Traditional Histological Techniques Research Articles

Structure of the ligule in American species of Selaginella P. Beauv. (Selaginellaceae)

The ligule is an important character in the taxonomic delimitation of Selaginella species. Here, the morphology of this organ is described in 11 American species. Four of these species are xerophytic: Selaginella convoluta, S. microphylla, S. peruviana, and S. sellowii, and seven are mesophytic: S. anceps, S. diffusa, S. flagellata, S. flexuosa, S. mollis, S. silvestris, and S. sulcata. Morphological data were obtained by traditional histological techniques. Observations were made by light and scanning electron microscopy. Variations in ligule size in vegetative leaves and sporophylls were determined using a correlation analysis with Spearman's coefficient. The ligule was found to have five body shapes: campanulate, linguiform, elliptic, fan-shaped, and rectangular. Each shape can have a lobed (bi-, tri-, or multi-lobed), rounded, or truncate apex. Size, shape, and apex are characteristic of each species and remain constant in young and mature leaves. No significant differences were observed between xerophytic and mesophytic species. The ligule of all species studied is described for the first time. Micrographs of all the species studied are presented, and the morphological results of this study have been mapped into the recent phylogeny of the genus.

Histological changes of the skin during postembryonic development of the crested newt Triturus ivanbureschi (Urodela, Salamandridae)

BackgroundAmphibian skin has been studied for many decades, especially the metamorphic changes in the skin of frogs. Less attention has been paid to salamander skin. Here, we describe changes in the skin structure during postembryonic development in a salamandrid species, the Balkan crested newt Triturus ivanbureschi. MethodUsing traditional histological techniques we examined the skin in the trunk region of three premetamorphic larval stages (hatchling, mid larval and late larval) and two postmetamorphic stages (juvenile, just after metamorphosis, and adult). ResultsIn larval stages, skin consists only of the epidermis, which gradually develops from the single epithelial cell layer in hatchlings, to a stratified epidermis with gland nests and characteristic Leydig cells at the late larval stage. During metamorphosis, Leydig cells disappear, and the dermal layer develops. In postmetamorphic stages, skin is differentiated on stratified epidermis and the dermis with well-developed glands. Three types of glands were observed in the skin of the postmetamorphic stages: mucous, granular and mixed. Gland composition appears to be stage- and sex-specific, with juveniles and adult female being more similar to each other. In juveniles and adult female, there are a similar proportion of glands in both dorsal and ventral skin, whereas in adult male granular glands dominated the dorsal skin, while mixed glands dominated the ventral skin. ConclusionOur results provide a baseline for future comparative research of skin anatomy in salamanders.

Enhanced contrast synchrotron X-ray microtomography for describing skeleton-associated soft tissue defects in zebrafish mutants.

Detailed histological analyses are desirable for zebrafish mutants that are models for human skeletal diseases, but traditional histological techniques are limited to two-dimensional thin sections with orientations highly dependent on careful sample preparation. On the other hand, techniques that provide three-dimensional (3D) datasets including µCT scanning are typically limited to visualizing the bony skeleton and lack histological resolution. We combined diffusible iodine-based contrast enhancement (DICE) and propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SRµCT) to image late larval and juvenile zebrafish, obtaining high-quality 3D virtual histology datasets of the mineralized skeleton and surrounding soft tissues. To demonstrate this technique, we used virtual histological thin sections and 3D segmentation to qualitatively and quantitatively compare wild-type zebrafish and nkx3.2 -/- mutants to characterize novel soft-tissue phenotypes in the muscles and tendons of the jaw and ligaments of the Weberian apparatus, as well as the sinus perilymphaticus associated with the inner ear. We could observe disrupted fiber organization and tendons of the adductor mandibulae and protractor hyoideus muscles associated with the jaws, and show that despite this, the overall muscle volumes appeared unaffected. Ligaments associated with the malformed Weberian ossicles were mostly absent in nkx3.2 -/- mutants, and the sinus perilymphaticus was severely constricted or absent as a result of the fused exoccipital and basioccipital elements. These soft-tissue phenotypes have implications for the physiology of nkx3.2 -/- zebrafish, and demonstrate the promise of DICE-PPC-SRµCT for histopathological investigations of bone-associated soft tissues in small-fish skeletal disease models and developmental studies more broadly.

