Intraspecific and Interspecific Phenotypic Differences Confirm the Absence of Cryptic Speciation in Triatoma sordida (Hemiptera, Triatominae).

Triatoma sordida is an endemic Chagas disease vector in South America, distributed in Brazil, Bolivia, Paraguay, and Uruguay. Chromosomal, molecular, isoenzymatic, and cuticular hydrocarbon pattern studies indicate cryptic speciation in T. sordida. Recently, T. rosai was described from specimens from Argentina initially characterized as T. sordida. Although several authors assume that the speciation process that supports this differentiation in T. sordida is the result of cryptic speciation, further morphological and/or morphometric studies are necessary to prove the application of this evolutionary event, because the only morphological intraspecific comparison performed in T. sordida is based on geometric morphometry and the only interspecific comparison made is between T. rosai and T. sordida from Brazil that evaluated morphological and morphometric differences. Based on this, morphological analyses of thorax and abdomen using Scanning Electron Microscopy and morphometric analyses of the head, thorax, and abdomen among T. sordida from Brazil, Bolivia, and Paraguay, as well as T. rosai, were performed to assess whether the evolutionary process responsible for variations is the cryptic speciation phenomenon. Morphological differences in the thorax and female external genitalia, as well as morphometric differences in the head, thorax, abdomen, pronotum, and scutellum structures, were observed. Based on this, the evolutionary process that supports, so far, these divergences observed for T. sordida populations/T. sordida subcomplex is not cryptic speciation. Moreover, we draw attention to the necessity for morphological/morphometric studies to correctly apply the cryptic species/speciation terms in triatomines.

Size, shape, and direction matters: Matching secondary genital structures in male and female mites using multiple microscopy techniques and 3D modeling.

Studies of female genital structures have generally lagged behind comparable studies of male genitalia, in part because of an assumption of a lower level of variability, but also because internal genitalia are much more difficult to study. Using multiple microscopy techniques, including video stereomicroscopy, fluorescence microscopy, low-temperature scanning electron microscopy (LT-SEM), and confocal laser scanning microscopy (CLSM) we examined whether the complex sperm transfer structures in males of Megalolaelaps colossus (Acari: Mesostigmata) are matched by similarly complex internal structures in the female. While both LT-SEM and CLSM are well suited for obtaining high-quality surface images, CLSM also proved to be a valuable technique for observing internal anatomical structures. The long and coiled sperm transfer organ on the chelicera of the males (spermatodactyl) largely matches an equally complex, but internal, spiral structure in the females in shape, size, and direction. This result strongly suggests some form of genital coevolution. A hypothesis of sexual conflict appears to provide the best fit for all available data (morphology and life history).

Penetration mechanics of elongated female and male genitalia of earwigs.

We unveiled the penile penetration mechanics of two earwig species, Echinosoma horridum, whose intromittent organ, termed virga, is extraordinarily long, and E. denticulatum, whose virga is conversely short. We characterised configuration, geometry, material and bending stiffness for both virga and spermatheca. The short virga of E. denticulatum has a material gradient with the stiffer base, whereas the long virga of E. horridum and the spermathecae of both species are homogeneously sclerotised. The long virga of E. horridum has a lower bending stiffness than the spermatheca. The virga of E. denticulatum is overall less flexible than the spermatheca. We compared our results to a previous study on the penetration mechanics of elongated beetle genitalia. Based on the comparison, we hypothesised that the lower stiffness of the male intromittent organ comparing to the corresponding female structure is a universal prerequisite for the penetration mechanics of the elongated intromittent organ in insects.

Coevolution between female seminal receptacle and sperm morphology in the semiaquatic measurer bug Hydrometra stagnorum L. (Heteroptera, Hydrometridae).

The coevolution between sperm length and size of the female sperm-storage organs is described for the first time within Heteroptera. The long sperm of the measurer bug Hydrometra stagnorum is characterized by the unusually long acrosome with its anterior region helically arranged, and by a very short nucleus. The sperm flagellum has a 9 + 9+2 conventional axoneme and crystallized mitochondrial derivatives. The female spermatheca consists of an extraordinarily long spermathecal duct ending with an apical spermathecal bulb into which flows also the secretions of a relatively short spermathecal gland. Both spermathecal duct and gland have a thin epithelium lined by a cuticle, beneath which a complex of secretory and duct forming cells are present. The secretions of these two structures flow into the apical spermathecal bulb. A thick layer of muscle fibers surrounds the epithelium. These results confirm the opinion that the dimensions of the female reproductive sperm-storage organs are able to drive the sperm morphology.

