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Toxicon 53 (2009) 367–374

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Toxicon

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Comparative morphological study of the venom glands of the centipedeCryptops iheringi, Otostigmus pradoi and Scolopendra viridicornis

Marta M. Antoniazzi a, Catia M. Pedroso a, Irene Knysak b, Rosana Martins b,Samuel P.G. Guizze b, Carlos Jared a, Katia C. Barbaro c,*

a Laboratorio de Biologia Celular, Instituto Butantan, Sao Paulo, Brazilb Laboratorio de Artropodes, Instituto Butantan, Sao Paulo, Brazilc Laboratorio de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, Butantan, CEP 05503-900 Sao Paulo, SP, Brazil

a r t i c l e i n f o

Article history:Received 21 August 2008Received in revised form 4 December 2008Accepted 8 December 2008Available online 14 December 2008

Keywords:CentipedesVenom glandsMorphologyScolopendraCryptopsOtostigmus

* Corresponding author. Tel.: þ55 11 37267222; faE-mail address: kbarbaro@butantan.gov.br (K.C.

0041-0101/$ – see front matter � 2008 Elsevier Ltddoi:10.1016/j.toxicon.2008.12.010

a b s t r a c t

Centipedes are widely distributed over all the continents. As they are well adapted tourban areas they can often cause accidents to humans by injecting venom produced in theglands located inside their maxillipeds. The fine morphology of the centipede venomglands is practically unknown. This present study is the first comparative report on thehistology, histochemistry and ultrastructure of the venom glands of the centipede speciesresponsible for the majority of accidents to humans in Brazil: Scolopendra viridicornis,Cryptops iheringi and Otostigmus pradoi. In all species the glands are basically composed ofcolumnar secretory cells radially disposed side by side, individually opening through poresin a central chitinous duct. Each secretory cell is covered by striated muscular fibres. Thesecretion has the form of small PAS positive granules and large hyaline secretory bro-mophenol blue positive vacuoles, indicating the presence of neutral polysaccharides andprotein. The secretion is conducted through the secretory cell necks to the pores, whichopen into the central chitinous duct. The results indicate a great similarity both inmorphology and primary chemical composition of the venom among the studied species,except for the size of the glands, which is proportional to the body dimensions of eachspecies.

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1. Introduction

Centipedes are arthropods belonging to the Chilopodaclass, widely distributed over all continents, except for theAntarctica (Attems, 1930; Bucherl, 1971; Knysak andMartins, 1999). They are characterized by the external bodysegmentation, with a pair of articulated legs connected toeach segment (Barnes et al., 1995). Connected to the fore-head is a pair of forceps, the maxillipeds, with venomousfang-like tips (Jangi, 1984). The venom is produced by a pairof glands one of each located in each maxilliped, withdiameters varying from 2.0 mm to 7.5 mm, depending on

x: þ55 11 37261505.Barbaro).

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the species size (Bucherl, 1946). The secretion of theseglands is used to kill or immobilize prey (Stankiewicz et al.,1999) by inoculation through horizontal movements of themaxillipeds. During daylight, centipedes look for refuge indark and humid spots such as litter, under rocks anddecomposing trunks, or inside subterranean galleries(Jangi, 1984). Because they are well adapted to urban areasand are commonly found in gardens and other peri-domiciliar areas (Knysak et al., 1994) they often causeaccidents to humans by injecting venom as a defence.

In Brazil, epidemiological data on accidents with centi-pedes are very scarce. However, two retrospective studiescomprising occurrences registered at the Butantan Insti-tute, Sao Paulo, Brazil, from 1980 to 2007, showed that mostaccidents with centipedes were caused by the generaCryptops and Otostigmus (around 90%) and only 4% by

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Scolopendra (Knysak et al., 1998; Medeiros et al., 2008).Hands and feet are usually the most affected body regions(Knysak et al., 1998; Barroso et al., 2001; Medeiros et al.,2008). Symptoms are characterized by burning pain,paresthesia and edema, discrete local haemorrhage, andsometimes can evolve to superficial necrosis (Knysak et al.,1998; Bush et al., 2001; Barroso et al., 2001; Medeiros et al.,2008). Systemic reaction, although rare, can occur and mayevolve to serious envenomation (Mumcuoglu and Leibocivi,1989; Harada et al., 1998; Rodriguez-Acosta et al., 2000;Ozsarac et al., 2004; Wang et al., 2004; Hasan and Hassan,2005; Yildiz et al., 2006).

