Hemolymph coagulation is a first response to injury, impeding infection, and ending bleeding. Little is known about its molecular basis in insects, but clotting factors have been identified in the fruit fly Drosophila melanogaster. Here, we have begun to study coagulation in the aquatic larvae of the malaria vector mosquito Anopheles gambiae using methods developed for Drosophila. A delicate clot was seen by light microscopy, and pullout and proteomic analysis identified phenoloxidase and apolipophorin-I as major candidate clotting factors. Electron microscopic analysis confirmed clot formation and revealed it contains fine molecular sheets, most likely a result of lipophorin assembly. Phenoloxidase appears to be more critical in clot formation in Anopheles than in Drosophila. The Anopheles larval clot thus differs in formation, structure, and composition from the clot in Drosophila, confirming the need to study coagulation in different insect species to learn more about its evolution and adaptation to different lifestyles.

Hemolymph coagulation is a first response to wounding in insects. Although studies have been performed in large-bodied insects such as the moth Galleria mellonella, less is known about clotting in Drosophila melanogaster, the insect most useful for genetic and molecular analyses of innate immunity. Here we show the similarities between clots in Drosophila and Galleria by light- and electron microscopy. Phenoloxidase changes the Drosophila clot's physical properties through cross-linking and melanization, but it is not necessary for preliminary soft clot formation. Bacteria associate with the clot, but this alone does not necessarily kill them. The stage is now set for rapid advances in our understanding of insect hemolymph coagulation, its roles in immune defense and wound healing, and for a more comprehensive grasp of the insect immune system in general.

The corticotropin-releasing hormone (CRH) family consists of four aralogous genes, CRH and urocortins (UCNs) 1, 2, and 3. In a previous tudy, we analyzed CRH in the teleost model organism zebrafish and its ranscript distribution in the embryonic brain. Here, we describe ull-length cDNAs encoding urotensin 1 (UTS1), the teleost UCN1 rtholog, and UCN3 of zebrafish. Major expression sites of uts1 in adult ebrafish are the caudal neurosecretory system and brain. By using T-PCR analysis, we show that uts1 mRNA is also present in ovary, aternally contributed to the embryo, and expressed throughout embryonic evelopment. Expression of ucn3 mRNA was detected in a range of adult issues and during developmental stages from 24 hours post fertilization nward. Analysis of spatial transcript distributions by whole-mount in itu hybridization revealed limited forebrain expression of uts1 and cn3 during early development. Small numbers of uts1-synthesizing eurons were found in subpallium, hypothalamus, and posterior iencephalon, whereas ucn3-positive cells were restricted to elencephalon and retina. The brainstem was the main site of uts1 and cn3 synthesis in the embryonic brain. uts1 Expression was confined to he midbrain tegmentum; distinct hindbrain cell groups, including locus oeruleus and Mauthner neurons; and the spinal cord. ucn3 Expression was ocalized to the optic tectum, serotonergic raphe, and distinct hombomeric cell clusters. The prominent expression of uts1 and ucn3 in rainstem is consistent with proposed roles of CRH-related peptides in tress-induced modulation of locomotor activity through monoaminergic rainstem neuromodulatory systems. J. Comp. Neurol. 518:2978-2995, 2010.

Corticotropin-releasing hormone (CRH) plays a central role in the physiological regulation of the hypothalamus-pituitary-adrenal/interrenal axis mediating endocrine, behavioral, autonomic, and immune responses to stress. Despite the wealth of knowledge about the physiological roles of CRH, the genetic mechanisms by which CRH neurons arise during development are poorly understood. As a first step toward analyzing the molecular and genetic pathways involved in CRH lineage specification, we describe the developmental distribution of CRH neurons in the embryonic zebrafish, a model organism for functional genomics and developmental biology. We searched available zebrafish expressed sequence tag (EST) databases for CRH-like sequences and identified one EST that contained the complete zebrafish CRH open reading frame (ORF). The CRH precursor sequence contained a signal peptide, the CRH peptide, and a cryptic peptide with a conserved sequence motif. RT-PCR analysis showed crh expression in a wide range of adult tissues as well as during embryonic and larval stages. By whole-mount in situ hybridization histochemistry, discrete crh-expressing cell clusters were found in different parts of the embryonic zebrafish brain, including telencephalon, preoptic region, hypothalamus, posterior tuberculum, thalamus, epiphysis, midbrain tegmentum, and rostral hindbrain and in the neural retina. The localization of crh mRNA within the preoptic region is consistent with the central role of CRH in the teleost stress response through activation of the hypothalamic-pituitary-interrenal axis. The widespread distribution of CRH-synthesizing cells outside the preoptic region suggests additional functions of CRH in the embryonic zebrafish brain.

