Influence of size and spatial competition on the bioactivity of coral reef sponges

Sponges biosynthesize a wealth of secondary metabolites, many with novel structures and strong biological activity. Such compounds may serve multiple ecological roles including anti-predation, anti-fouling functionalities and are implicated in border defense or attack during spatial competition. Relative size of benthic organisms may also play an important role in competitive interactions. To determine if a relationship exists between individual size and bioactive metabolite production in the context of spatial competition, we examined three sponge species with different morphologies: the massive Coscinoderma matthewsi, the club-shaped branching Hyrtios erecta, and the fan-shaped Ianthella basta. Extracts from sponges of various sizes and competitive environments were examined using a cell based bioassay as a proxy of bioactivity. For I. basta, sponge size was correlated with bioactivity; the largest individuals generally being the most bioactive. In contrast, there was no correlation between size and bioactivity for either C. matthewsi or H. erecta. Bioactivity of sponges in this study were however highly variable among individuals, regardless of levels of competition. The prevalence of encroaching organisms was not correlated with sponge size for any of the three sponge species, suggesting that potential bioactivity is not influenced by surrounding competition.     

Metabolic fingerprinting as an indicator of biodiversity: towards understanding inter-specific relationships among Homoscleromorpha sponges

Sponges are an important source of secondary metabolites showing a great diversity of structures and biological activities. Secondary metabolites can display specificity on different taxonomic levels, from species to phylum, which can make them good taxonomic biomarkers. However, the knowledge available on the metabolome of non-model organisms is often poor. In this study, we demonstrate that sponge chemical diversity may be useful for fundamental issues in systematics or evolutionary biology, by using metabolic fingerprints as indicators of metabolomic diversity in order to assess interspecific relationships. The sponge clade Homoscleromorpha is particularly challenging because its chemistry has been little studied and its phylogeny is still debated. Identification at species level is often troublesome, especially for the highly diversified Oscarella genus which lacks the fundamental characters of sponge taxonomy. An HPLC–DAD–ELSD–MS metabolic fingerprinting approach was developed and applied to 10 Mediterranean Homoscleromorpha species as a rapid assessment of their chemical diversity. A first validation of our approach was to measure intraspecific variability, which was found significantly lower than interspecific variability obtained between two Oscarella sister-species. Interspecific relationships among Homoscleromorpha species were then inferred from the alignment of their metabolic fingerprints. The resulting classification is congruent with phylogenetic trees obtained for a DNA marker (mitochondrial COI) and demonstrates the existence of two distinct groups within Homoscleromorpha. Metabolic fingerprinting proves a useful complementary tool in sponge systematics. Our case study calls for a revision of Homoscleromorpha with further phylogenetic studies and identification of additional chemical synapomorphic characters.     

Efforts to develop a cultured sponge cell line: revisiting an intractable problem

Residents of the marine environment, sponges (Porifera) have the ability to produce organic compounds known as secondary metabolites, which are not directly involved in the normal growth, development, or reproduction of an organism. Because of their sessile nature, the production of these bioactive compounds has been interpreted as a functional adaptation to serve in an important survival role as a means to counter various environmental stress factors such as predation, overgrowth by fouling organisms, or competition for limited space. Regardless of the reasons for this adaptation, a variety of isolated compounds have already proven to demonstrate remarkable anticancer, fungicidal, and antibiotic properties. A major obstacle to the isolation and production of novel compounds from sponges is the lack of a large, reliable source of sponge material. Sponge collection from the sea would be environmentally detrimental to the already stressed and sparse sponge populations. Sponge production in an aquaculture setting might appear to be an ideal alternative but would also be cost-ineffective and sponge growth is extremely slow. A third approach involves the development of a sponge cell culture system capable of producing the necessary cell numbers to harvest for research purposes as well as for the eventual commercial-scale production of promising bioactive compounds. Unfortunately, little progress has been made in this direction other than the establishment of temporary cultures containing aggregates and fragments of cells. One impediment toward successful sponge cell culture might be ascribed to a lack of published knowledge of failed methodologies, and thus, time and effort is wasted on continued reinvention of the same methods and procedures. Consequently, we have undertaken here to chart some of our unsuccessful research efforts, our methodology, and results to provide the sponge research community with knowledge to assist them to better avoid taking the same failed pathways.     

