Rev. Biol. Trop., 44(1): 153-157, 1996

The genome oC biolleyi (), a Costa rucan onychophoran

Marielos Mora!·2, Alvaro Herrera! and Pedro León! .3 I Centro de Investigación en Biología Celular y Molecular,'Facultad de Microbiología, 'Escuela de Medicina,Universidad de Costa Rica, San José, Costa Rica.

(Rec. 4-XI-1993. Rev. 23-IX-94. Acc. 14-XII-1994)

Abstraet: Cytologic studies of Epiperipatus biolleyi, a Costa Rican onychophoran, reveal !he presence of a large ge­ nome (C-value=4.3 pg/C) with many chromosomes, and a bimodal karyotype. Analysis of maje meiosis indicates lha! all chromosomes are rod-shaped, without distinct primary constrictions. Two large bivalents are consistently associa­ ted with nucleoli. Meiosis proceeds through the pachytene stage, with a few thick bivalents Ihat break up into many small and a few large chromosomes at the diplotene stage. At metaphase ¡he rod-shaped bivalents seem to be pulled from the telomeresinto opposite poles of !he spindle, lacking well defined kinetochores. DNA isolated from E. biolleyi presents a complex profiJe wi!h very low GC-content in isopycnic cesium gradients. Digestion with restriction enzy­ mes produces a homogenous spread of molecules, except wi!h Rae III, Hind m and Bgl n, which generate distinct bands of fluorescence. A Bgl n repeat of 1.9 Kb, has been cloned for evolutionary studies. Based on these results, many repetitive DNA familíes should be found in this genome.

Key words: Onychophorans, cytogenetics, evolution, C-value, repeated DNA familíes.

Cytogenetic studies have provided useful in­ me information. sights into the evolution of diverse groups of Here we present the first genomic study of a , since chromosome morphology and New World onychophoran, including the first DNA content are relatively conserved traits observations of maJe meiosis in this species within most species (Makino 1953; and the first C-value determination in the phy­ White 1977; Achar 1987; Alegria and León, lum. In E. biolleyi we find a large genome, 1991).Very Httle research has been done on very A+T rich, with a complex karyotype. onychophoran genomes, probably because Much more work is needed on onychophorans these invertebrates are rare, secretive animals. in general to establish trends in chromosome The only modern study on onychophoran cyto­ evolution, and with American species, in parti­ genetics, so far, was recentIy reported on Aus­ cular, to determine if the karyotype of E. bio­ tralian onychophorans from the family Peripa­ lleyi is representative of the whole genus and topsidae (Rowell et al. 1995). Peripatopsids similar to other genera in the New World. present a range of chrornosome numbers 2N=18 to 42. Early work by Montgomery MATERIAL AND METHODS (1900) with tissue sectioning techniques of spermatocytes from peripatopsids provide the Animais: During the last ten years we ha­ only information 011 chromosomes from Afri­ ve been studying a population of E. biolleyi can species. Montgomery reported a karyotype from Las Nubes de Cascajal, near San José, with N=14 chromosomes, but tissue sections Costa Rica, found almost exclusively on moss arenotoriously unreliable sources of chromoso- embankments along rural roads. The animals 154 REVISTA DE BIOLOGIA TROPICAL

can be kept in humid moss terraria for long pe­ independent determination of the buoyant den­ riods of time, feeding on small insects. Further sity. Standard used was DNA from phage C2 details on the habitat of this population and on (1.742 g/cm3) • the species are offered in Monge-Nájera et al. (1993) and Monge-Nájera and Brenes (1994). Digestion witb restriction enzymes: Diges­ tions were done using the conditions described Chromosoine preparations: Meiotic chro­ by distributors of the restriction enzymes BgI mosomes were observed in testicular squashes, TI, Alu 1, Bam HI, Eco RI, Hae nI, Hind nI, Pst with material fixed in 3:1 (ethanol:acetic acid) 1 and Sma 1, al! obtained from Bethesda Re­ and treated as described elsewhere (León and search Labs., Bethesda, MA. USA. After over­ Kezer 1978, Macgregor and Varley 1983). A 2 night digestion with a lO-foId excess of enzy­ min hypotonic treatment with distilled water me (10 units/ug), DNA samples were analyzed was attempted without useful results. in 1-2% agarose gels, stained with ethidium Preparations were stained with Giemsa and bromide and photographed with Polaroid film photographed with Panatomic-X film using a 667 with an ultraviolet transilluminator (Sam­ Zeiss microscope (MC63A). brook et al. 1989).

