The RivMar Webpage
 

    Welcome to the Rivulus marmoratus webpage. This page is intended as a resource for the research community and for those in the general fish-loving public who may be interested.. In addition to providing a convenient source of information, it is my hope that this page may inspire additional research on this incredibly interesting killifish species., for despite the 35 years which have passed since the discovery of its unusual reproductive mode, large gaps remain in our knowledge of its most basic biology. Many of these gaps, particularly those in the general area of reproductive endocrinology, seem to be unnecessary: they could undoubtedly be easily filled in by straightforward extension of knowledge or experimental paradigms developed in studies of other fish species. Such gaps are probably a signal that a "critical mass" of investigators has not yet appeared, and if this page does anything to hasten that appearance, it will have done its job.

    The site consists of three sections. First, some dramatic figures which I hope will capture your attention. Then, an abbreviated review article which I believe cites more than 95% of papers that have been published on R. marmoratus. Lastly, there is a list of the most frequently asked questions about the species and answers to them. These questions have been gleaned partly from those asked at seminar presentations but mostly from comments from reviewers of grant proposals. In fact, one might say that this section is a small attempt by me to prevent history from repeating itself!

    I thank my colleagues Will Davis, Scott Taylor and John Grizzle for permitting the use of their photographs. The Biological Sciences Initiative at Virginia Tech provided server space and webpage maintenance. Mr. William Zabronik kindly scanned several of the images and prepared the jpeg files. The page was assembled and guided through the arcane terrain of html and image editing by my son, Jonathan F. ("Jack" ) Turner.

    Finally, I would like to dedicate this page to two friends/colleagues who are no longer with us, to Vlad Walters, who would have enjoyed reading it, and to Bob Harrington, virtually the "inventor" of Rivulus marmoratus, who probably would have argued with me over it, at great length... --- Bruce J. Turner, August, 1998.

 
 
Fig. 1. Rivulus marmoratus, hermaphrodite with typical color pattern. Belize, 1992.		  
Fig. 2 Rivulus marmoratus, field-caught male, Twin Cays, Belize. 1990. Males differ in the intensity of the orange pigment and the dark markings on body and fins; in some older males, the dark markings disappear altogether, leaving an orange-washed fish with clear fins. 
 
 
 
Fig. 3. Section through the "unrestricted" (unpartitioned) ovotestis of R. marmoratus, (trichrome stain). Four nucleoli (dark red bodies) can be seen in the nucleus of the ooctye in the center of the section, with part of a larger oocyte (or mature ova) above it. Immediately to its right, two tubules, arranged vertically, are visible with primary and secondary spermatocytes. Note the anatomical proximity of testicular and ovarian tissues. In these gonads, mature eggs and sperm will be "ovulated" into a common lumen, where fertilization will occur.
 
 
 
 
 
 
Fig. 4. Ms. Barbara Lubinski demonstrating an efficient method for collecting live R. marmoratus.; Belize, 1992. The barb is removed from the tiniest hook obtainable. The hook is then attached by a thin line to a twig or branch and baited with a small worm, beetle, etc. The bait is jiggled or "bounced" on the surface of the water in a crab hole (or other likely spot). Eventually, a fish is attracted by the movement and takes the bait. Traps of various designs (but not conventional minnow traps) are also effective; these include small funnel traps, inserted directly into the crab hole and left in place for a tidal cycle. Conventional fish collecting techniques (seines, rotenone), are not very effective for this species, and it has been overlooked in particular areas even though extensive fish collections were made in its magrove forest habitat. This, in turn, has sometimes lead to the generally mistaken impression that the species is "rare" or "of special concern." 		  
Fig. 5. Emergent specimen of R. marmoratus, found on moist substrate under a rotting log on the mangrove forest floor. Belize, 1991. 
 
 
 
 
 
 
 
 
Fig. 6 Clonal inheritance of DNA fingerprint patterns in R. marmoratus.. All specimens shown here are descended from a single individual collected in Florida (locality lost) in the mid 1970’s and maintained initially by H. Grimm (University of Hamburg). A single individual of this stock was obtained by Dr. E.-H. Park, Hanyang University, Seoul, South Korea, in 1981 and bred for 20 additional generations. After the first generation, approx. 100 individuals were maintained per generation, and 20 — 30 of these were used as progenitors of the next generation. This sample is from the 19th Seoul generation. Although a few mutations can be detected with some hypervariable sequences as probes (see Laughlin et al. 1995) the overall stability of these patterns is a striking demonstration of clonality in this species.
 
 
Fig. 7. DNA fingerprinting of a sample of 6 field-caught R. marmoratus hermaphrodites, No Name Key, FL, 1986; specimens labeled 1 — 6, analysis repeated with two restriction enzymes, probe used is (GACA)4. Note that there are 5 different genotypes (clones) among the 6 individuals (specimens 2 and 6 have identical fingerprints). This level of variation is typical of very nearly all natural populations surveyed thus far. None of the clones shown here were detected in two subsequent surveys at/near this site. The gels also show a progeny test of specimen 3; note the identity of F1 and F2 fingerprints. From Turner et al. (1990).
 
 
 
 
 
 
 
Fig. 8. Progeny tests of 4 additional field-caught hermaphrodites, showing clonal inheritance of DNA fingerprint patterns. Line I. Everglades National Park (1989). Line II Miami (Harrington Clone M). Line III. Marco Island/Rookery Bay (1984). Line IV. No Name Key (1990). Restriction enzyme: Pal I, probes as indicated. From Lubinski, et al. (1995). Some 29 field-caught hermaphrodites have now been progeny tested in this manner, other than those from Twin Cays, Belize (see below), and, without exception, the pattern of inheritance has proven to be clonal.
 
 
Fig. 9. Detection of heterozygosity, and therefore presumably of outcrossing, in a single natural population. Progeny tests of field-caught hermphrodites from I, Pelican Cay 1991. and II and III, Twin Cays, 1991. Note the clonal inheritance in line I and the obvious segregation in lines II and III. Pal I digests, A and B are the same gel, sequentially reprobed as indicated. From Lubinski et al. (1995). The Twin Cays population contained a high frequency of males (nearly 25%), and these presumably participate in the mating system by fertilizing viable but unfertilized ova emitted by adult hermaphrodites, or, possibly, by immature hermaphrodites which lack testicular tissue. All 24 of the hermaphrodites collected on Twin Cays in 1991 were heterozygotes.
 
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