Experiencias del mantenimiento en cautiverio de escorpiones en Costa Rica para la extraccion de veneno.
Approximately 2 000 species of scorpions can be found around the world, and are classified in 15 families (Fet & Soleglad, 2005; Rein, 2013). Very few of these animals are considered "dangerous" for human beings, and the main scorpionism cases have been reported for regions such as: Australia (Isbister, Volschenk, & Seymour, 2004), Asia (Dehghani, Vazirianzadeh, Nasrabadi, & Moravvej, 2010), Africa (Laid et al., 2012), and America: especially Latin America (Borges, Miranda, & Pascale, 2012). An approximate number of 30 species of scorpions are potentially harmful for human beings, where 19 species belong to the family Buthidae, and the genus Centruroides and Tityus, found in America (Chippaux & Goyffon, 2008). The Centruroides genus is distributed from the North part of the American continent to Central America, and the Tityus genus is found from Central America through South America (Borges et al., 2012).
The highest annual incidence of scorpion stings in America occurs in Mexico, with 2 050 scorpion stings by 100 000 inhabitants (Chippaux & Goyffon, 2008). Other Latin American countries have lower scorpion sting incidence, for instance Brazil, Venezuela and Colombia with values of 142, 40, 5 scorpion sting by 100 000 inhabitants, respectively. It is worth mentioning that some of these scorpion stings end up in fatalities, especially in children (Chippaux & Goyffon, 2008). Due to the scorpionism occurrence in Latin America, the need for an antivenom elaboration has emerged. Thus, several commercial antivenoms are produced in different countries such as Brazil (Candido & Lucas, 2004), Mexico (Chippaux & Goyffon, 2008) and Venezuela (Poggioli De Scannone, 1996).
In Central America the studies on scorpions are scarce, probably due to the reduced amount of species that inhabit these countries. Costa Rica reports 14 species within seven genera and four families; with the genus Centruroides and Tityus presenting medical importance due to their envenomation features. Moreover, these two genera represent an emerging potential problem to the public health system, for Costa Rica and the other Central American countries (Leveridge, 2000).
In Costa Rica, the Clodomiro Picado Institute has a remarkable work on research and elaboration of snakebite antivenoms (Rojas, Jimenez, & Gutierrez, 1993); however, the scorpionism was not been attended until now. Furthermore, the National Center of Intoxication has reported an increased number of scorpion stings in the country since the 90's decade: only 24 cases for 1992, 206 cases in 1998, and 405 cases in 1999, practically a 100% increase (Leveridge, 2000). The current problem is that there is no proper antivenom for its treatment, and the gap between the scorpionism stings problem and an effective treatment became evident. Considering the scorpion stings relevance in public health and the potential harm that it may cause, the Clodomiro Picado Institute decided to study the toxins of the major scorpion species inducing pathologic effects, as well as the establishment of a live animal maintenance installation for venom extraction purposes.
Nowadays, the establishment of collections in captivity of animals that may cause a public health impact is fundamental. The elaboration of scorpion antivenoms requires the establishment of a collection in order to maintain the animals and for venom extraction. A previous effort was made by Bucherl of the Butantan Institute, that described the techniques for keeping a live collection of scorpions in laboratory conditions for venom extraction purposes (Whittemore, Keegan, Fitzgerald, Bryant, & Flanigan, 1963). Thus, the aim of this work was to report the experiences gathered in keeping a live collection of Costa Rican scorpions, for venom extraction, at the Clodomiro Picado Institute.
GENERAL PROCEDURES AND PRELIMINARY FINDINGS
An emergence and importance of the scorpionism in Latin America has increased in the last few years, especially in countries such as Costa Rica, where hundreds of scorpion stings have been reported, including one fatality in 1995 (Leveridge, 2000). For this reason, a group of scientists of the Clodomiro Picado Institute, started a research project on the characterization and, eventually, of a pilot antiscorpion serum batch elaboration.
