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The elimination of the spermatid cytoplasm during spermiogenesis enables the sperm to acquire a streamlined architecture, which allows for unhindered swimming. While this process has been well described in vertebrates, it has rarely been reported in invertebrates. In this study, we observed the process of cytoplasm elimination during spermiogenesis in Octopus tankahkeei (Mollusca, Cephalopoda) using light microscopy, transmission electron microscopy, and immunofluorescence. In the early spermatid, the cell is circular, and the nucleus is centrally located. With spermatid development, the cell becomes polarized. The nucleus gradually elongates and moves toward the end of the cell where the tail is forming. As a result, the cytoplasm moves past the nucleus at the anterior region of the future sperm head (the foreside of the acrosome). Following this, during the late stage of spermiogenesis, the cytoplasm condenses and collects on the foreside of the acrosome until finally the residual body is discarded from the top of the sperm head. This represents a distinct directionality for the development of cytoplasmic polarity and discarding of residual body compared with that reported for vertebrates (in which the cytoplasm of the elongating spermatids is polarized toward the caudal region). The fact that the cytoplasm also becomes concentrated suggests that water pumps may be involved in the elimination of water from the cytoplasm before the residual body is discarded. Furthermore, we found that microtubules, forming a manchette-like structure, are involved not only in reshaping of the nucleus but also in the transport of mitochondria and vesicles to the foreside of the acrosome, subsequently allowing them to be discarded with the residual body. This study broadens our understanding of the development of polarization and elimination of cytoplasm from spermatids in animals.

Animals with superior feed efficiency (FE) may also have an improved nitrogen use efficiency (NUE), which would be beneficial to economic profitability while reducing environmental impacts. When genetically selecting animals on FE, it is preferable to use residual traits [e.g. residual feed intake (RFI) or residual body gain (RG)] rather than ratios because of their predictable genetic outcomes. We studied the relationship of RFI and RG with NUE, estimated from the validated 15N abundance in plasma, across two contrasted diets based on corn or grass silages. We evaluated FE of 588 (half by diet) Charolais bulls (545 ± 57 kg BW) from 12 experimental cohorts (different farms and periods) over 200 days. Before the end of the FE test, plasma was sampled and analyzed for δ 15N. NUE was related to FE through simple-linear models with variables previously corrected for the cohort and diet effects. The models’ slopes were standardized according to FE deviation in order to compare the response of NUE to FE between indices. Higher NUE was related to higher FE (P < 0.001), showing positive correlations with RG (r=-0.40) and negative with RFI (r=0.29). However, the standardized slope of NUE to RG was significantly higher (+28%; P < 0.05) than that of NUE to RFI. This stronger NUE relation to RG compared to RFI could reflect a higher potential of RG animals to deposit N as compared to a more conservational N metabolism in RFI individuals. Regarding diets, and despite the correction of NUE and FE for this effect, the slopes of NUE to FE were numerically (P > 0.05) higher (-16% and +36%; for RG and RFI) in corn-based diets, which agrees with superior NUE observed in corn-vs-grass diets. Results suggested that superior RG animals may present proportionally higher NUE than superior RFI animals, with even better results in corn-vs-grass diets.

Spermatids eliminate excess cytoplasm to form streamlined sperm during spermiogenesis, which mechanism is insufficiently elucidated in fish. In this study, we investigated the cytoplasmic elimination procedure in spermatid during spermiogenesis in the large yellow croaker (Larimichthys crocea) using transmission electron microscopy. The early spermatid is subrotund with a centrally located nucleus. With further development, nucleus polarizes into one side of the cell while the cytoplasm with numerous vesicles near the membrane migrates to the caudal region. Furthermore, exocytosis-like structures were detected in middle spermatid. In late spermatid, the vesicles are reduced and rarely observed. These findings indicate that vesicles may be involved in cytoplasmic elimination possibly via exocytosis. In the later spermatid, a double-membrane, autophagosome-like structure envelopes the cytoplasm, which may develop into a single-membrane structure, and gets discarded from the cell as a residual body from the caudal region. This suggests its potential functions in the formation of residual body and cytoplasmic elimination. Overall, our results revealed that polarized development of spermatid causes polarized distribution of cytoplasm necessary for cytoplasmic elimination. Moreover, they provide ultrastructure evidence for vesicles and double-membrane structures involved in discarding spermatid cytoplasm in large yellow croaker, thus offering novel insights into cytoplasmic elimination during spermiogenesis in fish.