The toxinological study of centipede venom has alsobeen neglected. The scarce literature usually refers tospecies from the Scolopendridae family, specially the genusScolopendra (Gomes et al., 1983; Mohamed et al., 1980,1983; Menez et al., 1990). Recently, Malta et al. (2008)studied the biochemistry and pharmacological effects ofthe venoms of Scolopendra viridicornis, Cryptops iheringiand Otostigmus pradoi. The authors observed differences incomposition and toxicity among these venoms.

In relation to the morphological study of centipedevenom glands the scenario is even poorer. Although theirgeneral morphology has been known since the 19thcentury (Jangi, 1984), practically only the genus Scolopendrahas raised much interest. Even then, papers on this subjectare rare. Besides the descriptions of Barth (1967) andBucherl (1946, 1971), only a few authors have given atten-tion to this theme (Dass and Jangi, 1978; Nagpal and Kan-war, 1981; Jangi, 1984; Menez et al., 1990). From theultrastructural viewpoint, Dass and Jangi (1978) and Menezet al. (1990) made the most detailed description of theseglands in Scolopendra morsitans and Ethmostigmus rubripes,respectively. Based on this previous data, the present workcompares the histology, histochemistry and ultrastructureof the venom glands of representative species of the threegenera that cause the most accidents to humans in Brazil:S. viridicornis, C. iheringi and O. pradoi.

2. Material and methods

2.1. Animals

Three specimens of each one of the following specieswere collected: S. viridicornis Newport, 1844 (family Sco-lopendridae) (Fig. 1A), from Tocantins State, C. iheringiBrolemann, 1902 (family Cryptopidae) (Fig. 2A) andO. pradoi Bucherl, 1939 (family Scolopendridae) (Fig. 2C)both from Sao Paulo State.

2.2. Histology

The animals were sacrificed by anoxia inside a chambersaturated with carbonic gas. The maxillipeds were removedfrom the body and the chitin was cut longitudinally. Afterfixation in Karnovsky’s fixative, pH 7.2 (Karnovsky, 1965)for 24 h, the glands were dissected, dehydrated in ethanol,and processed for histology using glycol methacrylate(Leica) as the embedding medium. Sections about 2 mmthick were stained by toluidine blue–fuchsine(Junqueira, 1995).

2.3. Histochemistry

The sections were submitted to the methods of peri-odic-acid Schiff (PAS) and Alcian blue, pH 2.5, for identifi-cation of neutral and acidic mucosubstances, respectively,bromophenol blue, for protein identification and Sudanblack B for lipids identification (Bancrof and Stevens, 1996).

2.4. Photomicrography

Photomicrographs were obtained in an Olympus BX51microscope and in an Olympus SZ stereomicroscope,equipped with a digital camera and Image-Pro Expresssoftware (Media Cybernetics).

2.5. Transmission electron microscopy

Transversal sections of the glands were post-fixed in 1%osmium tetroxide, contrasted in 1% uranyl acetate, dehy-drated and embedded in spurr resin. Ultra thin sections(60 nm) were obtained in a Sorvall MT6000 ultramicro-tome, contrasted in 2% uranyl acetate and lead citrate, andexamined in a LEO 906E, operating in 100 kV.

3. Results

3.1. General morphology

In the three species the glands are located inside theexternal lateral face of each maxilliped and are completelyinvolved by the musculature. In the three species theirlength represents 5–6% of the total adult animal length,which varies between 40 and 80 mm in O. pradoi,60–100 mm in C. iheringi and 160–200 mm in S. viridicornis.Depending on the species, the glands vary in shape. Theyare cylindrical in S. viridicornis (Fig. 1C), while in C. iheringiand O. pradoi they show a polyhedrical shape (Fig. 2B and D,respectively). They communicate with the exterior bya chitinous duct, opening subterminally at the pointed tipof each one of the maxillipeds, resembling a syringe needle(Fig. 1B).

