Living without mitochondria: spermatozoa and spermatogenesis in two species of Urodasys (Gastrotricha, Macrodasyida) from dysoxic sediments

Abstract. The spermatozoa of two species of Macrodasyida (Gastrotricha), Urodasys anorektoxys and U. acanthostylis , show an ultrastructural organization diverging from one another and from other gastrotrichs: their main peculiarity is in the absence of mitochondria. In U. anorektoxys , the acrosome is a long, twisted column inserted into the nucleus, which is basally cylindrical, and the flagellum shows rows of peculiar, large globules parallel to the axonemal doublets. In U. acanthostylis , the acrosome is completely cork-screwed and surrounds the nucleus, and the tail shows columnar accessory fibers. At present, the absence of mitochondria in the mature sperm, and the peculiar fingerprint aspect of condensed chromatin are the only traits shared by the two species. The features of the spermatozoa of these two species of Urodasys widen the range of different models of gastrotrich spermatozoa, and place the genus in a peculiar position, from the spermatological point of view, within the Macrodasyida. The loss of mitochondria in mature spermatozoa is possibly related to either the dysoxic habitat of the two species or a peculiar fertilization mechanism.     

Macrodasyida (Gastrotricha): a cladistic analysis of morphology

Abstract. A cladistic analysis based on 33 morphological characters was performed for the 31 genera currently assigned to the order Macrodasyida (Gastrotricha). Outgroup analysis indicated that the order is monophyletic and that it is defined by the structure of the pharynx and the complex distribution of duo-gland adhesive organs. Of the 6 currently recognized families in Macrodasyida, our analysis confirmed that 4 families are monophyletic: Dactylopodolidae, Macrodasyidae, Thaumastodermatidae and Turbanellidae. Dactylopodolidae was further confirmed as the most basal family within the order based on the retention of several plesiomorphies. The other three families have well-defined autapomorphies but will require further investigation to increase inter- and intrafamilial phylogenetic resolution. Planodasyidae appeared to be a paraphyletic taxon with no obvious autapomorphies; genera clustered among members of a polyphyletic family, Lepidodasyidae. We recommend that future research on macrodasyidan phylogeny focus on issues of comparative morphology and ultrastructure in lesser-known taxa such as the Dactylopodolidae, and on the taxa Lepidodasyidae and Planodasyidae.     

The musculature of Draculiciteria tessalata (Chaetonotida, Paucitubulatina): implications for the evolution of dorsoventral muscles in Gastrotricha

The muscular system of the marine interstitial gastrotrich Draculiciteria tessalata (Chaetonotida, Paucitubulatina) was analyzed with fluorescent phalloidin. Muscles in circular, longitudinal, helicoidal and dorsoventral orientations were found. Circular muscles were present as discreet rings on the pharynx only. Five pairs of longitudinal muscles were found in dorsal, lateral and ventral positions. One of the two pairs of lateral muscles is newly described for the species. Helicoidal muscles, external to the circular muscles and some longitudinal bands, spiraled around the pharynx and anterior portion of the intestine. Two pairs of segmentally-arranged dorsoventral muscles were also present. Lateral dorsoventral muscles extended from the base of the pharynx to the anterior part of the caudal furca. Medial dorsoventral muscles extended from the pharyngeal-intestinal junction into each ramus of the caudal furca. A hypothesis on the evolution of dorsoventral muscles in D. tessalata is proposed which includes a splitting of circular muscles into separate somatic and splanchnic components with a further displacement of both muscle sets into a dorsoventral orientation.     

Architecture of the nervous system in two Dactylopodola species (Gastrotricha, Macrodasyida)

