A Drosophila model targets Eiger/TNFα to alleviate obesity-related insulin resistance and macrophage infiltration.

Obesity is associated with various metabolic disorders, such as insulin resistance and adipose tissue inflammation (ATM), characterized by macrophage infiltration into adipose cells. This study presents a new Drosophila model to investigate the mechanisms underlying these obesity-related pathologies. We employed genetic manipulation to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy and high circulating glucose levels. Moreover, we observed significant infiltration of immune cells (hemocytes) into the fat bodies, accompanied by insulin resistance. We found that attenuation of Eiger/TNFα signaling reduced ATM and improved insulin sensitivity. Furthermore, using metformin and the antioxidants anthocyanins, we ameliorated both phenotypes. Our data highlight evolutionarily conserved mechanisms allowing the development of Drosophila models for discovering therapeutic pathways in adipose tissue immune cell infiltration and insulin resistance. Our model can also provide a platform to perform genetic screens or test the efficacy of therapeutic interventions for diseases such as obesity, type 2 diabetes and non-alcoholic fatty liver disease.

A Novel Drosophila Model to Investigate Adipose Tissue Macrophage Infiltration (ATM) and Obesity highlights the Therapeutic Potential of Attenuating Eiger/TNFα Signaling to Ameliorate Insulin Resistance and ATM.

Obesity is a global health concern associated with various metabolic disorders including insulin resistance and adipose tissue inflammation characterized by adipose tissue macrophage (ATM) infiltration. In this study, we present a novel Drosophila model to investigate the mechanisms underlying ATM infiltration and its association with obesity-related pathologies. Furthermore, we demonstrate the therapeutic potential of attenuating Eiger/TNFα signaling to ameliorate insulin resistance and ATM. To study ATM infiltration and its consequences, we established a novel Drosophila model (OBL) that mimics key aspects of human adipose tissue and allows for investigating ATM infiltration and other related metabolic disorders in a controlled experimental system. We employed genetic manipulation to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic, and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy, and high levels of circulating glucose. Moreover, we observed a significant infiltration of immune cells (hemocytes) in the fat bodies accompanied by insulin resistance and systemic metabolic dysregulation. Furthermore, we found that attenuation of Eiger/TNFα signaling and using metformin and anti-oxidant bio-products like anthocyanins led to a reduction in ATM infiltration and improved insulin sensitivity. Our data suggest that the key mechanisms that trigger immune cell infiltration into adipose tissue are evolutionarily conserved and may provide the opportunity to develop Drosophila models to better understand pathways critical for immune cell recruitment into adipose tissue, in relation to the development of insulin resistance in metabolic diseases such as obesity and type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD). We believe that our OBL model can also be a valuable tool and provide a platform either to perform genetic screens or to test the efficacy and safety of novel therapeutic interventions for these diseases.

Reduction of nucleolar NOC1 leads to the accumulation of pre-rRNAs and induces Xrp1, affecting growth and resulting in cell competition.

NOC1 is a nucleolar protein necessary in yeast for both transport and maturation of ribosomal subunits. Here, we show that Drosophila NOC1 (annotated CG7839) is necessary for rRNAs maturation and for a correct animal development. Its ubiquitous downregulation results in a dramatic decrease in polysome level and of protein synthesis. NOC1 expression in multiple organs, such as the prothoracic gland and the fat body, is necessary for their proper functioning. Reduction of NOC1 in epithelial cells from the imaginal discs results in clones that die by apoptosis, an event that is partially rescued in a Minute/+ background, suggesting that reduction of NOC1 induces the cells to become less fit and to acquire a 'loser' state. NOC1 downregulation activates the pro-apoptotic Eiger-JNK pathway and leads to an increase of Xrp1, which results in the upregulation of DILP8, a member of the insulin/relaxin-like family known to coordinate organ growth with animal development. Our data underline NOC1 as an essential gene in ribosome biogenesis and highlight its novel functions in the control of growth and cell competition.

Author Correction: Probing spermiogenesis: a digital strategy for mouse acrosome classification.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Postnatal Changes in K+/Cl- Cotransporter-2 Expression in the Forebrain of Mice Bearing a Mutant Nicotinic Subunit Linked to Sleep-Related Epilepsy.