Matrix Metalloproteinase 9 Expression by Immunohistochemistry in Intratumor Macrophages as Tumor-Associated Macrophage Marker of Papillary Thyroid Carcinoma

Papillary thyroid carcinoma (PTC) comprises 80% of well-differentiated thyroid carcinomas. PTC progression is determined by a variety of biological markers, some of which are connected to the activities of Tumor-Associated Macrophage (TAM). TAM activity is difficult to observe using simply traditional histology techniques. Matrix Metalloproteinase 9 (MMP-9) is an important marker for identifying the development of PTC. However, no studies have linked the expression of MMP-9 to intratumoral macrophages in PTC patients, demonstrating that these macrophages are TAMs implicated in the development of PTC. Through MMP-9 expression on intratumor macrophages, the objective of this work was to evaluate TAM's involvement as a progression determinant of PTC. This cross-sectional study analyzed 40 samples, which included 21 PTC patients with intratumor macrophages and 19 PTC instances without intratumor macrophages. Medical records and paraffin blocks of the Biomedical Laboratory, Faculty of Medicine and Healthcare, Warmadewa University were used to collect samples. The proportion of MMP-9 expression in both groups' macrophages was then determined by immunohistochemical labeling and evaluated using Chi-Square with a significance level (a) of p<0.05. The results revealed a statistically significant difference in MMP-9 expression between the PTC groups with and without intratumor macrophages, with p-value = 0.001 (p<0.05) and OR = 11.9.

Investigating Interactions Between Endothelial Cells and Parasitized Red Blood Cells in Skin and Subcutaneous Tissue.

In this chapter we present the methods for using biopsies of skin or subcutaneous tissue to examine the interactions between parasitized red blood cells and endothelial cells in patients with malaria infection. Punch biopsy can be used to obtain all skin layers and needle biopsy to obtain subcutaneous tissue. Smears are useful for spreading vessels on a slide for immunofluorescence staining. Specimens can be fixed and embedded for sectioning and traditional histological or immunostaining techniques or confocal microscopy with three-dimensional reconstruction. Finally, endothelium can be dissociated, allowing individual cells to be isolated for culture and ex vivo assays or used for immunophenotyping.

State-of-Art in the Age Determination of Venous Thromboembolism: A Systematic Review.

Venous thromboembolism (VTE), consisting of deep vein thrombosis (DVT) and pulmonary embolism (PE), requires a forensic age determination to ascertain their causal relationship with recent events, such as trauma or medical treatment. The main objective of this systematic review is to identify the current state-of-the-art immunohistochemical methods for age determination of fatal VTE. A literature search was performed through different databases, according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Within the study, we have selected only cases represented by deceased patients for DVT and/or PTE in which thromboembolic material was collected during an autoptic examination and then subjected to a histological and an immunohistochemical investigation. Studies based on animal models were not included. We assessed bias risk. A database-based search produced a total of 19 articles. After excluding duplicate items from the selection, 14 articles were reviewed. Ten articles were excluded because they did not meet the inclusion criteria. The results have pointed out 4 studies that were included in the present analysis for a total of 157 samples of DVT and 171 PTE samples. These were analyzed using traditional histological and immunohistochemical techniques. The results must be interpreted with a critical eye because of their heterogeneity in terms of time, geography, and study design. The present review highlights the importance of associating specific immunohistochemical markers with a histological analysis for the timing of DVT/PTE fatal events. Further future experiences will hopefully endorse actual knowledge on the subject to increase the accuracy in the assessment of thrombus-embolus age.

