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The urticating apparatus in the caterpillar of Lonomia obliqua (Lepidoptera: Saturniidae)
Toxicon 119 (2016) 218e224
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The urticating apparatus in the caterpillar of Lonomia obliqua (Lepidoptera: Saturniidae) Diva Denelle Spadacci-Morena a, *, Magna Aparecida Maltauro Soares a, Roberto Henrique Pinto Moraes b, Ida Sigueko Sano-Martins a, Juliana Mozer Sciani c ~o Paulo, SP, Brazil rio de Fisiopatologia, Instituto Butantan, Sa Laborato ~o Paulo, SP, Brazil rio Especial de Coleço ~es Zoolo gicas, Instituto Butantan, Sa Laborato c ~o Paulo, SP, Brazil rio de Bioquímica e Biofísica, Instituto Butantan, Sa Laborato a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2016 Received in revised form 9 June 2016 Accepted 14 June 2016 Available online 16 June 2016
The presence of specialized cells for venom production in the Lonomia obliqua caterpillar has long been a controversial topic. In this study, we identify a cell inside the spine that specializes in the production of toxins. Our histological study showed that this glandular cell was inserted at the subapical region of the spine, in a constricted region like a ring. This cell type was not observed in all spines of the scolus. The constricted region of the spine observed by scanning electron microscopy displayed a circular groove in which the apical portion of the spine fits perfectly; however, some spines in the same scolus lacked this groove. After breaking off the spine at the most apical region, a small drop of orange or green liquid was observed to flow from its tip. These secretions were analysed by MALDI-ToF and found to possess biochemically different compositions. The green secretion demonstrated greater similarity to the haemolymph of the caterpillar than the orange secretion. Based on our findings, the spines with a groove probably contain the venom glands and produce an orange secretion. However, it is also possible that both secretions play an important role in envenoming because all spines in contact with the skin of the accidental victim should break regardless of whether they are present in a groove. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Caterpillar Saturniidae Lonomia obliqua Gland cell Urticating scolus Morphology
1. Introduction Worldwide, the order Lepidoptera comprises approximately 160.000 described species of moths and butterflies (Gullan and Cranston, 2008) with a larval stage that is commonly defined as a caterpillar. In Brazil, some Megalopygidae and Saturniidae (families within Lepidoptera) are known to cause adverse reactions in humans, most of which are due to exposure to the larval form. Lonomia obliqua Walker, 1855 (Saturniidae, Hemileucinae) caterpillars have been found in fruit trees in rural regions of south and southeast Brazil (Lorini, 1997). Human accidents involving this caterpillar was first reported in 1989 (Kelen et al., 1995). These caterpillars present gregarious and mimetic habits and they climb down from the treetops to the trunk, increasing the likelihood of such accidents. Contact between people and these caterpillars occur mainly on the dorsal scoli of the animals. These structures are formed by the central axis from which the spines originate.
* Corresponding author. E-mail address: [email protected] (D.D. Spadacci-Morena). http://dx.doi.org/10.1016/j.toxicon.2016.06.008 0041-0101/© 2016 Elsevier Ltd. All rights reserved.
Collectively, a series of scoli symmetrically cover the caterpillar body. In South America, two species, Lonomia achelous Cramer, 1777 and Lonomia obliqua, have been reported to be responsible for severe accidents and some fatal cases. The majority of the affected patients present with mild burning pain, nausea, and headache, but progression to a severe haemorrhagic syndrome characterized by ecchymoses, haematuria, bleeding from scars and mucous membranes, intracerebral bleeding and acute renal failure frequently ~ ango and Guerrero, 2001; Carrijo-Carvalho and occurs (Arocha-Pin Chudzinski-Tavassi, 2007). This type of accident is defined as lonomism. A bleeding syndrome caused by contact with L. obliqua presents as a haemostatic disorder with moderate consumption coagulopathy without significant thrombocytopenia (Zannin et al., 2003). However, a severe haemorrhagic syndrome associated with acute renal failure has been observed in approximately 5% of patients (Chan et al., 2008; Duarte et al., 1990, 1996; Fan et al., 1998; Gamborgi et al., 2006; Riella et al., 2008). Lethality occurs in approximately 1.5e2.0% of contacts, and the growing annual
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incidence rate in southern Brazil has led to the production of an antivenom (Dias da Silva et al., 1996) that has been shown to be effective (Caovilla and Barros, 2004; Gonçalves et al., 2007). Currently, several biological activities have been described for the extracts of the spines or bristles of this caterpillar (ChudzinskiTavassi et al., 2001; Fritzen et al., 2003; Reis et al., 1999, 2001, 2006; Rocha-Campos, 1999; Seibert et al., 2003, 2004; Veiga et al., 2005). However, the biology of these animals remains poorly understood (Lorini, 1999; Lorini et al., 2004), as the morphology of the gland or tissue that is specialized for the production of this caterpillar venom (Veiga et al., 2001). The urticating apparatus of various lepidopterans has been studied (Barth, 1954a, 1954b, 1956; Barth and Junqueira, 1954; Foot, 1922; Gilmer, 1925; Lamdin et al., 2000; Deml and Dettner, 2002). All these studies concluded that the venoms are typically produced by glandular cells located in the epidermis in contact with the haemocoel or inside the spines. It has been concluded that the L. obliqua caterpillar does not possess specialized cells for venom production, and secretory epithelial cells are responsible for producing the toxins that are stored in the subcuticular space (Veiga et al., 2001). However, in Automeris incisa (Saturniidae, Hemileucinae), a caterpillar of the same subfamily of L. obliqua, the venom gland is present inside the spines (Barth, 1954a). Thus, the location and description of the venom apparatus of the L. obliqua caterpillar remain an outstanding question that requires confirmation (Vegliante and Hasenfuss, 2012). In this study, we performed a morphological study of the spines of L. obliqua caterpillars and we identified a cell in the subapical portion of the spines that is specialized for the production of toxins. 2. Materials and methods Larvae of L. obliqua caterpillars used in this study were origi and S~ nated from the Parana ao Paulo states of Brazil, that were sent to Instituto Butantan for the production of anti-lonomic serum. rio Especial de Coleço ~ es Caterpillars were identified in the Laborato gicas (Instituto Butantan). We selected only those insects that Zoolo were in the fifth and sixth instar stages and that were moving and feeding well. The caterpillars were anaesthetized under a CO2 atmosphere (Dias da Silva et al., 1996), and only the pair of dorsal scoli from the 3rd thoracic segment and the 1st to 8th abdominal segments were collected. 2.1. External morphology of the scoli Whole caterpillars, their scoli and spines were observed and photographed under a M165C stereomicroscope (Leica Microsystems Inc., Wetzlar, Germany) with a DFC420 (Leica) digital camera and with Leica Application Suite 3.1.0 imaging software. For the scanning electron microscopy (SEM) observations were used four caterpillars. The pair of dorsal scoli (3rd thoracic and 1st to 8th abdominal segments) were dissected and isolated, cleaned, kilndried at 50 C and coated with a thin layer of gold (Sputtering Balzers Union - Model 010). Preparations were examined using a LEO 435VP SEM at 20 kV. 2.2. Histology of scoli Caterpillars were fixed in Bouin’s fixative and processed for routine historesin embedding (Leica Microsystems Inc.). The fixed samples were embedded in a flat plastic mould to obtain crosssections of the animals. Because scoli resemble a tree-shape and their axis is perpendicular to the integument, we obtained mainly obliqua and transverse spines sections. Some spines were
