Herpetolog?ca, 40(1), 1984, 82-88 ? 1984 by The Herpetologists' League, Inc. ESTIMATING PREY SIZE AND NUMBER IN CRAYFISH-EATING SNAKES, GENUS REGINA J. S. GODLEY, R. W. McDiARMID, AND N. N. ROJAS ABSTRACT: Snakes of the genus Regina feed almost exclusively on crayfish. The paired, sym- metrical gastroliths of crayfish are not digested and are detectable from x-rays of the snake. Gastrolith length is directly proportional to carapace length and can be obtained from x-rays. Carapace length can be converted to kcal of ingested energy. Using these relationships and repeated captures of radio-telemetered Regina, estimates of food consumption and energy intake by free- living snakes are feasible. New information on prey selectivity, feeding behavior, and predator- prey size relations in Regina grahami and R. septemvittata are presented and compared with similar data for other snakes. Key words: Reptilia; Serpentes; Colubridae; Regina; Food habits; Predator-prey size relation- ships; Crayfish THE foraging ecology of snakes is less often studied than many aspects of snake biology. Some species are rare and others are secretive; consequently, obtaining ad- equate samples is a common problem (Turner, 1977). In addition, most snakes eat relatively large prey at infrequent in- tervals (e.g.. Beavers, 1976; Fitch, 1960, 1965; Godley, 1980; Greene, 1983a,fe; Schoener, 1977; Seib, 1981), and their digestive tracts seldom contain food. Be- cause of the prey's advanced state of digestion in the hindgut (Brown, 1958; Godley, 1980; Henderson, 1970; Skoczy- las, 1970), positive identification of prey species and important measures of prey size often are obtainable only while the prey is in the snake's stomach. Here we describe an accurate, non-in- vasive technique for estimating the num- ber and sizes of prey in the entire diges- tive tracts of crayfish-eating snakes, genus Regina. The technique is designed specif- ically for dietary studies of R. grahami and R. septemvittata, two species that feed almost exclusively on soft, freshly molted crayfish (Branson and Baker, 1974; Burg- hardt, 1968; Hall, 1969; Kofron, 1978; Mushinsky and Hebrard, 1977; Strecker, 1926; Wood, 1949). We also provide new information on prey selectivity, feeding behavior, and predator-prey size relations in these species and summarize similar data for other species of snakes. The technique requires an understand- ing of specific events in the crayfish molt cycle and the relationship of these events to prey selection in R. grahami and R. septemvittata. Briefly, individuals of var- ious species of crayfish molt one to 12 times per year depending upon sex, age and en- vironment. Usually less than 15% of a population is molting at any one time (Ca- pelli and Magnuson, 1975; Drach, 1939; Prins, 1968; Stein, 1977; Stevenson, 1975). Prior to molt, calcium is extracted from the exoskeleton and stored in paired, sym- metrical gastroliths located in the cardiac stomach (McWhinnie, 1962; Richards, 1951; Stein and Murphy, 1976; Travis, 1960). The gastroliths are resorbed shortly after molt and contribute to the formation of a new exoskeleton. Gastroliths reappear upon initiation of the next molt cycle. Gastrolith growth and r?sorption are dis- tinct events and are tightly synchronized with other identifiable stages in the molt cycle (McWhinnie, 1962; Stevenson, 1975). As we shall show, these gastroliths provide a convenient, quantitative "marker" for estimating rates of feeding and energy in- take in R. grahami and R. septemvittata, and perhaps in other species that feed ex- tensively on molting crayfish (Neill, 1951; Penn, 1950). MATERIALS AND METHODS We estimated the number, size and feeding sequence of crayfish ingested by 36 R. grahami (USNM 12864-65, 12891, 82 March 1984] HERPETOLOGICA 83 12940-70, 13038-39) from New Orleans, Orleans Parish, Louisiana, and two (197638) from Badger Lake, Monona County, Iowa, by exposing identifiable prey with a single ventro-medial incision through the gut wall. The orientation (head or abdomen first) and the ingestion position (dorsal, ventral or lateral) of the crayfish relative to the snake's skull were recorded prior