REFERENCES AND NOTES 18. E. Kikawa and J. E. Pariso, Proc. Ocean Drill. as a steroidal alkaloid. Prog. Sci. Results 118. 285 (1991 ) Feathers, skin, striated muscle, uropy- 1. C. G. A. Harrison, in The Sea, C. Emiliani, Ed. 19. H. J. B. Dick et a/., ibid., p. 439. (Wiley, New York, 1981), vol. 7, pp. 219-239. 20. N. D. Opdyke and R. Hekinian, J. Geophys. Res. gial gland, heart-liver (combined), and 2. S. K. Baneriee. Tectono~hvsics105. 15 (1984) 72, 2257 (1967) stomach with contents were separated from 3. C. G. A. ~arrison.~nn;, kev. Earth planet. ~ci. 21. B. P. Luyendyk and W. G. Melson, Nature 215. individual hooded pitohuis, variable pito- 147 (19671. 15, 505 (1987). \ -- , huis (P. kirhocephalus), and rusty pitohuis 4. F. J. Vine and D. H. Matthews, Nature 199, 947 22. E. Irving, W. A. Robertson, F. Aumento, Can. J. (1963). Earth Sci. 7, 226 (1970) (P. ferrugineus) (2). Each tissue was stored 5. L. W. Morley and A. Larochelle, R. Soc. Can. 23. D. Stakes, C. Mevel. T. Chaput. Proc. Ocean Drill. separately in 100% ethanol and later was Prog. Sci. Results 118, 153 (1 991) Spec. Publ. 8, 512 (1964); L. W. Morley, in The macerated and washed with 100% ethanol. Sea, C. Emiliani, Ed. (Wiley. NewYork, 1981),vol. 24. Peridotite initially has no magnetite. During the 7. pp. 1717-1719. process of serpentinization, which is considered These crude ethanol extracts were concen- to occur over a long time scale, it acquires chem- 6. F. J. Vine and E. M. Moores, Geol. Soc. Am. Mem. trated so that 100 k1 of extract were equiv- ical remanent magnetization with the formation of 132. 195 (1972) magnetite. alent to 100 mg of tissue. 7. P. J. Fox and N. D. Opdyke. J. Geophys. Res. 78, 25. The relatively large amount of Fe-Ti oxide gabbro We conducted bioassays by injecting 51 39 (1 973). recovered from hole 7358 was not what we ex- D. V. Kent, B. M. Honnorez, N. D. Opdyke, P. J. ethanol extracts subcutaneously into the 8. pected to obtain from the oceanic gabbroic layer Fox, Geophys. J. R. Astron. Soc. 55, 51 3 (1978) before drilling. hindquarters of mice (3). The effect of the 9. S. K. Banerjee. J. Geophys. Res. 85. 3557 (1980). 26. We use arithmetic means in the discussion, as we injection was monitored for 3 hours or until 10. K. E. Davis [Earth Planet. Sci. Lett. 55, 190 (1981)] are primarily concerned with the application of the death. These assays showed that in all three also showed that isotropic gabbros from the up- average magnetization of these unique gabbros per part of layer 3 have thin magnetite rods that to magnetic anomalies, which result from the species the skin and feathers of the pitohuis have grown within plagioclase crystals. linear superposition of individual magnetic source were most toxic. the striated muscle was 11. D. J. Dunlop and M. Prevot, Geophys. J. R. contributions much less toxic, and the heart-liver, stom- Astron. Soc. 69, 763 (1982) 27. K. D. Klitgord, S. P. Huestis, J. D. Mudie, R. L. ach, intestines, and uropygial gland were 12. B. A. Swifi and H. P. Johnson. J. Geophys. Res. Parker, Geophys. J. R. Astron. Soc. 43, 387 89, 3291 (1984). (1975) least toxic (Table I). Concentrations of the 13. M. Talwani. C. C. Windisch, M. G. Langseth, Jr.. 28. K. L. Hayling and C. G. A. Harrison, J. Geophys. toxin varied interspecifically, and of the ibid. 76, 473 (1971). Res. 91, 12423 (1 986). three Pitohui species the hooded pitohui was 14. T. Atwater and J. D. Mudie, ibid. 78, 8665 (1973). 29. All of the samples used for magnetic measure- 15. Oceanic layer 1 is sediment that has very weak, ments in this study were taken by one of the most toxic, the variable pitohui was less negligible magnetization for a marine magnetic authors (E.K.) as his personal samples. toxic, and the rusty pitohui was least toxic anomaly source. Below layer 3 is the Moho, which 30. We thanks. Uyeda, T. Hilde, W. Sager, L. Stokking. (Table 1). In the variable pitohui, tissues of is underlain by serpentinized peridotite (layer 4). and D. Merrill for reading the earlier version of the 16. Layer 2A also exhibits overlapping of different manuscript; J. Watkins and the Institute for Rock an adult were more toxic than tissues of an magnetic polarities in vertical section, which Magnetism. University of Minnesota, for support; immature bird. would lower the effective magnetization responsi- and two anonymous reviewers for helpful com- After fractionation of extracts of feath- ble for marine magnetic anomalies. ments and suggestions that improved the paper. 