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Feeding behavior and venom toxicity of coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity
Comparative Biochemistry and Physiology, Part C 138 (2004) 485 – 492 www.elsevier.com/locate/cbpc
Feeding behavior and venom toxicity of coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity Aldo H. Urdanetaa, Federico Bolan˜osb, Jose´ Marı´a Gutie´rreza,* a
Instituto Clodomiro Picado, Facultad de Microbiologı´a, Universidad de Costa Rica, San Jose´, Costa Rica b Escuela de Biologı´a, Universidad de Costa Rica, San Jose´, Costa Rica Received 23 June 2004; received in revised form 16 August 2004; accepted 22 August 2004
Abstract The feeding behavior and venom toxicity of the coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity were investigated. Coral snakes searched for their prey (the colubrid snake Geophis godmani) in the cages. Once their preys were located, coral snakes stroke them with a rapid forward movement, biting predominantly in the anterior region of the body. In order to assess the role of venom in prey restraint and ingestion, a group of coral snakes was dmilkedT in order to drastically reduce the venom content in their glands. Significant differences were observed between snakes with venom, i.e., dnonmilkedT snakes, and dmilkedT snakes regarding their behavior after the bite. The former remained hold to the prey until paralysis was achieved, whereas the latter, in the absence of paralysis, moved their head towards the head of the prey and bit the skull to achieve prey immobilization by mechanical means. There were no significant differences in the time of ingestion between these two groups of coral snakes. Susceptibility to the lethal effect of coral snake venom greatly differed in four colubrid species; G. godmani showed the highest susceptibility, followed by Geophis brachycephalus, whereas Ninia psephota and Ninia maculata were highly resistant to this venom. In addition, the blood serum of N. maculata, but not that of G. brachycephalus, prolonged the time of death of mice injected with 2 LD50s of M. nigrocinctus venom, when venom and blood serum were incubated before testing. Subcutaneous injection of coral snake venom in G. godmani induced neurotoxicity and myotoxicity, without causing hemorrhage and without affecting heart and lungs. It is concluded that (a) M. nigrocinctus venom plays a role in prey immobilization, (b) venom induces neurotoxic and myotoxic effects in colubrid snakes which comprise part of their natural prey, and (c) some colubrid snakes of the genus Ninia present a conspicuous resistance to the toxic action of M. nigrocinctus venom. D 2004 Elsevier Inc. All rights reserved. Keywords: Micrurus nigrocinctus; Geophis sp.; Ninia sp.; Coral snake venom; Colubrid snake; Venom toxicity; Myonecrosis; Neurotoxicity; Natural resistance
1. Introduction Feeding behavior of snakes in nature involves two main strategies: (a) wide foraging and (b) ambushing prey from a stationary location (Greene, 1997). The first strategy is characteristic, among others, of species of the families Colubridae and Elapidae, whereas the latter is mostly used by species of the families Boidae and Viperidae (Greene,
* Corresponding author. Tel.: +506 2293135; fax: +506 2920485. E-mail address: [email protected] (J.M. Gutie´rrez). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.08.018
1997). Because most snakes feed on relatively large prey, they have to rely on various mechanisms of restrain, thus minimizing the risk of prey escape and of being harmed by prey or potential predators (Greene, 1983). The most important restrain mechanisms are (a) blunt trauma and mechanical reduction, (b) constriction, and (c) injection of a toxic secretion (venom) (Cundall and Greene, 2000). Constriction is performed by boids, colubrids, and some elapids, although a number of colubrids and elapids combine constriction with venom injection (Greene and Burghardt, 1978). Some colubrids, and all elapids and viperids, on the other hand, synthesize powerful venoms
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which are injected through effective venom-delivery systems and play a relevant role in prey immobilization and, as demonstrated in the case of viperid species, digestion (Meier and Stocker, 1995). Coral snakes (genera Micrurus, Leptomicrurus, and Micruroides) are, together with the pelagic sea snake Pelamis platurus, the American representatives of the family Elapidae (Campbell and Lamar, 1989; Roze, 1996). Their venoms contain low-molecular mass neurotoxins of the three-finger type which block the neuromuscular junction by binding with high affinity to the cholinergic receptor at the neuromuscular junction (Vital-Brazil, 1987; Rosso et al., 1996; Alape-Giro´n et al., 1996; de Oliveira et al., 2000), thereby promoting a paralytic effect. In addition, Micrurus sp. venoms contain abundant phospholipases A2, which induce myotoxicity (Gutie´rrez et al., 1983; Arroyo et al., 1987; Alape-Giro´n et al., 1999), hemorrhage (Francis et al., 1997), inflammation (Tambourgi et al., 1994), and presynaptic neurotoxicity (Vital-Brazil and Fontana, 1983/ 84; Goularte et al., 1995); a basic phospholipase A2 has been cloned from Micrurus corallinus venom gland (de Oliveira et al., 2003). Micrurus nigrocinctus is the most widely distributed coral snake in Central America, inhabiting lowland and premontane forest areas, and is found under debris, in pastures, in coffee plantations, and even in urban areas (Savage, 2002). Its venom exerts neurotoxicity and myotoxicity in mice (Gutie´rrez et al., 1986; Goularte et al., 1995; Alape-Giro´n et al., 1994, 1996, 1999). The effects of snake venoms on natural prey have been largely neglected, a trend that needs to be reverted in order to understand the role of such complex toxic secretions in their biological context. M. nigrocinctus feeds mostly on ectothermic prey because analysis of stomach contents of this species revealed caecilians, reptile eggs, many kinds of lizards, and abundant snakes, including those of the genera Anomalepis, Coniophanes, Geophis, and Ninia, which are common forest litter inhabitants (Greene and Seib, 1983; Roze, 1996). Coral snakes forage by crawling and pocking their heads under surface litter, recognizing prey by chemical and visual cues (Greene and Seib, 1983). A comprehensive study on the comparative toxicity of the venoms of various Micrurus species on different prey showed that, as a general rule, venoms are more toxic towards the natural prey than to other types of vertebrates (Jorge da Silva and Aird, 2001). However, the toxicity of M. nigrocinctus venom on its natural prey has not been investigated, nor the role played by venom on immobilization of their prey. The objectives of this study were three-fold: (a) to describe the feeding behavior of M. nigrocinctus on one of its natural prey (the colubrid snake Geophis godmani) in captivity, emphasizing on the role of venom in prey immobilization and ingestion; (b) to investigate the toxicity of M. nigrocinctus venom on four colubrid species of the genera Geophis and Ninia, in order to characterize the pharmacology of this coral snake venom on natural prey and
to explore the potential resistance of such prey to venom; and (c) to study the ability of the blood serum of snakes of the genera Ninia and Geophis to neutralize M. nigrocinctus venom.
