Genome size estimation in two populations of the Northern Chilean scallop, Argopecten purpuratus, using fluorescence image analysis

Journal of Shellfisheries Research, Jan, 2005 by Cristian Gallardo-Escarate, Josue Alvarez-Borrego, Elisabeth von Brand-Skopnik, Miguel Angel del Rio-Portilla

ABSTRACT Genome size (the C-value) is known to vary considerably among organisms, but is relatively constant among individuals of the same species. To determine the genome size in the Northern Chilean scallop Argopecten purpuratus, two populations were analyzed by measuring the fluorescence signal in hemocyte nuclei stained with DAPI. The fluorescence intensity was measured during a fluorescence-fading period by image analysis (fluorescence fading method). The area under the curve during a fading period was associated with the genome size, thus we determined the C-value in the Arica population as 1.057 [ or ] 0.057 pg of DNA and 1.139 [ or -] 0.066 pg of DNA for the Tongoy population. The interindividual variation was smaller that 6% for both populations. Variance analysis was performed to detect the effects of organisms and population in the genome size of A. purpuratus. Significant differences were detected between Arica and Tongoy populations (P = 0.0011). The genome size of Tongoy population was larger statistically compared with the Arica population. Several hypotheses are discussed about genome size variation.

KEY WORDS: genome size, Argopecten purpuratus, image analysis, fluorescence fading

INTRODUCTION

The Northern scallop Argopecten purpuratus (Lamarck, 1819) is a functional hermaphrodite inhabiting sedimentary substrates in sheltered bays along the Pacific Ocean from Paita, Peru (5[degrees] South) to Tongoy, Chile (30[degrees] South) (yon Brand-Skopnik & Ibarra-Humphries 2002). Since the 1980s, A. purpuratus has been cultured successfully and currently represents an important aquaculture product in Chile (Gajardo & Nunez 1992). Because of its economic importance in Northern Chile, several studies have been carried out to enhance the production of this scallop species (Navarro et al. 2000, Martinez & Perez 2003). Furthermore, the management reached in hatchery and larvae production has permitted to apply methodologies like ploidy manipulation to produce triploid organisms (Canello et al. 1992, Winkler et al. 1993) analyzed, using the chromosome count described by yon Brand et al. (1990). However, the knowledge about the genetic characteristics in populations of A. purpuratus along the Pacific coast is sparse (von Brand & Kijima 1990, yon Brand-Skopnik & Ibarra-Humphries 2002).

Genome size analyses are important to define genetic characteristics of a species and to estimate the ability of that species to undergo evolutionary changes. This type of analysis may be especially useful in biodiversity and conservation studies, as well as determination of ploidy levels in genetically manipulated species (Hinegardner 1974, Thorgaard et al. 1982, Komaru et al. 1988, Rodriguez-Juiz et al. 1995).

Diverse relationships on the haploid genome size (C-value) have been studied to determine the evolutionary role of the genome size (Gregory 2001 a). However, the genome size implication and its evolutionary role is a very complex topic, because of the variability of the genome sizes among eukaryotic species (Gregory 2002). The eukaryotic genome size varies more than 200,000-fold (Gregory 2001b). Nevertheless, whereas genome size varies significantly among species, the gene number varies considerably less.

Several methods have been used to quantify genome size. Densitometric techniques and flow cytometry have been used mainly because they allow to obtain estimations of genome size in many samples in a short time. However, the possible disadvantage of these techniques involves the Feulgen stain protocol in densitometry and the requirement of nuclei in suspension in flow cytometry. Recently, image analysis methods have been introduced in cytogenetic research (Uozu et al. 1997). Furthermore, we reported a new method for genome size estimation based on the fluorescent decay lifetime when the fluorochrome is exposed to excitation light. According to our results (Gallardo-Escarate et al. 2004), the DNA-fluorescent fading shows a relationship with the nuclear DNA content. To establish a fading unit we measured the fluorescence intensity during a blanching period using image analysis. The area under the curve (IF) was related with the genome size of several species. Our results showed that the IF in spermatozoa and red blood cell nuclei of Oreochromis mossambicus, spermatozoa of Haliotis rufescens, and red blood cell nuclei of Oncorhynchus mykiss showed a linear relationship with genome size reported for these species (r = 0.99), therefore we proposed a linear equation:

y = 3358.8x 801.82

to estimate other genome sizes. This work demonstrated the feasibility to estimate genome sizes by quantification of fluorescent fading.

In this study, we applied the method mentioned earlier for genome size estimation in two populations of the northern Chilean scallop Argopecten purpuratus.

MATERIALS AND METHODS

Biologic Material

Thirty organisms of Argopecten purpuratus were used to estimate the genome size in two scallop populations. Fifteen scallops were collected from a wild population in Arica (18[degrees]28'S) and 15 were collected from the cultured population in Tongoy Bay (30[degrees]15'S), both areas are located in the north of Chile. Shell length (SL), width (SW), height (SH) of each scallop were measured and SW/SL, SH/SL ratios were obtained.