Extracellular vesicles from Fasciola gigantica induce cellular response to stress of host cells.

Extracellular vesicles (EVs) from parasitic helminths play an important role in immunomodulation. However, EVs are little studied in the important parasite Fasciola gigantica. Here the ability of EVs from F. gigantica to induce cellular response to stress (reactive oxygen species generation, autophage and DNA damage response) in human intrahepatic biliary epithelial cells (HIBEC) was investigated. F. gigantica-derived EVs were isolated by ultracentrifugation, and identified with transmission electron microscopy, nanoparticle size analysis and parasite-derived EV markers. Internalization of EVs by HIBEC was determined by confocal immunofluorescence microscopy and flow cytometry. ROS levels in HIBEC were detected by molecular probing. EVs-induced autophagy and DNA-damaging effects were determined by evaluating expression levels of light chain 3B protein (LC3B), phosphor- H2A.X and phosphor-Chk1, respectively. Results revealed that EVs with sizes predominately ranging from 39 to 110 nm in diameter were abundant in adult F. gigantica and contained the parasite-derived marker proteins enolase and 14-3-3, and EVs were internalized by HIBEC. Further, uptake of EVs into HIBEC was associated with increased levels of reactive oxygen species, LC3Ⅱ, phosphor-H2A.X and phosphor-Chk1, suggesting EVs are likely to induce autophagy and DNA damage & repair processes. These results indicate F. gigantica EVs are associated with modulations of host cell responses and have a potential important role in the host-parasite interactions.

Fasciola gigantica: Ultrastructural cytochemistry of the tegumental surface in newly- excysted metacercariae and in vitro-penetrated juvenile flukes informs a concept of parasite defence at the interface with the host.

Cytochemical staining techniques were carried out en bloc with in vitro excysted and gut-penetrated Fasciola gigantica larvae in order to visualise the glycocalyx of the tegument, a structure which comprises the parasite component of the host-parasite interface, yet is incompletely preserved by conventional fixation and preparation techniques for electron microscopy. Positive reactivity with ruthenium red and periodic acid-thiocarbohydrazine-osmium (PATCO) techniques revealed that the glycocalyx is polyanionic and carbohydrate-rich throughout its depth. It comprises a trilaminate arrangement, with a thin dense zone and fibrillar layer closely apposed to the outer aspect of the apical plasma membrane, invested by an irregular thick mucopolysaccharide capsule. The latter, not recorded in adult flukes, may represent a specific adaptation to facilitate invasion in the face of host immunity, and may also protect the parasite surface from the action of host- and parasite-derived proteases. Early in the invasion of a naïve host, the glycocalyx may be partly responsible for triggering the responses of innate immunity, while later in infection, or when an anamnestic response is initiated in an immunocompetent host, the antibodies and activated lymphocytes of specific acquired immunity are invoked to interact with the parasite surface. The cytochemical properties of the glycocalyx, together with its potential for dynamic turnover due to exocytosis of the T0 tegumental secretory bodies, are likely to aid neutralisation of potentially damaging immune effectors and ensure their removal from the vicinity of the parasite by sloughing in complex with glycocalyx components.

Fasciola gigantica: Comparison of the tegumental ultrastructure in newly excysted metacercariae and in vitro penetrated juvenile flukes indicates intracellular sources of molecules with vaccinal and immunomodulatory potential.

Using in vitro procedures to prepare newly excysted metacercariae and gut-penetrated juvenile Fasciola gigantica, the ultrastructural features of the tegumental syncytium and perikarya of these ephemeral stages in the host-invasion process were compared. The T0-type tegumental cells in newly excysted metacercariae are packed with stored T0 granules which, following transport to the surface membrane of the syncytium, discharge by exocytosis to maintain the glycocalyx. The T0 cells become depleted of T0 granules during the penetration process, shrink in size, and initiate autophagy in the cytoplasm to facilitate metamorphosis from a storage function to active biosynthesis. The novel products appear to include lysosomes which contribute to the autophagosomes, and T1 granules, necessary for maintenance of the glycocalyx and immunoprotection, as the invasion process continues into the host liver. Residual bodies, the end-products of autophagy, are eliminated from the apical membrane of the tegumental syncytium into the host-parasite interface. There they may present a transient source of parasite-derived molecules, including lysosomal cathepsin-type proteases, with potential for interaction with the host's immune system, and so might be exploited as targets for vaccinal and immunomodulatory studies.

