Structure of the pecten neuropil pathway and its innervation by bimodal peg afferents in two scorpion species.

The pectines of scorpions are comb-like structures, located ventrally behind the fourth walking legs and consisting of variable numbers of teeth, or pegs, which contain thousands of bimodal peg sensillae. The associated neuropils are situated ventrally in the synganglion, extending between the second and fourth walking leg neuromeres. While the general morphology is consistent among scorpions, taxon-specific differences in pecten and neuropil structure remain elusive but are crucial for a better understanding of chemosensory processing. We analysed two scorpion species (Mesobuthus eupeus and Heterometrus petersii) regarding their pecten neuropil anatomy and the respective peg afferent innervation with anterograde and lipophilic tracing experiments, combined with immunohistochemistry and confocal laser-scanning microscopy. The pecten neuropils consisted of three subcompartments: a posterior pecten neuropil, an anterior pecten neuropil and a hitherto unknown accessory pecten neuropil. These subregions exhibited taxon-specific variations with regard to compartmentalisation and structure. Most notable were structural differences in the anterior pecten neuropils that ranged from ovoid shape and strong fragmentation in Heterometrus petersii to elongated shape with little compartmentalisation in Mesobuthus eupeus. Labelling the afferents of distinct pegs revealed a topographic organisation of the bimodal projections along a medio-lateral axis. At the same time, all subregions along the posterior-anterior axis were innervated by a single peg's afferents. The somatotopic projection pattern of bimodal sensillae appears to be common among arachnids, including scorpions. This includes the structure and organisation of the respective neuropils and the somatotopic projection patterns of chemosensory afferents. Nonetheless, the scorpion pecten pathway exhibits unique features, e.g. glomerular compartmentalisation superimposed on somatotopy, that are assumed to allow high resolution of substrate-borne chemical gradients.

Characterization of spermatogonial cells and niche in the scorpion mud turtle (Kinosternon scorpioides).

Undifferentiated spermatogonia (Aund) or spermatogonial stem cells (SSCs) are committed to the establishment and maintenance of spermatogenesis and fertility throughout a male's life and are located in a highly specialized microenvironment called niche that regulates their fate. Although several studies have been developed on SSCs in mammalian testis, little is known about other vertebrate classes. The present study is the first to perform a more detailed investigation on the spermatogonial cells and their niche in a reptilian species. Thus, we characterized Aund/SSCs and evaluated the existence of SSCs niche in the Kinosternon scorpioides, a freshwater turtle found from Mexico to northern and central South America. Our results showed that, in this species, Aund/SSCs exhibited a nuclear morphological pattern similar to those described for other mammalian species already investigated. However, in comparison to other spermatogonial cell types, Aund/SSCs presented the largest nuclear volume in this turtle. Similar to some mammalian and fish species investigated, both GFRA1 and CSF1 receptors were expressed in Aund/SSCs in K. scorpioides. Also, as K. scorpioides Aund/SSCs were preferentially located near blood vessels, it can be suggested that this niche characteristic is a well conserved feature during evolution. Besides being valuable for comparative reproductive biology, our findings represent an important step towards the understanding of SSCs biology and the development of valuable systems/tools for SSCs culture and cryopreservation in turtles. Moreover, we expect that the above-mentioned results will be useful for reproductive biotechnologies as well as for governmental programs aiming at reptilian species conservation.

Mechanisms of karyotype evolution in the Brazilian scorpions of the subfamily Centruroidinae (Buthidae).

The recently-revised subfamily Centruroidinae is part of the New World clade of buthid scorpions. In this study, we analyzed the cytogenetic characteristics of nine of the 10 Brazilian centruroidines, and one undescribed species of the genus Ischnotelson, using a phylogenetic approach to determine the chromosomal rearrangements responsible for the differentiation of karyotypes among the species. The cytogenetic data recorded in the present study supported the new taxonomic arrangement of the Centruroidinae, with all the species of the same genus sharing the same or similar diploid numbers, i.e., 2n = 20 or 22 in Troglorhopalurus lacrau and T. translucidus, 2n = 25 or 26 in Ischnotelson sp., I. guanambiensis and I. peruassu, and 2n = 28 in Jaguajir agamemnon, J. pintoi and J. rochae. The karyotype modelling in the ChromEvol software indicated 2n = 18 as the ancestral diploid number of the Centruroidinae. The differentiation of karyotypes among the centruroidine genera was based on increasing chromosome numbers resulting from progressive fission events. These changes probably occurred prior to the diversification of the genera Ischnotelson, Jaguajir, Physoctonus and Rhopalurus, and appear to have played a more important role in karyotype evolution at the intergeneric level than the interspecific one. However, the observed increase in diploid numbers was not accompanied by changes in the number or location of ribosomal genes or telomeric sequences. The identification of meiotic cells in female specimens also allowed us to discuss the mechanisms of achiasmatic meiosis in scorpions.

