Chemoecology 18: 181 – 190 (2008) 0937-7409/08/030181-10 Birkhuser Verlag, Basel, 2008 DOI 10.1007/s00049-008-0404-5
CHEMOECOLOGY
Maternal provisioning of sequestered defensive steroids by the Asian snake Rhabdophis tigrinus Deborah A. Hutchinson1, Alan H. Savitzky1, Akira Mori2, Jerrold Meinwald3, and Frank C. Schroeder4 1
Old Dominion University, Department of Biological Sciences, Norfolk, VA 23529, USA Kyoto University, Department of Zoology, Graduate School of Science, Sakyo, Kyoto 606-8502, Japan 3 Cornell University, Department of Chemistry and Chemical Biology, Ithaca, NY 14853, USA 4 Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, USA 2
Summary. Rhabdophis tigrinus obtains defensive steroids (bufadienolides) from its diet and sequesters those compounds in specialized structures on its neck known as nuchal glands. Hatchling snakes lacking these steroids must acquire them from toads consumed as prey. Here we show that females provision bufadienolides to their offspring in amounts correlated to the quantity in their own nuchal glands; thus, chemically protected mothers produce defended offspring. Bufadienolides can be provisioned to embryos via deposition in yolk and by transfer across the egg membranes within the oviducts. Maternally provisioned bufadienolides persist in the nuchal glands of juvenile snakes from the time of hatching in late summer until the following spring, when toads of ingestible size become abundant. Therefore, maternal provisioning may provide chemical protection from predators for young R. tigrinus in the absence of dietary sources of bufadienolides. Key words. Dietary toxins – Bufo – bufadienolide – nuchal glands – antipredator defense
Introduction Chemically defended animals may synthesize their defensive toxins from nontoxic precursors or sequester intact toxins from an exogenous source, such as their diet (Duffey 1980; Brower 1984; Daly et al. 1994; Eisner et al. 1997; Nishida 2002; Dumbacher et al. 2004; Saporito et al. 2004; Williams et al. 2004). Regardless of how adults become toxic, their newly emergent offspring could enjoy a selective advantage if they were defended by maternal provisioning of defensive chemicals. When toxins are Correspondence to: Deborah A. Hutchinson, email: dhutchin@odu. edu and Alan H. Savitzky, email:
[email protected]
deposited in the eggs of oviparous species, those toxins may serve as deterrents against egg predation (oophagy). Subsequently, the toxins may be incorporated into the developing embryos tissues. If embryos incorporate the toxins, the young also may be chemically defended for a period of time after hatching. There are many examples of defensive toxins in the eggs of chemically defended species. Eggs of roughskinned newts (Taricha granulosa) and a Costa Rican harlequin frog (Atelopus chiriquiensis) are provisioned with tetrodotoxin, a potent neurotoxin (Pavelka et al. 1977; Hanifin et al. 2003). Toxic eggs are known from toads (Licht 1968) and moths in the genus Utethesia (Eisner and Meinwald 1995; Eisner et al. 2000). Eggs of fireflies (Photuris) are endowed with betaine synthesized by the female, and also with sequestered lucibufagins when available (Gonzlez et al. 1999a). Toxins that provide chemical defense for eggs may or may not become incorporated into the developing embryos. Among invertebrates, provisioning of toxins in eggs can result in chemically defended larvae (Pasteels et al. 1986; Blum and Hilker 2002; Eisner et al. 2002). Among vertebrates, some chemically defended eggs gradually lose their toxicity as development ensues (Phisalix 1922; Twitty 1937). Larvae of Anaxyrus boreas (formerly Bufo boreas; see Frost et al. 2006) have been shown to lack defensive bufadienolides (Benard and Fordyce 2003), demonstrating a loss of provisioned toxins that had protected the eggs. Rhabdophis tigrinus is an oviparous Asian snake (Colubridae: Natricinae) that possesses unusual defensive glands in the skin of its neck (Nakamura 1935; Smith 1938). Each individual has a series of 10 – 20 pairs of these nuchal glands that extends caudally behind the head (Nakamura 1935; Smith 1938; Toriba and Sawai 1990). The contents of the nuchal glands irritate mucous membranes, cause corneal injuries (Nakamura 1935; Kitazume 1953; Kawashima 1957, 1959; Suzuki 1960; Asahi et