Identification of Fasciola species based on mitochondrial and nuclear DNA reveals the co-existence of intermediate Fasciola and Fasciola gigantica in Thailand.

Molecular techniques were used to identify Fasciola species collected from Chiang Mai Thailand. Morphometrically, 65 stained and 45 fresh worms collected from cattle suggested the possible occurrence of both F. gigantica and F. hepatica. Twenty-two worms comprising 15 from cattle and 7 from human patients, were identified subsequently based on three genetic markers: mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1), mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS2). All of them presented the F. gigantica type in maternally inherited mitochondrial sequences (nad1 and cox1), with six types in each sequence (FgNDI-CM1 to FgNDI-CM6 and FgCOI-CM1 to FgCOI-CM6, respectively). Remarkably, the predominant nad1 type, FgNDI-CM6, was identical to that of aspermic Fasciola sp. formerly reported from Thailand, Japan, Korea, China, Vietnam, and Myanmar. ITS2 sequences were analyzed successfully in 20 worms. Fifteen worms showed the F. gigantica type and five (including one worm from a patient) had mixed ITS2 sequences of both F. gigantica and F. hepatica in the same worms, with additional heterogeneity within both ITS2 types. This study revealed the intermediate form of Fasciola coexisting with F. gigantica for the first time in Thailand.

Time-dependent tegumental surface changes in juvenile Fasciola gigantica in response to triclabendazole treatment in goat.

Triclabendazole (TCBZ), the anthelmintic drug active against both mature and immature liver flukes, was used to investigate the effect of in vivo treatment on the tegumental surface of juvenile Fasciola gigantica. Five goats were infected with 150 F. gigantica metacercariae each by oral gavage. Four of them were treated with single dose of TCBZ at 10mg/kg at four weeks post-infection. They were euthanized at 0 (untreated), 24, 48, 72 and 96h post treatment. Juvenile flukes were manually retrieved from the goat livers and processed for scanning electron microscopy. In control flukes, the anterior region was adorned with sharply pointed spines projecting away from the surface, while in the posterior region, spines become shorter and narrower, loosing serration and with the appearance of distinct furrows and papillae. The dorsal surface retained the same pattern of surface architecture similar to that of ventral surface. Flukes obtained from 24h post-treatment did not show any apparent change and were still very active. However, there were limited movements and some blebbing, swelling, deposition of tegumental secretions and some flattening displayed by the flukes of 48h post-treatment. All the worms were found dead 72h post-treatment and showed advanced level of tegumental disruptions, consisting of severe distortion of spines, sloughing off the tegument to expose the basal lamina, formation of pores and isolated patches of lesions. By 96h post-treatment, the disruption was extremely severe and the tegument was completely sheared off causing deeper lesions that exposed the underlying musculature. The disruption was more severe at posterior than anterior region and on ventral than dorsal surface. The present study further establishes the time-course of TCBZ action in vivo with 100% efficacy against the juvenile tropical liver fluke.

The in vitro anthelmintic effects of plumbagin on newly excysted and 4-weeks-old juvenile parasites of Fasciola gigantica.

The effect of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) against newly excysted juveniles (NEJs) and 4-weeks-old immature parasites of Fasciola gigantica were compared with triclabendazole (TCZ). The anthelmintic efficacy of 1, 10 and 100µg/ml of PB or TCZ following incubation in vitro for 1-24h was compared using a combination of relative motility (RM), survival index (SI) and larval migration inhibition (LMI) assays for parasite viability. The RM and SI values of the PB-treated group decreased at a more rapid rate than the TCZ-treated group. For NEJs, the decreased RM values were first observed at 1h incubation with 1µg/ml PB, and 90% of flukes were killed at 24h. In contrast, in TCZ-treated groups a 10-fold higher concentration of TCZ (10µg/ml) resulted in only 9% dead parasites after 24h incubation. In 4-weeks-old juvenile parasites, PB reduced the RM value at 10µg/ml with 100% of flukes dead after 3h, while TCZ decreased RM values at the concentration of 100µg/ml but with only 5% of flukes killed at 24h. NEJs treated with PB exhibited 88%, 99% and 100% of LMIs at the concentrations of 1, 10 and 100µg/ml, respectively. NEJs incubated with TCZ have an LMI of only 32% at the highest concentration of 100µg/ml. Similarly PB had a significantly greater killing of immature 4weeks juvenile stages than TCZ at all concentrations; however, 4-weeks-old juvenile parasites were more resistant to killing by PB or TCZ at all concentrations when compared to NEJs. Further studies were carried out to investigate the alterations of the parasite tegument by scanning electron microscope (SEM). PB caused similar tegumental alterations in 4-weeks-old juveniles as those observed in TCZ treatment but with greater damage at comparative time points, comprising of swelling, blebbing and rupture of the tegument, loss of spines, and eventual erosion, lesion and desquamation of the total tegument. These data indicate that PB had a greater fasciolicidal effect against immature stages of F. gigantica parasites than TCZ and warrant further studies for use as a potential new anthelmintic against Fasciola infections.