The Na+/K+/Cl- cotransporter-1 (NKCC1) and the K+/Cl- cotransporter-2 (KCC2) set the transmembrane Cl- gradient in the brain, and are implicated in epileptogenesis. We studied the postnatal distribution of NKCC1 and KCC2 in wild-type (WT) mice, and in a mouse model of sleep-related epilepsy, carrying the mutant ß2-V287L subunit of the nicotinic acetylcholine receptor (nAChR). In WT neocortex, immunohistochemistry showed a wide distribution of NKCC1 in neurons and astrocytes. At birth, KCC2 was localized in neuronal somata, whereas at subsequent stages it was mainly found in the somatodendritic compartment. The cotransporters' expression was quantified by densitometry in the transgenic strain. KCC2 expression increased during the first postnatal weeks, while the NKCC1 amount remained stable, after birth. In mice expressing ß2-V287L, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8), with no concomitant change in NKCC1. Consistently, the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying ß2-V287L. At P60, the amount of KCC2 was instead higher in mice bearing the transgene. Irrespective of genotype, NKCC1 and KCC2 were abundantly expressed in the neuropil of most thalamic nuclei since birth. However, KCC2 expression decreased by P60 in the reticular nucleus, and more so in mice expressing ß2-V287L. Therefore, a complex regulatory interplay occurs between heteromeric nAChRs and KCC2 in postnatal forebrain. The pathogenetic effect of ß2-V287L may depend on altered KCC2 amounts in PFC during synaptogenesis, as well as in mature thalamocortical circuits.

Anthocyanins Function as Anti-Inflammatory Agents in a Drosophila Model for Adipose Tissue Macrophage Infiltration.

Epidemiological and preclinical studies have demonstrated that bioactive foods like flavonoids, polyphenolic compounds derived from fruits and vegetables, exert a protective action against obesity, cardiovascular disorders, and Adipocyte Tissue Macrophage infiltration (ATM). All these pathologies are characterized by increase in reactive oxygen species (ROS) and in proinflammatory cytokines that have been shown to favor the migration of immune cells, particularly of macrophages, in metabolically active organs like the liver and adipose tissue, that in Drosophila are constituted by a unique organ: the fat body. This study, using a unique Drosophila model that mimics human ATM, reveals the beneficial effects of flavonoids to reduce tissue inflammation. Our data show that anthocyanin-rich food reduces the number of hemocytes, Drosophila macrophages, infiltrating the fat cells, a process that is associated with reduced production of ROS and reduced activation of the JNK/SAPK p46 stress kinase, suggesting a fundamental function for anthocyanins as antioxidants in chronic inflammation and in metabolic diseases.

In vitro evidence for the participation of Drosophila melanogaster sperm ß-N-acetylglucosaminidases in the interactions with glycans carrying terminal N-acetylglucosamine residues on the egg's envelopes.

Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm-associated ß-N-acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid-phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well-defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal ß-N-acetylglucosamine, but not core ß-N-acetylglucosamine residues. The capacity of sperm ß-N-acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when ß-N-acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide ß-N-acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm-egg interactions in Drosophila.

Probing spermiogenesis: a digital strategy for mouse acrosome classification.

Classification of morphological features in biological samples is usually performed by a trained eye but the increasing amount of available digital images calls for semi-automatic classification techniques. Here we explore this possibility in the context of acrosome morphological analysis during spermiogenesis. Our method combines feature extraction from three dimensional reconstruction of confocal images with principal component analysis and machine learning. The method could be particularly useful in cases where the amount of data does not allow for a direct inspection by trained eye.

The Stearoyl-CoA Desaturase-1 (Desat1) in Drosophila cooperated with Myc to Induce Autophagy and Growth, a Potential New Link to Tumor Survival.