As the tachyzoite form of Toxoplasma gondii divides inside the parasitophorous vacuole, the daughter cells remain attached to each other at the posterior end through the so-called residual body (RB). Here, we studied this process using field emission scanning electron microscopy of dry scraped infected cells, transmission electron microscopy of random ultrathin sections, X-ray microanalysis, and 3-D modelling of tomographic volumes and slice and view series obtained by FIB SEM at 7, 24, and 48 h post infection. Combining these methods of observation, we traced a timeline of events for the formation, development, and fate of the RB. The RB is formed as the first endodyogenic division is complete. Before that, finger-like invaginations at the posterior end of the tachyzoite secrete tubules from the intravacuolar network. The RB is roughly spherical and measures 1 μm in diameter at random. Its size does not vary considerably as the division cycles that form the rosette proceed. The contents of the RB are similar to the cytoplasm of the parasites. It contains ER membranous profiles and vacuolar structures identified as acidocalcisomes. This was confirmed by microanalysis. Mitochondrial profiles seen inside the RB are actually branches of mother cell mitochondrion not yet split between the two daughter cells. Acidocalcisomes of a mother cell are distributed between the two daughter cells, but as the rosette of parasites grow, acidocalcisomes seem to concentrate inside the RB where they are usually larger and tend to fuse to each other, filling most of the space in the RB. Here we hypothesize that, upon egress, the acidocalcisomes would ultimately fuse with the RB membrane liberating its contents inside the parasitophorous vacuole (PV) and, by doing so; the RB would disintegrate, releasing its contents in the PV.

Heavy metals are known to cause deleterious effects on biota because of their toxicity, persistence and bioaccumulation. Here, we briefly document the ultrastructural changes observed in the miliolid foraminifer Pseudotriloculina rotunda (d'Orbigny in Schlumberger, 1893) and in the perforate calcareous species Ammonia parkinsoniana (d'Orbigny, 1839) induced by exposure to one of three heavy metals (zinc, lead, or mercury). The exposure of these two benthic foraminiferal species to the selected heavy metals appears to promote cytological alterations and organelle degeneration. These alterations include a thickening of the inner organic lining, an increase in number and size of lipid droplets, mitochondrial degeneration, and degradation vacuoles and residual body proliferation. Some of these alterations, including the thickening of the inner organic lining and the proliferation of lipids, might represent defense mechanisms against heavy metal-induced stress.

The nematode spermatozoa represent a highly modified (aberrant) type of male gametes that lack a flagellum but for which the process of spermatogenesis culminates in the production of a crawling spermatozoon on the basis of the cytoskeletal component known as "major sperm protein", or MSP. MSP is also known as an important hormone triggering oocyte maturation and ovulation in the model nematode Caenorhabditis elegans, where this protein was first identified. However, direct evidence of MSP localization and of its fate in nematode spermatogenic cells is rare. In this study, the spermatogenesis and sperm structure in the rhabditid nematode Acrobeles complexus (Rhabditida: Tylenchina: Cephalobomorpha: Cephaloboidea: Cephalobidae) has been examined with electron microscopy. Morphological observations were followed by high-pressure freezing and freeze-substitution fixation which allows post-embedding immunogold localization of MSP in all stages of sperm development using antibodies raised for MSP of C. elegans. In spermatocytes, synthetic activity results in the development of specific cellular components, fibrous bodies (FB) and membranous organelles (MO), which appear as FB-MO complexes where the filamentous matter of FB has been MSP-labeled. The spermatids subdivide into a residual body with superfluous cytoplasm, and a main cell body which contains nucleus, mitochondria and FB-MO complexes. These complexes dissociate into individual components, MO and FB, with the MSP being localized in FB. Immature spermatozoa from testes are opaque cells where a centrally located nucleus is surrounded by mitochondria, MO and FB clustered together, the MSP still being localized only in FB. Cytoplasm of mature spermatozoa from spermatheca is segregated into external pseudopods lacking organelles and a central cluster of mitochondria with intact MO surrounding the central nucleus. The FB ultimately disappear, and the MSP labeling becomes concentrated in the filamentous content of pseudopods and cytoplasm of the main cell body. Although the spermatogenesis and sperm structure of A. complexus is similar to that of many other rhabditid nematodes, their intact MO makes the morphology of the mature spermatozoa distinct from the "rhabditid pattern" and may be considered as a synapomorphy. The MSP localization in spermatogenic cells of A. complexus also follows the "rhabditid pattern" described in C. elegans and Ascaris spp. Our results and techniques of MSP labeling of A. complexus spermatogeneous cells reveal new possibilities to elucidate different research questions on MSP localization in nematodes related to C. elegans. Furthermore, the laboratory-cultured A. complexus strains can be used as a new and fascinating model to study MO and MSP functions in nematode reproduction.