Immunohistochemical stainings have become standard tools to describe the nervous system, but usually only singular or few markers are used and consequently show only subsets of neurons within the nervous system. We investigated two species of Dactylopodola (Gastrotricha, Macrodasyida) with a broad set and combination of markers, to represent the nervous system in a more holistic approach. We suggest that markers for both neurotubuli (tubulin) and neurotransmitters (e.g. serotonin, FMRF-amides, histamine) should be used. Combinations with markers for the musculature (phalloidin) and nuclei (propidiumiodide or other markers) help to reveal spatial patterns and when used with TEM can provide a more precise picture of the spatial relationships of particular nerves. Species of Dactylopodola have a brain consisting of a solid dorsal commissure and a fine ventral commissure. Cell somata of brain cells are arranged lateral to the dorsal commissure and form a dumbbell-like brain. Additionally, projections into the head region, head sensory organs, one pair of lateroventral nerve cords with three commissures and stomatogastric nerves are described. Obviously, some longitudinal transmitter-specific fibres run in parallel to the main longitudinal nerve and represent additional longitudinal fibres. In comparison with the nervous system architecture of other gastrotrich species and that of different bilaterian animals it is speculated that the gastrotrich nervous system retains several ancestral features, such as being commissural and not a compact brain.     

Ultrastructure of the sensory palps ofTetranchyroderma papii (Gastrotricha,Macrodasyida)

Summary The sensory palps of the macrodasyoid gastrotrichTetranchyroderma papii contain processes from two types of cell: 22–23 bipolar primary sensory cells and two to three support cells. In the proximal region of the palp each sensory cell contains a short ciliary segment with a basal body and from this ciliary segment a longer distal segment lacking axonemal microtubules extends through the major part of the length of the palp. Each support cell process bears microvilli and contains a conspicuous bundle of microtubules running the entire length of the process. The cell bodies of both cell types are situated in the epidermis of the head region. The palps are interpreted as having a chemosensory function. They are considered to be homologous to the posterior cephalic sensory organ ofTurbanella cornuta, but not the head tentacles ofChordodasys antennatus or nematode amphids.     

Ultrastructure of spermatogenesis and spermatozoa of Cephalodasys maximus (Gastrotricha,Macrodasyida)

Summary Spermatogenesis and sperm ultrastructure of the macrodasyidan gastrotrich Cephalodasys maximus are described by means of transmission electron microscopy. The filiform sperm consists of an acrosomal accessory structure and an acrosomal vesicle, both being surrounded by spiralled material. The successive nuclear helix encloses the spiral-shaped mitochondrion and the axoneme of the flagellum is accompanied by dense strings, three helical elements and peripheral microtubules. During spermiogenesis the acrosomal accessory structure develops first and moves into a cell projection, where the spiral around this acrosomal rod forms. A nuclear section with condensed chromatin and one single fused large mitochondrion follow into the extension, becoming helical. A connecting clasp between nucleus and flagellum shortens to a cap-like structure. Parallel to the acrosomal and nuclear projection the flagellum develops where the spiralled elements and the basal plate form in succession, while the basal body shrinks.     

Anatomy and ultrastructure of the reproductive organs in Dactylopodola typhle (Gastrotricha: Macrodasyida) and their possible functions in sperm transfer

Abstract. The reproductive anatomy of gastrotrichs is well known for several species, especially for the marine taxon Macrodasyida. However, there is little information on the reproductive organs and the modes of mating and sperm transfer in putative basal taxa, which is necessary for accurate reconstruction of the ground pattern of the Gastrotricha. We present the first detailed morphological investigation of the reproductive system of a putative basal gastrotrich, Dactylopodola typhle , using transmission and scanning electron microscopy, histology, and microscopic observations of living specimens. Dactylopodola typhle is a hermaphrodite that possesses paired female and male gonads, an unpaired uterus with an outlet channel that we call the cervix, and an additional accessory reproductive organ, the so-called caudal organ. We hypothesize that the hollow, secretory caudal organ serves for picking up autospermatozoa (self-sperm), for spermatophore formation, and finally for transferring the autospermatophore to a mating partner. The allospermatophore (foreign spermatophore) is stored within the uterus where fertilization occurs. We think that the mature and fertilized egg is released through the cervix and the dorsolateral female gonopore, and not by rupture of the body wall. Based on the morphology, we provide a plausible hypothesis for spermatophore formation and transfer in D. typhle . Preliminary phylogenetic considerations indicate that the stem species of Macrodasyida, perhaps that of all Gastrotricha, had paired ovaries and paired testes, an unpaired uterus, and only one accessory reproductive organ.     

