The innervation of the male copulatory organ of spiders (Araneae) - a comparative analysis.

BACKGROUND: Nervous tissue is an inherent component of the many specialized genital structures for transferring sperm directly into the female's body. However, the male copulatory organ of spiders was considered a puzzling exception. Based on the recent discovery of nervous tissue in the pedipalps of two distantly related spider species, we investigated representatives of all major groups across the spider tree of life for the presence of palpal nerves. We used a correlative approach that combined histology, micro-computed tomography and electron microscopy. RESULTS: We show that the copulatory organ is innervated in all species investigated. There is a sensory organ at the base of the sperm transferring sclerite in several taxa and nervous tissue occurs close to the glandular tissue of the spermophor, where sperm are stored before transfer. CONCLUSIONS: The innervation of the copulatory organ by the bulb nerve and associated efferent fibers is part of the ground pattern of spiders. Our findings pave the way for unraveling the sensory interaction of genitalia during mating and for the still enigmatic mode of uptake and release of sperm from the male copulatory organ.

Primary processing neuropils associated with the malleoli of camel spiders (Arachnida, Solifugae): a re-evaluation of axonal pathways.

BACKGROUND: Arachnids possess highly specialized and unorthodox sense organs, such as the unique pectines of Scorpiones and the malleoli of Solifugae. While the external morphology, numbers, and shapes of sensory organs are widely used in taxonomic studies, little is known about the internal anatomy of these organs and their associated processing neuropils in the central nervous system. Camel spiders (Solifugae) possess pedipalps and first walking legs heavily endowed with sensory structures, as well as conspicuous malleoli located ventrally on the proximal fourth walking legs. Malleoli are fan-shaped organs that contain tens of thousands of presumptive chemoreceptor neurons, but mechanoreceptive structures are absent. RESULTS: Here, we examine the organization of the synganglion based on microCT analysis, 3D reconstruction of serial paraffin sections, and backfill preparations to trace the malleolar pathway. The projection area of malleolar afferents is intriguingly located in the most anterior ventral nerve cord, located in between the pedipalpal neuromere hemispheres. However, malleolar axon bundles are separated by a thin soma layer that points to an anteriad projection of the fourth walking leg neuromere. A conspicuous projection neuron tract that may receive additional input from pedipalpal sensory organs connects the malleolar neuropil with the mushroom bodies in the protocerebrum. CONCLUSION: Arthropod chemosensory appendages or organs and primary processing neuropils are typically located in the same segment, which also holds true in Solifugae, although the malleolar neuropil is partially shifted towards the pedipalpal neuromere. A comparison of the malleoli in Solifugae and the pectines in Scorpiones, and of their primary processing neuropils, reveals certain similarities, while striking differences are also evident. Similarities include the ventral arrangement of peg-shaped sensory structures on the respective segmental appendage, exposing dense arrays of chemoreceptive sensilla, and projections to a primary processing neuropil with glomerular subdivision. Differences are, e.g., the lack of mechanoreceptive afferents and an associated processing neuropil.

First evidence of neurons in the male copulatory organ of a spider (Arachnida, Araneae).

Spider males have evolved a remarkable way of transferring sperm by using a modified part of their pedipalps, the so-called palpal organ. The palpal organ is ontogenetically derived from tarsal claws; however, no nerves, sensory organs or muscles have been detected in the palpal bulb so far, suggesting that the spider male copulatory organ is numb and sensorily blind. Here, we document the presence of neurons and a nerve inside the male palpal organ of a spider for the first time. Several neurons that are located in the embolus are attached to the surrounding cuticle where stresses and strains lead to a deformation (stretching) of the palpal cuticle on a local scale, suggesting a putative proprioreceptive function. Consequently, the male copulatory organ of this species is not just a numb structure but likely able to directly perceive sensory input during sperm transfer. In addition, we identified two glands in the palpal organ, one of which is located in the embolus (embolus gland). The embolus gland appears to be directly innervated, which could allow for rapid modulation of secretory activity. Thus, we hypothesize that the transferred seminal fluid can be modulated to influence female processes.