External female genitalia of Triatoma jatai, Triatoma costalimai and Triatoma williami (Hemiptera: Reduviidae: Triatominae).

BACKGROUND: Taxonomic identification of triatomines is generally performed based on aspects of their external morphology. However, the use of a multidisciplinary approach, considering morphological aspects of the external genitalia, morphometry, genetics, and phylogeography has been suggested, especially for similar and/or cryptic species. The rupestral species Triatoma jatai Gonçalves et al., 2013, Triatoma costalimai Verano & Galvão, 1959 and Triatoma williami Galvão et al., 1965, which are morphologically similar, have been found naturally infected with Trypanosoma cruzi (Chagas, 1909) in wild, peridomestic, and intradomestic environments, representing a risk of new outbreaks of Chagas disease. This study presents morphological description complementation of these species, with an emphasis on the structures of the female external genitalia, using scanning electron microscopy. METHODS: The females of T. jatai and T. costalimai (n = 10 of each) were captured in the Brazilian municipalities of Paranã and Aurora do Tocantins and were identified with the use of a dichotomous key for the Matogrossensis subcomplex. Females of T. williami (n = 5), were obtained from a laboratory colony. The females were cut transversely at the sixth abdominal segment and examined under scanning electron microscopy (SEM) at the Oswaldo Cruz/Fiocruz Institute Electronic Microscopy Platform. RESULTS: It was possible to differentiate the three species based on the characteristics of urotergites VII, VIII and IX and urosternite VII, as well as the genital plaques, gonocoxites, and gonapophyses. To our knowledge, morphological differences in the spines present on gonapophysis 8 in triatomines are described here for the first time. CONCLUSIONS: The results show that external genitalia of females are useful structures to differentiate T. costalimai, T. jatai and T. williami. SEM analysis contributes to and corroborates, together with other tools morphological and molecular, the distinction of the three species.

Adaptations for matrotrophy in the female reproductive system in the pseudoscorpion Chelifer cancroides (Chelicerata: Pseudoscorpiones, Cheliferidae).

Pseudoscorpiones (pseudoscorpions, false scorpions) is an order of small terrestrial chelicerates. While most chelicerates are lecithotrophic, that is, embryos develop due to nutrients (mostly yolk) deposited in the oocyte cytoplasm, pseudoscorpions are matrotrophic, that is, embryos are nourished by the female. Pseudoscorpion oocytes contain only a small amount of yolk. The embryos develop within a brood sac carried on the abdominal site of the female and absorb nutrients by a pumping organ. It is believed that in pseudoscorpions nutrients for developing embryos are produced in the ovary during a postovulatory (secretory) phase of the ovarian cycle. The goal of our study was to analyze the structure of the female reproductive system during the secretory phase in the pseudoscorpion Chelifer cancroides, a representative of the family Cheliferidae, considered to be one of the most advanced pseudoscorpion taxa. We use diverse microscopic techniques to document that the nutritive fluid is produced not only in the ovaries but also by the epithelial cells in the oviducts. The secretory active epithelial cells are hypertrophic and polyploid and release their content by fragmentation of apical parts. Our observations also indicate that fertilization occurs in the oviducts. Moreover, in contrast to previous findings, we show that secretion of the nutritive material starts when the fertilized oocytes reach the brood sac and thus precedes formation of the pumping organ. Summing up, we show that C. cancroides exhibits traits of advanced adaptations for matrotrophy due to coordinated secretion of the nutritive fluid by the ovarian and oviductal epithelial cells, which substantially increases the efficiency of nutritive fluid formation. Since the secretion of nutrients starts before formation of the pumping organ, we suggest that the embryos are able to absorb the nutritive fluid also in the early embryonic stages.

Genital morphology and the mechanics of copulation in the millipede genus Pseudopolydesmus (Diplopoda: Polydesmida: Polydesmidae).

