INFOWEB 15 :
A major symposium focused on the hermaphroditic Killifish Kryptolebias marmoratus

 

From Jean H. Huber
Private address: 7 Bd Flandrin, 75116 Paris, France
M.N.H.N., Ichthyology, PARIS, France.

 

Paris, September 15. 2013 [updated with an addendum on January 17. 2014]

 

Beware ! this newsletter is long, dense and technical, take time to read it, possibly along several days.

 

Dear Colleague, dear Aquarist !

 

A major new dedicated Symposium.

 

No surprise… the unique Killifish, Kryptolebias marmoratus (alias the mangrove Rivulus) has not finished to disclose its secrets and its fantastic specificities.
A full day symposium on January 4. 2012 has been dedicated to review our present knowledge since more than 50 years and future desirable prospects for the study of this uncomparable animal (and Killi-Hobbyists are extremely lucky to have it in their luggages, in comparison to other groups of aquarists) ; the symposium has been presented at the annual meeting of the Society for Integrative and Comparative Biology (from January 3. to 7.) at Charleston, South Carolina, USA and the results have been published as a special issue in their own scientific magazine, monthly since 2007 (Integrative and Comparative Biology, formerly known as American Zoologist, in volume 52, number 6) published in December 2012, to serve as a new major synthesis of our escalating knowledge on this fascinating hermaphroditic fish today named Kryptolebias marmoratus (there had been some scientific meetings specializing on the fish in the past, but not with such a dimension and not with such dynamics) ; the well-attended symposium has comprised 11 speakers, of which 4 women and 7 men, with academic ranks ranging from postdoctoral fellow to full professor, who have come from 4 countries with the objective of summarizing our present knowledge and of helping drive future research within this taxon and facilitating collaborations among researchers.

Within the genus Kryptolebias there are some species (not all the known congeners) that are the only vertebrates known to self-fertilize (i.e. an hermaphrodite alone fertilizes its own eggs-ovules with its own spermatozoids that are laid over substratum, then develop until offspring). This is the case of marmoratus described from Cuba in 1880 (originally as a Rivulus species), by the Cuban ichthyologist, Felipe Poey (26 May 1799 - 28 January 1891), who also described the genus Rivulus, just 20 years before, in 1860 (with the description of the species cylindraceus).

History of knowledge.

Although first described in 1880, the neotropical cyprinodontiformes fish K. marmoratus has avoided scientific focus up to 1961, when it is identified as the only known selfing hermaphroditic vertebrate (i.e., a single fish produces offspring from eggs), a unique behavior that has arised some 20 Million Years Ago (MYA) according to recent molecular findings ; the subsequent intense interest in this fish as a laboratory animal, continuing to today, might explain the paucity of wild collections (by distracting focus), but our collective knowledge now suggests that the inherent difficulty of wild collection is more a matter of looking in the "good" places (not the "wrong" places, derived from standard killifish fauna, like in the past) ; long thought to be rare in the mangroves, and, yes, it is rare in certain human-impacted habitats (with, then, conservation issues), K. marmoratus can be quite abundant, but in micro-habitats, not typically targeted by collectors : ephemeral pools at higher elevations in the swamp, crab burrows, and other fossorial or even terrestrial places (surprisingly) ; field studies of this enigmatic fish reveal an almost amphibious behavior and unparalleled adaptations to extreme situations among killifish ; during emersion (or aestivation) these fish tolerate extended dry periods (not only a few minutes, like all Rivulus species ; in water, they are exposed to temperature extremes, high levels of hydrogen sulfide (stinky smell), and depleted dissolved oxygen (breathless), a picture that any ordinary fish would consider as terribly unpleasant and finally not worth living !

As a matter of fact, our history of live collections for this fish can be rated as very poor with the exception of Florida (USA), Belize (Central America) and a few Caribbean islands ;

while the systematic and taxonomic study of K. marmoratus has been very limited (actually only 3 researchers have done some work, Lothar Seegers in 1984, Jean Huber in 1992, and Wilson Costa in 2004, 2006, 2011, with an intermediate ruling of the I.C.Z.N. Commission in-between in 2005, and the resulting situation is outstandingly confused with at least 5 names involved, marmoratus, bonairensis, ocellatus, caudomarginatus, hermaphroditus), on the other hand the biological research has been intense, spurred by the hermaphroditic characteristic of the fish which has involved 3 generations of researchers, Robert Whiting Harrington (1911-1975), first, as a pioneering innovator (usually publishing alone, or with Klaus D. Kallmann) during the years nineteen sixties and seventies, then a second generation mainly grown from a team of US researchers (Bruce J. Turner, Scott Taylor, William P. Davis, with occasionally some others, such as Mark Mackiewicz or John C. Avise) deepened the selfing, simultaneous hermaphroditism of the fish during the years nineteen eighties and nineties, and finally the picture has exploded with multiple publications from researchers of various origins (Europe, Russia, Japan, China, Brazil, and obviously USA), notably around Andrei Tatarenkov in molecular genetics, since the early 2000's ;

K. marmoratus routinely self-fertilizes and even if such a reproductive mode is common in plants and invertebrates, it is unique among vertebrates (to date !) ; still, hermaphroditism is taxonomically widespread among teleost fishes and, as evidence of sexual lability, it takes on many forms including simultaneous hermaphroditism (fish have both male and female sexual organs, but sex needs 2 of them), protogynous hermaphroditism (fish are born female and at some point in their lifespan change sex to male, e.g., reef wrasses), protandrous hermaphroditism (fish are born male and at some point in their lifespan change sex to female, e.g., reef clowns, i.e., the reverse of protogynous hermaphroditism), bidirectional sex change, and androdioecy (fish are composed of a male population and a distinct hermaphrodite population, as in K. marmoratus) [more general basic info on hermaphroditism : Wiki] ; actually the life history of K. marmoratus rather bears comparison with an invertebrate groundworm, named Caenorhabditis elegans (a free-living, transparent nematode, about 1 mm in length, which lives in temperate soil environments) in which the majority of individuals are hermaphroditic with preferential self-fertilization (but males occur, too !) ; that worm has been hence the subject of intense research with prizes at the end : the 2002 Nobel Prize in Physiology or Medicine has been awarded to Sydney Brenner, H. Robert Horvitz and John Sulston for their work on the genetics of its organ development and programmed cell death, the 2006 Nobel Prize in Physiology or Medicine has been awarded to Andrew Fire and Craig C. Mello for their discovery of RNA interference and the 2008 Nobel Prize in Chemistry has been awarded in part to Martin Chalfie shared for his work on green fluorescent protein ;

perhaps, one or several of our researchers on K. marmoratus will be regarded as candidates for a future Nobel prize !

 

hermaphrodite

 

Present state of the art from the symposium highlights.

 

Our present state of the art will be organized according to 25 themes (not according to the symposium agenda) [please note, each characteristics is not herein referenced to avoid heavyness of an already very technical text, but obviously they are referenced in the original articles listed in literature] :

1. Hermaphroditism. The historical knowledge of this unique sexual behavior for K. marmoratus starts with Harrington (1961, published in the major scientific magazine Nature) ; K. marmoratus is also the first fish species found to have environmental sex determination, whereby lower temperatures inhibit ovarian development, thus producing active males that may be useful for outcrossings (i.e., sexing males and hermaphrodites) and providing one potential route that avoids the hypothetical inbreeding degenerescence if only hermaphrodites would exist ; in the laboratory, individual adult fish can produce isogenic embryos (i.e., clones, genetically identical copies or highly homozygous lineages) ; in the wild, the fish exist as androdioecious populations in which both hermaphrodites and, although relatively rare (with exceptions), males are found, an extremely rare reproductive system otherwise known in only a few dozen extant plant and animal species (and no other fish outside Kryptolebias) ; under as yet unknown environmental conditions in the wild, males develop and outcrossing between the hermaphrodites and males occurs (i.e., not clones, genetically distinct offspring or heterozygous lineages) ; it is intriguing to consider the behavioral, neurological, and endocrinological (hormones) control necessary to accommodate this strange reproductive strategy ;

