Zoo Biology 32: 172176 (2013)

RESEARCH ARTICLE

Parthenogenesis in a Brazilian Rainbow Boa (Epicrates cenchria cenchria)

Matthew E. Kinney,1,2 Raymund F. Wack,2,3 Robert A. Grahn,4 and Leslie Lyons4
1 2

William R. Pritchard Veterinary Medical Teaching Hospital, University of California-Davis, Davis, California CA Sacramento Zoo, Sacramento, CA California 3 Wildlife Health Center, University of California Davis, Davis, CA California 4 Department of Population Health and Reproduction, University of California-Davis, Davis, California CA
A 22-year-old captive Brazilian rainbow boa (Epicrates cenchria cenchria) gave birth to four offspring after being housed with a vasectomized male. Sexual reproduction as a result of failed prior vasectomy, recanalization of the vas deferens, or prolonged sperm storage was ruled out using the clinical history, histopathology, and gross necropsy. Short tandem repeat (STR) DNA markers were genotyped in the male, female, and four offspring. None of the offspring possessed a diagnostic STR allele present in the potential sire. In addition, all offspring were homozygous at each STR locus evaluated, supporting parthenogenetic reproduction. This is the rst report of parthenogenesis in a Brazilian rainbow boa and has implications for the conservation of reptiles maintained in captive breeding programs. Zoo Biol. Biol. 32:172176, 2013. C 2012 2012Periodicals, Wiley Periodicals, 00:15, 2012. Wiley Inc. Inc.

Keywords: automixis; microsatellite; parentage; sperm storage; vasectomy

INTRODUCTION A 22-year-old captive Brazilian rainbow boa (Epicrates cenchria cenchria) gave birth to four snakes after being housed with a male rainbow boa which had been vasectomized 59 months prior. A single snake was still born. The remaining three snakes have continued to thrive during the 36 months since their birth. At the time of birth, four explanations were considered to elucidate the births; incomplete vasectomy, recanalization of the vas deferens, prolonged sperm storage, and parthenogenetic reproduction. To determine whether offspring were produced through sexual reproduction or parthenogenesis, DNA was isolated from the male, female, and all four offspring. Analysis of short tandem repeat (STR) markers demonstrated the offspring were the result of parthenogenetic cloning. Here, we explain how progeny production from incomplete vasectomy, recanalization of the vas deferens, and prolonged sperm storage were ruled out and provide evidence to support parthenogenetic reproduction.

MATERIALS AND METHODS Review of Specimen History The adult male and female Brazilian rainbow boas in this report were purchased at 3 months of age from a private facility and housed together in a zoological collection. These snakes were captive bred and since 3 months of age they have been housed as a pair with no other animals. The female snake gave birth to viable offspring four times prior to the males vasectomy. Multiple observations of copulation were noted before and after the males vasectomy while the pair remained in the same enclosure.
*Correspondence to: Matthew E. Kinney, Sacramento Zoo, 3930 West
Land Park Drive, to: Sacramento, CA 95822.Sacramento E-mail: mkinney@ucdavis. Correspondence Matthew E. Kinney, Zoo, 3930 West

edu Park Drive, Sacramento, CA 95822. E-mail: mkinney@ucdavis. Land edu Received 11 June 2012; Revised 11 September 2012; Accepted 21 September 2012 Received 11 June 2012; Revised 11 September 2012; Accepted 21 September 2012 DOI 10.1002/zoo.21050 Published online 19 October 2012 in Wiley Online Library DOI 10.1002/zoo.21050 (wileyonlinelibrary.com). Published online in Wiley Online Library (wileyonlinelibrary.com).