17. Leg 118 Shipboard Scientific Party, Nature 333. ers, skin, or muscle by acid-base partition- 115 (1988). 15 June 1992; accepted 25 August 1992 ing (4), only the alkaloid fraction was toxic to mice. Alkaloid fractions from skin and muscle were then examined by gas chro- matographic-mass spectral analysis, thin- Homobatrachotoxin in the Genus Pitohui: layer chromatography, and direct probe mass spectrometry. Thin-layer chromatog- Chemical Defense in Birds? raphy revealed the presence in skin of a single alkaloid that gave a blue color reac- John P. Dumbacher,* Bruce M. Beehler, Thomas F. Spande, tion with modified Ehrlich's reagent identi- H. Martin Garraffo, John W. Daly cal to that of homobatrachotoxin (5). That alkaloid cochromatographed with homoba- Three passerine species in the genus Pitohui, endemic to the New Guinea subregion, trachotoxin (Rf 0.50), and the mass spec- contain the steroidal alkaloid homobatrachotoxin,apparently as a chemical defense. Toxin trum (6) was also identical to that of ho- concentrations varied among species but were always highest in the skin and feathers. mobatrachotoxin; these results confirmed Homobatrachotoxinis a member of a class of compounds collectively called batrachotoxins the identity of the major Pitohui toxin in that were previously considered to be restricted to neotropical poison-dart frogs of the skin. Toxic effects of the alkaloid fractions genus Phyllobates. The occurrence of hornobatrachotoxin in pitohuis suggests that birds from skin were virtually identical to those and frogs independently evolved this class of alkaloids. of homobatrachotoxin, causing partial pa- ralysis of hind limbs, locomotor difficulties, and prostration at low dosages (<0.01 kg of hornobatrachotoxin) and tonic convulsions A variety of organisms are known to pro- es. In 1990 we discovered that the hooded and death at higher doses (>0.03 kg of duce or sequester noxious compounds that pitohui, Pitohui dichrous, contained in its homobatrachotoxin) . can be used for defensive purposes. Until feathers and muscle tissue a toxic substance Homobatrachotoxin (Fig. 1) is a mem- recently, no examples of chemical defense that could function as a defensive chemical. ber of a family of steroidal alkaloids collec- were known among birds although there are The toxin caused numbness, burning, and tively called batrachotoxins. Batrachotoxin many examples in all other vertebrate class- sneezing on contact with human buccal and (Rf 0.45), a closely related member of the nasal tissues during collection and prepara- same family of toxins, was not present. J. P. Dumbacher, Department of Ecology and Evolu- tion. University of Chicago, Chicago, IL 60637. tion of specimens. Local New Guineans Homobatrachotoxin was also present in B. M. Beehler, Wildllfe Conservation International- referred to the bird as a "rubbish bird" that muscle tissue but at much lower concentra- Conservation International, Division of Birds, Smith- should not be eaten unless it was &inned tions, consonant with the lower toxicity of sonian Institution, Washington. DC 20560. T. F. Spande, H. M. Garraffo, J. W. Daly, Laboratoryof and specially prepared (I)' We have since muscle extracts. Batrachotoxins depolarize Bioorganic Chemistry, National Institutes of Health, collected tissue of three species of the ge- nerve and muscle cells by activating Na+ Bethesda, MD 20892. nus, and we report the results of bioassays of channels (7) and thus irritate sensory neu- *To whom correspondence should be addressed. toxicity and the identification of the toxin rons in buccal tissue (1).

