In volvocine algae, gametogenesis is triggered by different cues depending on the species and their sexual systems. In isogamous unicellular organisms such as Chlamydomonas reinhardtii, which produce gametes of equal size and morphology, nitrogen depletion induces gametogenesis. In contrast, in oogamous multicellular species of the genus Volvox, which produce large, immobile eggs and small motile sperm, male gametogenesis is induced by a sex pheromone secreted by sperm packets (SPs), i.e., bundles of male gametes. Eudorina, an anisogamous multicellular volvocine alga that produces motile gametes of different sizes, is known to form SPs under nitrogen-depleted conditions. Intriguingly, a pheromone-like factor, present in male conditioned medium (CM), has also been implicated in SP formation. To clarify the relative contributions of nitrogen starvation and pheromone signaling, we conducted semi-quantitative analyses of SP induction using synchronously cultured male colonies of Eudorina. When CM was added to male cultures during an early maturation stage, most colonies formed SPs regardless of nitrogen availability. However, when the CM was diluted 100- to 100,000-fold, SP formation was significantly more efficient under nitrogen-depleted conditions than under nitrogen-replete conditions. Notably, SPs never formed without the addition of CM, even in a nitrogen-depleted medium. The SP-inducing activity of the CM was found to markedly decrease with protease treatment. These findings suggest that spermatogenesis in Eudorina is induced by a proteinaceous sex pheromone secreted by male colonies, and that nitrogen depletion, while not essential, enhances this pheromone activity.

In volvocine algae, gametogenesis is triggered by different cues depending on the species and their sexual systems. In isogamous unicellular organisms such asChlamydomonas reinhardtii, which produce gametes of equal size and morphology, nitrogen depletion induces gametogenesis. In contrast, in oogamous multicellular species of the genusVolvox, which produce large, immobile eggs and small motile sperm, male gametogenesis is induced by a sex pheromone secreted by sperm packets (SPs), i.e., bundles of male gametes.Eudorina, an anisogamous multicellular volvocine alga that produces motile gametes of different sizes, is known to form SPs under nitrogen-depleted conditions. Intriguingly, a pheromone-like factor, present in male conditioned medium (CM), has also been implicated in SP formation. To clarify the relative contributions of nitrogen starvation and pheromone signaling, we conducted semi-quantitative analyses of SP induction using synchronously cultured male colonies ofEudorina. When CM was added to male cultures during an early maturation stage, most colonies formed SPs regardless of nitrogen availability. However, when the CM was diluted 100- to 100,000-fold, SP formation was significantly more efficient under nitrogen-depleted conditions than under nitrogen-replete conditions. Notably, SPs never formed without the addition of CM, even in a nitrogen-depleted medium. The SP-inducing activity of the CM was found to markedly decrease with protease treatment. These findings suggest that spermatogenesis inEudorinais induced by a proteinaceous sex pheromone secreted by male colonies, and that nitrogen depletion, while not essential, enhances this pheromone activity.

Abstract This study describes the reproductive systems of male and female Johngarthia lagostoma , a land crab endemic to South Atlantic oceanic islands, focusing on spermatozoa production and storage. Specimens from Trindade Island (Brazil) were analysed for anatomy, histology, and histochemistry. The male system includes a pair of tubular testes showing different stages of spermatogenesis and spermiogenesis, leading to mature spermatozoa. These move to the anterior vas deferens (AVD), which has proximal and distal portions, the latter containing coenospermic spermatophores surrounded by secretion type I, reactive to proteins and acidic and neutral polysaccharides. The median (MVD) and posterior (PVD) vas deferens produce type II (strongly protein-reactive) and type III (weakly protein-reactive) secretions. Accessory glands between the MVD and PVD produce the same secretions plus a secretion type IV, reactive to neutral polysaccharides. These mix with the spermatophores and other vas deferens secretions, increasing the PVD’s secretion volume, crucial for the initial release of spermatophores into the seminal receptacle. The female reproductive system features voluminous seminal receptacles connected to the ovary, comprising mesodermal and ectodermal regions classified as ventral-type connection. Plugs and sperm packets are absent, with the seminal receptacles filled with free spermatozoa, suggesting dehiscence occurs shortly after sperm transfer. The influence of male and female secretions on this process is suggested, alongside their roles in sperm maintenance and fertilization facilitation.

