Research at VSU on Kryptolebia marmoratus formally Rivulus marmoratus

  Rare white/amelanistic Krytolebias marmoratus produced in the laboratory.  White fish such as this produce normal colored offspring when selfing.  As they reach sexual maturity they begin to develop a nearly normal wildtype color pattern.  The fish pictured is beginning to develop pigment and began laying eggs shortly after developing a near normal wildtype pattern


Publication on the biology of Kryptolebia marmoratus conducted at VSU


Mark Mackiewicz, Andrey Tatarenkov, Andrew Perry, J. Ryce Martin, John F. Elder, Jr., David L. Bechler, and John C. Avise. 2006.  Microsatellite documentation of male-mediated outcrossing between laboratory strains of the self-fertilizing mangrove killifish (Kryptolebias marmoratus).  Journal of Heredity (September 2006)


Abstracts on the biology of Kryptolebia marmoratus conducted at VSU and presented at various meetings

(See Resume for specific meetings)


Innate verses learned behaviors in the clonal hermaphrodite Rivulus marmoratus. David L. Bechler and John f. Elder, Valdosta State University, Valdosta, GA 31698.  In a previous presentation, we presented an experimental regime that analyzed four clones of adult Rivulus marmoratus to determine if timidity may be associated with clonal lineage and to determine the possible effects of timidity on other behaviors such as feeding. Our findings indicated that levels of timidity were clonally related and were most likely innate. Using a similar experimental design, we have now tested juveniles to determine whether or not the rates or levels of timidity in adults are also present from time hatching or develop over time.  Data indicates that rates or levels of timidity seen in adults exists form the time of hatching. We also tested situations involving learning (habituation) in adults and found that the rate at which a particular clone learned was different form its levels of timidity suggesting that timidity and learning are not necessarily interdependent.

Interclonal Hybridization and White Morph Production in Rivulus marmoratus.  David L. Bechler, John F. Elder and Andrew Perry, Department of Biology, Valdosta State University, Valdosta, GA 31698¸USA.  Rivulus marmoratus, a clonal self-fertilizing hermaphroditic vertebrate, is distributed from Florida through the Caribbean to Brazil with occasional males occurring in the laboratory and field.  Interclonal hybridization is indicated by high levels of heterozygosity in wild Belizean fish.  Using males and hermaphrodites from Florida, Belizean and Honduran clones, we have attempted to produce interclonal hybrids.  While genetic analyses are not yet complete, we can report that fertile eggs from attempted crosses have produced white morphs in the F1 and F2 generations.  White morphs tend to possess black irises and pupils, a rosy abdominal coloration, and begin to develop irregular dark pigmented areas as they approach sexual maturity.  Dark pigments continue to develop until the fish develops a near wildtype pattern.  Two white morphs retained a rosy background color atypical of the wildtype background coloration and have also retained black irises and pupils in contrast to the green and yellow flecked iris of wildtype hermaphrodites.  Clonal offspring of the white morphs possess a typical wildtype pattern.

An Infra Red Detection System for Monitoring the Movements of Small Fish in Artificial Systems. Frank Flaherty1, David L. Bechler2, Kelly Luke2 (1 Department of Physics, Astronomy and Geosciences, Valdosta State University, Valdosta, GA., 31698;  2 Department of Biology, Valdosta State University, Valdosta, GA., 31698).  We present the following system for acquiring time stamped data on the motion of fish through an artificial crab burrow made from PVC pipe.  The burrow was U-shaped with an open pool at each end and a small chamber in each of the long arms of the burrow.  A sensor consisting of an infrared light emitting diode and photo transistor were mounted opposite each other in holes drilled through the pipe at each of three observation points.  Observation points were located at the two openings of the burrow and at its nadir.  An infrared rather than visible light emitter was chosen to avoid the possibility of visible light affecting the behavior of the fish.  The passage of a fish between the pair interrupted the beam which was detected by a PIC 168F4 microprocessor.  The microprocessor was programmed to send data to the serial port of a personal computer (PC). A program written in True Basic wrote the sensor number and time of day to a file on the PC.  Since the microprocessor handles the critical tasks even a very slow PC is more than adequate for recording the file. The data file can be imported into a spreadsheet program for detailed analysis. A key advantage of this system is that it permits the monitoring of movements of fish that are too small to have internal PIT tags placed in them.