Spermiogenesis and spermatozoa in Acanthodasys aculeatus (Gastrotricha, Macrodasyida): an ultrastructural study

The spermatogenesis, the spermiogenetic process and the structure of the mature spermatozoon of Acanthodasys aculeatus (Gastrotricha, Macrodasyida) are described from an ultrastructural point of view. Several spermatogonia in mitotic divisions were seen, proving that euthely of gastrotrichs does not concern gonads. Spermiogenesis is characterized by the early formation of both the acrosome and the axoneme, by the subsequent appearances of a perinuclear helix and of a complex axial tubular structure in the acrosome and by the late development of the peraxonemal striated cylinder. The mature spermatozoon is filiform, and composed of a spiralized acrosome, a helical nuclear–mitochondrial complex and a long flagellum. The acrosome contains an axial tubular structure and the spring-shaped nucleus delimits a single, long mitochondrion. A perinuclear helix formed by the pro-acrosome surrounds the nuclear–mitochondrial complex extending for its whole length. A monolayered, obliquely striated cylinder encloses the 9 × 2 + 2 axoneme; its terminal part is empty because of the shortness of the axoneme.     

Phylogeny of Thaumastodermatidae (Gastrotricha: Macrodasyida) inferred from nuclear and mitochondrial sequence data

Background

Phylogenetic relationships within Gastrotricha are poorly known. Attempts to shed light on this subject using morphological traits have led to hypotheses lacking satisfactory statistical support; it seemed therefore that a different approach was needed.

Methodology/Principal Findings

In this paper we attempt to elucidate the relationships within the taxonomically vast family Thaumastodermatidae (Macrodasyida) using molecular sequence data. The study includes representatives of all the extant genera of the family and for the first time uses a multi-gene approach to infer evolutionary liaisons within Gastrotricha. The final data set comprises sequences of three genes (18S, 28S rDNA and COI mtDNA) from 41 species, including 29 thaumastodermatids, 11 non-thaumastodermatid macrodasyidans and a single chaetonotidan. Molecular data was analyzed as a combined set of 3 genes and as individual genes, using Bayesian and maximum likelihood approaches. Two different outgroups were used: Xenotrichula intermedia (Chaetonotida) and members of the putative basal Dactylopodola (Macrodasyida). Thaumastodermatidae and all other sampled macrodasyidan families were found monophyletic except for Cephalodasyidae. Within Thaumastodermatidae Diplodasyinae and Thaumastodermatinae are monophyletic and so are most genera. Oregodasys turns out to be the most basal group within Thaumastodermatinae in analyses of the concatenated data set as well as in analyses of the nuclear genes. Thaumastoderma appears as the sister taxon to the remaining species. Surprisingly, Tetranchyroderma is non-monophyletic in our analyses as one group of species clusters with Ptychostomella while another appears as the sister group of Pseudostomella.

Conclusions/Significance

Results in general agree with the current classification; however, a revision of the more derived thaumastodermatid taxa seems necessary. We also found that the ostensible COI sequences from several species do not conform to the general invertebrate or any other published mitochondrial genetic code; they may be mitochondrially derived nuclear genes (numts), or one or more modifications of the mitochondrial genetic code within Gastrotricha.     

Morphology and function of reproductive organs in Neodasys chaetonotoideus (Gastrotricha: Neodasys) with a phylogenetic assessment of the reproductive system in Gastrotricha