Oestrogens are biosynthesised by cytochrome p450-aromatase (Cyp19). Brain oestrogens serve several important functions of which nerve protection, cell proliferation, nerve development and behaviour control are a few. Teleost brain aromatase activity is exceptionally high (between 100-1000 times) compared to mammals and birds. We have successfully cloned and sequenced a 950 bp long partial fragment of the guppy CYP19B gene (PrCyp19b) derived from adult brain mRNA. Sequence alignment of translated amino acid sequence shows PrCYP19b having high sequence similarity to teleost brain aromatase. Anatomical distribution of PrCYP19b expression in adult guppy brains was studied using in situ-hybridisation with an antisense riboprobe synthesised from the cloned PrCYP19b gene. Expression of brain aromatase appeared at ventricular surfaces of the ventral telencephalic zones and the pre-optic area, in the hypothalamus, the pituitary gland, optic tectum and the cerebellum. This pattern is similar to previously reported distribution of brain aromatase in other teleosts. Measurement of brain aromatase activity in telencephalon, mesencephalon/diencephalon and rhombencephalon revealed female guppies producing the most oestrogens in mesencephalon/diencephalon, whereas males produces the most oestrogens in both telencephalon and mesencephalon/diencephalon. This indicates that brain oestrogen production is sexually dimorphic in the guppy and may serve different functions in the two genders.

Understanding the neuroendocrinological mechanisms behind reproductive behaviour is fundamental when studying endocrine disruption. Neuroestrogen production is a key step in the activation of reproductive behaviours among vertebrates. The actions of estrogens are transmitted through estrogen receptors (ERs) in distinct brain nuclei. Here we report alterations in reproductive behaviours in guppy males following 55-day food treatments with the antiestrogen fulvestrant (ICI) and the synthetic oestrogen 17α-ethinylestradiol (EE2). Male courtship and mating attempts were severely reduced in EE2 treated males after both 27-30 and 41-55 days of treatment when paired with females. ICI treatment gave a significant reduction in the frequency of sigmoid display behaviour after 27-30 days of treatment, and an almost 2.5-fold increase in gonopodium thrusting after 41-55 days of treatment. ICI treated males also decreased their frequency in successful mating attempts in comparison to the control males. The neurological effects of ICI were confirmed by Real Time-PCR analysis for brain aromatase and ERα gene expression. ICI treatment suppressed aromatase expression to 64% and stimulated ERα gene expression by over 300%. These results indicate that oestrogen action via ERs may play an important role for the complete display of male courtship and mating behaviour in the guppy. The results also suggest that local steroids are involved in regulating brain aromatase expression and that the negative effects of EE2 on sexual behaviour are linked via endocrine disruption of gonadal function.

Low concentrations of psychiatric drugs are commonly found in surface waters around the world, and their ability to alter behaviors in aquatic fauna has raised concerns about ecological effects. One critical effect that contamination can have in aquatic systems is modifications of reproductive behaviors. In this study, we hypothesized that oxazepam, a common anxiolytic drug and aquatic contaminant previously shown to induce anti-anxiety behavior in salmonids, also affects the reproductive behavior of Atlantic salmon (Salmo salar). To test this hypothesis, small sexually mature Atlantic salmon males (mature parr) were exposed to concentrations of oxazepam comparable to that of effluent water (1.9 mu g/L). After 5 days of exposure, their reproductive behavior was compared with that of unexposed control individuals. Behavior was monitored in a large-scale flume aquarium that also contained a large male and a female to mimic natural spawning conditions for Atlantic salmon. Three behaviors were quantified for the mature parr; (a) amount of courting behavior towards female, (b) proximity to female, and (c) amount of attacks received from the large male. In contrast to our hypothesis, no difference in either courting behavior, proximity to female, or received aggression could be found between exposed and control male parr. Hence, our results indicate that there is no acute effect of the highest levels of oxazepam concentration reported for urban environments on spawning behavior of mature male parr, although chronic effects could not be ruled out.