2. Materials and methods 2.1. Protocol for the study of feeding behavior This study was performed at the serpentarium of Instituto Clodomiro Picado. A total of 39 adult specimens of M. nigrocinctus (20 females and 19 males) were collected from the following localities in Costa Rica: Guadalupe, Curridabat, San Pedro, Escazu´, and Moravia (province of San Jose´); San Jose´ and Orotina (province of Alajuela); Puntarenas (province of Puntarenas); Can˜as (province of Guanacaste), Santo Domingo (province of Heredia); and Cartago (province of Cartago). Snakes were individually housed in cages of 302515 cm, with water ad libitum in a plastic container; the floor of the cages was covered with paper. All individuals were weighed to the nearest 0.1 g (on a digital balance, Ohaus, model C505-S) and measured, and sex was identified. Snakes were kept at a temperature of 24–26 8C. For this study, each coral snake was fed five times, at intervals of approximately 2 months, although some of them were fed only four times. The first three times snakes were fed without previous extraction of the venom, whereas, in the last two feedings, venom was extracted prior to feeding. For venom extraction, snakes were manually hold and the maxillary region was gently squeezed in order to obtain most of the venom stored in the gland without causing significant mechanical harm to the snake. Thus, although this treatment does not assure a complete depletion of venom, i.e., traces of venom may remain in fangs and in the venom gland, it does remove the great majority of the venom. For the sake of simplicity in describing the results, snakes subjected to this procedure will be referred to as dmilkedT snakes. Experiments to study feeding behavior were carried out 24 hr after venom extraction. To study feeding behavior, individual adult specimens of G. godmani, previously weighed and measured as described, were placed in the cages containing coral snakes, as far from the coral snake as possible (see dimensions of the cage above). G. godmani specimens were collected in the area of Las Nubes de Coronado (San Jose´). The following information was collected: (a) strategy used to capture the prey; (b) time to achieve paralysis, i.e., time interval between bite and immobilization of the prey, that is, when significant body movements of the prey ceased; (c) region of the body where bite occurred, and for this purpose, four distinct anatomical regions (1, 2, 3, and 4) were defined, each one comprising 25% of the body length, starting from the head; (d) if paralysis was achieved by a single bite or if the coral snake had to move towards the head to accomplish prey immobilization; (e) if, after the
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bite, the coral snake moved towards the head to start ingestion or if it moved towards the tail; and (f) time required for ingestion, i.e., the time lapse between initiation of swallowing the prey and the complete ingestion of it. A total of 181 tests, using 39 specimens of M. nigrocinctus, were performed during this study.
processing, tissues were embedded in Spurr resin, and thick (1.5 Am) sections were collected and stained with toluidine blue for histological observation.
2.2. Study of the toxic effects of venom
Blood was collected from five individuals of G. brachycephalus and four individuals of N. maculata. After clotting, the serum was separated by centrifugation at 2000g. For inhibition studies, 75 Ag M. nigrocinctus venom, dissolved in 200 AL PBS, and 300 AL of blood serum of the colubrids were incubated at 37 8C for 30 min. Then, 100 AL of the mixture (containing 15 Ag venom, which corresponds to two intravenous Median Lethal Doses (LD50), Bolan˜os, 1972) was injected intravenously in groups of four CD-1 mice (16–18 g body weight). Control mice were injected with (a) 2 LD50 of venom incubated with PBS without serum, (b) serum incubated with PBS instead of venom, and (c) PBS alone. Mice were observed every hour afterwards, and deaths were recorded.
2.2.1. Venom and snakes Venom from approximately 50 adult specimens of M. nigrocinctus from the provinces of San Jose´, Alajuela, Heredia, and Cartago were obtained. Immediately after collection, venom was frozen at 20 8C and then lyophilized and stored at 20 8C. Venom toxicity was studied in adult specimens of G. godmani, Geophis brachycephalus, Ninia psephota, and Ninia maculata, collected in the regions of Las Nubes de Coronado (San Jose´; the two species of Geophis and N. psephota), San Antonio de Coronado (San Jose´) and Santo Domingo (Heredia; N. maculata); snakes were kept at the serpentarium of Instituto Clodomiro Picado.
2.3. Inhibition of venom toxicity by the blood serum of colubrid snakes
2.4. Statistical analyses 2.2.2. Study of lethality Groups of individuals of G. godmani, G. brachycephalus, N. psephota, and N. maculata were weighed, and injected subcutaneously, in the thoracic region, with solutions containing various venom concentrations, dissolved in phosphate-buffered saline solution, pH 7.2 (PBS). A total of 10 snakes were used for each venom dose tested. The dose of venom injected was expressed as microgram (Ag) venom per gram (g) body weight and was adjusted for each animal by varying the volume of injection of venom solutions of various concentrations; in general, volumes of injection ranged between 80 and 200 AL. An additional control group of 10 individuals was injected with 100 AL of PBS. Snakes were observed and all deaths occurring during a 72-hr observation period were recorded. The Median Lethal Dose (LD50) was determined by the Spearman– Karber method (World Health Organization, 1981) and was defined as the venom dose that induced death in 50% of the injected snakes. 2.2.3. Study of pathological effects To investigate the pathological alterations induced by M. nigrocinctus venom, groups of three specimens of G. godmani were injected subcutaneously, as described above, with either a sublethal dose (4 Ag venom per g body weight) or a lethal dose (10 Ag/g) of M. nigrocinctus venom. Control snakes were injected with PBS under otherwise identical conditions. At 4 and 24 hr after injection, animals were sacrificed and tissue samples were obtained from muscles located near the injected site, as well as from heart and lungs. Tissues were immediately placed in Karnovsky’s fixative and were then postfixed with osmium tetroxide, as previously described (Moreira et al., 1992). After routine
To determine the influence of venom injection and sex on the parameters studied on feeding behavior, tests of homogeneity were performed. For the analysis of the effect of venom injection on time of paralysis and time of ingestion, a three-way Analysis of Variance was used. Linear regression was employed to determine the relationship between time of ingestion and length of the prey.
3. Results 3.1. Experiments on feeding behavior 3.1.1. Search for the prey In the vast majority of experiments, a dsearchingT behavior was observed; in these cases, coral snakes moved slowly, moving their heads forwardly and laterally, and actively flicking their tongue until they found their prey in the cage. In few cases, coral snakes remained in the same location until the prey moved close to them. 3.1.2. Capture and paralysis Once prey were located, coral snakes approached them up to a distance of 1–3 cm, from where they stroke with a rapid forward movement. Bites occurred predominantly in the 25% most anterior part of the body of G. godmani (region I); this region was bitten in 45% of the experiments (X 2=34.45, df=3, Pb0.001; Fig. 1). No significant differences were observed in the anatomical region where bites occurred between male and female coral snakes (X 2=2.53, df=3, P=0.470) nor between snakes with venom and dmilkedT snakes (X 2=3.82, df=3, P=0.282; Fig. 1). In
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Pb0.001). In addition, the time to achieve prey immobilization was more prolonged in dmilkedT snakes (mean=1273.1 sec, S.D.=800.5, range=463–3660 sec) than in snakes with venom (mean=671.4 sec, S.D.=352.3, range=164–2102 sec; F (1,138)=45.70, Pb0.005). 3.1.3. Events occurring before swallowing the prey In 97.8% of the experiments, coral snakes started to move towards the head of the prey, without releasing it, whereas, in only 2.2% of the cases, the movements were directed towards the tail (X 2=165.36, df=1, Pb0.001). In this regard, no differences were found between snakes with venom and dmilkedT snakes (X 2=0.19, df=1, P=0.661) nor between males and females (X 2=0.01, df=1, P=0.938), except that snakes with venom waited until paralysis ensued to perform these movements. In the few cases where coral snakes started moving their heads towards the tail of the prey, the snakes stopped this movement at a certain point and reverted its direction towards the head of the prey. Snakes reached the head of the prey through alternating jaw movements. Fig. 1. Anatomical location of bites by M. nigrocinctus in G. godmani. The body of G. godmani was divided into four anatomical regions (1, 2, 3, and 4), each region comprising 25% of the body length; region 1 corresponds to the most anterior part of the body. Results are presented as the frequency (number) of bites. The upper figure compares the frequency between males and females, whereas the lower figure compares the frequency of bites between coral snakes that were not dmilkedT (A) and those that were dmilkedT (B) before the experiments. Region 1 is the site where bites predominantly occurred, whereas there were no significant differences between males and females and between dmilkedT and dnonmilkedT snakes regarding the anatomical site of the bite (see text for details).