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al. 1985), and contain cardiotonic steroids known as bufadienolides (Akizawa et al. 1985a,b; Hutchinson et al. 2007). Bufadienolides act by inhibiting the sodiumpotassium pump in a digitalis-like fashion (Melero et al. 2000). The steroidal toxins in the nuchal glands are similar to those found in the integumentary secretions of toads (Bufonidae; Erspamer 1994), which often are consumed, in addition to other anuran prey, by R. tigrinus (Mori and Moriguchi 1988). Whereas toads are capable of synthesizing their defensive bufadienolides from cholesterol (Doull et al. 1951; Siperstein et al. 1957; Porto and Gros 1971; Porto et al. 1972), R. tigrinus is dependent on a diet of toads from which it can sequester these compounds (Hutchinson et al. 2007). Although toads are the ultimate source of bufadienolides in R. tigrinus, a previous experiment suggested that hatchlings may either lack or possess these defensive compounds, depending upon the level of toxins in the mothers nuchal glands (Hutchinson et al. 2007). Having demonstrated previously that R. tigrinus depends upon a diet of toads to supply its bufadienolides (Hutchinson et al. 2007), we sought to determine whether chemically defended dams (mothers) could provision sequestered bufadienolides to their offspring. In the present study, we conducted a dietary experiment on 12 gravid, wild-caught R. tigrinus and their offspring to answer this question. We maintained eight gravid R. tigrinus on nontoad diets and fed toads to four other gravid females. We analyzed samples of nuchal gland fluid from the gravid females and their unfed, fish-fed, and toad-fed offspring, as well as selected samples of yolk. We present evidence that maternal provisioning of toxins in R. tigrinus occurs through two mechanisms: by deposition in yolk and by transfer across the egg membranes within the oviduct, late in gestation. Furthermore, maternal provisioning of bufadienolides results in a gradation of chemical defense among clutches, in accordance with the level of toxins in the dams.
Methods and Materials Experimental Design and Sample Collection. Twelve gravid Rhabdophis tigrinus were collected in May and June, 2005 on the Japanese islands of Honshu, Kyushu, and Shikoku. Nine of the snakes were collected on Honshu, from Okayama Prefecture: Tomi-son (n = 1), Kagamino-cho (n = 1), Mikamo-son (n = 2), Shinjo-son (n = 1); Hiroshima Prefecture: Shohbara City (n = 1); and Kyoto Prefecture: Miyama-cho (n = 3). On Kyushu, one snake was collected from Kumamoto Prefecture: Yamato-cho. Two snakes were collected on Shikoku from Tokushima Prefecture: Naka-gun (n = 1) and Ishima Island (n = 1). The gravid females were fed nonbufonid frogs, primarily Pelophylax nigromaculatus (formerly Rana; Frost et al. 2006; Che et al. 2007), but also Hyla japonica and Rhacophorus arboreus. The females also were fed pre-killed fishes (Plecoglossus altivelis, Sardinops sagax, Oncorhynchus mykiss, Hypomesus nipponensis, and Pseudorasbora parva). Additionally, four dams were fed a single, live adult toad (Bufo japonicus) from Niijima (Tokyo Prefecture: Izu Islands) prior to oviposition, and two other dams were fed a live toad shortly after oviposition.
CHEMOECOLOGY
Samples of nuchal gland fluid were collected from the R. tigrinus dams upon capture (2–6 weeks prior to oviposition), and again after oviposition, at which point they had been fed in the laboratory. The nuchal gland fluid was collected by expressing a few glands onto a portion of Kimwipe (Kimwipes wipers S-200; Kimberly-Clark, Dallas, TX, USA) while wearing powder-free NBR nitrile gloves (AS ONE, Edobori, Nishiku, Osaka, Japan). The Kimwipe was then placed in a vial of methanol with forceps and covered with a teflon-lined cap. We were careful to express only a few glands to ensure that we could resample individuals at later dates; nuchal glands do not appear to regenerate, at least initially, after they have ruptured. We attempted to standardize the number of glands expressed (and thus the volume of fluid that was collected), so that samples could be compared through time and across individuals. Gloves were changed and forceps were rinsed in methanol between samplings. A control vial consisting of a Kimwipe in methanol was prepared at the end of each sampling session to ensure that cross contamination did not occur. All samples were stored at –208C. Oviposition took place at Kyoto University from June through July 2005; eggs hatched in August. The temperature of the enclosure housing the eggs and snakes was maintained between 25 and 308C. Five oviposited eggs from clutches 3, 10, 11, and 12 were dissected to collect yolk for chemical analyses. Yolk samples either were extracted in methanol and frozen, or were frozen without solvent, lyophilized, and later extracted in methanol. We assigned feeding treatments (toads or fish) to the hatchlings in a balanced, randomized design, so that each clutch was represented as equally as possible among the two treatment groups. However, for