Possible fasciocidal activity of methanol extract of Dregea volubilis leaves.

In eastern and southern part of India Dregea volubilis (Family Asclepediaceae) is widely used as anthelmintic in traditional system of medicine. The present study was conducted to evaluate the fasciocidal activity of the methanol extract of D. volubilis leaves (MEDV) and to observe the drug's effect on organisms through scanning electron microscopic (SEM) study. Live parasites (Trematode: Fasciola gigantica) were collected in 0.9% phosphate-buffered saline from the bile ducts of buffalo. Those were incubated in the said media at 37 ± 1 °C either as control, or with MEDV at 5, 10, 25, 50 and 100 mg/ml as test groups or with albendazole at 10mg/ml as standard group. The efficacy of the extract was determined on the basis of paralysis (temporary loss of spontaneous movement of the organisms) and/or death of the liver flukes. Death was confirmed when the organisms lost their motility permanently and their motility could not be revived even when vigorously shaken or dipped in warm water. MEDV at all concentration effectively paralyzed first and then killed the liver flukes (p < 0.001). Maximum fasciocidal activity was found with concentration of 100 mg/ml at 38.83 ± 3.41 min. Through SEM study, severe damages were observed in both the suckers as well as on the tegumental surfaces of the treated liver flukes. The study confirmed the fasciocidal activity of the MEDV.

Fasciola gigantica: the in vitro effects of artesunate as compared to triclabendazole on the 3-weeks-old juvenile.

The in vitro effect of artesunate (ATS) on the 3-week-old juveniles of Fasciola gigantica was compared with triclabendazole (TCZ) by incubating the parasites in M-199 medium containing the drugs at concentrations of 20, 40, and 80 µg/ml for 1, 3, 6, 12, and 24h. The anthelmintic activities of these drugs were evaluated based on the relative motility value (RM) and the alterations of the tegument as observed by scanning (SEM) and transmission (TEM) electron microscopy. The RM values of TCZ-treated flukes decreased significantly from 6 to 24h for all dosages. For ATS-treated flukes, RM value decreased markedly from 12 to 24h, but the rates of decline were less than TCZ at the same doses. When observed by SEM, the tegument showed similar sequence of morphological changes after treatments with both drugs, comprising of swelling of tegumental ridges, followed by blebbing and later rupturing of the blebs, leading to erosion and lesion, and disruption of the tegument. When examined by TEM, ultrastructural changes in the tegument and associated structures after treatments with TCZ and ATS were similar which comprised of swelling, blebbing of the tegument, dilation of basal infoldings, and depolymerization of the microtrabecular network. After a longer incubation time, the tegument was completely sloughed off and the tegument cell bodies became necrotic. Additionally, in ATS-treated flukes, mitochondria showed severe swelling, rupturing of outer membrane, and their interior filled with flocculent materials.

Tegumental alterations of Fasciola gigantica due to in vitro treatment with Ro-354.

Triclabendazole is the drug of choice against Fasciola infections in humans and animals. However, parasite resistance against triclabendazole is spreading in veterinary field, and there are no drugs of comparable activity currently available for the treatment and control of fascioliasis. The efficacy of a new rhodanine derivative Ro-354 against adult Fasciola gigantica in vitro was investigated. One hour post incubation, scanning and transmission electron microscopic examination revealed an evident disruption of the tegument of F. gigantica as blebbing, swelling and furrowing. Moreover, an increase in severity of tegumental damage as sloughing and absence of spines was observed. In conclusion, Ro-354 shows potent activity against F. gigantica in vitro, and, the authors recommend carrying out more studies to detect its efficacy in vivo.