2. Outcrossings. Self-fertilization (inbreeding) has resulted in naturally homozygous individuals and the hypothetical propagation of isogenic lineages in the wild ; while many wild-caught individuals are homozygous (clones), heterozygous individuals are also found, indicating that out-crossing in which a male breeds with an hermaphrodite, is also a banale occurrence (surprisingly) ; that outcrossing behavior, in which males presumably fertilize infertile eggs laid by hermaphrodites, is documented to occur frequently in some populations in Belize, and with less frequency in other populations (outcrossing rates in Florida are in the range 1%-9%, considerably lower than in Belize where they are as high as 60% at Twin Cayes and about 24% on a cay in Turneffe atoll) ; except for the offshore cays of Belize, males are rare (according to present still poor knowledge), but even in populations in which males are very rare or unknown, a certain amount of heterozygosity exists, suggesting possible hermaphrodite/hermaphrodite crossings (and/or another mechanism) ; hermaphrodite/male outcrossing has been documented in the laboratory and, given the general difficulties of observing this cryptic animal in its complex natural environment, it is not surprising that there has been no visual confirmation of outcrossing (or oviposition) in the field ; given that oviposition may take place out of the water (egg-stranding), there are obvious complications to outcrossing (close contact difficulties, among other difficulties), unless the egg is already fecundated by the primary male (which sounds strange) ; nevertheless, observations in aquaria, in which wild hermaphrodites from Florida have been presented with brightly colored, wild male fish from Belize, suggest that the innate behaviors for outcrossing are there ; when the male fish approaches the hermaphrodites near spawning "mops" at the top of the aquarium, the two sexes display many of the standard killifish courtship/spawning non-annual behaviors (e.g., in the related genus Rivulus) : recurved bodies coupling closely and vibrating (with converging fins) ; such behavior is rather incongruous, given that male phenotype is totally unknown in these nearly 100% hermaphroditic populations from Florida (males there are extremely rare, often less than 1%, and not in all spots) and those general intraspecific highly aggressive tendencies are normally seen between individuals in aquaria ; in total the overal picture appears to be complex, because all Florida and Belize western Caribbean populations (including Twin Cays) are phyletically indistinguishable and these western lineages share a common ancestor more recently than all other populations (eastern populations, e.g., in Brasil, being possibly divergent) ; therefore, the Twin Cays population is not a remnant ancestral outcrossing population and outcrossing is suspected to have evolved as a phenotypically plastic character, and its expression in K. marmoratus may be dormant unless triggered by some ecological factors that are poorly understood (or by artificial spur in aquarium) ;

3. Other reproductive traits. If in total the key issue for research is hermaphroditism, which allows to develop a clonal strain in the laboratory from a single hermaphroditic individual (and as a consequence several distinct clonal strains from several hermaphroditic individuals), the total picture of reproductive traits may be described as greatly opportunistic ; observed heterozygosities (non clonal situations) are severely constrained, as expected for a hermaphroditic species with a mixed-mating system and low rates of outcrossing and despite the pronounced population structure and the implied restrictions on effective gene flow due to hermaphrodites, isogenicity (genetic identity across individuals) within and among local inbred populations is surprisingly low (i.e., not above say 90%) even after factoring out probable de novo mutations (in a field survey of 12 populations with rare males in the Florida Keys, 75% of the fish are fully homozygous and another 15% are heterozygous at 3 or fewer of the surveyed loci) ; in the laboratory, the following have been demonstrated or hypothesized : standard reproduction by fertilization of eggs of hermaphrodite by male (and in that case, the population of hermaphrodites tends to loose its hermaphroditic power of producing spermatozoids), clonal reproduction by hermaphrodite, putative reproduction between hermaphrodites (as the single explanation of genomic heterozygocity in populations without males), and appearance of primary males (presumably rare with exceptions in the wild but easily obtained in the laboratory when water temperature is lowered between 18°C and 20°C, or by using 17alpha-methyltestosterone, an alkylated anabolic steroid used to treat men with a testosterone deficiency, bearing close structural similarity to testosterone, a natural hormone) and of secondary males, i.e. hermaphrodites with a male phenotype and non functional ovules (obtained when juveniles are submitted to high temperatures greater than 30°C) allowing to input heterozygocity when available (probably other water parameters could be involved in the wild since sunlight and oxygen contents have also been evaluated in the laboratory), but at this time, no proven ovo-viviparity and no proven availability of standard female types (the hermaphrodite externally looks like a female, like any Rivulus female with a supracaudal ocellus, but this is not biologically a female, this is both sexes at the same time and it has a complex reproductive organ, named the ovotestis) ;

4. Genome. K. marmoratus is diploid with 24 chromosomes ; the complete DNA sequence of the mitochondrial genome has been published as early as 2001, emblematic of the research priorities given to the fish ; using initial next-generation sequencing data, the genome size of K. marmoratus to be 900 Megabases (Mb) which is larger than the medaka genome (700 Mb) a related fish to Cyprinodontiformes, within the genus Oryzias (Oryz. latipes), well known to Japanese researchers ; no sex chromosome has been disclosed ; microsatellite divergence between isogenic lineages have been shown : the whole-genome sequencing of many lineages will probably reveal the amount of shared and unique single nucleotides (elementary units of genome) and the variation in insertions and deletions between lineages ; a genetic survey of 32 highly polymorphic loci, in 201 specimens from 12 populations in the Florida Keys (USA), reveals an unexpected extensive spatial genotypic diversity (150 specimens out of 201), i.e. heterozygosity, with a population-genetic structure on micro-spatial and micro-temporal scales, implying severe constraints on effective gene flow (androdioecy affects population genetics, but there may be other options, such as the hypothetical outcrossing between 2 hermaphrodites or even, let's be adventurous -and not too serious-, the internal fertilization… without gonopodium !) ;

5. Distribution. K. marmoratus has a huge geographical range, in fact the widest distribution of any inshore-dwelling coastal fish species in all of North, Central, and South America —about 52 degrees of north-south latitude, i.e. about 5 700 vertical kilometers from North to South or over 7 000 kilometers if airline kilometers (e.g., from Veracruz Mexico to Rio de Janeiro Brasil, 7 400 airline kilometers, or from Gainesville Florida to Rio de Janeiro Brasil, 7 200 airline kilometers) and obviously much more if the (sinuous) coast line is followed with the significant reservation that mangrove is not today available over these 7 000 plus kilometers (considering K. marmoratus in a large sense, i.e. including all 2, 3, 4 or 5 killifish names with an hermaphroditic focused behavior) ; this basically encompasses the entire tropical and subtropical western Atlantic basin, as far north as central Florida, USA, north through the Yucatan Peninsula and Bay of Campeche in Mexico, along almost the entire Central American, throughout all of the Antilles and Bahamas, including the island of Barbados, and northern South American coastline, and likely south to the mouth of the Amazon River, up to Rio ; however, records east and south of Venezuela are sparse (also those on the Caribbean coast of central America, except in Belize) ; this remarkable and widespread distribution is clearly a tribute to dispersal in shallow marine waters only (i.e., via the presently known or the previously historical seas or saline waters, to the contrary of vicariance meaning extension through land waters) ; more precisely, the apparent colonizing success of K. marmoratus can be attributed to a suite of biological characteristics including (1) a strong propensity to self-fertilize such that even a single individual can establish a new colony, (2) the physiological capacity to withstand diverse (top ends) temperatures, salinities, and hydrogen sulphide levels, (3) its cutaneous respiration (if need be) when temporarily emerged from water, (4) a habit of occupying hollowed mangrove logs, which might facilitate across-ocean dispersal of adults via drifting wood debris and (5) adhesive eggs that also might promote dispersal by sticking to flotsam ; the ultimate proof of these 3 latter hypotheses would be the presence of the fish on the island of Bermuda (off the Atlantic coast, but quite far today), the most distant extension of the tropical Atlantic and of the neotropical mangrove ecosystem ; however, the fish has yet to be recorded there, and in 2007 a brief survey within the very limited mangrove habitat of Bermuda has not documented this fish (yet unpublished data) ;

 

mangrove

 

6. Habitat. K. marmoratus is found in marine and brackish water or hypersaline pools, and is capable of surviving rapid and extreme salinity changes (e.g., following high tides) ; it is an extremely tolerant euryhaline (salt contents) and thermohaline (temperature contents) species, routinely enduring in the wild salinities ranging from freshwater to 114% and temperatures of 7°C to 38°C ; in spite of its extensive distribution, K. marmoratus has been frequently described by authors as rare in the wild, but recent data demonstrate that it is not true since the fish has not been searched at the proper places (where it can be abundant) ; hence, its special concern conservation status in Florida (USA) is over pessimistic ; still the fish are secretive and cryptic compared to the standard Killifish fauna in usual bitotopes (over 1000 traps have been set to collect fish, the traps being checked on average every 2.5 hours during daytime… overnight, 170 traps have been set, but have resulted in 8 fish only ; on average, 76.9 trap hours to capture 1 specimen of K. marmoratus while in comparison, in the same habitat, only 6.1 trap hours have been necesseray to capture other fish such as Fundulus confluentus, Gambusia sp., Fundulus xenicus, Poecilia latipinna, or Cyprinodon variegatus) ; actually, K. marmoratus is affiliated closely with and spends its entire life cycle within coastal mangroves, and the distribution of the fish fits closely with micro-habitats within the red mangrove, Rhizophora mangle ; recent experiments based on stable isotope data for K. marmoratus from Everglades, Florida, and Twin Cayes, Belize indicate a strong mangrove signal (yet unpublished data) ; these micro-habitats are intermittently dry shallow, stagnant pools, or terrestrial crab burrows, typically about 50 cm to 1 meter wide and about as deep as groundwater on site because the crab has to keep its gills moist at bottom (specifically, those of Cardisoma guanhumi, the great land bright blue crab, and Ucides cordatus, the mangrove land crab) or temporally flooded swales (including intertidal floods) or ditches (including anti-mosquito ditches) or even more surprisingly inside/under logs (some sorts of tunnels or galleries that are dug into dead mangrove wood by termites or beetle larvae) and also mangrove leaf litter or solution holes or empty beer cans and coconuts and even several meters inside anchialine cave systems (all being reclusive niches, being either fossorial, intermittently dry, or having adverse water quality conditions that preclude the establishment of other species of fish, notably with water turbid to opacity, and bottoms mostly treacherous, malodorous, marly muck) ;