96.7% of the tests, coral snakes stroke their prey and hold them; this pattern was significantly more frequent that the pattern based on bite, release, and relocation of the prey (only 3.3% of trials; X 2=157.8, df=1, Pb0.001). Both experimental groups of coral snakes behaved similarly in this regard (X 2=2.66, df=1, P=0.103), and no differences were observed between male and female coral snakes (X 2=0.54, df=1, P=0.463). A significant difference between snakes with venom and dmilkedT snakes was observed on the behavior after the bite. Snakes with venom remained hold to the prey at the site of initial bite and achieved paralysis, without having to move their heads along the body of the prey, in 75.4% of the experiments. In only 24.6% of the tests, they moved towards the head of the prey before the onset of paralysis. In contrast, 79.4% of coral snakes that had been dmilkedT moved their heads after the initial bite, without releasing the prey which had not been paralyzed, approaching the prey’s head and biting them in the skull. In only 20.6% of the bites, dmilkedT snakes remained hold to the prey at the site of the initial bite. The difference observed in this behavioral pattern after the bite between snakes with venom and dmilkedT snakes is statistically significant (X 2=1.83, df=1,
3.1.4. Ingestion of the prey Coral snakes ingested their prey head-first by performing alternating jaw movements which occur concomitantly with lateral movements of the head. The time of ingestion in coral snakes with venom was 1164.8 sec (S.D.=665.7, range=312–4357 sec), whereas the same parameter in dmilkedT snakes was 1114.5 sec (S.D.=603.5, range=435– 3625 sec); this difference was not statistically significant ( F (1,138)=0.19, P=0.664). No significant correlation was found between ingestion time and total length of the prey (r 2=0.01, P=0.216). 3.2. Toxicity of the venom 3.2.1. Lethality A large variation was observed in the values of LD50 among the various colubrid species injected with M. nigrocinctus venom by the subcutaneous route (Table 1). The highest toxicity was observed towards G. godmani Table 1 Lethality of M. nigrocinctus venom on various colubrid snake species and mice Species
LD50 (Ag/g)
Reference
Geophis godmani Geophis brachycephalus Ninia maculata Ninia psephota Mus musculus
7.67 (5.41–10.86) 21.69 (16.41–28.67) N50a N50a 2.06 (1.75–2.53)
This work This work This work This work Gutie´rrez et al. (1991)
Lethality is expressed as Median Lethal Dose (LD50), in terms of microgram (Ag) venom per gram (g) body weight, using the subcutaneous route; 95% confidence limits are included in parenthesis. a In these cases, no lethality was observed with the largest amount of venom tested (50 Ag/g).
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ance of these organs between envenomated snakes and snakes injected with PBS. Histological observation of skeletal muscles obtained from regions close to the injection site in the thoracic region evidenced conspicuous myonecrosis in snakes injected with both doses of M. nigrocinctus venom. Necrotic cells showed hypercontraction of myofilaments, leaving areas of the cell devoid of myofibrils (Fig. 2). Some muscle cells presented discrete lesions, known as ddelta lesionsT (Gutie´rrez et al., 1984), at the periphery. There was no microscopic evidence of hemorrhage because the structure of capillary vessels remained normal (Fig. 2). In samples collected 24 hr after venom injection, an inflammatory infiltrate was observed. Tissue sections from snakes injected with PBS were devoid of pathological alterations in skeletal muscle tissue (Fig. 2). No histopathological alterations were observed in samples obtained from heart and lungs from snakes injected with either PBS or coral snake venom. Furthermore, no local and systemic hemorrhage was observed. Thus, M. nigrocinctus venom induced predominantly neurotoxic and myotoxic effects in G. godmani. 3.3. Inhibition of venom toxicity by blood serum of colubrid snakes Fig. 2. Light micrographs of skeletal muscle of G. godmani 4 hr after subcutaneous injection of either 100 AL of PBS (A) or M. nigrocinctus venom (10 Ag venom/g body weight) dissolved in 100 AL of PBS (B). Tissue from snakes injected with PBS shows a normal histological pattern, whereas a number of necrotic muscle cells (N) are observed in tissue injected with venom. Necrotic cells are characterized by hypercontraction of myofibrillar material. Notice the presence of intact blood microvessels (arrow) in envenomated tissue, in agreement with the lack of hemorrhagic activity of this venom in G. godmani. Bar represents 50 Am.
Mice receiving an intravenous injection of the blood serum of G. brachycephalus or N. maculata did not show any evidence of toxicity nor any behavioral change when compared with mice injected with PBS by the same route. In
(LD50=7.67 Ag/g), whereas LD50 in G. brachycephalus was 21.69 Ag/g. In contrast, N. maculata and N. psephota showed a high resistance to the action of this venom because all injected snakes survived, even at the highest venom dose tested (50 Ag/g). When injected with lethal doses of venom, snakes of the two Geophis species presented signs of paralysis, with reduction in their movements before they died. Despite not dying, the species of Ninia receiving the highest dose of venom tested showed signs of neurotoxicity, i.e., reduction in their movements. It has been reported that the LD50 of M. nigrocinctus venom in mice, using the subcutaneous route, is 2.06 Ag/g (Gutie´rrez et al., 1991; Table 1). 3.2.2. Pathological effects Macroscopic observations of G. godmani specimens injected with lethal (10 Ag/g) or sublethal (4 Ag/g) doses of M. nigrocinctus venom revealed an inflammatory exudate, without hemorrhage, at the site of venom injection. Such exudate was not observed in snakes injected with PBS. No hemorrhagic lesions were observed in lungs and heart, and there were no differences in the macroscopic appear-
Fig. 3. Time-course of mortality in groups of four mice injected intravenously with either 2 LD50s of M. nigrocinctus venom incubated with PBS (n), or the same dose of venom incubated with blood serum of G. brachycephalus (.) or serum of N. maculata (E). Deaths were recorded every hour and survival expressed as percentage. The mortality curves of mice receiving venom alone or venom plus G. brachycephalus serum were identical and are, therefore, superimposed, whereas a prolongation in the time of death occurred when venom was incubated with N. maculata serum.
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contrast, mice receiving 2 LD50s of M. nigrocinctus venom showed rapid manifestations of neurotoxic envenomation, with hindlimb paralysis and respiratory difficulties, a process that ended in death within 3 hr (Fig. 3). When venom was incubated with the serum of G. brachycephalus before injection, mice showed the same pattern of toxic effects and lethality, and there was not a significantly delay in the time of death when compared with mice injected with venom alone (Fig. 3). In contrast, incubation of venom with the serum of N. maculata caused a partial inhibition of neurotoxic activity, reflected in a prolongation of the time of death (Fig. 3).
