with known cytometric DNA content, were used as reference cells. The blood ... Flow cytometric analysis was performed on blood ..... eagle and the gold eagle.
Cytometry 16:346-350 (1994)
G 1994 Wiley-Liss, Inc.
Evaluation of Interspecific DNA Content Variations and Sex Identification in Fakoniformes and Strigiformes by Flow Cytometric Analysis R. De Vita', D. Cavallo, P. Eleuteri, and G. Dell'Omo Environmental Biomedicine Division, ENEA Casaccia, and Section of Behavioral Pathophysiology, Lab F.O.S. Istituto Superiore di Sanita, Rome, Italy Received for publication August 17, 1993; accepted February 15, 1994
A high interspecific karyotype vari- DNA mass in picograms (pg) with respect ability has been evidenced in birds espe- to the standard value of 7.0 pg per human cially in Falconiformes and Strigiformes. lymphocyte nucleus. The results obAvian cytogenetic analysis, convention- tained showed an interspecific variabilally used for this study, presents several ity of DNA content and evidenced the difficulties. We used flow cytometric usefulness of FCM analysis as a rapid analysis in order to obtain further infor- and easy tool for studying the DNA patmation on the DNA patterns of different tern of different species of birds. Morespecies of birds belonging to the above- over, our results have confirmed and mentioned orders. Our study was per- extended the possibility of sex identificaformed on blood samples while chicken tion in species of birds characterized by erythrocytes and human lymphocytes, sexual monomorphism by evaluating the with known cytometric DNA content, small DNA content difference which exwere used as reference cells. The blood ists between males and females. samples of the birds under study were 0 1994 Wiley-Liss, Inc. stained, simultaneously to the reference cell, with a lysis-staining buffer containing propidium iodide. The nuclear DNA Key terms: DNA content, sex identificacontent of the bird samples was calcu- tion, birds of prey, Falconiformes, Strigilated as DNA index in relation to refer- formes, flow cytometry, avian erythroence cells, and was expressed as nuclear cytes
Nuclear DNA content determination in eukaryotic organisms is important for its evolutionary significance. While many studies are presently available on karyotype and nuclear DNA content for mammals, less data are available for other vertebrates. For birds, the few studies available, mainly regarding karyotype constitution, have revealed a high interspecific variability, especially evident in the orders of Falconiformes and Strigiformes. This variability essentially involves the 2n chromosome number and DNA content, and could be related to a n evolutionary process which is in agreement with several evolutionistic models (1,7,19). Avian cytogenetic analysis, conventionally used for studying the genome, presents several difficulties including the scarceness of karyological data, the presence of microchromosomes (characteristic of birds), and a high number of chromosomes (2). For these reasons, we deemed it of interest to investigate the nuclear
DNA content of several species of Falconiformes and Strigiformes, with the aim of obtaining further information on the DNA patterns of the different species belonging to each of the two orders. We performed our study on blood avian samples taken from some species of Falconiformes (Accipitridae and Falconidae families) and Strigiformes (Strigidae and Tytonidae) using Flow Cytometric (FCM) analysis. This technique allows rapid and accurate evaluation of cellular DNA content in thousands of interphase cells for each measurement and is advantageous with respect to both cytogenetics and static cytometry. In this study particularly, it is
'Address reprint requests to R.De Vita, Environmental Biomedicine Division, ENEA Casaccia, Via Anguillarese 301, 1-00060 Rome, Italy.
DNA VARIATIONS AND SEXING I N BIRDS BY F’CM
necessary to perform high-resolution FCM analysis to detect the small DNA content differences present among the species of same family and, especially, in three cases, among species of the same genus. The same approach could be used to monitor the small DNA content difference present between male and female birds with the aim of defining a sexing method as a n alternative to those presently in use. This is particularly important for the species characterized by sexual monomorphism.