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processed individually to obtain longitudinal sections. Threemicron-thick sections were stained with basic fuchsin and toluidine blue and then examined and photographed under a light microscope DM LS with a DFC420 digital camera and Leica Application Suite 3.1.0 imaging software (Leica Microsystems Inc.). 2.3. Biochemical analysis of the spine secretions and the haemolymph The dorsal scoli of the caterpillars were separated from the tegument, and then the spines were broken at their most apical portion (Fig. 1d). The released secretion was collected using a capillary tube. The haemolymph was collected from the haemocoel at a region distal from the spines using an insulin syringe. The secretions obtained from the different spines and the haemolymph were stored in phosphate buffer (0.1 M, pH 7.2) at 20 C until analysis. Secretions from spines of L. obliqua caterpillars and haemolymph were analysed by MALDI-ToF (Axima Performance, Shimadzu Corp., Kyoto, Japan) using both linear and reflectron modes. Samples were mixed with an appropriated matrix: sinapinic acid for the linear mode and a-cyano-4-hydroxycinnamic acid for the reflectron mode. Data were acquired under positive ionization mode within a range from 200 to 10,000 m/z (reflectron) and 10,000e100,000 m/z (linear). A peak list was obtained automatically by the software controlling the equipment. 3. Results and discussion Lonomia obliqua caterpillar presents scoli of the type “VII e urticating scolus” as described by Deml and Dettner (2002). Fifth and sixth instar larvae of L. obliqua caterpillars displayed scoli that were symmetrically distributed throughout the body of the animal in the dorsal, subdorsal, lateral and subventral regions (Fig. 1a). We analysed only the scoli from the two rows in the dorsal region (Fig. 1b) because they are probably the first scoli to come in contact with the skin of the victim. Scoli are composed of a central axis with side branches, resembling small trees. The ramifications, herein named spines, are rigid structures with colors that range from light to dark brown. The spines have a conical shape with a very thin tip. The size of the spines typically varies in the same scolus (Fig. 1b and c). The products of the gland secretion are inoculated into the skin of the victim through the spines. At the time of an accident, the most apical portion of the spines is broken off in the skin of the victims, and the poison is released (Gilmer, 1925). The mechanism by which the venom is expelled from the spine is not completely understood. One possibility is that local pressure is induced by the haemolymph, and the other possibility is that the release is caused by contraction of intersegmental muscles (Maschwitz and Kloft, 1971). Indeed, an intrinsic animal activity must be present for secretion release because, when we isolated one scolus and broke off the most distal portion of the spine, it was necessary to apply a mechanical pressure to release the secretion (Fig. 1d). After breaking off a spine, we observed a small drop of green or orange liquid flow from its tip (Fig. 1e and f). There was not a clear relationship between the spine colour and the colour of its secretion (i.e., both light and dark spines produced green and orange secretion). L. obliqua caterpillars in the 5th or 6th instar stage possessed scoli with closed spines, without communication with the external environment. However, when the tip was broken off, a canal was observed inside the spine (Fig. 2a). Furthermore, according to the external morphology, in the subapical region, some spines possessed a circular groove similar to a ring-like structure, within
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Fig. 1. Photomicrographs of the Lonomia obliqua caterpillar (Saturnidae) at the sixth larval instar stage. a- General view. b- Details of scoli from the median dorsal region. c- Scolus isolated from the median dorsal region. Note that the scolus has a central axis from which originate lateral branches (spines) with different colors and sizes. d- Apical region of the spine where it was broke off (scissors) and subapical region where it was pressured (arrow) to collect secretions. e- Details of a spine broken at its tip showing a secretion derived from the interior of the spine. f- Observe a small drop with green colour on the left and orange on the right that exuded from the interior of different spines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. SEM micrographs of spines from dorsal scoli from a sixth instar larva. a- Details of the fractured tip, including an internal canal (*). b and c- Spines with pointed and closed tips showing the presence (b) or absence (c) of a ring-like constriction around the spine (groove).
which the apical portion of the spine fits perfectly (Fig. 2b). This groove has been observed previously by Veiga et al. (2001). Barth (1956) described a similar structure in the spines of Sibine nesea that was considered the place destined for breakage during an
accident. In the present study, we observed spines with a similar circular groove and spines in the same scolus lacked a groove (Fig. 2c). Although many urticating apparatuses of caterpillars have been
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described by SEM, histological observations were essential to localize and describe the venom gland in our study. We generated transverse sections of the entire caterpillar body, as shown in Fig. 3a, to examine both the integument and the internal cavity of the caterpillar. This type of section was important to establish the relationship of the scolus with the body of the animal. Several serial sections were generated to evaluate the integument and spines along its length. The literature indicates that the venom gland can be found in different places in the body of the caterpillar, depending on the species studied. The venom gland can be found within structures called “funnel warts”, as in many representatives of the Lymantriidae family (currently Erebidae, Lymantriinae) (Deml and Dettner, 1997, 2001), or at the base of balloon hairs in the Lymantria dispar caterpillar, which also belongs to the Lymantriidae family (Deml and Dettner, 1995). By contrast, the venom gland is described inside of the scolus in some species of the Saturniidae (Barth,
Fig. 3. a- Localization and section plane used in this study, indicating the orientation of the histological sections. 3b- Schematic representations of the relationship of the scolus to the body of the caterpillar. In the haemocoel, muscle bundle (Mu), fatty body (Fb) and trachea (T) can be observed. The internal cavity is separated from the integument by a basal membrane (arrows). Along the integument, composed of epithelial cells (Ep) and chitin cuticle (cu), an evagination of the tegument itself is observed originating the scolus. Several spines (S) are formed from the central axis of the scolus. The central axis and the spines are covered with epithelial cells (Ep) and the cuticle (cu). Haemocytes (h) are observed throughout the body of the animal, even within the central axis of the scolus (*) but not within the spines.