Mate choice, copulation, genital morphology, and sperm storage are not very well understood in millipedes. The use of three-dimensional x-ray computed tomography (µCT) provides new morphological data regarding millipede reproductive systems in both the female and male, including chitinous sclerites and membranes, muscles, glands, oviducts, and sperm conduits. Here we present a complete integrated account of the morphology and function of the female genital organs in the family Polydesmidae (Diplopoda: Polydesmida) using µCT, UV fluorescence imaging, and scanning electron microscopy. These data allow us to consider competing hypotheses regarding millipede vulva formation. We additionally present the morphology of copulatory interface in Pseudopolydesmus Attems, 1898 using images of a mating pair in copula and by simulating the interface of the organs using 3D models from µCT, allowing us to tentatively identify a lock-and-key-like mechanism. Finally, we use µCT to reveal the topology of the seminal canal in the gonopod of male Pseudopolydesmus, a topic that has remained unresolved for nearly 80 years.

The fine structure of the germinal mass, brood cavity and birth canal of the rediae of the monoxenous hemiuroid digenean Bunocotyle progenetica Chabaud & Buttner, 1959.

Bunocotyle progenetica is a hemiuroid digenean whose sexual adults become fully developed and lay their eggs inside the rediae in the molluscan host. In this study, the fine structure of the germinal mass, brood cavity and birth canal in the B. progenetica rediae was examined using transmission electron and confocal microscopy. The large germinal mass attached to the body wall has a cellular composition typical for this organ. The characteristic traits of this germinal mass are weakly developed supporting tissue and the presence of deep lacunae opening into the brood cavity. These lacunae presumably participate in feeding the deeply lying embryos and facilitate their release into the brood cavity. The germinal mass is also characterized by intensive degeneration of cellular elements, which may represent a mechanism controlling the offspring number, limited in this species by the size of the redial brood cavity. The brood-cavity lining consists of flattened cells bearing lamellar projections and is connected anteriorly with the epithelium of the birth canal. The brood-cavity musculature, which is well developed in other hemiuroid digeneans, is significantly reduced in B. progenetica, most likely because their cystophorous cercariae remain inside the rediae, removing the need for muscle contractions pushing them through the brood cavity. The birth canal comprises three regions distinguished by the structure of the lining and muscle arrangement. The comparison of rediae of B. progenetica with parthenitae of other digeneans has shown that the organization of the redial reproductive apparatus in this species may have been influenced by life-cycle modification.

Morphology of the genital organs of the female red-rumped agouti (Dasyprocta leporina, Linnaeus, 1758) during estrous cycle phases and in advanced pregnancy.

The study investigated the gross and microscopic anatomy of the genital organs of 20 agoutis at different stages of the estrous cycle and four in the final trimester of pregnancy. Specimens were euthanized and their reproductive organs were fixed in a 4% paraformaldehyde or 2.5% glutaraldehyde solution and submitted to routine histological techniques for light and scanning electron microscopy. In the ovary, during the proestrus phase, we observed developing follicles and corpus luteum (CL) in regression; during estrus, there were Graafian follicles; during metestrus, there was a hemorrhagic corpus, whereas in diestrus, there was a mature CL. The uterus was partially double because the cervix was cranially septate but caudally, the septum disappeared, forming a single ostium that opened into the vagina. Changes occurred along the estrous cycle in the uterine and vaginal epithelia, that is, an increase in the uterine epithelium height accompanied by an increase of thickness of the vaginal epithelium during the follicular phase and a decrease of thickness of both epithelia during the luteal phase. The endometrial lining was composed of a simple cuboidal epithelium to simple columnar epithelium with basal nuclei. The vaginal mucosa consisted of epithelium that varied from nonkeratinized stratified squamous (luteal phase) to keratinized stratified squamous (follicular phase). The clitoris was external to the vagina. It presented two protruding lateral keratinized spicules and a centralized urethra, with no common parts between the urinary and genital tracts. Anatomical and histological changes were observed mainly in the cervix, vagina and spicules of the clitoris during the EC.

The female reproductive system and oogenesis in Thulinius ruffoi (Tardigrada, Eutardigrada, Isohypsibiidae).