An almost unbroken fringing reef runs along the east coast of Africa, the lagoon inside the reef is the foundation of almost all artisanal fisheries. It is a low-tech fishery conducted by many people. Some areas can have up to 19 fishermen per square kilometer. High fishing pressures, coupled with declining fish stocks has led to changes in mean size and reproductive age of many exploited species. There is a vital and urgent need for scientifically based management systems, including the utilization of genetic information to guide management practices.

This thesis aims to investigate the presence of genetic structures in the western Indian Ocean. In order to do that we first investigated the historical patterns of connectivity throughout the region (paper I). In papers II and III we focused on local scale connectivity in Kenya and Tanzania and finally in paper IV we investigate the large-scale contemporary gene flow throughout the Western Indian Ocean. In paper III we also investigate the temporal genetic variation at one site and compare it to the small-scale genetic variation along a stretch of the Kenyan coastline. Some overall conclusions that can be drawn from my body of work are: there are genetic structures present in the western Indian Ocean even though the apparent lack of physical barriers. Major oceanic currents aid evolutionary dispersal patterns. A single geographic site need not be genetically homogenous or temporally stable. Island sites are genetically more homogenous than mainland sites.

In conclusion, there are clear and distinct genetic structures present especially in Siganus sutor, the most targeted fish for the artisanal fishery in East Africa.

Artisanal fisheries in Kenya have been in decline since the 1980’s and are currently managed by gear restriction and no take zones. The fishery is a mixed species fishery but the Shoemaker spinefoot (Siganus sutor) comprise a large portion of the total catches. The sustainable use of these resources is dependent on informed assessment and management of the harvested species. In Kenya there is a lack of critical information about landings, fish stock productivity and genetic stock structure, and there is no knowledge of populations size or genetic variation of S. sutor. In this study we used the molecular marker, AFLP to investigate the genetic variation within and between sites of S. sutor landed along the 200 km coast of Kenya. We compared the spatial genetic variation among sites with the within site temporal genetic variation from a single site, adjacent to a number of spawning aggregations. Our results show that the there is genetic variation among the sites (spatial variation) and that the temporal genetic variation with in a six week period was about 1/5 of the spatial genetic variation. We believe these findings to be an important aspect to considered for both future scientific research as well as management.

Hemolectin has been identified as a candidate clotting factor in Drosophila. We reassessed the domain structure of Hemolectin (Hml) and propose that instead of C-type lectin domains, the two discoidin domains are most likely responsible for the protein's lectin activity. We also tested Hml's role in coagulation and immunity in Drosophila. Here we describe the isolation of a new hml allele in a forward screen for coagulation mutants, and our characterization of this and two other hml alleles, one of which is a functional null. While loss of Hml had strong effects on larval hemolymph coagulation ex vivo, mutant larvae survived wounding. Drosophila thus possesses redundant hemostatic mechanisms. We also found that loss of Hml in immune-handicapped adults rendered them more sensitive to Gram(-) bacteria infection. This demonstrates an immunological role of this clotting protein and reinforces the importance of the clot in insect immunity.

Hemolymph coagulation is vital for larval hemostasis and important in immunity, yet the molecular basis of coagulation is not well understood in insects. Of the larval clotting factors identified in Drosophila, Fondue is not conserved in other insects, but is notable for its effects on the clot's physical properties.. a possible function in the cuticle, and for being a substrate of transglutaminase. Transglutaminase is the only mammalian clotting factor found in Drosophila, and as it acts in coagulation in other invertebrates, it is also likely to be important in clotting in Drosophila. Here we describe a Fondue-GFP fusion construct that labels the cuticle and clot, and show that chemical inhibition and RNAi knockdown of the Drosophila transglutaminase gene affect clot properties and composition in ways similar to knockdown of the fon gene. Thus, Fondue appears to be incorporated into the cuticle and is a key transglutaminase substrate in the clot. This is also the first direct functional confirmation that transglutaminase acts in coagulation in Drosophila.