 

crab

 

7. Temperature. K. marmoratus is perhaps more tolerant of temperature extremes than are some other tropical species and it has been collected over a wide thermal range (7°C-38°C) ; there is evidence that the fish can survive 5°C when emersed (out of the water) when extreme cold has resulted in documented cold mortality in central Florida (i.e., at the present northern extreme of its geographic range) ; falling temperature seems to induce emersion in a laboratory setting, but the adaptive value of this is unclear (except to probably indicate that such emersion has survival value) ; clearly, fossorial niches (e.g., crab burrows on the forest floor, usually 8.0 cm in diameter and 33.9 cm long, yet unpublished data) offer a thermal refuge during cold events ; low temperature is probably the primary factor limiting northern distribution at the extremes of range in Florida, but ranges do not appear to be equivalent on both coasts (west and east) ; however, on average, water is warm (25°C-30°C) ;

8. Salinity. K. marmoratus is euryhaline and has been collected in the wild at salinities of 0-70 ppt and in the laboratory juveniles can tolerate 70-80 ppt (i.e., well over saline, the standard sea being at 35 ppt, or parts per thousand of salt) ; while it is noted that K. marmoratus can be reared and will reproduce in fresh water, collections at very low salinities in the wild probably reflect temporary conditions (not permanent) following heavy rainfall (yet unpublished data) ; in natural habitats where there is a gradient from pure fresh water to saline mangroves (e.g., Everglades, Florida), K. marmoratus were not found except in areas with salinity greater than 10 ppt (yet unpublished data) ; artificial attempts to introduce K. marmoratus into isolated freshwater pools in the field were unsuccessful, except in one instance where the site intermittently dried and no species of fish were present ; in contrast, extreme salinities are a common feature of mangroves during drought or periods of reduced tidal inundation ; adaptation to hypersaline conditions is linked to activity of chloride cells (chloride cells in the opercular epithelium and opercular skin sharply increase in size in K. marmoratus when raised at 100% and 200% seawater versus fish raised at 1% seawaterM; besides, in the laboratory, K. marmoratus can adapt to 114 ppt seawater ;

9. Hydrogen sulfide. H2S (Hydrogen sulfide) is widespread in marine environments, it is common in surface and porewaters of mangrove systems and it is well-known that it is toxic to any fish ; anyone venturing into the mangroves is familiar with the characteristic rotten egg smell ; besides its direct toxicity, H2S quickly phases out any available dissolved oxygen contents, presenting a double challenge to aquatic life ; H2S is likely a key driver of emersion behavior (jumping and staying outside water) in K. marmoratus, but there are few field studies on the concentrations of H2S in micro-habitats where the fish are collected (e.g., 0.4-0.7 ppm in inundated mangrove pools, or 51.1 ppm in pore water on Floridian mangrove islands, or 1.64 ppm in mangrove/saltmarsh wetlands in east central Florida, or 0.59 to 10.59 ppm within U. cordatus crab burrows in Belize) ; as a consequence, avoiding elevated levels may be a constant challenge for K. marmoratus ; during low-water periods, K. marmoratus within crab burrows and stagnant pools frequently develop a white coating on the skin and fins and if specimens displaying these features are captured and placed in fresh seawater, the coating disappears and attempts to scrape the material off the fish and preserve it have been unsuccessful (yet unpublished data) ; it seems likely that the coating is the actual growth of a sulfur-oxidizing bacterium (Beggiatoa sp.) ;

10. Ammonia. In Belizean U. cordatus crab burrows known to contain K. marmoratus, ammonia routinely averages about 1 mmol/l, a level greater than that typically found in most other aquatic habitats, fresh or saline ; since, in the laboratory, K. marmoratus can tolerate levels greater than 10 mmol/l, the observed levels would not appear to significantly stress the fish but, as with H2S, lack of tidal/rainfall flushing might allow levels to elevate further ; in addition, the pH of the water in burrows is found to be lower than in the adjacent open ocean (pH 7 versus pH 8) ; the lower pH observed in burrows may be the result of H2S production as a by-product from the metabolic activities of resident crab(s) and/or fish ; further complicating the chemical "soup" found within crab burrows is the reduced tolerance of fish to ammonia at lower salinities (salinities can drop drastically following heavy rainfall) ;

11. Dissolved oxygen. Other than H2S, another ubiquitous feature in mangroves is the common occurrence of low dissolved oxygen (DO) ; K. marmoratus have frequently been collected under conditions of very poor water quality, and have been documented to survive hypoxic conditions (less than 1.0 ppm in DO concentration) in the field (in isolated mangrove pools in Belize, ranging from 0.16 to 0.98) ; sinkhole habitats (where K. marmoratus are regularly found) had the lowest DO, followed by the transition zone (the higher elevations where K. marmoratus would typically be found in burrows and pools) which (partially) explains the paucity of other fish species in the internal mangroves ; finally, hypoxia (0.2 mg/L) is known to induce emersion in the laboratory ; survival in environments with low DO may require fish to be physiologically adapted for surface aerial respiration (SAR), in which the oxygen-enriched surface layer of the water is utilized in respiration ; K. marmoratus, with its flattened head, upturned mouth and small size exhibits the physical characteristics allowing SAR ; besides, an enhanced capillary network is found in the dorsum of the epidermis, within 1 mm of the surface (most dense on the nape, decreasing posteriorly until becoming absent past the Dorsal fin ; partial emersion, whereby K. marmoratus place themselves at the water's edge/surface, typically lodged against/under debris, is frequently seen in the field, as well as short-term bouts of SAR while actively swimming and skimming the surface (yet unpublished data) ; as a consequence, K. marmoratus have been observed "drowning" if retained in traps beneath the water, often only for very short periods (i.e., with classic symptoms of anoxia, with flared, pale gills, but the influence of H2S in these mortalities is unknown) ;

12. Diet. K. marmoratus in the wild is clearly a predator, with various terrestrial and aquatic invertebrates forming the bulk of the diet (e.g., mosquito larvae, gastropods, various crustaceans, dipterans, formicids, juvenile fishes, fish parts, polychaetes, copepods, and miscellaneous insects, like components of the related genus Rivulus) ; besides, it is cannibalistic, too ; in the most comprehensive study of diet to date, 111 specimens collected from the burrows of C. guanhumi in east-central Florida, 60% of fish guts are empty suggesting a paucity of food resources in the marginal, intermittently dry microhabitats where the fish is found ; periods of flooding by rainfall or tidal inundation may provide enhanced feeding opportunities and also allow K. marmoratus priority feeding, as other fish species would be moving from sources of permanent water, for example estuarine water itself or permanent ponds/creeks within the mangroves ; in addition, K. marmoratus leave the water (emersion) to capture food and return to the water to consume it ; crab burrows serve as pit-traps for insects, and some of the predation on insects by K. marmoratus has been observed at night ; mosquito larvae are a frequent dietary item because the saltmarsh mosquitoes, Aedes taeniorhynchus and Aedes sollicitans, are known to oviposit in some of the microhabitats occupied by K. marmoratus (e.g., intermittently dry pools and mosquito ditches) ; in the laboratory, K. marmoratus of 40mm standard length (SL) consume up to 80 larvae in 24 h, and even juvenile fish (1-day old) eat a maximum of 21 newly-hatched larvae (these feeding rates meet or exceed those of well-known mosquito larvivores such as Gambusia spp.) ; yet, natural populations of K. marmoratus never seem to reach the densities seen in larvivorous poeciliids (Gambusia), so their direct contribution to mosquito "control" is perhaps limited ;