Hybridization experiments indicate incomplete reproductive isolating mechanism between Fasciola hepatica and Fasciola gigantica.

Experiments on hybridization between Fasciola hepatica and Fasciola gigantica were carried out to clarify whether a reproductive isolating mechanism appears between the two Fasciola species. Molecular evidence for hybridization was based on the DNA sequence of the internal transcribed spacer 1 (ITS1) region in nuclear ribosomal DNA, which differs between the species. The results suggested that there were not pre-mating but post-mating isolating mechanisms between the two species. However, viable adults of the hybrids F1 and F2 were produced from both parental F. hepatica and F. gigantica. The hybrids inherited phenotypic characteristics such as ratio of body length and width and infectivity to rats from parental Fasciola hepatica and F. gigantica. These findings suggest that reproductive isolation is incomplete between Fasciola hepatica and F. gigantica. Adults of the hybrids F1 and F2 were completely different in mode of reproduction from aspermic Fasciola forms that occur in Asia and seem to be offspring originated from hybridization between F. hepatica and F. gigantica and to reproduce parthenogenetically.

Fasciola gigantica: Parasitological and scanning electron microscopy study of the in vitro effects of ivermectin and/or artemether.

OBJECTIVE: To evaluate the in vitro effects of different concentrations of ivermectin and/or artemether on Fasciolagigantica worms and to study the parasitological changes and tegumental alterations using scanning electron microscopy (SEM). METHODS: Fasciola gigantica worms were incubated in vitro for 24 and 48 h with three concentrations of either ivermectin or artemether (10, 20 and 50 microg/ml) or both in half concentration of either (5, 10 and 25 microg/ml). RESULTS: Exposure of Fasciola worms to 25+25 microg/ml of combined drug regimens or to 50 microg/ml of either ivermectin or artemether for 48 h led to 100%, 41.7% and 75% worm killing which were accompanied by a significant reduction in egg laying capacity and significant increase in dead eggs maximally recorded in combined drug regimens. SEM of the flukes incubated for 48 h with combined drug regimens showed maximal tegumental disruption with swelling of the worm body, roughness, blebbing, sloughing and complete loss of spines. Disruption to the tegument of the flukes induced by artemether was more than that of ivermectin. CONCLUSIONS: Artemether alone or combined with ivermectin in half doses had potent fasciocidal activities. Besides, half doses of combined drug regimens had higher ovicidal effects than each drug alone. In vivo studies are recommended to explore the efficacy of combined regimens against Fasciola infection.

Botanical study of Meryta denhamii Seem. and its antihelminthic activity against Fasciola gigantica.

The anthelmintic activity of the ethanolic extracts of stems and leaves of Meryta denhamii Seem. against adult liver flukes "Fasciola gigantica" was studied in vitro. Although leaves extract was inactive, stems extract exhibited anthelmintic activity and recorded LC50 and LC90 values, 16 and 26 gm/l respectively. The mode of action of the ethanolic extract of the stems on the adult flukes was evaluated by scanning electron microscopy (SEM). Tegumental sloughing, loss of spines and deformity of suckers were observed. These damages are responsible for the vermicidal effect of the ethanolic extract of the stems. The saponin contents of the stems (4.25%) and leaves (2.45%) were determined using haemolytic index. The plant was identified based on the macro and micromorphological features of the stem and leaf.

Fasciola gigantica: anthelmintic effect of the aqueous extract of Artocarpus lakoocha.

The effect of the crude extract of Artocarpus lakoocha (70% composition is 2,4,3',5'- tetrahydroxystilbene -THS) on adult Fasciola gigantica was evaluated after incubating the parasites in M-199 medium containing 250, 500, 750 and 1000 microg/ml of the crude extract, or triclabendazole (TCZ) at the concentrations of 80 and 175 microg/ml as the positive control, for 3, 6, 12 and 24h, using relative motility (RM) assay and observation by scanning electron microscope (SEM). Decreased contraction and motility were first observed after 3h incubation with TCZ at the concentration 80 and 175 microg/ml. TCZ markedly reduced the parasite's motility at the concentration of 175 microg/ml at 6h, and killed the worms after 12h exposure. The crude extract of A. lakoocha at all concentrations reduced the parasite's motility similar to TCZ at 3h incubation. In 250 and 500 microg/ml of the crude extract, the values were decreased from 3 to 12h, then they were stable between 12 and 24h and reduced to the level approximately 30-40% of the control. At 750 and 1000 microg/ml concentrations the crude extract rapidly reduced the RM values from the start to 12h and killed the parasites between 12 and 24h incubation. The crude extract also inhibited the larval migration by 75% and 100% at the concentrations of 250-500 and 750-1000 microg/ml, respectively. TCZ and the crude extract caused sequentially changes in the tegument including swelling, followed by blebbings that later ruptured, leading to the erosion and desquamation of the tegument syncytium. As the result, lesion was formed which exposed the basal lamina. The damage appeared more severe on the dorsal than the ventral surface, and earlier on the anterior part and lateral margins when compared to the posterior part. The severity and rapidity of the damages were enhanced with increasing concentration of the crude extract. Hence, the crude extract of A. lakoocha, may exert its fasciolicidal effect against adult F. gigantica by initially causing the tegumental damage.