SCIENCE VOL. 258 30 OCTOBER 1992 Table 1. Toxic effects in laboratory mice of extracts from tissues of three species of pitohuis

Equivalents of Species tissue injected Toxicity* (mg) Hooded pitohui (P. dichrous) Skin Convulsions and death in 18 to 19 min Feathers Convulsions and death in 15 to 19 min Striated muscle (breast) Convulsions and death in 35 to 70 min Heart and liver No convulsions or death Homobatrachotoxin Stomach No convulsions or death Fig. 1. Structure of homobatrachotoxin (3) Intestines Minimal or no effects Uropygial gland Minimal or no effects Variable pitohui (P. kirhocepha1us)t When a toxic species (a model) is avoided Skin Convulsions and death in 16 to 18 min by predators, other species (mimics) may Feathers Convulsions and death in 19 to 27 min avoid predation by resembling the model. Striated muscle (breast) No convulsions or death Four races of the variable pitohui resemble Heart and liver No convulsions or death Stomach No convulsions or death the coloration of the hooded pitohui, and Rusty pitohui (P. ferrugineus) both races of the variable pitohuis that we Skin Convulsions and death in 30 to 40 mln sampled (one resembles the coloration of Feathers No convulsions or death the hooded pitohui, and one does not) have Striated muscle (breast) No convulsions or death the same sour odor as the hooded pitohui *At the indicated dosage all extracts except those marked "minimal or no effects" induced hind limb paralysis, (14). In some regions, immature greater marked locomotor difficulties, and then profound prostration. Some extracts induced permanent paralysis in a melampittas, Melamgitta gigantea, also look hind limb. These effects are similar to those of 0.005 to 0.01 kg of homobatrachotoxin [compare with (3)]. similar to the hooded pitohui (15). Additional toxic sequelae are noted, indicating higher concentrations of toxin. Death in 18 to 19 min is commensurate with the presence of about 0.05 pg of hornobatrachotoxin, tExtracts of tissues from a juvenile appeared somewhat less toxic. REFERENCESANDNOTES

the skin, ranging from undetectable to about 1. I. S. Majnep and R. Bulmer, B~rdsof My Kalam Homobatrachotoxin was previously Country (Aukland Univ. Press. Aukland, New known only from the genus of poison-dart 3 kg. Levels of homobatrachotoxin in pito- Zealand, 1977). This compilation of folklore of the frogs, Phyllobates (Dendrobatidae), in which huis are thus low compared to levels in the Indigenous peoples of Kalam country, Central it occurs with nearlv eaual concentrations of frogs, particularly when the great difference Highlands Province, Papau New Guinea, is the . only publication that discusses the unpalatability batrachotoxin and with the less toxic, prob- in size is considered. Skin from a pitohui has of hooded pitohuis. The authors write that "some able precursor, batrachotoxinin A (3, 8). a mass of 4 to 5 g, whereas skin from the men say that the skln is bitter and puckers the The genus Phyllobates contains the three poison-dart frogs has a mass of 0.2 to 0.5 g, mouth" and that "substances Kalam regard as being 'bltter' make the mouth and lips feel rough toxic Colombian frogs (P. aurotaenia, P. depending on the species. and uncomfortable." bicolor, and P. terribilis) whose skins are used Batrachotoxins have been shown to de- 2. We collected two races of the variable pitohui, P. for poisoning blowgun darts (8). In Phyllo- polarize electrogenic membranes in a range kirhocephalus senex from Wau, East Sepik, Pa- pau New Guinea, and P, k, meridionalis from bates frogs, Naf channels are insensitive to of vertebrates and invertebrates (7). The Bonua, Central Province, in 1991. We collected the effects of batrachotoxins (9); the frog is most likely natural predators of pitohuis are rusty pltohuis from Bonua, Central Province, in thus ~rotectedfrom toxins that are released snakes, raptors, and potentially some arbo- 1991, and hooded pitohuis from Sogeri Plateau, from