Female control of mate plugging in a female-cannibalistic spider (Micaria sociabilis).

BACKGROUND: Sperm competition imposes a strong selective pressure on males, leading to the evolution of various physiological, morphological and behavioral traits. Sperm competition can be prevented by blocking or impeding the access to female genitalia by means of a mating plug. We investigated the factors responsible for plug production and function in the promiscuous female-cannibalistic spider Micaria sociabilis (Gnaphosidae). RESULTS: We performed mating trials using females with and without a plug that consists of an amorphous mass. The mating trials demonstrated that the probability of male plugging increased non-linearly with the duration of copulation. Copulation duration and plug production seem to be controlled by the female. We found that females terminated matings later if males were fast at genital coupling. Whereas incomplete plugs had disappeared on the day following copulation, complete plugs persisted (40%). In matings with females with complete plugs, only a small proportion of males (7%) were able to remove the plug, indicating the high effectiveness of plugging. Moreover, males ceased attempts to copulate with plugged females with higher probability. 3D X-ray microscopy of the female and male genitalia showed that the plug material can extend far into the female genital tract and that the plug material is produced by a massive gland inside the palpal organ of the modified male pedipalps. CONCLUSIONS: Our study demonstrates that the mating plug in M. sociabilis constitutes an effective male strategy to avoid sperm competition that seems to be under female control.

Potential and limitations of X-Ray micro-computed tomography in arthropod neuroanatomy: a methodological and comparative survey.

Classical histology or immunohistochemistry combined with fluorescence or confocal laser scanning microscopy are common techniques in arthropod neuroanatomy, and these methods often require time-consuming and difficult dissections and sample preparations. Moreover, these methods are prone to artifacts due to compression and distortion of tissues, which often result in information loss and especially affect the spatial relationships of the examined parts of the nervous system in their natural anatomical context. Noninvasive approaches such as X-ray micro-computed tomography (micro-CT) can overcome such limitations and have been shown to be a valuable tool for understanding and visualizing internal anatomy and structural complexity. Nevertheless, knowledge about the potential of this method for analyzing the anatomy and organization of nervous systems, especially of taxa with smaller body size (e.g., many arthropods), is limited. This study set out to analyze the brains of selected arthropods with micro-CT, and to compare these results with available histological and immunohistochemical data. Specifically, we explored the influence of different sample preparation procedures. Our study shows that micro-CT is highly suitable for analyzing arthropod neuroarchitecture in situ and allows specific neuropils to be distinguished within the brain to extract quantitative data such as neuropil volumes. Moreover, data acquisition is considerably faster compared with many classical histological techniques. Thus, we conclude that micro-CT is highly suitable for targeting neuroanatomy, as it reduces the risk of artifacts and is faster than classical techniques.

Ultrastructure of spermatozoa of Orsolobidae (Haplogynae, Araneae) with implications on the evolution of sperm transfer forms in Dysderoidea.

Haplogynae are highly diverse with respect to the primary male genital system and sperm characteristics. Additionally, all sperm transfer forms (STF) known for spiders are present. Besides individually transferred sperm (cleistospermia), sperm are transferred as conjugates, both primary (synspermia) and secondary sperm conjugates (coenospermia, rouleaux) occur. Nevertheless, the ultrastructure of spermatozoa and STF are described for few Haplogynae and often only one representative species was studied, resulting in a superficial insight in the evolution of these traits. To elucidate the evolution of STF within Haplogynae we investigated representatives of four genera of the dysderoid family Orsolobidae. Our data show the presence of synspermia (Orsolobus, Osornolobus, Hickmanolobus, and Tasmanoonops) and also cleistospermia (Osornolobus). The occurrence of different STF within one family or even genus has not been described for any other spider taxon so far. Moreover, the synspermia of species of Tasmanoonops and Hickmanolobus were not covered by a secretion sheath suggesting a previously unknown strategy of transferring sperm that is possibly related to sperm residency time or female triggered processes after copulation. Based on serial ultrathin sectioning and subsequent 3D-reconstruction, we obtained detailed measurements revealing remarkable size differences of STF. To evaluate the previously suggested correlation with the most distal region of the spermophor inside the embolus (intromittent part of the copulatory organ) we measured the diameter of the spermophor using micro-computed X-ray tomography data to obtain corresponding morphometric parameters. Based on these data only two species show similarity in STF and spermophor diameter.