clutches with only two hatchlings (clutches 5 and 12), we designated all offspring to the fish-fed group. Hatchlings were housed individually in screen-topped plastic containers with a paper towel as a substrate. Water was available ad libitum. Some offspring that hatched early were fed fish (Oryzias latipes) to sustain them until the assignment of feeding treatments. During the feeding trial, all hatchlings (3–6 weeks of age) were offered food on the same days. On each feeding day, hatchlings in the fish-fed group were given one live fish (O. latipes), which typically weighed between 0.07 and 0.14 g; hatchlings in the toad-fed group were offered three thawed metamorphic toads (Bufo japonicus), which weighed a total of 0.10–0.16 g. The metamorphic toads were raised from tadpoles collected in Chiba Prefecture and were frozen in the spring of 2005. The snakes were left with their food overnight, and any snakes that had not eaten by the following day were force-fed. Food was offered to the hatchlings on three dates, with any necessary force-feedings taking place on the following day. Samples of nuchal gland fluid were collected from 36 unfed hatchlings, 15 of which were resampled four days after the end of the feeding trial. Additionally, samples of nuchal gland fluid from 42 previously unsampled hatchlings were collected after the feeding trial to provide samples from a total of 29 fish-fed and 28 toad-fed snakes. To test for the persistence of bufadienolides, juvenile snakes that had not been exhausted of nuchal gland fluid by the end of the feeding trial (n = 22) were maintained in the laboratory on a diet of fish (Oryzias latipes and Pseudorasbora parva). Rhabdophis tigrinus is notoriously difficult to maintain for extended periods, and the average age at the time of death for the juveniles was 135 26 days; the longest lived juvenile died at 184 days of age. Additionally, a single dam was maintained on a diet of fish after being fed frogs (including two Bufo japonicus) in the laboratory. Samples of prey items fed to hatchling and adult R. tigrinus were analyzed chemically. Bufo japonicus metamorphs (Chiba Prefecture) and O. latipes, both of which were used as prey for hatchling R. tigrinus, were prepared as whole-body extracts in methanol. Skin secretions from Pelophylax nigromaculatus, individuals of which were fed to adult R. tigrinus, were collected using a transcutaneous amphibian stimulator (Grant and Land 2002). Parotoid gland secretions from adult B. japonicus (from Niijima Island) were collected by manually squeezing the glands. Skin secretions from anurans were collected on sections of Kimwipes and stored in methanol at –20 8C. Our animal use protocols conformed to institutional policies and practices.
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Chemical Analyses. Samples of nuchal gland fluid and prey extracts were shipped from Japan to the United States on dry ice for chemical analysis. Samples of nuchal gland fluid and skin secretions from anurans required no preparation prior to evaporation. The whole-body samples of prey in methanol (fish and metamorphic toads) were homogenized with a glass tissue grinder and filtered prior to chemical analysis. All samples were evaporated to dryness, reconstituted in deuterated methanol (CD3OD), and analyzed with 1H-NMR on a Unity INOVA 600 MHz spectrometer (Varian, Palo Alto, CA, USA) equipped with an Oxford magnet (Oxford Instruments, Eynsham, Witney, Oxon, UK) and a 5 mm inverse-detection HCN probe. Following analysis by 1H-NMR spectroscopy, we evaporated all samples to dryness and reconstituted them in a standardized volume of methanol for analysis with HPLC. Most samples were placed in 300 mL methanol, but this amount occasionally resulted in samples that were too concentrated or inadequately dissolved. To correct for this, we used 900 mL of methanol for yolk samples and 1500 mL for parotoid gland secretions of toads and nuchal gland fluid of adult R. tigrinus that contained large quantities of bufadienolides. Samples were analyzed with an Agilent (Santa Clara, CA, USA) 1100 Series HPLC equipped with a quaternary pump, diode array detector, and autosampler. The samples were fractionated through a reversed phase 25 cm x 10 mm Supelco (Bellefonte, PA, USA) Discovery HS C18 column. We used a solvent gradient starting with a mixture of methanol and water, the methanol content of which was held at 20 % (v/v) for two minutes and then increased linearly over a period of 38 minutes until reaching 100 % methanol. After two minutes at 100 % methanol, the solvent composition changed back to the initial 20: 80 methanol/water mixture. A flow rate of 3.4 ml/min was