The Arrangement of the Peripheral Olfactory System of Pleuragramma antarcticum: A Well-Exploited Small Sensor, an Aided Water Flow, and a Prominent Effort in Primary Signal Elaboration.

The olfactory system is constituted in a consistent way across vertebrates. Nasal structures allow water/air to enter an olfactory cavity, conveying the odorants to a sensory surface. There, the olfactory neurons form, with their axons, a sensory nerve projecting to the telencephalic zone-named the olfactory bulb. This organization comes with many different arrangements, whose meaning is still a matter of debate. A morphological description of the olfactory system of many teleost species is present in the literature; nevertheless, morphological investigations rarely provide a quantitative approach that would help to provide a deeper understanding of the structures where sensory and elaborating events happen. In this study, the peripheral olfactory system of the Antarctic silverfish, which is a keystone species in coastal Antarctica ecosystems, has also been described, employing some quantitative methods. The olfactory chamber of this species is connected to accessory nasal sacs, which probably aid water movements in the chamber; thus, the head of the Antarctic silverfish is specialized to assure that the olfactory organ keeps in contact with a large volume of water-even when the fish is not actively swimming. Each olfactory organ, shaped like an asymmetric rosette, has, in adult fish, a sensory surface area of about 25 mm2, while each olfactory bulb contains about 100,000 neurons. The sensory surface area and the number of neurons in the primary olfactory brain region show that this fish invests energy in the detection and elaboration of olfactory signals and allow comparisons among different species. The mouse, for example-which is considered a macrosmatic vertebrate-has a sensory surface area of the same order of magnitude as that of the Antarctic silverfish, but ten times more neurons in the olfactory bulb. Catsharks, on the other hand, have a sensory surface area that is two orders of magnitude higher than that of the Antarctic silverfish, while the number of neurons has the same order of magnitude. The Antarctic silverfish is therefore likely to rely considerably on olfaction.

Antarctic ecosystems in transition - life between stresses and opportunities.

Important findings from the second decade of the 21st century on the impact of environmental change on biological processes in the Antarctic were synthesised by 26 international experts. Ten key messages emerged that have stakeholder-relevance and/or a high impact for the scientific community. They address (i) altered biogeochemical cycles, (ii) ocean acidification, (iii) climate change hotspots, (iv) unexpected dynamism in seabed-dwelling populations, (v) spatial range shifts, (vi) adaptation and thermal resilience, (vii) sea ice related biological fluctuations, (viii) pollution, (ix) endangered terrestrial endemism and (x) the discovery of unknown habitats. Most Antarctic biotas are exposed to multiple stresses and considered vulnerable to environmental change due to narrow tolerance ranges, rapid change, projected circumpolar impacts, low potential for timely genetic adaptation, and migration barriers. Important ecosystem functions, such as primary production and energy transfer between trophic levels, have already changed, and biodiversity patterns have shifted. A confidence assessment of the degree of 'scientific understanding' revealed an intermediate level for most of the more detailed sub-messages, indicating that process-oriented research has been successful in the past decade. Additional efforts are necessary, however, to achieve the level of robustness in scientific knowledge that is required to inform protection measures of the unique Antarctic terrestrial and marine ecosystems, and their contributions to global biodiversity and ecosystem services.

Quantification of neurons in the olfactory bulb of the catsharks Scyliorhinus canicula (Linnaeus, 1758) and Galeus melastomus (Rafinesque, 1810).