Scorpions capture field trips: Scheduled field trips were conducted from 2005 through 2011 to the following locations: Santa Rosa National Park, Palo Verde National Park, Quepos, Playa Piro, Cabo Blanco and Carara in the Pacific versant; Hitoy Cerere National Park, Chiroles, La Selva Biological Station, Siquirres, Guayacan, Barbilla National Park and La Gamba in the Caribbean versant of Costa Rica (Fig. 1). Additionally, some scorpions were provided by particulars. The scorpions were collected using an ultraviolet flashlight and an intensive seeking strategy; captured scorpions were transported to the Clodomiro Picado Institute located in Dulce Nombre de Coronado, San Jose, Costa Rica; and were registered in the collection. The following species were collected during such field trips: Ananteris platnicky (Lourenco, 1982), Didymocentrus krausi (Francke, 1978), Centruroides edwardsii (Gervais, 1843), C. koesteri (Kraepelin, 1911), C. bicolor, C. limbatus, Tityus championi and T. pachyurus (Pocock, 1898), T. cerroazul (Lourenco, 1986), T. dedoslargos and T. ocelote (Francke & Stockwell, 1987).
[FIGURE 1 OMITTED]
On the other hand, 88% of the animals were collected by the Clodomiro Picado Institute staff during field trips. In Santa Rosa, Guanacaste, 800 scorpions were captured during several field trips. The most common species found in this locality was C. edwardsii, however, C. koesteri and D. krausi individuals were collected as well. Other collecting sites included Palo Verde National Park, located in Guanacaste; where the same species of scorpions were found. Additionally, Playa Piro, Peninsula de Osa, Puntarenas was visited, where the totality of C. bicolor and T. championi scorpions were found (Fig. 1).
The majority of specimens captured belong to the genus Centruroides, being C. edwardsii the most well-known scorpion in the country, especially by people in the North Pacific and Central Valley of Costa Rica. The species of this genus in Costa Rica can tolerate alterations in the forest and are even well adapted to urban environments (Viquez, 1999). On the other hand, C. limbatus is the most common species in the Caribbean, and C. bicolor the most common in the South Pacific (Viquez, 1999). Scorpions of the genus Tityus showed arboreal habits and can be found in unaltered to less altered forests (Borges et al., 2012); this made its observation and capture difficult, resulting in less encounters.
Maintenance in captivity: Considering the first and last register in the animal collection, the maintenance in captivity of these animals had a duration of nine years (2005-2013), resulting in 2 043 scorpions in total, classified in 11 species (Fig. 2A). The arrival of the animals was inconsistent throughout this period, showing highs and lows in the animal registration. Furthermore, since 2009, the scorpion's arrival showed a marked decrease with almost none in the last two years (Fig. 2B).
In the Clodomiro Picado Institute, within the Serpentarium section, a 15 [m.sup.2] laboratory was used to house the scorpions. The average temperature and relative humidity recorded during the day was 22.8[degrees]C and 76%, respectively. The scorpions were maintained individually in plastic cages (17 cm diameter and 5.5 cm height) to avoid cannibalism. Inside the cages, a Kraft[R] paper piece was included, providing shelter to the scorpions; in addition, a wet cotton ball was included as a water source. Each cage was identified with the general information of the individual, and was organized by species in the laboratory shelves.
[FIGURE 2 OMITTED]
On a weekly basis, each cage was subject to a general cleaning process, removing the remains of preys, and cleaning the feces with cotton, or replacing the container when necessary. The Kraft [R] paper and cotton ball were replaced weekly as well. Food was provided every 15 days with three types of preys: Tenebrio molitor larvae, Acheta domestica crickets and several species of cockroaches. The preys were also raised and maintained at the Institute.
