DNA in Amphibian and Reptile Venom Permits Access to Genomes Without Specimen Sacri fi ce

Amphibian defensive skin secretions and reptile venoms are rich sources of bioactive peptides with potential pharmacological/pharmaceutical applications. As amphibian and reptile populations are in rapid global decline, our research group has been developing analytical methods that permit generation of robust molecular data from non-invasive skin secretion samples and venom samples. While previously we have demonstrated that parallel proteome and venom gland transcriptome analyses can be performed on such samples, here we report the presence of DNA that facilitates the more widely-used applications of gene sequencing, such as molecular phylogenetics, in a non-invasive manner that circumvents specimen sacrifi ce. From this “surrogate” tissue, we acquired partial 12S and 16S rRNA gene sequences that are presented for illustration purposes. Thus from a single sample of amphibian skin secretion and reptile venom, robust and complementary proteome, transcriptome and genome data can be generated for applications in diverse scientifi c disciplines.


Introduction
On a global scale, amphibian and reptile populations have suffered widespread declines and extinctions in recent decades [1,2].While some declines are clearly due to factors including ultraviolet radiation, predation, habitat modifi cation, environmental acidity and toxicants, disease, changes in climate or weather patterns and interactions among these factors, in many others, the reasons are not clear [3,4,5,6].As part of the overall "biodiversity crisis", recent reports suggest that many amphibians and reptiles are undergoing range reduction and extinction, such as the Costa Rican golden toad (Bufo periglenes) and the CITES Appendix II protected helodermatid lizards (Heloderma suspectum and Heloderma horridum) [7,8,9,10].
Aside from the aesthetic value and functional roles these animals serve in their environs, they may offer properties of benefi t to human welfare, such as bioactive peptides.The structural diversity of polypeptides in amphibian skin granular gland and reptile venom gland probably refl ects a plethora of different roles, either in the regulation of physiological functions, defence against predators (amphibians) or in prey submission and capture (reptiles) [11,12,13,14].These venom and granular gland secretions are released following attack and stress (amphibians) or in striking prey (reptiles) and, in addition to polypeptides, they also contain various components (proteins, biogenic amines) resulting in a wide spectrum of bioactivities [15,16,17,18].Despite intensive study of frog skin peptides and lizard peptides for several decades, the vast majority remain uncharacterized and the vast majority of amphibian species remain to be studied.The extraordinary complexity and diversity of frog skin and lizard venom peptides renders intriguing resources for novel drug lead discovery-a fact that is particularly relevant at present as interest in peptides as therapeutics undergoes a renaissance in the pharmaceutical industry [19,20].In the past, the study of frog skin secretions and lizard venoms necessitated the sacrifi ce of the specimens and extraction of the dissected skin or venom gland, respectively.This procedure was highly destructive and often required several tens, hundreds or even thousands of specimens and the choice of chemical extraction medium was highly selective in terms of component solubility [15,21].The advent of the non-invasive mild transdermal electrical stimulation technique for frog skin secretion [22] and a modifi ed gentle acquisition technique for lizard venom [23,24], obviated the need for endangered specimen sacrifi ce and produced a more defi ned and molecularly complete product for proteomic analysis.However, molecular studies related to cloning of messenger RNAs encoding bioactive peptides required sacrifi ce of specimens and library construction from excised tissues [25,26,27,28].Recently, our research group has reported the presence of granular gland and venom gland transcriptomes in these secretions [29,30].Consistent with contemporary biological science ideology, we have further examined and modifi ed the technologies employed in the study of these secretion samples.
Here we describe the identifi cation and isolation of genomic DNA from such with original data presented on the partial structure of 12S and 16S ribosomal RNA genes from the skin secretion of the Chinese toad (Bombina maxima), and the venom of the Mexican beaded lizard (Heloderma horridum).As the technique is easily performed in the fi eld, ecological and ethical aspects of live animal research have been completely addressed.