347
Flow Cytometry The samples were analyzed by a PAS I1 flow cytometer (Partec, Munster, Germany). Excitation wavelengths around 488 nm of a 100 W mercury arc lamp were selected by a n FITC 488 EX filter. A TK 530 nm long-pass barrier filter, a 560 nm dichroic filter, and a n RG 610 step filter were placed before the photomultiplier tube. A dichroic mirror was used to separate the incident from emission light. Signals were collected and accumulated on a 512-channel memory. The quality and accumulation of the events were checked using a n MATERIALS AND METHODS oscilloscope and by display on video screen. A total of a t Samples least 2.104events was accumulated for each histogram. Flow cytometric analysis was performed on blood The histograms were recorded on floppy disks and ansamples taken from 45 birds of prey (38 Falconiformes) alyzed using the PASIFLOW software (Partec) to evaland (7 Strigiformes) kept by Legambiente Lazio in the uate the DNA Index (D.I.), the coefficient of variation “Centro per lo Studio ed il recupero dell’Avifauna,” (CV), and the cellular frequencies. The CV of the fluorescence distributions from stained avian erythrocytes Rome, and by Parco-Zoo del Garda, Verona, Italy Falconiformes. Specimens of the following species and human lymphocyte cells ranged from 1.0-3.0%. were analyzed: Family Accipitridae: Buteo buteo (5 These CV values were obtained by setting the flow cases); Neophron pernocterus (16 cases) which included cytometer to have the GI,, modal value of human lym9 subspecies pernocterus and 7 subspecies ginginianus, phocytes peak at channel 100 and the internal standard Aegypius monachus (1 case); Circus aeruginosus (3 (chicken erythrocytes) at channel 35. These setting concases); Circus cyaneus (1 case); Circus pygargus (1 ditions showed the best results in terms of histogram case); Milvus migrans (1 case); Pernis apivorus (2 quality. For each specimen a t least six measurements cases). Family Falconidae: Falco vespertinus (4cases); were performed to assess the reproducibility of results. Falco tinnunculus (3 cases); Falco peregrinus (1 case). The nuclear DNA content of the bird samples was calStrigiformes. Specimens of the following species culated as DNA Index value, which represents the ratio were analyzed: Family Strigidae: Strix aluco (3 cases); of GI,, modal value of avian cells to that of human Athene noctua (2 cases). Family Tytonidae: Tyto alba (2 reference cells, according to the formula D.I. = (XIS,) x (SBIH)where X is the fractional mode channel of the cases). avian samples under study, S, is the fractional mode For each case a few microliters of blood were colchannel of the internal reference cells (chicken erythlected by wing vein puncture and directly dropped into several milliliters of phosphate buffer. The cellular rocytes) in the X-SA mixture, and SBis the fractional suspension was then stored at + 4°C. With this proce- mode channel of the internal reference cells (chicken dure, the blood cells remain suitable for analysis for erythrocytes) in SB-H mixture with H as the fractional almost 4 weeks. Male chicken erythrocytes and male mode channel of human male lymphocytes. The DNA content was expressed as mass relative to human lymphocytes separated from peripheral blood, with known cytometric DNA content, were used a s ref- a standard value of 7.0 pg per human male lymphocyte nucleus according to the formula pg DNA = 7.0 x erence cells. (XIS,) x (SBIH), where X, SA,SB, and H have been previously described. Flow Cytometric Staining The technique of Krishan (ll),with modifications RESULTS (15), was used for DNA staining. Reference cells and In all forty-five cases analyzed, evaluable histograms blood cells from birds under study were suspended in 1 ml of lysis-staining buffer which consisted of 0.1% so- were obtained (Fig. 1). The reproducibility of the meadium citrate, 0.1% Triton X 100, and 50 pgIml propid- surements demonstrated a good reliability of the ium iodide. Fifty microliters of RNase (1 mgiml) were method employed. In fact, the values obtained in the added just prior to the addition of cells. The stained six different measurements from the same specimen suspension was then filtered trough a 53-pm nylon did not evidence significant differences, with a mean mesh, kept a t +4“C and in the dark, and analyzed standard deviation (S.D.) value of 0.03. For each case, within 15 min. For each specimen, male chicken eryth- the mean value of the six measurements was considrocytes were used as internal reference cells and ana- ered. The nuclear DNA content was expressed as pgI lyzed simultaneously to erythrocytes from the birds nucleus. This value was expressed for each species as studied. Moreover, a mixture of male chicken erythro- the mean value of the analyzed cases k S.D. The recytes and male human lymphocytes was analyzed to sults obtained from the different species of Falconicalibrate the flow cytometer before each series of mea- formes are shown in Table 1. The data relative to Strigiformes are reported in Table 2. surements.