Cytotoxic activity of Androctonus australis hector venom and its toxic fractions on human lung cancer cell line

Several studies have showed that animal venoms are a source of bioactive compounds that may inhibit the growth of cancer cells, which makes them useful agents for therapeutic applications. Recently, it was established that venom toxins from scorpions induced cytotoxic, antiproliferative and apoptogenic effects on cancer cells. Therefore, the present study aims to investigate the cytotoxic activity of Androctonus australis hector (Aah) scorpion venom and its toxic fractions (FtoxG-50 and F3) on NCI-H358 human lung cancer cells. Methods: The cytotoxic and antiproliferative activities were estimated using MTT assay, lactate dehydrogenase release and clonogenic assays. Apoptosis was evaluated by Hoechst 33258 staining, DNA fragmentation assay and caspase-3 activity. Oxidative stress was analyzed by reactive oxygen species, nitric oxide, malondialdehyde and protein carbonyl levels along with assessment of antioxidant status. In addition, alteration of mitochondrial membrane potential was analyzed by JC1 fluorescent dye. Results: The present findings showed that F3 fraction was more cytotoxic towards NCI-H358 lung cancer cells with an IC50 of 27.05 ± 0.70 g/mL than venom alone (396.60 ± 1.33 g/mL) and its toxic fraction FtoxG-50 (45.86 ± 0.91 g/mL). Nevertheless, F3 fraction was not cytotoxic at these concentrations on normal human lung fibroblast MRC-5 cells. Inhibition of NCI-H358 cell proliferation after F3 fraction exposure occurred mainly by apoptosis as evidenced by damaged nuclei, significant DNA fragmentation level and caspase-3 activation in a dose dependent manner. Moreover, F3 fraction enhanced oxidative and nitrosative stress biomarkers and dissipated mitochondrial membrane potential in lung cancer cells along with significant depletion in cellular enzymatic and non-enzymatic antioxidants. Further, the apoptosis induced by F3 fraction was markedly prevented by the antioxidant N-acetylcysteine (NAC) suggesting the potential mechanism of oxidative stress. Conclusion: These findings suggest that F3 fraction could induce apoptosis in lung cancer cells through involvement of oxidative stress and mitochondrial dysfunction. Hence, these properties make F3 fraction a promising candidate for development of new anticancer agents.

Organization and behavior of the synaptonemal complex during achiasmatic meiosis of four buthid scorpions.

Testicular cells of 4 buthid scorpions, Rhopalurus agamemnon (2n = 28), R. rochai (2n = 28), Tityus bahiensis (2n = 6), and T. fasciolatus (2n = 14), which show different types of chromosomal configurations in meiosis I, were subjected to cellular microspreading in order to (1) obtain knowledge about the organization and behavior of the synaptonemal complex (SC), and (2) acquire data about the mechanisms responsible for inter- and intraindividual chromosomal variation within Buthidae. Ultrastructural analysis of microspread nuclei revealed SCs with a well-preserved structure until late substages of prophase I, but did not detect kinetochore plates and recombination nodules. Pachytene cells of R. agamemnon, R. rochai and T. bahiensis exhibited single and unsynapsed axes continuous with totally synapsed SCs, indicating the occurrence of heterozygous chromosomal rearrangements. Although chromosome chains were not observed in T. fasciolatus, the presence of gaps and interlocks points out that this species also carries heterozygous rearrangements, involving a small chromosome segment. Especially in R. rochai, the cellular microspreading analysis was useful to clarify the origin of inter- and intraindividual variation in the number of bivalent-like elements and in the number of chromosomes involved in multivalent associations. It was found that more chromosomes were involved in rearrangements than previously established through investigations using light microscopy alone.