Generating aptamers towards human sperm cells using massively parallel sequencing

Determining the presence of sperm cells on an item or swab is often a crucial component of sexual offence investigation. However, traditional histological staining techniques used for the morphological identification of spermatozoa lack both specificity and sensitivity, making analysis a complex and time-consuming process. New methods for the detection of sperm cells based on aptamer recognition may be able to overcome these issues. In this work, we present the selection of ssDNA aptamers against human sperm cells using Cell-SELEX and massively parallel sequencing technologies. A total of 14 rounds of selection were performed following a modified Cell-SELEX protocol, which included additional steps for the isolation of spermatozoa from seminal fluid. Massively parallel sequencing using the Illumina Miseq platform was conducted on enriched aptamer pools to elucidate the structure of potential binders. A custom bioinformatics pipeline was also developed using Galaxy for the automated processing of sequencing datasets. This data revealed several promising aptamer candidates, which were shown to selectively bind sperm cells through both microscale thermophoresis and enzyme-linked oligonucleotide assays. These aptamers have the potential to increase the efficiency of sexual offence casework by facilitating sperm detection.Graphical abstract

Reproduction of the Androgenetic Population of the Asian Corbicula Clam (Bivalvia: Cyrenidae) in the Northern Dvina River Basin, Russia

The Corbicula clam is one of the most successful invaders of aquatic ecosystems and has invaded all continents except Antarctica. The natural dispersion of Corbicula seems to be limited by low winter temperatures that fall below the lower lethal temperatures (0 to +2 °C). However, Corbicula can be found in colder regions, taking refuge in waters heated by thermal power plants. The purpose of this investigation was to study the gonadal histology, reproductive cycle, and the seasonal changes of shell size structure of the Corbicula clam populations in the warm water discharge of the Arkhangelsk thermal power plant (Northwest Russia). Samples were collected monthly from January 2017 to December 2018 and processed using traditional histological and morphological techniques. The number of reproductive periods varied from year to year. It was established that the Corbicula clam has a continuous reproduction period which may be adaptive in unstable environmental conditions. This reproductive strategy is probably aimed at increasing the reproductive success of the population. Our data expand the understanding of reproductive features of the Corbicula clam in harsh environmental conditions. These results could be applied to control, monitoring, and management measures.

A combined morphometric approach to feature mouse kidney vasculature

Physiological kidney function is closely related to the state of the vascular network. Disorders, such as capillary rarefaction, predispose to chronic kidney disease (CKD). In this context, deepening of the methodologies for studying the renal vascular network can be of basic importance. To meet this need, numerous animal models and, in parallel, several methods have been developed. In this work we propose a protocol to accurately feature kidney vasculature in mouse, however, the same protocol is suitable to be applied also to other animal models. The approach is multiparametric and mainly based on micro-computed tomography (μCT) technique. Micro-ct allows to study in detail the vascular network of any organ by exploiting the possibility to perfuse the sample with a contrast agent. The proposed protocol provides a fast and reliable method to extract quantitative information from the μCT scan by using only the basic functions of the software supplied by the scanner without any additional analysis. Through iterative cropping of the scanned ROI and calculation of a sample-specific threshold we calculated that the average volume of a female BALB/c kidney of eighth weeks is 147.8 mm3 (5.4%). We also pointed out that the average volume of the vascular network is 4.9% (0.3%). In parallel we performed traditional histological and immunofluorescence techniques to integrate the information gained via μCT and to frame them in the tissue context. Vessel count on histological sections showed a different density in the different regions of the organ parenchyma, in detail, vessel density in the cortex was 19.03 ± 2.51 vessels/ROI while in the medulla it was 10.6 ± 1.7 vessels/ROI and 5.4 ± 1.3 vessels/ROI in the outer and inner medulla, respectively. We then studied vessel distribution in the renal parenchyma which showed that the 55% of vascular component is included in the cortex, the 30% in the outer medulla and the 15% in the inner medulla.Collectively, we propose an integrated approach that can be particularly useful in the preclinical setting to characterize the vasculature of any organ accurately and rapidly.

General and fine structure of Aeolidia papillosa cnidosacs (Gastropoda: Nudibranchia).

