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Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose Q5

bastien Dutertre a, b, *, Ai-Hua Jin b, Paul F. Alewood b, Richard J. Lewis b Se Institut des Biomol ecules Max Mousseron, UMR 5247, Universit e Montpellier 2 e CNRS, Place Eug ene Bataillon, 34095 Montpellier Cedex 5, France b Institute for Molecular Bioscience, The University of Queensland, 4072 Queensland, Australia a

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 July 2014 Received in revised form 22 September 2014 Accepted 25 September 2014 Available online xxxx

Conus geographus is the most dangerous cone snail species known, with reported human fatality rates as high as 65%. Crude venom gland extracts have been used to determine animal LD50 and to aid the isolation of several potent paralytic toxins. However, not only is the composition of injected venoms known to differ significantly from that in dissected venom glands, but also to vary according to predatory or defensive stimuli. Therefore, to study the venom that is directly relevant to human envenomation, the defense-evoked venom of several specimens of C. geographus was collected and analyzed by standard LC eMS methods. The molecular composition of individual defense-evoked venom showed significant intraspecific variations, but a core of paralytic conotoxins including a-GI, a-GII, m-GIIIA, u-GVIA and u-GVIIA was always present in large amounts, consistent with the symptomology and high fatality rate in humans. Differences between injected and dissected venoms obtained from the same specimen were also evident. Interestingly, an apparent linear correlation between the dry weight/volume of injected venom and the size of the shell allowed extrapolation to a human lethal dose (0.038e0.029 mg/kg) from an historic fatal case of C. geographus envenomation, which may help in the management of future victims. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Venom Cone snails Conotoxin Lethal dose Milking

1. Introduction Cone snails are carnivorous marine gastropods that prey on worms, mollusks and fish (Lewis et al., 2012). They use potent venom to rapidly immobilize their prey, deter competitors and defend against predators (Olivera, 2002). A few unfortunate collectors have experienced the deadly effects of the defensive venom of cone snails while collecting shells for sale or as a hobby (McIntosh and Jones, 2001). A survey of the literature reveals that many

Q1

cules Max Mousseron, * Corresponding author. Institut des Biomole  Montpellier 2 e CNRS, Place Euge ne Bataillon, UMR 5247, Universite 34095 Montpellier Cedex 5, France. E-mail addresses: [email protected], dutertreseb@ yahoo.fr (S. Dutertre).

species have inflicted injuries to humans, including the larger Conus geographus (piscivorous), Conus textile, Conus aulicus, and Conus marmoreus (molluscivorous) as well as the smaller Conus obscurus (piscivorous) and Conus nanus (vermivorous) (Clench, 1946; Kiser, 1962; Wade, 1964). Therefore, all cone snails, regardless of their size or feeding habit, need to be treated with caution as all have the ability to sting humans. However, the only confirmed cone snail species responsible for human fatalities is C. geographus (Iredale, 1935; Over, 1965; Rice and Halstead, 1968), with at least 36 deaths documented from 1670 to 1998 (A. Kohn, personal communication). Two deaths attributed to C. textile are questionable, as injection of crude dissected extracts from the venom gland into vertebrate animals was shown to be without effect (Endean and Rudkin, 1963). Since juvenile C. geographus have a size and patterning

http://dx.doi.org/10.1016/j.toxicon.2014.09.011 0041-0101/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Dutertre, S., et al., Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose, Toxicon (2014), http://dx.doi.org/10.1016/j.toxicon.2014.09.011