2012 Wiley Periodicals, Inc.

Parthenogenesis in a Brazilian Rainbow Boa 173

No DNA was collected from offspring produced prior to vasectomy. Twenty-seven months following vasectomy of the male, the female gave birth to two live snakes. DNA was not isolated from these two snakes. Offspring (three live and one stillborn) were born 59 months following vasectomy of the male. The three viable offspring were determined to be female using cloacal probing at 30 months of age. Briey, a probe was inserted caudally into the cloacal opening after sterile lubrication. In all snakes, the depth of the probe was equal to or less than three subcaudal scutes, which is consistent with probe depth previously described for female rainbow boas (E. cenchria) (three scutes) [Ross and Marzec, 1990]. The male snake was found dead in the enclosure 16 months after the birth of the four offspring. Gross necropsy and histopathology revealed the cause of death to be chronic cardiac disease. Histological examination of the vas deferens demonstrated brosis, complete occlusion, and cystic changes bilaterally. No evidence of vas deferens recanalization was found after examination of multiple tissue sections. Marked testicular atrophy and minimal spermatogenesis was present. DNA Isolation DNA was isolated from a whole blood sample obtained from the tail vein of the adult male and female snakes following the nucleated erythrocyte protocol, using DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA). A 3-mm segment of tail was collected from the stillborn snake and the rst shed skin was collected from the three viable offspring. A 1-cm segment of shed skin from each viable offspring was minced and used for DNA isolation. All tissue (one tail tip and three shed skin) samples from the four offspring were digested overnight following the tissue extraction protocol of the DNeasy Blood and Tissue kit. To remove any undigested scales, the digested tissue was centrifuged prior to application of the solution to the wash column. To compensate for low yield, DNA isolated from shed skin was concentrated prior to polymerase chain reaction (PCR) amplication using Genomic DNA Clean and Concentrator Kit (ZYMO Research Corp., Irvine, CA). STR Amplication Thirty-nine STR markers previously characterized in three genera of boids were used for analysis [Matson et al., 2001; Ramanana et al., 2009; Tzika et al., 2008]. Forward primers were modied with a 5 addition of a sequence specic to a dye-labeled primer. Using approximately 10 ng of template DNA for each sample, the following 20 l PCR reaction conditions were followed: 1 pmol of each dye-labeled primer and STR-specic reverse primer, 0.1 pmol each STR-specic forward primer, 2.0 mM dNTP, 2.0 mM MgCl2 , 0.5% BSA, 1X PCR buffer II, and 0.375 U AmpliTaq polymerase (Applied Biosys-

tems, Foster City, CA). Each STR was amplied in a separate reaction using the following temperature prole: 3-min incubation at 94 C followed by 35 cycles of denaturation at 94 C for 30 sec, annealing at 55 C for 20 sec, and extension at 72 C for 45 sec. The amplication was followed by a nal extension step of 30 min at 72 C to ensure that all products were full length. All PCR amplication was performed on a GeneAmp PCR Systems 9700 (Applied Biosystems). Products were individually size separated on an ABI 3730 DNA analyzer and exact fragment sizes were determined using STRand analysis software [Toonen and Hughes, 2001]. RESULTS Thirty-nine STRs were tested for analysis. Eighteen STRs failed to produce products or generated nonspecic products. Twelve were homozygous in all samples and therefore nondiagnostic. The alleles for STR marker sat 3 were uninformative for this male-female pairing and the male shared an allele with the female for sat 24 that did not segregate in the offspring. The remaining nine STRs had diagnostic differences between the two adults that would have been present in the offspring with proper segregation (Table 1). All offspring were homozygous for all STR loci, always an allele present in the female snake. In no instance did any offspring possess a diagnostic allele from the sire. Offspring 1 possessed four markers that displayed only the maternal allele that was not present in the male, offspring 2 had seven exclusionary markers, offspring 3 and the nonviable offspring each had three markers with only a maternal allele. DISCUSSION Surgical vasectomy was rst reported in snakes (two garter snakes (Thamnophis sirtalis radix) in 1979 [Zwart et al., 1979]. In all species, following identication of the vas deferens, the procedure is relatively simple and involves bilateral removal of a portion of the vas deferens with ligation or plugging of the remaining vas deferens. If anatomy is not correctly identied, inadvertent removal of non-vas deferens tissue is possible, which may result in continued male fertility. In this case, histopathologic examination of the tissue removed at the time of the vasectomy veried removal of two 34 mm segments of tissue composed of brous tissue with multiple proles of tubules lined by pseudostratied columnar epithelium surrounded by smooth muscles, consistent with vas deferens and epididymis. Vas deferens recanalization has not been reported in reptilian species. However, in humans postvasectomy recanalization has been well studied. In general, recanalization is extremely rare and may represent granuloma formation with multiple small proliferating epithelial lined channels containing spermatozoa [Haldar et al., 2000].
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TABLE 1. STR genotypes of potentially parthenogenic Brazilian rainbow boas STR sat 1 sat 3 sat 11 sat 24 55HDZ314 5HDZ277 55HDZ554 55HDZ602 55HDZ617 5HDZ229 5HDZ317 5HDZ360 5HDZ667 55HDZ11 55HDZ220 55HDZ302 55HDZ314 55HDZ339 55HDZ557 55HDZ56 55HDZ600 314 213 274 272A 184A 204 180 214A 218A 288 164 208 154 222 226 180 184 182 222 368 190 Male 318A 229 274 292 184A 208A 182A 214A 220 288 164 208 154 222 226 180 184 182 222 368 190 Female 314 213 274 292 183B 204 176B 204B 220 288 164 208 154 222 226 180 184 182 222 368 190 364B 229 278B 292 183B 210B 180 212B 222B 288 164 208 154 222 226 180 184 182 222 368 190 Offspring 1 314 213 274 292 183B 210B 176B 204B 220 288 164 208 154 222 226 180 184 182 222 368 190 314 213 274 292 183B 210B 176B 204B 220 288 164 208 154 222 226 180 184 182 222 368 190 Offspring 2 364B 213 278B 292 183B 210B 176B 204B 222B 288 164 208 154 222 226 180 184 182 222 368 190 364B 213 278B 292 183B 210B 176B 204B 222B 288 164 208 154 222 226 180 184 182 222 368 190 Offspring 3 314 229 274 292 183B 204 180 212B 222B 288 164 208 154 222 226 180 184 182 222 368 190 314 229 274 292 183B 204 180 212B 222B 288 164 208 154 222 226 180 184 182 222 368 190 Offspring 4 (dead) 364B 213 274 292 183B 204 180 212B 220 288 164 208 154 222 226 180 184 182 222 368 190 364B 213 274 292 183B 204 180 212B 220 288 164 208 154 222 226 180 184 182 222 368 190