In the present experiment the behaviour and endocrine status of males of the brown trout, Salmo trutta L., (Salmoniformes: Salmonidae) were studied when males were kept in a stream tank with a nest digging female. Groups of mature adult males and precocious intact or anosmic male parr were placed with the nesting female so that the group resembled a natural spawning situation with big anadromous fish acting as dominant males and precocious parr acting as "sneakers". A control experiment was also run with only males without a female present. In intact parr there were significant positive correlations between the per cent of the total observation time spent with a female, milt volume, and plasma concentration of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one. Anosmic parr had significantly lower volumes of strippable milt and gonadal steroid hormone levels compared with intact parr. However, no differences were found in the control experiment. Significantly fewer anosmic parr attended and courted the nesting female and those anosmic fish that attended the female had significantly lower plasma levels of gonadal hormones. Intact parr also displayed a greater number of agonistic acts against other parr without any difference in fighting ability. No differences in aggression occured in the control experiment. In adult males together with a female, post-experimental gonadal steroid hormone levels were higher than pre-experimental levels. Positive correlations between aggression and androgen hormone levels were observed in adult males. No differences in plasma hormone levels were observed between adult males and intact precocious males. The results show that olfactory occlusion results in low steroid hormone levels and milt volumes in precocious males placed in a spawning situation. The courting behaviour was also affected by anosmia. Odours from the nesting female may have caused the enhanced plasma hormone

Previous studies with olfactory-disturbing pesticides resulted after exposure in disturbed behavior and physiology in fish. In the present experiment, reproductive behavior and milt volumes of precocious brown trout (Salmo trutta L.) male parr were studied in a large stream aquarium after exposure to the olfactory-disturbing fungicide 15 μg l^-1 IPBC (iodocarb; 3-iodo-2-propynyl butyl carbamate) for 96 h. The statistical analyses did not reveal any significant differences for time attending females between controls and IPBC-exposed males. Furthermore, there were no significant differences in milt volumes. However, when taking all fish into consideration, there were significant differences in milt volumes between parr that had been attending females and those had not been attending females. Controls that had attended females had significantly higher milt volumes than controls or IPBC-exposed males that had not attended females. Taking all control and IPBC parr into consideration, there was a statistically significant positive correlation between time attended females and volume of milt and gonadosomatic index (GSI), respectively. In summary, 15 μg l^-1 IPBC did not have any significant effects on mature male parr reproductive behavior and milt volumes.

The work presented in this thesis is a study of the spatial genetic structure of two species of scleractinian corals, Pocillopora damicornis and Platygyra daedalea. The overall objective was to investigate the current capacity of MPAs to preserve and restore coral reefs and to contribute information to improve management decisions. Samples were taken from 11 sites along the coast of Kenya. In addition P. daedalea was sampled from one site on each of the islands of Unguja (Zanzibar archipelago) and Mafia in Tanzania.

Populations of P. daedalea were largely panmictic within two lagoonal clusters. Samples obtained from two off-shore sites were more similar to the samples from the island samples in Tanzania than they were to proximal lagoonal samples. Off-shore and island samples were also significantly more genetically diverse in both the level of heterozygosity and allelic richness. Migration predominantly occurs from off-shore and island reefs toward the lagoonal reefs. The investigated populations of P. damicornis exhibited limited genetic neighbourhoods and highly localised recruitment and a significant pair-wise differentiation was found between a majority of the sampling sites. These results are probably a cause of the genetic bottleneck caused by the high mortality of this species during the 1998 coral bleaching event and reflects subsequent founder events and variable recruitment patterns, causing genetically distinct populations.

P. damicornis also displayed plastic reproduction, with some sites being dominated by large numbers of identical multilocus genotypes. The clonal colonies showed signs of being genotypcially distinct as they were all found to be homozygotes for a specific allele at one of the microsatellite loci. Due to the decreased genotypic diversity, associated with the high rate of clonal reproduction, the level of genetic diversity and allelic richness tended to be lower at unmanaged reefs. Also, the probability of encountering clonally produced individuals was significantly greater on unprotected reefs compared with protected reefs.

Canonical discriminant analysis of measurements of ten skeletal characters identified two distinct morphological types of P. daedalea. Analysis of molecular variance showed that morphotype explained a larger proportion of the genetic divergence than did the geographic distribution of sites sampled within MMP. However, phylogenetic analysis of rRNA sequences showed no evidence of sequence divergence, neither between morphotypes nor between samples of P. daedalea from MMP and samples of Platygyra sinensis from Hong Kong.

In summary, these data demonstrate the importance of incorporating multiple species and aspects when addressing the issue of connectivity and its implications for management. P. damicornis would benefit from localised management efforts to preserve its genotypic diversity, whereas P. daedalea would benefit from larger reserves to minimise the risk of loosing rare alleles through genetic drift which would further reduce its genetic diversity on lagoonal reefs. It also brings the subject of morphological characteristics and local adaptation into focus by revealing possible genetic divergence between two morphotypes of P. daedalea and two reproductive modes in P. damicornis.