3.2. Histology

The venom gland histology is similar in the threespecies. In transversal sections, the glands are observedinvolved by a layer of striated muscular cells, which is fol-lowed by columnar secretory cells, radially disposed side byside, parallel to the smaller gland axis (Figs. 1D and 3A). Thegland diameter in these sections measured 392 � 44 mm inC. iheringi, 448 � 118 mm in O. pradoi and 716 � 50 mm inS. viridicornis. The secretory cells open in the apical regionto a duct which runs eccentrically along the larger glandaxis. As a consequence the cells are not homogeneous inheight, varying from about 90 to 290 mm in C. iheringi, 130–360 mm in O. pradoi, 150–440 mm in S. viridicornis. Theirenlarged base contains the nuclei, which are spherical, witha predominance of loose chromatin and one or twoconspicuous nucleoli (Fig. 3A). The secretory cells areindividually inserted among slender walls composed ofcells with a very thin and scarce cytoplasm and longitudinal

Fig. 1. Scolopendra viridicornis (A) photo of a specimen. (B) Fang-like maxilliped showing the subterminal pore (arrow). (C) Cylindrical transversal section throughthe venom gland. Note the radial arrangement of the secretory cells (stars) and the central duct with a cylindrical lumen (L). Toluidine blue–fuchsine stain. (D)Higher magnification of (C) showing the columnar secretory cells (SC) with the cytoplasm full of hyaline secretion. The arrows indicate the nuclei of the secretorycells. L – lumen. Toluidine blue–fuchsine stain. (E) Part of a transversal section through the venom gland unpublished to PAS. Most of the cytoplasm of thesecretory cells is weakly positive to the reaction (stars) although not in a homogeneous form. The arrows indicate accumulations of small high-positive granules.L – lumen. (F) Part of a transversal section through the venom gland submitted to bromophenol blue. Most secretory cells are strongly reactive to the method(stars) demonstrating that the secretion is rich in protein material. Note the negative result in the cells’ basal cytoplasm (arrowheads). The heterogeneousresponse for the method indicates different grades of secretion maturation among the cells. The arrows indicate the existing space around the secretory cells.

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striated muscular fibres (Fig. 3E). These latter are connectedto the peripheral muscular layer. The secretion occupies thegreater part of the secretory cell volume and is observed indifferent maturation stages (Fig. 3B, D and E). The basalcytoplasm shows a predominance of strongly stained smallgranules (around 2 mm in diameter), which gradually coa-lesce and acquire a light coloration, forming large andhomogeneous secretory vacuoles occupying most of thecytoplasm volume (Fig. 3B). The mature content of the largevacuoles is discharged through perforations in thechitinous wall lining the eccentrical duct, which is cylin-drical (with diameters measuring about 30 mm in O. pradoi,45 mm in C. iheringi and 80 mm in S. viridicornis) and runsalong the longer axis of the gland towards the subterminal

pore (Fig. 3C). In the apical region, before reaching the duct,the secretory cells straighten, forming necks with smallspherical granules in the cytoplasm, which are sided byepidermal cells with ellipsoidal nuclei (Fig. 3C).

3.3. Histochemistry

Similar results were obtained for the three species. Forthis reason, despite the results being illustrated only forS. viridicornis venom glands, they are also valid for C. iher-ingi and O. pradoi. The secretory cells’ content showedpositive to bromophenol blue although in a very hetero-geneous manner (Fig. 1F). Differences in intensity to thismethod were observed not only among different cells but

Fig. 2. (A) Photo of Cryptops iheringi. (B) Polyhedrical transversal section through the venom gland. Note the radial arrangement of the secretory cells (stars) fullof coalescent granules and the chitinous duct with a cylindrical lumen (L). The arrows point to the epidermal cells among the secretory cells. Toluidineblue–fuchsine stain. (C) Photo of Otostigmus pradoi. (D) Transversal section through the venom gland. The secretory cells (stars) are radially arranged around theduct with a cylindrical lumen (L). Toluidine blue–fuchsine stain.

Fig. 3. (A) Part of a transversal section of Scolopendra viridicornis venom gland showing the high columnar secretory cells (SC), with basal round nuclei (arrows)and cytoplasm full of secretion differentially stained. The arrowheads point to the muscular fibres involving the gland. L - lumen. Toluidine blue–fuchsine stain.(B) Basal region of a Scolopendra viridicornis venom gland, where the secretory cells are more immature, with many small spherical granules within the cytoplasm(small arrows). The larger arrows point to the secretory cells nuclei. Toluidine blue–fuchsine. (C) Central region of Cryptops iheringi venom gland where part of thechitinous duct wall with many pores (stars) is shown. The secretory cells’ necks (arrowheads) are intercalated with epidermal cells, full of dense sphericalgranules (arrows). Mu - muscular fibres. Toluidine blue–fuchsine. (D and E) Higher magnifications of the gland of Scolopendra viridicornis showing details of thecolumnar secretory cells (SC), each one in a different maturation stage. Among them, the adjacent thin epidermal cells (N2) and the radial muscular fibres(E, arrows) are observed. N1 - nuclei of secretory cells; N3 - nucleus of a muscular fibre involving the gland. Toluidine blue–fuchsine stain.