to removal of prey from the snake's stomach. After removal, the crayfish's carapace length (CL = maxi- mum dorsal midline distance from tip of rostrum to posterior edge of carapace) and gastrolith length (GL = maximum longi- tudinal length of gastroliths) were mea- sured with vernier calipers to the nearest 0.1 mm. Only GL was obtained from in- testinal contents; paired, symmetrical gas- troliths were assumed to represent the di- gested remains of a single crayfish. The molt stage of the crayfish (Drach, 1939; McWhinnie, 1962; Stevenson, 1975) also was recorded. Molt stages pertinent to this study are as follows. A;?Exoskeleton is soft, gelatinous and easily torn; gastrolith is fully formed; duration is of a few hours. Aj?Exoskeleton is leathery; gastrolith is reduced to about 50% of former size by end of stage; duration is ca. 24 h. B? Rostrum and cephalic carapace are firmer but flexible; gastrolith is very small to ab- sent at end of stage; duration is of one to three days. Similar data were obtained from a se- ries of 36 R. septemvittata collected in Montgomery Co., Ohio (USNM 128974- 98, 129016-27). However, before dissec- tion, each specimen was radiographed in dorso-ventral view. The radiographs were exposed for 30 s at instrument readings of 5 mamp and between 20 and 23 kV de- pending on snake size. Gastrolith mea- surements were taken from the radio- graphs with a dial caliper. Because gastroliths are slightly oblong in shape and the x-rays provided only a single plane of view, the longest dimension of either gas- trolith of the pair was chosen to represent the GL. To avoid possible bias, GL mea- surements taken from the radiographs were examined after dissection measure- ments were recorded. Because methods varied between the snake species, results are presented separately. RESULTS Regina grahami Only the remains of crayfish, repre- senting at least three species, Orconectes palmeri croelanus (Creaser), Procamba- rus clarkii (Girard) and P. vioscai (Penn), were recovered from the stomachs of 38 R. grahami. Of these prey, measurements of both GL and CL were obtainable from 17 crayfish (all were in molt stages Ai or early Aj), CL only from two crayfish (one in stage B, the other in stage Aj or As but gastroliths broken during processing), GL only from six crayfish (all in stages Ai or As but carapace partially digested), and two crayfish were represented only by sin- gle, very large (57 and 63 mm total length) chaela, each in a different snake. The largest chaela from an intact crayfish (CL = 31.5 mm) in the sample was 36.0 mm, suggesting that the latter two snakes encountered crayfish too large to consume whole and instead removed the only large portion (chaela) that could be disarticu- lated. In addition, GL measurements from 20 crayfish were obtained from intestinal contents. All of these crayfish appeared to have been in molt stage Ai or early A2. Although most of the exoskeletons of crayfish were partially digested, the gas- troliths seemed normal and unaffected by passage through the snake's digestive tract. In fact, captives of all species of Regina defecate gastroliths which macroscopical- ly are indistinguishable from those dis- sected from live crayfish of a similar molt stage (J. S. Godley, personal observation). As is typical of freshly molted crayfish (McWhinnie, 1962 for Orconectes virilis), GL is a linear function of CL (Fig. 1) and provides an excellent estimate of crayfish size. Most of the variance about this line probably is caused by three sources of ex- perimental error: (1) differences in GL: CL ratios among the three species of cray- 84 HERPETOLOGICA [Vol. 40, No. 1 ? 