Glossiphoniids are iteroparous hirudineans that brood their offspring. The young are attached by ... more Glossiphoniids are iteroparous hirudineans that brood their offspring. The young are attached by the posterior sucker to the parent's ventral side until development is complete. The weight loss of the brooding adults is commonly attributed to their reduced possibility of feeding and to the increased metabolic expense for movements needed to ventilate the offspring. The present study showed that there is a passage of nutritive substrates between the parental body wall and the young's sucker. A possible correlation between this passage and the reabsorption of some muscle fibers that occurs in the adult's body wall is also analyzed.
In leeches, the botryoidal tissue is composed of two different cell types ± granular botryoidal c... more In leeches, the botryoidal tissue is composed of two different cell types ± granular botryoidal cells and flattened endothelial-like cells ± localized in the loose connective tissue between the gut and the body wall sac. We have observed that the botryoidal tissue undergoes functional and structural modifications in response to the different needs arising during the life-cycle of the animal. In healthy, untreated leeches, botryoidal cells are organized in cords or clusters, sometimes surrounding few, small lacunae. Conversely, in wounded animals we have observed the transition of the botryoidal tissue from cluster/cord-like structures to a hollow/tubular architecture, typical of pre-vascular structures.
Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and... more Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and endothelial markers, and which are able to differentiate into myogenic cells to contribute to muscle regeneration. In spite of intensive research efforts, numerous questions remain regarding the role of cytokine signalling on myoendothelial cell differentiation and muscle regeneration. Here we used Hirudo medicinalis (Annelid, leech) as an emerging new model to study myoendothelial cells and muscle regeneration. Although the leech has relative anatomical simplicity, it shows a striking similarity with vertebrate responses and is a reliable model for studying a variety of basic events, such as tissue repair. Double immunohistochemical analysis were used to characterize myoendothelial cells in leeches and, by injecting in vivo the matrigel biopolymer supplemented with the cytokine Vascular Endothelial Growth Factor (VEGF), we were able to isolate this specific cell population expressing myogenic and endothelial markers. We then evaluated the effect of VEGF on these cells in vitro. Our data indicate that, similar to that proposed for vertebrates, myoendothelial cells of the leech directly participate in myogenesis both in vivo and in vitro, and that VEGF secretion is involved in the recruitment and expansion of these muscle progenitor cells.
It is well documented that in the model system Aphidius ervi Haliday (Hymenoptera, Braconidae)/Ac... more It is well documented that in the model system Aphidius ervi Haliday (Hymenoptera, Braconidae)/Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) host regulation by the parasitoid larva induces in the aphid haemolymph major changes of the titer of nutritional compounds such as proteins, acylglycerols and free amino acids, in order to meet the stage-specific demands of the developing larva. Since little is known about how the larva absorbs these mobilized nutritional resources, nutrient absorption by larval stages of A. ervi was studied. In 2nd instar larvae, leucine was ten-fold accumulated in the haemocoel, and tyrosine and glutamine two-fold. Glucose and fructose were readily absorbed and fructose was extensively metabolized by larval tissues. In 3rd instars, the presence of a number of larvae that did not ingest the incubation medium enabled us to determine the respective amounts of substrate absorbed by the epidermis and the midgut. An accumulation of leucine in the haemocoel was observed only when midgut cells were involved in absorption, while the amino acid concentration within body fluids never exceeded that of the incubation medium when the uptake was performed only by epidermal cells. The immunofluorescence analysis, the mutual inhibition exerted on labeled glucose or fructose uptakes by a 100-fold excess of the sugars and the strong inhibition of uptakes induced by 0.2mM cytochalasin B support the expression of facilitative GLUT2-like transporters in the apical and basal cell membranes of midgut epithelial cells. Taken together, these results prove that both midgut and epidermis are involved in nutrient absorption throughout the parasitoid development, that GLUT2 transporters are responsible for glucose and fructose uptakes and that the chemical gradient that favors the passive influx of the two sugars is maintained by their conversion to other substrates.
The authors present the first TEM observations on the head organ, a multicellular structure housi... more The authors present the first TEM observations on the head organ, a multicellular structure housing solid inclusions discovered in 1996 by Rota & Erséus in the proximity of the brain in Grania. The cellular components, their spatial organisation and their relationships to the inclusions are described. The ultrastructural data confirm the sensory nature of the organ and corroborate its interpretation as a georeceptor. There are four receptor cells, each giving off a whorl of 50 cilia with (8+1)×2 microtubular pattern and no rootlets. The solid inclusions are opaque to the electrons and have the appearance of amorphous aggregations. They contact the sensory cilia within separate extracellular compartments lined by extensions of glial cells, whose perikarya are located on the lateral aspects of the organ. The head organ is unprecedented in annelids and its structure is probably unique among invertebrates. Cells with similarly modified cilia were known in the central nervous system of enchytraeids and also occur in Grania in the region between the head organ and the cerebral ganglion proper (here called "rostral territory"). The derivation of the head organ from this type of ciliated cells is suggested. Original light microscopy observations on the central nervous system of enchytraeids are also included.
Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) chitinase A (ChiA) is a protein ... more Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) chitinase A (ChiA) is a protein which promotes the final liquefaction of infected host larvae. The potential of this viral molecule as a new tool for insect control is explored here. The ChiA gene was isolated from the AcMNPV genome by PCR and expressed in E. coli. The recombinant protein, purified by affinity chromatography, showed both exo-and endo-chitinase activities and produced perforations on the peritrophic membrane (PM) of Bombyx mori larvae which increased in number and in size, in a dose-dependent manner. This structural alteration resulted into a significant increase of PM permeability to methylene blue and to the small neuropeptide proctolin. When the fifth instar larvae of B. mori were fed on a artificial diet supplemented with the recombinant ChiA, 100% mortality was observed at a dose of 1 mg/g of larval body weight (LW), while at sub-lethal doses of 0.56 mg/g LW, a reduced larval growth was recorded. These results indicate that AcMNPV-ChiA may offer interesting new opportunities for pest control. r
Brain-derived neurotrophic factor (BDNF) belongs to neurotrophin family, a class of molecules pla... more Brain-derived neurotrophic factor (BDNF) belongs to neurotrophin family, a class of molecules playing key roles in neuronal development, survival and regeneration, neurite growth and plasticity: memory processes are mainly affected, and mutations of the human BDNF gene are associated to cognitive and behavioural disturbances. All neurotrophins contain a highly conserved C-terminal domain and bind to the same receptor family. Both correct folding and post-translational processing of the entire preproprotein are pivotal for sorting to the extracellular space, dimerization and receptor binding. Evolutionary studies conducted so far demonstrate that a single ancestor gene underwent two independent duplication events at an early stage of vertebrate evolution, leading to the formation of the current neurotrophins. However, works focusing on BDNF evolution are scarce and fragmentary, mainly in lower vertebrates. In this work, we report cloning of eight DNA sequences from amphibians and teleosts, and analysis of the entire coding regions (cDNA sequences) of BDNF from 35 organisms, from teleosts to mammals. A phylogenetic tree was constructed and the analysis of non-synonymous-synonymous substitution rates performed for the different branches. Our results suggest that natural selection is acting on mammals, separating them from other classes. Since preproprotein cleavage and 3D structure of mature protein are important for functional activity of BDNF, we also propose a de novo prediction of the 3D structure of translates in at least one species for each class, in order to get hints about the functional constraints of the protein.
The tentacles of Sepia officinalis are muscular structures that can be quickly everted and 'super... more The tentacles of Sepia officinalis are muscular structures that can be quickly everted and 'super-elongated' to capture prey. The speed and super-elongation are achieved by the presence of both cross-striated and helical muscles. In the present study, the complex organization and differentiation of various fibers of the cuttlefish were examined from an early stage of development (stage 26), when the embryo is still inside the egg gelcoating, until the juvenile stage (two weeks after hatching). The muscles start to differentiate centrifugally from the area around the axial nervous system where two types of myoblasts can be recognized. Smooth fibers (referred to here as 'smooth-like' fibers because of their similarity to vertebrate smooth fibers) appear first, then bundles and layers of circomyarian helical and crossstriated fibers differentiate. In Sepia , two muscle-specific transcription factors (MRF), Myf5-like and MyoD-like, have been identified and they are differently expressed during development. Myf5 was detected at first in myoblasts, which give rise to helical smooth-like fibers, while MyoD was expressed later in the other population of myocytes from which circomyarian helical and cross-striated fibers derive. The effective role of these two MRF in tentacle muscle differentiation was confirmed by RNA interference experiments. Injection of double stranded (ds)RNA Myf5 inhibited differentiation of smooth-like fibers, whereas injection of dsRNA MyoD resulted in inhibition of cross-striated and circomyarian helical fibers.
The angiogenic process in vertebrates and hirudineans has been compared. The leech Hirudo medicin... more The angiogenic process in vertebrates and hirudineans has been compared. The leech Hirudo medicinalis, subjected to an angiogenic stimulus (surgical explant or cytokine treatment) responds, as a vertebrate, with the formation of an extensive network of new vessels accompanied by the production of circulating cells. The reviewed data confirm the surprising similarity between hirudinean and vertebrate processes in wound healing, and suggest that basic common events such as antigenic expressions of endothelial and hemopoietic cells, cytokine secretion and regulation as well as extracellular matrix interactions, are conserved and extended across diverse species, tissues and developmental phases.
We focused our studies on the leech, Hirudo medicinalis. This invertebrate has a relative anatomi... more We focused our studies on the leech, Hirudo medicinalis. This invertebrate has a relative anatomical simplicity and is a reliable model for studying a variety of basic events, such as tissue repair, which has a striking similarity with vertebrate responses. Hirudo is also a good invertebrate model to test the actions of drugs and gene products, since the responses evoked by the different stimuli are clear and easily detectable due to their small size and anatomical simplicity. Here we review the use of this invertebrate model to investigate muscle regeneration and the role of hematopoietic stem cells in this process.