In vertebrates, the olfactory bulb (OB) is the zone of the brain devoted to receiving the olfactory stimuli. The size of the OB relative to the size of the brain has been positively correlated to a good olfactory capability but, recently, this correlation was questioned after new investigation techniques were developed. Among them, the isotropic fractionator allows to estimate the number of neurons and non-neurons in a given portion of nervous tissue. To date, this technique has been applied in a number of species; in particular the OB was separately analyzed in numerous mammals and in a single crocodile species. Thus, a quantitative description of the OB's cells is available for a small portion of vertebrates. Main aim of this work was to apply isotropic fractionator to investigate the olfactory capability of elasmobranch fishes, whose traditional concept of outstanding olfaction has recently been scaled down by anatomical and physiological studies. For this purpose, the OB of two elasmobranch species, Galeus melastomus and Scyliorhinus canicula, was studied leading to the determination of the number of neurons vs non-neurons in the OB of the specimens. In addition, the obtained cell quantification was related to the olfactory epithelium surface area to obtain a new parameter that encapsulates both information on the peripheral olfactory organ and the OB. The analyzed species resulted in an overall similar quantitative organization of the peripheral olfactory system; slight differences were detected possibly reflecting different environment preference and feeding strategy. Moreover, the non-neurons/neurons ratio of these species, compared to those available in the literature, seems to place elasmobranch fishes among the vertebrate species in which olfaction plays an important role.

High mercury levels in Antarctic toothfish Dissostichus mawsoni from the Southwest Pacific sector of the Southern Ocean.

Mercury is a bioaccumulating toxic pollutant which can reach humans through the consumption of contaminated food (e.g. marine fish). Although the Southern Ocean is often portrayed as a pristine ecosystem, its fishery products are not immune to mercury contamination. We analysed mercury concentration (organic and inorganic forms - T-Hg) in the muscle of Antarctic toothfish, Dissostichus mawsoni, a long-lived top predator which supports a highly profitable fishery. Our samples were collected in three fishing areas (one seamount and two on the continental slope) in the Southwest Pacific Sector of the Southern Ocean during the 2016/2017 fishing season. Mercury levels and the size range of fish varied between fishing areas, with the highest levels (0.68 ± 0.45 mg kg-1 wwt) occurring on the Amundsen Sea seamount where catches were dominated by larger, older fish. The most parsimonious model of mercury concentration included both age and habitat (seamount vs continental slope) as explanatory variables. Mean mercury levels for each fishing area were higher than those in all previous studies of D. mawsoni, with mean values for the Amundsen Sea seamount exceeding the 0.5 mg kg-1 food safety threshold for the first time. It might therefore be appropriate to add D. mawsoni to the list of taxa, such as swordfish and sharks, which are known to exceed this threshold. This apparent increase in mercury levels suggests a recent contamination event which affected the Southwest Pacific sector, including both the Amundsen and Dumont D'Urville seas.

Cytogenetic characterization of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902) through analysis of mitotic chromosomes from early larvae.

This paper describes the cytogenetic features of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902), a keystone species of the Antarctic coastal marine ecosystem. Conventional cytogenetic analyses and physical mapping of repetitive DNA sequences were performed on metaphase plates obtained through direct chromosome preparation from P. antarctica early larvae. The Antarctic silverfish have a diploid number (2n) = 48, and a karyotype made up of a majority of two-armed chromosomes (karyotype formula36m/sm + 10st + 2a, fundamental number = 94). Major ribosomal gene repeats were detected on three chromosome pairs (20, 21, and 23), in correspondence of dim DAPI stained regions. Long Interspersed Nuclear Elements (LINEs) were abundant and wide spread over all chromosomes. Overall, the cytogenetic data presented herein are consistent with a long independent cytogenetic and evolutionary history for the species. The large number of two-armed chromosomes, indicative of highly-rearranged karyotype, coupled with a diploid number of 48, a presumed primitive character for this fish group, and the spread of the major ribosomal genes on three chromosome pairs, make the Antarctic silverfish distinct from all other notothenioid species.

Secondary Folds Contribute Significantly to the Total Surface Area in the Olfactory Organ of Chondrichthyes.