Eukaryotic flagella collectively form metachronal waves that facilitate the ability to cause flow or swim. Among such flagellated and planktonic swimmers, large volvocine genera such as Eudorina, Pleodorina and Volvox form bundles of small male gametes (sperm) called "sperm packets" for sexual reproduction. Although these sperm packets reportedly have flagella and the ability to swim, previous studies on volvocine motility have focused on asexual forms and the swimming characteristics of sperm packets remain unknown. However, it is important to quantify the motility of sperm packets and sperm in order to gain insights into the significance of motility in the sexual reproduction of planktonic algae. In this study, we quantitatively described the behavior of three flagellated forms of a male strain of Pleodorina starrii-asexual colonies, sperm packets, and single dissociated sperm-with emphasis on comparison of the two multicellular forms. Despite being smaller, sperm packets swam approximately 1.4 times faster than the asexual colonies of the same male strain. Body length was approximately 0.5 times smaller in the sperm packets than in asexual colonies. The flagella from sperm packets and asexual colonies showed asymmetric waveforms, whereas those from dissociated single sperm showed symmetric waveforms, suggesting the presence of a switching mechanism between sperm packets and dissociated sperm. Flagella from sperm packets were approximately 0.5 times shorter and had a beat period approximately twice as long as those from asexual colonies. The flagella of sperm packets were densely distributed over the anterior part of the body, whereas the flagella of asexual colonies were sparse and evenly distributed. The distribution of flagella, but not the number of flagella, appear to illustrate a significant difference in the speeds of sperm packets and asexual colonies. Our findings reveal novel aspects of the regulation of eukaryotic flagella and shed light on the role of flagellar motility in sexual reproduction of planktonic algae.

Dynamic changes characteristics of the spermatozoon during the reproductive process of mud crab (Scylla paramamosain): From spermatophore formation, transportation to dispersion

Volvocine green algae are a model for understanding the evolution of mating types and sexes. They are facultatively sexual, with gametic differentiation occurring in response to nitrogen starvation (-N) in most genera and to sex inducer hormone in Volvox. The conserved RWP-RK family transcription factor (TF) MID is encoded by the minus mating-type locus or male sex-determining region of heterothallic volvocine species and dominantly determines minus or male gametic differentiation. However, the factor(s) responsible for establishing the default plus or female differentiation programs have remained elusive. We performed a phylo-transcriptomic screen for autosomal RWP-RK TFs induced during gametogenesis in unicellular isogamous Chlamydomonas reinhardtii (Chlamydomonas) and in multicellular oogamous Volvox carteri (Volvox) and identified a single conserved ortho-group we named Volvocine Sex Regulator 1 (VSR1). Chlamydomonas vsr1 mutants of either mating type failed to mate and could not induce expression of key mating-type-specific genes. Similarly, Volvox vsr1 mutants in either sex could initiate sexual embryogenesis, but the presumptive eggs or androgonidia (sperm packet precursors) were infertile and unable to express key sex-specific genes. Yeast two-hybrid assays identified a conserved domain in VSR1 capable of self-interaction or interaction with the conserved N terminal domain of MID. In vivo coimmunoprecipitation experiments demonstrated association of VSR1 and MID in both Chlamydomonas and Volvox. These data support a new model for volvocine sexual differentiation where VSR1 homodimers activate expression of plus/female gamete-specific-genes, but when MID is present, MID-VSR1 heterodimers are preferentially formed and activate minus/male gamete-specific-genes.

Functional morphology of the seminal receptacle of Mithrax, Mithraculus and Omalacantha spider crabs (Brachyura: Mithracidae)

Galli, Loris, Capurro, Matteo, Colasanto, Elisa, Molyneux, Tony, Murray, Andy, Torti, Carlo, Zinni, Matteo (2019): A synopsis of the ecology of Protura (Arthropoda: Hexapoda). Revue suisse de Zoologie 126 (2): 155-164, DOI: 10.5281/zenodo.3463443

