DEVELOPMENT OF THE ZFX / ZFY 5′-EXONUCLEASE ASSAY, A NEW TOOL FOR SEX DETERMINATION IN CETACEANS,

AND ITS APPLICATION TO SPERM WHALES IN THE GULF OF CALIFORNIA

Nadia T. Rubio-Cisneros1,2, Sarah L. Mesnick3 , Diane Gendron4, Ricardo Vazquez-Juarez1, Aviva Nestler5, Kelly Robertson3,

Nathalie Jaquet6 and Phillip A. Morin3

(1) Centro de Investigaciones Biológicas del Noroeste (CIBNOR), La Paz, BCS, México(2) Scripps Institution of Oceanography (SIO), La Jolla, CA, USA

(3) Southwest Fisheries Science Center (SWFSC), La Jolla, CA, USA (4) Centro Interdisciplinario de Ciencias Marinas (CICIMAR), La Paz, BCS, México

(5) Applied Biosystems, CA, USA(6) Provincetown Center for Coastal Studies, Provincetown, MA, USA

Development of the ZFX/ZFY 5′-exonuclease assay for sex determination in cetaceans.

Application of the ZFX/ZFY 5′-exonuclease assay to determine the sex of sloughed skin samples from sperm whales in the Gulf of California

PART 1.

PART 2.

INTRODUCTION

Why is sex determination in cetaceans important?

BiologyPopulation ecologyConservation

see Ruiz-Cooley et al. (2004); Croll et al. (2002)

INTRODUCTION

Problems with sex determination of cetacean species in the field

Many species do not have visible sexual dimorphism Genitalia are located internally Animals are at the surface for only brief periods of time Exceptions: sexually dimorphic species (but only adults)

Polymerase Chain Reaction (= PCR)

Allows the use of sex-specific sequences SRY and ZFX/ZFY Page et al. (1987), Sinclair et al. (1990), Schneider-Gadicke et al. (1989), Palsboll et al. (1992)

BACKGROUND: Polymerase Chain Reaction (PCR)

~311 bp KERATIN

~152 bp SRY

Agarose gel

100bp

Develop a Zfx/Zfy 5’-exonuclease assay with a small PCR product (105 bp) for sex determination in cetaceans

OBJECTIVE: Part 1 ZFX/ZFY 5’-exonuclease assay

● Universal primers for all cetacean species

● Equal product size for X and Y chromosome products for equal amplification efficiency

● Amplification of both X and Y chromosome products with the same PCR primers to equalize PCR efficiency

● Small product size for amplification of degraded DNA

● Simple fluorescent method for rapid sexing of samples

METHODS: Field ZFX/ZFY 5’-exonuclease assay

Samples●Southwest Fisheries Science Center tissue archive●Biopsy samples from live animals and strandings●33 different species samples

DNA extraction●DNEASY KIT (Quiagen)

Sample preservation●DMSO/NaCl (Amos & Hoelzel 1991)●Frozen @ -20º

Ensayo de la 5’ exonucleasa

• Morin et al. (1999 y 2004): sondas y primers

Clonación de regiones ZFX/ZFY en cetáceos• Nestler y Mesnick (2001)

MÉTODOS : LABORATORIO

Primers

Primers

sonda Taq man

Emisión de fluorescencia

5’exonucleasa

sonda Taq man

METHODS: Laboratory ZFX/ZFY 5’-exonuclease assay

•DNA•Sonda Taq man CY5, FAM•Primers•Reactivos

MÉTODOS : LABORATORIOMETHODS: Laboratory ZFX/ZFY 5’-exonuclease assay

Probe cleaved by 5’exonucleaseFluorescence is produced

SNP: single nucleotide polymorphism

Cumulative assay results (N=77)

-2000

3000

8000

13000

18000

-500 500 1500 2500 3500 4500

Y (CY5)

X (

FA

M)

NTC/ f ailed Female Male

RESULTS: ZFX/ZFY 5’-exonuclease assay sex determination

Plot of normalized fluorescence values (dRn) for a subset of the samples tested with the 5’-exonuclease assay, compiled from five different experiments.

3. Test the assay 1. Known male and female samples 2. Watch fluoresence values displayed in a computer monitor

METHODS : Optimization of the ZFX/ZFY 5’-exonuclease assay for sex determination

2. Real Time PCR: Optimization Tests 1. Probe and primer concentration 2. Primer concentration 3. Taq vs. Hotstart Taq 4. BSA (Bovine serum albumin) 5. Sensitivity to DNA concentration 6. Human DNA contamination 7. Assay replicability

1. Regular PCR Amplify 105-bp region

Real time PCR machine

The ZFX/ZFY 5’-exonuclease assay

determines sex in known ♂ and ♀ samples.