used. The injection volumes of the samples were 25 mL each, and each run lasted 53 minutes. To identify bufadienolides in nuchal gland fluid, we compared the retention times of peaks to those produced by the 17 previously identified compounds from nuchal gland fluid of R. tigrinus (Fig. 1b; Hutchinson et al. 2007). Isolated samples of the previously identified compounds were used as standards (Fig. 2). Of the 17 bufadienolides, compounds 6 and 7 co-eluted and thus could not be distinguished from one another. To summarize the data for hatchlings with the same feeding histories, only those compounds that were present in every individual are displayed in the results. Individual bufadienolides were considered present only when the area of their HPLC peak measured greater than 15.0 mAU*s (milli-absorbance units times seconds). To determine the quantities of bufadienolides present in each sample, we calibrated our HPLC system using a series of dilutions of telocinobufagin. The equation obtained from the linear trend line fitted to the dilution curve was used to convert the areas (mAU*s) of bufadienolide peaks at 280 nm into quantities of bufadienolides. The wavelength of 280 nm was selected because bufadienolides have absorbance maxima at approximately 200 and 300 nm; the latter exhibits a more stable baseline and experiences less interference from the UV absorbance of methanol. Peaks produced by bufadienolides (both structurally known and unknown) were identified by their characteristic UV absorbance spectra, and only those peaks with areas greater than 15.0 mAU*s were used to calculate the total quantity of bufadienolides per sample of nuchal gland fluid. We identified the major bufadienolides in parotoid gland secretions from adult B. japonicus by using dqCOSY NMR and HPLC. Chain lengths of bufotoxins were confirmed by direct injection into an Esquire-LC electrospray ion trap mass spectrometer (Bruker, Billerica, MA, USA). We compared our findings to structures reported in the literature (Shimada et al. 1977). Compounds are referred to by numbers in bold type, and those found in toads are identified by the prefix T. Statistical Analysis. To analyze the relationship of bufadienolides in nuchal gland fluid of unfed R. tigrinus dams and their unfed offspring, we performed a nonparametric Spearman correlation analysis using SPSS version 11.0 (Chicago, IL, USA). The data were plotted using SigmaPlot version 9.0 (SYSTAT, San Jose, CA, USA).
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Results Prey Chemistry and Preference. The presence or absence of bufadienolides in species commonly fed to captive Rhabdophis tigrinus was confirmed by 1H-NMR spectroscopy and HPLC. Bufadienolides were absent in the fish (Oryzias latipes) fed to hatchlings and in a nonbufonid frog (Pelophylax nigromaculatus) fed to adults. Bufadienolides were abundant in the parotoid gland secretions of adult Bufo japonicus, the main components of which are shown in Fig. 1a, as determined by dqCOSY NMR, mass spectroscopy, and HPLC. Small quantities (less than 20 mg) of bufadienolides were present in the metamorphic B. japonicus fed to hatchlings. Most hatchlings in the toad-fed group readily consumed the toads, but hatchlings in the fish-fed group often rejected the fish and had to be force-fed. Overall, toads were rejected only 5.6 % of the time, whereas fish were rejected in 70 % of feedings. This preference for bufonid prey, as opposed to fish, is consistent with previous findings for R. tigrinus (Mori 2004). Dams Fed Nonbufonid Prey. Six gravid, wild-caught R. tigrinus females were fed only nonbufonid prey in the laboratory. Dams 7 and 9 possessed less than 5 mg of bufadienolides per sample of nuchal gland fluid and produced offspring that lacked these compounds (Fig. 1b, Table 1). Samples of nuchal gland fluid from dams 1 and 4 contained bufadienolides in quantities less than 50 mg and 0.25 mg, respectively. Unfed hatchlings from clutches 1 and 4 possessed less than 50 mg of bufadienolides per sample of nuchal gland fluid (Table 1). Samples from dams 2 and 6 at the time of their capture contained more than 1.1 mg (up to 1.9 mg) of bufadienolides. Unfed hatchlings from clutches 2 and 6 possessed more than 0.35 mg (up to 0.60 mg) of these compounds per sample of nuchal gland fluid. It is important to note that the absolute quantities of bufadienolides reported here represent only a portion of the total amount of bufadienolides in a snake. We expressed only a few nuchal glands at any one time to allow for repeated sampling of each individual. Among unfed and fish-fed offspring, compound 10 (gamabufotalin) was the principal bufadienolide in all