13. Social behavior. K. marmoratus in the wild is not well known by recent solid observations with regard to social intraspecific behavior, except the strange aggregate behavior when emersed for long times (almost sticking one another in the termites tunnels in order to, presumably, keep moist) ; indirectly it is known as aggressive with regard to social extraspecific behavior because any intruder of reasonable size (and not a potential predator) in the crab hole is severely pushed away through intimidation (and for the collector, it is an astute way to catch that cryptic fish by mickey-mousing a small bait in front of its eyes !) ; indirectly it is also known as cannibalistic in the laboratory (parents often feed on their fry), probably also in the wild (and their starvation may be a spur) ; on the contrary, the intraspecific social behavior in the laboratory is known with some details and it is very aggressive ; in K. marmoratus, several experiments have been undertaken to assess the social behavior of hermaphrodites (i.e. presumably with both male and female hormones) : individuals with higher levels of endogenous testosterone are more aggressive, quicker to attack and have a higher chance of winning ; besides, the relationships between aggressiveness (a fish's readiness to perform gill display to its mirror image) and (1) boldness (the readiness to emerge from a shelter), (2) exploratory tendency (the readiness to approach a novel shelter), and (3) learning performance (the probability of entering the correct reservoir in a T-maze test) have been tested in K. marmoratus with the hypothesis that these 4 behaviors are all modulated by cortisol and testosterone and with the background that, shortly after a previous winning or losing experience, aggressiveness would be altered (e.g., individuals that have recently won tend to behave more aggressively towards a new opponent and enjoy an elevated chance of winning again -winner effect- while individuals that have lost recently tend to behave more submissively and suffer a higher chance of losing again -loser effect-) ; actually aggressiveness is positively correlated with boldness and exploratory tendency, and these 3 behavioral traits are all positively correlated with pre-experience testosterone levels ; aggressiveness and boldness are also positively correlated with pre-experience cortisol levels (exploratory tendency is not) ; conversally, aggressiveness and boldness are stronglier correlated than either of them with exploratory tendency (probably then testosterone and cortisol play important roles in mediating the correlations between these behavioral traits) ; on the other hand, learning performance is not significantly correlated with the other behavioral traits or with the levels of testosterone or cortisol ; recent experience in contests influenced aggressiveness, tendency to explore, and learning performance, but not their boldness; individuals that received a winning experience are quicker to display to their mirror image (aggressiveness) and performe better in the learning task but are slower to approach anovel object than were individuals that lost ; finally, another puzzling observation is that, behaviorally, hermaphrodites prefer to associate with males rather than with other hermaphrodites (although the ratio is so much imbalanced) ; also, land crab burrows play an important role in their social interactions and preliminary observations in an aquarium-terrarium (not in the field) with an open-water area and holes that simulate crab burrows have shown that K. marmoratus spend 40% of the time in the burrow with hermaphrodites marking a preference for associating with males rather than other hermaphrodites (obviously such a large and complex palette of behaviors seems a consequence of both hemaphroditism and androdioecy, not hemaphroditism alone) ; finally, the crab itself and its one-to-one relationship with K. marmoratus should not be underestimated (but it has never been studied in details), but actually (from limited experience) the fish itself is quite cunning alone (being able to find holes in plexiglas and pass them to reach a presumably (artificial) crab hole ; finally another issue (unsolved) is to understand why in laboratory, aquarium bred hermaphrodites (presumably isogenic-homozygous) exhibit more agressive social behaviors than hermaphrodites from the wild (presumably heterozygous) ;

14. Emersion. In addition to environmental sex determination and androdioecious reproduction (hermaphrodites + outcrossings), K. marmoratus is also known to emerge from its aquatic surroundings and assume a transitory, terrestrial existence (somewhat like all Rivulus species in a large old sense with more than 100 species, but not quite and during much longer times, being then amphibious, as we shall see further) ; also named aestivation, emersion is is a well-known behavior and for K. marmoratus specifically it seems that life is equally worthwhile inside or outside the water ; this aestivation includes both active movement out of the water (wiggling/flipping across a damp substrate) or the more torpid state achieved during more long-term emersion ; one physical driver of emersion is excessive H2S : in the laboratory 50% of K. marmoratus leaves the water at an H2S concentration of 0.12 ppm, a level extraordinarily low compared to known field levels ; the duration of emergence in the laboratory is correlated with the concentration of H2S and at lower concentrations, fish tend to ventilate at the surface more but do not emerse ; no effect of hypoxia on emergence with high levels of hypoxia, while low levels (0.2 mg/L) are known to induce emersion ; intra-specific aggression can also lead to emersion in both the field and laboratory ; other parameters of water quality (ammonia, salinity, and temperature) has not been evaluated in depth ; in addition to the already mentioned white coating on the skin and fins, the ventral surface of head region has a thicker epidermis (skin) that contains goblet cells producing mucus ; both of these features possibly provide protection against abrasion and desiccation when emersed ; additionally, mucus enhances adhesion to vegetation and other surfaces during terrestrial movements ; emersion induces no overall change in metabolic rate compared to fish in water ; the emersion process is associated with an apparent knowledge of terrestrial landscapes by the fish (experiments show a good level of accuracy in jumps in switching between crab burrows, even with a plexiglas barrier opened with a hole ; in a limited laboratory study, K. marmoratus prefers being in water at 25°C, moving to land as temperatures dropped to 19°C-20°C, but even then fish would temporarily return to water ; re-flooding would then result in immediate resumption of aquatic activity, within the original occupied territory ; fish that emersed for 412 hours (more than 17 days !) are less responsive to mechanical stimulation, requiring prodding before movement ; K. marmoratus can survive at least 66 consecutive days out of the water in the laboratory, fish being obviously emaciated (31.4% weight loss), but upon reflooding they immediately resumed feeding and normal activity ; in many cases in the wild, emersion is clearly the result of drying of the habitat, often over an extensive period ; multiple fish are known to emerse inside rotting mangrove logs on the forest floor, resulting in very high concentrations of individuals ; this phenomenon, termed "log packing", occurs inside logs that have been galleried by beetle larvae and termites, which subsequently fall into shallow pools in the mangroves, the galleries being 10-20mm in diameter in many cases riddling the logs and full of a sawdust-like frass, the byproduct of insect activity, these logs remain spongy and water-saturated for a considerable period of time after dry-down (yet unpublished data) ; how and when fish enter the logs is unknown, but it likely occurs as the pools near final dry-down ; preliminary laboratory data show that fish enter artificial "logs" in aquaria as water levels decline, but often wait until the log is out of the water, necessitating traveling across a "dry" substrate ; however, in the wild, fish may move over a relatively considerable distance once inside the logs, with some individuals being found 20 cm from obvious entrance holes ; the densities observed inside these logs are remarkable : 57 individuals inside a section 1.5m long and 9 cm in diameter (this agglutination might be a safe belt to maintain humidity) ; individuals are closely packed together, exhibiting enhanced mucus production (yet unpublished data), but become active immediately upon being exposed, either flipping out of the galleries or attempting to slither further into their interiors ; cessation of intra-specific aggression when fish are emersed has been noted in K. marmoratus, which can be quite aggressive when in water ; laboratory rearing in "community" tanks (many individuals of a common lineage in the same tank) has also revealed differences in emersion behavior between "clones", with some clones emersing more frequently and others emersing in compact groups of multiple individuals ; however, the clear survival value of emersion is very apparent in the field when other fish species (e.g., Poeciliids) are found dead in drying pools and crab burrows known to contain K. marmoratus ; emersion is not only a good effective habit for K. marmoratus in its adverse environment, but it is useful to (lazily) avoid movement in water with tide (from fresh to saline and vice versa, e.g., by resting on a small branch at the air-water interface, as evidenced by using a remote video camera) and it does not preclude its life (the fish continues to grow and feed outside water !) ;