In this study, we describe the female reproductive system organization and oogenesis in the eutardigrade Thulinius ruffoi. Light, confocal and electron microscopy was used in this study. During oogenesis, three phases can be distinguished: previtellogenesis, vitellogenesis, and choriogenesis. Germ-line cells form cell clusters in which the cells are connected by intercellular (cytoplasmic) bridges. These structures are crucial for delivering the yolk materials, macromolecules, ribosomes, and organelles to the developing oocyte. Vitellogenesis is of a mixed type. Autosynthesis and heterosynthesis of the yolk material occur. Yolk precursors that have been synthesized outside the ovary are delivered to the oocyte via endocytosis. We also present data on cortical granules, and moreover, we describe the cortical reaction in tardigrades, possibly for the first time.

The sperm pump and genital coupling of Panorpodes kuandianensis (Mecoptera: Panorpodidae).

Males of Panorpodidae possess a special sperm pump, through which they directly transfer seminal fluid to the female spermatheca. However, the sperm pump has not been studied in Panorpodes to date. Here, the structure of the sperm pump and the internal coupling of genitalia were investigated in the short-faced scorpionfly Panorpodes kuandianensis Zhong, Zhang, and Hua, 2011 using light and scanning electron microscopy. The sperm pump mainly consists of a piston, a pumping chamber, the anterior region of the aedeagal complex, the posterior region of the ejaculatory sac, and associated muscles. The piston as a propulsion apparatus is controlled by levator and depressor muscles. Its posterior region connects dorsally to the aedeagus via a joint. The pumping chamber is located between the piston and the aedeagus. The dorsal and ventral parameres were attached by retractor muscles. During copulation, the male phallotreme connects to the female copulatory pore to transfer sperm. Male gonostyli and parameres grasp the female to restrict the genitalia movement and impede her medigynium from retreating. The sperm ejaculatory mechanism of Panorpodes and the evolution of sperm transfer mode in insects are briefly discussed based on the structure of the sperm pump and the internal coupling of genitalia.

Genital morphology and copulatory behavior in triatomine bugs (Reduviidae: Triatominae).

Triatomines (Heteroptera: Reduviidae) include around 139 species, widely known as vectors of Chagas disease. Our aim is to review the existing knowledge of the genital morphology and sexual behavior and provide some functional analysis of these traits in triatomines. A complex set of traits comprise genitalia and these are highly variable among species. The components of the phallus and seminal products (secreted by action of testes and two accessory glands) interact to allow successful sperm transfer to the female spermathecae (usually a pair of blind tubes that emerge from the common oviduct). Seminal products may inhibit female physiology and extend mating duration. Mating behavior in triatomines is best characterized as scramble competition. We suggest that males may evaluate female condition prior to copulation, given that female fitness is largely affected by food (blood) source. Although rearing several triatomine species may be difficult and discourage from undertaking studies on this group, any further investigation on sexual behavior and mating interactions may provide data for applicative studies including Chagas disease vectors control.

Traumatic insemination is not the case in three Orius species (Heteroptera: Anthocoridae).

Traumatic insemination (TI) is an extraordinary style of mating behavior wherein the female integument is pierced by the male extragenital structure to transfer the spermatozoa into the female's body through wounding. Flower bugs of the genus Orius belong to the family Anthocoridae (Heteroptera), which is referred to as the "TI family". Males possess sharp shaped extragenitalia, and females receive the extragenitalia using the copulatory tubes, which are specialized extragenital structures in Orius species. Since TI is not well studied in insects possessing the copulatory tube, we examined the genital structures and copulatory processes of three species, Orius strigicollis, O. sauteri, and O. minutus. Scanning electron microscopic observations revealed the positions of male extragenital structures during copulation. A needle-like flagellum was deeply inserted into the female intersegment between the abdominal VII and VIII segments, while the curved part of a sickle-like cone forced the intersegment to expand. No scars were detected around the copulation region after copulation. The copulatory tube adhered to the interior of segment VII, and the interior integument around the copulatory tube remained intact after copulation. On the basis of these results, TI does not occur in these Orius species. A pair of seminal conceptacles, which exists in typical TI insects, was found at the base of the oviducts in O. strigicollis. The distal end of the copulatory tube connected to a closed bag with a double-membrane, termed the sperm pouch. The sperm pouch was filled with filamentous structures after copulation and structures with equivalent forms were observed in adult male testis. These structures, considered to be spermatozoa, persisted in the pouch for at least two weeks after copulation, suggesting that the pouch is a long-term spermatozoa storage organ.

