Mary Hagedorn

Court and spark: electric signals in the courtship and mating of gymnotoid fish

Abstract By mimicking tropical rainy season conditions in aquaria, we stimulated two species of gymnotoid electric fish, Eigenmannia virescens and Apteronotus leptorhynchus , to spawn in captivity. Their courtship activity, breeding behaviour and electric social communication were monitored in several groups over 2 years. Groups of both species established dominance hierarchies correlated with electric organ discharge frequency, aggressiveness and size. Spawning was preceded by several nights of courtship during which the male modulated its electric organ discharge to produce ‘chirps’. Continual bouts of chirping lasted for hours on evenings prior to spawning. These electrical signals play a significant role in courtship and spawning, as gravid E. virescens females could be stimulated to spawn by playing back into the tank a tape recording of male courtship chirps. In both species the chirp invovves a slight increase in frequency followed by a cessation of the dominant frequency. This suggests a common mode of signal production in these two different genera of fish. Chirps are short and abrupt during aggressive encounters, but assume a softer and more raspy quality during courtship.

Single electrocytes produce a sexually dimorphic signal in South American electric fish, Hypopomus occidentalis (Gymnotiformes, Hypopomidae)

SummaryHypopomus occidentalis is a weakly electric Gymnotiform fish with a pulse-type electric organ discharge (EOD).Hypopomus used in this study were taken from one of the northernmost boundaries of this species, the Atlantic drainage of Panama where the animals breed at the beginning of the dry season (December). In normal breeding populations,Hypopomus occidentalis exhibit a sexual dimorphism in EOD and morphology. Mature males are large with a broad tail and have an EOD characterized by a low peak power frequency. Females and immature males are smaller, having a slender tail and EODs with higher peak power frequencies (Fig. 1). This study describes differences in the EOD and electric organ morphology between breeding field populations of male and femaleHypopomus. Changes in physiology, morphology and EOD shape which may accompany this seasonal change were examined in steroid injected fish, using standard histological and physiological techniques.A group of females were injected with hormones (5α-dihydrotestosterone (DHT), estrogen or saline) to assess changes in their morphology and EOD. Animals treated with DHT developed characteristics which mimicked the sexually dimorphic characteristics of a male, while the other groups did not (see Fig. 5). Tissue from the tails of breeding males and females, and females treated with DHT, were sampled to measure the size of the electrocytes in the tail. The broader tail of males and DHT-females is composed of large electrocytes, whereas the slender tail of normal females is composed of smaller electrocytes. Therefore, the increase in the tail width in the female DHT group is caused by an enlargement of the electrocytes in this area.Intracellular recordings from the electrocytes of saline and DHT injected females show a difference in the responses of the rostral faces of the electrocytes from the two groups, which reflect the differences in their EODs. Saline-treated animals had symmetrical EODs (the first and second phase of the EOD were equal in duration and amplitude), while the physiological responses from each face of the electrocytes yielded responses that were similarly equal in duration and amplitude. DHT-treated animals had asymmetrical EODs (the first phase of the EOD was similar to that of saline treated fish and larger in amplitude and shorter in duration than the second phase) and the physiological responses of the electrocytes reflected this asymmetry. The differential recordings across the caudal face were similar to those from saline treated fish, while the responses from the rostral face were longer in duration and smaller in amplitude.These data suggest that the effects of androgens underlie the changes in single electrocytes which produce the sexually dimorphic signals and morphology present in natural breeding populations ofHypopomus occidentalis.

Acoustic communication in an electric fish, Pollimyrus isidori (Mormyridae)

SummaryIt has been known since von Frischs work in the 1930s that mormyrid electric fishes are quite sensitive to sound. We now describe a repertoire of natural sounds produced by the mormyrid,Pollimyrus isidori, during breeding and aggression; reception of communication sounds is probably a major function for mormyrid audition.1.In aquaria,Pollimyrus isidori produce ‘grunts’, ‘moans’, ‘growls’, ‘pops’ and ‘hoots’ at various phases during nesting, courtship, and territory defense.2.All five sounds are produced primarily at night. Territorial males produce grunts, moans and growls during courtship. Vocalizing is stimulated by the presence of a gravid female on the males territory and decreases with the onset of spawning. Hoots and pops are given during agonistic behavior.3.Grunts are bursts of acoustic pulses, stereotyped for an individual, with the potential as individual signatures.4.The electric organ is silent during grunts and moans and is discharged at a reduced rate during growls.5.The courtship and spawning ofPollimyrus isidori is described.

Altering Fish Embryos with Aquaporin-3: An Essential Step Toward Successful Cryopreservation

Abstract Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.