15. Skin and gills. Living outside its natural element, not surprisingly, induces several major physiological changes for K. marmoratus in order to maintain homeostasis when out of water for days to weeks (observed maximum : 66 consecutive days !) : first, by being able to breathe without its gills when out of the water (it should be remembered that in the water extant fishes breathe through their gills, a very efficient organ at extracting oxygen because water is 800 times more dense, 60 times more viscous, and contains 30 times less oxygen than air) ; second, by being able to activate gas exchanges in a different mode : in K. marmoratus, gaseous exchange occurs across the skin (another unique specificity for the fish), as dramatic remodeling of the gill reduces its effective surface area for exchange ; ionoregulation and osmoregulation are maintained in air by exchanging Na+ (sodium ion), Cl- (chlore ion), and H2O (water) across skin that contains a rich population of ionocytes (the key site on skin for ion transport) of which the surface area besides become 45 times larger in air-exposed fish in contact with a moist hypersaline compared to freshwater ; third, in K. marmoratus, excretion of ammonia (a nitrogenous waste product) occurs in part by cutaneous NH3 volatilization (NH4+ ion) facilitated by ammonia transporters on the surface of the epidermis (normally in water, all fish eliminate urea through their gills, here K. marmoratus has developped a strange mecchanism that eliminates amonia, hence urea indirectly, through the skin !) ; fourth, cutaneous angiogenesis (stronger blood vacsularisation) occurs when K. marmoratus are emersed for a week, suggesting a higher rate of blood flow to surface vessels ; then the skin of K. marmoratus replaces fish gills in all the major concerned functions when emersed, allowing them to move between aquatic and terrestrial environments with (?) ease ; indeed, this ability to move between aquatic and terrestrial habitats is a fascinating strategy that requires a number of adaptations in the skin that enable gaseous exchange, regulation of ions, and elimination of nitrogenous wastes (NH4+ ion)… however how remodelling of respiratory morphology is poorly known ; in the laboratory an experiment has been conducted involving acute hypoxia (insufficient oxygen level) which has induced hyperventilation, a rapid compensatory mechanism, that has limitations ; thus, fish moving between aquatic and terrestrial habitats may benefit from cutaneously breathing oxygen-rich air, but upon return to water must compensate for a less efficient branchial morphology (mild hypoxia) or suffer impaired respiratory function (severe hypoxia) ; research is still underway because it appears that in the laboratory various clonal strains react differently in hypersaline water when emersed : some lineages remodel their gills and decrease the effective surface area for exchange, presumably limiting excessive ion uptake, whereas other lineages show no response (yet unpublished data) ; here these specific strains may help solving key questions are : if cutaneous respiration dominates during emersion, how does the structure and function of the skin and gills reversibly remodel to accommodate changing roles, how quick, and how all these major environmental and physiological changes are genetically mediated ;

16. Predation. There are only 2 records of predation by wading birds (wading birds do not seem to frequent the small stinky ponds and heavy mangrove cover where K. marmoratus is found, with the exception of smaller species like the green heron) ; given the extremely secretive, cryptic and fossorial nature of K. marmoratus, it is hard to hypothesize intense avian predation ; estuarine fish and crustacea predators (e.g., snapper, barracuda, and blue crab) do move into K. marmoratus habitat during periods of heavy tidal flooding and there may be incidental predation on K. marmoratus then ; other predators might sporadically be Gobies, Cichlasoma uropthalmus, Belenesox belizanus, Strongylura notata, and eleotrids (Guavina guavina, Dormitator maculatus, Eleotris pisonis) ; a more significant predator may be the mangrove water snake, Nerodia fasciata compressicauda, seen in crab burrows and mangroves in Florida and Belize ;

17. Parasitism. K. marmoratus has never been noted to display any of the "classic" teleost skin diseases (Ichthyophthirius multifiliis or other white-spot diseases) in either laboratory or field populations ; whether the fish is innately more resistant to external parasites than most other species, or the exposure to parasites is not as frequent in the marginal habitats that the fish occupies, remains to be studied ; within a Belizean population with varying rates of genetic heterozygosity, more heterozygous fish had fewer internal parasites than did homozygous fish (however, all fish were infected with one of 3 different parasites, bacterial cysts on the gills, a protozoan), or acanthocephalans) ; further, even if less parasitism is involved, the fish are still subject to pathogens inducing an adaptive immune response with pathogen-derived antigens, and several hypotheses have been raised whether parasite selection along the increased homozygozity influence characters of the produced strains (e.g., in alleles of microsatellites) with putative consequences on behavior, male proportion, outcrossings, mating process (etc.) [in order to maintain some sort of diversity despite hermaphroditic in-breeding over several generations ?] ;

18. Competition. As already noted, other species of fish are not commonly sympatric with K. marmoratus, presumably due to the demanding physical regimes of their preferred microhabitats ; only a few other species (e.g., Gambusia spp., Poecilia spp., Fundulus spp., Cyprinodon spp., and D. maculatus) have been collected in any significant number in the intermittently flooded swales/potholes/ditches where K. marmoratus may be found ; however, most, if not all of these fishes, leave with receding tides or do not survive periodic dry-down ; conversely, K. marmoratus is rarely taken in mangrove habitats with permanent water ; at the higher elevations K. marmoratus occupies, abiotic conditions are too extreme to allow other competitively superior small fishes (e.g., Gambusia) to survive ; a congener, Rivulus tenuis, is commonly found sympatric with K. marmoratus in crab burrows/potholes on mainland Belize (it is a dioecious, freshwater species, but it may be collected in salinities up to 10 ppt, not more) ; if sympatric, when salinity increases no evidence of R. tenuis is reported ;

19. Population density. In some places, K. marmoratus can no longer be considered as "rare", with a single crab burrow having produced 26 individuals and catches from other burrows and potholes sometimes in excess of 10 individuals not uncommon ; in a preliminary survey of K. marmoratus on a Belizean offshore cay (Twin Cayes) transition mangroves (an area of predominantly black mangroves) inland from the fringing red mangroves, a field experiment using small fiberglass screen enclosures set during low tide (the sites then being dry and all K. marmoratus being presumably undergound) removed and smapled at each tidal flooding has shown that the density varied from 7 to 26 fish/m2, remarkable numbers when considering that the transition is basically intertidal ; another field experiment that has utilized a modified 6 m2 bottomless lift-net in riverine mangroves in the Everglades (deployed 189 times at 3 locations over 7 years) captured 21 species of fish, numerically dominated by K. marmoratus with an overall density of K. marmoratus of 0.39 fish/m2 ; K. marmoratus is most abundant at the higher elevation site with the least tidal flooding, and with lower density of fishes in general and least overall diversity of fish ; conversely, K. marmoratus is least abundant where tidal flooding is greatest, that is, at lower elevations ; higher catches is associated with greater amounts of leaf litter ; no association is found between number of fish and abundance of crab burrows (fiddler crabs and mud crabs: Uca sp. and E. limosum) ; since no fish is observed among the leaf litter at low tide, it is assumed that they are retreating down crab burrows at that time ; the movement of individual K. marmoratus in the field remains largely unstudied, but a small scale capture-recapture study in this fish suggests a high population turnover : in Belize, 14 marked (unique fin-clip) fish have been released back in their burrows of origin (only 2 marked recaptures in the place and surroundings, although a total of 81 individuals have been collected in the recapture effort, one marked fish having moved a maximum of 7m from burrow of origin in 5 days, while the other had moved 1.5 m) ; the sizes of the fish captured from the same burrows over time were noted with little repeatability in size, indicating a high turnover of occupants, while the low recapture rate again attests to high population density (yet unpublished data) ;

20. Fertilization and embryonic development. Most individuals of K. marmoratus are hermaphroditic and possess an ovotestis (mixed gonads), giving them the potential to produce homozygous progeny (clones) ; in an unsuccessful laboratory attempt to create functional female, it has been demonstrated that exogenous treatment with high concentrations of 17alpha-ethinylestradiol depresses relevant gene expression, decreases fertility, increases sterility, and delays age of reproductive maturity (hence the development and maintenance of a simultaneous ovotestis may be particularly sensitive to its hormonal milieu) ; the ultra-structure of spermatozoa is of primitive type (mature spermatozoa are found in ducts of hermaphrodite where ovulation takes place and fertilized eggs can be held within the ducts for up to 77 hours, but 80% are released within 24 hours) ; the embryonic development can be achieved, in aquarium conditions, both in water and in damp air outside water (and in the latter case when ready to hatch emersed eggs do hatch after some water is sprinkled on them ; the embryonic development has been reported in details with 32 stages between one-cell stage and prehatching stage (photographic images) ; K. marmoratus display considerable variability in the number of eggs it lays (oviposits) by self-fertilization in a week or even through cyclical periods of time ; to increase egg production consistently, an experiment has shown that when feeding hermaphrodites with newly hatched brine shrimp -Artemia salina- (to 350 nauplii per ml), by increasing the food 10-fold the increased egg production per week is 2-fold ;