5,0 ? E Y.0.157X-0.5S2 r = 0.8 8 0 20 30 Carapace Length (mm) FIG. 1.?Relationship of gastrolith length and car- apace length from 17 crayfish removed from the stomachs of Regina grahami. fish represented in the sample, (2) differ- ences in crayfish molt stage, and (3) the difficulties of accurately measuring the CL of soft-bodied crayfish taken from a snake's stomach. In a sample of 18 Pro- cambarus fallax from Hillsborough Co., Florida, which were sacrificed within six h of molting, the correlation coefficient between GL and CL was 0.98 (J. S. God- ley, unpublished data). The slope of this line was not significantly different from that shown in Fig. 1 (F^ai = 2.48, F = 0.1253). Predator-prey size relationships in R. grahami can be examined by using GL as an estimate of crayfish size (GL estimated for two crayfish from CL) and snake snout-vent length (SVL) as an estimate of predator size (Fig. 2A). In our data set, the regression of GL on CL (Fig. 1) al- lowed estimates of size for 27 digested crayfish and increased the usable crayfish sample size 158.8% (from 17-44). Snake SVL explained a significant (F = 0.0029, Y = 1.436 + 0.0046X) but small percent- age (r^ = 0.193) of the variation in the size of prey taken by R. grahami (Fig. 2A). Although adult R. grahami consumed larger crayfish than juveniles, the mini- mum size of ingested prey did not change during ontogeny. Of the 25 snakes with food, 14 contained one crayfish, six had two crayfish each, two contained three, and three had eaten four crayfish. SVL was positively correlated with the total number of prey whether individuals with empty digestive tracts were included (Spearman rank correlation, r^ = 0.47; P = 0.0076, n = 38) or excluded (r, = 0.58, P = 0.0024, n = 25) from the analysis. Prey orientation could be determined for 26 of 27 crayfish recovered from the R. grahami stomachs; 12 were ingested head first and 14 abdomen first, suggest- ing no orientation preference (x^ = 0.08, F > 0.75). Vertical position of prey could be determined for 22 of the 27 prey and was not significantly different from ran- dom (x' = 4.0, 2 df, F > 0.10) with 12 crayfish ingested on their side with re- spect to the snake's skull, eight with ven- ters up, and two with venters down. Anal- ysis of covariance with GL as the dependent variable, SVL as the covariate, and prey orientation and position as in- dependent variables showed no significant effects for the latter two variables. Regina septemvittata In this species, specimens were x-rayed prior to dissection to determine if radio- graphs could provide reliable estimates of feeding activity. The two criteria we judged necessary for establishing reliabil- ity were: (1) radiographs must detect all crayfish in the digestive tract, and (2) measurements of GL taken from x-rays and those obtained from dissection must show high correspondence. Gastroliths representing 26 crayfish were identified from the radiographs of the 36 R. septemvittata. The same num- ber of prey was found when the snakes were dissected. A representative radio- graph is shown in Fig. 3. A strong corre- lation exists between GL based on radio- graphs and GL based on dissections (Fig. 4). Only four of the 26 crayfish in the R. septemvittata were in the stomachs. These four prey (Cambarus diogenes Girard) were partially digested and no CL mea- March 1984] HERPETOLOGICA 85 9 ? B . A ? 7 U "i , 1 6 ? ? - c S " = ? ? ? tJ ? t / ? :: 4 ? ? tD . ? ? 3 ? ? ? 2 r ? ? ? ? ? B 400 500 600 Snout-Vent Lengtti (mm) Regino gratiami 300 Snout-Vent Lengtti (mm) Regina septemvrttota FIG. 2.?Size relationship between snakes and their respective crayfish prey. (A) Regina grahami (n ?? 44). (B) Regina septemvittata (n = 26). surements were obtained; all were in molt stage Al and were ingested abdomen first. Judging from the condition of the gas- troliths (representing 22 crayfish) found in the intestines, these crayfish also were in molt stage A; or A2 when eaten. The scat- ter plot of crayfish GL on R. septemvit- tata SVL (Fig. 2B) was similar to that ob- served in jR. grahami (Fig. 2A), but the regression was not significant (r^ = 0.09, P = 0.139). DISCUSSION Our work has revealed an accurate, nondestructive technique for estimating the number and size of prey in two species of Regina based on the following obser- vations. (1) Regina feed almost exclusive- ly on crayfish. (2) The paired, symmetri- cal gastroliths of crayfish are not digested. (3) GL is directly proportional to CL. (4) GL is obtainable from x-rays of Regina that have fed on crayfish. Insight into several poorly known as- pects of snake foraging ecology and diges- tive physiology could be gained using