The reproductive system of the important basal gastrotrich Neodasys chaetonotoideus is described and reconstructed on the basis of light microscopy, serial ultrathin sections (ultrastructure) and scanning electron microscopy. Starting frontally, the hermaphroditic reproductive system consists of paired and tube shaped lateral testes that do not possess elongated seminal ducts but most likely open directly via paired ventral pores. The unpaired, medio‐dorsal ovary region contains early oogenic stages that mature caudally towards the uterus region, where the most mature egg is positioned laterally to the midgut. The ovary region is not covered with an epithelial lining whereas the uterus region possesses a distinct epithelial wall. Between ovary and uterus region, we have detected a conspicuous section of the female gonad, the vitellogenic oviduct that consists of a thick epithelial wall which forms cellular protuberances into the developing oocytes passing the oviduct. We interpret this as a special, hitherto undescribed mode of vitellogenesis in Gastrotricha. Further caudally, the uterus continues with the fronto‐caudal organ, a complex of two substructures that are apparently homologous to the frontal organ and the caudal organ of many species of the Gastrotricha Macrodasyida. Neodasys chaetonotoideus obviously engages in spermatophore formation and transfer. In this study we develop a morpho‐functional scenario for the gonads and accessory organs in terms of spermatophore production, exchange and oviposition. We compare our newly obtained data with already published results on the reproductive organs of several species of Gastrotricha by means of a species‐character matrix and provide a computer aided evaluation by a parsimonious character optimization. A reconstruction of the reproductive system of the stem species of Gastrotricha on the basis of three recent phylogenetic analyses is presented. These reconstructions give support for a Neodasys‐like reproductive system in the ground pattern of Gastrotricha with slight morphological differences and direct transfer of spermatozoa rather than spermatophore transfer. The evolution of selected characters is traced thus revealing some incidents of convergent evolution as well as the evolutionary replacement of the ancestral frontal organ by the derived frontal sac in at least two separated lineages.     

Two new interesting genera of Gastrotricha (Macrodasyida and Chaetonotida) from the Brazilian freshwater psammon

Two new genera and species of Gastrotricha are described from the psammon of a freshwater body in Brazil: Redudasys fornerise and Arenotus strixinoi. The former is the first undoubted member of the order Macrodasyida recorded from a freshwater environment. It is characterized by the reduction in number of adhesive tubes, the absence of male sexual organs and the presence of a well-developed protonephridial system. The latter belongs to the order Chaetonotida (family Chaetonotidae) and is characterized by the uniform body covering with a thick layer of soft homogeneous cuticle. A possible mode of colonization of fresh waters by marine Macrodasyida, involving colonization of freshwater areas underlying marine beaches, is discussed.     

The male postabdomen and reproductive system of Bibio marci Linnaeus, 1758 (Hexapoda: Diptera: Bibionidae)

The male postabdomen and the internal parts of the male genital system of Bibio marci (Bibionomorpha) were examined and reconstructed 3‐dimensionally. Several features differ from the presumptive dipteran groundplan. The bases of the gonopods are fused with each other and with tergite IX. The penis is not tube‐shaped and only sclerotized on the ventral side. The vasa deferentia are S‐shaped, and two pairs of accessory glands are present. In contrast to these characteristics, the arrangement of the internal parts is probably close to the ancestral condition. With its specific shape, the penis is well suited for the transfer of a spermatophore. The dorsal sclerite of the copulatory organ probably represents the medially fused parameres. A cladistic analysis of 27 characters of the postabdomen yielded two most parsimonious trees, with the strict consensus as follows: Nannochoristidae (outgroup) + (Culicidae [Culicomorpha] + ((Nymphomyiidae + (Tipulidae + Trichoceridae)) + (Tabanidae [Brachycera] + (Bibionidae, Anisopodidae, Axymyiidae [Bibionomorpha])))). Potential synapomorphies of Bibionomorpha (including Axymyiidae) and Brachycera are the fusion of sternum IX with the gonocoxites, the fusion of the parameres forming the dorsal sclerite and the presence of an entire series of postabdominal muscles (M4, M20, M23, M26, M27, M31, M35 and M37). The results of the analysis are preliminary as it is based on a single‐character system with a limited taxon sampling. However, the main result – a clade Bibionomorpha + Brachycera – is fully compatible with current hypotheses on dipteran phylogeny.     

A fresh look at Dinodasys mirabilis (Gastrotricha, Macrodasyida), with focus on the reproductive apparatus and sperm ultrastructure