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reminiscent of C. textile, these may be cases of misidentification. A death rate as high as 65% has been claimed for C. geographus, and dissected venom gland extracts have confirmed its venom is potent on the vertebrate neuromuscular system (Endean and Rudkin, 1963; Yoshiba, 1984). Symptoms reported for non-fatal stings include nausea, cephalgia and local paralysis, while life-threatening envenomations produce rapid cerebral edema, coma and respiratory failure within hours of envenomation (Fegan and Andresen, 1997). Early analyses of C. geographus venom identified 22 conotoxins from crude venom gland extracts, many showing potent paralytic effects (Olivera et al., 1985). These paralytic conotoxins form what is known as the “motor cabal”, which produces a flaccid paralysis in animals consistent with symptoms of human envenomation. This suite of toxins target an extensive array of ion channels involved in neuromuscular transmission in vertebrates, including presynaptic calcium channels (u-conotoxins), postsynaptic sodium channels (m-conotoxins) as well as muscle type nicotinic receptors (a-conotoxins). Interestingly, when the injected venom collected from various live Conus species was compared to dissected venom gland extracts, major differences were noted (Biass et al., 2009; Dutertre et al., 2010; Jakubowski et al., 2005). Furthermore, we recently demonstrated that the composition of the injected venom dramatically changes in response to prey or defensive stimuli (Dutertre et al., 2014), with surprisingly little overlap in venom composition. Therefore, only the defence-evoked venom is relevant for a better understanding of human envenomation. In this study, we collected the defence-evoked venom of three different specimens of C. geographus with varying shell length and characterized their composition using LCeMS methods. Our results indicate that despite of obvious intraspecific variations, the potent paralytic conotoxins are always injected in large amount during a defensive strike that are likely responsible for human fatalities. These paralytic peptides are highly abundant in the proximal part of the duct, consistent with earlier biochemical studies (Endean et al., 1974; Endean and Rudkin, 1963). A correlation was observed between the size of the shell and quantity of venom injected, allowing the first estimate of a human lethal dose. 2. Methods Cone collection e Three adult specimens of C. geographus (79.0, 89.5 and 99.5 mm) were collected from Gould reef (Queensland, Australia), by reef walking at low tide. All cone snails were housed in a unique 160 L aquarium maintained at 23e24  C with a light cycle of 12:12. Venom extraction e A milking procedure has been developed to collect defence-evoked venom from live C. geographus (Dutertre et al., 2014). C. geographus rarely uses its stinging apparatus in a predatory mode, using instead a “net strategy” to capture fish in its distended rostrum. Our milking strategy takes advantage of the aggressive behavior of C. geographus, where under threat, it will readily extend its proboscis and sting on contact. Only slight pressure applied to the shell induces this typical defensive behavior,

allowing venom to be collected in a tube covered with parafilm. Due to the serious health hazard associated with C. geographus envenomation, this milking procedure should be done using thick gloves and a long pair of forceps only by those experienced in cone snail behaviors. Following each milking, the collecting tube is briefly centrifuged and the volume of venom measured to the nearest 0.5 ml. After lyophilization, the dry weight of each milking was determined before storage at 20  C. Two specimens of C. geographus were sacrificed to allow direct comparison of milked versus extracted venom. Dissections were carried out on ice and the venom was stripped from the duct, lyophilized and stored at 20  C until use. LC-ESIeMS e All LCeMS experiments were performed on an AB Sciex QSTAR Pulsar, an electrospray time-of-flight (TOF) MS equipped with a Turbo-Spray ionization source. Each lyophilized venom sample was resuspended in 0.1% formic acid, and equivalent amounts of injected venom and dissected duct extracts were subjected to LC-ESIeMS in order to obtain a complete mass list of peptides. The LC separation was achieved using a Thermo C18 4.6  150 mm column eluted with a linear (1%) gradient of B (90% acetonitrile/0.1% formic acid (aq)) at a flow rate of 0.3 ml min1 over 80 min using an Agilent 1100 series HPLC system. A cycle of one full scan over the mass range (MS) (400e2000 m/z) was applied. Proteomic data analysis. LC-ESIeMS reconstruction was carried out using Analyst LCMS reconstruct BioTools over a mass range of 1000e8000 Da, with the mass tolerance set to 0.2 Da and the S/N threshold set to 10. Dedicated bioinformatics tools recently implemented in ConoServer (Kaas et al., 2012) were used to remove duplicate masses from each sample analyzed and to generate lists of unique masses for sample comparisons. The precision level for matching samples was set to 0.25 Da. All known conotoxin sequences isolated from C. geographus venom to date were retrieved from the ConoServer database and their monoisotopic mass specifically searched for in each MS data set.