The aim of this study was to analyse spermatogenesis in the African butterflyfish, Pantodon buchholzi, using transmission electron microscopy and scanning electron microscopy. P. buchholzi is the most basal teleost that exhibits insemination and produces a highly complex introsperm with the most elongate midpiece known in teleost fishes. Their early stages (spermatogonia and spermatocytes) do not differ greatly from those of other fishes, with the exception of Golgi apparatus degradation appearing as spindle-shaped bodies (SSBs). In round, early spermatids, the development of the flagellum begins after the migration of the centriolar complex towards the nucleus. Later, the elongation of the midpiece coincides with the displacement of the mitochondria and their fusion to produce nine mitochondrial derivatives (MDs). In these spermatids, the nucleus is situated laterally to the midpiece, with condensing chromatin in the centre of the nucleus. Within the midpiece, the flagellum is located within a cytoplasmic canal and is surrounded by a cytoplasmic sleeve containing fibres, MDs and a great amount of cytoplasm located on one side. During the next phase, nuclear rotation, the highly condensed chromatin is displaced to a position above the centriolar apparatus, whereas chromatin-free nucleoplasm is transferred to the cytoplasm. Later, this nucleoplasm, still surrounded by the nuclear membrane, is eliminated into the cyst lumen as the nucleoplasmic packet. Within the highly elongate spermatids, other excess organelles (SSBs, endoplasmic reticulum and mitochondria) are eliminated as residual bodies (RBs). Fully developed spermatozoa, which contain conical-shaped nuclei, eventually coalesce to form unencapsulated sperm packets (spermatozeugmata) that are surrounded by RBs at the level of the extremely elongate midpieces. Later, RBs are removed at the periphery of the cyst by means of phagocytosis by Sertoli cells.

Volvox sect. Volvox is an interesting group of green algae; it comprises mostly monoicous species, but evidence suggests an evolution towards dioicy. Based on cultured strains originating from Thailand, we describe Volvox longispiniferus, a novel species in Volvox sect. Volvox. This species is distinguished from others in the section by the large number of sperm packets in its monoicous sexual spheroids and by the long spines on its zygote wall. Phylogenetic analyses indicate that V. longispiniferus is distinct from the other species of two monophyletic groups within Volvox sect. Volvox. In addition, the novel species produces more zygotes when different cultures are combined compared with a single culture, suggesting a preference for outcrossing.

Volvox sect. Volvox is an interesting group of green algae; it comprises mostly monoicous species, but evidence suggests an evolution towards dioicy. Based on cultured strains originating from Thailand, we describe Volvox longispiniferus, a novel species in Volvox sect. Volvox. This species is distinguished from others in the section by the large number of sperm packets in its monoicous sexual spheroids and by the long spines on its zygote wall. Phylogenetic analyses indicate that V. longispiniferus is distinct from the other species of two monophyletic groups within Volvox sect. Volvox. In addition, the novel species produces more zygotes when different cultures are combined compared with a single culture, suggesting a preference for outcrossing.

Testis structure and sperm packet formation in the phylogenetically basal teleost Pantodon buchholzi

ABSTRACT A female terrestrial isopod in amber from Myanmar is described as Palaeoarmadillo microsoma gen. et sp. n. (Isopoda: Oniscidea: armadillidae). Placement in the family Armadillidae is based on the 2-segmented flagellum with the flagellum nearly as stout as the basal segments, the head with 2 small medial anterior processes, the fronto-caudally compressed cephalon, the large expanded protopodite with small exopodites dorsally attached and the possibility (based on the shape of the epimera) that the specimen could have formed a global shape. Autapomorphies of the fossil are the shape of the basal antennomeres, flagellum and shape and size of the uropod exopodites. The specimen is parasitized by fly larvae (Diptera) and two mite spermatophores with sperm packets are attached to the dorsum of the isopod.

ABSTRACTThe male reproductive system of Amazon freshwater crabs Sylviocarcinus pictus and Dilocarcinus pagei were described and compared in terms of anatomy and histochemistry In addition, sperm transfer experiments of D. pagei with sequential copulations were performed and the anatomy of the seminal receptacle was checked. The anatomy of the male reproductive system of both species is an inverted ‘U’ shape, connected by the testes, from which the pair of vas deferens divided into anterior, median and posterior regions depart. Sylviocarcinus pictus and D. pagei revealed coenospermia, which differed from the families of freshwater crabs in Southeast Asia. The histochemical composition of the seminal fluid is similar in both Trichodactylidae, following the pattern described for most Brachyura. Through the copulation experiments and anatomical results, these animals do not show a sperm plug, but rather form different sperm packs when engaging in multiple copulations with different males.