Metabolic phenotype mediates the outcome of competitive interactions in a response‐surface field experiment

Competition and metabolism should be linked. Intraspecific variation in metabolic rates and, hence, resource demands covary with competitive ability. The effects of metabolism on conspecific interactions, however, have mostly been studied under laboratory conditions. We used a trait‐specific response‐surface design to test for the effects of metabolism on pairwise interactions of the marine colonial invertebrate, Bugula neritina in the field. Specifically, we compared the performance (survival, growth, and reproduction) of focal individuals, both in the presence and absence of a neighbor colony, both of which had their metabolic phenotype characterized. Survival of focal colonies depended on the metabolic phenotype of the neighboring individual, and on the combination of both the focal and neighbor colony metabolic phenotypes that were present. Surprisingly, we found pervasive effects of neighbor metabolic phenotypes on focal colony growth and reproduction, although the sign and strength of these effects showed strong microenvironmental variability. Overall, we find that the metabolic phenotype changes the strength of competitive interactions, but these effects are highly contingent on local conditions. We suggest future studies explore how variation in metabolic rate affects organisms beyond the focal organism alone, particularly under field conditions.     

Long-term culture of sponge explants: conditions enhancing survival and growth,and assessment of bioactivity

Sponges are an important source of secondary metabolites with pharmaceutical interest. This is the main reason for the increasing interest of sponge culture recent years. The optimal culture system depends on the species to be cultured: while some species easily produce sponge aggregates after dissociation (primmorphs), others show a great capacity to regenerate after fragmentation (explants). Corticium candelabrum is a Mediterranean bacteriosponge that can undergo asexual reproduction. We have taken advantage of this capability and cultured C. candelabrum explants under several experimental conditions. To find the best conditions for obtaining functional explants, we assayed a range of conditions, including seasons of collection, culture temperature, filtered versus filtered-sterile seawater, addition of antibiotics and proportion of ectosome. We monitored the changes in shape and ultrastructure during the formation of explants. After 24 h, TEM images showed the aquiferous system disarranged, in particular at the sponge periphery. From 2 to 4 weeks later, the aquiferous system regenerated, and fragments became functional sponges (explants). Explants were cultured under two regimes: in vitro and in a closed aquarium system. Antibiotics were only added to the in vitro culture to assess their effect on the symbiotic bacteria, which remained healthy despite the presence of antibiotics. Two food regimens (marine bacteria and green algae) were assayed for their ability to satisfy the metabolic requirements of explants. We monitored explant survival and growth. Explants showed a high long-term survival rate (close to 100%). Growth rates were higher in the closed aquarium system, without antibiotic addition, and fed with algae. Explants cultures were hardly contaminated because manipulation was reduced to a minimum and we used sterilized seawater. C. candelabrum produces bioactive molecules, which may play a defensive role in the sponge and may have pharmaceutical interest. The bioactivity of the explants was similar to that of wild sponges.     

Impact of an invasive alga (Womersleyella setacea) on sponge assemblages: compromising the viability of future populations

The effects of invasive species on native fauna are understudied, even though their consequences should be taken into consideration for the proper conservation and management of marine systems. Furthermore, bioinvasions may have greater consequences if they affect key structural species with slow dynamics such as marine sponges. We propose that reproductive output could be used as a potential early warning signal to detect possible future changes in population trends of long-lived species (i.e. sponges) as a result of biological invasions. The aim of this study was to investigate the effects of invasive algal (Womersleyella setacea) overgrowth on sponge reproduction by comparing the presence of reproductive elements (spermatic cysts, oocytes, embryos, and larvae) in sponges covered by a thick carpet of the invasive algae and in sponges dwelling in the same habitat but without the invasive algae. Three variables were calculated to assess the impact of the invasive alga on sponge reproduction: the reproductive effort, the proportion of individuals in reproduction, and the size of the reproductive structures. We studied eight sponge species representing the main components of the deep rocky reefs of the area. Our results showed that W. setacea had a strong negative effect on sponge reproduction in six out of eight sponge species studied, with lower and even nil reproductive structures on the sponges subjected to the algal overgrowth. Thus, considering that sexual reproduction is necessary for the persistence of most sponge populations, a significant and constant reduction of the reproductive effort may compromise their viability and affect future trends in these benthic systems.     