3. Results 3.1. Defence-evoked venom Three specimens of C. geographus of varying size were maintained live in our aquarium for milking experiments (Fig. 1). As recently described (Dutertre et al., 2014), milked venom from C. geographus specimens obtained following defensive stimuli was milky and contained insoluble granular material. This is in contrast to the translucent predation-evoked venom of other piscivorous cone snails and more like the predation-evoked venom of molluscivorous cone snails (Dutertre et al., 2014). Venom yields were relatively constant for each individual but varied between specimens according to the size. The smallest specimen (79.0 mm) injected 21.5 ± 0.5 ml of venom on average, which is half the volume (42.5 ± 1.5 ml) injected by the medium size specimen (89.5 mm). The largest specimen (99.5 mm) injected a remarkable 84 ± 3 ml of venom, the largest “milking” yet obtained from a cone snail. Size of shell had a significant linear correlation with the volume

Please cite this article in press as: Dutertre, S., et al., Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose, Toxicon (2014), http://dx.doi.org/10.1016/j.toxicon.2014.09.011

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Fig. 1. Shells of the three specimens of C. geographus used in this study. The dimensions of the shells of the specimens used in this study can be directly compared to the size of the specimen responsible for the lethal case that occurred on Hayman Island, Australia, in 1935.

(r2 ¼ 0.95; Fig. 2A) and the dry weight of injected venoms (r2 ¼ 0.93; Fig. 2B). 3.2. Intraspecific variations of the defence-evoked venom

only the 10 most intense masses, the intraspecific variations were again obvious, yet there was a better overlap, with 40e90% of masses common between specimens. The most dissimilar regions were 18e25 min, with 32 mg in a single defensive sting (dash lines).

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Fig. 3. Intraspecific variations within the defence-evoked venom of three specimens of C. geographus. LCeMS profiles for the defence-evoked venom from the big (A), medium (B) and small (C) specimens. All three venoms appear highly complex, with a similar general distribution of masses (D). However, the overlap of the mass lists between the three specimens is only 20e30 %, whether the entire MS spectrum is considered (E) or only the top 200 most intense masses (F).

masses in common, followed by the most hydrophobic regions from 55e65 min and 65e80 min, which had 50e70% of masses in common. In contrast, section 25e35 min, which contains the vast majority of known paralytic conotoxins, and section 45e55 min had 80e90% of components in common. 3.3. Comparative proteomic analysis of injected venom and duct extracts from the same specimens To compare the composition of the defence-evoked and dissected venoms from the same specimen, the medium specimen was sacrificed and its whole venom duct extracted and analyzed by LCeMS to allow direct comparison to its injected venom. This comparison revealed that the TIC profile of the whole venom duct extract was clearly different from the milked venom (Fig. 4). In particular, peaks present in the most hydrophobic part of the milked

venom (>60 min) were absent from the dissected venom. While 1669 masses were detected in the dissected venom, a number that appears very close to the number of masses identified in the milked venom (1781), surprisingly only 341 (~20%) masses were common to both venoms. To analyze in more detail the differences between injected and dissected venom, the largest specimen was sacrificed and its venom duct divided into four equal parts (distal (close to the pharynx), distal central, proximal central and proximal (close to the bulb)) and the LCeMS profiles of each section compared (Fig. 5). This analysis revealed significant variation between different regions as reported previously for C. textile (Tayo et al., 2010) and C. geographus (Dutertre et al., 2014), with adjacent distal/ distal central and proximal central/proximal appeared more similar. The number of masses recovered from each section varied little and remained in the same range, with 2310 masses found in the distal part, 1771 in the distal

Please cite this article in press as: Dutertre, S., et al., Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose, Toxicon (2014), http://dx.doi.org/10.1016/j.toxicon.2014.09.011

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Table 1 Comparative analysis of the top 10 dominant masses in small, medium and big milked C. geographus venoms. Ranking based on MS Intensity is indicated in brackets. Small 0e18 min 2607.21 (GIIIA) 2612.22 2606.18 2642.21 2706.17 1306.11 2625.21 2644.18 2592.22 (GIIIC) 870.73 25e35 min 3313.47 (GVIIA) 1795.89 1436.47 (GI) 3329.39 2184.87 (GID) 3035.21 (GVIA) 4189.56 (GVIIIA) 1963.70 1033.49 (Con-G) 3297.50 45e55 min 1069.27 1067.27 975.35 3536.74 1055.26 1053.26 1068.78 2044.66 959.36 1156.30 65e80 min 2997.60 3177.66 2980.80 638.63 3027.60 4493.37 656.64 1937.98 640.63 405.28

Medium

Big

2607.23 (1) 2612.25 (5) Not found 2642.25 (3) 2706.21 (16) 1306.12 (8) 2624.24 (12) Not found 2594.26 (6) not found 70%

2607.21 (1) 2612.24 (7) 2606.19 (5) 2642.25 (3) 2706.19 (10) Not found 2625.26 (4) Not found 2592.24 (11) 869.0 (12) 80%