storage sites in skin. It is not yet known real marsupials. All are expected to be Central Province, in 1990 and 1991 3. T. Tokuyama, J. Daly, 6. Witkop, J. Am. Chem. how ~itohuistolerate homobatrachotoxin in sensitive to batrachotoxins. We therefore SOC.91, 3931 (1969). their own muscle tissue. Pitohui birds and believe that homobatrachotoxin provides 4. The ethanol extract was dlluted with an equal Phyllobates frogs are phylogenetically and the hooded, variable, and perhaps rusty volume of water and extracted three times with equal volumes of chloroform to separate an aque- geographically separated and are the only pitohuis some protection against predators. ous-ethanol fraction containing polar compounds. known organisms that contain batrachotox- Poisonous animals are often conspicuous The comblned chloroform extracts were extracted ins, suggesting independent evolutionary ac- (10, 1 l), and several studies have argued that three times w~thhalf volumes of 0.1 N HCI. The chloroform layer, containing nonbasic lipid-solu- quisition of this alkaloid in birds and frogs. conspicuousness enhances the effectiveness of ble compounds, was dried (Na2S04) and then On the basis of bioassavs of toxicitv., , we educating predators about prey noxiousness was concentrated in vacuo to dryness, and the estimate that a 65-g hooded pitohui con- (10, 12), that conspicuous color patterns star- residue was dissolved In ethanol for assay. The tle or cause hesitation in predators I), and combined HCI extracts were brought to pH 10 tains 15 to 20 ue of homobatrachotoxin in (1 with 1 N aqueous ammonia and were extracted the skin and ;:other 2 to 3 kg in the that some colors or color patterns are avoided three times with equal volumes of chloroform. The feathers, whereas muscle and other tissues by an instinctive or unlearned mechanism in combined chloroform extracts, containing ipid- contribute less than 1 kg. Skin of an adult predators (13). Both hooded and variable soluble alkaloids, were dried (Na2S04) and were concentrated in vacuo to dryness and the residue variable pitohui (85 to 95 g) is estimated to pitohuis emit a strong sour odor and are was dissolved in ethanol for assay. contain 6 to 10 kg of homobatrachotoxin, brightly colored. The wings, tail, and head of 5. Silica gel thln-layer chromatoplates were used whereas skin of a 100-g rusty pitohui is the hooded pitohui and of some races of the with chloroform-methanol (9.1) as the developing solvent, and modifled Ehrlich's reagent (0.1% estimated to contain 1 to 2 ,ue - of homoba- variable pitohui are black, and the remaining pdimethylaminocinnamaldehyde in 0.1 N HCI) trachotoxin. In contrast, the total amount portions of the body are a sharply contrasting was used for detection. of batrachotoxins in the skin of Colombian orange-brown. 6. Mass spectrometer VG7070F. Electron impact Although the exact function of homo- spectrum, direct probe (numbers in parentheses poison-dart frogs of the genus Phyllobates are intensities relative to the base oeak set eaual ranges from about 100 kg in two species (P. batrachotoxin in pitohuis remains un- to 100). mass-to-charge ratios (mh) of 399 (25), aurotaenia and P. bicolor) to 1000 kg in a known, the discovery of this toxic alkaloid 370 (201, 312 (43), 294 (1 7). 286 (15), 184 (54), third species (P. terribilis) (8). The two in pitohuis expands the known possible 153 (171, 138 (24), and 88 (100). Batrachotoxin and homobatrachotoxin do not give a significant Central American frog species (P. vittatus antipredator adaptations in birds to include molecular ion because of thermal decomposition. and P. lugubris) have much lower levels in chemical defense and perhaps mimicry. The exact mass of m/z 399 (measured 399.2410)

