Tina Keller-Costa

Chemical communication in cichlids: A mini-review.

The family Cichlidae is well-known for pair-formation, parental care, territoriality, elaborate courtship and social organization. Do cichlids use chemical communication to mediate any of these behaviours? Early studies suggest that parent cichlids can discriminate between conspecific and heterospecific wrigglers (but not eggs) using olfactory cues. Some species are able to discriminate between their own brood and other conspecific broods based on olfaction. The young recognise conspecific adults (although not necessarily their parents) through the odorants they release. In both scenarios, protection of the young from predation is the likely selective force. Some male cichlids use urinary pheromones during courtship and spawning to attract females and induce ovulation. Females--in their turn--may base their mate-choice in part on assessment of those self-same pheromones. The same pheromonal system may be involved in establishing and maintaining the social hierarchies in lek-breeding cichlids. Individual recognition is also mediated by chemical communication. Finally, there is ample behavioural evidence that cichlids--like ostariophysan fish--release alarm cues that alert conspecifics to predation danger. Although the effects of these cues may be similar (e.g., increased shelter use, tighter schooling), they are different substances which remain to be identified. Cichlids, then, use chemical communication associated with many different behaviours. However, given the diversity of cichlids, little is known about the mechanisms of chemical communication or the chemical identity of the cues involved. The aim of this mini-review is to persuade those working with cichlids to consider the involvement of chemical communication, and those working in chemical communication to consider using cichlids.

Identity of a Tilapia Pheromone Released by Dominant Males that Primes Females for Reproduction

Knowledge of the chemical identity and role of urinary pheromones in fish is scarce, yet it is necessary in order to understand the integration of multiple senses in adaptive responses and the evolution of chemical communication [1]. In nature, Mozambique tilapia (Oreochromis mossambicus) males form hierarchies, and females mate preferentially with dominant territorial males, which they visit in aggregations or leks [2]. Dominant males have thicker urinary bladder muscular walls than subordinates or females and store large volumes of urine, which they release at increased frequency in the presence of subordinate males or preovulatory, but not postspawned, females [3-5]. Females exposed to dominant-male urine augment their release of the oocyte maturation-inducing steroid 17α,20β-dihydroxypregn-4-en-3-one (17,20β-P) [6]. Here we isolate and identify a male Mozambique tilapia urinary sex pheromone as two epimeric (20α- and 20β-) pregnanetriol 3-glucuronates. We show that both males and females have high olfactory sensitivity to the two steroids, which cross-adapt upon stimulation. Females exposed to both steroids show a rapid, 10-fold increase in production of 17,20β-P. Thus, the identified urinary steroids prime the female endocrine system to accelerate oocyte maturation and possibly promote spawning synchrony. Tilapia are globally important as a food source but are also invasive species, with devastating impact on local freshwater ecosystems [7, 8]. Identifying the chemical cues that mediate reproduction may lead to the development of tools for population control [9-11].

Evidence for selective bacterial community structuring in the freshwater sponge "Ephydatia fluviatilis"

To understand the functioning of sponges, knowledge of the structure of their associated microbial communities is necessary. However, our perception of sponge-associated microbiomes remains mainly restricted to marine ecosystems. Here, we report on the molecular diversity and composition of bacteria in the freshwater sponge Ephydatia fluviatilis inhabiting the artificial lake Vinkeveense Plassen, Utrecht, The Netherlands. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints revealed that the apparent diversities within the domain Bacteria and the phylum Actinobacteria were lower in E. fluviatilis than in bulk water. Enrichment of specific PCR-DGGE bands in E. fluviatilis was detected. Furthermore, sponge- and bulk water-derived bacterial clone libraries differed with respect to bacterial community composition at the phylum level. E. fluviatilis-derived sequences were affiliated with six recognized phyla, i.e., Proteobacteria, Planctomycetes, Actinobacteria, Bacteroidetes, Chlamydiae and Verrucomicrobia, in order of relative abundance; next to the uncultured candidate phylum TM7 and one deeply rooted bacterial lineage of undefined taxonomy (BLUT). Actinobacteria, Proteobacteria, and Bacteroidetes were the dominant bacterial phyla in the freshwater clone library whereas sequences affiliated with Planctomycetes, Verrucomicrobia, Acidobacteria and Armatimonadetes were found at lower frequencies. Fine-tuned phylogenetic inference showed no or negligible overlaps between the E. fluviatilis and water-derived phylotypes within bacterial taxa such as Alphaproteobacteria, Bacteroidetes and Actinobacteria. We also ascertained the status of two alphaproteobacterial lineages as freshwater sponge-specific phylogenetic clusters, and report on high distinctiveness of other E. fluviatilis specific phylotypes, especially within the Bacteroidetes, Planctomycetes and Chlamydia taxa. This study supports the contention that the composition and diversity of bacteria in E. fluviatilis is partially driven by the host organism.