21. Oviposition and habitat of juveniles. In a laboratory setting, observations in the crab burrow microcosm (microniche) suggest that wild fish strand their embryos (laying eggs among vegetation at or above water surface), a trait rather common among the genera Rivulus and Kryptolebias, and in fact, much of this behavior may occur at night ; however, repeated searches for embryos in the field largely have proved fruitless, with only a few embryos found on the sides of crab burrows and several secreted within leaf litter at the exposed margins of stagnant pools ; embryos develop fully without standing water, provided they are kept damp ; embryos apparently enter a diapause state when fully developed (obviously nothing to do with annuals), holding at full development and then finally hatching when flooded (observed both in the laboratory and in the field) ; very large (40-55mm SL) wild gravid hermaphrodites in both Belize and Florida have been known to release large numbers of viable embryos (approximately 50 or more) in mass spawnings within collection containers after capture (this is also known in other groups of killifish) ; while this may be a response to the stress of capture, the prospect for deposition of large numbers of embryos in a single oviposition event in the wild seems likely ; the place of oviposition remains one of the more enduring mysteries because, given the apparent density of many populations, there must be many embryos present ; in the tropics, it is safe to assume that reproduction occurs year-round, except for prolonged droughts and/or low tides ; in the sub-tropics (e.g., Florida), there are some data on K. marmoratus gonads that support a hiatus in reproduction during cooler weather ; in 111 gonads from phenotypic hermaphrodites from east-central Florida, most fish (97%) contain developing or mature eggs within the ovotestes, but fish taken during late winter (February) have eggs that are smaller (less than 0.25mm in diameter) and fewer in number (40-60), while in April to May eggs are larger (greater than 1.0 mm) and more numerous (up to 75 total) ;

22. Juveniles. Both morphological characters and meristic counts indicate that K. marmoratus can be considered to be a juvenile after 9.8 mm in standard length (20 DAH, i.e. days after hatching) ; few juveniles (less than 10mm SL) have been found in the wild (only using poison, like rotenone), because collections during August in mosquito ditches in east-central Florida have only yielded 22 very small juveniles (7.5-11.1mm SL) ; given the propensity for cannibalism in K. marmoratus, the fact that no fish smaller than 7mm SL has ever been taken from a crab burrow suggests that juveniles may largely be occupying as-yet unidentified micro-habitats ;

23. Age and growth. Growth of laboratory-reared fish has been extensively documented, but only one study has looked at the growth of caged wild fish, placed in different small enclosures at varying depths in the water within the mangroves : growth, then, is related both to temperature and incidence of hypoxia, and inversely correlated with the number of fish in enclosures ; K. marmoratus grow more slowly when placed with species other than its own ; under laboratory settings, reared at 25°C plus or minus 1°C and 17 ppt salinity, the fish complete their life cycle from 0 to 100 days after hatching (DAH) when they reproduce themselves, and their early development has been evaluated by examining growth and morphometric parameters, meristic characters (vertebral and fin-ray counts), bone-cartilage development, and pigmentation : growth was isometric (i.e. proportional) for preanal length, head length, snout length, body depth, pectoral-fin length, dorsal-fin length, anal-fin length, and caudal-peduncle depth, while meristic counts for vertebrae (mean 34.2), Dorsal-fin (mean 8.6), Anal-fin (mean 11.4), and Caudal-fin rays (mean 30.2) are complete at 0 DAH, whereas Pectoral-fin rays are only complete by 30 DAH and Ventral-fin rays by 60 DAH, with full ossification of meristic elements proceeded by 30 DAH for vertebrae, by 60 DAH for Caudal-, Dorsal-, and Anal- fin rays, between 60 DAH and 100 DAH for Pectoral-fin rays, and by 100 DAH for Ventral-fin ray ; based on the presence of daily otolith increments up to 60 days age, a field experiment has to be undertaken to disclose aging wild fish ; similarly no data is available on the longevity of fish in the wild, but K. marmoratus can be longlived in the laboratory, with one specimen noted to survive 8.2 years (yet unpublished data) ; finally it should be noted that in the laboratory, late in their life cycle (3-4 years), approximately 60% of the hermaphroditic individuals transform into secondary males ;

24. Model for genomic, embryologic and other researches. Since the fish can be reared easily (as a non annual Killifish, mostly like Rivulus species, needing 2-3 weeks incubation time for the eggs) and quickly (an hermaphrodite can start reproduction at the age of 3-4 months, or to use an emblematic figure at 100 DAH), K. marmoratus has been maintained in laboratories for 50 years ; due to the preferential self-fertilization, wild-caught (rather heterozygous) lineages that are reared in the laboratory for several generations quickly become homozygous (theoretically 4 generations in the laboratory are already enough to confirm homozygosity) ; over 250 distinct genetic lineages are currently being maintained in laboratories, and that is obviously a strong basis to use them as a model for genomic, embryologic, behavioral, toxicological, carcinogenetic (etc.) experiments ;

however that is not fair enough… the fish have shown unexpected particularism ; e.g., behavioral and life-history traits are reproducible within isogenic lineages (clones), suggesting that many of the traits are heritable (transmitted by a single parent, then inherited by the offspring) and have a genetic component (gene-linked) ; for example, significant differences among lineages of K. marmoratus in aggression and in responses to fighting experience have been demonstrated ; yes, surprisingly, lineages are highly homozygous and highly differentiated, both genetically (microsatellite markers) and phenotypically (life-history and behavioral traits), which means that crossing lineages to create F2 (second generation offspring) or Fn (generation n) recombinant inbred lines will facilitate understanding the genetic basis of these traits and the manner in which they segregate (with possible outcomes for human genomic research) ; the naturally homozygous strains present distinct phenotypes that segregate between them, making this a tractable system for studying differentiation within populations and among populations ; for example, crossing 2 lineages with divergent patterns of growth results in hybrid F2s with intermediate characteristics… and similarly for behavior and those phenotypes ;

being an oviparous fish, K. marmoratus offers all the advantages of popular models such as zebrafish (Danio rerio) or medaka (Oryzias latipes), with the additional benefit of unique genetics and efforts have been undertaken in laboratories to fully establish it as a model species for embryological research by the manipulation of embryos (with 2 methodological approaches : handling of embryos with and without their chorionic membrane, for embryo manipulation and imaging, with a research interest in maternal contribution of RNA versus gene expression by the embryo) and also by chemical mutagenesis (a pilot study on 36 hermaphrodites, treated with varying doses of the common chemical mutagen, N-ethyl-N-nitrosourea, or ENU, and allowed to self-cross, has yielded zygotic mutants identifiable in the F2 generation, hence offering a promising screen to discover genes that regulate gonadal development and other developmental endpoints) ; as a consequence, the species is a very attractive and potentially powerful model for integrative and comparative biological research ;

for similar reasons, it is also a good model for toxicity, or for human oncology (cancers !) research : studying the genes involved in tumorigenesis and in the metabolism of toxins is a spur to develop K. marmoratus as a model for tumor development ; for example, in laboratory studies, K. marmoratus has shown susceptibility to a number of xenobiotics including carcinogens (toxicologists are interested in the mechanisms of action of environmental chemicals of contemporary interest such as endocrine disruptors and may consider this as a suitable fish model for the study of chemically induced carcinogenesis) since single short-term exposure to model chemical carcinogens is sufficient to induce tumourigenesis in K. marmoratus in a relatively short time span ; recently, sequences of the oncogene, ras, and tumour suppressor gene, p53 (both important in medical research because they play significant roles not only in carcinogenesis, but also in other signal transduction pathways) have been tested in K. marmoratus… which makes new paths for research on this fish with applications in cancer research, aquatic toxicology, endocrinology and genomics ;

finally, as it has been already mentioned herein, K. marmoratus is also a tool of research to understand the genetic basis for its biology and other adaptations (e.g. survival outside water), by working on the possibility of de novo assembly of its genome with sequencing and bioinformatics ; in total, K. marmoratus represents an attractive system for studying the genetic basis for many of the already defined phenotypes (gene bank data are readily available, though yet, not as extensively as for zebrafish or Japanese medaka, and gene sequencers are today quite affordable and quick : only live new populations are missing) ;

25. Current systematics. Presently, there are 2 very closely related species mentioned during the symposium to be hermaphroditic and worth testing for their validity ; their history is a series of hiccups ; historically, the first species is K. ocellatus described by Hensel, in 1868 (as a Rivulus species), then K. marmoratus has been described by Poey, in 1880 (also as a Rivulus species), the former from Brazil (Rio de Janeiro), the latter from Cuba (probably not far from Havana) ; they have been, subsequently to the hermaphroditism discovery, synonymized (or limited to subspecies status) and although the name marmoratus, as a junior synonym, should have become disused, the I.C.Z.N. (1995) decided the reverse by ruling out the name ocellatus as invalid in case it would be a synonym of marmoratus, on the basis that marmoratus was a name in prevailing usage (notably because of series of researches on hermaphroditism) ; if 2 very closely related hermaphroditic species have been evaluated as probably distinct (with our presently very poor knowledge and collections), the boundary in their respective distribution is completely confused ; however, there are 3 other names that have been described for fish related to those 2 : 2 acknowledged synonyms K. heyei, K. garciai, and also K. bonairensis, a name with a fluctuating status, more often as a synonym, and if a valid name, that would be only due to its intermediate range in the Netherlands Antilles, in-between Cuba and Rio de Janeiro (nothing else is mentioned in the symposium while participants are probably either not aware that a few months before Wilson Costa had reshuffled the cards or not willing/able to discuss the issue… see further !) ; and the confusing picture is even more complexified with the putative hermaphroditism (although preferably not hermaphroditic) of a related fish south of Rio de Janeiro !