bufadienolide-containing samples (Table 1). Among the dams, however, compound 10 was the most prevalent bufadienolide in only three of nine samples. Dams Fed Bufonid Prey. Four gravid, wild-caught dams (3, 5, 10, and 11) were fed one toad (Bufo japonicus) each, 9–19 days prior to oviposition. Samples of nuchal gland fluid from dams 10 and 11 at the time of their capture contained 8.3 and 2.4 mg of bufadienolides, respectively (Table 2). Unfed hatchlings from clutch 10 possessed more than 0.75 mg (up to 1.4 mg) of bufadienolides per sample of nuchal gland fluid, whereas unfed hatchlings from dam 11 possessed more than 0.25 mg of bufadienolides per sample (Table 2). At the time of their capture, dams 3 and 5 possessed more than 0.30 and 0.50 mg of
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Fig. 1 (a) Major bufadienolides in the parotoid gland secretion of Bufo japonicus, as determined by NMR, HPLC, and MS. Compounds T3, T5, and T7 are known also from skin secretions of North American toads. Note the corrected chain length of T5 (Hutchinson et al. 2007). (b) Bufadienolides in the nuchal gland fluid of Rhabdophis tigrinus. Compound 8 is 11ahydroxytelocinobufagin, compound 10 is gamabufotalin, compound 13 hellebrigenin, and compound 17 is telocinobufagenin.
bufadienolides, respectively, per sample of nuchal gland fluid (Table 2). Unfed hatchlings from clutches 3 and 5 possessed more than 0.15 mg (up to 0.30 mg) of bufadienolides per sample (Table 2). Some of the gravid dams that were fed a toad sequestered bufadienolides from that prey item that they did not possess previously. One of these newly accumu-
lated bufadienolides (compound 14) was detected in the nuchal gland fluid of their offspring as well. Dams 3 and 11 accumulated compound 14 from a toad consumed 12–19 days prior to oviposition; this bufadienolide was detected in the nuchal gland fluid of all of their offspring tested (Fig. 3, Table 2). Compound 14 was lacking in the sample of nuchal gland fluid from dam 5 at the time of her
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Fig. 2 HPLC profiles of nuchal gland fluid from R. tigrinus. (a) Unfractionated reference bufadienolides. (b) Dam 10 from this study, after being fed a toad in the laboratory.
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capture, but it was present in the nuchal gland fluid of her offspring (Table 2). Dam 5 escaped before she could be resampled, so it was not possible to determine whether she sequestered compound 14 in her nuchal gland fluid from the toad she consumed in captivity. Dam 10 was fed a toad nine days prior to oviposition, but did not sequester any bufadienolides from this toad meal that she did not already possess, so it was not possible to track newly sequestered compounds in her unfed hatchlings (Table 2). Two additional dams (8 and 12) were fed a toad in the laboratory after oviposition. Samples of nuchal gland fluid from dams 8 and 12 at capture contained more than 0.40 and 7.9 mg of bufadienolides, respectively. Dam 12 sequestered compound 14 from her toad meal in captivity (Table 2), whereas dam 8 already possessed this compound. However, the most prominent bufadienolide in the nuchal gland fluid of dam 8 changed from compound 15 at the time of her capture to compound 14 following her toad meal in captivity. Furthermore, dam 8 sequestered compounds 1, 3, 5, 9, 12, 13, and 17 from this toad meal (Table 2). HPLC profiles of bufadienolides in yolk from oviposited eggs closely resembled the profiles of nuchal gland fluid from their respective dams at the time of their capture. Yolk samples from dam 12, which was not fed a toad in the lab while gravid, contained all of the
Table 1 Bufadienolides in nontoad-fed dams and in their hatchlings fed controlled diets
Total quantities of bufadienolides in the majority of samples are represented by shades of gray: light gray, 1–100 mg; medium gray, 0.1–0.5 mg; dark gray, 0.5–1 mg; black, 1–9 mg; None, no bufadienolides; N/A, no samples available. Bufadienolides are identified by number (see Fig. 1b), and the most prominent bufadienolide in each category is underlined. N represents the number of hatchlings per clutch; n, number of samples analyzed (some hatchlings were sampled more than once, especially in the small clutches). Samples from unfed dams were collected at the time of capture; samples from fed dams were collected after oviposition. Localities of the clutches are abbreviated as follows: OP: T = Okayama Prefecture: Tomi-son; OP: K = Okayama Pref.: Kagamino-cho; OP: M = Okayama Pref.: Mikamo-son; HP: S = Hiroshima Pref.: Shohbara City; KP: Y = Kumamoto Pref.: Yamato-cho; KP: M = Kyoto Pref.: Miyama-cho.