Water distribution and permeability of zebrafish embryos, Brachydanio rerio

Teleost embryos have not been successfully cryopreserved. To formulate successful cryopreservation protocols, the distribution and cellular permeability to water must be understood. In this paper, the zebrafish (Brachydanio rerio) was used as a model for basic studies of the distribution to permeability to water. These embryos are a complex multi-compartmental system composed of two membrane-limited compartments, a large yolk (surrounded by the yolk syncytial layer) and differentiating blastoderm cells (each surrounded by a plasma membrane). Due to the complexity of this system, a variety of techniques, including magnetic resonance microscopy and electron spin resonance, was used to measure the water in these compartments. Cellular water was distributed unequally in each compartment. At the 6-somite stage, the percent water (V/V) was distributed as follows: total in embryo = 74%, total in yolk = 42%, and total in blastoderm = 82%. A one-compartment model was used to analyze kinetic, osmotic shrinkage data and determine a phenomenological water permeability parameter, Lp, assuming intracellular isosmotic compartments of either 40 or 300 mosm. This analysis revealed that the membrane permeability changed (P < 0.05) during development. During the 75% epiboly to 3-somite stage, the mean membrane permeability remained constant (Lp = 0.022 +/- 0.002 micron x min-1atm-1 [mean +/- S.E.M.] assuming isosmotic is 40 mosm or Lp = 0.049 +/- 0.008 micron x min-1atm-1 assuming isosmotic is 300 mosm). However, at the 6-somite stage, Lp increased twofold (Lp = 0.040 +/- 0.004 micron x min-1atm-1 assuming isosmotic is 40 mosm or Lp = 0.100 +/- 0.017 micron x min-1atm-1 assuming isosmotic is 300 mosm). Therefore, the low permeability of the zebrafish embryo coupled with its large size (and consequent low area to volume ratio) led to a very slow osmotic response that should be considered before formulating cryopreservation protocols.

Relative dominance among males is expressed in the electric organ discharge characteristics of a weakly electric fish

The spectral characteristics of the electric organ discharge (EOD) of a species of pulse-type electric fish, Hypopomus occidentalis, can rapidly change during aggressive interactions. Pairs of animals were matched in aggressive displacement tests to score for dominace. The winning males displayed a reduction in the peak-power frequency of their EODs within 2 days. In addition, a correlation was found between the maintenance of dominant status and the display of the largest-amplitude EOD. Thus, in an interacting group of this species, a male with an EOD characterized by a lowest peak-power frequency and largest-amplitude holds the dominant status. Mechanisms are discussed as to how aggressive behavioural interactions may rapidly effect physiological changes in the EOD.

Synodontid Catfish: A New Group of Weakly Electric Fish

Three species of synodontid catfish can produce weak biphasic electric discharges in either continuous or burst-like fashion. The peak-power frequency of these electric organ discharges is around 100 Hz. The electric organ appears to have originated from a sonic muscle: the organ lies dorsal to the swim bladder, contains modified muscle tissue, and is innervated from a motor nucleus located in the caudal medulla. It is possible that synodontid catfish use electric signals in communication and/or object detection.

Comparative cryobiological traits and requirements for gametes and gonadal tissues collected from wildlife species

A major challenge to retaining viability of frozen gametes and reproductive tissues is to understand and overcome species-specificities, especially because there is substantial diversity in cryobiological properties and requirements among cell types and tissues. Systematic studies can lead to successful post-thaw recovery, especially after determining: 1) membrane permeability to water and cryoprotectant, 2) cryoprotectant toxicity, 3) tolerance to osmotic changes, and 4) resistance to cooling and freezing temperatures. Although species-dependency ultimately dictates the ability of specific cells and tissues to survive freeze-thawing, there are commonalities between taxa that allow a protocol developed for one species to be useful information for another. This is the reason for performing comparative cryopreservation studies among diverse species. Our laboratory has compared cellular cryotolerance, especially in spermatozoa, in a diverse group of animals-from corals to elephants-for more than 30 yrs. Characterizing the biophysical traits of gametes and tissues is the most efficient way to develop successful storage and recovery protocols, but, such data are only available for a few laboratory, livestock, and fish species, with virtually all others (wild mammals, birds, reptiles, and amphibians) having gone unstudied. Nonetheless, when a rare animal unexpectedly dies, there is no time to understand the fundamentals of biophysics. In these emergencies, it is necessary to rely on experience and the best data from taxonomically-related species. Fortunately, there are some general similarities among most species, which, for example, allow adequate post-thaw viability. Regardless, there is a priority for more information on biophysical traits and freezing tolerance of distinctive biomaterials, especially for oocytes and gonadal tissues, and even for common, domesticated animals. Our colleague, Dr John Critser was a pioneer in cryobiology, earning that moniker because of his advocacy and devotion to understanding the differences (and similarities) among species to better store living genetic material.

Oxidative Stress in Zebrafish (Danio rerio) Sperm

Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line.

THE EMBRYOGENESIS OF ROD PHOTORECEPTORS IN THE TELEOST FISH RETINA, HAPLOCHROMIS BURTONI

Development of the retina, like that of other tissues, occurs via an orderly sequence of cell division and differentiation, producing the functional retina. In teleost fish, however, cell division and differentiation in the retina continue throughout the life of the animal in two distinct ways. Stem cells in a circumferential germinal zone at the periphery of the retina give rise to all retinal cell types and progenitor cells located throughout the retina in the outer nuclear layer (ONL) produce new rod photoreceptors. These processes in adult retina recapitulate in space the embryonic events responsible for forming the retina. Analysis of these events in an African cichlid fish, Haplochromis burtoni, confirmed that cone photoreceptors differentiate first, followed by rod photoreceptors. Correspondingly, at the margin of the eye, cone photoreceptors differentiate nearer to the margin than do rods. Control of photoreceptor production is not understood. Here we present the time of appearance and distribution pattern of GABA and vimentin which are candidates for the control of retinal cell division and differentiation. Antibody staining reveals that both GABA and vimentin exhibit unique patterns of expression during embryonic retinal development. Vimentin immunoreactivity is evident throughout the retina in a spoke-like pattern between developmental Days 4 and 7, as both cone and rod photoreceptors are being formed. GABA is expressed in horizontal cells between Days 5 and 7, corresponding to the onset of rod differentiation in time and in position within the retina. Moreover, the wave of GABAergic staining in the horizontal cells parallels the wave of rod differentiation across the embryonic retina of H. burtoni. Thus, GABA may play a role in the development of rod photoreceptors.