Workplan for BIC (POCTI/BIA-BDE/55463/2004)

Comunicação química na tilápia Moçambicana, Oreochromis mossambicus.

Chemical communication in the Mozambique tilapia, Oreochromis mossambicus.

Summary

Chemical communication is believed to play diverse and important roles in the biology of fishes. However, the number of species that have been studied in detail remains very low. Given their distinctive reproductive strategies and complex social behaviour, the cichlids have received surprisingly little attention in this respect. Over the past three years, our laboratory has made significant inroads into the understanding of chemical communication in the Mozambique tilapia, an African mouth-brooding cichlid. It is clear that this fish uses chemical signals both during reproduction and in the maintenance of social hierarchies. Thus the aim of the proposed project is to extend and embellish these initial findings, particularly with regard to the chemical cues that the females release, and answer some of the questions raised by previous research in both our and other laboratories. Firstly, the identification of putative pheromones released by pre-ovulatory females will be carried out in conjunction with IACR-Rothamsted (United Kingdom). We already know that pre-ovulatory females smell different from post-ovulatory to males, and that males behave differently towards them, depending on this olfactory cue. Our aim is to establish what these cues may be, and their likely source and routes of release. Once the likely site of phermone synthesis is established - the ovaries - we can assess the endocrine factors responsible for the regulation of pheromone production in vitro. This may also prove to be a convenient way to collect relatively large amounts of pheromones for identification. Thirdly, the effects of the putative pheromones on male physiology and behaviour will be investigated; we already have good evidence that female pheromones induce an increase in the urination rate of males as part of their courtship 'display' (male urine is a potent odorant to females). Lastly, we will begin to investigate how this pheromonal information is processed by the CNS using a combination of neuronal activity-dependent labelling and immunocytochemistry for the early response element c-fos; to where in the olfactory bulbs (and possibly beyond) this information is relayed. In the future, this will allow us to define how the pheromonal message is translated into the appropriate behavioural and physiological responses. This species has a number of advantages for this type of study; the social behaviour is well-described, males and females are easily recognisable and are reproductively active throughout the year. It is also a resilient fish and ameniable to the type of experimental manipulations outlined in this study. As such, it is an ideal introductory model for young scientists to learn how to formulate, and test, hypotheses. We think that the proposed project will establish the Mozambique tilapia as the model species for studies in chemical communication in cichlids and provide an important addition to the studies of this phenomenon in teleosts as a whole.

Description of the Project

i) Objectives

To identify chemicals released to the environment by pre-ovulatory females that are detected by males.

To identify the source and routes of release of these chemicals.

To investigate the effects of endocrine factors that govern the rate of synthesis of these putative pheromonal compounds by the ovaries (and/or other tissues) in vitro.

To identify the biological effects of these chemicals on male behaviour and physiology.

To trace the neural pathways of olfactory receptor neurones for these chemicals to the olfactory bulb.

vii) General Plan

Task 1; Chemical Identification of Putative Female Pheromone(s).

Investigadores; P.C. Hubbard, E.N. Barata, A.V.M. Canario (CCMar), BIC

John Pickett, Mike Birkett (IACR-Rothamsted).

Duração da Tarefa; 24 months

Person-months; 36

Expected Results;

The chemical identity of the substances released by pre-ovulatory females which males can detect will be established. The likely sites of synthesis (ovaries) and routes of release (urine) will be investigated.

Description of the task;

Previous work has shown that males are able to distinguish pre-ovulatory (ripe) females from post-ovulatory females, and that this is mediated by olfaction. Thus, we have both an electro-physiological assay (the electro-olfactogram or EOG) and behavioural assay (increase in urination rate of males) for the putative pheromones released by pre-ovulatory females. With the collaboration of colleagues in IACR-Rothamsted (United Kingdom; Missions and Services), we aim to identify what these substances are. Female tilapia of known sexual status (pre- or post-ovulatory) will be placed in isolation tanks (20 l water) for a period of 2 hours. At the end of this period all the water will be extracted using C-18 solid-phase extraction (SPE) columns (Sep-Paks) as previously described (Frade et al., 2002). We will also attempt to take urine from the fish (this is easy with males, who tend to store urine in the bladder, but less so in females). Then the fish will be killed and the ovaries will be taken. The ovaries will be homogenised, filtered and the filtrates will be subjected to a similar SPE procedure. The olfactory potency of these samples will be tested by EOG in males. It is expected that the eluates of the water from pre-olulatory females will evoke larger responses than those from post-ovulatory females. We strongly suspect that the same pattern will apply to the eluates of the urine and ovaries. The most potent fractions will be taken to IACR-Rothamsted for HPLC (Missions and Services). The fractions produced will be brought back to Faro and each tested for EOG potency. Those fractions containing significant differences in olfactory potency between pre- and post-ovulatory females will be further fractionated (to single peaks) and these will again be tested for olfactory potency by EOG. Those peaks giving strong olfactory responses will be purified (HPLC) and putatively identified by gas-chromatography linked mass-spectrometry (GC-MS). Confirmation of bio-activity of the putative pheromones identified will be by EOG of synthetic compounds (if commercially available) and behavioural testing on males in visual, but not chemical, contact with females (Task 3). The method for recording the EOG from tilapia is run routinely in our laboratory, as the assessment of urination rates (Almeida et al., 2003; Frade et al., 2003; Frade et al., 2002; Miranda et al., 2003). Furthermore, we have been collaborating with IACR-Rothamsted for over two years in a different, but related, project (POCTI/BSE/45843/2002).