Loci with no diagnostic value

Twenty-one STRs provided robust amplication products for analysis. Bolded types were diagnostically informative. A = female exclusionary alleles (alleles present only in the male), B = male exclusionary alleles (alleles present only in the female). STR = short tandem repeats.

In one study of 870 vasectomized men, 50 (5.7%) had at least one post vasectomy semen analysis that showed motile sperm, which the authors considered evidence of recanalization [Labrecque et al., 2003]. It is important to note that while early (<6 month) or late (>6 month) recanalization is a considerable risk, the pregnancy rate after vasectomy is about 1 in 2,000 (0.05%) likely due to reduced number of sperm in the ejaculate [Philp et al., 1984]. Upon the death of the potential sire in the case described in this report, recanalization of the vas deferens with subsequent male fertility was ruled out. Gross necropsy and histopathological evaluation concluded that the vas deferens was disrupted with adhesions (to the regional fat pads), brosis, and cystic changes bilaterally. The vas deferens were both occluded and testicular atrophy with minimal spermatogenesis was noted in both testicles. Therefore, recanalization of the vas deferens following vasectomy was eliminated as a possible mechanism of reproduction in the case reported here. Prolonged sperm storage has been documented in a number of snake species [Birkhead and Mller, 1993; Fox, 1976; Stewart, 1972]. Seven years and six months is the longest reported time period of suspected sperm storage in a snake [Magnusson, 1979]. However, in most reports of suspected prolonged sperm storage, parthenogenetic reproduction was not excluded as a mechanism for offspring production. A number of hypotheses have been proposed regarding the function of prolonged sperm storage includ-

ing; separation of reproductive events (copulation and fertilization) to optimize the timing in both sexes, insurance against not nding a partner (especially in species with a low likelihood of encountering members of the opposite sex), and enhanced opportunities for sperm competition, as sperm storage could extend the time period over which sperm from different males overlap in the reproductive tract [Birkhead and Mller, 1993]. In the present case, the male and female were housed in the same enclosure prior to vasectomy. Vasectomy was performed approximately ve years prior to birth of the four offspring making sperm storage unlikely. To denitively exclude prolonged sperm storage, genetic analysis was required to demonstrate the lack of male genetic contribution to the offspring. STR genotypes supported offspring parthenogenesis. There were two possible parents but no female exclusionary alleles (alleles present only in the potential sire) present in any of the offspring. The lack of the male only alleles and the observed allele homozygosity of the STR markers in the offspring support derivation from a parthenogenetic event. Parthenogenesis has been described in a variety of vertebrates including captive sh, amphibians, reptiles, birds, and mammals [Bartelmez and Riddle, 1924; Booth et al., 2011a, 2011b; Chapman et al., 2007; Feldheim et al., 2010; Groot et al., 2003; Kono et al., 2004; Lenk et al., 2005; Olsen and Marsden 1954; Robinson et al., 2011; Sarvella, 1973; Schut et al., 2008; Smith et al., 2000;

