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.

Organization of dermal glands and characteristic of secretion in the freshwater mite, Limnesia maculata (O. F. Müller, 1776) (Acariformes, Limnesiidae).

The presence of dermal glands is a synapomorphy in the freshwater mites-the large branch of the hyporder Parasitengonina. Dermal glands of the mite Limnesia maculata (O. F. Müller, 1776) (Acariformes, Limnesiidae) were studied using light and electron microscopy (TEM and SEM) for the first time. Two types of dermal glands were recognized in adult mites-14 pairs of the uniformly organized common dermal glands scattered throughout the entire body volume, and one pair of the characteristic so-called idiosomal dermal glands centrally located and stretched along the ventral body wall, supposedly corresponding to the epimeroglandularia 4. The common dermal glands are composed of a single large alveolus with the basally located columnar epithelium. An intra-alveolar lumen is positioned above the epithelium and packed with a secretion in the form of long curved electron-dense bands. These bands are directed to the excretory opening and released to the outside in the form of strongly coiled bands. The cytoplasm of the common dermal glands is filled with short RER cisterns and shows few characteristic Golgi bodies with round dense secretory granules at the distal pole. In contrast, a large elongated sac represents the idiosomal dermal glands having a relatively thin secretory epithelium on the periphery. The cuboidal epithelial cells contain areas with tightly packed RER cisterns and weakly recognized Golgi bodies. The apical cell surface bears irregular short microvilli. The large intra-alveolar lumen always contains an electron-dense secretion with myriads of small lighter particles. This kind of secretion was never seen released through the opening. The excretory opening of the common and the idiosomal dermal glands shows differences in its organization but basically is represented by a cuticular "bell" narrowing to the opening and showing a particular kind of valves at the inner side of the flaps of the orifice.

Ultrastructure of the intestinal system in unfed larvae of Limnesia maculata (O.F. Müller, 1776) (Acariformes, Limnesiidae).

The intestinal system of unfed fresh-water mite larvae Limnesia maculata (O.F. Müller, 1776) (Acariformes, Limnesiidae) has been studied with transmission electron microscopy. The intestinal system is composed of the foregut, including the pharynx and the esophagus, the sac-like blind midgut and the excretory organ. The pharynx begins with the mouth covered by the labrum. The pharynx runs along the bottom of the gnathosoma and shows ventral dilators. No valves are expressed between the pharynx and the esophagus. The esophagus possesses strongly plicate walls and, before entering the midgut, passes through the brain. The sac-like midgut does not reveal a well-pronounced lumen, developed epithelium and separate lobes. It consists of the two cell types of endoderm origin mixed in the midgut volume. The first type - the vacuolated cells - does not possess Golgi bodies and lysosomal apparatus and shows electron-lucent vacuoles with a granular inclusion inside. These cells apparently do not take part in digestion of the embryonic yolk. The second type - the non-vacuolated cells - shows both, a well developed Golgi complex and large heterolysosomes, and obviously digests the embryonic yolk. Consequently, they may be attributed as specialized vitellophages. Nevertheless, both cell types may take part in formation of the definitive midgut epithelium. The sac-like thin-walled excretory organ is strongly dilated and contains the embryonic wastes in the form of electron-dense globules and birefringent particles. No muscle envelope surrounds the excretory organ. The embryonic wastes together with wastes accumulated during feeding may be evacuated from the organ only after completion of feeding. The excretory canal on this developmental stage is not connected with the excretory organ. It opens to the outside with a simple slit-like excretory pore. Before feeding, larvae have to pass the process of the post-molt development before their midgut would be ready to receive nutrients.

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.

Ultrastructure and functional morphology of dermal glands in the freshwater mite Limnochares aquatica (L., 1758) (Acariformes, Limnocharidae).

This study is the first attempt to describe the ultrastructure and functional morphology of the dermal glands in Limnochares aquatica (L., 1758). The dermal glands were studied using light-optical, SEM and TEM microscopy methods during different stages of their activity. In contrast to the vast majority of other fresh water mites, dermal glands of the studied species are originally multiplied and scattered freely over the mite body surface. The opening of the glands is saddle-like, formed of several tight cuticular folds and oriented freely to the long axis of the mite body. Either a small cuticular spine or, rarely, a slim sensitive seta is placed on one pole of the opening. On the inside, the central gland portion is provided with a complex cuticular helicoid armature. The glands are composed of prismatic cells situated around the intra-alveolar lumen, variously present, and look like a fig-fruit with the basal surface facing the body cavity. The glands are provided with extremely numerous microtubules, frequently arranged in bundles, and totally devoid of synthetic apparatus such as RER cisterns and Golgi bodies. Three states of the gland morphology depending on their functional activity may be recognized: (i) glands without secretion with highly folded cell walls and numerous microtubules within the cytoplasm, (ii) glands with an electron-dense granular secretion in the expanded vacuoles and (iii) glands with the secretion totally extruded presenting giant empty vacuoles bordered with slim cytoplasmic strips on the periphery. Summer specimens usually show the first gland state, whereas winter specimens, conversely, more often demonstrate the second and the third states. This situation may depend on some factors like changes of the seasonal temperature, pH, or oxygenation of the ambient water. On the assumption of the morphological characters, dermal glands may be classified not as secretory but as a special additional excretory organ system of the body cavity. Despite the glands lack cambial cells, restoration of functions after releasing of 'secretion' looks possible. Organization of dermal glands is discussed in comparison to other water mites studied.

Dermal glands in freshwater mites Limnesia undulata (O.F. Müller, 1776) and Limnesia fulgida (C.L. Koch, 1836) (Acariformes, Limnesiidae).

Dermal glands in the water mites Limnesia undulata (O.F. Müller, 1776) and Limnesia fulgida (C.L. Koch, 1836) and their secretion were studied by means of light microscopical, transmission electron microscopical (TEM) and scanning electron microscopical (SEM) methods. These mites possess two types of dermal glands - the 'common' dermal glands in a number of 14 pairs and one pair of the so-called 'idiosomal' dermal glands. The common dermal glands are bi-lobed organs and consist of high prismatic secretory cells directed to the gland mouth and mostly replacing the intra-alveolar lumen. The cells contain numerous cisterns of rough endoplasmic reticulum (RER) and specifically organized Golgi bodies (GB) producing electron-dense elongated secretory granules. These granules are released from the cells via apocrine secretion and come to the gland mouth, where they are sometimes accompanied by secretory cell cytoplasm. The final secretion may show a fibrous character. The idiosomal glands are sac-like organs stretched along the ventral body wall in posterior direction from the gland orifice corresponding to the epimeroglandularia 4. The secretory epithelium leaves a large intra-alveolar lumen filled with an electron-dense secretory material. Golgi bodies are organized identically with those in the common glands, which indicates the general homology of these two types of dermal glands. The glands' orifices are organized similarly in all glands and possess an internal funnel-shaped sclerite with muscle armament, an internal valve, medial epicuticular flaps and an external circular cuticular ring. All glandularia, except for E4 and V1, are accompanied with a long and thin sensitive seta. During fixation, secretion of the common dermal glands is extruded to the exterior in the form of large amounts of convoluted tube-like structures. In the living organisms, being secreted in mass from the glands, this secretion acquires the form of long rigid mostly hollow un-branched threads comparable with the similar silken threads of other water arthropods. The function of the idiosomal glands secretion still remains unknown.