The olfactory organ of Chondrichthyes is characterized by a central support with several lamellae covered by a sensory olfactory epithelium. Although secondary folds are present on the lamellae in all the chondrichthyan species analyzed to date, their shape and size have not been described. We here analyze the olfactory organ of 13 elasmobranch and 1 holocephalan species, describe the shape of the secondary folds and evaluate how they contribute to the epithelial surface area. The secondary folds vary in shape and size, and they should always be considered when comparing the epithelial surface area among species; in fact, the increase of the area, due to the presence of the secondary folds, spans from 70 to 495% in the considered species. Because of the complexity of the shapes, we approach the description of the secondary folds by analyzing histological sections of the olfactory lamellae to obtain illustrative silhouettes. We introduce two indexes in order to describe a 2D-sectioned shape of the secondary folds. Considering the different numerical parameters which describe the morphology of the olfactory organ (secondary folds included), a principal component analysis elucidates the possible ecological role and phylogenetic relationship of the chondrichthyan olfactory organ.

Legacy persistent organic pollutants including PBDEs in the trophic web of the Ross Sea (Antarctica).

The ecological features of the Ross Sea trophic web are peculiar and different from other polar food webs, with respect to the use of habitat and species interactions; due to its ecosystem integrity, it is the world's largest Marine Protected Area, established in 2016. Polar organisms are reported to bioaccumulate lipophilic contaminant, viz persistent organic pollutants (POPs). Legacy POPs and flame retardants (polybrominated diphenyl ethers, PBDEs) were studied in key species of the Ross Sea (Euphausia superba, Pleuragramma antarctica) and their predators (Dissostichus mawsoni, Pygoscelis adeliae, Aptenodytes forsteri, Catharacta maccormicki, Leptonychotes weddellii). Gaschromatography revealed the presence of PCBs, HCB, DDTs, PBDEs in most of the samples; HCHs, dieldrin, Eldrin, non-ortho PCBs, PCDDs, PCDFs were detected only in some species. The average ∑PBDEs was 0.19-1.35 pg/g wet wt in the key-species and one-two order of magnitude higher in the predators. Penguins and skuas from an area where a long-term field camp is located showed higher BDE concentrations. The ΣDDTs was higher in the Antarctic toothfish (20 ± 6.73 ng/g wet wt) and in the South Polar skua (5.911 ± 3.425 ng/g wet wt). The TEQs were evaluated and the highest concentration was found in the Weddell seal, due to PCB169, 1,2,3,4,7,8-HxCDF, and 2,3,4,6,7,8-HxCDF. There was no significant relationship between the trophic level and the POP concentrations. Although low concentrations, organisms of the Ross Sea trophic web should be further studied: lack of information on some ecotoxicological features and human impacts including global change may distress the ecosystem with unpredictable effects.

Clarification of the Terminology of the Olfactory Lamellae in Chondrichthyes.

Several papers regard the anatomy of the peripheral olfactory organ, the olfactory rosette, in the class of Chondrichtyes. The complex shape of this organ and the differences among species give clues to functional, evolutionary, and ecological observations; data on a larger number of species are needed in order to have a more complete insight. The rosette is made up of a central support and of numerous lamellae, which are lined by the sensory epithelium. The size, shape and number of these lamellae, which are highly variable among species, are noteworthy because they affect the sensory surface area, the water dynamic within the olfactory chamber, and the organization of the olfactory bulb. In the literature about Chondrichthyes, the definition of olfactory lamella is confused, because authors use the same words for different structures. The average number of lamellae is distinctive for each species, and the meaning of this difference is not completely understood and, in this frame, a not unambiguous definition of lamella leads to difficulties in comparing data from different publications and analyzing them together. We aim to give here an unambiguous definition of olfactory lamella, that should be each single fold of tissue extending from the raphe, and of lamellar number of a given species, that should be the average number of lamellae in one olfactory organ. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:2039-2045, 2017. © 2017 Wiley Periodicals, Inc.

Gross anatomy and histology of the olfactory rosette of the shark Heptranchias perlo.