Serotonin-immunoreactive neurons in scorpion pectine neuropils: similarities to insect and crustacean primary olfactory centres?

The pectines of scorpions are a single pair of mechano- and chemosensory appendages located ventrally behind the most posterior pair of walking legs. They are used for probing the substrate in behaviours such as prey tracking and courtship. The sensory afferents on the pectines supply large segmental neuropils with a conspicuous glomerular structure. The pectine neuropils thus bear similarities to insect and crustacean deutocerebral chemosensory centres associated with the antennae, but they also possess idiosyncratic features. One characteristic property of many insect and decapod crustacean olfactory neuropils is their innervation by single, or very few, large serotonergic (inter-) neurons. This feature, among others, has been proposed to support homology of the olfactory lobes in the two arthropod groups. A possible serotonergic innervation of the scorpion pectine neuropils has not yet been studied, despite its apparent diagnostic and functional importance. We thus examined serotonin-immunoreactivity in the pectine neuropils of Androctonus australis and Pandinus imperator. Both scorpion species yielded similar results. The periphery of the neuropil and the matrix between the glomeruli are supplied by a dense network of serotonin-immunoreactive (5-HT-ir) arborisations and varicosities, while the glomeruli themselves are mostly free of 5-HT-ir fibres. The 5-HT-ir supply of the pectine neuropils has two origins. The first is a pair of neurons on each body side, up to 30 µm in diameter and thus slightly larger than the surrounding somata. These cell bodies are and associated with the neuromeres of the genital and pectine segments. The situation is reminiscent of the 5-HT supply of insect and crustacean olfactory and antennal neuropils. The second 5-HT innervation of the pectine neuropils is from a group of some 10-20 ipsilateral neuronal somata of slightly smaller size (15-20 µm). These are part of a much larger 5-HT-ir group comprising 70-90 somata. The whole group is located more anteriorly than the single soma mentioned above, and associated with the neuromere of the last (4th) walking leg. When compared to data from other arthropods, our findings may suggest that glomerular organisation is an ancestral feature of primary chemosensory centres innervated by arthropod appendages. This idea needs further scrutiny, although supporting evidence may have been overlooked previously, due to the small size of chemosensory neuropils in walking legs and in reduced segmental appendages.

A double role of sperm in scorpions: the mating plug of Euscorpius italicus (Scorpiones: Euscorpiidae) consists of sperm.

Mating plugs occluding the female gonopore after mating are a widespread phenomenon. In scorpions, two main types of mating plugs are found: sclerotized mating plugs being parts of the spermatophore that break off during mating, and gel-like mating plugs being gelatinous fluids that harden in the female genital tract. In this study, the gel-like mating plug of Euscorpius italicus was investigated with respect to its composition, fine structure, and changes over time. Sperm forms the major component of the mating plug, a phenomenon previously unknown in arachnids. Three parts of the mating plug can be distinguished. The part facing the outside of the female (outer part) contains sperm packages containing inactive spermatozoa. In this state, sperm is transferred. In the median part, the sperm packages get uncoiled to single spermatozoa. In the inner part, free sperm is embedded in a large amount of secretions. Fresh mating plugs are soft gelatinous, later they harden from outside toward inside. This process is completed after 3-5 days. Sperm from artificially triggered spermatophores could be activated by immersion in insect Ringer's solution indicating that the fluid condition in the females' genital tract or females' secretions causes sperm activation. Because of the male origin of the mating plug, it has likely evolved under sperm competition or sexual conflict. As females refused to remate irrespective of the presence or absence of a mating plug, females may have changed their mating behavior in the course of evolution from polyandry to monandry.

Complex meiotic configuration of the holocentric chromosomes: the intriguing case of the scorpion Tityus bahiensis.