Nudibranch mollusks (Gastropoda: Heterobranchia) are widely known for their ability to incorporate some active biochemical compounds of their prey, or even organelles and symbionts of the prey, which assured biological success of this group. At the same time, the process of nematocysts obtaining and incorporation into specific structures called cnidosacs by cladobranch mollusks remain poorly studied. This highlights a necessity of additional ultrastructural studies of cnidosac and adjacent organs in various aeolid mollusks using modern microscopic methods as they may provide new insight into the cnidosac diversity and fine-scale dynamics of nematocysts sequestration process. The present study is focused on the general and fine structure of the cnidosac area in cladobranch Aeolidia papillosa (Aeolidiidae). Specific goals of our study were to provide a detailed description of histological and ultrafine structure of epidermis, upper parts of the digestive glands and the cnidosac, its innervation and proliferation using standard histological techniques, confocal laser scanning microscopy (CLSM) and transmission electron microscopy. Our results clearly demonstrated that A. papillosa cnidosac is a much more complex structure, than it was thought, especially compared with simple cnidosacs found in flabellinids and facelinids. Using CLSM for functional morphological analysis provides a better resolution in visualization of structural elements within a cnidosac compared with traditional histological techniques. We revealed the presence of two cell types in the cnidophage zone: cnidophages and interstitial cells, which differ in ultrastructure and function. Our results also document the presence of a specific cnidopore zone, lined with differentiated cuboid epithelium bearing long microvilli, which likely provides a unidirectional flow of nematocysts during kleptocnides extrusion. For the first time, occurrence of vacuoles containing protective chitinous spindles in the cnidosac epithelium was shown.

Phenotyping Intact Mouse Bones Using Bone CLARITY.

The bone is typically studied using traditional histology techniques, that is, serial sectioning and staining. While effective, these techniques are laborious and destructive, as the native 3D environment of the bone is not maintained. Presented here is a bone-clearing methodology, termed Bone CLARITY, which combines published techniques for clearing soft tissues, including delipidation for the removal of light-scattering membranes, hydrogel-embedding for the stabilization of fragile epitopes, heme elution for the reduction of blood-based autofluorescence; as well as specialized steps, including decalcification and progressive refractive index matching, for addressing the unique challenges posed by osseous tissue. This method renders the bone transparent and enables the detailed visualization of an intact tissue specimen at multiple spatial scales.

Informatively of histological study by method of mapping ellipticity of polarization of microscopic myocardial images

Purpose of the study. To investigate the informational value of the histological examination of the human myocardium in case of sudden cardiac death and the possibility of a method of mapping ellipticity of polarization of microscopic images of the myocardium in the diagnosis and objective differentiation of acute coronary insufficiency (ACI) and chronic coronary heart disease (CIHD).
 Materials and methods. Samples of human myocardium from 89 people who died as a result of ACI and CIHD were investigated, using traditional histological and laser polarization techniques, with a standard-scheme laser polarimeter. Subsequently, statistical processing of the obtained results was conducted.
 Results. The results obtained show that the values of the statistical moments of the 3rd and 4th order, which characterize the coordinate distributions of the ellipticity of polarization of microscopic images of myocardial samples on the scale of optical magnification ×56, were the most informative for differential diagnosis of ACI and CIHD. Accuracy of ACI and CIHD detection by mapping the ellipticity of polarization of myocardial microscopic images at an optical magnification of ×56 reaches ~ 97%, as opposed to the traditional histological examination ~ 81%.
 Conclusion. On the basis of the obtained data, we confirm the diagnostic efficiency and accuracy of the method of mapping the values of ellipticity polarization of microscopic images of histological sections of the myocardium in the problem of diagnosis and objective differentiation of ACI and CIHD.

In vivo measurement of enhanced agouti-related peptide release in the paraventricular nucleus of the hypothalamus through Gs activation of agouti-related peptide neurons.