Muscular hypertrophy of urinary bladders in dominant tilapia facilitates the control of aggression through urinary signals

The urination pattern of the Mozambique tilapia (Oreochromis mossambicus) depends on social context, and the olfactory potency of urine released depends on social rank (males) and reproductive status (females). This strongly suggests that urine mediates chemical communication in this species. The current study tested, firstly, whether urine production rate depends on sex or social status and, secondly, whether differences in urination pattern and volume of urine stored are associated with variation in the morphology of the urinary bladder. Finally, the effect of urination during aggressive male–male interactions was assessed. Urine production in catheterized fish depended neither on sex nor social status (males). Nevertheless, males had larger kidneys than females. Dominant males had heavier urinary bladders than subordinate males or females, mainly due to enlarged muscle fibres, thicker urothelium and a thicker smooth muscle layer. In male pairs wherein urination was prevented by temporary constriction of the genital papillae, social interaction escalated to aggression (mouth-to-mouth fighting) more rapidly and frequently than control pairs. This was accompanied by elevated plasma testosterone and 11-ketotestosterone levels. In control encounters, the male that initiated the aggressive behaviour was usually the winner of the subsequent fight; this did not happen when the males could not urinate. These results suggest that the larger, more muscular bladder of dominant males is an adaptation, facilitating higher urination frequency, post-renal modulation and storage of larger urine volumes for longer. It is likely that urinary pheromones modulate aggression in male–male encounters by providing information on the social rank and/or motivation of the emitter; males are unlikely to invest in costly highly aggressive

The Freshwater Sponge Ephydatia fluviatilis Harbours Diverse Pseudomonas Species (Gammaproteobacteria, Pseudomonadales) with Broad-Spectrum Antimicrobial Activity

Bacteria are believed to play an important role in the fitness and biochemistry of sponges (Porifera). Pseudomonas species (Gammaproteobacteria, Pseudomonadales) are capable of colonizing a broad range of eukaryotic hosts, but knowledge of their diversity and function in freshwater invertebrates is rudimentary. We assessed the diversity, structure and antimicrobial activities of Pseudomonas spp. in the freshwater sponge Ephydatia fluviatilis. Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprints of the global regulator gene gacA revealed distinct structures between sponge-associated and free-living Pseudomonas communities, unveiling previously unsuspected diversity of these assemblages in freshwater. Community structures varied across E. fluviatilis specimens, yet specific gacA phylotypes could be detected by PCR-DGGE in almost all sponge individuals sampled over two consecutive years. By means of whole-genome fingerprinting, 39 distinct genotypes were found within 90 fluorescent Pseudomonas isolates retrieved from E. fluviatilis. High frequency of in vitro antibacterial (49%), antiprotozoan (35%) and anti-oomycetal (32%) activities was found among these isolates, contrasting less-pronounced basidiomycetal (17%) and ascomycetal (8%) antagonism. Culture extracts of highly predation-resistant isolates rapidly caused complete immobility or lysis of cells of the protozoan Colpoda steinii. Isolates tentatively identified as P. jessenii, P. protegens and P. oryzihabitans showed conspicuous inhibitory traits and correspondence with dominant sponge-associated phylotypes registered by cultivation-independent analysis. Our findings suggest that E. fluviatilis hosts both transient and persistent Pseudomonas symbionts displaying antimicrobial activities of potential ecological and biotechnological value.

Chemical communication in tilapia: a comparison of Oreochromis mossambicus with O. niloticus.