Sharks belonging to the family Hexanchidae have six or seven gill slits, unlike all other elasmobranchs, which have five gill slits. Their olfactory organs have a round shape, which is common for holocephalans, but not for elasmobranchs. Thus, the shape of the olfactory organ represents a further, less striking, peculiarity of this family among elasmobranchs. Despite that, the microscopic anatomy and histology of the olfactory organ have not yet been studied in any species of this family. Here, an anatomical and histological description of the olfactory organ of the sharpnose sevengill shark Heptranchias perlo is given. The organ is a rosette, with a central raphe and 31-34 primary lamellae, which bear secondary lamellae with a more or less branched shape. The elastic connective capsule which envelops the olfactory rosette possibly changes its shape along with water influx. In the olfactory epithelium, the supporting cells also have a secretory function, while no specialized mucous cells are visible; regarding this feature the olfactory epithelium of H. perlo differs from that of other chondrichthyan species. The immunohistochemical investigation of the sensory epithelium shows the absence of immunoreactivity for Gαolf in receptor neurons, which confirms previous observations in Chondrichthyes.

Evolution in an extreme environment: developmental biases and phenotypic integration in the adaptive radiation of antarctic notothenioids.

BACKGROUND: Over the past 40 million years water temperatures have dramatically dropped in the Southern Ocean, which has led to the local extinction of most nearshore fish lineages. The evolution of antifreeze glycoproteins in notothenioids, however, enabled these ancestrally benthic fishes to survive and adapt as temperatures reached the freezing point of seawater (-1.86 °C). Antarctic notothenioids now represent the primary teleost lineage in the Southern Ocean and are of fundamental importance to the local ecosystem. The radiation of notothenioids has been fostered by the evolution of "secondary pelagicism", the invasion of pelagic habitats, as the group diversified to fill newly available foraging niches in the water column. While elaborate craniofacial modifications have accompanied this adaptive radiation, little is known about how these morphological changes have contributed to the evolutionary success of notothenioids. RESULTS: We used a 3D-morphometrics approach to investigate patterns of morphological variation in the craniofacial skeleton among notothenioids, and show that variation in head shape is best explained by divergent selection with respect to foraging niche. We document further an accelerated rate of morphological evolution in the icefish family Channichthyidae, and show that their rapid diversification was accompanied by the evolution of relatively high levels of morphological integration. Whereas most studies suggest that extensive integration should constrain phenotypic evolution, icefish stand out as a rare example of increased integration possibly facilitating evolutionary potential. Finally, we show that the unique feeding apparatus in notothenioids in general, and icefish in particular, can be traced to shifts in early developmental patterning mechanisms and ongoing growth of the pharyngeal skeleton. CONCLUSION: Our work suggests that ecological opportunity is a major factor driving craniofacial variation in this group. Further, the observation that closely related lineages can differ dramatically in integration suggests that this trait can evolve quickly. We propose that the evolution of high levels of phenotypic integration in icefishes may be considered a key innovation that facilitated their morphological evolution and subsequent ecological expansion.

First Description of a Palatal Organ in Chimaera monstrosa (Chondrichthyes, Holocephali).

Chimaeroid fishes are the only extant Holocephali, a subclass of Chondrichthyes. We describe for the first time a well evidenced structure localized in the palate of the chimaeroid Chimaera monstrosa, here named a palatal organ (PO). Attention has been paid to the holocephalan head morphology, but there has been no mention of this particular organ in the literature. The PO is a soft-tissue mass located within a slight hollow in between the two vomerine toothplates, and it protrudes into the oral cavity, resembling the mammalian incisive papilla. It is characterized by dense connective tissue with abundant collagen and elastic fibers and no muscular tissue. The robust innervation but low density of taste buds suggest a role in gustation for the PO, but primary utility in general mechanical sensitivity likely implicated in food sorting. The presence of numerous multicellular serous glands in the anterior/dorsal part of the PO is quite surprising because, in gnathostome fish, the presence of multicellular glands within the mouth has been reported in only the rare case of teeth-associated venom glands. Hypothesized roles for these glands could include food lubrication, digestion and defense against pathogens. In the literature, the presence of a PO has been demonstrated in many published images of chimaeroid fishes, but has gone unnoticed. This trait could represent a peculiar characteristic of all or a subset of holocephalans.