Lipids are an important energy supply in our cells and can be stored or used to produce macromolecules during lipogenesis when cells experience nutrient starvation. Our proteomic analysis reveals that the Drosophila homologue of human Stearoyl-CoA desaturase-1 Desat1) is an indirect target of Myc in fat cells. Stearoyl-CoA desaturases are key enzymes in the synthesis of monounsaturated fatty acids critical for the formation of complex lipids such as triglycerides and phospholipids. Their function is fundamental for cellular physiology, however in tumors, overexpression of SCD-1 and SCD-5 has been found frequently associated with a poor prognosis. Another gene that is often upregulated in tumors is the proto-oncogene c-myc, where its overexpression or increased protein stability, favor cellular growth. Here, we report a potential link between Myc and Desat1 to control autophagy and growth. Using Drosophila, we found that expression of Desat1, in metabolic tissues like the fat body, in the gut and in epithelial cells, is necessary for Myc function to induce autophagy a cell eating mechanism important for energy production. In addition, we observed that reduction of Desat1 affects Myc ability to induce growth in epithelial cells. Our data also identify, in prostatic tumor cells, a significant correlation between the expression of Myc and SCD-1 proteins, suggesting the existence of a potential functional relationship between the activities of these proteins in sustaining tumor progression.

Dynamic of contribution of UBPy-sorted cargo to acrosome biogenesis: effects of its derailment in a mouse model of globozoospermia, the infertile Vps54 (L967Q) mutant.

The sperm acrosome is a specialized vacuole, a member of the family of cell-specific lysosome-related organelles. Its exocytosis, the acrosome reaction, is a crucial event during fertilization. The released acrosomal contents promote sperm penetration through the investments of the oocyte, whereas the membranous components of the acrosome are involved in sperm-oocyte interaction/fusion and oocyte activation. The way that these functionally distinct acrosomal costituents reach the vacuole during its biogenesis remains poorly understood. The biosynthetic pathway and a consistent supply from the endosomal system have recently been documented. We use immunogold electron microscopy to determine the contribution of endosome cargo-sorting during step-by-step mouse acrosomogenesis. The chosen proteins of this study were UBPy (ESCRT-DUB), together with endosome compartment markers EEA1 and pallidin. The latter is described here for the first time in male germ cells. This new insight expands our knowledge of acrosomogenesis, confirming the plasticity of the endosomal system in supporting cell-type-specific functions. We also study wobbler mice, whose Vps54 mutation causes motor neuron degeneration and male infertility. Use of electron/immunoelectron microscopy and immunofluorescence enabled us to establish that the lack of an acrosome in wobbler spermatozoa is attributable to an early block in acrosome biogenesis and that the mislocalization of acrosome-destined proteins, potentially involved in the signaling events leading to oocyte activation, is possibly responsible for wobbler infertility, even after intracytoplasmic sperm injection.

Drosophila sperm surface alpha-L-fucosidase interacts with the egg coats through its core fucose residues.

Sperm-oocyte interaction during fertilization is multiphasic, with multicomponent events, taking place between egg's glycoproteins and sperm surface receptors. Protein-carbohydrate complementarities in gamete recognition have observed in cases throughout the whole evolutionary scale. Sperm-associated α-L-fucosidases have been identified in various organisms. Their wide distribution and known properties reflect the hypothesis that fucose and α-L-fucosidases have fundamental function(s) during gamete interactions. An α-L-fucosidase has been detected as transmembrane protein on the surface of spermatozoa of eleven species across the genus Drosophila. Immunofluorescence labeling showed that the protein is localized in the sperm plasma membrane over the acrosome and the tail, in Drosophila melanogaster. In the present study, efforts were made to analyze with solid phase assays the oligosaccharide recognition ability of fruit fly sperm α-L-fucosidase with defined carbohydrate chains that can functionally mimic egg glycoconjugates. Our results showed that α-L-fucosidase bound to fucose residue and in particular it prefers N-glycans carrying core α1,6-linked fucose and core α1,3-linked fucose in N-glycans carrying only a terminal mannose residue. The ability of sperm α-L-fucosidase to bind to the micropylar chorion and to the vitelline envelope was examined in in vitro assays in presence of α-L-fucosidase, either alone or in combination with molecules containing fucose residues. No binding was detected when α-L-fucosidase was pre-incubated with fucoidan, a polymer of α-L-fucose and the monosaccharide fucose. Furthermore, egg labeling with anti-horseradish peroxidase, that recognized only core α1,3-linked fucose, correlates with α-L-fucosidase micropylar binding. Collectively, these data support the hypothesis of the potential role of this glycosidase in sperm-egg interactions in Drosophila.