The present work describes Aphidius ervi Haliday (Hymenoptera, Braconidae) larval anatomy and dev... more The present work describes Aphidius ervi Haliday (Hymenoptera, Braconidae) larval anatomy and development, focusing on time-related changes of body structure and cell ultrastructure, especially of the epithelial layers involved in nutrient absorption. Newly hatched 1st instar larvae of A. ervi are characterised by gut absence and a compact cluster of cells makes up their body. As the parasitoid larva develops, the central undifferentiated cell mass becomes hollowed out, leading to the formation of gut anlage. This suggests that absorption of nutrients at that stage may take place through the body surface, as more directly demonstrated by the occurrence on the epidermis of proteins associated with transepithelial transport, such as Na 1 /K 1 -ATPase and alkaline phosphatase (ALP). Second instar larvae show the presence of the gut with a well-differentiated brush border and a peritrophic membrane. Gut cells are ®lled by masses of glycogen granules and lipid droplets. The tracheal system starts to be visible. The haemocoel becomes evident in late 2nd instar, and contains large silk glands. Mature 3rd instar larvae are typically hymenopteriform. The midgut accounts for most of the body volume and is actively involved in nutrient absorption, as indicated by the well developed brush border and by the presence of Na 1 /K 1 -ATPase and ALP on the basolateral and luminal membrane respectively. At this stage, large lipid droplets have gradually replaced the cellular glycogen stores in the midgut cells. The tracheae are completely differentiated, but their internal lumen still contains ®brillar material, suggesting that they are not functional as long as host¯uids bath the parasitoid larva. In late 3rd instar larvae, silk glands, structurally similar to Malpighian tubules, show a very intense vesicular traf®c toward the internal lumen, which, eventually, results in being ®lled by secretion products, suggesting the possible recycling of metabolic waste products during mummy formation. q
Archives of Insect Biochemistry and Physiology, 1998
Fifth instar larvae of B. mori were topically or orally treated with increasing amounts of the In... more Fifth instar larvae of B. mori were topically or orally treated with increasing amounts of the Insect Growth Regulator (IGR) fenoxycarb in a single application, in order to determine its effects on the nutritional parameters, the midgut functional activities and the growth of the silk glands. The IGR affected in a dose-dependent manner the progress of the life cycle of the insect, causing a delay or inhibition of spinning, alteration of the feeding behaviour, decrease of the nutritional parameters, impairment of the growth of the silk glands, and an increased mortality during larval-pupal transformation. Measurement of leucine uptake into midgut brush border membrane vesicles and midgut histochemistry revealed a reduced absorption of leucine by the midgut and a large alteration of a number of midgut enzyme activities as a result of treatments with a high dose of fenoxycarb (2.5 mg). Treatments with a dose of 2.5 femto g/larva caused an increase in leucine uptake by the midgut, an increased weight of the cocoon shell, and a modification of some midgut enzyme activities. The lepidopteran midgut appears to be a larval organ that responds promptly to the exposure to fenoxycarb. The epithelial columnar cells modify their absorptive functions, at least with regard to amino acid uptake, as well as their metabolic activity, with a modification of the oxidative status of the cells that is detectable with a single dose of the chemical as low as few fg/larva. Arch. Insect Biochem. *Abbreviations used: AcP = acid phosphatase; AD = approximate digestibility; AlP = alkaline phosphatase; AM = anterior-middle; BBMV = brush border membrane vesicles; BrdU = 5-Bromodeoxyuridine; CI = Consumption index; CW = weight of the diet consumed; ECD = efficiency of conversion of digested food to body substance; ECI = efficiency of conversion of ingested food to body substance; FCCP = carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone; Fenoxycarb = Ethyl[2-(p-phenoxy-phenoxy) ethyl] carbamate; GR = growth rate; IGR = Insect Growth Regulator; JH = juvenile hormone; LD 50 = Dose wich is lethal to 50% of the organisms tested; Methoprene = 11-Methoxy-3,7,11-trimethyl-2,4-dodecadienoic acid 1-methylethyl ester; NADH-TR = NADH tetrazolium reductase; O.R.O = oil red O; P = posterior; PAS = periodic acid Schiff stain; ppm = part per million; SDH = succinic dehydrogenase.
AJP: Regulatory, Integrative and Comparative Physiology, 2007
Sugars are critical substrates for insect metabolism, but little is known about the transporters ... more Sugars are critical substrates for insect metabolism, but little is known about the transporters and epithelial routes that ensure their constant supply from dietary resources. We have characterized glucose and fructose uptakes across the apical and basolateral membranes of the isolated larval midgut of the aphid parasitoid Aphidius ervi. The uptake of radiolabeled glucose at the basal side of the epithelium was almost suppressed by 200 microM cytochalasin B, uninhibited by phlorizin, and showed the following decreasing rank of specificity for the tested substrates: glucose > glucosamine > fructose, with no recognition of galactose. These functional properties well agree with the expression of GLUT2-like transporters in this membrane. When the apical surface of the epithelium was also exposed to the labeled medium, a cation-dependent glucose uptake, inhibited by 10 microM phlorizin and by an excess of galactose, was detected suggesting the presence in the apical membrane of a cation-dependent cotransporter. Radiolabeled fructose uptakes were only partially inhibited by cytochalasin B. SGLT1-like and GLUT5-like transporters were detected in the apical membranes of the epithelial cell by immunocytochemical experiments. These results, along with the presence of GLUT2-like transporters both in the apical and basolateral cell membranes of the midgut, as we recently demonstrated, allow us to conclude that the model for sugar transepithelial transport in A. ervi midgut appears to be unexpectedly similar to that recently proposed for sugar intestinal absorption in mammals.