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also within the same cells, indicating different maturationgrades of the secretion, which, as a final step, tend to bevery positive to bromophenol blue. In relation to the PASmethod (Fig. 1E), positive results were observed for thesmall spherical granules present both in the basal regionand in the apical region adjacent to the duct (cell necks) ofthe secretory cells. The rest of the cells expressed a weakpositive result to this method, which varied depending onthe secretion grade of maturation. The methods of Alcianblue pH 2.5 as well as Sudan black B were totally negative inthe gland (data not shown).

3.4. Transmission electron microscopy

In the three species, the ultrastructural aspects of thevenom glands were also similar. For this reason thedifferent figures shown here can be considered valid for allspecies.

The gland is externally covered by striated muscularfibres, running both in longitudinal and circular directions(Fig. 4B and C). These cells are involved by a basal lamina ofmedium electron density (Fig. 4A and C). In these periph-eral muscular fibres other striated fibres are anchored,

Fig. 4. Transmission electron microscopy (A) Basal region of the venom glandheterochromatic nucleus (N1) and part of a secretory cell, with spherical euchromatiendoplasmic reticulum (RER). The arrows point to the electron-dense basal lamimusculature involving the gland (stars) and a radial muscular fibre (Mu) among thshows a magnification of the region indicated by the black square, where a transvepoint to the electron-dense basal lamina involving the gland. (C) Higher magnificatio(Mu2) in the peripheral muscular fibre (Mu1). Small arrows - electron-dense basal lamvenom gland of Scolopendra viridicornis showing a secretory cell, with the spherical e(RER), a neighbouring epidermal cell with ellipsoidal nucleus (N2), and a radial mu

running centripetally towards the glandular centre amongthe secretory cells, also involved by the basal lamina(Fig. 4C). While the muscular fibres nuclei are irregular inshape, with large areas of heterochromatin, the nuclei ofthe glandular cells are spherical, with a predominance ofeuchromatin and containing one or two well-developednucleoli (Fig. 4A and D). The basal cytoplasm of the glan-dular cells is very rich in rough endoplasmic reticulum.Mitochondria are also abundant (Fig. 4C). Among themuscular and secretory cells many neuronal terminationsare seen, containing small neurosecretory vesicles (Fig. 4B).Conspicuous Golgi apparatus was also observed (Fig. 5A).The material secreted by the glandular cells is observed inthe basal cytoplasm preferentially in the form of manysmall electron-dense spherical granules. In O. pradoi, thesegranules were observed containing material in the form ofrod-like inclusions (Fig. 5B). The granules tend to coalesceand, as secretion maturation occurs, it looses electrondensity, eventually forming very large secretory vacuoleswith homogeneous electron-luscent material, occupyingpractically the whole cytoplasm. Besides the muscularfibres, running parallel to the secretory cells, very thin cellsare observed, with elongated nuclei rich in

of Otostigmus pradoi showing a peripheral muscular fibre with irregularc nucleus (N2), with a conspicuous nucleolus (n), and cytoplasm rich in roughna. (B) Basal region of the venom gland of Otostigmus pradoi showing thee secretory cells, very rich in rough endoplasmic reticulum (RER). The insertrse section of a nervous fibre full of neurosecretion, is observed. The arrowsn of (B), evidencing the anchorage spot (large arrow) of radial muscular fibreina; m - mitochondria; RER - rough endoplasmic reticulum. (D) Detail of the

uchromatic nucleus (N1) and cytoplasm rich in rough endoplasmic reticulumscular fibre (Mu). Id - interdigitations joining the cells.