Abstract Our aim was to describe the reproductive system of males and the formation of sperm packages in the seminal receptacle (SR) of recently mated females of the arrow crab Stenorhynchus seticornis. The male reproductive system was analyzed, and was described using light microscopy and histological and histochemical methods. The first pair of gonopods was described by means of scanning electron microscopy. Additionally, the dehiscence of spermatophores was tested using samples obtained from the vas deferens of males and from the seminal receptacle of recently mated females. Testes were tubular type, and each vas deferens consisted of three regions: the anterior vas deferens (AVD), including a proximal portion that was filled with free spermatozoa and a distal portion contained developing spermatophores; the median vas deferens (MVD) that contained completely formed spermatophores; and the posterior vas deferens (PVD), which contained only granular secretions. The accessory gland, which was filled with secretions, was located in the transition region between the MVD and the PVD. The spermatophores from the MVD were of different sizes, and none of them showed dehiscence in seawater, whereas those spermatophores in contact with the seminal receptacle were immediately broken. The ultrastructure of the gonopods revealed the presence of denticles at the distal portion, which contribute to the mechanical rupture of the spermatophore wall during the transfer of sperm. The contents of the PVD and accessory gland of males are transferred together with the spermatophores, and are responsible for the secretions observed among the sperm packets in the SR of the female. We suggest that these secretions formed the layers found in the SR of recently mated females, and may play a role in sperm competition in arrow crabs.

BackgroundVolvox carteri f. nagariensis is a model taxon that has been studied extensively at the cellular and molecular level. The most distinctive morphological attribute of V. carteri f. nagariensis within V. carteri is the production of sexual male spheroids with only a 1:1 ratio of somatic cells to sperm packets or androgonidia (sperm packet initials). However, the morphology of male spheroids of V. carteri f. nagariensis has been examined only in Japanese strains. In addition, V. carteri f. nagariensis has heterothallic sexuality; male and female sexes are determined by the sex-determining chromosomal region or mating-type locus composed of a > 1 Mbp linear chromosome. Fifteen sex-specific genes and many sex-based divergent shared genes (gametologs) are present within this region. Thus far, such genes have not been identified in natural populations of this species.ResultsDuring a recent fieldwork in Taiwan, we encountered natural populations of V. carteri that had not previously been recorded from Taiwan. In total, 33 strains of this species were established from water samples collected in Northern Taiwan. Based on sequences of the internal transcribed spacer 2 region of nuclear ribosomal DNA and the presence of asexual spheroids with up to 16 gonidia, the species was clearly identified as V. carteri f. nagariensis. However, the sexual male spheroids of the Taiwanese strains generally exhibited a 1:1 to > 50:1 ratio of somatic cells to androgonidia. We also investigated the presence or absence of several sex-specific genes and the sex-based divergent genes MAT3m, MAT3f and LEU1Sm. We did not identify recombination or deletion of such genes between the male and female mating-type locus haplotypes in 32 of the 33 strains. In one putative female strain, the female-specific gene HMG1f was not amplified by genomic polymerase chain reaction. When sexually induced, apparently normal female sexual spheroids developed in this strain.ConclusionsMale spheroids are actually variable within V. carteri f. nagariensis. Therefore, the minimum ratio of somatic cells to androgonidia in male spheroids and the maximum number of gonidia in asexual spheroids may be diagnostic for V. carteri f. nagariensis. HMG1f may not be directly related to the formation of female spheroids in this taxon.