Basic studies on angiogenesis in normal and pathologic conditions, as well as research on drugs o... more Basic studies on angiogenesis in normal and pathologic conditions, as well as research on drugs or genes/proteins that stimulate or regulate the angiogenic process, can rely on an increasing number of experimental models. Among non-mammalian models, Zebrafish is adopted by an increasing number of research groups. Moreover, angiogenesis and vasculogenesis in invertebrates like the leech Hirudo medicinalis share a high degree of similarity with the same processes occurring in humans, both under the structural/functional and biochemical points of view. Interestingly, Hirudo angiogenic growth factor receptors respond to corresponding human/mammalian recombinant growth factors and cytokines; in addition, Hirudo endogenous angiogenic growth factors and receptors react with antibodies against their human/mammalian counterparts. Furthermore, as it will be shown in this review, Hirudo has the unique advantage of having a virtually avascular muscular body wall, whereas the reliability of such a peculiar feature as a model for physiologically vascularised mammalian tissues has to be thoroughly investigated. Hirudo has proven so far to allow unambiguous, clear-cut studies on the angiogenic potential of gene-products or drugs, as well as on the anti-angiogenic compounds. This article will review the biology of angiogenesis in Hirudo and the data so far collected on angiogenesis stimulation/modulation in this model; an example describing a study on the biological activity of a naked DNA vector for angiogenesis gene therapy will also be provided.
Functional and morphological modifications in the IPLB-LdFB insect cell line were examined follow... more Functional and morphological modifications in the IPLB-LdFB insect cell line were examined following a short treatment with a reversible inhibitor of mitochondrial ATP synthase, oligomycin A, and subsequent incubation for various times in oligomycin-A-free medium. Oncosis, apoptosis and autophagy at variable percentages were observed under the various experimental conditions. Together with oncotic and apoptotic pathways that lead directly to cell death, the insect cells responded to ATP depletion with autophagy. Our results revealed that, in most cases, autophagy failed to restore cellular homeostasis, probably because of a massive sequestration of mitochondria in autophagic vacuoles. This critical event was a “point of no return” and ultimately resulted in cell necrosis. However, cells with a misshapen body and nucleus resembling “resistant forms” were observed at the end of the experiments. Our findings indicate that oligomycin-A-induced autophagy can promote cell protection or cell destruction and is an open-ended process that can lead to survival or death depending on a combination of concomitant factors.
The cuticle of Gordius panigettensis (Sciacchitano, 1955) was studied by scanning electron micros... more The cuticle of Gordius panigettensis (Sciacchitano, 1955) was studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM).The cuticle is composed of 30–50 compact layers. The number of the layers is higher in the central part of the animal’s body and decreases at the extremities. Each layer is composed of parallel tightly packed fibres approximately
The wound healing process in leeches involves different types of cells like macrophages, NK-like ... more The wound healing process in leeches involves different types of cells like macrophages, NK-like cells and granulocytes. These cells that are involved in immune defence, can co-operate to attack and/or isolate the non self (de Eguileor et al ., 1999; de Eguileor et al ., 2000a; de Eguileor et al ., 2000b). In addition other types of cells, like fibroblasts
Toxoneuron nigriceps is an endophagous parasitoid of larval stages of the noctuid moth Heliothis ... more Toxoneuron nigriceps is an endophagous parasitoid of larval stages of the noctuid moth Heliothis virescens. As all parasitoids, this wasp avoid host immune reaction by a combination of several passive and active mechanisms. Secretions injected by ovipositing females, which contain venom, calyx fluid and polydnaviruses, are the most probably factors actively disrupting Heliothis virescens immune system. This paper describes the
An increasing body of evidence indicates that in the leech Hirudo medicinalis the angiogenic proc... more An increasing body of evidence indicates that in the leech Hirudo medicinalis the angiogenic process is finely regulated and coordinated by the botryoidal tissue. In this paper we provide evidence on the involvement of botryoidal tissue cells in angiogenesis induced in H. medicinalis by a variety of stimuli including surgical wounds or the administration of modulators of neovascularization. Interestingly, we show that either human activators of vascular cell growth, or anti-angiogenic peptides like angiostatin and endostatin, or the drug mitomycin, can induce a prompt biological response in H. medicinalis. We show as well that angiogenesis in this invertebrate shares a surprising degree of similarity with neovascularization in vertebrates, both at the biochemical and cellular levels, because it involves similar growth factors/growth factor receptors, and relies on analogous cell-cell or cell-matrix interactions. For these reasons we suggest that H. medicinalis can be used as a repro...