Toxoplasma gondii contains a limited subset of actin binding proteins. Here we show that ablation of the putative actin regulator cyclase-associated protein (TgCAP) leads to significant defects in some but not all actin dependent processes, including a defect in cell-cell communication, but surprisingly not synchronicity of division. Two CAP isoforms originate from alternative translational start sites and are beneficial for parasite fitness while a single isoform is sufficient for virulence in mice. Examination of the mutant parasites by 3D electron microscopy reveals that loss of CAP results in a defect to form a normal residual body, but all parasites remain connected within the vacuole. This dissociates synchronicity of division and parasite rosetting and reveals that establishment and maintenance of the residual body may be more complex than previously thought. These results highlight the different spatial requirements for actin turnover in Toxoplasma, controlled by a reduced subset of actin binding proteins.

Cyclospora cayetanensis is a coccidian parasite of humans of known and growing importance. However, we are surprisingly naïve as to our understanding of how to diagnose it and how it develops inside the human body. Here we provide details of the developmental stages of C. cayetanensis in the gallbladder of a 33-yr-old male with human immunodeficiency virus. The gallbladder was removed surgically in 2001 because of severe abdominal pain. For the present study, the archived paraffin block of gallbladder was processed for light microscopy and transmission electron microscopy (TEM). Histological sections were examined after staining with hematoxylin and eosin (HE) or using the periodic acid Schiff (PAS) reaction. Immature and mature asexual stages, gamonts, and oocysts were seen in epithelial cells, both in the superficial epithelium and in glands. The merozoites were present singly, in pairs, and 3 or more in a single parasitophorous vacuole in the host cytoplasm. Up to 6 nuclei were seen in immature schizonts without evidence of merozoite formation. Mature schizonts were 7.6 × 5.1 μm and contained up to 10, 3-4 μm long merozoites. Merozoites were 0.6 to 2.0 μm wide, and their shape varied from pear-shaped to slender. Merozoites were generally PAS-positive; however, some were intensely positive, some had only minute granules, while others were PAS-negative. The microgamonts (male) were 6.6 × 5.2 μm and contained fewer than 20 microgametes around a residual body. The microgametes were up to 2 μm long and were flagellated. Macrogamonts (female) contained distinctive eosinophilic wall-forming bodies that varied in size and were less than 1 μm in HE-stained sections. Macrogamonts were 5.8-6.5 × 5.3-6.5 μm. Oocysts in sections were unsporulated and had a diameter of 5.7-7.5 μm. The TEM examination confirmed the histologic findings. The DNA extracted from paraffin sections was confirmed as C. cayetanensis with real-time PCR. The detailed description of the life cycle stages of C. cayetanensis reported here in an immunosuppressed patient could facilitate histopathologic diagnosis of this parasite. We have shown that the parasite's development more closely resembles that of Cystoisospora than Eimeria and that the parasite has multiple nuclei per immature meront indicating schizogony, and we have undermined evidence for a Type II meront.

Spermatogenesis and sperm ultrastructure were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) in the longidorid Xiphinema theresiae. All germ cell stages, except spermatogonia, are present in the testes of young adult males. The nonflagellated, slightly elongated sperm displays little intraspecific variation and, although never polarized into a head and tail region, has a remarkably precise form, with a high degree of internal organization. Incipient fingerlike pseudopodia appear in the young spermatid and increase to such an extent that the adult sperm has a conspicuous "woolly" appearance. Microfilament bundles encircle the perinuclear mitochondria in the spermatid, and seem to be closely associated with the evaginated plasma membrane, especially in the spermatozoon. A large nucleus with nuclear envelope is prominent in the spermatocyte, but the envelope is absent in the young spermatid. Mitochondria are present in all germ cell stages and undergo certain morphological changes (e.g., in size and number, presence or absence of cristae), as well as changes in intracellular movements during spermatogenesis. Membranous organelles are prominent in the spermatocyte, but disappear in the older spermatid. Annulate lamellae and a residual body (i.e., cytophore) are conspicuous in the spermatocyte and spermatid, respectively; the spermatozoon clearly lacks a refringent body (i.e., acrosome).