In allopatric speciation species differentiation generally results from different selective pressures in different environments, and identifying the traits responsible helps to understand the isolation mechanism(s) involved. Male Mozambique tilapia (Oreochromis mossambicus) use urine to signal dominance; furthermore, 5β-pregnane-3α,17,20β-triol-3α-glucuronide (and its α-epimer, 5β-pregnane-3α,17,20α-triol-3α-glucuronide), in their urine is a potent pheromone, the concentration of which is correlated with social status. The Nile tilapia (Oreochromisniloticus) is a close relative; species divergence probably resulted from geographical separation around 6 million years ago. This raises the question of whether the two species use similar urinary chemical cues during reproduction. The olfactory potency of urine, and crude extracts, from either species was assessed by the electro-olfactogram and the presence of the steroid glucuronides in urine from the Nile tilapia by liquid-chromatography/mass-spectrometry. Both species showed similar olfactory sensitivity to urine and respective extracts from either species, and similar sensitivity to the steroid glucuronides. 5β-Pregnan-3α,17α,20β-triol-3α-glucuronide was present at high concentrations (approaching 0.5mM) in urine from Nile tilapia, with 5β-pregnan-3α,17α,20α-triol-3α-glucuronide present at lower concentrations, similar to the Mozambique tilapia. Both species also had similar olfactory sensitivity to estradiol-3-glucuronide, a putative urinary cue from females. Together, these results support the idea that reproductive chemical cues have not been subjected to differing selective pressure. Whether these chemical cues have the same physiological and behavioural roles in O. niloticus as O. mossambicus remains to be investigated.

A Multi-Component Pheromone in the Urine of Dominant Male Tilapia (Oreochromis mossambicus) Reduces Aggression in Rivals.

Males often use scent to communicate their dominance, and to mediate aggressive and breeding behaviors. In teleost fish, however, the chemical composition of male pheromones is poorly understood. Male Mozambique tilapia, Oreochromis mossambicus, use urine that signals social status and primes females to spawn. The urinary sex pheromone directed at females consists of 5β-pregnane-3α,17α,20β-triol 3-glucuronate and its 20α-epimer. The concentration of these is positively correlated with male social rank. This study tested whether dominant male urine reduces aggression in receiver males, and whether the pregnanetriol 3-glucuronates also reduce male-male aggression. Males were allowed to fight their mirror image when exposed to either: i) water control or a chemical stimulus; ii) dominant male urine (DMU); iii) C18-solid phase (C18-SPE) DMU eluate; iv) C18-SPE DMU eluate plus filtrate; v) the two pregnanetriol 3-glucuronates (P3Gs); or vi) P3Gs plus DMU filtrate. Control males mounted an increasingly aggressive fight against their image over time. However, DMU significantly reduced this aggressive response. The two urinary P3Gs did not replicate the effect of whole DMU. Neither did the C18-SPE DMU eluate, containing the P3Gs, alone, nor the C18-SPE DMU filtrate to which the two P3Gs were added. Only exposure to reconstituted DMU (C18-SPE eluate plus filtrate) restored the aggression-reducing effect of whole DMU. Olfactory activity was present in the eluate and the polar filtrate in electro-olfactogram studies. We conclude that P3Gs alone have no reducing effect on aggression and that the urinary signal driving off male competition is likely to be a multi-component pheromone, with components present in both the polar and non-polar urine fractions.

Olfactory sensitivity to steroid glucuronates in Mozambique tilapia suggests two distinct and specific receptors for pheromone detection

Cichlids offer an exciting opportunity to understand vertebrate speciation; chemical communication could be one of the drivers of African cichlid radiation. Chemical signals mediate key aspects in the lives of vertebrates and often are species specific. Dominant male Mozambique tilapia [Oreochromis mossambicus (Peters 1852)] release a sex pheromone, 5β-pregnan-3α,17α,20β-triol 3-glucuronate and its 20α-epimer, via their urine. The objective of this study was to assess the sensitivity, specificity and versatility of the olfactory system of O. mossambicus to other steroids and their conjugates using the electro-olfactogram. Oreochromis mossambicus was sensitive to several 3-glucuronidated steroids, but did not respond to prostaglandins, unconjugated steroids or 17- or 20-conjugated steroids. Stimulation of the olfactory epithelium with increasing concentrations (1 pmol l−1 to 10 μmol l−1) of 5β-pregnan-3α,17α,20β-triol 3-glucuronate, 5β-pregnan-3α,17α,20α-triol 3-glucuronate, 3α,17α-dihydroxy-5β-pregnan-20-one 3-glucuronate, etiocholanolone 3α-glucuronate and 17β-estradiol 3-glucuronate produced characteristic sigmoidal concentration–response curves. However, tilapia were most sensitive to 17β-estradiol-3-glucuronate, which also had the lowest apparent EC50 and maximal response amplitude. Cross-adaptation and binary mixture experiments suggested that 5β,3α-reduced pregnan- and androstan-3-glucuronates share (a) common olfactory receptor(s), whereas 17β-estradiol 3-glucuronate is detected via (a) distinct olfactory receptor(s). In conclusion, the Mozambique tilapia has evolved high olfactory sensitivity and specificity to 3-glucuronidated steroids through two distinct olfactory receptor types; one detecting a male sex pheromone and a second detecting 17β-estradiol 3-glucuronate, a putative female-derived signal. However, O. mossambicus differs markedly in its olfactory perception from the more recently derived East African cichlid Astatotilapia burtoni, suggesting that chemical communication could, indeed, be involved in speciation.

