Variation in venom yield and protein concentration of the centipedes Scolopendra polymorpha and Scolopendra subspinipes

Highlights

• We examined influences on venom yield in Scolopendra polymorpha and Scolopendra subspinipes.

• Volume yield was linearly related to centipede body length.

• Volume yield increased with increasing relative forcipule length and relative body mass.

• Venom volume and protein concentration were lower for previously milked animals.

• This is the most comprehensive study to date of venom yield in any centipede.

Abstract

Venom generally comprises a complex mixture of compounds representing a non-trivial metabolic expense. Accordingly, natural selection should fine-tune the amount of venom carried within an animal's venom gland(s). The venom supply of scolopendromorph centipedes likely influences their venom use and has implications for the severity of human envenomations, yet we understand very little about their venom yields and the factors influencing them. We investigated how size, specifically body length, influenced volume yield and protein concentration of electrically extracted venom in Scolopendra polymorpha and Scolopendra subspinipes. We also examined additional potential influences on yield in S. polymorpha, including relative forcipule size, relative mass, geographic origin (Arizona vs. California), sex, time in captivity, and milking history. Volume yield was linearly related to body length, and S. subspinipes yielded a larger length-specific volume than S. polymorpha. Body length and protein concentration were uncorrelated. When considering multiple influences on volume yield in S. polymorpha, the most important factor was body length, but yield was also positively associated with relative forcipule length and relative body mass. S. polymorpha from California yielded a greater volume of venom with a higher protein concentration than conspecifics from Arizona, all else being equal. Previously milked animals yielded less venom with a lower protein concentration. For both species, approximately two-thirds of extractable venom was expressed in the first two pulses, with remaining pulses yielding declining amounts, but venom protein concentration did not vary across pulses. Further study is necessary to ascertain the ecological significance of the factors influencing venom yield and how availability may influence venom use.

Introduction

Many animals depend on venoms to procure food, defend themselves, or deter competitors (Mebs, 2002). Maintaining a sufficient venom supply is essential to avoid the serious costs of venom depletion, including lost prey capture opportunities and diminished defensive capabilities (Currier et al., 2012, Haight and Tschinkel, 2003, Hayes, 2008, Malli et al., 1998). Because venom is generally a complex mixture of compounds (Fry et al., 2009, Rodriguez de la Vega et al., 2010, Undheim and King, 2011), representing a non-trivial metabolic expense (Billen, 1990, McCue, 2006, Nisani et al., 2012, Nisani et al., 2007, Pintor et al., 2010, Pintor et al., 2011), natural selection should fine-tune the amount of venom carried within an animal's venom gland(s) (Mirtschin et al., 2002). The amount of venom an animal possesses may be influenced by ultimate factors such as prey type, prey size, and rates of prey encounter and venom regeneration (Mirtschin et al., 2002).

One measure of the amount of venom an animal possesses is venom yield, the quantity of venom expelled, either voluntarily or involuntarily, from an intact, live animal. Venom yield in arthropods has been measured most commonly in terms of dry mass (Herzig, 2010, Herzig et al., 2008), volume (de Roodt et al., 2012, Fox et al., 2009), and wet mass (Rocha-e-Silva et al., 2009, Sahayaraj et al., 2006). Venom yield often refers to the maximum amount of venom that can be expelled using a given extraction technique, such as electrical milking (Herzig et al., 2004, McCleary and Heard, 2010), glandular massage (Mackessy, 1988), administration of saliva-inducing chemicals such as pilocarpine (Hill and Mackessy, 1997), and spontaneous ejection (Hopkins et al., 1995, Sahayaraj et al., 2006, Tare et al., 1986). Venom yield can be influenced by diverse factors, including body size (Fox et al., 2009, Vapenik and Nentwig, 2000), age (Brown, 1973, Malli et al., 1993), sex (Atkinson, 1981, Glenn and Straight, 1977, Rocha-e-Silva et al., 2009), season (Bücherl, 1953, Vieira et al., 1988, Wiener, 1956, Wiener, 1959), temperature (Gregory-Dwyer et al., 1986, Kochva, 1960, Morgan, 1969), humidity (Kristensen, 2005), geographic population (Binford, 2001, Mirtschin and Davis, 1992, Mirtschin et al., 2002), health (Brown, 1973, Klauber, 1997), and number and frequency of milkings (Kristensen, 2005, Perret, 1977, Sissom et al., 1990).