Agouti-related peptide (AgRP) neurons of the hypothalamus play a role in hunger-triggered food intake, stability of body weight, and long-term energy balance. A recent study showed that activation of the Gs-linked G protein-coupled receptors (GCPR) expressed by hypothalamic AgRP neurons promotes a sustained increase in food intake. Enhanced AgRP release has been the postulated underlying mechanism. Here, we confirmed that activation of Gs-coupled receptors expressed by AgRP neurons in the arcuate nucleus (ARC) of the hypothalamus, which is the primary brain region for the synthesis and release of AgRP, leads to increased release of AgRP in the paraventricular nucleus of the hypothalamus (PVN). We were unable to confirm changes in AgRP expression or intracellular content using traditional histological techniques. Thus, we developed an assay to measure AgRP in the extracellular fluid in the brain using large molecular weight cut-off microdialysis probes. Our technique enables assessment of brain AgRP pharmacokinetics under physiological conditions and in response to specific pharmacological interventions designed to modulate AgRP signaling.

Imaging Peripheral Nerve Regeneration: A New Technique for 3D Visualization of Axonal Behavior

BackgroundPeripheral nerve assessment has traditionally been studied through histological and immunological staining techniques in a limited cross-sectional modality, making detailed analysis difficult. A new application of serial section electron microscopy is presented to overcome these limitations. MethodsDirect nerve repairs were performed on the posterior auricular nerve of transgenic YFP-H mice. Six weeks postoperatively the nerves were imaged using confocal fluorescent microscopy then excised and embedded in resin. Resin blocks were sequentially sectioned at 100 nm, and sections were serially imaged with an electron microscope. Images were aligned and autosegmented to allow for 3D reconstruction. ResultsBasic morphometry and axonal counts were fully automated. Using full 3D reconstructions, the relationships between the axons, the Nodes of Ranvier, and Schwann cells could be fully appreciated. Interactions of individual axons with their surrounding environment could be visualized and explored in a virtual three-dimensional space. ConclusionsSerial section electron microscopy allows the detailed pathway of the regenerating axon to be visualized in a 3D virtual space in comparison to isolated individual traditional histological techniques. Fully automated histo-morphometry can now give accurate axonal counts, provide information regarding the quality of nerve regeneration, and reveal the cell-to-cell interaction at a super-resolution scale. It is possible to fully visualize and “fly-through” the nerve to help understand the behavior of a regenerating axon within its environment. This technique provides future opportunities to evaluate the effect different treatment modalities have on the neuroregenerative potential and help us understand the impact different surgical techniques have when treating nerve injuries.

3-dimensional visualization of implant-tissue interface with the polyethylene glycol associated solvent system tissue clearing method.

ObjectivesDental implants are major treatment options for restoring teeth loss. Biological processes at the implant‐tissue interface are critical for implant osseointegration. Superior mechanical properties of the implant constitute a major challenge for traditional histological techniques. It is imperative to develop new technique to investigate the implant‐tissue interface.Materials and methodsOur laboratory developed the polyethylene glycol (PEG)‐associated solvent system (PEGASOS) tissue clearing method. By immersing samples into various chemical substances, bones and teeth could be turned to transparent with intact internal structures and endogenous fluorescence being preserved. We combined the PEGASOS tissue clearing method with transgenic mouse line and other labelling technique to investigate the angiogenesis and osteogenesis processes occurring at the implant‐bone interface.ResultsClearing treatment turned tissue highly transparent and implant could be directly visualized without sectioning. Implant, soft/hard tissues and fluorescent labels were simultaneously imaged in decalcified or non‐decalcified mouse mandible samples without disturbing their interfaces. Multi‐channel 3‐dimensional image stacks at high resolution were acquired and quantified. The processes of angiogenesis and osteogenesis surrounding titanium or stainless steel implants were investigated.ConclusionsBoth titanium and stainless steel implants support angiogenesis at comparable levels. Successful osseointegration and calcium precipitation occurred only surrounding titanium, but not stainless steel implants. PEGASOS tissue clearing method provides a novel approach for investigating the interface between implants and hard tissue.

The use of fluorescent staining techniques for microscopic investigation of polymorphonuclear leukocytes and bacteria.

The use of fluorescent stains to visually investigate eukaryotic and/or prokaryotic cells is increasing quickly and manuscripts within all areas of research publish results using fluorescent staining techniques. However, in contrast to literature on traditional histological staining techniques, the literature on fluorescent stains and staining techniques does not offer as good an illustration of cellular morphology. The aim of this guideline is to illustrate different fluorescent stains and staining techniques for imaging immune cells, in particular the polymorphonuclear leukocytes (PMNs), in combination with infecting bacteria as seen in chronic bacterial infections. Thereby providing the first guideline for the morphology and identification of fluorescently stained PMNs, bacteria and biofilm.