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Table 2 Bufadienolides in toad-fed dams and in their hatchlings fed controlled diets
Total quantities of bufadienolides in the majority of samples are represented by shades of gray: light gray, 1–100 mg; medium gray, 0.1–0.5 mg; dark gray, 0.5 – 1 mg; black, 1 – 9 mg; N/A, no samples available. Bufadienolides are identified by number (see Fig. 1b), and the most prominent bufadienolide per category is underlined (inconsistent for clutch 12). N represents the number of hatchlings per clutch; n, number of samples analyzed (some hatchlings were sampled more than once, especially in the small clutches). The only adults fed toads prior to oviposition were dams 3, 5, 10, and 11; the number of days prior to oviposition that the toad was consumed is indicated in the top row for these four dams. Samples from unfed dams were collected at the time of capture; samples from fed dams were collected after oviposition. Localities of the clutches are abbreviated as follows: OP: M = Okayama Prefecture: Mikamo-son; OP: S = Okayama Pref.: Shinjo-son; KP: M = Kyoto Pref.: Miyama-cho; TP: N = Tokushima Pref.: Naka-gun; TP: I = Tokushima Pref.: Ishima Island.
bufadienolides found in her nuchal gland fluid (Fig. 4). Most of these bufadienolides also were detected in the nuchal gland fluid of her unfed hatchlings (Fig. 4). The yolks of the other sampled clutches (3, 10, and 11) contained all of the major bufadienolides found in the nuchal gland fluid of the respective unfed dams; these compounds were detected in the nuchal gland fluid of their offspring as well. Dams 3 and 11 sequestered compound 14 from a toad meal in captivity (consumed during gestation), and small quantities of this bufadienolide were detected in samples of yolk taken from their oviposited eggs. Comparison of Bufadienolides in Dams and Their Offspring. A Spearman correlation analysis revealed a significant, positive relationship between the quantities of bufadienolides in the nuchal gland fluid from unfed dams and their unfed offspring (r = 0.939; p < 0.0005; Fig. 5). The dominant bufadienolide in the dams was more variable than that among the hatchlings, and the bufadienolides sequestered from adult and metamorphic B. japonicus differed. The major bufadienolide in most unfed dams was compound 6 or 7 (n = 6), and the second most prominent bufadienolide was compound 10 (gamabufotalin; n = 3). In contrast, the dominant bufadienolide in
hatchlings, regardless of feeding treatment, was almost always compound 10 (72 of 83 samples; see Fig. 4). Among the dams that were fed a toad in captivity, compound 14 was the most frequently sequestered bufadienolide that was not possessed previously. In hatchlings, compounds 8, 11, and 13 were the most common newly acquired bufadienolides sequestered from metamorphic toads. Persistence of Bufadienolides in Hatchling and Adult Snakes. Samples collected from fish-fed juvenile snakes that were maintained in the laboratory for extended periods revealed that bufadienolides persist in the nuchal glands for at least six months. The two longest-lived hatchlings, which were fed only fish, were from a clutch laid by a female from toad-rich Ishima Island (clutch 12); they lived for 184 and 180 days. These individuals had up to 3.1 mg of bufadienolides in their nuchal glands post mortem. Snakes from mainland Shikoku and Honshu also exhibited lengthy persistence of bufadienolides in their nuchal gland fluid. Fish-fed juveniles from these locations that died at 107–150 days of age possessed up to 1.3 mg of bufadienolides. Despite the lengthy persistence of bufadienolides in general, compounds present in small quantities at the time of hatching diminished within a matter of weeks. Furthermore, the total quantity of bufadienolides in the
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Fig. 3 Aromatic region of 1H-NMR spectra of nuchal gland fluid from dam 3 and her offspring. The gray bars highlight the three regions diagnostic of bufadienolides. Nineteen days prior to oviposition, the dam was fed a toad, from which she sequestered two bufadienolides that she did not possess previously. One of the newly acquired bufadienolides (arrows; compound 14) was provisioned to her unfed offspring along with compounds that she possessed prior to that toad meal.