Serial block-face imaging and its potential for reconstructing diminutive cell systems: a case study from arthropods.

Until recently, three-dimensional reconstruction on an ultrastructural level was only possible using serial section transmission electron microscopy (ssTEM). However, ssTEM is highly challenging and prone to artifacts as, e.g., section loss and image distortions. New methods, such as serial block-face scanning electron microscopy (SBFSEM) overcome these limitations and promise a high lateral resolution. However, little is known about the usability of SBFSEM in diminutive, but highly complex cellular systems. We used spider sperm (~3 µm in diameter), which fulfills these conditions, to analyze the potential of SBFSEM compared with ssTEM. Our data suggest that the resolution obtained by SBFSEM allows depicting structures on a cellular level and is sufficient to discriminate subcellular components, but is highly dependent on previous staining procedures and electron density of the target structures.

The enigmatic Otway odd-clawed spider (Progradungula otwayensis Milledge, 1997, Gradungulidae, Araneae): Natural history, first description of the female and micro-computed tomography of the male palpal organ.

The recently described cribellate gradungulid Progradungula otwayensis Milledge, 1997 is endemic to the Great Otway National Park (Victoria, Australia) and known from only one male and a few juvenile specimens. In a recent survey we recorded 47 specimens at several localities across the western part of the Great Otway National park. Our field data suggest that this species is dependant on the microclimate in the hollows of old myrtle beech trees since other hollow trees were very much less inhabited. Furthermore, we describe the female for the first time and study the male palpal organ by using X-ray microtomography. The female genitalia are characterized by eight spermathecae which are grouped in two quartets. The spermophor resembles the general organization of gradungulids, but is similar to Kaiya Gray, 1987 by a convoluted appearance within the embolus. The muscle 30 is located in the cymbium and resembles the organization of other non-entelegyne Araneomorphae.

May salivary gland secretory proteins from hematophagous leeches (Hirudo verbana) reach pharmacologically relevant concentrations in the vertebrate host?

Saliva of hematophagous leeches (Hirudo sp.) contains bioactive proteins which allow the leech proper feeding and storage of ingested blood, but may also exert effects in the host. Leech therapy is used to treat many different ailments in humans, although only a small fraction of salivary proteins are characterized yet. Moreover, we do not know whether complete transfer of salivary proteins stored in the unicellular salivary glands in a leech to the host during feeding may generate concentrations that are sufficiently high to affect physiological processes in the host. Our 3D reconstruction of a portion of internal leech tissue from histological sections revealed that one leech contains approx. 37,000 salivary gland cells. Using tissue slices from pig liver and mouse skeletal muscle for reference, we obtained data for protein densities in leech salivary gland cells. As individual salivary cells are voluminous (67,000 µm(3)) and the stored proteins are densely packed (approx. 500 µg/mm(3)), we extrapolated that a single leech may contain up to 1.2 mg of salivary proteins. Analyzing protein extracts of unfed or fed leeches by 2D electrophoresis, we calculated the relative molar amounts of individual salivary proteins in the mass range of 17-60 kDa which may be released from a single leech during feeding. Distribution of these salivary proteins in the host (assumed plasma volume of 5 l) may result in concentrations of individual compounds between 3 and 236 pmol/l. Such concentrations seem sufficiently high to exert biochemical interactions with target molecules in the host.

Formation of primary sperm conjugates in a haplogyne spider (Caponiidae, Araneae) with remarks on the evolution of sperm conjugation in spiders.