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing.

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

3D Histological Evaluation of Growth Plate Cartilage using Confocal Laser Scanning Microscopy

Traditional histological processing techniques can subject tissue to distortion and artifacts and provide a limited view of cell dynamics. An evolving histological technique is the use of optical microscopy to acquire high resolution image stacks to visualize tissue in‐depth. Using the confocal laser scanning microscope (CLSM), it is possible to collect an image stack to process into a 3‐D perspective. In bone, autofluorescence is largely due to the collagen component. We aimed to use the autofluorescent properties of the collagen in the growth plate of murine femurs to characterize and evaluate the resting, proliferating and hypertrophic zones of articular cartilage. Murine distal femurs were sectioned along their longitudinal axis. The bones were allowed to incubate in refractive index mating solution (RIMS) media for two days prior to imaging. Z stacks of approximately 80–100 μm in depth were captured using ACS APO 40x/1.15 oil on Leica SPE confocal microscope. Acquisition of the z‐stacks were obtained in the blue‐green and green‐yellow emission spectrum. Incubation of tissue in RIMS solution reduced the opacity of the tissue and facilitated in‐depth optical imaging. Imaging was focused on the growth plate (Fig 1). Sequential image stacks were acquired with confocal microscope and Leica LAS software algorithms were used to compose 3‐D images. Image stacks were further manipulated in ImageJ and ICY to isolate and count the chondrocytes, respectively. 3D histology can be accomplished with minimal processing cost and can provide an alternate perspective of cell dynamics. Intrinsic biomolecules in tissue possess endogenous fluorophores that can at times be advantageous when evaluating intact tissue architecture. Confocal microscopy in conjunction with a refractive index matching solution rendered the murine femurs less opaque and allowed for greater photon penetration and thus resolution of spatial‐temporal structures in the bone matrix. Examination of the matrix and morphology of the cells within, provided an indication of the development of the cells and progression of ossification.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Techniques for Investigating the Anatomy of the Ant Visual System.

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye anatomy of insects. These include traditional histological techniques optimized for work on ant eyes and adapted to work in concert with other techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These techniques, although vastly useful, can be difficult for the novice microscopist, so great emphasis has been placed in this article on troubleshooting and optimization for different specimens. We provide information on imaging techniques for the entire specimen (photo-microscopy and SEM) and discuss their advantages and disadvantages. We highlight the technique used in determining lens diameters for the entire eye and discuss new techniques for improvement. Lastly, we discuss techniques involved in preparing samples for LM and TEM, sectioning, staining, and imaging these samples. We discuss the hurdles that one might come across when preparing samples and how best to navigate around them.

Gonadal cycle of Corbicula fluminea (Bivalvia: Corbiculidae) in Pampean streams (Southern Neotropical Region).

Corbicula fluminea is an aggressive invasive species of bivalve that arrived into the Río de la Plata River between the late 60’s and early 70’s, and dispersed widely throughout the Neotropical region, evidencing a great adaptive flexibility to different environmental conditions. This species is a functional hermaphrodite with larval incubation inside the inner demibranch. Despite its widespread distribution, there are no previous studies of complete gonadal histology and reproductive cycle for this species in the Neotropical region. In this study, the reproductive dynamics of C. fluminea in a temperate region, the Santa Catalina Pampean stream, Argentina, is described. Samples of 20–30 individuals were collected monthly from April 2003-April 2005 and processed using traditional histological techniques. During the two years of this study, seven spawning events were recognized. Three major spawns occurred in spring and summer, and other four minor ones during summer and autumn. Events of oocyte recovery were observed after spawning. A high number of incubating individuals was detected. The results stressed the difficulty of identifying a particular pattern of gamete release and of spawning behaviour in this invasive species, especially when inhabiting an unstable environment.