Hypocretin (orexin) regulates glutamate input to fast-spiking interneurons in layer V of the Fr2 region of the murine prefrontal cortex.

We studied the effect of hypocretin 1 (orexin A) in the frontal area 2 (Fr2) of the murine neocortex, implicated in the motivation-dependent goal-directed tasks. In layer V, hypocretin stimulated the spontaneous excitatory postsynaptic currents (EPSCs) on fast-spiking (FS) interneurons. The effect was accompanied by increased frequency of miniature EPSCs, indicating that hypocretin can target the glutamatergic terminals. Moreover, hypocretin stimulated the spontaneous inhibitory postsynaptic currents (IPSCs) on pyramidal neurons, with no effect on miniature IPSCs. This action was prevented by blocking 1) the ionotropic glutamatergic receptors; 2) the hypocretin receptor type 1 (HCRTR-1), with SB-334867. Finally, hypocretin increased the firing frequency in FS cells, and the effect was blocked when the ionotropic glutamate transmission was inhibited. Immunolocalization confirmed that HCRTR-1 is highly expressed in Fr2, particularly in layer V-VI. Conspicuous labeling was observed in pyramidal neuron somata and in VGLUT1+ glutamatergic terminals, but not in VGLUT2+ fibers (mainly thalamocortical afferents). The expression of HCRTR-1 in GABAergic structures was scarce. We conclude that 1) hypocretin regulates glutamate release in Fr2; 2) the effect presents a presynaptic component; 3) the peptide control of FS cells is indirect, and probably mediated by the regulation of glutamatergic input onto these cells.

Tet-Trap, a genetic approach to the identification of bacterial RNA thermometers: application to Pseudomonas aeruginosa.

Modulation of mRNA translatability either by trans-acting factors (proteins or sRNAs) or by in cis-acting riboregulators is widespread in bacteria and controls relevant phenotypic traits. Unfortunately, global identification of post-transcriptionally regulated genes is complicated by poor structural and functional conservation of regulatory elements and by the limitations of proteomic approaches in protein quantification. We devised a genetic system for the identification of post-transcriptionally regulated genes and we applied this system to search for Pseudomonas aeruginosa RNA thermometers, a class of regulatory RNA that modulates gene translation in response to temperature changes. As P. aeruginosa is able to thrive in a broad range of environmental conditions, genes differentially expressed at 37 °C versus lower temperatures may be involved in infection and survival in the human host. We prepared a plasmid vector library with translational fusions of P. aeruginosa DNA fragments (PaDNA) inserted upstream of TIP2, a short peptide able to inactivate the Tet repressor (TetR) upon expression. The library was assayed in a streptomycin-resistant merodiploid rpsL(+)/rpsL31 Escherichia coli strain in which the dominant rpsL(+) allele, which confers streptomycin sensitivity, was repressed by TetR. PaDNA fragments conferring thermosensitive streptomycin resistance (i.e., expressing PaDNA-TIP2 fusions at 37°C, but not at 28°C) were sequenced. We identified four new putative thermosensors. Two of them were validated with conventional reporter systems in E. coli and P. aeruginosa. Interestingly, one regulates the expression of ptxS, a gene implicated in P. aeruginosa pathogenesis.

The ESCRT-deubiquitinating enzyme USP8 in the cervical spinal cord of wild-type and Vps54-recessive (wobbler) mutant mice.

Usp8 is a deubiquitinating enzyme that works as regulator of endosomal trafficking and is involved in cell proliferation. "In vivo" USP8 is predominantly expressed in the central nervous system and testis, two organs with highly polarized cells. Considering that neuronal cell functionality is strictly dependent on vesicular traffic and ubiquitin-mediated sorting of the endocytosed cargo, it could be of relevance to investigate about USP8 in neuronal cells, in particular motor neurons. In this study, we found that USP8 is expressed in the gray and white matter of the spinal cord, labeling neuronal cell bodies, axonal microtubules and synaptic terminals. The glia component is essentially USP8-immunonegative. The partial colocalization of USP8 with EEA1 in motor neurons indicates that USP8 is involved in early endosomal trafficking while that with Vps54 suggests an involvement in the retrograde traffic. The variant Vps54(L967Q) is responsible for the wobbler phenotype, a disorder characterized by motor neuron degeneration. We searched for USP8/Vps54 in wobbler spinal cord. The most worth-mention result was that wobbler oligodendrocytes, in contrast to the wild-type, are heavily USP8-immunoreactive; no significant modification was appreciated about the cellular expression of mutated Vps54. On the other hand, as to the neuronal intracellular localization, both USP8 and Vps54(L967Q) did not show the typical spot-like distribution, but seemed to accumulate in proteinaceous aggregates. Collectively, our study suggests that in neuronal cells USP8 could be involved in endosomal trafficking, retrograde transport and synaptic plasticity. In disorders leading to neurodegeneration USP8 is upregulated and could influence the neuron-oligodendrocyte interactions.