Acquisition of toad and lizard venom
Two adult specimens of large-webbed bell toads, Bombina maxima, were obtained from a commercial source, housed in a vivarium under a 12 h/12 h light/dark cycle at 22 o C and fed multivitaminloaded crickets three times per week.Under these conditions, toads have remained in good health in excess of 4 years.Defensive skin secretions were obtained by the gentle electrical stimulation method and under these conditions, secretions were most pronounced from paired paratoid and tibial glands.Firstly, the skin surface was moistened with distilled water followed by three periods of transdermal electrical stimulation (5 V, 100 Hz, 140 ms pulse width), each of 10s duration [22].Skin secretions were washed from the skin with distilled water, snap-frozen in liquid nitrogen and lyophilized.
Adult captive specimens of the Mexican beaded lizard (Heloderma horridum) were housed in the Arizona-Sonora Desert Museum.After properly restraining the animal [23], venom was collected by inserting a plastic reinforced rubber hose into the mouth and then collecting venom in a glass beaker as it was expressed from the glands into the oral cavity via the bite pressure of the lizard [24].The beaker was then chilled and subsequently snap-frozen with liquid nitrogen prior to lyophilization.
Both techniques mentioned above caused no harm and minimal stress to the animals.As Heloderma horridum is a CITES Appendix II designate [31], all necessary permits for export and import of venom were obtained from both the United States Department of the Interior-Fish and Wildlife Service and the British Customs and Excise, in accordance with the terms of the international treaty.
Total genomic DNA for PCR was extracted from twenty milligrams of lyophilized lizard venom using a Wizard ® genomic DNA purifi cation kit (Promega Corporation, U.S.A).The Heloderma horridum ribosomal RNA genes were amplifi ed using a sense primer (S2-12, 5'-CAGAGAACTACGAGT-GAAAA-3') and an antisense primer (AS2-12, 5'-TTTACTACTAAATCCGCCTT-3') for the 12S fragment by thermostable polymerase (Invitrogen, U.S.A).These primers were complementary to a domain of nucleotide sequences in the 5' and 3' regions of the 12S ribosomal RNA genes (named HS-12S) from Heloderma suspectum (EMBL accession Nos.AF004474).The PCR cycling procedure was as follows: an initial denaturation step for 5min at 94 °C followed by 35cycles consisting of denaturation for 30s at 94 °C, primer annealing for 30s at 55 °C and fi nally extension for 5min at 72 °C.The resulting PCR products were purifi ed by a gel extraction system (Life and Technologies, U.K.) (Fig. 1C) and cloned using a pGEM-T vector system (Promega Corporation, U.S.A.) and sequenced using an ABI BigDye Terminator v3.1 cycle sequencing kit and an ABI 3100 automated DNA sequencer (Applied Biosystems, U.S.A).

Results
Approximately From Heloderma horridum, a novel nucleotide sequence of a 305-bp segment of the 12S rRNA gene was determined by the PCR-clone sequencing method from the venom-derived library (Fig. 1C).The novel 12S rRNA gene sequence has been CTTAAAACCCAAAGGACTTGGCGGTGCCCCACCCACCTAGAGGAGCCTGT BO-12S  deposited in the EMBL Nucleotide Sequence Database under the accession code AJ563605.Alignment of nucleotide sequences of Heloderma horridum 12S rRNA (HH-12S) and Heloderma suspectum 12S rRNA (HS-12S) (Fig. 4), using the AlignX programme of the Vector NTI Bioinformatics suite (Informax), revealed a very high degree of similarity between ribosomal RNAs.The NCBI-BLAST search found that HH-12S showed 94% nucleotide sequence identity with HS-12S.