nuchal gland fluid either remained the same or decreased after the offspring were fed fish. Among defended hatchlings, toad-fed individuals typically possessed the same quantity of bufadienolides as the fish-fed hatchlings, although the diversity of bufadienolide compounds often was greater in the toad-fed group. Hatchlings that were fed nonbufonid diets did not synthesize bufadienolides over time. For example, fishfed hatchlings from clutch 9, an undefended clutch, still lacked bufadienolides at the time of their deaths at up to 155 days of age. Bufadienolides also showed persistence in the single adult snake from Ishima Island (dam 12) that was maintained in captivity. A sample collected from this individual 286 days after she consumed her last toad meal in the laboratory contained 16 mg of bufadienolides. Another sample taken 405 days after her last toad meal contained 9.9 mg of bufadienolides.
Discussion Our results show that Rhabdophis tigrinus females provision their offspring with sequestered bufadienolides. Hatchlings from dams with less than 100 mg of bufadie-
Fig. 4 HPLC profiles of nuchal gland fluid and yolk from clutch 12. The dams nuchal gland fluid contained a large quantity of bufadienolides, all of which were found in the yolks of her oviposited eggs. Most of these bufadienolides also were evident in the nuchal gland fluid of her unfed hatchlings. Note the prominence of compound 10 (gamabufotalin) in the nuchal gland fluid of the hatchling, as compared to its prevalence in the yolk and nuchal gland fluid of the dam.
nolides per sample of nuchal gland fluid either lacked bufadienolides completely or possessed small quantities that are unlikely to be biologically relevant in antipredator defense. In contrast, dams with large quantities of bufadienolides in their own nuchal gland fluid consistently produced offspring with high levels of these defensive compounds. Despite the positive relationship in the total quantities of bufadienolides between dams and their offspring, maternal provisioning in R. tigrinus appears to be a selective process. Selectivity in the sequestration of defensive compounds has been reported in chrysomelid beetles that store specific pyrrolizidine alkaloids from plants (Hartmann et al. 1997). Although quantities of individual bufadienolides in adult R. tigrinus serve as
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Fig. 5 Quantities of bufadienolides in nuchal gland fluid samples of dams (prior to feeding in the laboratory) and their unfed hatchlings, showing mean and standard deviation for each clutch. The Spearman correlation coefficient is 0.939 (p < 0.0005).
general predictors of the compounds that will appear in hatchlings, the provisioning of gamabufotalin (compound 10) followed an atypical pattern. Gamabufotalin was the dominant bufadienolide in 87 % of samples from hatchlings, whereas this compound typically was not the principal bufadienolide in the nuchal gland fluid of the dams. There are several possible explanations for the prominence of gamabufotalin in the hatchlings. Gamabufotalin may be provisioned preferentially in yolk over other compounds. However, our data do not support this explanation because samples of yolk tended to possess the same relative quantities of individual bufadienolides as the nuchal gland fluid of the dams. Another possible explanation is that the most prominent bufadienolide of hatchlings represents the most abundant compound in the dam at the time of vitellogenesis, perhaps reflecting her diet at that time. However, it is unlikely that all of the dams would have had gamabufotalin as their most prominent bufadienolide during vitellogenesis, given the variation in the composition of nuchal gland fluid among dams in this study. A more likely hypothesis is that gamabufotalin may be absorbed from the yolk and transported to the nuchal glands of embryos more readily than other bufadienolides. Other processes cannot be ruled out, however, such as the modification of provisioned bufadienolides by embryos, resulting in the conversion of other compounds to gamabufotalin. Regardless of the reason for the pattern, it may be beneficial to hatchling R. tigrinus to have gamabufotalin as their dominant bufadienolide. This compound is approximately 10 times more effective at inhibiting the sodium-potassium pump in canine kidney than ouabain (Azuma et al. 1986), and thus may be more effective as an antipredator compound than other bufadienolides. However, the physiological activities of most of the other bufadienolides remain to be determined.