Insulin sensitivity is reflected by characteristic metabolic fingerprints--a Fourier transform mass spectrometric non-targeted metabolomics approach

Background

A decline in body insulin sensitivity in apparently healthy individuals indicates a high risk to develop type 2 diabetes. Investigating the metabolic fingerprints of individuals with different whole body insulin sensitivity according to the formula of Matsuda, et al. (ISI_Matsuda) by a non-targeted metabolomics approach we aimed a) to figure out an unsuspicious and altered metabolic pattern, b) to estimate a threshold related to these changes based on the ISI, and c) to identify the metabolic pathways responsible for the discrimination of the two patterns.

Methodology and Principal Findings

By applying infusion ion cyclotron resonance Fourier transform mass spectrometry, we analyzed plasma of 46 non-diabetic subjects exhibiting high to low insulin sensitivities. The orthogonal partial least square model revealed a cluster of 28 individuals with alterations in their metabolic fingerprints associated with a decline in insulin sensitivity. This group could be separated from 18 subjects with an unsuspicious metabolite pattern. The orthogonal signal correction score scatter plot suggests a threshold of an ISI_Matsuda of 15 for the discrimination of these two groups. Of note, a potential subgroup represented by eight individuals (ISI_Matsuda value between 8.5 and 15) was identified in different models. This subgroup may indicate a metabolic transition state, since it is already located within the cluster of individuals with declined insulin sensitivity but the metabolic fingerprints still show some similarities with unaffected individuals (ISI >15). Moreover, the highest number of metabolite intensity differences between unsuspicious and altered metabolic fingerprints was detected in lipid metabolic pathways (arachidonic acid metabolism, metabolism of essential fatty acids and biosynthesis of unsaturated fatty acids), steroid hormone biosyntheses and bile acid metabolism, based on data evaluation using the metabolic annotation interface MassTRIX.

Conclusions

Our results suggest that altered metabolite patterns that reflect changes in insulin sensitivity respectively the ISI_Matsuda are dominated by lipid-related pathways. Furthermore, a metabolic transition state reflected by heterogeneous metabolite fingerprints may precede severe alterations of metabolism. Our findings offer future prospects for novel insights in the pathogenesis of the pre-diabetic phase.     

Richness and bioactivity of culturable soil fungi from the Fildes Peninsula,Antarctica

Since the discovery of penicillin, fungi have been an important source of bioactive natural products. However, as a specific resource, the bioactive potentiality and specificity of fungal metabolites from the Antarctic region have had little attention. In this paper, we investigated the diversity patterns and biological activities of cultivable fungi isolated from soil samples in Fildes Peninsula, King George Island, Antarctica. Fungal communities showed low abundance and diversity; a total of 150 cultivable fungi were isolated from eight soil samples. After being dereplicated by morphological characteristics and chemical fingerprints, 47 fungal isolates were identified by ITS-rDNA sequencing. We confirmed that these isolates belonged to at least 11 different genera and clustered into nine groups corresponding to taxonomic orders in the phylogenetic analysis. Using two different fermentation conditions, 94 crude extracts acquired from the abovementioned different metabolite characteristic isolates were screened by bioactivity assay and 18 isolates produced biologically active compounds. Compared with HPLC–DAD–UV fingerprint analysis of culture extracts and standard compounds, two bioactive components secalonic acid and chetracins were identified. Our research suggests that the abundance and diversity of Antarctic cultivable fungal communities exhibit unique ecological characteristics and potential producers of novel natural bioactive products.     

Metagenomic approaches to exploit the biotechnological potential of the microbial consortia of marine sponges

Natural products isolated from sponges are an important source of new biologically active compounds. However, the development of these compounds into drugs has been held back by the difficulties in achieving a sustainable supply of these often-complex molecules for pre-clinical and clinical development. Increasing evidence implicates microbial symbionts as the source of many of these biologically active compounds, but the vast majority of the sponge microbial community remain uncultured. Metagenomics offers a biotechnological solution to this supply problem. Metagenomes of sponge microbial communities have been shown to contain genes and gene clusters typical for the biosynthesis of biologically active natural products. Heterologous expression approaches have also led to the isolation of secondary metabolism gene clusters from uncultured microbial symbionts of marine invertebrates and from soil metagenomic libraries. Combining a metagenomic approach with heterologous expression holds much promise for the sustainable exploitation of the chemical diversity present in the sponge microbial community.     