Determination of DNA content: Feulgen Cloning repetitive sequences: Bands were reagent was used to stain endolymph smears fi­ excised from gels with a razor blade and fro­ xed in methanol-acetic acid as described by zen. To obtain clonable sequences the frozen Macgregor and Varley (1983). Human lympho­ bands were squeezed and the DNA solution cytes included in the same slides were used as was clarífied by centrifugatíon to eliminate standard. Qne hundred cells of each species small pieces of agarose (Sambrook et al. 1989). were measured in a Reickert integrating micro­ A 1.9 Kb sequence produced by BgI n diges­ densitometer and the average value was used to tion was cloned into pUC13 previously lineari­ estimate C-value (DNA contentlhaploid nucle­ zed with Bam HI, as described by Sambrook et us), assuming 3,4 pg for the human C-value. al (1989). Twenty recombinant colonies (Viei­ ra and Messing 1982) were tested for inserts of Isolation of DNA: DNA was isolated from the appropriate size, after growing ovemight in carcasses frozen in liquid nitrogen, as described 10 mI LB-broth cultures containing ampicillin in detail elsewhere (Mora 1991). Briefly, pow­ or carbenicillin (60 ug/ml). T4 DNA ligase was dered carcasses were resuspended in STE (0.01 used for the ligation reactions at lSoC. Host M Tris [pH=8.5]; 0.1 M NaCl,; 0.02 M ED­ cells used were JMI03, a strain capable of 0:­ TA); sodium dodecyI sulfate (to a final concen­ complementation (Sambrook et al. 1989). tration of 1 %) and proteinase K (100 ug/ml, fi­ nal concentration) were added. After overnight digestion at 530 C, the solution was extracted RESULTS with phenol and chloroform as usual (Sam­ brook et al. 1989), and the DNA in the aqueous Genomic DNA studies: Genomic DNA phase was precipitated with two volumes of from E. biolleyi is very A+T fich (70% A+T), ethanol and washed twice with 70% ethanol. with a complex profile containing several dis­ tinct components on the light and heavy side of Isopycnic gradients: DNA was dissolved in the main band in the isopycnic gradients (Fig. TE buffer (O.OlM Tris-CI [pH=7.4], 1 mM ED­ 1). The main band peak is close to 30% G+C, TA), mixed with concentrated solutions of based on the refractive index of the peak frac­ CsCl to a final concentratíon of 1.7 g/mI, de­ tions, and on the analytical ultracentrifugation termined by measuring the refractive index results. There is at least one very large satellite with a Bausch and Lomb refractometer. Centri­ component on the heavy side of the gradient fugatíon and fractionation of the gradients was (Fig. 1). Digestion of DNA with a lO-fold ex­ done as described elsewhere (León and Gámez cess of restriction enzyme usually produces dif­ 1981). Gabriel Macaya kindly analyzed this fuse fluorescence along gel Janes, after staining DNA preparation in an analytical ultracentrifu­ with ethidium bromide. However, three enzy­ ge at the University of París VII, to obtain an mes- BgI n, Hae nI, and Hind 1lI- out of eight MORA et al.: The genome of Epiperipatus biolleyi 155

the same slide as reference. E. biolleyi cells consistently had higher UV absorbance values. We estimated that E. biolleyi diploid nuclei contain about 8.6 pg of DNA, consequently the C-value is about 4.3 pg (compared to 3.4 pg in haploid human cells). Consistent with tbis high C-value, E. biolle­ yi presents many chromosomes in metaphase cells, with a wide range of sizes, from very small members that could be considered mi­ crochromosomes, to intermediate and large size

Fig. 1. DNA from E. biolleyi centrifuged to equilibrium in members of the set (Fig. 2A). A striking obser­ ese\. The top of the gradient is lo the right. The peak of vation is the absence of primary constrictions at absorbance has a Ge-content of 30%. metaphase or during other stages of meiosis in these chromosomes. In testicular squashes, pachytene stage cells are abundant, containing that were tested were found to generate repeti­ a few tbick "bivalents" (Fig. 2B and C). These tious sequences and produce distinctly visible structures break up into individual chromoso­ bands over the diffuse background. We have mes as diplotene stage begins. lt appears that cloned the 1.9 Kb. Bgl n digestion product, in­ several different chromosomes are packed into to the pUC13 vector. Of twenty white colonies each of these individual structures, con se­ tested, two had 1.9 Kb inserts. These were de­ quently, these are not pachytene bivalents in signated pEb1 and pEM. According to prelimi­ the traditional sense (White 1977, Kezer and nary tests with restriction enzymes and sequen­ Macgregor 1973). Nucleoli are often observed cing studies (M. Mora, unpublished), both clo­ attached to two large chromosomes in pachyte­ nes have identical sequences. The sequence is ne cells (Fig. 2B, arrows). We have counted the AT-rich and contains restriction sites for Eco number of chromosomes in 10 metaphase pla­ RI, Hind In and Bam ID (Mora 1991). tes, with number ranging in diplotene and me­ taphase cells between N= 28 to N=32. The dis­ Cytologic and cytophotometric studies: crepancies are due to variation in thenumber of The average fluorescence of 100 Feulgen-stai­ small chromosomes. We cannot offer a definiti­ ned endolymph cells measured with a micros­ ve karyotype for this species, without primary pectrophotometer, was used to estimate DNA constrictions to define arm ratios and without content, using 100 human white blood cells on well defined G-bands.