4. Discussion In captivity, M. nigrocinctus uses a feeding behavior based predominantly on searching for prey in the cage, a pattern that has been also described for other elapid species (Radcliffe and Chiszar, 1980; Greene, 1984; Mattison, 1995). Micrurus fulvius probes leaf litter in search for its natural prey, constituted mostly by burrowing colubrid snakes (Greene, 1984). The search for prey was associated in our experiments with forward and lateral movements of the head, and with frequent tongue flicking, evidencing the relevance of chemical cues in this search. In addition, visual cues may be also important (Greene, 1984, 1997). After localization of the prey, M. nigrocinctus bit mostly in the cephalic and thoracic regions of G. godmani. In the case of specimens that had not been depleted of venom, the snakes hold to their prey at the site of the bite until they were paralyzed, a pattern also described for M. fulvius (Greene, 1984) and other elapids (Radcliffe and Chiszar, 1980; Kardong, 1982); in only few cases did coral snakes release their prey and relocalized them again. In the case of coral snakes that had been dmilkedT before feeding, the bite occurred at the same anatomical locations, but because paralysis was not achieved, the snakes moved their head, without releasing their prey, towards the anterior end, reaching the head and biting in the cephalic region, often causing mechanical damage to the skull of G. godmani. It is likely that prey movements inhibit preingestion maneuvers (Greene, 1984). In addition, our observations suggest that, if preys are not paralyzed within the first minutes after the bite, a second strategy for prey immobilization is employed, based on approaching and biting the head of the prey, inflicting evident mechanical damage, a strategy that has been described in snakes that do not rely on constriction or envenomation to restrain their prey (Cundall and Greene, 2000). The adaptive value of this alternative behavioral pattern is probably based on a reduction of the time spent before the beginning of ingestion, with the consequent reduced risk of predation or damage by the prey being attacked. Hence, venom injection by M. nigrocinctus plays a relevant role in prey immobilization, contributing to rapid paralysis and early prey ingestion. In the absence of venom,
M. nigrocinctus has to rely on mechanical damage to immobilize prey, requiring more time to initiate ingestion. In the vast majority of observations, M. nigrocinctus swallowed G. godmani head-first after the onset of paralysis, a pattern commonly observed in coral snakes (Roze, 1982, 1996) and other elapids (Voris et al., 1978). However, in many experiments, G. godmani specimens were still alive, although paralyzed, when ingestion started, in agreement with the observations of Greene (1984) with M. fulvius. In the few cases in which head movements after the bite were directed towards the tail of G. godmani, coral snakes stopped at a certain point, reversed this trend and started moving their heads toward the head of G. godmani. Detection of the orientation of scale superposition has been proposed as a mechanism to identify the prey’s head (Greene, 1976). Once ingestion started, no differences were observed in the time of ingestion between the two experimental groups, suggesting that, although venom plays a role in paralysis, it does not contribute to a more rapid ingestion. The times recorded for prey ingestion are similar to those previously reported by Roze (1982) for coral snakes. The paralytic effect induced in G. godmani agrees with the experiments performed by injecting M. nigrocinctus venom in this colubrid species. In both cases, G. godmani specimens reduced their movements until they became fully paralyzed before dying. Hence, observations clearly demonstrate a neurotoxic effect of M. nigrocinctus venom on snakes of the genus Geophis. Various short-chain neurotoxins have been isolated from this venom (Rosso et al., 1996; Alape-Giro´n et al., 1996), and the complete amino acid sequence of one of them has been determined (Rosso et al., 1996). a-Neurotoxins block the nicotinic cholinergic receptor at the motor end plate of mammalian skeletal muscle, originating a flaccid paralysis that culminates in respiratory arrest (Vital-Brazil, 1987; Rosso et al., 1996). A conspicuous variation in susceptibility to M. nigrocinctus venom was observed in G. godmani, G. brachycephalus, N. psephota, and N. maculata because the latter two species resisted a venom dose as high as 50 Ag/g body weight, and G. brachycephalus required a significantly higher dose than G. godmani to die. It is noteworthy that this venom is more toxic to mice, on a weight per weight basis and using the subcutaneous route, than to these four colubrid species, although mice do not constitute a natural prey for this coral snake (Greene and Seib, 1983). Our observations indicate that M. nigrocinctus venom does not follow the general trend, described for various Micrurus species, of being more toxic to their natural prey than to other vertebrates that do not constitute normal prey (Jorge da Silva and Aird, 2001). It is likely that a natural resistance to M. nigrocinctus venom has evolved in the colubrid species included in the present study, particularly in Ninia species. Notwithstanding, our unpublished observations indicate that M. nigrocinctus fed with N. maculata are able to paralyze this colubrid before ingestion, thereby indicating
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that the amount of venom delivered in a bite is sufficient to kill even this highly resistant prey. The venom yield of M. nigrocinctus is 5.76 mg (Bolan˜os, 1972). In general terms, natural resistance of animals to the action of snake venoms relies on two different mechanisms: (a) the presence of inhibitory proteins in serum (Domont et al., 1991; Lizano et al., 2003) and (b) modifications in the structure of cholinergic receptor, in such a way that the affinity of neurotoxins for this receptor is reduced (Takacs et al., 2004). It is noteworthy that, among the four colubrid species studied, the one showing the highest resistance was N. maculata, being also the most sympatric with M. nigrocinctus in Costa Rica (Savage, 2002), suggesting that a selective pressure may have been at work in the acquisition of resistance of this colubrid to the venom of M. nigrocinctus. Interestingly, the blood serum of N. maculata was able to prolong the time of death of mice injected with M. nigrocinctus venom, whereas no such effect was observed with the serum of G. brachycephalus. This suggests that the observed resistance may be at least partially due to inhibitory serum factors to venom neurotoxic components. In addition to many isoforms of a-neurotoxins, M. nigrocinctus venom is rich in phospholipases A2 (AlapeGiro´n et al., 1994, 1996, 1999; Rosso et al., 1996). Venom phospholipases A2 display a wide variety of pharmacological effects owing to a process of accelerated evolution (Kini, 1997, 2003; Ohno et al., 2003). Many elapid venoms contain extremely potent neurotoxic phospholipases A2 which act presynaptically at the neuromuscular junction, an effect described for M. nigrocinctus venom (Goularte et al., 1995). In addition, M. nigrocinctus phospholipases A2 exert potent myotoxicity in mice (Arroyo et al., 1987; Alape-Giro´n et al., 1994, 1999). Our histopathological observations clearly evidence a myotoxic effect of this venom on thoracic skeletal muscles of G. godmani. As previously described for mouse skeletal muscle (Gutie´rrez et al., 1986; Arroyo et al., 1987), myonecrosis in G. godmani is of rapid onset and is characterized by hypercontraction of myofibrils and by the presence of cells with ddelta lesionsT, which have been associated with early plasma membrane disruption (Gutie´ rrez et al., 1984). Such observation suggests that M. nigrocinctus myotoxins act at the plasma membrane of muscle cells, promoting a calcium influx responsible of hypercontraction. Preliminary ultrastructural observations corroborated the presence of interruptions in the continuity of G. godmani muscle cell plasma membrane (our unpublished data). Early plasma membrane damage characterizes the action of several myotoxic phospholipases A2 isolated from various elapid venoms (Harris and Cullen, 1990). Results suggest that M. nigrocinctus venom act in a similar way in mouse and G. godmani skeletal muscles, the plasma membrane being the first site of action. As described in mice (Gutie´rrez et al., 1980, 1986), M. nigrocinctus venom does not induce local nor systemic hemorrhage in G. godmani, nor does it affect cardiac muscle and pulmonary
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tissue. Thus, the pathophysiology of envenomation by M. nigrocinctus venom in G. godmani is characterized by neurotoxicity and myotoxicity. The biological role of myotoxicity induced by M. nigrocinctus phospholipases A2s is not clear at present because it does not seem to be required for prey immobilization, the latter being achieved by the action of neurotoxins. This venom has a very low proteolytic action on a variety of substrates (Lomonte and Gutie´rrez, 1983; Jorge da Silva and Aird, 2001); hence, digestion of proteins has to be achieved by proteinases present in gastric and pancreatic secretions. It is suggested that widespread muscle degeneration in colubrid snakes due to envenomation by M. nigrocinctus may facilitate the digestion of the muscle mass by such proteinases, a hypothesis that needs to be tested in future studies. Various Micrurus sp. venoms have been reported to induce hemorrhage upon intravenous injection in mice (Jorge da Silva and Aird, 2001), and a hemorrhagic phospholipase A2 was isolated from Micrurus frontalis frontalis venom (Francis et al., 1997). No hemorrhagic effect was observed in G. godmani injected with M. nigrocinctus venom. Lack of hemorrhage was also described in mice injected with this venom (Gutie´rrez et al., 1980) and with a purified myotoxic phospholipase A2 (Arroyo et al., 1987). In conclusion, M. nigrocinctus venom exerts neurotoxic and myotoxic effects on G. godmani and plays a key role in prey immobilization, reducing the time lapse between bite and ingestion. A drastic variation in susceptibility to M. nigrocinctus venom occurs between different colubrid snake species, and a significant natural resistance to M. nigrocinctus venom was observed in N. maculata, a prominent component in the diet of this coral snake.
Acknowledgements The authors thank Rodrigo Aymerich, Danilo Chaco´n, Gerardo Serrano, Santos Rodrı´guez and Randall Valverde for their valuable collaboration in various aspects of this study. Thanks are also due to Mahmood Sasa for fruitful discussions and for critical review of the manuscript, and to Javier Nu´n˜ez for collaboration in the histological work. A. Urdaneta received support from the Deutscher Akademischer Austauschdienst (DAAD) for his graduate studies. This work was carried out in partial fulfillment of the requirements for the M.Sc. degree of A. Urdaneta at the University of Costa Rica.