Pulsed-field gel electrophoresis fingerprinting for identification of Azospirillum species

Pulsed-field gel electrophoresis (PFGE) was used to obtain macrorestriction fingerprints of restriction enzyme-cut DNA of natural isolates of Azospirillum spp. Metabolic profiles, along with other phenotypic characteristics, were compared with these fingerprints to differentiate among the azospirilla isolates. A wide diversity of phenotypes (e.g., colony color, motility, and accumulation of poly--hydroxybutyrate granules) was observed among the natural isolates of azospirilla. PFGE revealed that TCTAGA, the sequence recognized by Xba1, is rare in the genome of azospirilla. The PFGE fingerprint revealed that azospirilla associated with different crops have a very similar genetic background. PFGE fingerprints were more consistent in the identification of azospirilla isolates from specific hosts than the metabolic fingerprints. For further differentiation at strain level, metabolic, physiological, and morphological profiles provide additional information.Journal No. J-2920 of the Montana Agricultural Experiment Station.     

Primmorphs from archaeocytes-dominant cell population of the sponge hymeniacidon perleve: improved cell proliferation and spiculogenesis

Marine sponges (Porifera) possess an extraordinary diversity of bioactive metabolites for new drug discovery and development. In vitro cultivation of sponge cells in a bioreactor system is very attractive for the sustainable production of sponge-derived bioactive metabolites; however, it is still a challenging task. The recent establishment of sponge primmorphs, multicellular aggregates from dissociated mixed-cell population (MCP), has been widely acknowledged to hold great promise for cultivation in vitro. Here we present a new method to establish an in vitro sponge primmorph culture from archaeocyte-dominant cell population (ADCP) enriched by a Ficoll gradient, rather than a mixed-cell population (MCP). Our rationale is based upon the totipotency (the ability of a cell to differentiate into other cell types) of archaeocyte cells and the different biological functions of various sponge cell types. A sponge, Hymeniacidon perleve collected from the China Yellow Sea was used as a model system for this investigation. Distinct dynamics of primmorph formation were observed while significant increases in DNA synthesis, cell proliferation (up to threefold), and cell growth (up to fourfold) were achieved. Furthermore, a time-dependent spiculogenesis was clearly demonstrated in our longterm culture, indicating high metabolic activity of primmorphs from the ADCP. This new method represents an important step forward to advance sponge cell culture in vitro that may lead to commercial exploitation of sponge-derived drugs.     

A rare continual flowering strategy and its influence on offspring quality in a gynodioecious plant

The majority of angiosperms have a single annual bout of reproduction; species that flower continually throughout the year are rare. Ochradenus baccatus is a gynodioecious, desert shrub whose principal flowering period is associated with the winter rains, although large individuals also remain in flower during the hot, dry summer. The goal of this study was to examine the reproductive consequences of continual flowering in a large population of O. baccatus in Israel. Over the two years of this study, 60% of individuals flowered continuously. The number of fruit and seeds per fruit were greater in winter. Winter seeds were ～12% heavier than summer seeds and had significantly higher germination rates (80 vs. 60%, respectively). Although summer seeds were smaller and less likely to germinate, we propose that the benefit derived from their production lies in their ability to capitalize on the first winter rains. These early rain events provide a head start on establishment and growth in the hostile desert environment. Plants that delay reproduction until the onset of rains risk having their offspring face the dry conditions of spring and summer.     

Diversity and biotechnological potential of the sponge-associated microbial consortia

Sponges are well known to harbor diverse microbes and represent a significant source of bioactive natural compounds derived from the marine environment. Recent studies of the microbial communities of marine sponges have uncovered previously undescribed species and an array of new chemical compounds. In contrast to natural compounds, studies on enzymes with biotechnological potential from microbes associated with sponges are rare although enzymes with novel activities that have potential medical and biotechnological applications have been identified from sponges and microbes associated with sponges. Both bacteria and fungi have been isolated from a wide range of marine sponge, but the diversity and symbiotic relationship of bacteria has been studied to a greater extent than that of fungi isolated from sponges. Molecular methods (e.g., rDNA, DGGE, and FISH) have revealed a great diversity of the unculturable bacteria and archaea. Metagenomic approaches have identified interesting metabolic pathways responsible for the production of natural compounds and may provide a new avenue to explore the microbial diversity and biotechnological potential of marine sponges. In addition, other eukaryotic organisms such as diatoms and unicellular algae from marine sponges are also being described using these molecular techniques. Many natural compounds derived from sponges are suspected to be of bacterial origin, but only a few studies have provided convincing evidence for symbiotic producers in sponges. Microbes in sponges exist in different associations with sponges including the true symbiosis. Fungi derived from marine sponges represent the single most prolific source of diverse bioactive marine fungal compounds found to date. There is a developing interest in determining the true diversity of fungi present in marine sponges and the nature of the association. Molecular methods will allow scientists to more accurately identify fungal species and determine actual diversity of sponge-associated fungi. This is especially important as greater cooperation between bacteriologists, mycologists, natural product chemists, and bioengineers is needed to provide a well-coordinated effort in studying the diversity, ecology, physiology, and association between bacteria, fungi, and other organisms present in marine sponges.     