The class Chilopoda, part of the subphylum Myriapoda, is divided into five living orders (and 1 extinct): Scutigeromorpha, Lithobiomorpha, Craterostigmomorpha, Scolopendromorpha, and Geophilomorpha (Edgecombe and Giribet, 2007). This diverse group of terrestrial arthropods (an estimated 3500 species) serves an ecologically important role as soil and leaf litter predators (Edgecombe and Giribet, 2007, Robertson et al., 1994, Trucchi et al., 2009, Undheim and King, 2011, Wallwork, 1976). Despite their importance, our knowledge of the natural history of centipedes is very limited (Forti et al., 2007, Molinari et al., 2005). Anatomically, centipedes possess a long, segmented body with one pair of legs per segment, and a head containing a pair of long antennae (Lewis, 1981). The legs of the first trunk segment are modified to form the characteristic forcipules that are used to grasp and envenomate prey (Bonato et al., 2010, Lewis, 1981, Undheim and King, 2011). The forcipules are also employed in defense (Davis, 1993, Demange, 1981, Maschwitz et al., 1979, Neck, 1985). Although prey immobilization (Undheim and King, 2011) comprises the primary role of the relatively complex venom (Liu et al., 2012a, Liu et al., 2012b, Undheim and King, 2011), a digestive function has also been suggested (Jangi, 1984, Martin, 1971, Minton, 1974), but remains unclear (Bücherl, 1971a, Dugon and Arthur, 2012b). To date, we understand very little about venom yields and the factors influencing them in centipedes.

Of the five extant orders of centipedes, the fleet-footed Scolopendromorpha, ranging in adult length from 1 to 30 cm (Edgecombe and Koch, 2008), contains the largest (Mundel, 1990), most fiercely predatory (Edgecombe and Koch, 2008), and most medically important (Balit et al., 2004, Jangi, 1984) of all centipedes. Within the Scolopendromorpha, the family Scolopendridae comprises powerfully muscled and stoutly built centipedes that potentially pose serious health hazards to humans due to their venomous sting (Jangi, 1984). In this study we investigated venom yields of two scolopendrids, Scolopendra polymorpha and Scolopendra subspinipes.

S. polymorpha inhabits desert, dry grassland, and forest habitats from the Great Plains westward to California, ranging up the Pacific states into Oregon, and throughout the desert southwest into northern Mexico (Crabill, 1960, Crawford and Riddle, 1974, Shelley, 2002). The sting of S. polymorpha causes temporary sharp pain in humans (Baerg, 1924, Maldonado, 1998, Turk, 1951). S. subspinipes is cosmopolitan in tropical and subtropical regions of the world (Kronmuller, 2012, Lewis et al., 2010). The sting of S. subspinipes reportedly causes intense pain, burning, swelling, and erythema (Bouchard et al., 2004, Bush et al., 2001, Mohri et al., 1991, Veraldi et al., 2010).