Glossiphoniids are iteroparous hirudineans that brood their offspring. The young are attached by ... more Glossiphoniids are iteroparous hirudineans that brood their offspring. The young are attached by the posterior sucker to the parent's ventral side until development is complete. The weight loss of the brooding adults is commonly attributed to their reduced possibility of feeding and to the increased metabolic expense for movements needed to ventilate the offspring. The present study showed that there is a passage of nutritive substrates between the parental body wall and the young's sucker. A possible correlation between this passage and the reabsorption of some muscle fibers that occurs in the adult's body wall is also analyzed.
In leeches, the botryoidal tissue is composed of two different cell types ± granular botryoidal c... more In leeches, the botryoidal tissue is composed of two different cell types ± granular botryoidal cells and flattened endothelial-like cells ± localized in the loose connective tissue between the gut and the body wall sac. We have observed that the botryoidal tissue undergoes functional and structural modifications in response to the different needs arising during the life-cycle of the animal. In healthy, untreated leeches, botryoidal cells are organized in cords or clusters, sometimes surrounding few, small lacunae. Conversely, in wounded animals we have observed the transition of the botryoidal tissue from cluster/cord-like structures to a hollow/tubular architecture, typical of pre-vascular structures.
Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and... more Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and endothelial markers, and which are able to differentiate into myogenic cells to contribute to muscle regeneration. In spite of intensive research efforts, numerous questions remain regarding the role of cytokine signalling on myoendothelial cell differentiation and muscle regeneration. Here we used Hirudo medicinalis (Annelid, leech) as an emerging new model to study myoendothelial cells and muscle regeneration. Although the leech has relative anatomical simplicity, it shows a striking similarity with vertebrate responses and is a reliable model for studying a variety of basic events, such as tissue repair. Double immunohistochemical analysis were used to characterize myoendothelial cells in leeches and, by injecting in vivo the matrigel biopolymer supplemented with the cytokine Vascular Endothelial Growth Factor (VEGF), we were able to isolate this specific cell population expressing myogenic and endothelial markers. We then evaluated the effect of VEGF on these cells in vitro. Our data indicate that, similar to that proposed for vertebrates, myoendothelial cells of the leech directly participate in myogenesis both in vivo and in vitro, and that VEGF secretion is involved in the recruitment and expansion of these muscle progenitor cells.
It is well documented that in the model system Aphidius ervi Haliday (Hymenoptera, Braconidae)/Ac... more It is well documented that in the model system Aphidius ervi Haliday (Hymenoptera, Braconidae)/Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) host regulation by the parasitoid larva induces in the aphid haemolymph major changes of the titer of nutritional compounds such as proteins, acylglycerols and free amino acids, in order to meet the stage-specific demands of the developing larva. Since little is known about how the larva absorbs these mobilized nutritional resources, nutrient absorption by larval stages of A. ervi was studied. In 2nd instar larvae, leucine was ten-fold accumulated in the haemocoel, and tyrosine and glutamine two-fold. Glucose and fructose were readily absorbed and fructose was extensively metabolized by larval tissues. In 3rd instars, the presence of a number of larvae that did not ingest the incubation medium enabled us to determine the respective amounts of substrate absorbed by the epidermis and the midgut. An accumulation of leucine in the haemocoel was observed only when midgut cells were involved in absorption, while the amino acid concentration within body fluids never exceeded that of the incubation medium when the uptake was performed only by epidermal cells. The immunofluorescence analysis, the mutual inhibition exerted on labeled glucose or fructose uptakes by a 100-fold excess of the sugars and the strong inhibition of uptakes induced by 0.2mM cytochalasin B support the expression of facilitative GLUT2-like transporters in the apical and basal cell membranes of midgut epithelial cells. Taken together, these results prove that both midgut and epidermis are involved in nutrient absorption throughout the parasitoid development, that GLUT2 transporters are responsible for glucose and fructose uptakes and that the chemical gradient that favors the passive influx of the two sugars is maintained by their conversion to other substrates.
The authors present the first TEM observations on the head organ, a multicellular structure housi... more The authors present the first TEM observations on the head organ, a multicellular structure housing solid inclusions discovered in 1996 by Rota & Erséus in the proximity of the brain in Grania. The cellular components, their spatial organisation and their relationships to the inclusions are described. The ultrastructural data confirm the sensory nature of the organ and corroborate its interpretation as a georeceptor. There are four receptor cells, each giving off a whorl of 50 cilia with (8+1)×2 microtubular pattern and no rootlets. The solid inclusions are opaque to the electrons and have the appearance of amorphous aggregations. They contact the sensory cilia within separate extracellular compartments lined by extensions of glial cells, whose perikarya are located on the lateral aspects of the organ. The head organ is unprecedented in annelids and its structure is probably unique among invertebrates. Cells with similarly modified cilia were known in the central nervous system of enchytraeids and also occur in Grania in the region between the head organ and the cerebral ganglion proper (here called "rostral territory"). The derivation of the head organ from this type of ciliated cells is suggested. Original light microscopy observations on the central nervous system of enchytraeids are also included.
Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) chitinase A (ChiA) is a protein ... more Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) chitinase A (ChiA) is a protein which promotes the final liquefaction of infected host larvae. The potential of this viral molecule as a new tool for insect control is explored here. The ChiA gene was isolated from the AcMNPV genome by PCR and expressed in E. coli. The recombinant protein, purified by affinity chromatography, showed both exo-and endo-chitinase activities and produced perforations on the peritrophic membrane (PM) of Bombyx mori larvae which increased in number and in size, in a dose-dependent manner. This structural alteration resulted into a significant increase of PM permeability to methylene blue and to the small neuropeptide proctolin. When the fifth instar larvae of B. mori were fed on a artificial diet supplemented with the recombinant ChiA, 100% mortality was observed at a dose of 1 mg/g of larval body weight (LW), while at sub-lethal doses of 0.56 mg/g LW, a reduced larval growth was recorded. These results indicate that AcMNPV-ChiA may offer interesting new opportunities for pest control. r
Brain-derived neurotrophic factor (BDNF) belongs to neurotrophin family, a class of molecules pla... more Brain-derived neurotrophic factor (BDNF) belongs to neurotrophin family, a class of molecules playing key roles in neuronal development, survival and regeneration, neurite growth and plasticity: memory processes are mainly affected, and mutations of the human BDNF gene are associated to cognitive and behavioural disturbances. All neurotrophins contain a highly conserved C-terminal domain and bind to the same receptor family. Both correct folding and post-translational processing of the entire preproprotein are pivotal for sorting to the extracellular space, dimerization and receptor binding. Evolutionary studies conducted so far demonstrate that a single ancestor gene underwent two independent duplication events at an early stage of vertebrate evolution, leading to the formation of the current neurotrophins. However, works focusing on BDNF evolution are scarce and fragmentary, mainly in lower vertebrates. In this work, we report cloning of eight DNA sequences from amphibians and teleosts, and analysis of the entire coding regions (cDNA sequences) of BDNF from 35 organisms, from teleosts to mammals. A phylogenetic tree was constructed and the analysis of non-synonymous-synonymous substitution rates performed for the different branches. Our results suggest that natural selection is acting on mammals, separating them from other classes. Since preproprotein cleavage and 3D structure of mature protein are important for functional activity of BDNF, we also propose a de novo prediction of the 3D structure of translates in at least one species for each class, in order to get hints about the functional constraints of the protein.
The tentacles of Sepia officinalis are muscular structures that can be quickly everted and 'super... more The tentacles of Sepia officinalis are muscular structures that can be quickly everted and 'super-elongated' to capture prey. The speed and super-elongation are achieved by the presence of both cross-striated and helical muscles. In the present study, the complex organization and differentiation of various fibers of the cuttlefish were examined from an early stage of development (stage 26), when the embryo is still inside the egg gelcoating, until the juvenile stage (two weeks after hatching). The muscles start to differentiate centrifugally from the area around the axial nervous system where two types of myoblasts can be recognized. Smooth fibers (referred to here as 'smooth-like' fibers because of their similarity to vertebrate smooth fibers) appear first, then bundles and layers of circomyarian helical and crossstriated fibers differentiate. In Sepia , two muscle-specific transcription factors (MRF), Myf5-like and MyoD-like, have been identified and they are differently expressed during development. Myf5 was detected at first in myoblasts, which give rise to helical smooth-like fibers, while MyoD was expressed later in the other population of myocytes from which circomyarian helical and cross-striated fibers derive. The effective role of these two MRF in tentacle muscle differentiation was confirmed by RNA interference experiments. Injection of double stranded (ds)RNA Myf5 inhibited differentiation of smooth-like fibers, whereas injection of dsRNA MyoD resulted in inhibition of cross-striated and circomyarian helical fibers.
The angiogenic process in vertebrates and hirudineans has been compared. The leech Hirudo medicin... more The angiogenic process in vertebrates and hirudineans has been compared. The leech Hirudo medicinalis, subjected to an angiogenic stimulus (surgical explant or cytokine treatment) responds, as a vertebrate, with the formation of an extensive network of new vessels accompanied by the production of circulating cells. The reviewed data confirm the surprising similarity between hirudinean and vertebrate processes in wound healing, and suggest that basic common events such as antigenic expressions of endothelial and hemopoietic cells, cytokine secretion and regulation as well as extracellular matrix interactions, are conserved and extended across diverse species, tissues and developmental phases.
We focused our studies on the leech, Hirudo medicinalis. This invertebrate has a relative anatomi... more We focused our studies on the leech, Hirudo medicinalis. This invertebrate has a relative anatomical simplicity and is a reliable model for studying a variety of basic events, such as tissue repair, which has a striking similarity with vertebrate responses. Hirudo is also a good invertebrate model to test the actions of drugs and gene products, since the responses evoked by the different stimuli are clear and easily detectable due to their small size and anatomical simplicity. Here we review the use of this invertebrate model to investigate muscle regeneration and the role of hematopoietic stem cells in this process.