This study aimed to evaluate and document the excystation process of Cryptosporidium muris oocysts in various incubation media, and to monitor the behaviour of excysting and freshly excysted sporozoites. A test of oocyst viability, using fluorescent double staining with fluorescein diacetate and propidium iodide, was performed prior to each experimental assay. Light microscope observations confirmed that relatively often only three sporozoites were released; the fourth one either left the oocyst later together with a residual body or remained trapped within the oocyst wall. These results suggest that successful oocyst excystation is not limited by the viability of all four sporozoites. Darkening of oocysts to opaque and their specific movement (the so-called "oocyst dancing") preceded the final excystation and liberation of sporozoites, while the dormant oocysts appeared refractive. The process of excystation in C. muris is not gradual as generally described in cryptosporidia but very rapid in an eruptive manner. Experiments were performed using oocysts stored at 4 °C for various time periods, as well as oocysts freshly shed from host rodents (Mastomys coucha) of different ages. The most suitable medium supporting high excystation rate (76 %) and prolonged motility of sporozoites was RPMI 1640, enriched with 5 % bovine serum albumin (BSA). Our results emphasize that to reliably evaluate the success of in vitro excystation of cryptosporidia, not only the number of released sporozoites in a set time period should be taken into consideration but also their subsequent activity (motility), as it is expected to be essential for the invasion of host cells.

Permethrin, a pyrethroid chemical, is widely used as a pesticide because of its rapid insecticidal activity. Although permethrin is considered to exert very low toxicity on mammalian reproductive organs, the effects of early low-level and chronic exposure on adult spermatogenesis are unclear. We exposed mice to environmentally relevant concentrations of permethrin (0, 0.3, and 30ppm) in drinking water during prenatal and postnatal period and examined the effects on the testis in mice offspring when they reached maturity (12 weeks of age). Using methyl green-pyronin staining, we found an abnormal accumulation of RNA in the seminiferous epithelia of mice treated with permethrin; this accumulation may be derived from an enlargement of the residual body. Additionally, permethrin may cause a decline of Sertoli cell functions. Our findings demonstrate that low-level and chronic permethrin exposure during prenatal and postnatal period has distinct effects on male reproductive system in mice.

The Egyptian skinks Mabuya quinquetaeniata were investigated for haemogregarines. The intracellular parasites were found in three developmental forms; trophozoite measured 4 µm; the intermediate form (a newly formed gamont) measured 14 µm and the elongated crescent-shaped one that measured 18 µm and considered as a mature gamont. Meronts were found only in the parenchyma cells of the liver of the infected skinks. The merogonic process began when the free parasites invaded the parenchyma cells, then they became more rounded or oval and began to grow in size. Meanwhile, the parasitophorous vacuole appeared enclosing the parasite and grows in size during the growth of the meronts. The formation of merozoites occurred by ectomerogony. The areas of the surface, overlying the peripheral nuclei, soon become elevated. At a later stage, these elevations become more prominent forming a conical protruding merozoite anlage, which contains tubular structure at the apical pole indicating the formation of the conoid. Moreover, they form subpellicular microtubules, rhoptries, micronemes, mitochondria and also Golgi apparatus. The cytoplasm remaining, after the separation of the merozoites, constituted the residual body, which decreased with the progress of the merogonic phase. The present meronts were found in two forms; the first form (macromeront) measured 18×12 µm and produces about 43 micromerozoites, each measured 13 µm. The second one (micromeront) measured 9×7 µm and producing 11 macromerozoites, each measured 17 µm. On the ultrastructural level, each parasite has a pellicle, which consists of outer and inner membranes, conoid, rhoptries, few micronemes and some of dense bodies.

Two asexual generations were developed before the commencement of the sexual phase. First generation schizont containing 12-16 randomly scattered merozoites masured 19.5 x 16.3 um, supranuclear and ectomerogonic with residual body. Each merozoite being vermiform and measured 7.7 x 2.4 um. The merozoites formation was completed within 48 hours. post-infection (p.i). Meanwhile, mature second generation schizont measured 17.5 x 15 um. It conained 4-20 merozoites, each measured 9.5 x 2.8 um, and the process was completed within 84 hours p.i. The present study showed all changes in the ultrustructural forms associated with the development, starting from uninuclear schizont to multinuclear schizont. The process of merozoite formation, recorded in the present study, termed "ectomerogony" in which the merozoites developed at the periphery of each schizont. This method is common and recorded among many species of Eimeria. The electron microscope study revealed that the pellicle consists of two membranes, an outer thin unit membrane and an inner complex one usually formed of two membranes. During this stage, the merozoites of the first generation invaded new uninfected epithelial cells. The same events, recorded in the formation of the first generation schizonts and merozoites were carried out in the second generation, except that the second has specific arrangement of merozoites, and it was rich with endoplasmic reticulum. The outer pellicular unit membrane of merozoites was continous enclosing the whole merozoite without interruptions except an invagination at the site of micropore.