3313.50 (1) 1795.92 (4) 1436.49 (2) 3329.47 (7) 2184.89 (5) 3035.25 (3) 4189.63 (6) Not found 1033.50 (10) Not found 80%

3313.50 (1) 1795.89 (6) 1436.47 (5) Not found 2184.88 (7) 3035.23 (3) 4189.55 (9) 1963.73 (11) 1033.49 (19) Not found 80%

1069.28 (1) 1067.28 (2) 975.35 (3) 3536.74 (4) 1055.27 (6) 1053.27 (9) 1068.79 (8) 2044.67 (7) Not found 1156.32 (16) 90%

1069.27 (1) 1067.27 (2) 975.34 (3) 3536.74 (5) 1055.26 (4) 1053.26 (6) 1068.79 (9) 2044.66 (7) Not found Not found 80%

2997.57 (2) 3177.63 (1) Not found 638.62 (5) 3027.52 (3) 4493.37 (4) Not found 1937.95 (6) Not found 405.27 (14) 70%

2997.58 (4) 3177.68 (8) Not found 638.63 (1) Not found Not found 656.64 (2) Not found Not found 405.27 (5) 50%

central part, 2264 in the proximal central part and 1923 in the proximal part. Interestingly, when the mass lists are compared with the list extracted from the injected venom of the same animal, only 21e43% overlap is observed. 3.4. Distribution of the known conotoxins in the injected venom and along the venom duct Since there are a number of well-characterized conotoxins isolated from the venom of C. geographus, it was of interest to determine their occurrence in the injected venom and partitioning in venom gland sections. Despite >40 years of research on the venom of C. geographus, only 22 conotoxin sequences have been confirmed at the peptide/pharmacological level (Table 2) in contrast to >2500

Small 18e25 min 3030.27 2986.27 2417.95 2433.95 3002.26 1452.51 (G5.1) 3046.26 3288.46 (GVIIB) 3332.48 2928.27 35e45 min 3038.52 3330.40 3492.79 975.36 3022.52 3370.31 3374.43 1665.20 1665.70 3370.80 55e65 min 2778.73 1055.26 1053.27 3144.46 3606.36 3650.35 3686.29 3261.77 3729.28 1675.02

Medium

Big

Not found Not found 2417.97 (5) 2433.97 (7) Not found 1452.52 (9) Not found 3288.47 (2) 3332.50 (3) Not found 50%

Not found Not found 2417.97 (6) 2433.94 (5) Not found Not found Not found 3288.44 (1) 3332.45 (2) Not found 40%

3038.53 (3) 3330.44 (4) 3493.70 (7) 975.35 (9) 3022.50 (1) Not found 3374.45 (11) 1665.22 (6) 1665.72 (15) Not found 80%

Not found 3330.45 (7) 3492.77 (14) 975.34 (1) Not found 3370.83 (5) Not found 1665.21 (9) 1665.71 (10) Not found 60%

2778.72 (1) 1055.27 (9) 1053.27 (12) Not found 3606.38 (6) 3650.37 (14) 3685.31 (2) Not found Not found 1675.03 (7) 70%

2778.73 (4) 1055.25 (1) 1053.26 (2) Not found 3606.37 (9) 3650.36 (7) Not found Not found Not found Not found 50%

masses detected in single milkings. A significant number of these characterized conotoxins are paralytic to vertebrates, including inhibitors of calcium channels (u-GVIA, u-GVIB, u-GVIC, u-GVIIA and u-GVIIB), sodium channels (m-GIIIA, m-GIIIB and m-GIIIC) and muscle nicotinic acetylcholine receptors (a-GI, a-GIA and a-GII). High levels of these paralytic toxins were detected in the injected venoms of all three specimens investigated in this study. The distribution of the known toxins along the venom duct shows a clear pattern, with most of the paralytic toxins highly expressed in the proximal parts, whereas non-paralytic such as conopressin G are only detected in the distal parts (Table 2), consistent with recent proteomic and transcriptomic studies (Dutertre et al., 2014; Hu et al., 2012). However, some apparent inconsistencies seem to contradict this new

Please cite this article in press as: Dutertre, S., et al., Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose, Toxicon (2014), http://dx.doi.org/10.1016/j.toxicon.2014.09.011