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1954a; Deml and Dettner, 1990, 1993, 1997, 2003) and Nymphalidae families (Osborn et al., 1999). Moreover, in some cases the venom gland can be found in the cuticle without reaching the body cavity, as in Sibinea nesea (Limacodidae) (Barth, 1956), or under the tegument of larvae, as in some caterpillars of the Megalopygidae (Barth, 1954b; Eagleman, 2008) and Saturniidae families (Deml and Dettner, 1994). The haemocoel of the caterpillar contained muscles, fatty bodies, haemocytes and the tracheal system characteristic of insects (Figs. 3b, 4a and b). The surface of the caterpillar body was covered by the chitinous cuticle and traversed by wide canals called scoli (Figs. 3b and 4a). The scolus is an evagination of the epithelial cells of the tegument formed by a main central calibre axis and the lateral branches that form spines (Figs. 3b and 4a). When a caterpillar was sectioned in its middle region (Fig. 3b), the relationship between the animal’s body and scolus was clearly visualized, supporting the studies of Gilmer (1925) regarding the origin of the scolus. The central axis cuticle was covered by an epithelium with a morphology similar to the epithelial wall of the animal (Fig. 3b). Along this central axis and in the internal cavity of the caterpillar, there were several different lineages of haemocytes with variable morphology (Figs. 3b and 4b). However, haemocytes were not observed within the spines. The epidermis, a single layer of epithelial, cuboidal and juxtaposed cells with a slightly basophilic cytoplasm, was present just beneath the cuticle. These cells contained a central nucleus with one or more evident nucleoli, with punctiform chromatin clumps (Fig. 4a and b). A basal membrane (Figs. 3b, 4a and b) separated the epidermis from the general body cavity of the caterpillar (haemocoel). The epidermis region and cuticle were carefully examined and no complex of secretory cells related to venom production was observed at the base of the scoli or even attached to the animal’s body cavity, similar to the arrangement described for L. obliqua (Veiga et al., 2001). The spines were also covered by chitinous cuticle and flat epithelial cells, which exhibited nuclear chromatin that was grouped into small clumps. These cells, like those of the animal wall, were regularly arranged and encircling the lumen of the spines (Fig. 4cee). The apical region of the spines was covered with cuticle and had a central canal (Fig. 4d), and no cells were found in this region. In the longitudinal sections, we observed a constriction in the subapical region of some spines, in which the most apical part fits perfectly into the other part of the spine (Fig. 4d) as previously mentioned for the SEM figure (Fig. 2b). Our histological sections of the spines revealed a large glandular cell with a morphology that was similar to the glandular cell of the Automeris incisa caterpillar (Barth, 1954a), which is phylogenetically close to L. obliqua. This unique cell with a saccular structure lies parallel to the chitin wall of the spine and has irregular and extensive cytoplasmic and nuclear areas that can be observed in the transverse (Fig. 4c, e) and longitudinal sections (Fig. 4d). As shown in Fig. 4d, this cell, which is located in the subapical region of the spine, occupied almost the entire region of the spine, leaving only a narrow strip between this cell and the epithelium lining the spine. Compared with the epithelial cells, this cell was clearly much larger and displayed a very peculiar morphology. Its irregular nucleus displayed clumped chromatin, a feature that is characteristic of the defensive glandular cells of the Lepidoptera (Gilmer, 1925; Snodgrass, 1993). We observed nuclei alternatively displaying finely dispersed chromatin containing nucleoli (Fig. 4c) or increased chromatin clumping and a more compact nucleus (Fig. 4e). These morphological changes in glandular cells were due to cells being in different stages of gland secretion (Barth, 1954a). Accordingly, Fig. 4e represent resting glands with condensed nuclear chromatin. In contrast, Fig. 4c shows a cell with smooth chromatin containing nucleoli, typical of a cell in the synthesis
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Fig. 4. Photomicrographs of the integument of L. obliqua. 4a and 4b- Transverse section of the 6th and 5th instar caterpillar integument respectively. The epithelial cells (Ep) present a slightly basophilic cytoplasm. Separating the integument of the haemocoel there is a well-developed basal membrane (arrowhead). Fatty body (Fb). Trachea (T). Haemocytes (h). Cuticle (cu). Observe the local formation of a scolus in 4a (*). 4c and 4e- Transverse section of a spine. Epithelial cells (arrows) juxtaposed parallel to the cuticle (cu) internally cover the lumen of the spine where a single glandular cell is present. The chromatin morphology varies according to the functional state of the cell. In 4c, the nucleus (*) is sinuous with dispersed chromatin and nucleolus (nu). In 4e, the chromatin is compacted and cytoplasm (cy) consists of a narrow band around the nucleus (N) d- Longitudinal section of the subapical region of a spine. The chitinous wall is lined internally by epithelial cells (arrows). Inside the lumen of the spine there is the poison apparatus comprising a single large cell (*) with a sac-like form, inserted into the subapical region of the spine (dotted rectangle). The apical region consists only of chitin and has a central canal (c). e- Transverse section of a spine. The nucleus (N) of the urticating cell contains compacted chromatin and cytoplasm consisting of a narrow band around the nucleus. Epithelial cells (arrows). Toluidine blue/ basic fuchsin stain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
phase. The weakly eosinophilic cytoplasm of this cell was quite irregular, exhibiting numerous invaginations (Fig. 4c). Near the subapical of the spine, the cytoplasm was observed to be inserted in the constricted region like a ring, which appeared to have an important role in the anchorage (docking) of the gland, as shown in Fig. 4d. This region is believed to represent a modification of the cuticle wall, with the appearance of an insertion cup, in which the apical portion of the spine fits perfectly to the spine reminder. A narrow canal also noted, where the substance produced by the gland was stored. Perhaps this alteration increases the endurance of the spine, facilitating penetration of the skin of the victim.