Regulation of glutamate release by heteromeric nicotinic receptors in layer V of the secondary motor region (Fr2) in the dorsomedial shoulder of prefrontal cortex in mouse.

We studied how nicotinic acetylcholine receptors (nAChRs) regulate glutamate release in the secondary motor area (Fr2) of the dorsomedial murine prefrontal cortex, in the presence of steady agonist levels. Fr2 mediates response to behavioral situations that require immediate attention and is a candidate for generating seizures in the frontal epilepsies caused by mutant nAChRs. Morphological analysis showed a peculiar chemoarchitecture and laminar distribution of pyramidal cells and interneurons. Tonic application of 5 µM nicotine on Layer V pyramidal neurons strongly increased the frequency of spontaneous glutamatergic excitatory postsynaptic currents. The effect was inhibited by 1 µM dihydro-ß-erythroidine (which blocks α4-containing nAChRs) but not by 10 nM methyllicaconitine (which blocks α7-containing receptors). Excitatory postsynaptic currents s were also stimulated by 5-iodo-3-[2(S)-azetidinylmethoxy]pyridine, selective for ß2-containing receptors, in a dihydro-ß-erythroidine -sensitive way. We next studied the association of α4 with different populations of glutamatergic terminals, by using as markers the vesicular glutamate transporter type (VGLUT) 1 for corticocortical synapses and VGLUT2 for thalamocortical projecting fibers. Immunoblots showed higher expression of α4 in Fr2, as compared with the somatosensory cortex. Immunofluorescence showed intense VGLUT1 staining throughout the cortical layers, whereas VGLUT2 immunoreactivity displayed a more distinct laminar distribution. In Layer V, colocalization of α4 nAChR subunit with both VGLUT1 and VGLUT2 was considerably stronger in Fr2 than in somatosensory cortex. Thus, in Fr2, α4ß2 nAChRs are expressed in both intrinsic and extrinsic glutamatergic terminals and give a major contribution to control glutamate release in Layer V, in the presence of tonic agonist levels.

Failure of acrosome formation and globozoospermia in the wobbler mouse, a Vps54 spontaneous recessive mutant.

The acrosome is a unique organelle that plays an important role at fertilization and during sperm morphogenesis and that is absent in globozoospermia, an inherited infertility syndrome in humans. At the light of recent experimental evidence, the acrosome is considered a lysosome-related organelle to whose biogenesis both the endocytic and biosynthetic pathways contribute. Vps54 is a vesicular sorting protein involved in the retrograde traffic; the recessive Vps54(L967Q) mutation in the mouse results in the wobbler phenotype, characterized by motor-neuron degeneration and male infertility. Here we have investigated the spatio-temporal occurrence/progression of the wobbler fertility disorder starting from mice at post-natal day 35, the day of the first event of spermiation. We show that the pathogenesis of wobbler infertility originates at the first spermiogenetic wave, affecting acrosome formation and sperm head elongation. Vps54(L967Q)-labeled vesicles, on the contrary of the wild-type Vps54-labeled ones, are not able to coalesce into a larger vesicle that develops, flattens and shapes to give rise to the acrosome. Evidence that it is the malfunctioning of the endocytic traffic to hamper the development of the acrosome comes out from the study on UBPy. UBPy, a deubiquitinating enzyme, is a marker of acrosome biogenesis from the endocytic pathway. In wobbler spermatids UBPy-positive endosomes remain single, scattered vesicles that do not contribute to acrosome formation. As secondary defect of wobbler spermiogenesis, spermatid mitochondria are misorted; moreover, with the progression of the age/disease also Sertoli-germ cell adhesions are compromised suggesting a derailment in the endocytic route that underlies their restructuring.