Q3

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Fig. 4. Comparison of defence-evoked and dissected venom for Medium specimen. LCeMS profiles of both the defense-evoked venom (injected; A) and dissected venom (B) from the medium specimen show significant differences, in particular in the most hydrophobic region (>40 min). Although a similar number of masses were detected in both venoms, the overlap was only ~20% (C).

paradigm. Indeed, GIA, a well characterized paralytic peptide, is detected in the distal parts only, but at a very low intensity (ranking 1206e1505). As a consequence of this low intensity, no MS/MS data could be obtained to confirm the sequence of GIA, and the possibility that this mass corresponds to another yet unrelated peptide remains since it is not detected in any of the milked venoms. Contulakin-G shows a 3 to 4 times higher intensity in the distal parts compared to the proximal parts, consistent with its predatory role. On the contrary, Conantokin-G seems to be highly expressed along the entire duct and is logically detected in the defence-evoked venom, although not as a major component. 4. Discussion In this study, we investigated the defence-evoked venom of three specimens of C. geographus. The potency of C. geographus venom is thought to be mainly due to only a few paralytic toxins, most of which have already characterized based on bioassays-guided fractionation of crude venom (notably m-GIIIA, u-GVIA, a-GI and u-GVIIA). Most, if not all of these paralytic toxins were present at similar levels in the injected venom of all three specimens (Tables 1 and 2), suggesting that despite the overall high levels of intraspecific variation identified, a core of lethal toxins is

always injected. This core of paralytic toxins makes most of the large volume of venom injected, providing a novel argument to explain the lethality of C. geographus. Indeed, the injected volumes measured in this study are by far the largest ever reported for a cone snail. Therefore, this large volume of venom means higher concentration of toxins in the circulatory system of victims, likely explaining the rapid collapse of vital functions. Our results also suggest that the toxicity of C. geographus venom is not significantly affected by these intraspecific variations, mainly because they only concern these minor components. Therefore, larger animals will inject more venom (including more of the deadly toxins), hence the suggested correlation between size and dangerousness (the larger the animal, the poorer the prognosis for the victim). C. geographus is rather unusual among cone snails, being a particularly aggressive species. Indeed, most cone snails are shy and will retract into their shell when manipulated, but C. geographus readily extends its proboscis as a defensive mechanism. About three dozen deaths have been attributed to C. geographus stings (AL Kohn, personal communication), and the potent paralytic toxins that have been isolated from dissected gland extracts likely account for this lethality. However, some elements of the ecology of C. geographus may also explain the high potency of this venom. This species hunts at night, like most cone snails,

Please cite this article in press as: Dutertre, S., et al., Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose, Toxicon (2014), http://dx.doi.org/10.1016/j.toxicon.2014.09.011

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Fig. 5. Comparison of defence-evoked and extracts from four sections of the Big specimen venom duct. To account for the regionalization of the venom duct (F), extracts from distal (B), distal central (C), proximal central (D) and proximal (E) were analyzed by LCeMS together with the defence-evoked venom (A) of the big specimen. The mass lists from the different sections show an overlap of 15e35 % with the mass list of the defence-evoked venom (G).

Please cite this article in press as: Dutertre, S., et al., Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose, Toxicon (2014), http://dx.doi.org/10.1016/j.toxicon.2014.09.011

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Name

Sequence

Medium MV

Big MV

2262.94 3615.49

2263.03 (1583) 2262.99 (1075) 2263.02 (155) nf nf nf

2068.97 1622.58 1451.54 3322.24 1436.48 1622.58 1608.58 2184.85 1415.5 2607.12 2638.14 2592.18 3035.15 3093.16 2873.07 3313.38 3287.33 4188.42

nf nf nf nf 1436.47 nf nf 2184.87 1415.53 2607.18 nf 2592.22 3035.20 3093.24 2873.04 3313.45 nf 4189.53

1033.49 3301.53

1033.48 (31) 3301.47 (718)

(2)

(5) (115) (27) (186) (8) (49) (96) (6) (11)

nf nf 1451.60 nf 1436.48 nf nf 2184.91 1415.54 2607.21 2638.26 2592.24 3035.27 3093.26 2873.07 3313.48 3287.46 4189.61

nf nf (1733) 1451.61 nf (1) 1436.46 nf nf (4) 2184.88 (40) 1415.48 (31) 2607.19 (205) 2638.01 (291) 2592.25 (137) 3035.25 (18) 3093.24 (265) 2873.36 (10) 3313.49 (174) 3287.42 (2) 4188.52