 

 

Future propects of biological and systematic research on Kryptolebias.

 

On the biological side, the symposium has pinpointed the major uncertainties that need to be tackled in order to boost our knowledge on this fascinating fish :

  1. the factors that lead to parental cannibalism in the laboratory (and probably also in the wild) ;
  2. the typical habitat of the juveniles (unbelievably rare in the field according to present poor knowledge) ;
  3. the adult oviposition sites (and the time for it, guessingly at night ??) ;
  4. the factors (such as parasites or various environmental conditions, or even physiology) that might impact selfing rates (vs. outcrosses) ;
  5. the factors that might impact the number of males in the wild (from nearly none to nearly a quarter of a population, and even much more in a single case, bearing in mind that lower temperatures, e.g., 18°C-20°C, inducing males in laboratory are usually unknown in the wild… except in Florida where males are nearly absent !) ;
  6. the reasons why to-date males are found only in the wild in significant numbers in the Belize Cays and nowhere else (to make it straight, why the hell a sexual reproduction is used there while hermaphroditic reproduction is very succesfully -and selfishly- achieved with no male elsewhere ?) ;
  7. the demonstration, or not, of sexing between hermaphrodites ;
  8. the detailed mechanisms that allows survival outside water (and their reversibility) ;
  9. the complete biological and genetic study of several units in the intermediate geographical positions of the chain of populations (outside the 2 geographical extremes of Florida and Brasil) ;
  10. the complete biological and genetic study of the third direct component of the superspecies living at the Southern dead end of the distribution, south of Rio de Janeiro (being hermaphroditic or not) ;
  11. the complete biological and genetic study of the related congeners that are not hermaphroditic (brasiliensis, campelloi (unknown live), gracilis (if valid), sepia (notably for the latter the understanding of its unique melanistic color pattern according to mood).
 

On the systematic side, as already mentioned the symposium remained uncertain (even ambiguous… the focus being elsewhere) ; from the scattered samples that have been molecularly studied (mostly at either end of the distribution of all hermaphroditic lineages), researchers seems to consider 2 extreme sets of populations (Florida and southern, on the one hand, Rio de Janeiro and northern, on the other hand, and total confusion in-between, the former being K. marmoratus, the latter being… un-named or named as ocellatus or named as hermaphroditus depending on the lecturer ; in addition, a team has introduced a major systematic uncertainty by showing that at least 4 distinct and not vicariant populations (Turks and Caicos, Panama, and southern Cuba and Puerto Rico) among the Caribbean populations that are now molecularly analysed are clearly very close to that of the Rio de Janeiro samples, i.e. not close by any mean to K. marmoratus ; then the species definition and the biological history for these populations covering today more than 7 200 airline kilometers are incredibly complex (while the type locality of K. marmoratus is in northern Cuba, there is a phenotype in southern Cuba much closer to that of the Rio de Janeiro samples, about 7 000 airline kilometers southerly !) ;

besides, a third species, with a standard breeding behavior, i.e., preferably not hermaphroditic, but, according to Costa also hermaphroditic (an observation not confirmed at the symposium !) has been the subject of a systematic turmoil since a publication by Costa at the end of 2011 ; after having examined the unique type of ocellatus, Costa considers that the previous identification of ocellatus by Seegers (who also has studied that single type) in 1984 is erroneous and the description by Seegers of caudomarginatus corresponds to the true ocellatus, then caudomarginatus would be a junior synonym of ocellatus and ocellatus in the previous sense is described as hermaphroditus : he supports his argumentation on 3 points vs. caudomarginatus, the number of scales of the longitudinal series equal to 43 and lower in ocellatus, depth of body in male lower in ocellatus, Ventral fins in male of ocellatus short, not reaching the base of the second or third Anal-fin ray ;

and that is not only a question of LL scales since the only morphometric study (back in 2003) performed by a single researcher over the entire range (and not by several researchers with different methods and experience) has shown differences, yes, but minor and with little geographic rationals (a study among 12 widespread populations and among individual clonal lineages reared in the laboratory from 2 populations, Belize and Florida, with 32 meristic and morphometric characters in 187 fish ranging from Brasil to Florida, and with univariate and multivariate statistics tools for determination of overall differences among populations that shows significantly heteromorphy for all characters, notably number of precaudal vertebrae, distance from Ventral fins origin to Anal fin origin, Pectoral fin length, and number of branched Caudal fin rays, with the exception of Pectoral fin rays, also in clones, also in individuals raised at the same water temperature, 25°C, suggesting the heteromorphy is genetically related) ;

finally, let us add, with some provocation, that the mangrove being today segmented and having being segmented since a long period, on the one hand, and the hermaphroditic fish being able to produce clones in a few generations (with, inside each population, heterozygosities, yes), on the other hand, a logical (still provocative) answer would hypothetize that there should be dozens of distinct species, each for each biogeographical unit of mangroves, if isolation would be warranted… or (reversibly and still provocative) a single species with a chaotic continuum of variations ;

what we know with a certain comfort (is there any comfort in systematics and taxonomy ?) is the following :

  1. up to now 7 available names have been described for the superspecies : K. ocellatus (Hensel, 1868), K. marmoratus (Poey, 1880), K. heyei (Nichols, 1914), K. garciai (Cruz & Dubitsky, 1976), K. bonairensis (Hoedeman, 1958), K. caudomarginatus (Seegers, 1984), K. hermaphroditus Costa, 2011 (a smart name, if confirmed) ;
  2. if 1 only preferably hermaphroditic species is due to remain valid, then the name marmoratus must only be used because the I.C.Z.N. (1995) has decided so by ruling out the name ocellatus as invalid in case it would be a synonym of marmoratus (although ocellatus is more senior than marmoratus), on the basis that marmoratus has been a name in prevailing usage ; on the other hand if 2 or more preferably hermaphroditic species are due to remain valid, then the name marmoratus is still valid and "other" names will be resurrected ;
  3. the supposedly distinctive osteological and morphological characters between marmoratus and the southern populations near Rio de Janeiro in Brasil, with then a redescription and revalidation of ocellatus (in line with the prior I.C.Z.N. ruling) as disclosed by Costa in 2004 and 2006 appear somewhat useless (the phenotypes in southern Cuba and the 3 other nearby populations genetically closer to ocellatus are not morphologically distinguishable from marmoratus) ;
  4. the preferably standard and maybe also hermaphroditic (evidenced only from gonadal tissues that are similar to those of the Floridan populations with proven biological hermaphroditism and that are distinctive from the strictly standard, not hermaphroditic, but related and vicariant K. brasiliensis) species living from south Rio de Janeiro to Santos (at least), in southeastern Brasil is also valid but it is not stably named at the present time, either K. caudomarginatus following Seegers (1984) or K. ocellatus following Costa (2011), both using contradictory observations on the same morphological characters (a face-to-face conflict) ;
  5. the preferably hermaphroditic species living from around Rio de Janeiro (and northerly with no clear boundary), if also valid (as per point 2) is not stably named at the present time, either K. ocellatus following Seegers (1984) or K. hermaphroditus following Costa (2011), both using contradictory observations on the same morphological characters (the same face-to-face conflict) ;
  6. there is an urgent need of the restudy of the unique type of K. ocellatus (Hensel, 1868) to clear out the crucial and thorny issue (as per points 4 and 5) by a third, preferably German speaking, party (for the precise understanding of the German texts), because both Seegers is a renown amateur morphologist (with a Ph.D. thesis on central African Killifishes, precisely on morphology) and Costa is a renown professional specialist of morphology and osteology of South American fishes with more than 200 new fish names since 1989 (mainly, but not only, Killifish).
 