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Spurway, 1953; Watts et al., 2006]. A necessary requirement for viable parthenogenetic reproduction is the maintenance of ploidy levels of offspring. Three mechanisms have been described to maintain ploidy levels; apomixis, premeiotic doubling of chromosomes, and automixis [Lampert, 2008]. In the absence of mutations, apomixis and premeiotic doubling of chromosomes produces offspring genetically identical to the mother. In the present case, the heterozygous state of the female is not observed at any of the STR in the offspring, thus excluding apomixis and premeiotic doubling. Automixis can produce genetically variable offspring because of segregation and recombination of nonidentical homologous chromosomes. The degree of variability depends on the exact mechanism [Lampert, 2008]. Automixis is the only reported mechanism of parthenogenesis in reptilian species [Lampert, 2008]. Apomixis (oocyte production by mitosis) and premeiotic doubling (genome doubling prior to meiosis) in a Burmese python could not be excluded but was not proven [Groot et al., 2003]. In the ZW heterogametic sex determination system, automixis has the potential to result in male or female progeny. If automictic terminal fusion (fusion of the second polar body with the egg nucleus during meiosis) occurs, male (ZZ) or female (WW) progeny can result. Until recently, it was thought that female WW vertebrates were nonviable, however a recent report in a Boa Constrictor (Boa constrictor imperators) suggests WW females produced from terminal fusion automixis can be viable [Booth et al., 2011a]. In contrast to terminal fusion, central fusion (fusion of the rst polar body with the oocyte during meiosis) results in female (ZW) only progeny and maintains maternal heterozygosity [Lampert, 2008]. In the present study, the sex of all three viable offspring was determined to be female using cloacal probing. Offspring produced in this case report are either ZW or WW. Because maternal homozygosity is maintained in the offspring, automictic terminal fusion with production of WW progeny is considered most likely. Parthenogenesis, a rare event with unknown evolutionary signicance, has fascinated scientist for a number of years. To the authors knowledge, parthenogenetic reproduction in vertebrates has only been documented in captive populations with females isolated from males for an extended period of time. Recently, parthenogenesis has been identied as a possible problem for genetic management of Komodo dragons (Varanus komodoensis) [Watts et al., 2006]. Parthenogenisis has the potential to bias the sex ratio (depending on both the mechanism used to maintain ploidy levels and gametic sex determination system of the species) and instantaneously results in homozygosity of the genome, which results in an increased risk for lower overall tness [Watts et al., 2006]. Reproductive plasticity has been conrmed in Komodo dragons using genetic ngerprinting. In a single case, a female was documented to reproduce asexually through parthenogenesis and later sexually after the introduction of a male.

This observation led to the concern that housing female Komodo dragons in exhibits without males could induce parthenogenesis with potential negative impacts on the populations genetic pool. The present case is the rst to document parthenogenesis in the presence of a conspecic vasectomized male. This nding is signicant as it can no longer be assumed that the presence of a male prevents parthenogenesis in all species. Unfortunately, the offspring that were produced prior to vasectomy of the male are unavailable for genetic analysis and therefore we can not determine if this female reproduced parthenogenetic offspring while being housed with a sexually capable male. We are also unable to document reproductive plasticity, as the mechanism of reproduction (sexual vs. parthenogenic) for the offspring produced prior to the vasectomy of the male was not determined. The documentation of parthenogenesis in an individual housed with a vasectomized conspecic is important, as it has the potential to inuence housing decisions in species that have breeding recommendations to promote genetic diversity within a population. Genetic ngerprinting of offspring produced within populations of endangered species may be needed to maintain maximum genetic diversity as it may not be accurate to assume offspring produced by pairs are the result of sexual reproduction. CONCLUSIONS 1. Parthenogenesis, most likely as a result of automictic terminal fusion, was demonstrated for the rst time in the Brazilian rainbow boa (E. cenchria cenchria) through analysis of STR genotypes. 2. Incomplete vasectomy of the male cagemate, recanalization of the vas deferens, and sperm storage were excluded using histopathology and genetic analysis. 3. This report of parthenogenesis in an individual housed with a conspecic male, calls for more complete investigation into the triggers of parthenogenesis and the potential genetic consequences of parthenogenesis for captive populations. REFERENCES
Bartelmez GW, Riddle O. 1924. On parthenogenic cleavage and on the role of water adsorption on the ovum in the formation of the subgerminal cavity in the pigeons egg. Am J Anat 33:5766. Birkhead TR, Mller AP. 1993. Sexual selection and the temporal separation of reproductive events: sperm storage data from reptiles, birds, and mammals. Biol J Linn Soc 50:295311. Booth W, Johnson DH, Moore S, Schal C, Vargo EL. 2011a. Evidence for viable, non-clonal but fatherless Boa constrictors. Biol Lett 7:253 256. Booth W, Million L, Reynolds GR, Burghardt GM, Vargo EL, Schal C, Tzika AC, Schuett GW. 2011b. Consecutive virgin births in the new world boid snake, the Colombian Rainbow Boa, Epicrates maurus. J Heredity 102:759763. Chapman DD, Shivji MS, Louis E, Sommer J, Fletcher H, Prodohl P. 2007. Virgin birth in a hammerhead shark. Biol Lett 3:425427. Feldheim KA, Chapman DD, Sweet D, Fitzpatrick S, Prodohl PA, Shivji MS, Snowden B. 2010. Shark virgin birth produces multiple, viable offspring. J Heredity 101:374377.