In vitro effect of artemether and triclabendazole on adult Fasciola gigantica.

Triclabendazole "Fasinex" is the drug of choice against fasciolosis because of its high efficacy against both mature and immature flukes, however parasite resistance against this drug is increasing. Hence, there is pressing need for new fasciolicidal drugs. In the present study, the in vitro effect of artemether on adult flukes was evaluated using scanning electron microscopy. After 24 h incubation with 10 microg/ml artemether, the tegument of the apical cone appeared to be slightly more swollen than normal. This swelling became so severe and the spines appeared sunken, with their tips protruding from a swollen and blebbed base, on increasing the concentration to 20 microg/ml. With the higher concentration of 30 microg/ml, extensive and severe tegumental swelling occurred in the apical cone region of the flukes. There were many blebs around ventral sucker, a number of which appeared to have burst causing lesion. The tegumental changes occurred following incubation in artemether were comparable with those observed with triclabendazole in its active sulphoxide metabolite form (TCBZ-SX).

The surface ultrastructure of the Egyptian liver fluke Fasciola gigantica.

Scanning electron microscopy (SEM) of adult Fasciola gigantica, a known liver fluke in Egypt, shows the presence of tegumental spines and folds giving the appearance of a rough surface. The spines are small and closely-spaced anteriorly, increasing in size and number on the in the mid-lateral aspect of the ventral surface. Towards the posterior end the spines progressively decrease in both size and number. Tegumental transverse folds or invaginations are interspaced between the spines. There are two types of sensory papillae on the tegumental surface. The dorsal surface exhibits similar features but the spines and papillae are less numerous with less extensive surface folds than on the ventral surface of the body. The oral and ventral suckers, together with the excretory pore, appear spineless, bearing ciliate sensory papillae. Further studies should be directed at deciphering the genetic codes and the synthesis of some of these antigens by recombinant DNA technology to be used in serodiagnosis, vaccination and for drug receptors.

An in vitro effect of propolis on adult worms of Fasciola gigantica.

The effect of Siwa propolis on adult flukes was evaluated using scanning electron microcopy. It gave an overview of the surface architecture of the tegument of Fasciola gigantica apical cone. The base of the spines appeared to be "flaking off" and showed severe blebbing after 24h incubation with 10 micro/ml propolis. This swelling became so sever and the spines were barely visible, on increasing the concentration to 20 micro/ml. Besides, there were many large blebs on the apical cone, a number of which appeared to have burst, causing lesions and the tegument was marked by a number of pits caused by the loss of spines. With the higher concentration of 30 micro/ml, erosion of the surface had occurred to such extent that no tegument remained, only a mass of fibrous structures. The tegumental changes occurred following incubation in propolis were compared with those observed with triclabendazole (TCBZ) "Fasinex" because of its high efficacy against both mature and immature flukes.

Distribution of 28.5 kDa antigen in the tegument of adult Fasciola gigantica.

Monoclonal antibody (MoAb) specific to 28.5 kDa tegumental antigen (TA) was used to localize this antigen in various tissues of adult Fasciola gigantica by means of indirect immunofluorescence, immunoperoxidase and immunogold techniques. The indirect immunofluorescence and immunoperoxidase detections revealed that this antigen was concentrated in the tegument particularly in its outer rim, tegumental cells and their processes, epithelial linings of the oral sucker and the proximal part of digestive tract. It was also detected at a moderate concentration in spermatogenic cells in the testes, cells of Mehlis' gland, oocytes within the ovary, and ovum within the egg of adult parasites. At TEM level, the immunogold detection showed deposit of gold particles specifically in G(2) tegumental granules and on the surface membrane. Thus, this antigen is expressed in the tegument and associated structures of adult parasites, and it could be a major component of the G(2) granules which are shown to fuse with the surface membrane and contribute material to replace the casted-off membrane. This process is a part of membrane turnover that prevents the parasite from being attacked by the host immune effector cells.