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Feeding behavior and venom toxicity of coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity Aldo H. Urdanetaa, Federico Bolan˜osb, Jose´ Marı´a Gutie´rreza,* a
Instituto Clodomiro Picado, Facultad de Microbiologı´a, Universidad de Costa Rica, San Jose´, Costa Rica b Escuela de Biologı´a, Universidad de Costa Rica, San Jose´, Costa Rica Received 23 June 2004; received in revised form 16 August 2004; accepted 22 August 2004
Abstract The feeding behavior and venom toxicity of the coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity were investigated. Coral snakes searched for their prey (the colubrid snake Geophis godmani) in the cages. Once their preys were located, coral snakes stroke them with a rapid forward movement, biting predominantly in the anterior region of the body. In order to assess the role of venom in prey restraint and ingestion, a group of coral snakes was dmilkedT in order to drastically reduce the venom content in their glands. Significant differences were observed between snakes with venom, i.e., dnonmilkedT snakes, and dmilkedT snakes regarding their behavior after the bite. The former remained hold to the prey until paralysis was achieved, whereas the latter, in the absence of paralysis, moved their head towards the head of the prey and bit the skull to achieve prey immobilization by mechanical means. There were no significant differences in the time of ingestion between these two groups of coral snakes. Susceptibility to the lethal effect of coral snake venom greatly differed in four colubrid species; G. godmani showed the highest susceptibility, followed by Geophis brachycephalus, whereas Ninia psephota and Ninia maculata were highly resistant to this venom. In addition, the blood serum of N. maculata, but not that of G. brachycephalus, prolonged the time of death of mice injected with 2 LD50s of M. nigrocinctus venom, when venom and blood serum were incubated before testing. Subcutaneous injection of coral snake venom in G. godmani induced neurotoxicity and myotoxicity, without causing hemorrhage and without affecting heart and lungs. It is concluded that (a) M. nigrocinctus venom plays a role in prey immobilization, (b) venom induces neurotoxic and myotoxic effects in colubrid snakes which comprise part of their natural prey, and (c) some colubrid snakes of the genus Ninia present a conspicuous resistance to the toxic action of M. nigrocinctus venom. D 2004 Elsevier Inc. All rights reserved. Keywords: Micrurus nigrocinctus; Geophis sp.; Ninia sp.; Coral snake venom; Colubrid snake; Venom toxicity; Myonecrosis; Neurotoxicity; Natural resistance
1. Introduction Feeding behavior of snakes in nature involves two main strategies: (a) wide foraging and (b) ambushing prey from a stationary location (Greene, 1997). The first strategy is characteristic, among others, of species of the families Colubridae and Elapidae, whereas the latter is mostly used by species of the families Boidae and Viperidae (Greene,
* Corresponding author. Tel.: +506 2293135; fax: +506 2920485. E-mail address: [email protected] (J.M. Gutie´rrez). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.08.018
1997). Because most snakes feed on relatively large prey, they have to rely on various mechanisms of restrain, thus minimizing the risk of prey escape and of being harmed by prey or potential predators (Greene, 1983). The most important restrain mechanisms are (a) blunt trauma and mechanical reduction, (b) constriction, and (c) injection of a toxic secretion (venom) (Cundall and Greene, 2000). Constriction is performed by boids, colubrids, and some elapids, although a number of colubrids and elapids combine constriction with venom injection (Greene and Burghardt, 1978). Some colubrids, and all elapids and viperids, on the other hand, synthesize powerful venoms
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which are injected through effective venom-delivery systems and play a relevant role in prey immobilization and, as demonstrated in the case of viperid species, digestion (Meier and Stocker, 1995). Coral snakes (genera Micrurus, Leptomicrurus, and Micruroides) are, together with the pelagic sea snake Pelamis platurus, the American representatives of the family Elapidae (Campbell and Lamar, 1989; Roze, 1996). Their venoms contain low-molecular mass neurotoxins of the three-finger type which block the neuromuscular junction by binding with high affinity to the cholinergic receptor at the neuromuscular junction (Vital-Brazil, 1987; Rosso et al., 1996; Alape-Giro´n et al., 1996; de Oliveira et al., 2000), thereby promoting a paralytic effect. In addition, Micrurus sp. venoms contain abundant phospholipases A2, which induce myotoxicity (Gutie´rrez et al., 1983; Arroyo et al., 1987; Alape-Giro´n et al., 1999), hemorrhage (Francis et al., 1997), inflammation (Tambourgi et al., 1994), and presynaptic neurotoxicity (Vital-Brazil and Fontana, 1983/ 84; Goularte et al., 1995); a basic phospholipase A2 has been cloned from Micrurus corallinus venom gland (de Oliveira et al., 2003). Micrurus nigrocinctus is the most widely distributed coral snake in Central America, inhabiting lowland and premontane forest areas, and is found under debris, in pastures, in coffee plantations, and even in urban areas (Savage, 2002). Its venom exerts neurotoxicity and myotoxicity in mice (Gutie´rrez et al., 1986; Goularte et al., 1995; Alape-Giro´n et al., 1994, 1996, 1999). The effects of snake venoms on natural prey have been largely neglected, a trend that needs to be reverted in order to understand the role of such complex toxic secretions in their biological context. M. nigrocinctus feeds mostly on ectothermic prey because analysis of stomach contents of this species revealed caecilians, reptile eggs, many kinds of lizards, and abundant snakes, including those of the genera Anomalepis, Coniophanes, Geophis, and Ninia, which are common forest litter inhabitants (Greene and Seib, 1983; Roze, 1996). Coral snakes forage by crawling and pocking their heads under surface litter, recognizing prey by chemical and visual cues (Greene and Seib, 1983). A comprehensive study on the comparative toxicity of the venoms of various Micrurus species on different prey showed that, as a general rule, venoms are more toxic towards the natural prey than to other types of vertebrates (Jorge da Silva and Aird, 2001). However, the toxicity of M. nigrocinctus venom on its natural prey has not been investigated, nor the role played by venom on immobilization of their prey. The objectives of this study were three-fold: (a) to describe the feeding behavior of M. nigrocinctus on one of its natural prey (the colubrid snake Geophis godmani) in captivity, emphasizing on the role of venom in prey immobilization and ingestion; (b) to investigate the toxicity of M. nigrocinctus venom on four colubrid species of the genera Geophis and Ninia, in order to characterize the pharmacology of this coral snake venom on natural prey and
to explore the potential resistance of such prey to venom; and (c) to study the ability of the blood serum of snakes of the genera Ninia and Geophis to neutralize M. nigrocinctus venom.
2. Materials and methods 2.1. Protocol for the study of feeding behavior This study was performed at the serpentarium of Instituto Clodomiro Picado. A total of 39 adult specimens of M. nigrocinctus (20 females and 19 males) were collected from the following localities in Costa Rica: Guadalupe, Curridabat, San Pedro, Escazu´, and Moravia (province of San Jose´); San Jose´ and Orotina (province of Alajuela); Puntarenas (province of Puntarenas); Can˜as (province of Guanacaste), Santo Domingo (province of Heredia); and Cartago (province of Cartago). Snakes were individually housed in cages of 302515 cm, with water ad libitum in a plastic container; the floor of the cages was covered with paper. All individuals were weighed to the nearest 0.1 g (on a digital balance, Ohaus, model C505-S) and measured, and sex was identified. Snakes were kept at a temperature of 24–26 8C. For this study, each coral snake was fed five times, at intervals of approximately 2 months, although some of them were fed only four times. The first three times snakes were fed without previous extraction of the venom, whereas, in the last two feedings, venom was extracted prior to feeding. For venom extraction, snakes were manually hold and the maxillary region was gently squeezed in order to obtain most of the venom stored in the gland without causing significant mechanical harm to the snake. Thus, although this treatment does not assure a complete depletion of venom, i.e., traces of venom may remain in fangs and in the venom gland, it does remove the great majority of the venom. For the sake of simplicity in describing the results, snakes subjected to this procedure will be referred to as dmilkedT snakes. Experiments to study feeding behavior were carried out 24 hr after venom extraction. To study feeding behavior, individual adult specimens of G. godmani, previously weighed and measured as described, were placed in the cages containing coral snakes, as far from the coral snake as possible (see dimensions of the cage above). G. godmani specimens were collected in the area of Las Nubes de Coronado (San Jose´). The following information was collected: (a) strategy used to capture the prey; (b) time to achieve paralysis, i.e., time interval between bite and immobilization of the prey, that is, when significant body movements of the prey ceased; (c) region of the body where bite occurred, and for this purpose, four distinct anatomical regions (1, 2, 3, and 4) were defined, each one comprising 25% of the body length, starting from the head; (d) if paralysis was achieved by a single bite or if the coral snake had to move towards the head to accomplish prey immobilization; (e) if, after the
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bite, the coral snake moved towards the head to start ingestion or if it moved towards the tail; and (f) time required for ingestion, i.e., the time lapse between initiation of swallowing the prey and the complete ingestion of it. A total of 181 tests, using 39 specimens of M. nigrocinctus, were performed during this study.