800 SCIENCE VOL. 258 30 OCTOBER 1992 corresponds to the expected empirical formula of has selective value for pitohuis, but it offers an months, 18 were measured semiweekly for 4 C,,H,,NO, (calculated 399 2319) alternative explanation for the original evolution of to 18 months, and 3 infants were measured 7 E. X. Albuquerque, J. W. Daly, B. Wltkop, Science Miillerian mimicry in this group. A phylogenetlc 172, 995 (1971). analysls of P~tohwand the Interracial variation in dailv for 4 months. 8. C. W Myers, J. W. Daly, B. Malkin, Bull. Am. Mus. the variable pitohui will clarify thls issue. ~ecumbentlength, weight, and head Nat. Hist. 161 , 307 (1978) 15. B. M. Beehler, T. K. Pratt. D. A. Zimmerman, Birds circumference were assessed according to 9. J. W. Daly, C. W. Myers. J. E. Warnick, E. X. of New Guinea (Princeton Univ. Press, Princeton. Albuquerque, Science 208, 1383 (1980) NJ, 1986). standard techniques (1 0) ; the serial length 10. T. Guilford. Am. Nat. 131, 57 (1988). 16. We thank N. Wahlberg, B, lova, and the many measurements are the focus of this report. 11. R. R. Baker and G. A. Parker, Philos. Trans. R. other workers who contributed to our field efforts, Total recumbent length was measured to N. Whittaker for the mass spectral determinations, Soc. London Ser. B 287, 63 (1979). the nearest 0.05 cm by two observers with a 12. T. Guilford, Anim. Behav 34, 286 (1986). and S. Arnold, J. Diamond, H. Fales, K. Kelley, K. 13. W. Schuler and E. Hesse, Behav. Ecol. Sociob~ol. Kubzdela, H. Landel, and S. Pruett-Jones for specially designed infant measuring board 16, 249 (1985). comments on earlier drafts of this manuscript. (1 1) during home visits; 80% of the mea- Fieldwork was supported by National Geographic 14. It could be argued that, if variable and hooded Society grant 4026-89 and Sigma Xi, the Scientific surements were revlicates. pitohuis are sister taxa, the mimetic resemblances Research Society. may be historical artifacts due to shared primitive Sources of measurement error and varia- traits. This possibility does not refute that mimicry 5 May 1992; accepted 30 July 1992 tion include the equipment, repeated mea- surements, the technique of the observer, and the cooperation of individual subjects. Saltation and Stasis: A Model of Human Growth The technical errors of repeated measure- ment (12) for length were significantly dif- M. Lampl,* J. D. Veldhuis, M. L. Johnson ferent between children, reflecting individ- ual variability in cooperation. A pooled Human growth has been viewed as a continuous process characterized by changing intra-observer error of 0.124 cm (1729 reo- velocity with age. Serial length measurements of normal infants were assessed weekly (n licates) and an inter-observer pooled error bf = 1 O), semiweekly (n = 18),and daily (n = 3) (1 9 females and 12 males) during their first 0.11 cm (with an independent rater), paral- 21 months. Data show that growth in length occurs by discontinuous, aperiodic saltatory lel reports of technical errors of replicate spurts. These bursts were 0.5 to 2.5 centimeters in amplitude during intervals separated measurement (0.1 14 to 0.145 cm) recently by no measurable growth (2 to 63 days duration). These data suggest that 90 to 95 percent published (1 3). Because the technical error of normal development during infancy is growth-free and length accretion is a distinctly of repeated observations cannot account for saltatory process of incremental bursts punctuating background stasis. all errors of measurement. the auantitative analytic methods were designed to take into account a wider range of possible measure- ment error inherent in the data. The present assumptions regarding the bi- focus on the structure of the individual time Analytical methods developed in part ology of human growth are based primarily course of growth is unprofitable. Sporadic for the evaluation of evisodic hormonal on height and weight data collected in reports suggest that traditional studies may pulses (14) were modified for the analysis of auxological studies. Individuals have been overlook important aspects of individual the serial bodv measurements as an ad hoc