1033.50 (23) 3301.52 (344)

Big DV (D)

Big DV (DC)

Big DV (PC)

Big DV (P)

2262.95 (9) 3615.64 (2)

2262.96 (19) nf

2262.96 (6) 3615.67 (8)

2262.98 (4) nf

2263.03 (24) 3615.67 (200)

nf 1622.97 (1491) nf nf (6) 1436.53 1622.97 1608.97 (1) 2184.97 (12) 1415.49 (10) 2607.25 (982) 2638.28 (293) nf (266) 3035.25 (30) 3093.25 (79) (4) 3313.50 (28) nf (18) 4189.58

1033.48 (40) 3301.52 (613)

Q4

Medium DV

2069.03 (1235) 1622.72 nf nf (33) nf (1235) 1622.72 (1620) 1608.74 (318) 2184.97 (791) nf (78) nf (1143) nf nf (1) 3035.25 (15) 3093.29 2873.36 (14) 3313.51 nf (25) 4189.67

1033.54 (162) nf

(120) 2069.04 (48) (1505) 1622.77 (1206) nf nf nf (1505) 1622.77 (1206) (1418) nf (226) 2184.94 (428) nf nf nf nf (12) 3035.28 (78) (32) 3093.33 (334) (224) nf (625) 3313.54 (166) 3287.49 (181) (289) nf

1033.49 (31) nf

1033.52 (360) nf

2069.06 nf nf nf 1436.53 nf nf 2184.92 1415.54 2607.26 nf 2592.31 3035.27 3093.33 2873.31 3313.53 3287.48 4189.62 nf nf

(169) 2069.07 (420) nf nf nf (57) 1436.56 (40) nf nf (9) 2184.92 (6) (38) 1415.55 (30) (12) 2607.26 (9) 2638.29 (252) (391) 2592.3 (130) (5) 3035.31 (34) (28) 3093.31 (22) (196) 2873.30 (120) (3) 3313.52 (18) (1) 3287.51 (2) (6) 4188.69 (63) nf 3301.54 (274)

g, gamma carboxyglutamate; *, amidation; O, hydroxyproline; gT, glycosylated threonine; w, bromotryptophane; Z, pyroglutamate nf, not found.

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GEggLQgNQgLIRgKSN* ACSGRGSRCOOQCCMGLRCGRGNPQKCIG AHgDV Contulakin-G ZSEEGGSNA(gT)KKPYIL G1.1 ECCNPACGRHYSCKG G5.1 QGWCCKENIACCV G6.1 DDECEPPGDFCGFFKIGPPCCSGWCFLWCA GI ECCNPACGRHYSC* GIA ECCNPACGRHYSCGK GIC GCCSHPACAGNNQHIC* GID IRDgCCSNPACRVNNPHVC* GII ECCHPACGKHFSC* GIIIA RDCCTOOKKCKDRQCKOQRCCA* GIIIB RDCCTOORKCKDRRCKOMKCCA* GIIIC RDCCTOOKKCKDRRCKOLKCCA* GVIA CKSOGSSCSOTSYNCCRSCNOYTKRCY* GVIB CKSOGSSCSOTSYNCCRSCNOYTKRCYG GVIC CKSOGSSCSOTSYNCCRSCNOYTKRC GVIIA CKSOGTOCSRGMRDCCTSCLLYSNKCRRY GVIIB CKSOGTOCSRGMRDCCTSCLSYSNKCRRY GVIIIA GCTRTCGGOKCTGTCTCTNSSKCGCRYNV HPSGwGCGCACS* Conopressin G CFIRNCPKG* Scratcher KFLSGGFKgIVCHRYCAKGIAKEFCNCPD* peptide Conantokin-G Conotoxin-GS

Mass (Da) Small MV

S. Dutertre et al. / Toxicon xxx (2014) 1e10

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Table 2 Known conotoxins from C. geographus. Matching masses from milked (MV) and dissected (DV) venoms (Intensity ranking is indicated in brackets).