this technique. For example, little is known about the effects of different feeding, temperature and activity regimes on clearance times and rates of digestion in snakes of different size, sex and reproduc- tive condition (see Dandrifosse, 1974; Godley, 1980; Skoczylas, 1978 for re- views). Here a major difficulty has been the lack of a convenient, natural marker to follow the course of prey digestion. X-rays made at appropriate intervals of crayfish-eating snakes would provide such a system under controlled laboratory con- ditions. Perhaps the greatest use of this technique lies in its potential field appli- cation. By using the relationships devel- oped above, a portable x-ray unit, and re- peated captures of Regina equipped with transmitters that measure heart rate or body temperature, accurate estimates of the energy budget of free-living snakes are possible (the energy content of crayfish is known, see Godley, 1980; Stein and Murphy, 1976). Concurrent x-rays of non- telemetered animals from the same pop- ulation could serve as a control for possi- ble effects of experimental procedures. Our results show that crayfish are sub- ject to pr?dation by Regina grahami and R. septemvittata only while they are in molt stages Ai or A2. Further, the low vari- ance in GL for crayfish of any size (Fig. 86 HERPETOLOGICA IVol. 40, No. 1 FIG. 3.?Radiograph of Regina septemvittata (USNM 128998) from Ohio. This snake contains three pairs of gastroHths and a pebble (arrow). 1) and the rapid temporal decline in GL following molt (McWhinnie, 1962) sug- gest that most crayfish are eaten by these snakes within 6 h of molt (stage A,). This extreme molt-stage selectivity may have important ecological consequences. In three species of crayfish found within the range of R. grahami or ?. septemvittata, freshly molted individuals comprise less than 15% of the population averaged over the year (Prins, 1968; Stein, 1977). In ad- dition, all of these species seem to have a "mass, synchronized molt" (Prins, 1968: 678) which would further limit the bio- mass of snakes that could be supported on a given population of crayfish. In contrast, morphological and behavioral adaptations of Regina alleni (Franz, 1977; Godley, 1980) and R. rigida (Kofron, 1978) enable these species to exploit crayfish in all stages of their molt cycle. Available density es- timates for R. septemvittata (Branson and Baker, 1974) and R. alleni (Godley, 1980) support this contention. Variation in molt-stage selectivity among species of Regina also is reflected in their feeding behavior. Covariance analysis suggests that in R. grahami, ori- entation and positioning of crayfish is ran- dom with respect to the snake's skull and 3 4 5 Gastrolith Length (mm)-X-ray FIG. 4.?Relationship between gastrolith length as measured from gastroliths removed from gut and those measured from x-rays of Regina septemvittata (n = 25); one gastrolith was damaged during pro- cessing and excluded from this comparison. is independent of crayfish size (see also Hall, 1969). However, R. septemvittata, which feeds on crayfish of similar molt stages, apparently ingests crayfish only abdomen first (see also Branson and Ba- ker, 1974; Wood, 1949), suggesting that these two closely related species (Ross- man, 1963) have diverged behaviorally while maintaining similar molt-stage se- lectivity. R. alleni consumes crayfish ab- domen first and lateral with respect to the snake's skull regardless of crayfish size, molt-stage or snake feeding experience (Franz, 1977; Godley, 1980 and unpub- lished). The feeding behavior of R. rigida is unknown but probably is similar to that of R. alleni. Snake SVL proved to be a relatively poor predictor of the size of crayfish eaten by Regina grahami (r^ = 0.19, P = 0.0029) or R. septemvittata (r^ = 0.09, P = 0.139) (Fig. 2). In reviewing similar data for 17 other species of snakes distributed among five families (Beavers, 1976; Godley, 1980; Greene, 1983a,?; Mushinsky et al, 1982; March 1984] HERPETOLOGICA 87 Reynolds and Scott, 1982; Seib, 1981; Shine, 1977; Voris and Moffett, 1981), we found four common, intraspecific trends. (1) Larger snakes can consume