traditionally measured at quarterly intervals growth patterns because undulations in first approximation descriptor. Individual during infancy, and annually or biannually growth rates shorter than the period of serial -growth data were modeled as a series during childhood and adolescence. Physio- measurement go undetected (5). Descrip- of putative, distinct, stepwise (saltatory) logical- data are mathematicallv smoothed tive studies suvvort.. this conclusion with increases or jumps separated by variable and growth is represented as a continuous data on nonlinearity (6) or short-term ve- intervals of no change. Using replicate curve of three sequential stages: infancy, locity oscillations in serial height as well as measurements and an error estimate, we with growth progressing at a rapidly decel- total body or lower leg length (7). The exvress serial increments as standard nor- erating rate from birth; childhood, as general dictum, however, is that while mal deviates. These deviates are assessed at growth approaches a relatively constant some oscillation occurs in the -growth rate an experimentally defined probability (or P but slow rate; and adolescence, when the of some children, growth is a continuous value) of falsely rejecting the null hypoth- pubertal growth spurt propels the body and generally constant process (I), and that esis of no difference in serial length mea- toward final adult form with a sharp in- the most satisfactory assessment of chil- sures. crease and final rapid decrease in growth dren's growth is still considered to be made The growth in length of all subjects in velocity (1, 2). over annual intervals (8). this study occurred by saltatory increments Although undulations in growth veloci- The availability of human growth hor- with a mean amplitude of 1.01 cm identi- tv vatterns have been described in individ- mone and the resulting clinical potential fied at the P < 0.05 level. A plot of this 1. ual data as early as the 18th century (3), for treatment of growth disorders, as well as growth punctuates intervals when no statis- they have most often been assumed to advances in molecular biology describing tically significant growth occurred (Table reflect measurement error (4). The consen- normal cellular growth control mecha- 1). We found that the growth saltations sus for most of the centurv has been that a nisms, underscore the importance of clari- were not identifiably periodic but episodic. fying normal growth dynamics. Information on the vrecise temvoral struc- M. Lampl, Department of Anthropology, University of The present study further investigates ture of a growth saltation is constrained by Pennsylvania, Philadelphia, PA 19104. the nature of normal infant -growth with the measurement interval. the smallest J. D. Veldhuis, National Science Foundation Center for time-intensive data and an analytic descrip- window for incremental growth documen- Biological Timing and Division of Endocrinology and Metabolism, Department of Internal Medicine, Univer- tor. Thirty-one clinically normal (9) Cau- tation. When assessed weekly, length in- sity of Virginia Health Sciences Center, Charlottesville, casian American infants (19 females and 12 crements from 0.5 to 2.5 cm ~unctuated7- VA 22908. males) were studied between the ages of 3 to 63-day intervals of no growth. Semi- M. L. Johnson, National Science Foundation Center for Biological Timing and Department of Pharmacology, davs and 2 1 months after varental informed weekly assessments showed saltatory length University of Virginia Health Sciences Center, Char- consent of an institutionally approved hu- increments of 0.5 to 2.5 cm punctuating 3- lottesville, VA 22908. man subjects protocol. Ten of these infants to 60-day intervals of no growth. Daily 'To whom correspondence should be addressed. were measured weekly for periods of 4 to 12 measurements documented length incre-

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