Functional anatomy of the female reproductive system of the American lobster (Homarus americanus).

Light microscopy studies of the female American lobster Homarus americanus reproductive system are essentially nonexistent or outdated. Based on samples taken in the spring, summer, and autumn from the southern Gulf of St. Lawrence between 1994 and 2014, and using a combination of histological and scanning electron microscope techniques, we propose an ovarian cycle with 10 stages, identifying for the first time a recovery stage. Also, an atypical resorption stage, characterized by massive reabsorption of mature oocytes, is occasionally observed during summer months. The oviducts are composed of connective tissue (elastic and collagen fibers) with no muscle or secretory activities. Their epithelium shows a cyclic pattern and phagocytosis activities linked to spawning. Although the role of the seminal receptacle is to store and protect semen, free spermatozoa (i.e., without the spermatophoric wall and the acellular gelatinous substance that constitute the semen) were also observed in its posteriolateral grooves immediately prior to spawning, which is consistent with an external fertilization mechanism at the seminal receptacle. Unexpectedly, free spermatozoa were observed externally near two pore-like structures located on the gonopore's operculum, not at the seminal receptacle, after spawning; hence, more work is needed to fully understand the fertilization mechanism for the American lobster.

Copulatory mechanism and functional morphology of genitalia and anal horn of the scorpionfly Cerapanorpa dubia (Mecoptera: Panorpidae).

The scorpionfly genus Cerapanorpa is characterized by the male possessing a single finger-like anal horn on the posterior portion of tergite VI. However, the functional morphology of this anal horn and the genitalia have not been studied to date. Herein, we investigated the functional morphology of the genitalia and the nongenital structures of the scorpionfly Cerapanorpa dubia by observing the mating process and dissecting the freeze-fixated pairs in copula to reveal the copulatory mechanism. The male C. dubia provides a solid salivary mass to the female as a nuptial gift prior to copulation. When the female starts to feed on the gift, the male uses his notal organ and complex genital structures to control the closest wing and genitalia of the female to establish a V-shaped mating position. In the maintenance phase of copulation, the male uses his anal horn in cooperation with the basally-constricted abdominal segment VII to clamp female abdominal segment VIII. The male hypovalves grasp female cerci, and move up and down rhythmically. The paired parameres clasp both sides of female tergite IX. The basal processes on male gonostyli grip the pleural membranes of the female genital chamber. In the sperm transfer phase, the male aedeagus directly couples with the female medigynium to transmit sperm by connecting his phallotreme to the female's copulatory pore. The evolution of the male complex grasping structures in Panorpidae is also briefly discussed.

The functional anatomy of external genitalia in the black tiger prawn (Penaeus monodon) studied by micro-computed tomography and scanning electron microscopy.

Although artificial insemination has been used for decades in Penaeus monodon aquaculture, the interaction of male and female external genitalia during spermatophore transfer has not been fully documented. As a result, studying the functional anatomy of this process may help to better refine the insemination technique. The sexual act in penaeoid prawns is virtually impossible to observe directly; as a result, this study aimed to describe the functional anatomy and interaction of external genitalia, such as the petasma, appendices masculinae and genital papillae of the male, with that of the thelycum and genital lobes of the female, using a combination of micro-computed tomography and scanning electron microscopy. We hypothesise that the spermatophores are ejaculated into the ventromedial groove of the petasma and squeezed by abdominal flexure into the spermathecae of female's thelycum. During this process, the 'spike-like' setae observed on the petasma and appendices masculinae are speculated to control the transferring direction of the spermatophores. The approach of three dimensional remodelling and animation reported in this study may prove useful in the examination of further hypotheses related to the functional anatomy of external genitalia and/or appendages for other crustacean species.

Chonopeltis australis (Crustacea: Branchiura); the female reproductive system.

The female reproductive system has been described for Dolops ranarum (Stuhlman, 1891) and various Argulus spp. but, there is no description of the reproductive system for Dipteropeltis spp. Calman, 1912 or Chonopeltis spp. Thiele, 1900. This paper describes the female reproductive system and egg laying behaviour of Chonopeltisaustralis Boxshall, 1976 using histology, light microscopy, scanning electron microscopy and observations. The histological study of six specimens showed that the organ structure is similar to that of Argulus spp. and D. ranarum. The oocytes therefore develop in the gonocoel, are ovulated into the lumen of the ovary which is continuous with the functional oviduct and eventually the gonopore. Females of C. australis deposit eggs on the surrounding substrate while the fish is at rest, without leaving the host, thereby mitigating the risk of not locating a host again.