The present work describes Aphidius ervi Haliday (Hymenoptera, Braconidae) larval anatomy and dev... more The present work describes Aphidius ervi Haliday (Hymenoptera, Braconidae) larval anatomy and development, focusing on time-related changes of body structure and cell ultrastructure, especially of the epithelial layers involved in nutrient absorption. Newly hatched 1st instar larvae of A. ervi are characterised by gut absence and a compact cluster of cells makes up their body. As the parasitoid larva develops, the central undifferentiated cell mass becomes hollowed out, leading to the formation of gut anlage. This suggests that absorption of nutrients at that stage may take place through the body surface, as more directly demonstrated by the occurrence on the epidermis of proteins associated with transepithelial transport, such as Na 1 /K 1 -ATPase and alkaline phosphatase (ALP). Second instar larvae show the presence of the gut with a well-differentiated brush border and a peritrophic membrane. Gut cells are ®lled by masses of glycogen granules and lipid droplets. The tracheal system starts to be visible. The haemocoel becomes evident in late 2nd instar, and contains large silk glands. Mature 3rd instar larvae are typically hymenopteriform. The midgut accounts for most of the body volume and is actively involved in nutrient absorption, as indicated by the well developed brush border and by the presence of Na 1 /K 1 -ATPase and ALP on the basolateral and luminal membrane respectively. At this stage, large lipid droplets have gradually replaced the cellular glycogen stores in the midgut cells. The tracheae are completely differentiated, but their internal lumen still contains ®brillar material, suggesting that they are not functional as long as host¯uids bath the parasitoid larva. In late 3rd instar larvae, silk glands, structurally similar to Malpighian tubules, show a very intense vesicular traf®c toward the internal lumen, which, eventually, results in being ®lled by secretion products, suggesting the possible recycling of metabolic waste products during mummy formation. q
Archives of Insect Biochemistry and Physiology, 1998
Fifth instar larvae of B. mori were topically or orally treated with increasing amounts of the In... more Fifth instar larvae of B. mori were topically or orally treated with increasing amounts of the Insect Growth Regulator (IGR) fenoxycarb in a single application, in order to determine its effects on the nutritional parameters, the midgut functional activities and the growth of the silk glands. The IGR affected in a dose-dependent manner the progress of the life cycle of the insect, causing a delay or inhibition of spinning, alteration of the feeding behaviour, decrease of the nutritional parameters, impairment of the growth of the silk glands, and an increased mortality during larval-pupal transformation. Measurement of leucine uptake into midgut brush border membrane vesicles and midgut histochemistry revealed a reduced absorption of leucine by the midgut and a large alteration of a number of midgut enzyme activities as a result of treatments with a high dose of fenoxycarb (2.5 mg). Treatments with a dose of 2.5 femto g/larva caused an increase in leucine uptake by the midgut, an increased weight of the cocoon shell, and a modification of some midgut enzyme activities. The lepidopteran midgut appears to be a larval organ that responds promptly to the exposure to fenoxycarb. The epithelial columnar cells modify their absorptive functions, at least with regard to amino acid uptake, as well as their metabolic activity, with a modification of the oxidative status of the cells that is detectable with a single dose of the chemical as low as few fg/larva. Arch. Insect Biochem. *Abbreviations used: AcP = acid phosphatase; AD = approximate digestibility; AlP = alkaline phosphatase; AM = anterior-middle; BBMV = brush border membrane vesicles; BrdU = 5-Bromodeoxyuridine; CI = Consumption index; CW = weight of the diet consumed; ECD = efficiency of conversion of digested food to body substance; ECI = efficiency of conversion of ingested food to body substance; FCCP = carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone; Fenoxycarb = Ethyl[2-(p-phenoxy-phenoxy) ethyl] carbamate; GR = growth rate; IGR = Insect Growth Regulator; JH = juvenile hormone; LD 50 = Dose wich is lethal to 50% of the organisms tested; Methoprene = 11-Methoxy-3,7,11-trimethyl-2,4-dodecadienoic acid 1-methylethyl ester; NADH-TR = NADH tetrazolium reductase; O.R.O = oil red O; P = posterior; PAS = periodic acid Schiff stain; ppm = part per million; SDH = succinic dehydrogenase.
AJP: Regulatory, Integrative and Comparative Physiology, 2007
Sugars are critical substrates for insect metabolism, but little is known about the transporters ... more Sugars are critical substrates for insect metabolism, but little is known about the transporters and epithelial routes that ensure their constant supply from dietary resources. We have characterized glucose and fructose uptakes across the apical and basolateral membranes of the isolated larval midgut of the aphid parasitoid Aphidius ervi. The uptake of radiolabeled glucose at the basal side of the epithelium was almost suppressed by 200 microM cytochalasin B, uninhibited by phlorizin, and showed the following decreasing rank of specificity for the tested substrates: glucose > glucosamine > fructose, with no recognition of galactose. These functional properties well agree with the expression of GLUT2-like transporters in this membrane. When the apical surface of the epithelium was also exposed to the labeled medium, a cation-dependent glucose uptake, inhibited by 10 microM phlorizin and by an excess of galactose, was detected suggesting the presence in the apical membrane of a cation-dependent cotransporter. Radiolabeled fructose uptakes were only partially inhibited by cytochalasin B. SGLT1-like and GLUT5-like transporters were detected in the apical membranes of the epithelial cell by immunocytochemical experiments. These results, along with the presence of GLUT2-like transporters both in the apical and basolateral cell membranes of the midgut, as we recently demonstrated, allow us to conclude that the model for sugar transepithelial transport in A. ervi midgut appears to be unexpectedly similar to that recently proposed for sugar intestinal absorption in mammals.