processing, tissues were embedded in Spurr resin, and thick (1.5 Am) sections were collected and stained with toluidine blue for histological observation.
2.2. Study of the toxic effects of venom
Blood was collected from five individuals of G. brachycephalus and four individuals of N. maculata. After clotting, the serum was separated by centrifugation at 2000g. For inhibition studies, 75 Ag M. nigrocinctus venom, dissolved in 200 AL PBS, and 300 AL of blood serum of the colubrids were incubated at 37 8C for 30 min. Then, 100 AL of the mixture (containing 15 Ag venom, which corresponds to two intravenous Median Lethal Doses (LD50), Bolan˜os, 1972) was injected intravenously in groups of four CD-1 mice (16–18 g body weight). Control mice were injected with (a) 2 LD50 of venom incubated with PBS without serum, (b) serum incubated with PBS instead of venom, and (c) PBS alone. Mice were observed every hour afterwards, and deaths were recorded.
2.2.1. Venom and snakes Venom from approximately 50 adult specimens of M. nigrocinctus from the provinces of San Jose´, Alajuela, Heredia, and Cartago were obtained. Immediately after collection, venom was frozen at 20 8C and then lyophilized and stored at 20 8C. Venom toxicity was studied in adult specimens of G. godmani, Geophis brachycephalus, Ninia psephota, and Ninia maculata, collected in the regions of Las Nubes de Coronado (San Jose´; the two species of Geophis and N. psephota), San Antonio de Coronado (San Jose´) and Santo Domingo (Heredia; N. maculata); snakes were kept at the serpentarium of Instituto Clodomiro Picado.
2.3. Inhibition of venom toxicity by the blood serum of colubrid snakes
2.4. Statistical analyses 2.2.2. Study of lethality Groups of individuals of G. godmani, G. brachycephalus, N. psephota, and N. maculata were weighed, and injected subcutaneously, in the thoracic region, with solutions containing various venom concentrations, dissolved in phosphate-buffered saline solution, pH 7.2 (PBS). A total of 10 snakes were used for each venom dose tested. The dose of venom injected was expressed as microgram (Ag) venom per gram (g) body weight and was adjusted for each animal by varying the volume of injection of venom solutions of various concentrations; in general, volumes of injection ranged between 80 and 200 AL. An additional control group of 10 individuals was injected with 100 AL of PBS. Snakes were observed and all deaths occurring during a 72-hr observation period were recorded. The Median Lethal Dose (LD50) was determined by the Spearman– Karber method (World Health Organization, 1981) and was defined as the venom dose that induced death in 50% of the injected snakes. 2.2.3. Study of pathological effects To investigate the pathological alterations induced by M. nigrocinctus venom, groups of three specimens of G. godmani were injected subcutaneously, as described above, with either a sublethal dose (4 Ag venom per g body weight) or a lethal dose (10 Ag/g) of M. nigrocinctus venom. Control snakes were injected with PBS under otherwise identical conditions. At 4 and 24 hr after injection, animals were sacrificed and tissue samples were obtained from muscles located near the injected site, as well as from heart and lungs. Tissues were immediately placed in Karnovsky’s fixative and were then postfixed with osmium tetroxide, as previously described (Moreira et al., 1992). After routine
To determine the influence of venom injection and sex on the parameters studied on feeding behavior, tests of homogeneity were performed. For the analysis of the effect of venom injection on time of paralysis and time of ingestion, a three-way Analysis of Variance was used. Linear regression was employed to determine the relationship between time of ingestion and length of the prey.
3. Results 3.1. Experiments on feeding behavior 3.1.1. Search for the prey In the vast majority of experiments, a dsearchingT behavior was observed; in these cases, coral snakes moved slowly, moving their heads forwardly and laterally, and actively flicking their tongue until they found their prey in the cage. In few cases, coral snakes remained in the same location until the prey moved close to them. 3.1.2. Capture and paralysis Once prey were located, coral snakes approached them up to a distance of 1–3 cm, from where they stroke with a rapid forward movement. Bites occurred predominantly in the 25% most anterior part of the body of G. godmani (region I); this region was bitten in 45% of the experiments (X 2=34.45, df=3, Pb0.001; Fig. 1). No significant differences were observed in the anatomical region where bites occurred between male and female coral snakes (X 2=2.53, df=3, P=0.470) nor between snakes with venom and dmilkedT snakes (X 2=3.82, df=3, P=0.282; Fig. 1). In
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Pb0.001). In addition, the time to achieve prey immobilization was more prolonged in dmilkedT snakes (mean=1273.1 sec, S.D.=800.5, range=463–3660 sec) than in snakes with venom (mean=671.4 sec, S.D.=352.3, range=164–2102 sec; F (1,138)=45.70, Pb0.005). 3.1.3. Events occurring before swallowing the prey In 97.8% of the experiments, coral snakes started to move towards the head of the prey, without releasing it, whereas, in only 2.2% of the cases, the movements were directed towards the tail (X 2=165.36, df=1, Pb0.001). In this regard, no differences were found between snakes with venom and dmilkedT snakes (X 2=0.19, df=1, P=0.661) nor between males and females (X 2=0.01, df=1, P=0.938), except that snakes with venom waited until paralysis ensued to perform these movements. In the few cases where coral snakes started moving their heads towards the tail of the prey, the snakes stopped this movement at a certain point and reverted its direction towards the head of the prey. Snakes reached the head of the prey through alternating jaw movements. Fig. 1. Anatomical location of bites by M. nigrocinctus in G. godmani. The body of G. godmani was divided into four anatomical regions (1, 2, 3, and 4), each region comprising 25% of the body length; region 1 corresponds to the most anterior part of the body. Results are presented as the frequency (number) of bites. The upper figure compares the frequency between males and females, whereas the lower figure compares the frequency of bites between coral snakes that were not dmilkedT (A) and those that were dmilkedT (B) before the experiments. Region 1 is the site where bites predominantly occurred, whereas there were no significant differences between males and females and between dmilkedT and dnonmilkedT snakes regarding the anatomical site of the bite (see text for details).