There is a growing body of knowledge regarding scolopendromorphs and their venoms (reviewed in Undheim and King, 2011). While there have only been a few studies focusing on scolopendromorph behavioral use of venom (e.g., Dugon and Arthur, 2012b, Formanowicz and Bradley, 1987), recent studies have shed light on the venom apparatus (Antoniazzi et al., 2009, Chao and Chang, 2006, Dugon and Arthur, 2012a, Dugon et al., 2012, Jarrar, 2010), venom transcriptome (Liu et al., 2012a, Liu et al., 2012b, Undheim et al., 2012, Yang et al., 2012), and venom components and biochemistry (Gonzalez-Morales et al., 2009, Guo et al., 2013, Jarrar, 2010, Liu et al., 2012a, Liu et al., 2012b, Malta et al., 2008, Peng et al., 2010, Rates et al., 2007, Yang et al., 2012, Yang et al., 2013). Other recent studies reported the effects of venom on invertebrates (Rates et al., 2007, Yang et al., 2012), non-human vertebrates (Malta et al., 2008, Menez et al., 1990), and humans (Chaou et al., 2009, Haddad et al., 2012, Lewis et al., 2010, Othong et al., 2012, Uzel et al., 2009, Veraldi et al., 2010). With the exception of incidental observations described by Malta et al. (2008), no quantitative data have been published regarding the quantity of venom centipedes have at their disposal (Minton, 1974), or the factors that influence venom yield or protein concentration, despite venom being a key element in the predatory behavior of centipedes (Dugon and Arthur, 2012b, Quistad et al., 1992).

Knowledge of venom yields and factors influencing them in centipedes are important for a fuller understanding of centipede venom use, and may prove useful to the medical community. The amount of venom an animal produces likely influences the severity of envenomation following a sting or bite (Janes et al., 2010, Mirtschin et al., 2002), and thus understanding venom yields may give insight into the range of prey types and sizes that a given centipede might be capable of taking (Wigger et al., 2002), as well as the effectiveness of defense it could mount. While most centipede stings of humans are not life threatening (Burnett et al., 1986, Bush et al., 2001), scolopendromorph stings can be medically significant (Balit et al., 2004, Jangi, 1984, Lewis, 1981, Logan and Ogden, 1985, Mohri et al., 1991). If knowledge of venom yields in S. polymorpha and S. subspinipes serves as a foundation for an understanding of yields in other scolopendromorphs, then perhaps the characteristics of an offending centipede can help guide physicians in their treatment of the patient (Hayes and Mackessy, 2010, Janes et al., 2010).

Evidence from centipede anatomy supports the hypothesis that venom yields in scolopendromorphs relate to body size. The venom glands are located inside the external lateral face of each forcipule (Antoniazzi et al., 2009) and extend from the trochanteroprefemoral (Bonato et al., 2010, Lewis et al., 2005) segment of the forcipule to the basal part of the tarsungulum (Jangi, 1984, Lewis et al., 2005, Menez et al., 1990), although exceptions exist (Edgecombe and Koch, 2008). In the three scolopendromorphs studied by Antoniazzi et al. (2009), venom gland length represented 5–6% of the total adult animal length.

Circumstantial evidence from the effects of centipede stings in humans further suggests that larger centipedes possess and deploy more venom than smaller centipedes. Reports indicate that larger centipedes cause more painful stings (Balit et al., 2004, Harwood et al., 1979, McFee et al., 2002, Norris, 2007, Undheim and King, 2011) with a higher incidence of swelling (Balit et al., 2004, Maldonado, 1998). Duration of pain also reportedly varies with centipede size (Gomes et al., 1982). However, with the exception of the comments of Maldonado (1998) and Gomes et al. (1982), which indicate an intraspecific relationship between centipede size and sting-symptom severity, it remains unclear whether sting severity varies with size within a given species or whether venom differences between species of differing size are contributing to the reported size-symptom relationship. Without further intraspecific studies it is difficult to draw firm conclusions regarding the relationship between size of centipede of a given species and quantity of venom possessed or injected.

We designed the present study to determine if and how size, specifically body length, influenced yield and protein concentration of electrically extracted venom in the centipedes S. polymorpha and S. subspinipes. We also compared venom yields and venom protein concentrations between these two species, and investigated additional potential sources of venom yield and protein concentration variation in S. polymorpha, including relative forcipule size, relative mass, geographic origin, sex, time in captivity, and milking history.