Zoo Biology

176 Kinney et al.

Fox H. 1976. The urinogenital system of reptiles. In: Gans C, Parson TS, editors. Biology of reptiles, vol 6. London: Academic Press. p 1157. Groot TVM, Bruins E, Breeuwer JAJ. 2003. Molecular genetic evidence for parthenogenesis in the Burmese python, Python molurus bivittatus. Hereditary 90:130135. Haldar N, Cranston D, Turner E, MacKenzie I, Guillebaud J. 2000. How reliable is a vasectomy? Long-term follow-up of vasectomised men. Lancet 365:4344. Kono T, Obata Y, Wu Q, Niwa K, Ono Y, Yamamoto Y, Sung Park F, Seo J-S, Ogawa H. 2004. Birth of parthenogenetic mice that can develop to adulthood. Nature 428:860864. Labrecque M, Hoang DQ, Turcot L. 2003. Association between the length of the vas deferens excised during vasectomy and the risk of postvasectomy recanalization. Fertil Steril 79:10031007. Lampert KP. 2008. Facultative parthenogenesis in vertebrates: reproductive error or chance? Sex Dev 2:290301. Lenk P, Eidenmueller B, Stuadter H, Wicker R, Wink M. 2005. A parthenogenetic Varanus. Amph Rep 26:507514. Magnusson WE. 1979. Production of an embryo by an Acrochoruds javanicus isolated for years. Copeia 4:744745. Matson CW, Williamson JE, Huebinger RM, Louis Jr EE. 2001. Characterization of polymorphic microsatellite loci from the two endemic genera of Madagascan Boids, Acrantophis and Sanzinia. Mol Ecol Notes 1:4143. Olsen MW, Marsden SJ. 1954. Natural parthenogenesis in turkey eggs. Science 120:545546. Philp T, Guillebaud J, Budd D. 1984. Late failure of vasectomy after two documented analyses showing azoospermic semen. Brit Med J 289:7779. Ramanana MA, Bailey CA, Shore GD, Ramilijaona O, Brenneman RA, Louis Jr EE. 2009. Characterization of 20 microsatellite marker loci in the Malagasy Tree Boa (Sanzinia madagascariensis madagascariensis). Conserv Gen 10:19531956. Robinson DP, Baverstock W, Al-Jaru A, Hyland K, Khazanehdari KA. 2011. Annually recurring parthenogenesis in zebra shark Stegostoma fasciatum. J Fish Bio 79:13761382. Ross RA, Marzec G. 1990. Reproductive husbandry. In: Ross RA, Marzec G, editors. The reproductive husbandry of pythons and boas. Stanford, CA: The Institute for Herpetological Research. p 39 74. Sarvella P. 1973. Adult parthenogenetic chickens. Nature 243:171. Schut E, Hemmings N, Birkhead TR. 2008. Parthenogenesis in a passerine bird, the Zebra Finch Taeniopygia guttata. Ibis 150:197 199. Smith HM, Thiss ET, Chiszar D. 2000. Further observation on a merolepid (partially scaleless) water snake (Nerodia sipedon). Bull Maryland Herp Soc 36:914. Spurway H. 1953. Spontaneous parthenogenesis in sh. Nature 171:437. Stewart GR. 1972. An unusual record of sperm storage in a female garter snake (genus Thamnophis). Herpetologica 28:346347. Toonen RJ, Hughes S. 2001. Increased throughput for fragment analysis on an ABI Prism R 377 Automated Sequencer using a membrane comb and STRand software. BioTechniques 31:13201324. Tzika AC, Koenig S, Miller R, Garcia G, Remy C, Milinkovitch MC. 2008. Population structure of an endemic vulnerable species, the Jamaican boa (Epicrates subavus). Mol Ecol 17:53344. Watts PC, Buley KR, Sanderson S, Boardman CC, Gibson R. 2006. Parthenogenesis in Komodo dragonsshould males and females be kept together to avoid triggering virgin birth in these endangered species? Nature 444:10211022. Zwart P, Dorrestein GM, Stades FC, Broer BH. 1979. Vasectomy in the garter snake (Thamnophis sirtalis radix). J Zoo An Med 10:1721.

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