooks. The functional roles of haptoral muscles and patterns of neurotransmitters are discussed. In D. crucifer, the confocal reflectance microscopy revealed an additional sclerite (accessory dorsal bar) that had never been described previously in this or any other species of Dactylogyrus, suggesting that the reflected-light technique might be useful in identifying sclerites undetectable by conventional methods.

Comparative morphology of the bursal nozzles in acoels (Acoela, Acoelomorpha).

Systematics of the Acoela is particularly difficult because of the paucity of readily discernible morphological features. In other soft-bodied worms, sclerotized structures, such as copulatory stylets, provide important characters that can be seen in whole mounts, but acoels generally lack such features. Among the few sclerotized structures in acoels are bursal nozzles-tubiform outlets on the seminal bursae that are believed to be conduits (spermatic ducts) through which allosperm are transported to the oocytes. Early classifications of the Acoela used features of the female reproductive system, including bursal nozzles, for distinguishing major groups, but the current system essentially ignores them as too plastic to provide higher-level distinctions. We used confocal and electron microscopy to further characterize bursal nozzles in five acoel species, and found all composed of actin-reinforced extensions of stacked, flat mesenchymal cells. In Notocelis gullmarensis, Aphanostoma bruscai, and Daku woorimensis, the nozzle is a stiffened region of the same cells forming the wall of the bursa. By contrast, in Wulguru cuspidata cells forming the nozzle are distinct from those of the bursa. The so-called bursal cap of A. bruscai and D. woorimensis has small sclerotized disjunct units within it, also composed of stacked, flat, actin-reinforced cells. The nozzle of W. cuspidata, prominent like that of other convolutid acoels, is relatively complex, its actin-reinforced cells sandwiched with secretory cells and its base bearing a "sorting apparatus" of egg-shaped cells that send narrow processes inside the spermatic duct. Cases of sperm inside the nozzle corroborate its assumed role in reproduction. Whereas most nozzles sit at the end of the bursa facing the ovary, in species of Pseudmecynostomum and purportedly in a few other acoels, they sit between the female pore and the bursa, constituting what we call a vaginal nozzle. All bursal nozzles of acoels show a common ground pattern indicating common ancestry, but certain features discerned through electron and confocal microscopy show promise of providing synapomorphies for grouping some species.