Fig. 5. Transmission electron microscopy (A) Detail of a venom secretory cell of Scolopendra viridicornis, where the rough endoplasmic reticulum (RER) and theGolgi apparatus (Go) are well developed. G1 and G2 - secretory granules of different sizes; m - mitochondrium; arrowheads - coated vesicles. (B) Part of a venomsecretory cell in Otostigmus pradoi gland, showing granules (G) containing material in the form of rod-like inclusions. (C) Central region of the venom gland ofScolopendra viridicornis showing the epidermal cells, with irregular heterochromatic nuclei (N), and the secretory cell necks with electron-dense sphericalgranules (arrows) or with an electron-transparent appearance (stars). (D) Two pores of the venom gland in Scolopendra viridicornis transpassing the chitinouselectron-dense cuticle (stars) lining the duct. The electron-transparent secretory cells’ necks (sn) are involved by an electron-dense ring (arrows) and open in thepores, where membranous electron-dense structures are observed (arrowheads). N - epidermal cell nucleus.

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heterochromatin and cytoplasm prolongations frequentlydifficult to identify, except for the interdigitations joiningthem with the secretory cells (Fig. 4D).

The analysis of a more central glandular region closer tothe duct, observed in transversal sections to the gland,shows that the apical region of the secretory cells is totallyfilled with low homogeneous electron-dense secretion. Inthis region, the thin cells are more visible, with ellipsoidalor irregular nuclei rich in heterochromatin (Fig. 5C).Spherical electron-dense granules are often observed inthis region (Fig. 5C). The secretion is eliminated throughthe straightened cell apical region, which is frequentlyobserved as an empty space surrounded by a thick elec-tron-dense ring and is connected to the pores in thechitinous duct wall (Fig. 5D). The pores are always partiallyclosed by a membranous electron-dense structure whichalso seems to be composed of chitinous material.

4. Discussion

Although the general morphology of centipede venomglands has been known for a long time, studies about thissubject are scarce and most of the existing papers are onlyabout the species of genus Scolopendra, or about other

genera of family Scolopendridae (Bucherl, 1946, 1971; Dassand Jangi, 1978; Jangi, 1984; Menez et al., 1990; Nagpal andKanwar, 1981). This paper presents a comparativemorphological study of the venom glands in the genusScolopendra (family Scolopendridae) in relation to othertwo common genera of Brazilian fauna, Otostigmus,belonging to the same Scolopendridae family, and Cryptops,from the Cryptopidae family.

The most evident difference noted in the three specieswas in relation to the gland dimensions, which areproportional to the body size of each species, and theanatomical glandular shape, which is cylindrical in S. vir-idicornis and polyhedrical in C. iheringi and O. pradoi.Histological and histochemical results, in a general manner,did not show significant differences among the threestudied species and were similar to the already existingdata for other species. Histochemical results demonstrateda heterogeneous distribution of PAS and bromophenol bluepositive material within the gland. The only differencenoted so far relates to the granules containing rod-likestructures observed in the gland of O. pradoi, which arevery similar to what was described by Menez et al. (1990) inthe Australian species E. rubripes and can reflect similarityin secretion components of these two species.

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On the other hand, recent toxinological data demon-strated that there are differences in some biological andenzymatic venom effects (Malta et al., 2008). All threevenoms induce nociception, edema and myotoxicity, withdiscrete differences among them. However, only S. vir-idicornis induces hemorrhagic activity. On the other hand,C. iheringi venom was devoid of hemolytic activities (Maltaet al., 2008). Among the three species, S. viridicornisshowed the most toxic activities which must be poten-tialized by the larger amount of venom injected due to itslarger size (Malta et al., 2008). Since centipede venom isprimary used in capturing preys (Demange, 1981; Lewis,1981; Edgecombe and Giribet, 2007), the differences foundby Malta et al. (2008) are a consequence of the species-specific adaptation to kill available prey. These differences,however, have not been detected in the present study at themorphological level. This could be explained by the factthat chemical differences are subtle and the venom issynthesized by the same organelles in the three species.

In relation to the musculature, the ultrastructureobservation shows the presence of fibres involving thegland in both longitudinal and circular directions. Thesefibres, working together with the internal radial fibresthrough nervous stimulation, make it possible for theanimal to squeeze the gland in different directions, indi-vidually discharging the secretory cells’ content inside theduct and consequently liberating the venom through theexternal pore.

