614 



Fishery Bulletin 88(3), 1990 



Hind-]Slk 



23,130 



9,419 



6,557 



4,317 



2,322 



2,028 — 



564- 



EcoR-1 



EcoR-l 



Hin6-m 



Figure 2 



Top; Restriction digests with Eco- 

 RI (if mlDNA from Spanish sar- 

 dine SiirdiiieUn aurita visualized 

 on a routine Hoechst/agarose gel 

 photographed in transilluminated 

 IIV light. Lane 1 is the //i«d-III 

 digested A-DNA standard. Bottom: 

 Eco-Rl (left) and Hind-lU digests 

 of mtDNA of threadherring Opis- 

 thonema ogiinum. All digests were 

 carried out on ~0.3 ng of mtDNA 

 for 2.5 hours, then electro- 

 phoresed for 3 hours at 50 volts. 

 The Hiiid-lU digest of S. aurita 

 mtDNA (not shown) produces five 

 fragments compared with two for 

 that of 0. ogiinum. 



accomplished through low-volume density-gradient 

 separation in a relatively inexpensive tabletop ultracen- 

 trifuge which is essentially portable. This simple over- 

 night ultracentrifugation followed by extraction of the 

 mtDNA and subsequent ethanol precipitation would 

 allow mtDNA isolation from at least 30 individuals in 

 a normal work-week by a single worker. Five grams 

 of fresh, ripe, ovarian tissue should produce enough 

 mtDNA per individual to be digested separately by 20 

 restriction endonucleases and still be resolved on 

 Hoechst 33258/agarose gels. 



Agarose gels incorporating the Hoechst 33258 fluo- 

 rochrome (DeFlaun and Paul 1986) is a good substitute 

 for staining of restriction fragments using ethidium 

 bromide. Whereas Hoechst 33258 binds specifically and 

 quantitatively to AT-rich mtDNA (Latt and Stetten 

 1976) ethidium bromide has mixed RNA-DNA specifi- 

 city (Le Pecq and Paoletti 1966) reducing its resolving 

 power and making visualization of small fragments 

 (<500 bp) difficult. The suggestion of Lansman et ai. 

 (1981), that minimally 0.5 t^g of mtDNA be used per 

 digestion to insure visualization of fragments with 

 ethidium-bromide staining, limits its resolving power 



to about 20 ng of DNA. In contrast, a zone of fragments 

 containing only 7.0 ngof DNA (~ 1.8 ^g per digestion) 

 should be visible on Hoechst 33258/agarose gels, and 

 these can be viewed immediately following electro- 

 phoresis on a UV transilluminator. 



Our estimate of the sizes of the mtDNA genomes of 

 Sardinella aur-ita and Opisthoncma ogiinum is close 

 to those obtained for other clupeids (Kornfield and 

 Bogdanowicz 1987, Avise et al. 1989). Ours are prob- 

 ably very good estimates since the pattern-recognition 

 system used here greatly reduces measurement error 

 of fragment mobility inherent in the use of stereo- 

 microscopes and other manual methods. This survey 

 of only a few individuals did not reveal any polymor- 

 phisms at Eco-Rl or Hind-Ill in either species, but 

 the restriction fragments produced at both restriction 

 sites are quite distinct between the species (Fig. 2). 

 With new microextraction methods for mtDNA avail- 

 able (Graves et al. 1990), it should be possible to even- 

 tually separate or identify even the eggs and very 

 young larvae of ^S. nurita and 0. ogiinum obtained in 

 field samples. The ability to do so could be used to pro- 

 vide estimates of egg and larval mortality rates in the 