Discussion
Molecular cloning of bioactive polypeptide mRNAs from amphibian skin or reptile venom gland provides much useful information on endogenous propolypeptide convertase specifi cities, co-encoded peptides and can be a vital step in the initiation of studies designed to map genomic organization of respective genes [16,20].Until now, whilst isolation and structural characterization of proteins/peptides could be achieved using lyophilized venom, construction of transcriptome cDNA and genomic DNA libraries for the purpose of molecular cloning of precursors and gene mapping, respectively, required the use of tissue samples that necessitated sacrifi ce of living specimens [25,26,27,28].Amphibians and reptiles are two groups of vertebrates that are suffering global declines due to a combination of factors, including habitat destruction and pollution on one hand and pathogenic viral and fungal diseases on the other [1,2].These factors however, do not explain all observed population crashes [6,32,33].The acquisition of a wide range of amphibian/reptile species for venom studies is not trivial and every effort has to be made to address the well-being of rare captive specimens.We were thus faced with the problem of obtaining structural information on skin/venom gland peptide cDNAs and genomic DNA without recourse to specimen sacrifi ce.To achieve this goal, we attempted to obtain this information by a novel and highly speculative route-cloning from the skin secretion or venom itself-essentially using the secretion or venom as a "surrogate" tissue for this purpose.
In initial studies performed on several different species, we unambiguously demonstrated that the skin or venom gland transcriptomes are present in stimulated defensive secretions or venoms as evidenced by the successful cloning and sequencing of an array of full-length polyadenylated mRNAs encoding venom gland secretion peptides [29,30].
In this study, we focused our attention to determine if genomic DNA was present in a PCR appropriate format in such lyophilized venoms.It was indeed found to be and approximately 50-250 ng of genomic DNA was present.We successfully obtained partial but expected (from primers employed) DNA nucleotide sequence of the mitochondrial gene for 12S rRNA and 16S rRNA from a single lyophilized sample of amphibian skin secretion and reptile venom.The skin secretion of large-webbed bell toads (Bombina maxima) and the venom of the Mexican beaded lizard (Heloderma horridum) from which the mitochondrial gene nucleotide sequences for 12S rRNA and 16S rRNA have not been reported, were employed as representative model systems.Ribosomal DNA (rDNA) sequences have been aligned and compared in a number of living organisms, and this molecule has been wellrecognized as an extremely robust, accurate and consistent marker for phylogenetic analyses and for species identifi cation [34].The reasons for the systematic versatility of rDNA include the numerous rates of evolution among different regions of rDNA (both among and within genes), the presence of many copies of most rDNA sequences per genome, and the pattern of concerted evolution that occurs among repeated copies.An interesting fi nding in the present study was the extremely high degree of nucleotide sequence conservation between the 12S rRNA of Bombina maxima and Bombina orientalis, with only nine sites of nucleotide substitution and one site of nucleotide deletion observed.However, in the case of 16S rRNA, twenty-six sites of nucleic acid substitution were observed.12S or 16S rRNA gene sequences have been applied to examine the phylogenetic relationships among higher taxa such as genera or families [35].Bombina orientalis, as it name suggests, is found in the Far East, specifi cally in North East China and Korea.However Bombina maxima, only occurs in a very discrete region of South West China.Bombina maxima and Bombina orientalis have very close phylogenetic relationships [36,37].For helodermatid lizards, herpetologists have long recognized that the two extant species, the Gila monster (Heloderma suspectum) and the Mexican beaded lizard (Heloderma horridum), have an extremely close relationship, as they are of restricted, essentially common global distribution, they are morphologically similar, they represent the two known venomous lizards of medical importance to humans and where they occur sympatrically, they hybridize [15,20,38].The high degree of nucleotide sequence conservation between the 12S rRNAs of Heloderma horridum and Heloderma suspectum found in this study, with only 16 sites of nucleotide substitution and 4 sites of nucleotide deletion, substantiates the previous assertion that Heloderma horridum and Heloderma suspectum have a very close phylogenetic relationship.Molecular genetic studies are being employed more frequently in the fi eld of taxonomy as adjuncts to the more classical approach of phenotypic characteristics, such as skeletal structure [39].The present data may thus provide amphibian and reptile systematists with an additional insight into the relationships of bombinid toads and helodermatid lizards.

Conclusions
To our knowledge, this study is the fi rst reported where a single sample of amphibian skin secretion and reptile venom has been shown to contain elements of the animal's genome that when amplified and sequenced, can provide useful information for a variety of applications such as in molecular taxonomy.This is of course, in addition to the global venom gland proteome and transcriptome.Such a fi nding offers a rapid, noninvasive and non-lethal approach to obtaining molecular genetic data in a manner that does not compromise scientific robustness and that additionally, offers the possibility of performing and serially-repeating such studies on the same individual specimens.The development of this technique and hopefully its adoption by researchers in the fi eld, is a signifi cant contribution to the evolving ethical framework regarding the usage of live animals for scientifi c research, especially those that are representative of threatened biodiversity.Thus the application of technologies such as described in the present study should preserve biodiversity for future biotechnological exploitation and systematic studies.

Figure 1 .
Figure 1.Gel electropherograms of PCR products generated using primers to (A) 12S rRNA, (B) 16S rRNA from genomic DNA libraries constructed from lyophilized skin secretion of Bombina maxima and (C) 12S rRNA from genomic DNA libraries constructed from lyophilized venom of Heloderma horridum.(D) Lane 1, 2 and 3 represent no template control for each PCR reaction.The left hand lane in each case contains a standard ladder of 100bp increment DNA fragments as annotated.