Sperm storage is common in internally fertilizing animals, but is also present in several external fertilizers, such as many cephalopods. Cephalopod males attach sperm packets (spermatangia) to female conspecifics during mating. Females of eight externally fertilizing families comprising 25% of cephalopod biodiversity have sperm-storage organs (seminal receptacles) in their buccal area, which are not in direct physical contact with the deposited spermatangia. The mechanism of sperm transmission between the implantation site and the storage organ has remained a major mystery in cephalopod reproductive biology. Here, jumbo squid females covering almost the entire life cycle, from immature to a laboratory spawned female, were used to describe the internal structure of the seminal receptacles and the process of sperm storage. Seminal fluid was present between the spermatangia and seminal receptacles, but absent in regions devoid of seminal receptacles. The sperm cellular component was formed by spermatozoa and round cells. Although spermatozoa were tracked over the buccal membrane of the females to the inner chambers of the seminal receptacles, round cells were not found inside the seminal receptacles, suggesting that spermatozoa are not sucked up by the muscular action of the seminal receptacles. This finding supports the hypothesis that spermatozoa are able to actively migrate over the female skin. Although further experimental support is needed to fully confirm this hypothesis, our findings shed light on the elusive process of sperm storage in many cephalopods, a process that is fundamental for understanding sexual selection in the sea.

Abstract We examined the female reproductive system of the yellowline arrow crab Stenorhynchus seticornis by means of histological and histochemical techniques. Mature specimens obtained in the field were kept in the laboratory for mating experiments. After 24 h, newly mated females were dissected, and their reproductive trait routinely processed for embedding in historesin. The specimens examined each possessed a pair of kidney‐shaped seminal receptacles (SR), and these we classified as ventral type, based on the location of the oviduct opening. The mesodermal dorsal region (DR) of SR consisted of a stratified epithelium with scaly cells, while the ectodermal ventral region (VR) was composed of a simple epithelium covered by a cuticle. The oviduct opened at the transition region (TR) between DR and VR and had no velum. The simple epithelium of TR had more folds on the face of the oviduct opening. The vagina exhibited the same features as the TR epithelium and was contiguous to VR, anchored by muscles. In the lumen, from one to three strata of sperm packets were observed, the dorsal one containing free sperm, and the most ventral stratum, spermatophores. An acidophilic glycoprotein layer enclosed the strata. Spermatophores in the ventral stratum were enclosed in a voluminous secretion, composed by acid polysaccharides most likely from the last male mated. The ventral‐type receptacle, stratified sperm packets, and polyandry, usually observed in females of Majoidea, suggest the occurrence of sperm competition in S. seticornis, favoring the sperm of the last male mated, as its sperm mass is located near the opening of the female oviduct.

:Volvox capensis was recorded previously only from South Africa. Here we established culture strains of this species from a sample collected in Montana, USA. Morphological details of asexual and sexual spheroids and molecular phylogeny of these strains were studied. The present alga was identified as V. capensis on the basis of morphological characteristics of asexual spheroids and zygotes. However, differences between the Montana and South African materials were recognized in number of sperm packets in a sexual, monoecious spheroid as well as in mode of gametic union between sperm and eggs. Possible polyspermy was observed in eggs of V. capensis by 4′-6-diamidino-2-phenylidole staining. Genetic difference between these two entities was small based on sequences of internal transcribed spacer 2 region of nuclear ribosomal DNA.

Lifetime spermatophore (sperm packet) production, which can provide valuable information about male energetics and mating, is not known for any species of copepod. Spermatophore production could limit reproduction in species in which females require multiple matings to remain fertilized and in populations that often have adult sex ratios highly skewed toward females. Spermatophore production rates were measured in the laboratory for two species of calanoid copepods, Acartia tonsa and Acartia hudsonica, from time of maturation until death, and under three food regimes (high, low, and no food). Rates of spermatophore production in A. tonsa were independent of food treatment. By contrast, in A. hudsonica, spermatophore production was significantly greater under high food than in low or no food. Spermatophore production decreased significantly with age in both species and the majority of males ceased producing spermatophores halfway through their adult lives. This pattern occurred regardless of food treatment, except for in A. hudsonica under low-food conditions: low production rates in younger males were compensated by continued reproduction into old age. Therefore, there was no difference in the total lifetime number of spermatophores produced by males of A. hudsonica fed at high- or low-food conditions. In the absence of food, however, significantly fewer spermatophores were produced. In A. tonsa, there was no difference among food treatments in the total number of spermatophores produced over a male’s life. The effect of these low rates of spermatophore production on fertilization was evident in the field. During 2 years of weekly collections, at no point in time were all females in the population fertilized. We conclude that low rates of spermatophore production over a lifetime and the short reproductive period of males contribute to the low frequencies of mated females in field populations.