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L)E VITA ET AL.
Table 1 Nuclear D N A Content of Some Species of Falconiformes
A Family Species Subspecies Accipitridae Circus cyaneus Circus aeruginosus Circus pygargus Miluus migrans Pernis apiuorus Buteo buteo Aegypius monachus gingintanus Neophron pernocterus pernocterus Falconidae Falco uespertinus Falco tinnunculus Falco peregrinus Internal reference Gallus domesticus
No. of cases
DNA content (pginucleus; means S.D.)
1 3 1 1 2 5 1 7 9
2.84 2.86 t 0.03 2.91 2.93 3.05 t 0.02 3.06 t 0.01 3.12 3.17 ? 0.09 3.20 t 0.09
4 3 1
2.93 0.05 3.08 t 0.03 2.89
8
2.50 t 0.07
Table 2 Nuclear D N A Content of Some Species
B
Familv SDecies Strigidae Strix aluco Athene noctua Tytonidae Tyto alba Internal reference Gallus domesticus
of
*
*
Strigiformes
Common name
No. of cases
DNA content (pginucleus; means ? S.D.)
Tawnyowl Little owl
3 2
3.18 0.06 3.34 i 0.04
Barn owl
2
3.45
2
0.01
8
2.50
?
0.07
*
content difference between the two sexes with DNA content of females being less than that of males. The differences ranged from 1.0 to 5.8%among the six pairs of Falconiformes and were 0.6 and 3.2% for the two pairs of Strigiformes analyzed. These compare with 2.4% for Gallus domesticus which were used as the internal reference. FIG. 1. Flow cytometric DNA content dlstributions of domestic chicken internal reference cells (first peak) together with erythrocyt?s of each studied species (second peak) in Strigiformes. A. Tawny owl; B: barn owl.
This analytical approach was applied also to evaluate the possibility of discriminating, by FCM analysis, the small DNA content difference present in birds between the heterogametic (ZW) female and the homogametic (ZZ) male with W smaller than Z. Among the birds analyzed, we examined the results obtained from 8 pairs of seven different species for which the sex was known. For each specimen the mean of six repeated measurements -t S.D. values were reported (Table 3). Our approach evidenced a small but consistent DNA
DISCUSSION The importance of genome size as well as chromosome complement, as a fundamental characteristic of a species, has become apparent in recent years. Whereas several studies are available on the karyotype constitution of birds, very few data are actually present on genome size. These latter evidence a n evolutionary significance of the specific amount of nuclear DNA and, in particular, the independence of chromosome number among related species, its relative constancy in many natural groups of animals with no evident trend in the evolution of the major groups, and the tendency of advanced or specialized forms to have genomes smaller than ancestral forms (1,8,18).These observations suggest that the genome size is under evolutionary control.
DNA VARIATIONS AND SEXING IN BIRDS BY FCM
349
Table 3 Nuclear D N A Content of Males US.Females of Some Species of Falconiformes and Strigiformes
DNA content (pginucleus; means of repeated measurements S.D.)