90 unique samples (116 PCRs)

82 successful amplifications

33 species (9 families)

RESULTS: ZFX/ZFY 5’-exonuclease assay sex determination in different cetacean species

The 9 FAMILIES

Balaenidae

Balaenopteridae

Delphinidae

Eschrichtidae

Kogiidae

Monodontidae

Phocoenidae

Physeteridae

Ziphiidae

Fam (X chromosome) standard curve for T. truncatus DNA

y = -3.2058x + 43.619R2 = 0.9899

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5

Log DNA amount (pico grams)

Ct

Cy-5 (Y chromosome) standard curve for T. truncatus DNA

y = -3.1138x + 43.683R2 = 0.9793

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5

Log DNA amount (pico grams)C

t

RESULTS: ZFX/ZFY 5’-exonuclease assay sensitivity to DNA concentration

Ct value: calculated fractional cycle at which the PCR product crosses a threshold of detection (Galluzzi et al., 2004) .

RESULTADOS: 2. PCR en Tiempo Real6. Contaminación con ADN humano.

Se obtuvieron resultados similares en los 60° y 64° C

Regular PCR sex determination

A unique protocol has not been proven in a large number of species.

Amplified products are >150 bp.

Less sensitivity in product detection.

Qualitative results.

Post-PCR process

ZFX/ZFY 5’-exonuclease assay

Rapid and efficient for 33 cetacean species.

105 bp. product.

Higher sensitivity in product detection,

useful different DNA quality samples.

Qualitative & quantitative results.

No post-PCR process

CONCLUSIONS: ZFX/ZFY 5’-exonuclease assay

Part 2.

APPLICATION OF THE ZFX/ZFY 5′-EXONUCLEASE ASSAY TO SLOUGHED

SKIN SAMPLES OF SPERM WHALES

FROM THE GULF OF CALIFORNIA.

INTRODUCTION: sperm whales in the Gulf of California

Gulf of CaliforniaJaquet and Gendron 2002

Sperm whale samples field seasons (1999-2004)

INTRODUCTION

Kasuya & Oshumi (1966): dorsal fin callus (N = 32 ♂ and 76 ♀)

sexual character: absent in mature ♂ occasionally in immature ♂ frequently in pregnant or lactating ♀

Clarke & Paliza (1994): dorsal fin callus (N = 1,473 ♂ and 1,204 ♀).

Cyclic phenomenon caused by estrogens and androgens.

Callus reported in ♂ and ♀ of different age classes.

INTRODUCTION: the dorsal fin “callus” in sperm whales

Application of the ZFX/ZFY 5′-exonuclease assay to sloughed skin

of sperm whale samples in the Gulf of California.

Investigate the sex of sperm whales that have callus information – report on preliminary findings.

OBJECTIVES: Part 2

Sightings● groups followed with acoustical probes, using a 8-13 m research boat.

● Groups followed for hours to days.

Data collection●Cluster composition●Age class of individuals in cluster: adult male, adult females, juveniles, calves●Photo Identification●Callus information

Sloughed skin sample collection●Dip net (30 × 20 cm; 1-mm mesh)

METHODS: Field investigation (Gulf of California)

PCRRESULTS: ZFX/ZFY 5’-exonuclease assay slough skin sex determination results

66 Sloughed skin samples collected from sightings of single

animals (1999-2004)

56 Amplified

10 Failed amplification

83% efficiency

2 days of laboratory work

216 Biopsies (Rubio-Cisneros et al. 2004; modification of Richard et al., 1996)

191 Amplifications

RESULTS: ZFX/ZFY 5’-exonuclease assay vs regular PCR sex determination techniques

66 Sloughed skin samples of single animals (this study)

56 Amplifications 83% efficiency 2 days of laboratory work

88% efficiency 3 months of laboratory work

RESULTS: Sex of individuals with the dorsal fin “callus”

56 samples with sex determination

16 samples with callus information

10 callus present 1 ♂ (juvenile) 9 ♀(8 adults, 1 juvenile)

6 callus absent 3 ♂ (adult male, adult, juvenile) 3 ♀ (adults)

CONCLUSIONS:

Application of the ZFX/ZFY 5′-exonuclease assay to sloughed skin ofsperm whale samples in the Gulf of California was: highly successful reliable less time consuming

The high resolution of the ZFX/ZFY 5′-exonuclease assay in sperm whale sloughed skin will allow:

Use of a non-invasive method for sampling tissue of free-living sperm whales

Testing of questions of biological and ecological significance for which sex is an important variable

CONACYTSWFSC Molecular Ecology LaboratoryCIBNORCICIMAR: Raul & FabiolaSCRIPPS