Adults of Dinodasys mirabilis were studied for the first time. The specimens, collected from the west coast of Sweden, were investigated alive and with electron microscopy. Sexually mature specimens attain a total length of 450?C490???m; the adhesive apparatus is made up of anterior, lateral, ventrolateral, dorsal and posterior tubes; and one pair of ??cirrata?? type tubes is also present. The reproductive apparatus is hermaphroditic, paired testes extend rearward from the pharyngeo-intestinal junction to 3/4 of the trunk, and sperm ducts bend anteriorly at U52 and join together into a common, mid-ventral pore at U33. Two ovaries lie along the sides of the caudal intestine, extending anteriorly from U68. Frontal organ present on the right side of the body centered a U70; caudal organ was absent; a gland organ surrounding the terminal intestine may be present, but its homology with other organs in a similar position is uncertain. The spermatozoon is a filiform cell, formed by a long acrosome, a spring-like nucleus and a flagellum. The acrosome is divided into two regions: the anterior-most is thin and corkscrew-shaped, and the posterior one is rectilinear; both regions are delimited by a continuous external layer of thick, dense material, which in longitudinal section appears obliquely striated and surrounds a long pile of stout, electron-dense cylinders; the nucleus contains condensed chromatin and is coiled around an elongate mitochondrion; the flagellum possesses a 9?×?2?+?2 axoneme devoid of striated cylinder. Within Macrodasyida, U-bend sperm ducts and the peculiar ultrastructure of the acrosome are characteristics shared by other Turbanellidae studied so far, providing a foundation for the current systematization of Dinodasys.     

Reproductive system of the genus <Emphasis Type=Italic>Crasiella</Emphasis> (Gastrotricha,Macrodasyida)

Crasiella diplura from Sweden and Crasiella sp. from Italy were studied alive and with transmission electron microscopy (TEM). The two species are simultaneous hermaphrodites and share the same reproductive system lay-out: paired ovaries extend along the posterior part of the intestine and join mid-dorsally, while bilateral, club-shaped testes lie at the sides of the anterior gut, extending as deferentia that fuse on the mid-ventral plane and open into a single pore; gametes mature in a caudocephalic and centripetal direction. The bulky, sac-like, frontal organ is lined by a simple epithelium and lies dorsolaterally to the intestine, on the left side of the body. The spindle-shaped caudal organ is musculo-glandular and is located ventrolaterally to the gut on the right side. It is characterized by the presence of a roughly Y-shaped internal channel that opens into two pores close to each other, which function for the intake and outlet of the (auto)sperm, respectively. The spermatozoa, which are peculiar and similar in the two species, are characterized by a long and complex acrosome consisting of four ultrastructurally distinct regions, three of which find equivalence in other gastrotrich species. The flagellum lacks a striated cylinder. Anatomy and ultrastructure enable us to hypothesize a modality of sperm transfer in Crasiella that is similar to that observed in Macrodasys.     

Internal anatomy and ultrastructure of the male reproductive system of the Norway lobster Nephrops norvegicus (Decapoda: Astacidea)

The Norway lobster (Nephrops norvegicus) is economically important in Europe. However, apart from the female reproductive system, very little is known about its internal anatomy. This article focuses on studying the internal anatomy and ultrastructure of the male reproductive system. This system follows the general pattern found among decapod crustaceans, with several peculiarities. Testes are composed of lobular sperm ducts in which the spermatozoa are fully constituted. The spermatozoa present three lateral arms and a long acrosome, which gives a false appearance of flagellated spermatozoa. The two testes form a double H under the heart, and the vas deferens (VD) arise from each side at the posterior edge of the double H. The main characteristic of the VD is the presence of a sphincter in the enlarged area of the distal end of the middle VD. The MVD here shows an increase in musculature of the wall as compared to the VD, which regulates the passage of the sperm cord to the distal VD (DVD) and thence to the thelycum of the female. The wall of the spermatophore is formed in the distal part of the proximal VD, which surrounds the unique sperm cord present in the VD. Isolated spermatophores are not observed in the VD. The sperm cord is pinched off during copulation by the musculature of the DVD. Then, a portion of the sperm cord is transferred from each VD to form the isolated spermatophores. The wall of the spematophores and the spermatozoa that are observed inside the thelycum have the same morphology as those observed in the VD. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

The muscular system of Musellifer delamarei (Renaud-Mornant, 1968) and other chaetonotidans with implications for the phylogeny and systematization of the Paucitubulatina (Gastrotricha)