A demonstration of nesting in two antarctic icefish (genus Chionodraco) using a fin dimorphism analysis and ex situ videos.

Visual observations and videos of Chionodraco hamatus icefish at the "Acquario di Genova" and histological analyses of congeneric species C. hamatus and C. rastrospinosus adults sampled in the field provided new anatomical and behavioral information on the reproductive biology of these white blooded species that are endemic to the High-Antarctic region. During the reproductive season, mature males of both species, which are different from females and immature males, display fleshy, club-like knob modifications of their anal fin that consisted of a much thicker epithelium. Histology indicated that the knobs were without any specialized glandular or sensorial organization, thus suggesting a mechanical and/or ornamental role of the modified anal fin. In addition, the occurrence of necrotic regions at the base of the thickened epithelium and the detachment of the knobs in post-spawning C. hamatus males indicated the temporary nature of the knobs. The role of these structures was confirmed as mechanical and was clarified using visual observations and videos of the behavior of two C. hamatus during a reproductive event that occurred in an exhibit tank at the "Acquario di Genova". The reproductive process included pre-spawning activity, preparation of the nest, egg guarding and successfully ended with egg hatching. When the spawning event approached, the male prepared the nest. The nest was constructed on an accurately selected bottom surface, which was flattened and maintained free from sand or debris by a combination of radial body movements and continuous anal fin sweeping, thus demonstrating the important mechanical/abrasive function of the anal fin knobs. The present data are the first records of active nesting in icefish and clarify the meaning of dimorphic temporary structures, whose function would have been difficult to obtain in the field.

First detection of taste buds in a chimaeroid fish (Chondrichthyes: Holocephali) and their Gαi-like immunoreactivity.

The mucosa covering the tongue of the Chimaera monstrosa has been investigated with histological and immunohistochemical methods allowing to describe, for the first time, gustatory structures (taste buds) in this subclass of cartilaginous fish. G-protein-alpha-subunit-inhibitory-like (Gαi-like) immunoreactivity has been detected in the taste buds of C. monstrosa, as described in other vertebrates. In order to gain confidence on the antiserum used, able to recognize three Gαi proteins in mammals, alignments of the antigenic sequence in mammals and other vertebrates were performed. The data were used for a research of putative genes in the genome of the holocephalan Callorhinchus milii, to date the only cartilaginous fish with a sequenced genome; the highlighted sequences could suggest the presence of all three genes (gnai1, gnai2 and gnai3) in holocephalans. The sequences of the predicted proteins present a high identity with the mammalian proteins.

Neuronal nitric oxide synthase (nNOS) immunoreactivity in the olfactory system of a cartilaginous fish.

Nitric oxide is a regulative molecule with important roles in the olfactory system of vertebrates. Chondrichtyans have a key position in vertebrate evolution and nothing is known about nitric oxide in their olfactory system. Aim of this work was to investigate the neuronal nitric oxide synthase (nNOS) immunoreactivity in the olfactory system of the shark Scyliorhinus canicula. Because nitric oxide is often related to GABA in the olfactory system, also the distribution of GABA and its synthesis enzyme GAD has been investigated. In the olfactory epithelium scattered cells in the basal and medial zone of the epithelium thickness presented nNOS-like immunoreactivity. In the olfactory bulb the nNOS-like immunoreactivity has been highlighted in nerve fibers around some blood vessels and in scattered GABAergic granule cells. The presence of nNOS in the olfactory system of S. canicula is overall lesser than that described in other vertebrates, even if nitric oxide probably keeps some essential functions.

First evidence of a leptin-like peptide in a cartilaginous fish.