Recombinant expression of Drosophila melanogaster α-L-fucosidase in Trichoplusia ni cells.

A cDNA encoding an α-l-fucosidase from Drosophila melanogaster was obtained from the recombinant plasmid named pGEM-DmFuca and inserted into the pBacHTeGFPT vector to construct the recombinant donor plasmid which was transposed to the target AcBacmid in Escherichia coli (DH10) by Tn7 transposition function. The AcBacmid-GFP-DmFuca plasmid was used to transfect Tn-5B1-4 cells of the Cabbage looper Trichoplusia ni. SDS-PAGE analysis revealed a band of about 80kDa. Using a polyclonal antiserum raised against α-l-fucosidase protein from D. melanogaster Western blotting analysis confirmed that the fusion protein eGFP-DmFuca has been successfully expressed in a biologically active form in Tn-5B1-4 cells. The recombinant protein, containing the histidine-tag motif, was purified using an affinity chromatography column. In vitro binding assays the purified eGFP-DmFuca interacts with α-l-fucose residues present on the micropyle of the D. melanogaster eggshell, confirming that the α-l-fucosidase is a good candidate as receptor involved in gamete interactions in fruit fly.

Cloning, sequence identification and expression profile analysis of α-L-fucosidase gene from the Mediterranean fruit fly Ceratitis capitata.

The Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae) is one of the most destructive agricultural pests, a polyphagus insect of relevant economic importance and is widespread in many regions around the world. It is the best-studied fruit fly pest at genetic and molecular level and much has been learned on its ecology and behaviour. An α-L-fucosidase has been recently hypothesized to be involved in sperm-egg interactions in Drosophila melanogaster and in other Drosophila species. Here, a complete cDNA encoding a putative α-L-fucosidase of the medfly was amplified using the reverse polymerase chain reaction (RT-PCR) with degenerate based on the conserved coding sequence information of several insect α-L-fucosidases, cloned and sequenced (GenBank accession no. FJ177429). The coding region consisted of 1482 bp which encoded a 485-residues protein (named CcFUCA) with a predicted molecular mass of 56.1 kDa. The deduced protein sequence showed 75% amino acid identity to D. melanogaster α-L-fucosidase, and in fact the phylogenetic tree analysis revealed that CcFUCA had closer relationships with the α-L-fucosidases of drosophilid species. The tissue expression analysis indicated that CcFuca was expressed in a single transcript in all tissues, suggesting a ubiquitous localization pattern of the encoded protein. Our findings provide novel insights on a gene encoding a protein potentially involved in primary gamete interactions in C. capitata.

Glycosidases in the plasma membrane of Ceratitis capitata spermatozoa.

Fruit flies in the family Tephritidae are rated among the world's most destructive agricultural pests. The Mediterranean fruit fly Ceratitis capitata is emerging as a model organism to study the fertilization in Insects. Three integral proteins with glycosidase activity are present in the plasma membrane of spermatozoa. The glycosidases have been purified and characterized. We have demonstrated the presence of three enzymes, a ß-N-acetylhexosaminidase, an α-mannosidase and an α-l-fucosidase. The molecular mass of the native enzymes estimated by gel filtration was 160 kDa for ß-N-acetylhexosaminidase, 310 kDa for α-mannosidase and 140 kDa for α-l-fucosidase. SDS-PAGE showed that ß-N-acetylhexosaminidase is a dimer of a single protein of 73 kDa, α-mannosidase consists of six subunits with different molecular weights and α-l-fucosidase is a dimer made up by two different monomers. Characterization of the purified enzymes included glycosylation pattern, pI, optimal pH, substrate preference, kinetic properties and thermal stability. Soluble forms similar to the sperm associated glycosidases are present. Polyclonal antibodies raised against synthetic peptides designed from the predicted products of the Drosophila melanogaster genes encoding ß-N-acetylhexosaminidase and α-l-fucosidase were used. Immunofluorescence labelling of spermatozoa showed that the enzymes are present in the sperm plasma membrane overlying the acrosome and the tail. This work represents the first report on the characterization in C. capitata of sperm proteins that are potentially involved in primary gamete recognition.