Unfortunately the certainties are by far outweighted by uncertainties (please read carefully !) :
  1. the definition of the species concept is herein exceptionally uneasy (whatever definition is chosen among many) because the genetics are only known at both ends but with little help (they are distinctive at both ends and also distinctive in one -the northern- end, at least !) while the morphology somewhat varies (on alas few studied populations with poorly known variations, as a continuum, as an artefact, as a methodology bias or as local events ?) and the color pattern is broadly uniform along the distribution (knowing that the color pattern is strongly labile according to mood within a population of any Kryptolebias) ;
  2. the osteo-morphology is known from various points but the alleged differences are not congruent with genetics (and palaeo-history) ;
  3. the distribution is scattered over a huge continuum (there is yet no support in palaeo-history for a neotropical species extending by dispersion northerly from the south of Rio de Janeiro around 20 MYA, then diversifying in a much smaller area around Cuba, even if Cuba is acknowledged to have been a fragmented series of islands in palaeo-history) ;
  4. the punctual event of hermaphroditism is not congruent with such a (very old) distribution and only 2 species (if several biological species are involved, the speciation would have taken place after that event and then a north-south polarity at the 2 extremes seems unlikely, rather a multiple speciation within that huge distribution, since 20 MYA, seems more likely, but then the compulsory morphological differentiation with biogeography is missing) ;
  5. the presence of a distinct phenotype with standard breeding behavior (and also hermaphroditism, at least histologically) at the southern dead end is not simplifying the situation and might even be in contradiction with some of the above argumentation (reversal or less old speciation or original stem but without northern outcome or a vanished one !), even if molecular data suggest it is more primitive (while the standard sexual behavior is more primitive than hermaphroditism !) ;
  6. if the molecular study of populations from southern central America and from northern south America reveals some heterogeneity that might imply isolation (a significant hypothesis), then the systematic situation will become even more obscure and the practical move will be either for a single polytypic species (unavoidable but satisfactory) or for a series of cryptic unseparable species (tasting modern and with systematic value, but with little practical interest) ;
 

unfortunately again, in terms of systematics, the issue is not simply one or several hermaphroditic-focused species… the issue is also how to name the phenotype with standard breeding behavior from the extreme south, either ocellatus described by Hensel or caudomarginatus described by Seegers ;

in total the present taxonomic and systematic situation is so ambiguous and unclear that it should be given full priority (to review the unique type of ocellatus by a third party, to study molecularly several populations outside the dead ends of distribution and disclose their relationships to either of the named taxa if any rational appears) : pending clarification, Costa's proposal has been followed in Killi-Data as the latest published scientific evidence (with reservation) ;

yes, that embarrassing situation is clearly a fact, but we can live with it if not too lengthened, can't we ?

and in any case, the scientific momentum is very positive and encouraging (the publication of the whole genome -not only mitochondrial- of K. marmoratus is expected soon, many new publications are in the pipe line and many herein arguments produced on the symposium will be outdated), a little bit more dynamism in collections and cooperation between aquarist collectors and scientists would change the face of that (small) world ; hence, if you are a Brasilian aquarist collector, do collect the fish from anywhere between North of Amazon to Bahia, if you are a French aquarist collector, do collect the fish from anywhere in Guyane (there are already records, but no DNA's), if you are a Dutch aquarist collector, do collect the fish from anywhere in the Netherlands Antilles or in Suriname, if you are a Spanish aquarist collector, do collect the fish from anywhere in central America to Venezuela (there is already a record from Maracaibo city, but no DNA's), if you are an aquarist collector from anywhere, do collect the fish from anywhere between Guatemala to eastern Brasil ! This is not very demanding since you have more than 7 000 straight line kilometers of virgin territories (but not easy, admittedly… and with necessary mangrove) ; you just need to spare a few live (or if impossible 95% ethanol preserved) hermaphrodites for researchers attending to that symposium (of course Killi-Data can be your ambassador) ;

and by the way if you are an 'ordinary' aquarist after reading this difficult technical text (congratulations for your efforts), do you still consider that this type of research on such an ugly fish is worthless to you and your aquarium practise (i.e., only appealing to a bunch of naked skull brained scientists) ? Well, let's think of it over a minute : first, as an 'ordinary' person you might imagine a little bit, even without prior knowing, that huge progresses on human cancer research has been already achieved (and is still in progress) with ras and p53 targets that our fish helps to understand better as a model ; second, as an 'ordinary' killi-hobbyist with sometimes harshy problems of in-breeding issues, of unbalanced sexation, of strange aggressiveness, of moody-about-sex females (or males) in your strain (while your pal in the killifish association next door is enjoying no such problems with 'exactly' the same water, the same processes, the same strain), you might imagine, even without prior knowing, that this basic research on this ugly fish has already set paths of better understanding those harshy issues, simply because it is a unique mix-up of differentiated clones that react differently by behavior, by sexation, by breathing, by tolerance to pollution, by hormonal, chemical or parasitic contents in water (etc.) ; third, as an 'ordinary' but very experienced aquarist, you can see the huge scientific progresses achieved on this ugly and unique fish (but not so unique, just at the very end of a continuum… how about the related Rivulus ?) Mysteries, yes, yes, yes, but extremely appealing mysteries !

 

Addendum on January 17. 2014 : following this newsletter, Frans Vermeulen, a dedicated specialist on South American killifish who lives in Aruba an island of the Venezuelan coast has decided to go and collect the fish in his island based on very old collecting reports and he has been successful (see the YouTube report with perfect biotope illustration) and he has sent fish for DNA study to researchers, and he is going on another collecting trip in January 2014 to Bonaire with the same goals (Bonaire being the type locality of the taxon, bonairensis).

 

For today, let's celebrate this unique and primitive fish and all the researchers dedicated to it during the last 5 decades.

In total a very important and sensitive newsletter !
Also, very technical… and several K-D-I members have reviewed this text so that it is not untouchably too difficult to standard aquarist readers

Hopefully a boost to our community and a spur to speed up knowledge progress on our (beloved) fishes !

 

Take care and enjoy the scientific or aquaristic complexity of killifish !

Do not hesitate to ask questions for future Newsletters.

Visit frequently the website www.killi-data.org !

Thank you for your support over the years.

With my kindest regards.

Jean (and the K-D-I admin team who prepared the report and contacted the authors, among which the following are especially thanked for their solid cooperation : notably Brian Earley, who manages a full bibliography of more than 400 papers on the species, then Scott Taylor and Andrey Tatarenkov, who kindly answered specific questions, and Patricia A. Wright, Joanna L. Kelley, and last but not least Edward F. Orlando who provided copies of their works and encouragements to the team).

Bibliographic references of the symposium (special issue of Integrative and Comparative Biology, Volume 52, Issue 6, [pages] published December 2012) :
* Mangrove Killifish: An Exemplar of Integrative Biology: Introduction to the Symposium. - Orlando, Edward F. [721-723]
* Twenty-Four Years in the Mud: What Have We Learned About the Natural History and Ecology of the Mangrove Rivulus, Kryptolebias marmoratus ? - Taylor, D. Scott [724-736]
* The Possibility of De Novo Assembly of the Genome and Population Genomics of the Mangrove Rivulus, Kryptolebias marmoratus. - Kelley, Joanna L.; Yee, Muh-Ching; Lee, Clarence; Levandowsky, Elizabeth; Shah, Minita; Harkins, Timothy; Earley, Ryan L.; Bustamante, Carlos D. [737-742]
* Microevolutionary Distribution of Isogenicity in a Self-fertilizing Fish (Kryptolebias marmoratus) in the Florida Keys. - Tatarenkov, Andrey; Earley, Ryan L.; Taylor, D. Scott; and John C. Avise [743-752]
* The Effects of Salinity on Acute Toxicity of Zinc to Two Euryhaline Species of Fish, Fundulus heteroclitus and Kryptolebias marmoratus - Bielmyer, Gretchen K.; Bullington, Joseph B.; DeCarlo, Carri A.; Chalk, Stuart J.; Smith, Kelly [753-760]
* Manipulation and Imaging of Kryptolebias marmoratus Embryos. - Mourabit, Sulayman; Kudoh, Tetsuhiro [761-768]
* Creating Females? Developmental Effects of 17[alpha]-Ethynylestradiol on the Mangrove Rivulus' Ovotestis. - Farmer, Jennifer L.; Orlando, Edward F. [769-780]
* Establishing Developmental Genetics in a Self-fertilizing Fish (Krytolebias marmoratus). - Moore, Ginger L.; Sucar, Sofia; Newsome, Jennifer M.; Ard, Melissa E.; Bernhardt, Lynda; Bland, Michael J.; Ring, Brian C. [781-791]
* Environmental Physiology of the Mangrove Rivulus, Kryptolebias marmoratus, A Cutaneously Breathing Fish That Survives for Weeks Out of Water. - Wright, Patricia A. [792-800]
* Aggression and Related Behavioral Traits: The Impact of Winning and Losing and the Role of Hormones. - Chang, Ching; Li, Cheng-Yu; Earley, Ryan L.; Hsu, Yuying [801-813]
* Phenotypic Plasticity and Integration in the Mangrove Rivulus (Kryptolebias marmoratus): A Prospectus. - Earley, Ryan L.; Hanninen, Amanda F.; Fuller, Adam; Garcia, Mark J.; Lee, Elizabeth A. [814-827]


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