Importantly, the terminal region of the spine was acellular, and the cuticle present therein was thicker than the rest of cuticle to facilitate the introduction of this region of the spine into the victim’s skin. The serial sections were essential to the location of the venom gland inside the spine and also to show that this gland was not present in all spines of the scolus. Additionally, we also observed similar gland cells in the spines of the scoli from caterpillars in other instars. Further studies to characterize the morphophysiology of this gland in younger larvae are on going. A small amount of poisonous secretion was obtained from each spine. An analysis performed with MALDI identified seven similar
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Fig. 5. Comparison among spines secretions (green and orange) and haemolymph of the L. obliqua determined by MALDI-ToF. (a) General profile from 3000 to 90,000 m/z and (b) a higher magnification of 5000e20,000 m/z showing the region containing the peaks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
substances between the green-coloured secretion and the haemolymph. By contrast, we observed four substances in the orange secretion that were similar to those found in the green secretion, but only one substance in common with the haemolymph (Fig. 5a, b). The other substances that were not found in the haemolymph but were present in the green secretion of the spines could be responsible for the synthesis of cuticle because some substances present in the haemolymph are known to be transported to the tegument to form the cuticle (see Crossley, 1979). These substances that were present in the haemolymph but, not in the green secretion might be related to the general metabolism of the animal. Another potential explanation for these differences could be the method used to collect the haemolymph. The haemolymph was frozen immediately after collection and centrifuged only after thawing for the biochemical analysis. Therefore, frozen haemocytes present in the haemolymph could have been triggered to release substances during the thawing procedure. Notably, the green secretions of some spines were similar in colour to the haemolymph of the caterpillar. L. obliqua caterpillars feed on green pigment-containing leaves of various tree species (Lorini, 1999) that probably supply the green colour to the haemolymph of these caterpillars. The haemolymph circulates through the scolus and spines to nourish the cells on the top of the spine. When a spine is broken off, it can release this green substance. Additionally, we noticed that the green secretion was slightly viscous compared with the orange secretion. This phenomenon may be related to the coagulant activity of the haemolymph, the goire and function of which is already established in insects (Gre Goffinet, 2009). This secretion is clearly produced in a separate microenvironment (gland), in which only some molecules can move through the plasma membrane of the cell. This result, in addition to those obtained during the histological examinations, supports the claim that this secretion is the poisoning substance. In conclusion, our studies demonstrate that the scoli of L. obliqua caterpillars possess some spines with venom-producing cells and others lacking such cells. Second, there are two types of spines (with and without grooves). Third, some spines produce orange secretions and others produce green secretions. The green
secretion has a greater similarity to the haemolymph than to the orange secretion. In our study, it was not possible to establish a relationship between the different types of spines and the different secretions. However, we hypothesize that the spines with a groove that produce the orange secretion are likely to be the venom glands. By contrast, the green secretion that is very similar to haemolymph is found in spines without grooves that lack a toxin-producing cell. However, the possibility of contamination with haemolymph can not be discarded. In addition, the detailed biochemical study of these secretions in progress may clarify this point. Alternatively, both secretions may have an important role in envenoming because, when placed in contact with the skin of the victim, all spines should break off regardless of the presence of grooves. Ethical statement The research project that derived this manuscript was submitted to the Instituto Butantan Animal Care and Use Committee (CEUAIB 525/08) but was exempted from the analysis because the animal (Lonomia obliqua - Caterpillar) is not a vertebrate. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments We would like to thank Mrs. Joanita Lopes and Mr. Bruno de Carvalho Trindade, for their support at Instituto Butantan Library. ~o de Amparo This study was partially supported by the Fundaça a ~o Paulo (FAPESP, Brazil; grant #01/07643Pesquisa do Estado de Sa ~o Butantan. 7) and Fundaça Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2016.06.008.
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Gilmer, P.M., 1925. A comparative study of the poison apparatus of certain lepdopterous larvae. Ann. Entomol. Soc. Am. 18, 203e239. Gonçalves, L.R.C., Sousa-e-Silva, M.C.C., Tomy, S.C., Sano-Martins, I.S., 2007. Efficacy of serum therapy on the treatment of rats experimentally envenomed by bristle extract of the caterpillar Lonomia obliqua: comparison with epsilonaminocaproic acid therapy. Toxicon 50, 349e356. goire, C., Goffinet, G., 2009. Controversies about the coagulocyte. In: Gupta, A.P. Gre (Ed.), Insect Hemocytes: Development, Forms, Functions, and Techniques. Cambridge University Press, New York, pp. 189e229. Gullan, P.J., Cranston, P.S., 2008. Os insetos: um resumo de entomologia. Rocca, S~ ao Paulo. Kelen, E.M.A., Picarelli, Z.P., Duarte, A.C., 1995. Hemorragic syndrome induced by contact with caterpillars of genus Lonomia (Saturniidae, Hemileucinae). J. Toxicol. Toxin. Rev. 14, 283e308. Lamdin, J.M., Howell, D.E., Kocan, K.M., Murphey, D.R., Arnold, D.C., Fenton, A.W., Odell, G.V., Ownby, C.L., 2000. The venomous hair structure, venom and life cycle of Lagoa crispata, a puss caterpillar of Oklahoma. Toxicon 38, 1163e1189. Lorini, L.M., 1997. Lonomia obliqua. In: Nicolella, A., Barros, E., Torres, J.B., Marques, M.G. (Eds.), Acidentes Com Animais Peçonhentos: Consulta R apida. Hospital de Clínicas de Porto Alegre, Porto Alegre, pp. 51e53. gicos e morfolo gicos da Lonomia obliLorini, L.M., 1999. A taturana: aspectos biolo qua. EDIUPF, Passo Fundo, Brazil. Lorini, L.M., Rebelato, G.S., Bonatti, J., 2004. Reproductive parameters of Lonomia obliqua Walker, 1855 (Lepidoptera: Saturniidae) in laboratory. Braz. Arch. Biol. Technol. 