absolutely larger prey than smaller individuals. (2) The variance in prey size tends to increase with increasing snake size. (3) A least squares regression of prey size on snake size usually yields a positive, significant slope but rarely accounts for more than 50% of the variation. (4) The slope of the boundary line for minimum prey size is much lower than that for maximum prey size such that the size of the smallest prey is similar for adults and juveniles of the same species. As did Voris and Moffett (1981), we define prey boundary lines as the approximate lower and upper size limits of prey eaten by a snake during its ontogeny. We are concerned with the increasing tendency among some workers to inter- pret the shape of predator-prey size tra- jectories in snakes as a reflection of prey size selection seemingly without con- sideration of alternative hypotheses. Con- straints imposed by head morphology alone would restrict the range of prey sizes ingested by gape-limited snakes (Gans, 1961; Greene, 1983a) and could produce each of the four intraspecific trends noted above. A morphological-constraint hy- pothesis also could explain why, in our opinion, experimental attempts to dem- onstrate prey selection based on size in snakes have failed (Czaplicki and Porter, 1974; Godley, 1980; Reynolds and Scott, 1982; Smith and Watson, 1972). Although we do not doubt that ecological factors influence prey choice in snakes, it remains to be shown that any species of snake se- lects a smaller range of prey sizes than the limits imposed by its morphology. Acknowledgments.?Margaret Daniel and Horton H. Hobbs, Jr. assisted with the crayfish identifica- tions; Claudia Angle prepared some of the illustra- tions; Susan Jewett assisted with the x-rays; and Ma- rianne Scott typed the manuscript. Harry Greene and Henry Mushinsky reviewed an early draft of the manuscript. To all of these people, we extend our thanks. LITERATURE CITED BEAVERS, R. A. 1976. Food habits of the western diamondback rattlesnake, Crotalus atrox, in Texas (Viperidae). Southwest. Nat. 10:503-515. BRANSON, B. A., AND E. C. BAKER. 1974. An eco- logical study of the queen snake, Regina septem- vtttata (Say) in Kentucky. Tulane Stud. Zool. Bot. 18:153-171. BROWN, E. E. 1958. Feeding habits of the northern watersnake, Natrix sipedon sipedon Linnaeus. Zool?gica 43:55-71. BURGHARDT, G. M. 1968, Chemical preference studies on newborn snakes of three sympatric species of Natrix. Copeia 1968:732-737. CAPELLI, G. M., AND J. J. MAGNUSON. 1975. Re- production, molting, and distribution of Orco- nectes propinquus (Girard) in relation to temper- ature in a northern mesotrophic lake. Pp. 415-428, In J, W, Avault, Jr, (Ed.), Second International Symposium on Freshwater Crayfish. Louisiana State University, Div, Continuing Education, Baton Rouge, CZAPLICKI, J, A., AND R, H. PORTER. 1974. Visual cues mediating the selection of goldfish (Carassius auratus) by two species of Natrix. J, Herpetol, 8: 129-134, DANDRIFOSSE, G, 1974. Digestion in reptiles, Pp, 249-275. In M. Florkin and B. T. Scheer (Eds.), Chemical Zoology, Vol. 9. Academic Press, New York, DRACH, P, 1939, Mue et cycle d'intermue chez les crustac?s d?capodes, Ann, Inst, Oceanogr, Monaco 19:103-391, FITCH, H. S, 1960, Autecology of the copperhead, Univ, Kansas Publ, Mus, Natur, Hist. 13:85-288, , 1965. An ecological study of the garter snake, Thamnophis strtalis. Univ. Kansas Publ. Mus. Nat, Hist, 15:493-564, FRANZ, R, 1977, Observations on the food, feeding behavior and parasites of the striped swamp snake, Regina alleni. Herpetologica 33:91-94, GANS, C, 1961. The feeding mechanism of snakes and its possible evolution. Am. Zool. 1:217-227. GODLEY, J. S. 1980. Foraging ecology of the striped swamp snake, Regina alleni, in southern Florida, Ecol, Monogr. 50(4):411-436, GREENE, H. W, 1983?I. Dietary correlates of the origin and radiation of snakes. Am, Zool, 23:431- 441, . 