CHEMOECOLOGY
Sequestration and provisioning of bufadienolides by R. tigrinus may involve chemical modification, which has been reported for other species that rely on sequestered defensive compounds. Sequestered lucibufagins are hydroxylated by fireflies (Gonzlez et al. 1999b), and the toxicity of sequestered alkaloids is enhanced by hydroxylation in dendrobatid frogs (Daly et al. 2003). In R. tigrinus, gamabufotalin (T7 and 10; Fig. 1) may be sequestered unaltered by adults and hatchlings, or it may be derived from side-chain hydrolysis of a bufotoxin (T3). Similarly, compound 14, which was sequestered by several dams after their toad meal in the laboratory, may result from side-chain hydrolysis and subsequent hydroxylation of T11. Side-chain hydrolysis and hydroxylation of T5 may give rise to compounds 7 and 11; all three are hydroxyketones. Bufadienolides present in the nuchal gland fluid of fish-fed hatchlings, representing maternally provisioned compounds, persist for at least six months. This finding suggests that maternal provisioning alone can provide chemical protection from the time of hatching in late summer until the following spring. By the time R. tigrinus hatch, the toads that had metamorphosed the previous spring may be too large to be consumed (Urano and Ishihara 1987). Recent field observations suggest that hatchlings can, in at least some cases, consume juvenile toads (DAH, AHS and AM, personal observation). However, hatchling snakes that are unable to locate or consume juvenile toads must rely on bufadienolides provisioned by the dam for chemical defense until newly metamorphosed toads become available the following spring. Such reliance would be possible based upon the persistence of bufadienolides demonstrated here. Furthermore, our results indicate that metamorphic toads can serve as a source of bufadienolides for young R. tigrinus, although the quantities of those compounds in metamorphs are small. Importantly, our results demonstrate that toads consumed by a female during gestation can supply bufadienolides to her late-stage embryos, as well as to her own nuchal gland fluid. The eggs of oviparous squamates generally undergo about one-third of their development in the oviducts, prior to oviposition (Shine 1983; Andrews and Mathies 2000). In the present study, newly acquired bufadienolides were provisioned to offspring 12–19 days before oviposition. At that stage of gestation, the eggs presumably are in the oviducts of the female, so any transfer of toxins at that time would likely occur by diffusion across the egg membranes, and probably the intact eggshell, which is laid down during passage through the oviducts (Andrews and Mathies 2000). The presence of large quantities of bufadienolides in yolks of fertile eggs indicates that those compounds are provisioned by deposition in yolk as well, affording two opportunities for provisioning to occur. Our results suggest that maternal provisioning of sequestered bufadienolides is a common phenomenon, but not a universal one, among R. tigrinus. Provisioned buf-
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adienolides become incorporated into embryonic tissues, so that hatchlings are likely to be chemically defended against predators. The presence of bufadienolides in yolk indicates that dams can transmit dietary bufadienolides to their offspring during vitellogenesis, although it is unknown whether the compounds must be ingested during vitellogenesis or whether they can be stored from earlier meals and mobilized later, when the yolk is deposited in the developing ova. In addition, some provisioning can occur long after vitellogenesis is complete, when the fully yolked eggs pass through the oviducts. Hatchlings are capable of sequestering additional bufadienolides from ingested toads, if such prey items are available to them. If toads are unavailable, maternally provisioned bufadienolides will play an especially important role in the ability of juvenile snakes to deter predators. Given the vulnerability of hatchling snakes to predation (Burger 1998), the presence of defensive toxins presumably is an important contributor to offspring survival, and thus maternal provisioning is expected to be subject to strong selective pressure.
Acknowledgments We thank Mark J. Butler IV, Dayanand N. Naik, and Christopher A. Binckley for statistical advice; Stephen T. Deyrup for assistance with mass spectroscopy; Thomas F. Spande and John W. Daly for supplying telocinobufagin; Yohei Kadota, Akira Katayama, Noriko Kidera, Toshiro Kuroki, Masami Hasegawa, Koji Mochida, Aya Nakadai, Sumio Okada, and Hirohiko Takeuchi for collecting animals in the field; Yohei Kadota, Noriko Kidera, Takashi Haramura, and Koji Tanaka for collecting some samples of nuchal gland fluid; Tatsuya Hishida and Koji Tanaka for husbandry; R. James Swanson for use of a vacuum pump; and two anonymous reviewers for helpful comments that greatly improved the manuscript. This work was supported by the National Science Foundation (IBN0429223 and IOB-0519458 to AHS and JM); Old Dominion University Dissertation Fellowship (to DAH); the Society for Integrative and Comparative Biology and Sigma Xi, The Scientific Research Society (Grants-in-Aid of Research to DAH); The Honor Society of Phi Kappa Phi (Love of Learning Award to DAH); Kyoto University Grants for the 21st Century COE A14 and the Global COE Program A06 (to AM); and the Japan Society for the Promotion of Science (to AM).
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Received 20 January 2008; accepted 4 March 2008 Published Online First 19 March 2008
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