47, 575e577. Maschwitz, U.W.J., Kloft, W., 1971. Morphology and fuction of the venom apparatus of insects e bees, wasps, ants, and caterpillars. In: Bücherl, W., Buckley, E.E. (Eds.), Venomous Animals and Their Venoms. Academic Press, New York, pp. 1e60. , K., 1999. Ultrastructure of the spines and neck gland of Osborn, F., S anchez, F., Jaffe Abananote hylonome Doubleday, 1844 (Lepidoptera: Nymphalidae). Int. J. Insect Morphol. Embryol. 28, 321e330. Reis, C.V., Andrade, S.A., Ramos, O.H.P., Ramos, C.R.R., Ho, P.L., Batista, I.C., Chudzinski-Tavassi, A.M., 2006. Lopap, a prothrombin activator from Lonomia obliqua belonging to the lipocalin family: recombinant production, biochemical characterization and structure-function insights. Biochem. J. 398, 295e302. Reis, C.V., Kelen, E.M.A., Farsky, S.H.P., Portaro, F.C.V., Sampaio, C.A.M., Fernandes, B.L., Camargo, A.C.M., Chudzinski-Tavassi, A.M., 1999. A Caþþ activated serine protease (LOPAP) could be responsible for the haemorrhagic syndrome caused by the caterpillar Lonomia obliqua. Lancet 353, 1942e1942. Reis, C.V., Portaro, F.C.V., Andrade, A.S., Fritzen, M., Fernandes, B.L., Sampaio, C.A.M., Camargo, A.C.M., Chudzinski-Tavassi, A.M., 2001. A prothrombin activator serine protease from the Lonomia obliqua caterpillar venom (Lopap): biochemical characterization. Thromb. Res. 102, 427e436. Riella, M.C., Chula, D., Freitas, S., Mazza, M.M., Pachaly, M.A., 2008. Acute renal failure and haemorrhagic syndrome secondary to toxin of caterpillars (Lonomia obliqua). NDT Plus 6, 445e446. ~o biolo gica e imunoquímica do extrato Rocha-Campos, A.C.M., 1999. Caracterizaça de cerdas de Lonomia obliqua. Produç~ ao do soro policlonal específico para uso ~o Paulo. humano. PhD thesis. Universidade de Sa Seibert, C.S., Guerra-Shinohara, E.M., Sano-Martins, I.S., 2003. In vitro hemolytic of Lonomia obliqua caterpillar bristle extract on human and wistar rat erythrocytes. Toxicon 41, 831e839. Seibert, C.S., Oliveira, M.R.L., Gonçalves, L.R.C., Santoro, M.L., Sano-Martins, I.S., 2004. Intravascular hemolysis induced by Lonomia obliqua caterpillar bristle extract: an experimental model of envenomation in rats. Toxicon 44, 793e799. Snodgrass, R.E., 1993. Principles of Insect Morphology. Cornell University Press, New York. Vegliante, F., Hasenfuss, I., 2012. Morphology and diversity of exocrine glands in Lepidopteran larvae. Annu. Rev. Entomol. 57, 187e204. Veiga, A.B., Blochtein, B., Guimar~ aes, J.A., 2001. Structures involved in production, secretion and injection of the venom produced by the caterpillar Lonomia obliqua (Lepidoptera, Saturniidae). Toxicon 39, 1343e1351. ~es, J.A., Francischetti, I.M., 2005. A catalog for the Veiga, A.B., Ribeiro, J.M., Guimara transcripts from the venoms structures of the caterpillar Lonomia obliqua: identification of the proteins potentially involved in the coagulation disorder and hemorrhagic syndrome. Gene 355, 11e27. Zannin, M., Lourenço, D.M., Motta, G., Dalla Costa, L.R., Grando, M., Gamborgi, G.P., Noguti, M.A., Chudzinski-Tavassi, A.M., 2003. Blood coagulation and fibrinolytic factors in 105 patients with hemorrhagic syndrome caused by accidental contact with Lonomia obliqua caterpillars in Santa Catarina, Southern Brazil. Thromb. Haemost 89, 355e364.
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The urticating apparatus in the caterpillar of Lonomia obliqua (Lepidoptera: Saturniidae) Diva Denelle Spadacci-Morena a, *, Magna Aparecida Maltauro Soares a, Roberto Henrique Pinto Moraes b, Ida Sigueko Sano-Martins a, Juliana Mozer Sciani c ~o Paulo, SP, Brazil rio de Fisiopatologia, Instituto Butantan, Sa Laborato ~o Paulo, SP, Brazil rio Especial de Coleço ~es Zoolo gicas, Instituto Butantan, Sa Laborato c ~o Paulo, SP, Brazil rio de Bioquímica e Biofísica, Instituto Butantan, Sa Laborato a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2016 Received in revised form 9 June 2016 Accepted 14 June 2016 Available online 16 June 2016
The presence of specialized cells for venom production in the Lonomia obliqua caterpillar has long been a controversial topic. In this study, we identify a cell inside the spine that specializes in the production of toxins. Our histological study showed that this glandular cell was inserted at the subapical region of the spine, in a constricted region like a ring. This cell type was not observed in all spines of the scolus. The constricted region of the spine observed by scanning electron microscopy displayed a circular groove in which the apical portion of the spine fits perfectly; however, some spines in the same scolus lacked this groove. After breaking off the spine at the most apical region, a small drop of orange or green liquid was observed to flow from its tip. These secretions were analysed by MALDI-ToF and found to possess biochemically different compositions. The green secretion demonstrated greater similarity to the haemolymph of the caterpillar than the orange secretion. Based on our findings, the spines with a groove probably contain the venom glands and produce an orange secretion. However, it is also possible that both secretions play an important role in envenoming because all spines in contact with the skin of the accidental victim should break regardless of whether they are present in a groove. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Caterpillar Saturniidae Lonomia obliqua Gland cell Urticating scolus Morphology
1. Introduction Worldwide, the order Lepidoptera comprises approximately 160.000 described species of moths and butterflies (Gullan and Cranston, 2008) with a larval stage that is commonly defined as a caterpillar. In Brazil, some Megalopygidae and Saturniidae (families within Lepidoptera) are known to cause adverse reactions in humans, most of which are due to exposure to the larval form. Lonomia obliqua Walker, 1855 (Saturniidae, Hemileucinae) caterpillars have been found in fruit trees in rural regions of south and southeast Brazil (Lorini, 1997). Human accidents involving this caterpillar was first reported in 1989 (Kelen et al., 1995). These caterpillars present gregarious and mimetic habits and they climb down from the treetops to the trunk, increasing the likelihood of such accidents. Contact between people and these caterpillars occur mainly on the dorsal scoli of the animals. These structures are formed by the central axis from which the spines originate.
* Corresponding author. E-mail address: [email protected] (D.D. Spadacci-Morena). http://dx.doi.org/10.1016/j.toxicon.2016.06.008 0041-0101/© 2016 Elsevier Ltd. All rights reserved.
