Venomic analyses of Scolopendra viridicornis nigra and Scolopendra angulata (Centipede, Scolopendromorpha): Shedding light on venoms from a neglected group
Introduction
Centipedes are elongated and dorso-ventrally flattened arthropods, which belong to the Chilopod class. Their body is divided in head and thorax. Each thorax segment carries one pair of ambulatory legs except for the one in the hindmost segment where these structures were modified for mechanical defense and/or for sensory purposes. Another pair of modified legs, the forcipules, is located in the post-cephalic segment. The femoral part of the forcipule contains a venom gland, which communicates with the exterior through an orifice in the tip of these structures. The venom is used both to subjugate prey and in the defense against predators (Lewis, 1981).
There are five Chilopod orders (Geophilomorpha, Scutigeromorpha, Lithobimorpha, Craterostigmomorpha and Scolopendromorpha), all of them possessing venom apparati, with the scolopendromorphs being those most frequently involved in human accidents (Lewis, 1981).
Generally, the venom of scolopendromorphs is not lethal to humans, although a few lethal accidents have been reported (Pineda, 1923; Remington, 1950; Serinken et al., 2005). Symptoms of the sting are restricted to intense local or irradiating pain, redness, edema, local hyperthermia, and superficial necrosis. Amongst the observed systemic symptoms which have been reported are nausea, emesis, sudoresis, anxiety and depression (Knysak et al., 1998; Barroso et al., 2001; Bush et al., 2001). Myonecrosis, leading to rhabdomyolysis and acute renal insufficiency (Logan and Ogden, 1985) and proteinuria (Hasan and Hassan, 2005) have also been associated with centipede accidents.
Scolopendrism—the name by which the accidents with scolopendromorphs are known—figures prominently amongst the accidents caused by poisonous animals. For instance, between 1998 and 2000 the accidents involving scolopendromorphs corresponded to 16.8% of all registered accidents in the Toxicological Information Center of Belém (Pará, Brazil) (Barroso et al., 2001). Also, amongst the 3000 annual cases of accidents caused by venomous animals reported to the Vital Brazil Hospital of the Butantan Institute (HVB-IB), which is considered to be the main Brazilian medical center for notification of such cases, 5% are caused by centipedes (Knysak et al., 1998).
Despite this medical importance, information about the composition of venoms of scolopendromorphs is very limited. This is probably due to the lack of severe systemic symptoms and fatalities in adults. Early analyses of crude extracts obtained from the venom glands have shown the presence of serotonin, histamine, lipids, polysaccharides and polypeptides (Welsh, 1963; Gomes et al., 1982; Mohamed et al., 1983). Protease activities in Scolopendra species have also been reported (Zaid, 1958; Mohamed et al., 1983; You et al., 2004).
Other studies have focused on the pharmacological properties of the crude venom or its fractions. Extracts of the venom gland from Scolopendra morsitans were capable of arresting the muscle heart of toads and to provoke relaxation in a number smooth-muscle preparations, besides being capable of evoking hyperglycemia together with liver glycogenolysis (Mohamed et al., 1980). In another study, it was demonstrated that the application of a Scolopendra sp. venom fraction, named SC1, on the cockroach giant axon induced an increase in the leak current, correlated with a decrease in the membrane resistance (Stankiewicz et al., 1999). The authors supported the idea that components in SC1 were able to open non-specific pores in the axonal membrane. The presence of muscarinic agonists in SC1 has also been demonstrated. More recently, an arthropod-toxic fraction from the venom of Scolopendra sp. was shown to increase the basal release of neurotransmitters in the ventral ganglia of crustaceans (Gutierrez et al., 2003).
Several of these authors comment on the paucity of information available about the venom from the scolopenders. Others cite the biotechnological potential of the toxins from these Chilopods as bioinsecticides (Stankiewicz et al., 1999; Gutierrez et al., 2003) and as tools for the study of the insect nervous system (Gutierrez et al., 2003). Also it is relevant to note that centipede extracts have been used in Chinese traditional medicine for the treatment of tetanus, convulsions and acute heart attacks (Noda et al., 2001).
In this study, we have used a structure to function proteomic approach, combining 2D-LC, ESI-Q-TOF/MS, N-terminal sequencing and similarity searching to better understand the complexity and biotechnological potential of the venoms from two Brazilian Scolopendra species: Scolopendra viridicornis nigra and Scolopendra angulata. Also, fractions from S. viridicornis venom were screened for toxicity towards insects.
Section snippets
Reagents and chemicals
All reagents and chemical products were of analytical or spectroscopic grade and were obtained from the following sources: sodium acetate, sodium chloride, 4-vinylpyridine and bovine serum albumin (BSA) were from Sigma-Aldrich (Saint Louis, USA). PTH-Amino acids mobile phase, 5% phenyl isothiocyanate n-heptane solution and 12% trimethylamine solution were from Wako (Osaka, Japan). Trifluroacetic acid was from Vetec (Rio de Janeiro, Brazil). Ethyl acetate and 1-chlorobutane were from TEDIA (Rio
Two-dimensional chromatography and mass spectrometry analyses
In order to assess the complexities of the venoms and to purify the largest possible number of proteins to be N-terminally sequenced, venoms from both species were subjected to two-dimensional liquid chromatography. This technique consists of the sequential use of ion-exchange fractionation (first dimension), followed by further purification by RPC (second dimension) of the fractions obtained in the first step.
The CIEX profile of both venoms is shown in Fig. 1. The CIEX from S. viridicornis
Conclusions
The venoms studied in this work are quite diverse, as may be expected from a generalist feeding species such as the representatives of the Scolopendra genus (Lewis, 1981). However, the venoms of S. viridicornis nigra and S. angulata are very similar to each other, in chromatographic behavior, mass composition and protein sequences.
In this study, the identification of the toxin classes in the venoms of these two Brazilian centipede species was aimed. In order to achieve this goal, at least one
Acknowledgments
We would like to acknowledge the technical support work performed by Julio Cesar dos Reis in keeping the animals and in venom extraction. We would also like to acknowledge the help of Adriano Barbosa, Armando Menezes, Daniel Carvalho, Estevam Bravo, Isabella Sander and Rodrigo Pacífico in the capture of S. viridicornis nigra specimens.
We are thankful to Dr. Irene Knysak and Samuel Paulo Gioia Guizze for the taxonomical work and Dr. Gloria R. Franco and Dr. Marcelo M. Santoro for fruitful
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