The morphology of the reproductive system in the crab Percnon gibbesi (Decapoda: Brachyura: Grapsoidea) reveals a new combination of characters in Thoracotremata.

Recent studies revealed a high diversity of reproductive structures in heterotreme brachyurans, while those of Thoracotremata seem rather uniform. Yet, there still is a huge lack of data in this group as only few species have been studied with respect to their reproductive system. The phylogenetic position of Percnidae is ambiguous. Recent molecular studies place it within polyphyletic grapsoids. We herein study the reproductive morphology of Percnon gibbesi using histology, scanning electron microscopy, micro-computed tomography and 3D-reconstructions to test whether this species shows the characteristic thoracotreme pattern. Our results reveal that the male copulatory system conforms to other thoracotremes. It is composed of two pairs of pleopods (gonopods) and likewise paired penes. The first gonopod is relatively long. It possesses a bent terminal process with a distal opening of the ejaculatory canal, a character also present in other grapsoids. The second gonopod is short and terminates in an apical girdle. The female reproductive system reveals a combination of characters, so far unknown for thoracotremes. The paired oviducts do not lead into the seminal receptacles, but run into separate cuticular ducts joined with the vaginae. Accessory sperm storage organs, the bursae, are also connected to the vaginae. Bursae have previously only been described in heterotreme crabs. The data presented in this study reveals a higher diversity of thoracotreme reproductive systems than anticipated.

The ontogeny of the female reproductive system in the parasitic castrator pea crab Calyptraeotheres garthi: Implications for its mating system.

The knowledge of the mating system of pea crabs is still fragmentary as it remains dubious whether females copulate in the juvenile and free-living 'hard' or in the obligatory symbiotic stages (adult stage 'V' or intermediate stages II to IV). To discriminate between these two possibilities, we analysed the female seminal receptacles, vagina and opercula, and the sperm content in different stages of the pea crab Calyptraeotheres garthi. Our histology and scanning electron microscopy results revealed that in the hard stage the seminal receptacle is simple without secretory epithelia, and vagina and opercula are not controlled by musculature. In stages II to IV, the seminal receptacles, vagina, and opercula are under development and these structures reach maturity in stage V. These results suggest that females become receptive in stage V and not during predating stages. We found no spermatozoa in SR of 'hard' and stage II to IV females while these structures were loaded of sperm in most stage V, indicating that females start to mate in stage V. Our results support the notion that males of C. garthi roam among hosts in search for sedentary stage V females, as predicted by Baeza and Thiel's () model of mating systems for symbiotic crustaceans. Nevertheless, we failed to reveal whether females mate repeatedly: the accumulation of sperm in larger females might indicate occurrence of multiple copula or a high variability in male sperm transfer.

The role of lysosomes in the phenomenon of concentration of aluminum and indium in the female reproductive system. An ultrastructural study.

The female reproductive system is one of the most complex systems in the body taking into account the hormonal fluctuations associated with ovarian and uterine menstrual cycles. The purpose of this work was to study the impact of aluminum nitrate and indium sulfate on the uterus and the ovary of a pregnant rat. The experiment was performed on adult female rats of Wistar strain weighing approximately 250g. The Transmission Electron Microscopy (TEM) showed the presence of electron-dense material in lysosomes of both uterine cells (myometrium and endometrium cells) and in the cells of the ovary (internal theca and granulosa cells). In addition to the presence of aluminum and indium deposits in the uterine and ovarian tissue, impaired endoplasmic reticulum, mitochondria and vacuolation were also identified. We concluded that lysosomes of uterus and ovary cells had the function to extract aluminum and indium introduced into the body in a soluble form. Then, the two elements were sequestrated within these organelles in an insoluble form most probably as phosphate salts such as reported for other kind of cells; kidney, liver, bone morrow Berry, 1996 [1]. This mechanism seems to be a defense one in which the lysosome would play a central role. Our results concerning the impact of the aluminum or indium presence in the lysosome of female reproductive system will be further used in order to assess their effects on the fertility and viability of oocytes in the pregnant treated rats.