Collectively, a series of scoli symmetrically cover the caterpillar body. In South America, two species, Lonomia achelous Cramer, 1777 and Lonomia obliqua, have been reported to be responsible for severe accidents and some fatal cases. The majority of the affected patients present with mild burning pain, nausea, and headache, but progression to a severe haemorrhagic syndrome characterized by ecchymoses, haematuria, bleeding from scars and mucous membranes, intracerebral bleeding and acute renal failure frequently ~ ango and Guerrero, 2001; Carrijo-Carvalho and occurs (Arocha-Pin Chudzinski-Tavassi, 2007). This type of accident is defined as lonomism. A bleeding syndrome caused by contact with L. obliqua presents as a haemostatic disorder with moderate consumption coagulopathy without significant thrombocytopenia (Zannin et al., 2003). However, a severe haemorrhagic syndrome associated with acute renal failure has been observed in approximately 5% of patients (Chan et al., 2008; Duarte et al., 1990, 1996; Fan et al., 1998; Gamborgi et al., 2006; Riella et al., 2008). Lethality occurs in approximately 1.5e2.0% of contacts, and the growing annual
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incidence rate in southern Brazil has led to the production of an antivenom (Dias da Silva et al., 1996) that has been shown to be effective (Caovilla and Barros, 2004; Gonçalves et al., 2007). Currently, several biological activities have been described for the extracts of the spines or bristles of this caterpillar (ChudzinskiTavassi et al., 2001; Fritzen et al., 2003; Reis et al., 1999, 2001, 2006; Rocha-Campos, 1999; Seibert et al., 2003, 2004; Veiga et al., 2005). However, the biology of these animals remains poorly understood (Lorini, 1999; Lorini et al., 2004), as the morphology of the gland or tissue that is specialized for the production of this caterpillar venom (Veiga et al., 2001). The urticating apparatus of various lepidopterans has been studied (Barth, 1954a, 1954b, 1956; Barth and Junqueira, 1954; Foot, 1922; Gilmer, 1925; Lamdin et al., 2000; Deml and Dettner, 2002). All these studies concluded that the venoms are typically produced by glandular cells located in the epidermis in contact with the haemocoel or inside the spines. It has been concluded that the L. obliqua caterpillar does not possess specialized cells for venom production, and secretory epithelial cells are responsible for producing the toxins that are stored in the subcuticular space (Veiga et al., 2001). However, in Automeris incisa (Saturniidae, Hemileucinae), a caterpillar of the same subfamily of L. obliqua, the venom gland is present inside the spines (Barth, 1954a). Thus, the location and description of the venom apparatus of the L. obliqua caterpillar remain an outstanding question that requires confirmation (Vegliante and Hasenfuss, 2012). In this study, we performed a morphological study of the spines of L. obliqua caterpillars and we identified a cell in the subapical portion of the spines that is specialized for the production of toxins. 2. Materials and methods Larvae of L. obliqua caterpillars used in this study were origi and S~ nated from the Parana ao Paulo states of Brazil, that were sent to Instituto Butantan for the production of anti-lonomic serum. rio Especial de Coleço ~ es Caterpillars were identified in the Laborato gicas (Instituto Butantan). We selected only those insects that Zoolo were in the fifth and sixth instar stages and that were moving and feeding well. The caterpillars were anaesthetized under a CO2 atmosphere (Dias da Silva et al., 1996), and only the pair of dorsal scoli from the 3rd thoracic segment and the 1st to 8th abdominal segments were collected. 2.1. External morphology of the scoli Whole caterpillars, their scoli and spines were observed and photographed under a M165C stereomicroscope (Leica Microsystems Inc., Wetzlar, Germany) with a DFC420 (Leica) digital camera and with Leica Application Suite 3.1.0 imaging software. For the scanning electron microscopy (SEM) observations were used four caterpillars. The pair of dorsal scoli (3rd thoracic and 1st to 8th abdominal segments) were dissected and isolated, cleaned, kilndried at 50 C and coated with a thin layer of gold (Sputtering Balzers Union - Model 010). Preparations were examined using a LEO 435VP SEM at 20 kV. 2.2. Histology of scoli Caterpillars were fixed in Bouin’s fixative and processed for routine historesin embedding (Leica Microsystems Inc.). The fixed samples were embedded in a flat plastic mould to obtain crosssections of the animals. Because scoli resemble a tree-shape and their axis is perpendicular to the integument, we obtained mainly obliqua and transverse spines sections. Some spines were
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processed individually to obtain longitudinal sections. Threemicron-thick sections were stained with basic fuchsin and toluidine blue and then examined and photographed under a light microscope DM LS with a DFC420 digital camera and Leica Application Suite 3.1.0 imaging software (Leica Microsystems Inc.). 2.3. Biochemical analysis of the spine secretions and the haemolymph The dorsal scoli of the caterpillars were separated from the tegument, and then the spines were broken at their most apical portion (Fig. 1d). The released secretion was collected using a capillary tube. The haemolymph was collected from the haemocoel at a region distal from the spines using an insulin syringe. The secretions obtained from the different spines and the haemolymph were stored in phosphate buffer (0.1 M, pH 7.2) at 20 C until analysis. Secretions from spines of L. obliqua caterpillars and haemolymph were analysed by MALDI-ToF (Axima Performance, Shimadzu Corp., Kyoto, Japan) using both linear and reflectron modes. Samples were mixed with an appropriated matrix: sinapinic acid for the linear mode and a-cyano-4-hydroxycinnamic acid for the reflectron mode. Data were acquired under positive ionization mode within a range from 200 to 10,000 m/z (reflectron) and 10,000e100,000 m/z (linear). A peak list was obtained automatically by the software controlling the equipment. 3. Results and discussion Lonomia obliqua caterpillar presents scoli of the type “VII e urticating scolus” as described by Deml and Dettner (2002). Fifth and sixth instar larvae of L. obliqua caterpillars displayed scoli that were symmetrically distributed throughout the body of the animal in the dorsal, subdorsal, lateral and subventral regions (Fig. 1a). We analysed only the scoli from the two rows in the dorsal region (Fig. 1b) because they are probably the first scoli to come in contact with the skin of the victim. Scoli are composed of a central axis with side branches, resembling small trees. The ramifications, herein named spines, are rigid structures with colors that range from light to dark brown. The spines have a conical shape with a very thin tip. The size of the spines typically varies in the same scolus (Fig. 1b and c). The products of the gland secretion are inoculated into the skin of the victim through the spines. At the time of an accident, the most apical portion of the spines is broken off in the skin of the victims, and the poison is released (Gilmer, 1925). The mechanism by which the venom is expelled from the spine is not completely understood. One possibility is that local pressure is induced by the haemolymph, and the other possibility is that the release is caused by contraction of intersegmental muscles (Maschwitz and Kloft, 1971). Indeed, an intrinsic animal activity must be present for secretion release because, when we isolated one scolus and broke off the most distal portion of the spine, it was necessary to apply a mechanical pressure to release the secretion (Fig. 1d). After breaking off a spine, we observed a small drop of green or orange liquid flow from its tip (Fig. 1e and f). There was not a clear relationship between the spine colour and the colour of its secretion (i.e., both light and dark spines produced green and orange secretion). L. obliqua caterpillars in the 5th or 6th instar stage possessed scoli with closed spines, without communication with the external environment. However, when the tip was broken off, a canal was observed inside the spine (Fig. 2a). Furthermore, according to the external morphology, in the subapical region, some spines possessed a circular groove similar to a ring-like structure, within
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Fig. 1. Photomicrographs of the Lonomia obliqua caterpillar (Saturnidae) at the sixth larval instar stage. a- General view. b- Details of scoli from the median dorsal region. c- Scolus isolated from the median dorsal region. Note that the scolus has a central axis from which originate lateral branches (spines) with different colors and sizes. d- Apical region of the spine where it was broke off (scissors) and subapical region where it was pressured (arrow) to collect secretions. e- Details of a spine broken at its tip showing a secretion derived from the interior of the spine. f- Observe a small drop with green colour on the left and orange on the right that exuded from the interior of different spines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. SEM micrographs of spines from dorsal scoli from a sixth instar larva. a- Details of the fractured tip, including an internal canal (*). b and c- Spines with pointed and closed tips showing the presence (b) or absence (c) of a ring-like constriction around the spine (groove).