Task 2; Endocrine Regulation of Putative Pheromone Production in Females.

Investigadores; BIC, P.C. Hubbard, E.N. Barata, A.V.M. Canario (CCMar).

Duração da Tarefa; 24 months

Person-months; 36

Expected Results;

Those endocrine factors affecting the olfactory potency of incubates from ovaries and testes isolated in vitro will be established. This will allow us to predict which circulating factors regulate the rate of pheromone production in vivo. This may allow some predictions of the cellular pathways involved.

Description of the task;

Given that the release of the putative female pheromones is related to the ovulatory cycle, and assuming that the ovaries are the site of synthesis of these chemicals, the regulation of their synthesis is likely to be under endocrine control. This will be tested by incubating ovaries from pre- and post ovulatory females in vitro and exposing them to likely regulatory endocrine factors (e.g. GnRH, 11-keto testosterone, 17-oestradiol). Females of known sexual status (pre- or post-ovulatory) will be killed by a sharp blow to the head, and the gonads dissected out and weighed. These will be placed in organ chambers with aerated fish Ringer's solution at 27ºC (pump; Equipment) and allowed to equilibrate for one hour with frequent changes of Ringer's solution. One of each pair of gonads (the other acting as control) will be incubated with a candidate endocrine factor at concentrations chosen to reflect circulating levels in vivo. The control will receive vehicle only. After an hour's incubation, the incubate will be collected and subjected to SPE using C-18 Sep-Paks, as described above. The eluates of these incubates will then be assessed for olfactory potency by EOG on males. Those endocrine factors causing a clear concentration-dependent increase (or decrease) in the olfactory potency of incubates from ovaries or testes, within the physiological range, will be the likely candidate endocrine factors regulating pheromone production in vivo. Depending on the results from EOG experiments, we may also check the bio-activity of these eluates in the appropriate behavioural assays (see above). The eluates of these experiments can also be stored (in ethanol at -20ºC) for measurement of the putative pheromones once their chemical identity is known, and the appropriate assays have been developed. This may also provide a method by which we can produce relatively large amounts of the putative pheromones at a high concentration; this may help in the chemical identification steps (see above).

Task 3; Effects of Putative Female Pheromones on Male Behaviour and Physiology.

Investigadores; BIC, P.C. Hubbard, E.N. Barata, A.V.M. Canario (CCMar).

Duração da Tarefa; 24 months

Person-months; 36

Expected Results

The effect of chemicals identified in Task 1 on the urination rates of males will be established. Their effects on male courtship behaviour will also be investigated.

Description of the task

Previous work has shown that substances released by pre-ovulatory females cause a dramatic increase in the urination rate of males, and that this is mediated by olfaction (Almeida et al., 2003; Miranda et al., 2003). We will test which of those chemicals identified in Task 1 are responsible for this effect whilst identifying other possible effects of these substances on male courtship behaviour. Males will be placed in an aquarium which is divided down the middle by both an opaque plate and a Perspex plate; a female will be placed in the other half of the aquarium. To assess their urination rate, they will be injected with a dye that is excreted in the urine, and thus makes urination visible (Almeida et al., 2003; Appelt and Sorensen, 1999; Miranda et al., 2003). Their behaviour and urination rate will be recorded over 45 minutes. The opaque divider will then be removed; the male and female will be in visual, but not chemical, contact with each-other. Subsequent changes in behaviour and urination rate of the male will be recorded. Behaviour will be analysed using "The Observer" software (Noldus Information Technology); this is routinely used in our laboratory. A week later, the experiment will be repeated except that this time, eluate of pre-ovulatory female water (Task 1) will be added to the water on the male's side (at a concentration based on olfactory sensitivity as assessed by EOG; Task 1). Urination rates and behaviour will again be recorded and compared to previous experiments without chemical stimuli. The candidate female pheromones identified in Task 1 can then be tested in the same way to find which of them cause a change in urination rate and/or courtship behaviour of the male. Pilot experiments suggest that the male needs to be in visual contact with the female for the chemical stimuli to have a clear behavioural effect. Time permitting, we can investigate the relative importance of visual and chemical stimuli in evoking male courtship behaviour.

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