Population-specific metabolic phenotypes of Arabidopsis lyrata ssp. petraea

Plant populations growing at the margin of their range may exhibit traits that indicate genetic differentiation and adaptation to their local abiotic environment. Here, it was investigated whether geographically separated marginal populations of Arabidopsis lyrata ssp. petraea have distinct metabolic phenotypes within the plant foliage. Seeds of A. petraea were obtained from populations along a latitudinal gradient (49-64 N), namely Germany, Wales, Sweden and Iceland and grown in a controlled cabinet environment. Targeted metabolic profiles and fingerprints were obtained at the same initial developmental stage. The free amino acid compositions were population specific, with fold differences in arginine, aspartic acid, asparagines, glycine, phenylalanine, alanine, threonine, histidine, serine and gamma-aminobutyric acid (GABA) concentrations. Sucrose, mannose and fructose concentrations were also different between populations but polyhydric alcohol concentrations were not. Principal component analysis (PCA) of metabolite fingerprints revealed metabolic phenotypes for each population. It is suggested that glucosinolates were responsible for discriminating populations within the PCA. Metabolite fingerprinting and profiling has proved to be sufficiently sensitive to identify metabolic differences between plant populations. These findings show that there is significant natural variation in metabolism among populations of A. petraea.     

Bioactivity-guided isolation of antimicrobial metabolite from Xylaria sp.

Symbiotic plant-microbe metabolic interactions not only have beneficial effects on plants but also contribute to rich, unmatched and complex chemical biodiversity with biological potential. Systematic delineated bioprospecting of fungal diversity associated with Ficus pumila Linn (Moraceae) for antimicrobial metabolite revealed Xylaria sp. FPL-25(M). The present study describes bioactivity guided fractionation prioritized for antimicrobial potential. Thus, chemical investigation of culture broth of Xylaria sp. FPL-25(M) by bioactivity guided fractionation with spectroscopic techniques revealed bioactive metabolite xylobovide-9-methyl ester. The xylobovide-9-methyl ester exhibited broad-spectrum antimicrobial activity. However, Gram-positive bacteria and fungi were more susceptible than Gram-negative bacteria. The present study results represent bioassay-based screening strategy which facilitates rapid, efficient and reliable approach for endophytic strain prioritization for novel bioactive molecules.     

海绵共附生微生物的研究新进展

海绵是最原始的一类后生动物,已被作为海洋活性化合物的重要来源之一,其独特的孔状结构使其成为许多海洋微生物的优良宿主。近年来国内对海绵及其共附生微生物的研究主要集中在它们产生的活性物质方面,但对海绵共附生微生物的分布、多样性及其对宿主海绵作用的研究鲜有报道,就国内外研究进展进行了综述。     

Variation in metabolic rate between populations of a geographically widespread lizard

In geographically widespread ectotherms, variation in life history phenotypes may be caused by differences in maintenance metabolism of individuals. I estimated daily and annual maintenance metabolism of eastern fence lizards, Sceloporus undulatus, from two populations with markedly different life histories; lizards in South Carolina grow faster, mature earlier, and have greater annual reproductive output than lizards in New Jersey. I measured diel cycles of resting metabolic rate (RMR) at four temperatures (20 degrees, 30 degrees, 33 degrees, and 36 degrees C) during spring, summer, and fall. In all seasons, RMR increased significantly from 20 degrees to 33 degrees C but did not differ significantly between 33 degrees and 36 degrees C. Adults from New Jersey had a higher RMR than adults from South Carolina in summer and fall but not in spring. Juveniles from South Carolina had a higher RMR than juveniles from New Jersey in summer but not in spring or fall. Annual maintenance metabolism of New Jersey lizards (53.7 kJ) was greater than that of South Carolina lizards (45.8 kJ), despite the shorter duration of activity in New Jersey. I conclude that the difference in maintenance metabolism between populations contributes to the greater production by S. undulatus in South Carolina.     

Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up < 1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.     

Temporal variations in growth and reproduction of Tedania anhelans and Chondrosia reniformis in the North Adriatic Sea

Most works concerning growth and reproduction of Mediterranean sponges have been performed in the oligotrophic western Mediterranean while little is known about sponge dynamics in the North-western Adriatic Sea, a basin characterized by low winter temperature and eutrophy. In order to deepen our understanding of sponges in the North Adriatic Sea and verify how its peculiar trophic and physical conditions affect sponge life cycles, temporal trend of sponge cover (%) and reproductive timing of Chondrosia reniformis and Tedania (Tedania) anhelans were studied over a 1-year period looking for a possible relation with variations of temperature or food availability. In C. reniformis, although little variations of sponge cover were evidenced around the year, the number of individuals and their size increase during spring. Asexual reproduction, via drop-like propagules, mainly occurs in spring and summer, while sexual reproduction is characterized by a maximum number of oocytes in August. T. anhelans progressively grows from spring to summer and develops propagules on its surface that reach their maximum size in July. In autumn, the sponge undergoes a process of progressive shrinkage and almost disappears in winter when temperature reaches 7–8°C. Larvae occur during summer. In the North Adriatic Sea sponges have larger sizes, higher density and a wider period of oocytes production compared with the same species from the Mediterranean Sea, suggesting these differences could be due to high food availability characterizing the eutrophic Adriatic basin. On the contrary, the sharp water temperature variations and the very low winter temperature, 5–6°C lower than what has been reported for the Mediterranean Sea, regulate temporal variations in abundance and cause the disappearance of thermophile species during winter.     

银鲫两个雌核发育克隆间两性生殖子代的遗传多样性分析

Based on the discovery of gonochoristic reproductive mode in silver crucian carp (Carassius auratus gibelio), 18 individuals from the mated offspring between clone F and clone D and their parents were analyzed using 11 RAPD primers. The mated offspring differentiated into three phenotypes. One phenotype is similar to that of clone F (SF). The second is similar to that of clone D (SD). The third shows a novel longer and thinner body type (NL). Electrophoretogram results show abundant polymorhic DNA fragments among individuals in the FD mated offspring. Obviously, the polymorphic DNA fragments originate from the genome recombination owing to the gonochoristic reproductive mode. The FD mated offspring amplify different fingerprints from maternal, and their fingerprints are different from each other. These different DNA fragments can be divided into four groups according to their origin. The average genetic distance among individuals from the FD offspring (0.23 +/- 0.123) is much higher than that of allogynogenetic offspring, which is only about 0.01. Of the three phenotypes in the FD offspring, the average distances among SD is the highest (0.235 +/- 0.097). The next is SF phenotype, in which the average distances is 0.148 +/- 0.073. The average distances among NL is the lowest (0.094 +/- 0.083). The gonochoristic reproductive mode introduces high genotypic variability and underlines genetic diversity. A dendrogram was constructed by NJTREE cluster analysis based on a total of 2320 distinguishable fragments (116 per individual). NL3 and maternal are most closely related, and SD3 and paternal are most closely related. The average genetic distances between SD and SF or NL (> 0.31) is higher than that of between SF and NL (0.124). Consistent with previous study, the genetic distance between maternal and paternal is 0.35. The average genetic distances between FD offspring and parents are both about 0. 32. But the similarities among the three phenotypes in mated group FD offspring and parents are different. The similarity between SF and maternal (71%) is higher than similarities among other phenotypes and maternal. The similarity between SD and paternal (70%) is higher than similarities among other phenotypes and paternal. The results suggest the similarities among three phenotypes in the FD offspring and parents are related with their phenotypes because some DNA fragments specific for phenotypes were amplified in this study. The revelation about reproductive diversity will be able to open the door in which complicated mechanism has been locked in unisexual organisms for long time, and establish the important roles of silver crucian carp in the studies on evolutionary genetics in unisexual or polyploid vertebrates.