which the apical portion of the spine fits perfectly (Fig. 2b). This groove has been observed previously by Veiga et al. (2001). Barth (1956) described a similar structure in the spines of Sibine nesea that was considered the place destined for breakage during an
accident. In the present study, we observed spines with a similar circular groove and spines in the same scolus lacked a groove (Fig. 2c). Although many urticating apparatuses of caterpillars have been
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described by SEM, histological observations were essential to localize and describe the venom gland in our study. We generated transverse sections of the entire caterpillar body, as shown in Fig. 3a, to examine both the integument and the internal cavity of the caterpillar. This type of section was important to establish the relationship of the scolus with the body of the animal. Several serial sections were generated to evaluate the integument and spines along its length. The literature indicates that the venom gland can be found in different places in the body of the caterpillar, depending on the species studied. The venom gland can be found within structures called “funnel warts”, as in many representatives of the Lymantriidae family (currently Erebidae, Lymantriinae) (Deml and Dettner, 1997, 2001), or at the base of balloon hairs in the Lymantria dispar caterpillar, which also belongs to the Lymantriidae family (Deml and Dettner, 1995). By contrast, the venom gland is described inside of the scolus in some species of the Saturniidae (Barth,
Fig. 3. a- Localization and section plane used in this study, indicating the orientation of the histological sections. 3b- Schematic representations of the relationship of the scolus to the body of the caterpillar. In the haemocoel, muscle bundle (Mu), fatty body (Fb) and trachea (T) can be observed. The internal cavity is separated from the integument by a basal membrane (arrows). Along the integument, composed of epithelial cells (Ep) and chitin cuticle (cu), an evagination of the tegument itself is observed originating the scolus. Several spines (S) are formed from the central axis of the scolus. The central axis and the spines are covered with epithelial cells (Ep) and the cuticle (cu). Haemocytes (h) are observed throughout the body of the animal, even within the central axis of the scolus (*) but not within the spines.
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1954a; Deml and Dettner, 1990, 1993, 1997, 2003) and Nymphalidae families (Osborn et al., 1999). Moreover, in some cases the venom gland can be found in the cuticle without reaching the body cavity, as in Sibinea nesea (Limacodidae) (Barth, 1956), or under the tegument of larvae, as in some caterpillars of the Megalopygidae (Barth, 1954b; Eagleman, 2008) and Saturniidae families (Deml and Dettner, 1994). The haemocoel of the caterpillar contained muscles, fatty bodies, haemocytes and the tracheal system characteristic of insects (Figs. 3b, 4a and b). The surface of the caterpillar body was covered by the chitinous cuticle and traversed by wide canals called scoli (Figs. 3b and 4a). The scolus is an evagination of the epithelial cells of the tegument formed by a main central calibre axis and the lateral branches that form spines (Figs. 3b and 4a). When a caterpillar was sectioned in its middle region (Fig. 3b), the relationship between the animal’s body and scolus was clearly visualized, supporting the studies of Gilmer (1925) regarding the origin of the scolus. The central axis cuticle was covered by an epithelium with a morphology similar to the epithelial wall of the animal (Fig. 3b). Along this central axis and in the internal cavity of the caterpillar, there were several different lineages of haemocytes with variable morphology (Figs. 3b and 4b). However, haemocytes were not observed within the spines. The epidermis, a single layer of epithelial, cuboidal and juxtaposed cells with a slightly basophilic cytoplasm, was present just beneath the cuticle. These cells contained a central nucleus with one or more evident nucleoli, with punctiform chromatin clumps (Fig. 4a and b). A basal membrane (Figs. 3b, 4a and b) separated the epidermis from the general body cavity of the caterpillar (haemocoel). The epidermis region and cuticle were carefully examined and no complex of secretory cells related to venom production was observed at the base of the scoli or even attached to the animal’s body cavity, similar to the arrangement described for L. obliqua (Veiga et al., 2001). The spines were also covered by chitinous cuticle and flat epithelial cells, which exhibited nuclear chromatin that was grouped into small clumps. These cells, like those of the animal wall, were regularly arranged and encircling the lumen of the spines (Fig. 4cee). The apical region of the spines was covered with cuticle and had a central canal (Fig. 4d), and no cells were found in this region. In the longitudinal sections, we observed a constriction in the subapical region of some spines, in which the most apical part fits perfectly into the other part of the spine (Fig. 4d) as previously mentioned for the SEM figure (Fig. 2b). Our histological sections of the spines revealed a large glandular cell with a morphology that was similar to the glandular cell of the Automeris incisa caterpillar (Barth, 1954a), which is phylogenetically close to L. obliqua. This unique cell with a saccular structure lies parallel to the chitin wall of the spine and has irregular and extensive cytoplasmic and nuclear areas that can be observed in the transverse (Fig. 4c, e) and longitudinal sections (Fig. 4d). As shown in Fig. 4d, this cell, which is located in the subapical region of the spine, occupied almost the entire region of the spine, leaving only a narrow strip between this cell and the epithelium lining the spine. Compared with the epithelial cells, this cell was clearly much larger and displayed a very peculiar morphology. Its irregular nucleus displayed clumped chromatin, a feature that is characteristic of the defensive glandular cells of the Lepidoptera (Gilmer, 1925; Snodgrass, 1993). We observed nuclei alternatively displaying finely dispersed chromatin containing nucleoli (Fig. 4c) or increased chromatin clumping and a more compact nucleus (Fig. 4e). These morphological changes in glandular cells were due to cells being in different stages of gland secretion (Barth, 1954a). Accordingly, Fig. 4e represent resting glands with condensed nuclear chromatin. In contrast, Fig. 4c shows a cell with smooth chromatin containing nucleoli, typical of a cell in the synthesis