Mitotic and meiotic chromosomes of Tityus bahiensis were investigated using light (LM) and transmission electron microscopy (TEM) to determine the chromosomal characteristics and disclose the mechanisms responsible for intraspecific variability in chromosome number and for the presence of complex chromosome association during meiosis. This species is endemic to Brazilian fauna and belongs to the family Buthidae, which is considered phylogenetically basal within the order Scorpiones. In the sample examined, four sympatric and distinct diploid numbers were observed: 2n = 5, 2n = 6, 2n = 9, and 2 = 10. The origin of this remarkable chromosome variability was attributed to chromosome fissions and/or fusions, considering that the decrease in chromosome number was concomitant with the increase in chromosome size and vice versa. The LM and TEM analyses showed the presence of chromosomes without localised centromere, the lack of chiasmata and recombination nodules in male meiosis, and two nucleolar organiser regions carrier chromosomes. Furthermore, male prophase I cells revealed multivalent chromosome associations and/or unsynapsed or distinctly associated chromosome regions (gaps, less-condensed chromatin, or loop-like structure) that were continuous with synapsed chromosome segments. All these data permitted us to suggest that the chromosomal rearrangements of T. bahiensis occurred in a heterozygous state. A combination of various factors, such as correct disjunction and balanced segregation of the chromosomes involved in complex meiotic pairing, system of achiasmate meiosis, holocentric nature of the chromosomes, population structure, and species dispersion patterns, could have contributed to the high level of chromosome rearrangements present in T. bahiensis.

Spermatozoa and sperm packages of the European troglophylous scorpion Belisarius xambeui Simon, 1879 (Troglotayosicidae, Scorpiones).

Studies on the sperm morphology in scorpions are rare, but the existing investigations already revealed a remarkable interfamiliar diversity. The present study reports for the first time on the spermatozoa and sperm packages of a representative of the family Troglotayosicidae, the troglophylous species Belisarius xambeui. The spermatozoa are characterized by (1) a thread-like nucleus, which is slightly bent anteriorly; (2) an asymmetrical cap-like acrosomal vacuole, which encloses the anterior tip of the nucleus; an acrosomal filament is absent; (3) an axoneme with a 9+0 microtubular pattern; (4) a midpiece consisting of elongated mitochondria coiling around the axoneme; the number can vary between 3 and 6 (mostly 4). At the end of spermiogenesis, the spermatozoa aggregate in order to form oval-shaped sperm packages in which all sperm cells show the same orientation. A single package consists of approximately 150 sperms. A secretion sheath is always absent. The present results might provide new characters for further systematic studies and their phylogenetic implications are briefly discussed.

Morphology and function of male genitalia (spermatophores) in Euscorpius italicus (Euscorpiidae, Scorpiones): complex spermatophore structures enable safe sperm transfer.

The structure and function of the spermatophore of Euscorpius italicus are analyzed. We show how the spermatophore gets shaped from two hemispermatophores and for the first time the sperm transfer mechanism is shown in detail, illustrating function and importance of all complex lobe structures of an euscorpiid spermatophore. A detailed description of the interaction of spermatophore and female genitalia is given. The capsular region of the spermatophore bears different lobes: The distal and basal lobes hook into two cavities on the inner side of the female's genital operculum. A so-called "crown-like structure" hooks into a membranous area in the genital atrium. During sperm transfer, these crown-like structures move backwards, in this way widening the female's genital opening. The sperm duct of the spermatophore is coated with numerous spicules on its outer side, which could serve as a sealing mechanism and/or may stimulate the female. Furthermore, we conclude that "safeguarding of sperm transfer" is one driving force for evolution of male genital complexity in scorpions, but also sexual selection by cryptic female choice could partly play a role.

Neurosecretory cells in the circadian-clock system of the scorpion, Androctonus australis.

The somata of the efferent neurosecretory fibers that control the circadian sensitivity rhythm in the median eyes of the scorpion, Androctonus australis, were detected in the brain by retrograde labeling with Lucifer Yellow CH. A total of 20-40 neurons are arranged in two groups displaying a bilaterally symmetrical, marginal position near the circumesophageal connectives. Half the cells in each group send fibers into the ipsilateral optic nerve; the fibers from the other half enter the contralateral optic nerve.

The sperm and its formation in the scorpion Centruroides vittatus.

The development of sperm from a spermatid in the scorpion, Centruroides vittatus (Say), is described. The mature sperm is short with helical nucleus and a peculiar structure of the sperm tail. This peculiarity consists of alternating mitochondrial derivatives and membranous or network elements wrapped about the flagellum. We note the absence both of microtubules in the sperm of Centruroides and also of the centriole adjunct, which is present in other scorpion spermatids.