62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61

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but it reveals a brightly colored foot, which may attract predators. Whereas the shell of cone snails offers the first line of protection against predation and constitutes an inviolable fortress for many predators, many crustaceans and fish are able to break the shells of gastropod mollusks to feed on the soft parts. Cones of the Gastridium clade, such as C. geographus, or Conus tulipa, are thought to be among the most dangerous species to human, and interestingly, they possess the weakest protection, having the lightest and thinnest shells. As an evolutionary hypothesis, we propose that some cone snails evolved a potent venom to compensate for the lack of efficient protection from their shell (Dutertre et al., 2014). This hypothesis is supported by the unusual aggressive behavior of the Gastridium Conus. In conclusion, the large size, thin shell and aggressive behavior rather than its piscivorous diet make C. geographus the most dangerous species of extant cone snail. As indicated in Table 1, the proportion of particular toxins into the milked venom compared to the venom duct can be very different. More surprisingly, the proportions can be completely inverted. For instance, conantokin-G is highly present in the venom duct, while only traces of it could be detected in the injected venom. In contrast, a-GI is one of the major toxins in the injected venom, whereas it is less abundant in the venom duct. These results are consistent with the predatory use of conantokin-G and defensive use of a-GI. Indeed, conantokin-G is a major component of C. geographus predatory venom (Dutertre et al., 2014). This also raises the question to whether the LD50 values previously estimated from dissected material are really relevant to human envenomation. A pertinent example of this discrepancy is the lack of activity of C. textile dissected venom on mammals, while this species has been involved in several lethal accidents. Misidentification of C. textile for C. geographus has been put forward to explain this difference. Yet, in light of our results, the defence-evoked venom of C. textile may also prove more potent compared to the dissected venom. 4.1. Historic case and estimation of the lethal dose Almost 80 years ago, a young man picked up a shell of a live C. geographus on the shore of Hayman Island and died in just under five hours as a direct consequence of cone snail envenomation. This was the first well-documented fatal case to be reported and for which the culprit was caught and preserved, allowing unambiguous identification to be made (voucher Mo 1689, the Queensland Museum, Brisbane, Australia). Measurement of the shell of this killer snail revealed a length of 85 mm. The size of this shell fits within the sizes of the shells used in this study, for which we have demonstrated a linear correlation between the dry weight of venom injected and the size of the shell. Therefore, we can calculate within a reasonable estimate the dose of venom that killed this man in 1935. According to our data, a snail of 85 mm will inject on average a volume of 32 ml of venom, corresponding to a dry weight of 2.5 mg. Since the victim was 27 year old at the time of his death, playing football regularly, he must have been relatively fit, with a body weight likely comprised between 65 and 85 kg. We therefore estimate the lethal dose to be comprised

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between 0.038 mg/kg and 0.029 mg/kg. Our result is significantly different from previous estimate of the human lethal dose of C. geographus. In 1984, Shigeo Yoshiba has calculated the lethal dose in humans to be 1e3 mg/kg (Yoshiba, 1984), values that are 10e30 times lower than ours. This discrepancy mainly arises from the author's wrong assumption that C. geographus injects its venom only in the volume contained within a single radula tooth. It is well known that a cone snail's radula acts as delivery and tethering device, being both hollowed and barbed. However, through the lumen of the radula will be pushed a larger volume of venom than it can hold, a volume most likely determined by the size of the venom reservoir, which remains to be unambiguously identified. Clearly, the amount of venom injected has been underestimated by Shigeo Yoshiba, leading to erroneous values. In addition, animal toxicity data based on dissected venom extracts have been used to calculate this lethal dose, and we have demonstrated significant differences in molecular composition between dissected and injected venoms. These differences most likely translate into different pharmacological effects. It is also interesting to note that the LD50 of the world most venomous snakes are in the range of 0.025e0.030 mg/kg (subcutaneous injection in mouse), indicating that the venom of C. geographus is at least equally potent in human (Broad, 1979). In conclusion, we believe that our data, based on experimental measurements of defence-evoked venoms, represent the most accurate human lethal dose to date and may help in the management of future victims of C. geographus stings. Acknowledgments We thank members of the Brisbane Shell Club for collecting the three specimens of C. geographus used in this study. We also thank Dr John Healy, Curator of the Molluscs section at the Queensland Museum, for allowing the first author to examine the preserved specimen of Conus geographus responsible for the 1935 fatal case. Cedric Rubrecht is also acknowledged for preliminary analysis of LCeMS data. This work was supported by a NHMRC Program grant (R.J.L, P.F.A.) and a UQ postdoctoral fellowship (S.D). Conflict of interest None. References Biass, D., Dutertre, S., Gerbault, A., Menou, J.L., Offord, R., Favreau, P., Stocklin, R., 2009. Comparative proteomic study of the venom of the piscivorous cone snail Conus consors. J. Proteomics 72, 210e218. Broad, A.J., Sutherland, S.K., Coulter, A.R., 1979. The lethality in mice of dangerous Australian and other snake venom. Toxicon 17, 661e664. Clench, W.J., 1946. The Poison Cone Shell. In: Occasional Papers on Mollusks 1, pp. 49e80. Dutertre, S., Biass, D., Stocklin, R., Favreau, P., 2010. Dramatic intraspecimen variations within the injected venom of Conus consors: an unsuspected contribution to venom diversity. Toxicon 55, 1453e1462. Dutertre, S., Jin, A.H., Kaas, Q., Jones, A., Alewood, P.F., Lewis, R.J., 2013. Deep venomics reveals the mechanism for expanded Peptide diversity in cone snail venom. Mol. Cell. Proteomics 12, 312e329. Dutertre, S., Jin, A.H., Vetter, I., Hamilton, B., Sunagar, K., Lavergne, V., Dutertre, V., Fry, B.G., Antunes, A., Venter, D.J., Alewood, P.F.,