The feeding behavior observed in the genus Centruroides and Didymocentrus was predominantly active, capturing the prey immediately after it was deposited in the cage. The scorpions captured the prey with the pedipalps and inoculated venom rapidly, stinging the prey as many times as necessary to subdue it; they ate it later. On the other hand, the Tityus genus was not prone to subdue the prey immediately; but the preys were eaten in a similar way as the other genera. Additionally, the genus Tityus tended to stand more frequently on the wet cotton balls. The feeding behavior needs further investigation, since a methodological design applicable to each species was considered necessary. It is possible that scorpions of the genus Centruroides and Didymocentrus are active predators, while the genus Tityus might be sit-and-wait or ambush predators (Polis, 1990).
The environmental conditions, substratum, feeding programs, feeding items and hydration we used were the same as those employed in the maintenance of collections of scorpions, spiders, insects and other arthropods worldwide. Nevertheless, most of the Costa Rican species tend to fall into cannibalism when kept in captivity, requiring individual maintenance. This feature is contradictory to the reports of the Butantan Institute, where up to 300 scorpions could be maintained in one single box, provided with corrugated cardboard acting as shelter and preventing stress conditions and cannibalism (Candido & Lucas, 2004).
The maintenance conditions of the live collection were carefully controlled, yet a high number of deaths were registered during the entire captivity period. One of the main problems related to this issue was inanition, either because the scorpion rejected the prey, or because the use of the wrong size of prey to feed the animals. When the scorpion did not eat the prey, a decomposition process began inside the cage, which could trigger the appearance of fungus, which might be another cause of death.
Additionally, the presence of other arthropods such as mites, spiders, collembolans and psocoptherans, whose origin is unknown, could affect the scorpions. One of the mites identified was Tyrophagus putrescentiae, may have contacted the scorpions through the grains used to feed the preys, such as crickets and mealworm beetles.
On the other hand, scorpions showed a high dehydration tolerance, especially those who live in desert conditions. The most important water source in the wild is obtained from preys (Polis, 1990); nonetheless, dehydration could be considered a death cause in scorpions. However, in laboratory conditions, scorpions drink water from other sources, especially when food items are not adequate for them (Polis, 1990).
Additionally, the major death cause in collections to gather venom is precisely the venom extraction processes and the stress it causes. We observed that scorpions that were not subject to venom extraction processes lived longer (i.e. D. krausi, 4.47 years average) than those submitted to repeated venom extraction processes (Oukkache, Chgoury, Lalaoui, Cano, & Ghalim, 2013).
Newborns and longevity: The animals born in captivity were separated from their mothers when they fell off the mother's back. They were placed together in a terrarium that was provided with water, and were fed with the first larval stages of Tenebrio molitor. By the time of the second molt, the newborns were placed in individual cages and were registered in the collection.
The implementation of a digital database allowed the analysis of general information about the status of the collection through the years. Thus, information such as: amount of animals registered by species, number of species, locations, newborns, number of deaths and longevity was analyzed. Additionally, a distribution map was made, that included the locations from which the animals were collected (using the geographic coordinates of each site and geo-referenced using Qgis v2.8.2 software).
A Non-parametric Kruskal-Wallis test was used to assess the longevity in captivity of the different genus, as well as the differences in longevity of the genus Tityus born in captivity. In addition, a post-hoc test was used to evidence the main differences among the groups. All the tests were performed using R v3.2.1 software, and values of P < 0.05 were considered as statistically significant.
The adaptability to captivity was analyzed by the longevity of each individual of the different species (time since it was first registered in the collection until its death). For these analyses, information concerning 1 614 scorpions was employed. Thus, significant differences in the longevity in captivity of the different genus was found ([H.sub.3] = 353.8; P < 0.001), with the genus Didymocentrus showing a longer time in captivity with an average of 4.47 years, followed by Centruroides with an average of 2.99 years, and finally Tityus, with only 1.07 years in captivity.
In addition, the different species born in captivity showed significant differences in their longevity ([H.sub.3] = 94.32; P < 0.001); for which, T. pachyurus had a higher life expectancy with an average of 1.79 years in captivity when compared to other species.