Activity Patterns of the Mangrove Killifish, Kryptolebias marmoratus, in an Artificial Crab Burrow Kelly Luke1, David L. Bechler1, Frank Flaherty2 (1 Department of Biology, Valdosta State University, Valdosta, GA., 31698;  2 Department of Physics, Astronomy and Geosciences, Valdosta State University, Valdosta, GA., 31698).  The mangrove killifish uses burrows of the land crab, Cardisoma guanhumi as shelters.  Using a detection system (see associated poster) consisting of three pairs of infrared sensors fed to a PIC 168F4 microprocessor linked to a PC, we studied the movement patterns of three clones of Kryptolebias marmoratus in an artificial crab burrow.  The burrow system was made of PVC pipe, was U-shaped with an open pool at each end and a small chamber in each of the long arms of the burrow.  Sensors were located at the openings of the burrow and its nadir.  Distance moved within the burrow and time spent resting at each sensor was computed using Excel Logic statements. Clones moved significantly greater distances through the burrow during the day time than at night, but no interclonal differences were found.  All clones spent more time at sensors at the mouths of the burrow, but less time at any one sensor during the night.  Intraclonal analyses revealed that clones Hon 7 and Hon 11 were more active at night moving from one end of the burrow to the other than was clone 50.91, which spent more time at the burrow mouths.  Interclonal analyses of time spent at sensors showed no differences.  From these analyses we conclude that K. marmoratus spent more time at night in the open pools and that in general complex behaviors involving movement and resting in the artificial burrow masks potential differences due to the high variability exhibited by the clones.

Microsatellite Documentation of Outcrossing Between Inbred Laboratory Strains of the Self-fertilizing Mangrove Killifish (Kryptolebias marmoratus). Mark Mackiewicz1, Andrey Tatarenkov1, Andrew Perry2, J. Ryce Martin3, John F. Elder,  Jr.3, David L. Bechler3, and John C. Avise1.  (1Department of Ecology and Evolutionary Biology, University of California, Irvine, CA., 92697; 2Deaprtment of Biology, University of Louisiana-Monroe, Monroe, LA;  3Department of Biology, Valdosta State University, Valdosta, GA., 31698.)  At EEEF 2004, Iceland, Bechler, Elder and Perry in the presentation “Interclonal Hybridization and White Morph Production in Rivulus marmoratus” reported on the occurrence of white Kryptolebias (formerly Rivulus) marmoratus morphs, and speculated that they might result from inter-strain hybridizations.  Here we report that hybridizations did occur, but white morphs were not a product of hybrid events.  Primers for 36 microsatellite loci were developed and employed to characterize genetic stocks and detect possible outcrossing between inbred laboratory strains of Kryptolebias marmoratus.  The molecular markers readily distinguished various strains and proved useful for diagnosing inter-strain hybridizations.  From attempted crosses involving hermaphrodites from particular geographic strains and gonochoristic males from others, two among a total of 32 surveyed progeny (6.2%) displayed multi-locus heterozygosity clearly indicative of inter-strain gametic syngamy.  One of these outcross hybrids was allowed to resume normal self-fertilization, and microsatellite assays of the resulting progeny demonstrated that heterozygosity decreased by approximately 50% after one generation, as expected.  Although natural and laboratory populations of K. marmoratus consist mostly of synchronous hermaphrodites with efficient mechanisms of internal self-fertilization, these microsatellite findings demonstrate conclusively that conspecific males can mediate occasional outcross events, and that this process can release extensive genic heterozygosity which then becomes available (upon resumption of regular self-fertilization) for rapid conversion into plethoras of new recombinant inbred lines.  The fact that one white morph was not a hybrid proves that the occurrence of white morphs is not genetically controlled, but possibly and artifact of raising K. marmoratus in the laboratory.

The Role of a Brown Diatom and a Bluegreen Alga on the Culture and Emersion of the Mangrove Killifish, Kryptolebias (Rivulus) marmoratus.  Dustin Shockley1, M Jason Sanders1, David L. Bechler1, James Nienow1, D. Scott Taylor2  (1Department of Biology, Valdosta State University, Valdosta, GA 31698;  2Brevard County Environmentally Endangered Lands Program, 5560 N. US 1, Melbourne, FL 32940).  During the culture of the mangrove killifish, Kryptolebias marmoratus, specimens with lordosis, dysfunctional swim bladders and deformed opercula occasionally occur.  Survival rates of eggs and offspring are also often highly variable.  During the culture process it is common to have a brown diatom form a biofilm on the sides of the glass and plastic culture chambers only later to have it replaced by a bluegreen alga.  Speculation has been that the brown diatom has had a negative impact on fish resulting in the maladies observed.  At VSU, we have tentatively identified two microbes in our system as the brown diatom Achnanthes sp. and the bluegreen alga, Leptolyngbya sp, a cyanobacterium .  Using these microbes, we inoculated culture dishes and test chambers with either Achnanthes or Leptolyngbya or no microbe and conducted a series of experiments involving hatching, growth rates and adult emersion (the behavior of sitting out of the water frequently seen in this species) on objects projecting out of the water in the laboratory. Results of the experiments indicate that the cyanobacterium, Leptolyngbya sp, produced poorer hatching rates and higher death rates during the first two weeks after hatching.  Once fish reach a minimum size of ~5.5 mm neither microbe had any significant effect on growth or perching rate.  While perching rates were not affected by either microbe, it was found that the different clones used in the experiment perched at significantly different rates.