The gorgonian coral Eunicella labiata hosts a distinct prokaryotic consortium amenable to cultivation

ABSTRACT Microbial communities inhabiting gorgonian corals are believed to benefit their hosts through nutrient provision and chemical defence; yet much remains to be learned about their phylogenetic uniqueness and cultivability. Here, we determined the prokaryotic community structure and distinctiveness in the gorgonian Eunicella labiata by Illumina sequencing of 16S rRNA genes from gorgonian and seawater metagenomic DNA. Furthermore, we used a ‘plate‐wash’ methodology to compare the phylogenetic diversity of the ‘total’ gorgonian bacteriome and its ‘cultivatable’ fraction. With 1016 operational taxonomic units (OTUs), prokaryotic richness was higher in seawater than in E. labiata where 603 OTUs were detected, 68 of which were host‐specific. Oceanospirillales and Rhodobacterales predominated in the E. labiata communities. One Oceanospirillales OTU, classified as Endozoicomonas, was particularly dominant, and closest relatives comprised exclusively uncultured clones from other gorgonians. We cultivated a remarkable 62% of the bacterial symbionts inhabiting E. labiata: Ruegeria, Sphingorhabdus, Labrenzia, other unclassified Rhodobacteraceae, Vibrio and Shewanella ranked among the 10 most abundant genera in both the cultivation‐independent and dependent samples. In conclusion, the E. labiata microbiome is diverse, distinct from seawater and enriched in (gorgonian)‐specific bacterial phylotypes. In contrast to current understanding, many dominant E. labiata symbionts can, indeed, be cultivated. &NA; Graphical Abstract Figure. In contrast to current understanding, the phylogenetically distinct prokaryotic community inhabiting the gorgonian Eunicella labiata is highly amenable to cultivation, opening new avenues to study coral‐microbe interactions

Variation in Urinary Amino Acids in the Mozambique Tilapia: A Potential Signal of Dominance or Individuality?

The urine of male tilapia (Oreochromis mossambicus) contains a pheromone which is—at least in part—composed of two steroid glucuronates. However, the polar fraction of urine (not containing steroids) also contains potent odorants, the identity and relevance of which are unknown. Amino acids are polar, potent odorants for fish, and are often found at high concentrations in fish urine. We therefore measured urinary amino acid concentrations (by gas-chromatography/mass-spectrometry) and assessed their contribution to the odor of male urine (by electro-olfactogram)—do amino acids form part of the “dominance pheromone” in tilapia? Urine was taken from males of different social status (dominant and subordinate) and passed through C18 solid-phase extraction cartridges. The polar fraction (aqueous flow-through) contained highly variable levels (up to 17 mM) of l-arginine, l-glutamate, and l-phenylalanine. Urinary [l-arginine] was higher, and [l-phenylalanine] lower, in urine from dominant males than that from subordinates. Although no statistical differences were seen for urinary [l-glutamate], a weak positive correlation with the donor’s social rank was found. In addition, comparison of olfactory responses to an artificial mixture of amino acids, based on the concentrations in a pool of urine from dominant males, showed that these three constitute most—but not all—of the olfactory potency of the polar fraction. These results show that (1) urinary amino acids constitute part of the olfactory potency of male urine, (2) levels of l-arginine, l-phenylalanine and, to a lesser extent l-glutamate, depend on social status, and (3) odorants other than amino acids are present in the aqueous polar fraction. We suggest that amino acids contribute to a urinary “signature mixture”; this could be important in individual recognition during aggressive male–male interactions, wherein urine has previously been shown to play a modulatory role.