We studied comparatively the muscle organization of several gastrotrich species, aiming at shedding some light on the evolutionary relationships among the taxa of the suborder Paucitubulatina. Under confocal laser scanning microscope, the circular muscles were present in the splanchnic position as incomplete circular rings in Musellifer delamarei (Chaetonotidae) and Xenotrichula intermedia (Xenotrichulidae) and as dorsoventral bands in Xenotrichula punctata, Heteroxenotrichula squamosa and Draculiciteria tesselata (Xenotrichulidae); in the somatic position, M. delamarei shares the presence of dorsoventral muscles with all the Xenotrichulidae, in contrast with the remaining Chaetonotidae that lack these muscles. Maximum parsimony analysis of the muscular characters confirmed monophyly of Paucitubulatina and Xenotrichulidae, while the Chaetonotidae was paraphyletic, with the exclusion of Musellifer , which is the most basal genus within the Paucitubulatina. Xenotrichulidae is the sister taxon to Chaetonotidae, which in turn has Polymerurus as the most basal taxon. In general, the results agree with recent phylogenetic inferences based on molecular characters and support the hypothesis that, within Paucitubulatina, dorsoventral muscles are plesiomorphies retained in marine, interstitial, hermaphroditic gastrotrichs. Dorsoventral muscles were subsequently lost during changes in lifestyle and reproduction modality that took place with the invasion of the freshwater environment. This new information prompted us to reconsider the systematization of Chaetonotidae, proposing the establishment of Muselliferidae fam. nov. to include the genera Musellifer and Diuronotus .  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 379–398.     

Male reproductive system in Neorossia caroli (Joubin 1902) (Cephalopoda: Sepiolidae) from Sardinian waters (western Mediterranean Sea) with particular reference to sexual products

The male reproductive system of the bobtail squid Neorossia caroli (Cephalopoda: Sepiolidae) is described in detail, based on observations of 90 mature males caught from 500 to 1600?m depth in Sardinian waters (western Mediterranean Sea). Reproductive organs in mature specimens accounted for up to 6% of total body weight. Of this, 70% was represented by the spermatophoric complex. Up to 83 spermatophores were found inside the Needham's sac. Mean spermatophore length was 16.7?mm. Sperm mass, cement body, and ejaculatory apparatus represented 63.2%, 13.1%, and 23.7% of the total spermatophore length, respectively. Inverted spermatophores, empty spermatophore sheaths, and spermatangia were also found in the sac, and their presence is discussed. Spermatangia implanted in several parts of the bodies of males (e.g., head, funnel, and eyes) were recorded and their occurrence is discussed. The spermatophoric reaction was induced in the laboratory by submerging spermatophores in seawater, and it is described briefly.     

A new non-naked species of Ptychostomella (Gastrotricha) from Brazil

A new species of marine Gastrotricha from Brazil is described and discussed. Ptychostomella lamelliphorasp. n. is one of the several new taxa that were found during an extensive survey of the gastrotrich fauna carried out in 2002 and 2003 along the coastline of the State of São Paulo. The new species is unique in that it possesses cuticular ornamentations in the form of plate-like structures (scales) along the lateral borders of the body and two massive clusters of densely packed adhesive tubes on the ventral surface, near the ano-genital opening. Both these features appear to be adaptations to challenge the high energy waters that characterize the species’ microhabitat: the coarse sublittoral sand in the channel between the mainland and the largest island in the State, Ihlabela. Additionally, a key to the described Ptychostomella species of the world is provided.     

Analysis of 18S rRNA gene sequences suggests significant molecular differences between Macrodasyida and Chaetonotida (Gastrotricha)

Partial 18S rRNA gene sequences of four macrodasyid and one chaetonotid gastrotrichs were obtained and compared with the available sequences of other gastrotrich species and representatives of various metazoan phyla. Contrary to the earlier molecular data, the gastrotrich sequences did not comprise a monophyletic group but formed two distinct clades, corresponding to the Macrodasyida and Chaetonotida, with the basal position occupied by the sequences of Tetranchyroderma sp. and Xenotrichula sp., respectively. Depending on the taxon sampling and methods of analysis, the two clades were separated by various combinations of clades Rotifera, Gnathostomulida, and Platyhelminthes, and never formed a clade with Nematoda. Thus, monophyly of the Gastrotricha is not confirmed by analysis of the presently available molecular data.