Classification of the parenchymal cells in Fasciola gigantica based on ultrastructure and their expression of fatty acid binding proteins (FABPs).

Parenchymal cells in adult Fasciola gigantica can be classified into three types based on their ultrastructural features and different quantities of fatty acid binding protein (FABP) being stored. Parenchymal cell type 1 (Pc1) has pale cytoplasm consisting largely of a loose network of fine fibers, and it contains few mitochondria but numerous glycogen particles. This cell type may be specialized in the storage and metabolism of glycogen and glucose. Parenchymal cell type 2 (Pc2) has similar cytoplasmic features as Pc1 but contains more numerous mitochondria, and high concentration of FABP as reflected by high density of immunostaining and immunogold labeling using specific monoclonal antibody (MoAb) to FABP as probe. Pc2 may, thus, specialize in the storage and metabolism of fatty acids and other lipids. Parenchymal cell type 3 (Pc3) has dense cytoplasm containing large amount of rough endoplasmic reticulum, Golgi complex and mitochondria, which is typical of a secretory cell. Furthermore, Pc3 has very little glycogen particles and is not stained by MoAb against FABP. It could, thus, be concerned with the synthesis of fibers, which form the scaffold of the parenchyma.

Immunolocalization of cytoskeletal components in the tegument of the 3-week-old juvenile and adult Fasciola gigantica.

Components of three cytoskeletal elements, namely, microtubule, intermediate and actin filaments have been localised in the tegument of the 3-week-old juvenile and adult Fasciola gigantica by means of immunofluorescence and immunoperoxidase techniques, using mouse monoclonal anti-alpha-tubulin, anti-cytokeratin antibodies and biotinylated-phalloidin, respectively. The immunostaining with the above probes were also performed in adult Schistosoma mansoni for comparison. The presence of tubulin, indicative of microtubules, was demonstrated in the tegumental cell bodies, their cytoplasmic processes, and the basal layer of the tegumental syncytium of F. gigantica. While in S. mansoni, tubulin appeared as vertical lines stretching across the whole thickness of the syncytium. Cytokeratin, representing one type of intermediate filaments, was detected in the tegumental cell bodies, their cytoplasmic processes, tegumental syncytium and spines of F. gigantica. In contrast, cytokeratin was evident only in the syncytium of S. mansoni, but not in the spines. Phalloidin, which could bind to actin, a subunit of microfilament, was detected in the tegumental cell bodies, their processes, and the microtrabecular network which form the scaffold of the tegumental syncytium of F. gigantica. In S. mansoni, actin was localized in similar tissues except the syncytium was not stained while spines exhibited intense staining. In F. gigantica, the presence of microtubules and actin filaments in the tegumental cells, their processes and in the syncytium could mediate the movement of secretory granules from the cell bodies towards the basal as well as the apical layer of the tegument. Cytokeratin filaments may serve to reinforce the integrity of the tegumental syncytium as well as the spines.

Spermiogenesis and ultrastructure of the spermatozoon of the liver fluke Fasciola gigantica Cobbold, 1856 (Digenea: Fasciolidae), a parasite of cattle in Senegal.

The present paper describes the spermiogenesis and the ultrastructure of the spermatozoon of Fasciola gigantica, as revealed by transmission electron microscopy. Spermiogenesis in F. gigantica begins with the formation of a differentiation zone containing 2 centrioles with associated striated roots and an intercentriolar body between them. Each centriole develops a flagellum. Proximodistal fusion of these flagella with the median cytoplasmic extension gives rise to the spermatozoon. Spermiogenesis in F. gigantica is characterized by the formation of a dorsolateral cytoplasmic expansion, an external ornamentation of the cell membrane, and spinelike bodies. These 3 structures were also observed in the anterior part of the spermatozoon. Our study describes for the first time the simultaneous presence of dorsolateral cytoplasmic expansion, external ornamentation of the plasma membrane, and spinelike bodies in the spermatozoon of a trematode.