Fig. 2. Spermatocyte squashes of E. biolleyi stained with Giemsa and photographed under the 40x objective lens fOI a final magnification of x800. A metaphase 1 ceJl is shown in (A) with 2N=60. In (8) a late pachytene cell is shown, displaying two nucleoli attached to the ends of two large bivaIents (arrows). An early pachytene is observed in the rniddle of (e), with a lep­ totene cell to the right of it. 156 REVISTA DE BIOLOGlA TROPICAL

DISCUSSION trophoresis of the digested products and clo­ ning in small vectors. If repetitive families are A vást literature is available on the chromo­ present and have a particular restriction site, somes of insects, myriapods and annelids bands can be easily observed over the diffuse (White 1977, Achar and Chowdaiah 1980, fluorescent background in each lane of the gel. Achar 1986, 1987, Colmagro et al. 1986, Sing­ Digestion of E. biol1eyi DNA with restriction hal et al. 1986, Fischer 1987). Comparisons of enzymes has again disclosed the existence of these chromosomes with the E. biolleyi kar­ repetitive DNA families that we have cloned yotype describedhere, revea1 morphological si­ for phylogenetic studies. milarities with chilopods, in which many spe­ cies lack primary constrictions and have similar bimodal karyotypes. For instance, the primitive RESUMEN chilopods in the genus Lithobius (Achar 1984, 1986; Colmagro et al. 1986) have many chro­ Estudios citológicos del onicóforo costarri­ mosomes with small, medium and large mem­ cense Epiperipatus biolleyi demostraron la pre­ bers in the seto similar to E. biolleyi. The more sencia de un genoma grande (valor C= 4.3 derived diplopods have fewer, more homoge­ pglC) conmuchos cromosomas en un cariotipo nous chromosomes (2N=12 to 2N=30). The bimodal. El análisis de la meiosis en machos few pauropods that have been studied have indica que todos los cromosomas tienen forma chromosomes that vary in number from homogénea, sin constricciones primarias, aun­ 2N=12 to 2N=28. Annelid chromosomes, ho­ que dos de estos presentan constricciones se­ wever, are also variable in number, particu­ cundarias asociadas a nucleolos. Durante el pa­ larly in polychaetes (Vitturi et al 1984), alt­ quitene de la meiosis aparecen todos los biva­ hough they show little resemblance, otherwi­ lentes asociados entre sí en cilindros gruesos se, with the E. biolleyi chromosomes. Alto­ que al entrar en diplotene se desinregran para gether, the greatest resemblance that we have dar origen a muchos cromosomas pequeños y encountered is with the Chilopoda. Clearly, a algunos cromosomas grandes. Durante la meta­ single karyotype is of limited value for phylo­ fase meiótica los cromosomas aparentemente genetic comparisons, until more is known son separados a polos opuestos, sin que pueda about karyologic trends in the family Peripa­ observarse un cinetocoro bien definido. El tidae and in the whole phylum . ADN de esta especie presenta un complejo per­ It is noteworthy that chilopods and onychop­ fil con un alto contenido A+T, en gradientes horans also present similar embryologic pro­ isopícnicos de cloruro de cesio. La digestión perties (Anderson, 1973) and other evolutio­ con enzimas de restricción produce una distri­ nary similarities (Ballard et al, 1992; Emerson bución homogénea de moléculas, excepto and Schram, 1990;Tiegs and Manton, 1958;). cuando se utilizan las enzimas Hae ID,Hind ID Peripatopsids present a range of chromoso­ y Bgl n, las cuales generan bandas bien defini­ me numbers 2N=18 to 42 (Rowell et al. 1992). das, por la presencia de secuencias repetidas. No morphological data were presented and the Una de estas secuencias de 1.98 Kb producida position of centromeres was not reported. con la enzima Bgl II fue clonada en un plásmi­ Montgomery's (1900) initial report described a do bacteriano para estudios evolutivos. Con ba­ karyotype with N=14 chromosomes, but as al­ se en estos resultados esperamos encontrar mu­ ready indicated, tissue sections are unreliable chas familias de ADN repetitivo en este orga­ sources of chromosome information. His came­ nismo. ra lucida drawings, however, show chromoso­ mes without well defined primary constric­ tions, as we have found. ACKNOWLEDGEMENTS A shotgun approach to the study of repetiti­ ve families has been previously applied to ge­ We are grateful with the Castillo family of Las nomic studies in vertebrate (Alegría and León Nubes de Cascajal for their field support. We also 1991). This approach consists of digesting total thank the Villalobos family from Finca La Dibu­ DNA with a battery of restriction endonuclea­ jada for providing specimens for this research. ses, and searching for distinct bands after elec- We thank Bernal Morera for identification of MORA et al.: The genome of Epiperipatus biolleyi 157

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