Most of the scorpions born in captivity die rapidly, which is a common feature within the juvenile scorpions with survival rates reported of just 5% (Gopalakrishnakone, Cheah, & Gwee, 1995). This is mainly due to difficulties in facilities, food sources (i.e. prey sizes), and more specialized professionals to take care of the collection (Candido & Lucas, 2004). On the other hand, in Costa Rica, the genus Tityus shows a dependency towards more humid habitats, since they can be found in very humid forests and are associated to vegetation. Thus, the laboratory conditions could not be suitable for this particular genus; nonetheless, the scorpions T pachyurus born in captivity tolerated the existing laboratory conditions and lived longer than those captured in the wild. This feature could be important in the establishment of a well-developed animal colony for venom extraction purposes.
Venom extraction and yield: The venom extraction processes were conducted three times per year only on C. edwardsii; whilst for C. bicolor, T. championi and T. pachyurus, the venom extraction process took place once a year. This procedure employed the same individuals in each process, and the new incoming animals were added to the procedures, mainly in the case of venom extractions of C. edwardsii.
The device used as electro-stimulator was composed of an electric converter unit (General Electric[R]), where the alternate electric current (120 V) was converted and regulated to provide the micro-volts needed to perform the electrostimulation, a plate supporting the electrodes and a pedal switch to activate the electrostimulus (both homemade). The venom was obtained by electrostimulation of the venom glands located at the telson. For this, each individual was held by the last segment of the tail, and was placed between two electrodes, producing an electro-stimulus of 0.7-0.8 mV. The released venom was collected in a capillary tube and deposited in an Eppendorf tube (0.5 mL), that was kept cold (4-8[degrees]C) during the extraction process.
On the other hand, for C. edwardsii three venom extraction processes per year were conducted during the first six years, with a total of 19 extractions (just one venom extraction for last year); resulting in the highest venom yield (Table 1). For the rest of the species, the venom extraction processes were less frequent, and few species were not even extracted due to its availability.
This process of electro-stimulation has proved to be the most effective technique in terms of quantity and quality of the venom extracted (Oukkache et al., 2013). The venom yield of each species is consequent with the body mass of the scorpions. Thus, the highest venom yield (1.07 mg) corresponded to C. bicolor, which is a large scorpion (reaching 12 cm in total length); the lowest venom yield (0.11 mg) was obtained from T. pachyurus, whose average body size is 5 cm. These amounts of venoms were suitable for the development of antivenoms, since a minimum of 0.4 mg are required for the scorpion antivenom elaboration (Candido & Lucas, 2004).
Venom storage, characterization and evaluation: The venom collected was pooled by species, and kept at -20[degrees]C in 2.0 mL Eppendorf tubes, until it was processed. The venom procedures consisted in centrifuging the venom at 12 000 rpm (15 294 g) for five minutes; then, the supernatant was collected and the pellet discarded. The supernatant was frozen at -90[degrees]C and lyophilized for 24 hours. The solid venom was weighed and stored at 20[degrees]C until it was used.
The venom was characterized by total protein content using a Thermo Scientific NanoDrop 2000 C [R] Spectrophotometer; using the software NanoDrop 2 000 / 2 000 C version 1.4.2, Thermo Fisher Scientific (copyright 2009). The total protein content was determined by absorbance at 280 nm with equivalence of 1 abs unit = 1 mg/mL. We found similar values for all the species studied, with the exception of T. pachyurus, whose total protein content was relatively lower (Table 1). Likewise, the highest total protein content was found in C. bicolor and the lowest protein content in T. pachyurus, which is in correlation with the body mass and the amount of venom yielded.
Additionally, we evaluated the venoms toxicity by the median lethal dose ([LD.sub.50]) as described by Solano et al. (2010). Our results showed that C. bicolor venom had a higher toxicity, and T. pachyurus and C. edwardsii a lower toxicity (Table 1) than the other species. Toxicity of venoms can be related to the type of preys (Polis, 1990); C. bicolor showed the highest toxicity among the species studied, and this might be in correlation with the size and type of preys it feeds. This particular feature may be useful in preying on larger animals, where a high quantity and more lethal venom is required.