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Fig. 4. Photomicrographs of the integument of L. obliqua. 4a and 4b- Transverse section of the 6th and 5th instar caterpillar integument respectively. The epithelial cells (Ep) present a slightly basophilic cytoplasm. Separating the integument of the haemocoel there is a well-developed basal membrane (arrowhead). Fatty body (Fb). Trachea (T). Haemocytes (h). Cuticle (cu). Observe the local formation of a scolus in 4a (*). 4c and 4e- Transverse section of a spine. Epithelial cells (arrows) juxtaposed parallel to the cuticle (cu) internally cover the lumen of the spine where a single glandular cell is present. The chromatin morphology varies according to the functional state of the cell. In 4c, the nucleus (*) is sinuous with dispersed chromatin and nucleolus (nu). In 4e, the chromatin is compacted and cytoplasm (cy) consists of a narrow band around the nucleus (N) d- Longitudinal section of the subapical region of a spine. The chitinous wall is lined internally by epithelial cells (arrows). Inside the lumen of the spine there is the poison apparatus comprising a single large cell (*) with a sac-like form, inserted into the subapical region of the spine (dotted rectangle). The apical region consists only of chitin and has a central canal (c). e- Transverse section of a spine. The nucleus (N) of the urticating cell contains compacted chromatin and cytoplasm consisting of a narrow band around the nucleus. Epithelial cells (arrows). Toluidine blue/ basic fuchsin stain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
phase. The weakly eosinophilic cytoplasm of this cell was quite irregular, exhibiting numerous invaginations (Fig. 4c). Near the subapical of the spine, the cytoplasm was observed to be inserted in the constricted region like a ring, which appeared to have an important role in the anchorage (docking) of the gland, as shown in Fig. 4d. This region is believed to represent a modification of the cuticle wall, with the appearance of an insertion cup, in which the apical portion of the spine fits perfectly to the spine reminder. A narrow canal also noted, where the substance produced by the gland was stored. Perhaps this alteration increases the endurance of the spine, facilitating penetration of the skin of the victim.
Importantly, the terminal region of the spine was acellular, and the cuticle present therein was thicker than the rest of cuticle to facilitate the introduction of this region of the spine into the victim’s skin. The serial sections were essential to the location of the venom gland inside the spine and also to show that this gland was not present in all spines of the scolus. Additionally, we also observed similar gland cells in the spines of the scoli from caterpillars in other instars. Further studies to characterize the morphophysiology of this gland in younger larvae are on going. A small amount of poisonous secretion was obtained from each spine. An analysis performed with MALDI identified seven similar
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Fig. 5. Comparison among spines secretions (green and orange) and haemolymph of the L. obliqua determined by MALDI-ToF. (a) General profile from 3000 to 90,000 m/z and (b) a higher magnification of 5000e20,000 m/z showing the region containing the peaks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
substances between the green-coloured secretion and the haemolymph. By contrast, we observed four substances in the orange secretion that were similar to those found in the green secretion, but only one substance in common with the haemolymph (Fig. 5a, b). The other substances that were not found in the haemolymph but were present in the green secretion of the spines could be responsible for the synthesis of cuticle because some substances present in the haemolymph are known to be transported to the tegument to form the cuticle (see Crossley, 1979). These substances that were present in the haemolymph but, not in the green secretion might be related to the general metabolism of the animal. Another potential explanation for these differences could be the method used to collect the haemolymph. The haemolymph was frozen immediately after collection and centrifuged only after thawing for the biochemical analysis. Therefore, frozen haemocytes present in the haemolymph could have been triggered to release substances during the thawing procedure. Notably, the green secretions of some spines were similar in colour to the haemolymph of the caterpillar. L. obliqua caterpillars feed on green pigment-containing leaves of various tree species (Lorini, 1999) that probably supply the green colour to the haemolymph of these caterpillars. The haemolymph circulates through the scolus and spines to nourish the cells on the top of the spine. When a spine is broken off, it can release this green substance. Additionally, we noticed that the green secretion was slightly viscous compared with the orange secretion. This phenomenon may be related to the coagulant activity of the haemolymph, the goire and function of which is already established in insects (Gre Goffinet, 2009). This secretion is clearly produced in a separate microenvironment (gland), in which only some molecules can move through the plasma membrane of the cell. This result, in addition to those obtained during the histological examinations, supports the claim that this secretion is the poisoning substance. In conclusion, our studies demonstrate that the scoli of L. obliqua caterpillars possess some spines with venom-producing cells and others lacking such cells. Second, there are two types of spines (with and without grooves). Third, some spines produce orange secretions and others produce green secretions. The green
secretion has a greater similarity to the haemolymph than to the orange secretion. In our study, it was not possible to establish a relationship between the different types of spines and the different secretions. However, we hypothesize that the spines with a groove that produce the orange secretion are likely to be the venom glands. By contrast, the green secretion that is very similar to haemolymph is found in spines without grooves that lack a toxin-producing cell. However, the possibility of contamination with haemolymph can not be discarded. In addition, the detailed biochemical study of these secretions in progress may clarify this point. Alternatively, both secretions may have an important role in envenoming because, when placed in contact with the skin of the victim, all spines should break off regardless of the presence of grooves. Ethical statement The research project that derived this manuscript was submitted to the Instituto Butantan Animal Care and Use Committee (CEUAIB 525/08) but was exempted from the analysis because the animal (Lonomia obliqua - Caterpillar) is not a vertebrate. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments We would like to thank Mrs. Joanita Lopes and Mr. Bruno de Carvalho Trindade, for their support at Instituto Butantan Library. ~o de Amparo This study was partially supported by the Fundaça a ~o Paulo (FAPESP, Brazil; grant #01/07643Pesquisa do Estado de Sa ~o Butantan. 7) and Fundaça Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2016.06.008.
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