Basic studies on angiogenesis in normal and pathologic conditions, as well as research on drugs o... more Basic studies on angiogenesis in normal and pathologic conditions, as well as research on drugs or genes/proteins that stimulate or regulate the angiogenic process, can rely on an increasing number of experimental models. Among non-mammalian models, Zebrafish is adopted by an increasing number of research groups. Moreover, angiogenesis and vasculogenesis in invertebrates like the leech Hirudo medicinalis share a high degree of similarity with the same processes occurring in humans, both under the structural/functional and biochemical points of view. Interestingly, Hirudo angiogenic growth factor receptors respond to corresponding human/mammalian recombinant growth factors and cytokines; in addition, Hirudo endogenous angiogenic growth factors and receptors react with antibodies against their human/mammalian counterparts. Furthermore, as it will be shown in this review, Hirudo has the unique advantage of having a virtually avascular muscular body wall, whereas the reliability of such a peculiar feature as a model for physiologically vascularised mammalian tissues has to be thoroughly investigated. Hirudo has proven so far to allow unambiguous, clear-cut studies on the angiogenic potential of gene-products or drugs, as well as on the anti-angiogenic compounds. This article will review the biology of angiogenesis in Hirudo and the data so far collected on angiogenesis stimulation/modulation in this model; an example describing a study on the biological activity of a naked DNA vector for angiogenesis gene therapy will also be provided.
Functional and morphological modifications in the IPLB-LdFB insect cell line were examined follow... more Functional and morphological modifications in the IPLB-LdFB insect cell line were examined following a short treatment with a reversible inhibitor of mitochondrial ATP synthase, oligomycin A, and subsequent incubation for various times in oligomycin-A-free medium. Oncosis, apoptosis and autophagy at variable percentages were observed under the various experimental conditions. Together with oncotic and apoptotic pathways that lead directly to cell death, the insect cells responded to ATP depletion with autophagy. Our results revealed that, in most cases, autophagy failed to restore cellular homeostasis, probably because of a massive sequestration of mitochondria in autophagic vacuoles. This critical event was a “point of no return” and ultimately resulted in cell necrosis. However, cells with a misshapen body and nucleus resembling “resistant forms” were observed at the end of the experiments. Our findings indicate that oligomycin-A-induced autophagy can promote cell protection or cell destruction and is an open-ended process that can lead to survival or death depending on a combination of concomitant factors.
The cuticle of Gordius panigettensis (Sciacchitano, 1955) was studied by scanning electron micros... more The cuticle of Gordius panigettensis (Sciacchitano, 1955) was studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM).The cuticle is composed of 30–50 compact layers. The number of the layers is higher in the central part of the animal’s body and decreases at the extremities. Each layer is composed of parallel tightly packed fibres approximately
The wound healing process in leeches involves different types of cells like macrophages, NK-like ... more The wound healing process in leeches involves different types of cells like macrophages, NK-like cells and granulocytes. These cells that are involved in immune defence, can co-operate to attack and/or isolate the non self (de Eguileor et al ., 1999; de Eguileor et al ., 2000a; de Eguileor et al ., 2000b). In addition other types of cells, like fibroblasts
Toxoneuron nigriceps is an endophagous parasitoid of larval stages of the noctuid moth Heliothis ... more Toxoneuron nigriceps is an endophagous parasitoid of larval stages of the noctuid moth Heliothis virescens. As all parasitoids, this wasp avoid host immune reaction by a combination of several passive and active mechanisms. Secretions injected by ovipositing females, which contain venom, calyx fluid and polydnaviruses, are the most probably factors actively disrupting Heliothis virescens immune system. This paper describes the
An increasing body of evidence indicates that in the leech Hirudo medicinalis the angiogenic proc... more An increasing body of evidence indicates that in the leech Hirudo medicinalis the angiogenic process is finely regulated and coordinated by the botryoidal tissue. In this paper we provide evidence on the involvement of botryoidal tissue cells in angiogenesis induced in H. medicinalis by a variety of stimuli including surgical wounds or the administration of modulators of neovascularization. Interestingly, we show that either human activators of vascular cell growth, or anti-angiogenic peptides like angiostatin and endostatin, or the drug mitomycin, can induce a prompt biological response in H. medicinalis. We show as well that angiogenesis in this invertebrate shares a surprising degree of similarity with neovascularization in vertebrates, both at the biochemical and cellular levels, because it involves similar growth factors/growth factor receptors, and relies on analogous cell-cell or cell-matrix interactions. For these reasons we suggest that H. medicinalis can be used as a repro...
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