A group of five rabbits were used in the development of a pilot anti-scorpion serum. The immunization scheme was as follow: six inocula every 15 days using Complete Freund's coadjutant, and two boosters every 22 days employing Incomplete Freund's coadjutant. Decreasing amounts of venom (300, 150, 75, 37.5, 18.75, and 9.5ug) were used in each inoculum; and a venom solution of 500 uL (w / v) in a final solution (venom-adjuvant); the final volume of inoculation was 2 mL via subcutaneous and multiple sites of injections. Nevertheless, the anti-scorpion serum resulted unable to neutralize the lethal dose used to challenge the venom toxicity. The elaboration of a pilot anti-scorpion serum showed several difficulties in order to raise an acceptable titer of antibodies capable to neutralize the venom toxicity. Previous studies have also found that the anti-scorpion sera have a relative low effective action (Borges et al., 2012). Several factors may have influenced this result: small doses of venoms, frequency of inoculation, use of adjuvants and immunogenicity of the proteins in venom, among others. Additional, research should contribute to find effective protection against scorpion venom toxicity.
The scorpion collection kept along the nine-year period showed to be efficient in terms of venom extraction processes, venom yield and relatively good survival of animals. However, several weaknesses were found, leaving aside important information on nutrition, behavior, growth and development, and environmental conditions. In spite that there is vast information concerning scorpionism, its toxins and the effect of the stings in public health (Lourenco et al., 1996; Batista et al., 2007; Chippaux & Goyffon, 2008), there is scarce information regarding the collection and establishment of scorpion colonies for venom extraction purposes. Although, there are several countries (i.e. Brazil, Mexico, Venezuela) currently working in the development of a scorpion antivenom, we concluded that a deeper investigation on the factors causing deaths in scorpion collections in captivity is necessary. Besides, the improvement of maintenance conditions in laboratories and the breeding procedures of scorpions are imperative, in order to produce the venom required for antivenom elaboration.
This work was partially funded by the Vicerrectoria de Investigacion, University of Costa Rica with the projects: 741-A5048 and 741-B2-081. The Clodomiro Picado Institute partially funded the maintenance of the live collection of scorpions. For the contribution in collecting and maintaining the collection, the authors thank: Jazmin Arias, Fabian Bonilla, Danilo Chacon and Santos Rodriguez, staff members of the Clodomiro Picado Institute. Special thanks to interns and volunteers: Carolina Vega, Carmen Vasquez, Jennifer Rivera, Olga Duran, Rebeca Hidalgo and Mauricio De la O. Also, the authors thank Pamela Zuniga for reviewing the English grammar of the manuscript.
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Emanuel Brenes (1) * & Aaron Gomez (2)
(1.) Escuela de Ciencias Biologicas, Universidad Nacional, 3000 Heredia, Costa Rica; firstname.lastname@example.org
(2.) Instituto Clodomiro Picado, Facultad de Microbiologia, Universidad de Costa Rica, 2060 San Jose, Costa Rica; email@example.com
Received 09-IX-2015. Corrected 03-III-2016. Accepted 04-IV-2016.
TABLE 1 Total venom yield, total protein content and median lethal dose of the main scorpion species maintained at the Clodomiro Picado Institute, Universidad de Costa Rica Number of Average of Venom yield Species extractions individuals (mg) Centruroides edwardsii 19 200 1 269.3 Tityus championi 5 71 137.6 Centruroides bicolor 5 23 122.8 Tityus pachyurus 3 15 5.1 Average venom yield Total by Protein [LD.sub.50] individual content (mg/ ([micro]g/g Species (mg) mL) mice) Centruroides edwardsii 0.40 1.11 4.4 Tityus championi 0.39 1.07 3.9 Centruroides bicolor 1.07 1.43 3.4 Tityus pachyurus 0.11 0.93 4.1
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|Title Annotation:||texto en ingles|
|Author:||Brenes, Emanuel; Gomez, Aaron|
|Publication:||Revista de Biologia Tropical|
|Date:||Sep 1, 2016|
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