96.7% of the tests, coral snakes stroke their prey and hold them; this pattern was significantly more frequent that the pattern based on bite, release, and relocation of the prey (only 3.3% of trials; X 2=157.8, df=1, Pb0.001). Both experimental groups of coral snakes behaved similarly in this regard (X 2=2.66, df=1, P=0.103), and no differences were observed between male and female coral snakes (X 2=0.54, df=1, P=0.463). A significant difference between snakes with venom and dmilkedT snakes was observed on the behavior after the bite. Snakes with venom remained hold to the prey at the site of initial bite and achieved paralysis, without having to move their heads along the body of the prey, in 75.4% of the experiments. In only 24.6% of the tests, they moved towards the head of the prey before the onset of paralysis. In contrast, 79.4% of coral snakes that had been dmilkedT moved their heads after the initial bite, without releasing the prey which had not been paralyzed, approaching the prey’s head and biting them in the skull. In only 20.6% of the bites, dmilkedT snakes remained hold to the prey at the site of the initial bite. The difference observed in this behavioral pattern after the bite between snakes with venom and dmilkedT snakes is statistically significant (X 2=1.83, df=1,
3.1.4. Ingestion of the prey Coral snakes ingested their prey head-first by performing alternating jaw movements which occur concomitantly with lateral movements of the head. The time of ingestion in coral snakes with venom was 1164.8 sec (S.D.=665.7, range=312–4357 sec), whereas the same parameter in dmilkedT snakes was 1114.5 sec (S.D.=603.5, range=435– 3625 sec); this difference was not statistically significant ( F (1,138)=0.19, P=0.664). No significant correlation was found between ingestion time and total length of the prey (r 2=0.01, P=0.216). 3.2. Toxicity of the venom 3.2.1. Lethality A large variation was observed in the values of LD50 among the various colubrid species injected with M. nigrocinctus venom by the subcutaneous route (Table 1). The highest toxicity was observed towards G. godmani Table 1 Lethality of M. nigrocinctus venom on various colubrid snake species and mice Species
LD50 (Ag/g)
Reference
Geophis godmani Geophis brachycephalus Ninia maculata Ninia psephota Mus musculus
7.67 (5.41–10.86) 21.69 (16.41–28.67) N50a N50a 2.06 (1.75–2.53)
This work This work This work This work Gutie´rrez et al. (1991)
Lethality is expressed as Median Lethal Dose (LD50), in terms of microgram (Ag) venom per gram (g) body weight, using the subcutaneous route; 95% confidence limits are included in parenthesis. a In these cases, no lethality was observed with the largest amount of venom tested (50 Ag/g).
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ance of these organs between envenomated snakes and snakes injected with PBS. Histological observation of skeletal muscles obtained from regions close to the injection site in the thoracic region evidenced conspicuous myonecrosis in snakes injected with both doses of M. nigrocinctus venom. Necrotic cells showed hypercontraction of myofilaments, leaving areas of the cell devoid of myofibrils (Fig. 2). Some muscle cells presented discrete lesions, known as ddelta lesionsT (Gutie´rrez et al., 1984), at the periphery. There was no microscopic evidence of hemorrhage because the structure of capillary vessels remained normal (Fig. 2). In samples collected 24 hr after venom injection, an inflammatory infiltrate was observed. Tissue sections from snakes injected with PBS were devoid of pathological alterations in skeletal muscle tissue (Fig. 2). No histopathological alterations were observed in samples obtained from heart and lungs from snakes injected with either PBS or coral snake venom. Furthermore, no local and systemic hemorrhage was observed. Thus, M. nigrocinctus venom induced predominantly neurotoxic and myotoxic effects in G. godmani. 3.3. Inhibition of venom toxicity by blood serum of colubrid snakes Fig. 2. Light micrographs of skeletal muscle of G. godmani 4 hr after subcutaneous injection of either 100 AL of PBS (A) or M. nigrocinctus venom (10 Ag venom/g body weight) dissolved in 100 AL of PBS (B). Tissue from snakes injected with PBS shows a normal histological pattern, whereas a number of necrotic muscle cells (N) are observed in tissue injected with venom. Necrotic cells are characterized by hypercontraction of myofibrillar material. Notice the presence of intact blood microvessels (arrow) in envenomated tissue, in agreement with the lack of hemorrhagic activity of this venom in G. godmani. Bar represents 50 Am.
Mice receiving an intravenous injection of the blood serum of G. brachycephalus or N. maculata did not show any evidence of toxicity nor any behavioral change when compared with mice injected with PBS by the same route. In
(LD50=7.67 Ag/g), whereas LD50 in G. brachycephalus was 21.69 Ag/g. In contrast, N. maculata and N. psephota showed a high resistance to the action of this venom because all injected snakes survived, even at the highest venom dose tested (50 Ag/g). When injected with lethal doses of venom, snakes of the two Geophis species presented signs of paralysis, with reduction in their movements before they died. Despite not dying, the species of Ninia receiving the highest dose of venom tested showed signs of neurotoxicity, i.e., reduction in their movements. It has been reported that the LD50 of M. nigrocinctus venom in mice, using the subcutaneous route, is 2.06 Ag/g (Gutie´rrez et al., 1991; Table 1). 3.2.2. Pathological effects Macroscopic observations of G. godmani specimens injected with lethal (10 Ag/g) or sublethal (4 Ag/g) doses of M. nigrocinctus venom revealed an inflammatory exudate, without hemorrhage, at the site of venom injection. Such exudate was not observed in snakes injected with PBS. No hemorrhagic lesions were observed in lungs and heart, and there were no differences in the macroscopic appear-
Fig. 3. Time-course of mortality in groups of four mice injected intravenously with either 2 LD50s of M. nigrocinctus venom incubated with PBS (n), or the same dose of venom incubated with blood serum of G. brachycephalus (.) or serum of N. maculata (E). Deaths were recorded every hour and survival expressed as percentage. The mortality curves of mice receiving venom alone or venom plus G. brachycephalus serum were identical and are, therefore, superimposed, whereas a prolongation in the time of death occurred when venom was incubated with N. maculata serum.
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contrast, mice receiving 2 LD50s of M. nigrocinctus venom showed rapid manifestations of neurotoxic envenomation, with hindlimb paralysis and respiratory difficulties, a process that ended in death within 3 hr (Fig. 3). When venom was incubated with the serum of G. brachycephalus before injection, mice showed the same pattern of toxic effects and lethality, and there was not a significantly delay in the time of death when compared with mice injected with venom alone (Fig. 3). In contrast, incubation of venom with the serum of N. maculata caused a partial inhibition of neurotoxic activity, reflected in a prolongation of the time of death (Fig. 3).