In the present study, besides the external and radialmuscular fibres, two basic types of cells were clearlyidentified in the gland: the columnar secretory cells andvery thin cells, forming cellular walls involving each one ofthe secretory cells. These thin cells can be easily identifiedin the internal portion of the gland, among the secretorycells’ necks, forming epidermal pockets which have alreadybeen described by Jangi (1966). Dass and Jangi (1978)studying the Indian species S. morsitans named these thincells as hypodermal cells which would be responsible forthe reposition of degenerated secretory cells by mitosis .Differently, Nagpal and Kanwar (1981), working with theIndian Otostigmus ceylonicus, considered the thin cells asbeing involved in the deposition of the chitinous cuticlewhich covers the glandular duct. The morphologicalobservation of the three species included in this study didnot indicate any evidence of degeneration of secretory cells,cell division or cell migration as suggested by Dass andJangi (1978). On the contrary, our data seem to supportthose of Nagpal and Kanwar (1981) since the constantpresence of the thin cells around the secretory cells’ necksmay indicate their relationship with the production of thechitinous duct walls.

The cytoplasm of the secretory cells showed to bepositive to bromophenol blue especially inside the largevacuoles indicating that the mature secretion is rich inprotein material. These results confirm those found byMalta et al. (2008) who found many proteins by SDS-PAGEwith a range of 7–205 kDa. In relation to PAS, the resultseems to be the opposite to that observed for bromophenolblue: while the reaction was more intense within the smallsecretion granules in the basal cytoplasm and in the cellnecks near the pores, the large vacuoles are weakly

positive, indicating small amounts of neutral poly-saccharides in the mature secretion. The presence ofnumerous small secretory granules in the secretory cells’apical region indicates, however, that the secretion dis-charged in the duct must be a mixture of polysaccharidesand protein material. The negative results for Alcian blue aswell as Sudan black B indicate, respectively, the absence ofacid polysaccharides and lipid material. These histochem-ical results are in accordance with the findings of Nagpaland Kanwar (1981) for O. ceylonicus. These authors,however, using specific methods for lipids, were also ableto identify lipids in the immature secretion which graduallydisappear during the maturation process.

Ultrastructural results are in accordance with ourhistochemical findings. The nuclei of secretory cells aremostly constituted of euchromatin and present verydeveloped nucleoli, indicating ribosomal RNA and proteinsynthesis. This is confirmed by the huge rough endoplasmicreticulum and the conspicuous Golgi apparatus in thecytoplasm. The small secretory granules, more frequent inthe basal cytoplasm, are more electron-dense than thelarge vacuoles, which may be explained by the probablesimultaneous synthesis of polysaccharides. Dass and Jangi(1978) also affirm that in S. morsitans, before the venom isready to be discharged into the duct, the secretory vesiclesgo through a stage of solubilization which can explain theirelectron-luscent appearance.

In relation to the glandular mechanism of secretionrelease, our observation of thick electron-dense ringsaround the secretory cell necks and membranous electron-dense structures partially closing the pores corresponds towhat has already been described by Dass and Jangi (1978)and Menez et al. (1990). According to Dass and Jangi (1978),these structures would be part of a mechanism composedof a sphincter (the electron-dense ring), and a nozzle-likenon-return valve (the membranous electron-dense struc-ture closing the pore), and would both work together,under neuromuscular control, in order to regulate thevenom discharge from each one of the secretory cells to theduct. Such a complex regulatory discharge mechanism maycause a variation in the amount of secretion released and,together with the differences in biological and enzymaticvenom effects observed by Malta et al. (2008), may have aninfluence on the varied envenomation clinical picturesobserved in humans (Medeiros et al., 2008), even for acci-dents caused by the same species.

The present work is the first comparative report on thehistology, histochemistry and ultrastructure of Braziliancentipedes’ venom glands. The comparison of the presentdata with that already obtained for Australian and Asianspecies shows that the general morphology and histo-chemistry of the group are very conservative. Differences inbiological activity may reflect subtle differences in chem-ical composition as a consequence of species-specificadaptation for capturing their preys.

Acknowledgements

This work was supported by FAPESP (2003/04527-1).The authors thank Simone G.S. Jared and Tatiana L. Fernandesfor their technical assistance. We also thank CNPq for the

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grant of Katia C. Barbaro (306158/2004-3) and Carlos Jared(300170/96-3). IBAMA provided animal collection permitsno. 02027.002695/2004 and CGEN provided the license forgenetic patrimony access (221/2006).

Conflict of interest

The authors declare that there is no conflict of interest.

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