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Q2

62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122

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Lewis, R.J., 2014. Evolution of separate predation- and defenceevoked venoms in carnivorous cone snails. Nat. Commun. 5, 3521. Endean, R., Parish, G., Gyr, P., 1974. Pharmacology of the venom of Conus geographus. Toxicon 12, 131e138. Endean, R., Rudkin, C., 1963. Studies of the venoms of some conidae. Toxicon 1, 49e64. Fegan, D., Andresen, D., 1997. Conus geographus envenomation. Lancet 349, 1672. Hu, H., Bandyopadhyay, P.K., Olivera, B.M., Yandell, M., 2012. Elucidation of the molecular envenomation strategy of the cone snail Conus geographus through transcriptome sequencing of its venom duct. BMC Genomics 13, 284. Iredale, T., 1935. Fatal case of attack by cone. Naut. 49, 41. Jakubowski, J.A., Kelley, W.P., Sweedler, J.V., Gilly, W.F., Schulz, J.R., 2005. Intraspecific variation of venom injected by fish-hunting Conus snails. J. Exp. Biol. 208, 2873e2883. Kaas, Q., Yu, R., Jin, A.H., Dutertre, S., Craik, D.J., 2012. ConoServer: updated content, knowledge, and discovery tools in the conopeptide database. Nucleic Acids Res. 40, D325eD330. Kiser, J., 1962. Conus obscurus stings collector. Hawaii. Shell News 10, 3.

Lewis, R.J., Dutertre, S., Vetter, I., Christie, M.J., 2012. Conus venom Peptide pharmacology. Pharmacol. Rev. 64, 259e298. McIntosh, J.M., Jones, R.M., 2001. Cone venomefrom accidental stings to deliberate injection. Toxicon 39, 1447e1451. Olivera, B.M., 2002. Conus venom peptides: reflections from the biology of clades and species. Annu. Rev. Ecol. Syst. 33, 25e47. Olivera, B.M., Gray, W.R., Zeikus, R., McIntosh, J.M., Varga, J., Rivier, J., de Santos, V., Cruz, L.J., 1985. Peptide neurotoxins from fish-hunting cone snails. Science 230, 1338e1343. Over, R., 1965. Geography cone strikes again. Hawaii. Shell News 13, 1e2. Rice, R.D., Halstead, B.W., 1968. Report of fatal cone shell sting by Conus geographus Linnaeus. Toxicon 5, 223e224. Tayo, L.L., Lu, B., Cruz, L.J., Yates 3rd, J.R., 2010. Proteomic analysis provides insights on venom processing in Conus textile. J. Proteome Res. 9, 2292e2301. Wade, U., 1964. Conus sponsalis stings boy. Hawaii. Shell News 12, 2. Yoshiba, S., 1984. An estimation of the most dangerous species of cone shell, Conus (Gastridium) geographus Linne, 1758, venom's lethal dose in humans. Nihon Eiseigaku Zasshi 39, 565e572.

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Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose.

Conus geographus is the most dangerous cone snail species known, with reported human fatality rates as high as 65%. Crude venom gland extracts have be...
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