*
Order Species Falconiformes Circus aeruginosus Buteo b uteo Neophron pernocterus pernocterus Neophron pernocterus ginginianus Falco vespertinus Falco tinnunculus Strigiformes Strix aluco Tyto alba Internal reference Gallus domesticus
Male
Female
O/c
difference male vs. female
2.88 1 0 . 0 3 3.07 0.02 3.29 ? 0.05 3.28 i 0.04 2.99 k 0.05 3.12 t 0.04
2.82 3.04 3.12 3.09 2.87 3.07
t 0.04
2.1 1.0 5.2 5.8 4.0 1.6
3.21 i_ 0.03 3.46 t 0.01
3.11 2 0.08 3.44 k 0.05
3.2 0.6
2.50 i 0.02
2.44 2 0.03
2.4
*
The results obtained in this study on bird genome size by FCM analysis extend those reported in another study that use the same analytical approach to investigate the relation between genome size and taxonomic groups in birds (18). In particular our study showed interspecies DNA content differences among the studied cases, especially evident in the Falconiformes, confirming data in the literature. In fact, previous studies with cytogenetic analysis (3,4,14) and with static cytometry (19) have revealed substantial difference, in genome constitution, particularly among the species belonging to the Falconiforrnes and, within the Strigiformes, between the two families of Strigidae and T y tonidae. Such variability involves both the chromosome diploid number (2x1) and the nuclear DNA content (pginucleus). Cytogenetic analysis within Falconiformes has shown a karyotype variability in the family of the Accipitridae with the chromosome diploid number (2n) ranging from 62 to 78 and in that of Falconidae with 2n in the range 48-52 for subfamily falconinae and 84 for poliborinae (3). Within the Strigiformes, the variability is evident between the two families of Strigidae and Tytonidae with chromosome diploid numbers of 2n = 82 and 2n = 92 (2,141, respectively. The data furnished by static cytornetry has shown a large range of DNA content with low and moderate DNA values in Falconiformes, ranging from 3.08 to 4.5 pg per nucleus, and a smaller range with moderate and high DNA values in Strigiformes, ranging from 3.92 to 4.97 pginucleus (19). A comparison of our data and the values reported by Venturini e t al. (19) for a few species examined in both studies, has evidenced, in our results, lower DNA content values. However, in terms of relative DNA content values, the results obtained in the two studies show a similar trend, with low and moderate values in Falconiformes and moderate and high values in Strigiformes. The different value ranges observed in the two studies are probably due to the different species
2 0.04 2 0.04 I 0.03
i 0.01 k
0.03
examined and to the different methods of investigation. The presence of a large range for genome size in the Falconiformes could mean, according to Hinegardner’s ( 8 )model, that this order is in a n evolutionary phase in which, after a n initial increase of genome size, the unused DNA begins to be lost. On the other hand, the different findings for Strigiformes, with a moderate range and high DNA values, could be explained by the fact that this order could be in a n evolutionary phase immediately after the genome size increase and before the DNA loss due to specialization (19). The results obtained in this study demonstrate the utility of FCM analysis as a rapid, simple, and statistically reliable technique in characterizing different species of birds on the basis of nuclear DNA content, employing only a few microliters of blood for each specimen. In fact, this technique makes it possible to perform several measurements of different specimens in one day, unlike either cytogenetic analysis which requires time for culturing and metaphase scoring, or static cytometry which requires longer staining and measuring times. Another important advantage of FCM is that a large number of measurements, each of which evaluates almost %lo4cells, can be done by taking only a few microliters of blood from each animal. Other studies have evidenced the possibility of estimating, by FCM, nuclear DNA mass in species belonging to the different vertebrate classes in relation to reference species with known cytometric DNA content (9,12,15). In this study, in particular, it was necessary to perform high-resolution FCM analysis. In fact, the possibility of detecting small DNA content differences using FCM analysis mainly depends on the quality of sample preparation and the efficiency of the cytometric equipment (5,20). In our study, carefully controlled and standardized conditions of sample preparation, staining and measurement were established. Our results have confirmed and extended the results
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reported by Nakamura et al. (13) about the possibility of assessing, by FCM, the small DNA content difference present in birds between heterogametic females (ZW) and homogametic males (22) with W