4. Discussion In captivity, M. nigrocinctus uses a feeding behavior based predominantly on searching for prey in the cage, a pattern that has been also described for other elapid species (Radcliffe and Chiszar, 1980; Greene, 1984; Mattison, 1995). Micrurus fulvius probes leaf litter in search for its natural prey, constituted mostly by burrowing colubrid snakes (Greene, 1984). The search for prey was associated in our experiments with forward and lateral movements of the head, and with frequent tongue flicking, evidencing the relevance of chemical cues in this search. In addition, visual cues may be also important (Greene, 1984, 1997). After localization of the prey, M. nigrocinctus bit mostly in the cephalic and thoracic regions of G. godmani. In the case of specimens that had not been depleted of venom, the snakes hold to their prey at the site of the bite until they were paralyzed, a pattern also described for M. fulvius (Greene, 1984) and other elapids (Radcliffe and Chiszar, 1980; Kardong, 1982); in only few cases did coral snakes release their prey and relocalized them again. In the case of coral snakes that had been dmilkedT before feeding, the bite occurred at the same anatomical locations, but because paralysis was not achieved, the snakes moved their head, without releasing their prey, towards the anterior end, reaching the head and biting in the cephalic region, often causing mechanical damage to the skull of G. godmani. It is likely that prey movements inhibit preingestion maneuvers (Greene, 1984). In addition, our observations suggest that, if preys are not paralyzed within the first minutes after the bite, a second strategy for prey immobilization is employed, based on approaching and biting the head of the prey, inflicting evident mechanical damage, a strategy that has been described in snakes that do not rely on constriction or envenomation to restrain their prey (Cundall and Greene, 2000). The adaptive value of this alternative behavioral pattern is probably based on a reduction of the time spent before the beginning of ingestion, with the consequent reduced risk of predation or damage by the prey being attacked. Hence, venom injection by M. nigrocinctus plays a relevant role in prey immobilization, contributing to rapid paralysis and early prey ingestion. In the absence of venom,
M. nigrocinctus has to rely on mechanical damage to immobilize prey, requiring more time to initiate ingestion. In the vast majority of observations, M. nigrocinctus swallowed G. godmani head-first after the onset of paralysis, a pattern commonly observed in coral snakes (Roze, 1982, 1996) and other elapids (Voris et al., 1978). However, in many experiments, G. godmani specimens were still alive, although paralyzed, when ingestion started, in agreement with the observations of Greene (1984) with M. fulvius. In the few cases in which head movements after the bite were directed towards the tail of G. godmani, coral snakes stopped at a certain point, reversed this trend and started moving their heads toward the head of G. godmani. Detection of the orientation of scale superposition has been proposed as a mechanism to identify the prey’s head (Greene, 1976). Once ingestion started, no differences were observed in the time of ingestion between the two experimental groups, suggesting that, although venom plays a role in paralysis, it does not contribute to a more rapid ingestion. The times recorded for prey ingestion are similar to those previously reported by Roze (1982) for coral snakes. The paralytic effect induced in G. godmani agrees with the experiments performed by injecting M. nigrocinctus venom in this colubrid species. In both cases, G. godmani specimens reduced their movements until they became fully paralyzed before dying. Hence, observations clearly demonstrate a neurotoxic effect of M. nigrocinctus venom on snakes of the genus Geophis. Various short-chain neurotoxins have been isolated from this venom (Rosso et al., 1996; Alape-Giro´n et al., 1996), and the complete amino acid sequence of one of them has been determined (Rosso et al., 1996). a-Neurotoxins block the nicotinic cholinergic receptor at the motor end plate of mammalian skeletal muscle, originating a flaccid paralysis that culminates in respiratory arrest (Vital-Brazil, 1987; Rosso et al., 1996). A conspicuous variation in susceptibility to M. nigrocinctus venom was observed in G. godmani, G. brachycephalus, N. psephota, and N. maculata because the latter two species resisted a venom dose as high as 50 Ag/g body weight, and G. brachycephalus required a significantly higher dose than G. godmani to die. It is noteworthy that this venom is more toxic to mice, on a weight per weight basis and using the subcutaneous route, than to these four colubrid species, although mice do not constitute a natural prey for this coral snake (Greene and Seib, 1983). Our observations indicate that M. nigrocinctus venom does not follow the general trend, described for various Micrurus species, of being more toxic to their natural prey than to other vertebrates that do not constitute normal prey (Jorge da Silva and Aird, 2001). It is likely that a natural resistance to M. nigrocinctus venom has evolved in the colubrid species included in the present study, particularly in Ninia species. Notwithstanding, our unpublished observations indicate that M. nigrocinctus fed with N. maculata are able to paralyze this colubrid before ingestion, thereby indicating
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that the amount of venom delivered in a bite is sufficient to kill even this highly resistant prey. The venom yield of M. nigrocinctus is 5.76 mg (Bolan˜os, 1972). In general terms, natural resistance of animals to the action of snake venoms relies on two different mechanisms: (a) the presence of inhibitory proteins in serum (Domont et al., 1991; Lizano et al., 2003) and (b) modifications in the structure of cholinergic receptor, in such a way that the affinity of neurotoxins for this receptor is reduced (Takacs et al., 2004). It is noteworthy that, among the four colubrid species studied, the one showing the highest resistance was N. maculata, being also the most sympatric with M. nigrocinctus in Costa Rica (Savage, 2002), suggesting that a selective pressure may have been at work in the acquisition of resistance of this colubrid to the venom of M. nigrocinctus. Interestingly, the blood serum of N. maculata was able to prolong the time of death of mice injected with M. nigrocinctus venom, whereas no such effect was observed with the serum of G. brachycephalus. This suggests that the observed resistance may be at least partially due to inhibitory serum factors to venom neurotoxic components. In addition to many isoforms of a-neurotoxins, M. nigrocinctus venom is rich in phospholipases A2 (AlapeGiro´n et al., 1994, 1996, 1999; Rosso et al., 1996). Venom phospholipases A2 display a wide variety of pharmacological effects owing to a process of accelerated evolution (Kini, 1997, 2003; Ohno et al., 2003). Many elapid venoms contain extremely potent neurotoxic phospholipases A2 which act presynaptically at the neuromuscular junction, an effect described for M. nigrocinctus venom (Goularte et al., 1995). In addition, M. nigrocinctus phospholipases A2 exert potent myotoxicity in mice (Arroyo et al., 1987; Alape-Giro´n et al., 1994, 1999). Our histopathological observations clearly evidence a myotoxic effect of this venom on thoracic skeletal muscles of G. godmani. As previously described for mouse skeletal muscle (Gutie´rrez et al., 1986; Arroyo et al., 1987), myonecrosis in G. godmani is of rapid onset and is characterized by hypercontraction of myofibrils and by the presence of cells with ddelta lesionsT, which have been associated with early plasma membrane disruption (Gutie´ rrez et al., 1984). Such observation suggests that M. nigrocinctus myotoxins act at the plasma membrane of muscle cells, promoting a calcium influx responsible of hypercontraction. Preliminary ultrastructural observations corroborated the presence of interruptions in the continuity of G. godmani muscle cell plasma membrane (our unpublished data). Early plasma membrane damage characterizes the action of several myotoxic phospholipases A2 isolated from various elapid venoms (Harris and Cullen, 1990). Results suggest that M. nigrocinctus venom act in a similar way in mouse and G. godmani skeletal muscles, the plasma membrane being the first site of action. As described in mice (Gutie´rrez et al., 1980, 1986), M. nigrocinctus venom does not induce local nor systemic hemorrhage in G. godmani, nor does it affect cardiac muscle and pulmonary
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tissue. Thus, the pathophysiology of envenomation by M. nigrocinctus venom in G. godmani is characterized by neurotoxicity and myotoxicity. The biological role of myotoxicity induced by M. nigrocinctus phospholipases A2s is not clear at present because it does not seem to be required for prey immobilization, the latter being achieved by the action of neurotoxins. This venom has a very low proteolytic action on a variety of substrates (Lomonte and Gutie´rrez, 1983; Jorge da Silva and Aird, 2001); hence, digestion of proteins has to be achieved by proteinases present in gastric and pancreatic secretions. It is suggested that widespread muscle degeneration in colubrid snakes due to envenomation by M. nigrocinctus may facilitate the digestion of the muscle mass by such proteinases, a hypothesis that needs to be tested in future studies. Various Micrurus sp. venoms have been reported to induce hemorrhage upon intravenous injection in mice (Jorge da Silva and Aird, 2001), and a hemorrhagic phospholipase A2 was isolated from Micrurus frontalis frontalis venom (Francis et al., 1997). No hemorrhagic effect was observed in G. godmani injected with M. nigrocinctus venom. Lack of hemorrhage was also described in mice injected with this venom (Gutie´rrez et al., 1980) and with a purified myotoxic phospholipase A2 (Arroyo et al., 1987). In conclusion, M. nigrocinctus venom exerts neurotoxic and myotoxic effects on G. godmani and plays a key role in prey immobilization, reducing the time lapse between bite and ingestion. A drastic variation in susceptibility to M. nigrocinctus venom occurs between different colubrid snake species, and a significant natural resistance to M. nigrocinctus venom was observed in N. maculata, a prominent component in the diet of this coral snake.
Acknowledgements The authors thank Rodrigo Aymerich, Danilo Chaco´n, Gerardo Serrano, Santos Rodrı´guez and Randall Valverde for their valuable collaboration in various aspects of this study. Thanks are also due to Mahmood Sasa for fruitful discussions and for critical review of the manuscript, and to Javier Nu´n˜ez for collaboration in the histological work. A. Urdaneta received support from the Deutscher Akademischer Austauschdienst (DAAD) for his graduate studies. This work was carried out in partial fulfillment of the requirements for the M.Sc. degree of A. Urdaneta at the University of Costa Rica.
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