1983i>. Feeding behavior and diet of the eastern coral snake, Micrurus fulvius. Spec, Publ, Mus. Nat, Hist. Univ. Kansas: In press. HALL, R. J. 1969. Ecological observations on Gra- ham's water snake, Regina grahami (Baird and Girard). Am. Midi. Nat, 18:156-163. HENDERSON, R. W. 1970. Feeding behavior, diges- tion, and water requirements of Diadophis punc- tatus arnyi Kennicott, Herpetologica 16:520-526, KoFRON, C. P, 1978. Food and habitats of aquatic snakes (Reptilia, Serpentes) in a Louisiana swamp. J. Herpetol. 12:543-554. 88 HERPETOLOGICA [Vol. 40, No. 1 MCWHINNIE, M. A. 1962. Gastrolith growth and calcium shifts in the freshwater crayfish, Orco- nectes virilis. Comp. Biochem. Physiol. 7; 1-14. MusHiNSKY, H. R., AND J. J. HEBRARD. 1977. Food partitioning by five species of water snakes in Lou- isiana. Herpetologica 33:162-166. MusHiNSKY, H. R., J. J. HEBRARD, AND D. S. VODOPICH. 1982. Ontogeny of water snake for- aging ecology. Ecology 63:1624-1629. NEILL, W. T. 1951. Notes on the role of crawfishes in the ecology of reptiles, amphibians, and fishes. Ecology 32:764-766. PENN, G. H. 1950. Utilization of crawfishes by cold- blooded vertebrates in the eastern United States. Am. Midi. Nat. 44:643-658. PRINS, R. 1968. Comparative ecology of the cray- fishes Orconectes rusticus rusticus and Cambarus tenebrosus in Doe Run, Meade County, Kentucky. Int. Rev. Ges. Hydrobiol. 53:667-714. REYNOLDS, R. P., AND N. J. SCOTT, JR. 1982. Use of a mammalian resource by a Chihuahuan snake community. Pp. 99-118. In N. J. Scott, Jr. (Ed.), Herpetological Communities. U.S. Dept. Int. Fish Wildlife Ser., Wildlife Research Report 13. RICHARDS, A. G. 1951. The integument of arthro- pods. University of Minnesota Press, Minneapolis. ROSSMAN, D. A. 1963. Relationships and taxonomic status of North American natricine snake genera Liodytes, Regina, and Clonophis. Occ. Pap. Mus. Zool., Louisiana State Univ. 29:1-29. SCHOENER, T. W. 1977. Competition and the niche. Pp. 35-136. In C. Gans and D. W. Tinkle (Eds.), Biology of the Reptilia. Vol. 7. Academic Press, New York. SEIB, R. L. 1981. Size and shape in a neotropical burrowing colubrid snake, Geophis nasalis, and its prey. Am. Zool. 21:933. SHINE, R. 1977. Habitats, diets, and sympatry in snakes: a study from Australia. Can. J. Zool. 55: 1118-1128. SKOCZYLAS, R. 1970. Influence of temperature on gastric digestion in the grass snake, Natrix natrix L. Comp. Biochem. Physiol. 33:793-804. . 1978. Physiology of the digestive tract. Pp. 589-717. In C. Gans and K. A. Gans (Eds.), Biol- ogy of the Reptilia. Vol. 8. Academic Press, New York. SMITH, G. C, AND D. WATSON. 1972. Selection patterns of corn snakes, Elaphe guttata, of differ- ent phenotypes of the house mouse. Mus muscu- lus. Copeia 1972:529-532. STEIN, R. A. 1977. Selective pr?dation, optimal for- aging, and the predator-prey interaction between fish and crayfish. Ecology 58:1237-1253. STEIN, R. A., AND M. L. MURPHY. 1976. Changes in proximate composition of the crayfish Orco- nectes propinquus with size, sex, and life stage. J. Fish. Res. Board Can. 33:2450-2458. STEVENSON, J. R. 1975. The molting cycle in the crayfish: recognizing the molting stages, effects of ecdysone, and changes during the cycle. Pp. 255- 267. In J. W. Avault, Jr. (Ed.), Second Internation- al Symposium on Freshwater Crayfish. Louisiana State Univ., Div. Continuing Education, Baton Rouge. STRECKER, J. K. 1926. On the habits of some south- ern snakes. Baylor Univ. Mus. Contri. 4:3-11. TRAVIS, D. F. 1960. Matrix and mineral deposition in skeletal structures of the decapod Crustacea. Pp. 57-116. In R. F. Sognnaes (Ed.), Calcification in Biological Systems. Publ. 64, Am. Assoc. Adv. Sei., Washington, D.C. TURNER, F. B. 1977. The dynamics of populations of squamates, crocodilians and rhynchocephalians. Pp. 157-264. In C. Gans and D. W. Tinkle (Eds), Biology of the Reptilia. Vol. 7. Academic Press, New York. VORIS, H. K., AND M. W. MOFFETT. 1981. Size and proportion relationship between the beaked sea snake and its prey. Biotropica 13:15-19. WOOD, J. T. 1949. Observations on Natrix septem- vittata (Say) in southwestern Ohio. Am. Midi. Nat. 42:744-750. Accepted: 25 August 1983 Associate Editor: Kentwood Wells JSG and NNR: Department of Biology, University of South Florida, Tampa, FL 33620, USA; JSG and RWM: U.S. Fish and Wildlife Service, National Museum of Natural History, Washington, DC 20560, USA