Sperm conjugation, where two or more sperm are physically united, is a rare but widespread pheno-menon across the animal kingdom. One group well known for its different types of sperm conjugation are spiders. Particularly, haplogyne spiders show a high diversity of sperm traits. Besides individual cleistospermia, primary (synspermia) and secondary (coenospermia, "spermatophore") sperm conjugation occurs. However, the evolution of sperm conjugates and sperm is not understood in this group. Here, we look at how sperm are transferred in Caponiidae (Haplogynae) in pursuit of additional information about the evolution of sperm transfer forms in spiders. Additionally, we investigated the male reproductive system and spermatozoa using light- and transmission electron-microscopy and provide a 3D reconstruction of individual as of well as conjugated spermatozoa. Mature spermatozoa are characterized by an extremely elongated, helical nucleus resulting in the longest spider sperm known to date. At the end of spermiogenesis, synspermia are formed by complete fusion of four spermatids. Thus, synspermia might have evolved early within ecribellate Haplogynae. The fused sperm cells are surrounded by a prominent vesicular area. The function of the vesicular area remains still unknown but might be correlated with the capacitation process inside the female. Further phylogenetic and functional implications of the spermatozoa and sperm conjugation are discussed.

Comparative analysis of deutocerebral neuropils in Chilopoda (Myriapoda): implications for the evolution of the arthropod olfactory system and support for the Mandibulata concept.

BACKGROUND: Originating from a marine ancestor, the myriapods most likely invaded land independently of the hexapods. As these two evolutionary lineages conquered land in parallel but separately, we are interested in comparing the myriapod chemosensory system to that of hexapods to gain insights into possible adaptations for olfaction in air. Our study connects to a previous analysis of the brain and behavior of the chilopod (centipede) Scutigera coleoptrata in which we demonstrated that these animals do respond to volatile substances and analyzed the structure of their central olfactory pathway. RESULTS: Here, we examined the architecture of the deutocerebral brain areas (which process input from the antennae) in seven additional representatives of the Chilopoda, covering all major subtaxa, by histology, confocal laser-scan microscopy, and 3D reconstruction. We found that in all species that we studied the majority of antennal afferents target two separate neuropils, the olfactory lobe (chemosensory, composed of glomerular neuropil compartments) and the corpus lamellosum (mechanosensory). The numbers of olfactory glomeruli in the different chilopod taxa ranged from ca. 35 up to ca. 90 and the shape of the glomeruli ranged from spheroid across ovoid or drop-shape to elongate. CONCLUSION: A split of the afferents from the (first) pair of antennae into separate chemosensory and mechanosensory components is also typical for Crustacea and Hexapoda, but this set of characters is absent in Chelicerata. We suggest that this character set strongly supports the Mandibulata hypothesis (Myriapoda + (Crustacea + Hexapoda)) as opposed to the Myriochelata concept (Myriapoda + Chelicerata). The evolutionary implications of our findings, particularly the plasticity of glomerular shape, are discussed.

Gross morphology, histology, and ultrastructure of the alimentary system of Ricinulei (Arachnida) with emphasis on functional and phylogenetic implications.

Ricinuleid functional mouthparts are the cucullus, the chelicerae, the pedipalps, and the labrum. These structures are movably jointed to the rest of the prosoma, most likely protruded upon hydrostatic hemolymph pressure and retracted by prosomal muscles. Seta-like protrusions from the labrum and the pedipalpal coxae form a sieve-like filter inside the preoral cavity and the mouth. Although the tip of the labrum can be elevated upon muscle constriction, ingestion of large, solid food particles is unlikely. The mouth has a crescent-shaped cross section. The cuticle-lined, also crescent-shaped pharynx is equipped with a large dilator muscle but lacks antagonistic constrictor muscles. It represents a precerebral sucking pump. The triangular to Y-shaped, cuticle-lined esophagus is equipped with constrictor and dilator muscles. Its posterior part represents a postcerebral sucking pump. Four blind ending diverticula ramify from the anterior prosomal part of the entodermal midgut tube. Two of these diverticula remain inside the prosoma and form few short branches. The other two extend through the pedicel into the opisthosoma and ramify and coil there. A stercoral pocket protrudes ventrally out of the midgut tube. The most distal part of the midgut tube is modified into a contractile rectal gland. Its secretions may have defensive or physiological functions. A short anal atrium is formed by the cuticle-lined ectodermal hindgut which opens at the end of the three-segmented metasoma. The telescoping segments of the metasoma are protruded by hemolymph pressure and retracted by muscles.