Leptin is a hormone involved in food intake. Although leptin is evolutionarily conserved, no studies have investigated its presence in cartilaginous fish. Here, we report the presence of leptin-like immunoreactivity in the gastro-intestinal tract and liver of the cartilaginous fish Scyliorhinus canicula using western blot and immunohistochemical analyses. A leptin-like immunoreactive band of 16 kDa was detected in the homogenate of the stomach, whereas no immunoreactivity was observed in the intestine or the liver. Immunohistochemistry of the gastric mucosa revealed leptin-like staining localized to mucous-secreting cells and endocrine cells. This is the first report of a leptin-like peptide in a cartilaginous fish.

Cell proliferation and apoptosis in the olfactory epithelium of the shark Scyliorhinus canicula.

To date, no study has been published on cell renewal in the olfactory epithelium of Chondrichthyes. Our work aimed at detecting proliferating cells (by Proliferating Cell Nuclear Antigen - PCNA immunohistochemistry) and apoptotic cells (by terminal uridine deoxynucleotidyl transferase nick end labeling method) in the olfactory epithelium of the shark Scyliorhinus canicula. PCNA immunoreactivity and mitotic figures were localized almost exclusively at the basal and apical thirds of the epithelial thickness. Double immunofluorescence for PCNA and OMP (a marker of mature olfactory neurons) showed that PCNA immunoreactivity is lacking in mature olfactory neurons, with the exception of crypt neurons. Crypt neurons, a cell type peculiar to fish, often showed PCNA immunoreactivity in the nucleus and may be involved in repair processes. The role of PCNA in mature crypt neurons requires further investigation to be clarified. Apoptosis was observed in sensory neurons and in basal cells. Our data highlight the presence of cell proliferation at different levels within the epithelium and the occurrence of apoptosis in both mature and proliferating cells.

G protein alpha subunits in the olfactory epithelium of the holocephalan fish Chimaera monstrosa.

Receptor neurons in the olfactory and vomeronasal epithelia of vertebrates have dendritic specialization that is correlated to the receptor gene family they express and the G protein coupled with that receptor (in particular the G protein alpha subunit). There are not very many data in the literature about the morphological and molecular features of the olfactory epithelium of Chondrichthyes. In this work, the presence and distribution of different types of G protein alpha subunits (Galpha(o), Galpha(q) and Galpha(olf)) were investigated in the olfactory epithelium of the holocephalan Chimaera monstrosa using immunohistochemistry. Only Galpha(o)-like immunoreactivity was detected in the microvillous receptor neurons and in numerous axon bundles of the fila olfactoria. These preliminary data are in agreement with studies showing G protein alpha subunits in elasmobranchs and support the data present in the literature about putative odorant receptor families in the sequenced genome of the holocephalan Callorhinchus milii.

Molecular pedomorphism underlies craniofacial skeletal evolution in Antarctic notothenioid fishes.

BACKGROUND: Pedomorphism is the retention of ancestrally juvenile traits by adults in a descendant taxon. Despite its importance for evolutionary change, there are few examples of a molecular basis for this phenomenon. Notothenioids represent one of the best described species flocks among marine fishes, but their diversity is currently threatened by the rapidly changing Antarctic climate. Notothenioid evolutionary history is characterized by parallel radiations from a benthic ancestor to pelagic predators, which was accompanied by the appearance of several pedomorphic traits, including the reduction of skeletal mineralization that resulted in increased buoyancy. RESULTS: We compared craniofacial skeletal development in two pelagic notothenioids, Chaenocephalus aceratus and Pleuragramma antarcticum, to that in a benthic species, Notothenia coriiceps, and two outgroups, the threespine stickleback and the zebrafish. Relative to these other species, pelagic notothenioids exhibited a delay in pharyngeal bone development, which was associated with discrete heterochronic shifts in skeletal gene expression that were consistent with persistence of the chondrogenic program and a delay in the osteogenic program during larval development. Morphological analysis also revealed a bias toward the development of anterior and ventral elements of the notothenioid pharyngeal skeleton relative to dorsal and posterior elements. CONCLUSIONS: Our data support the hypothesis that early shifts in the relative timing of craniofacial skeletal gene expression may have had a significant impact on the adaptive radiation of Antarctic notothenioids into pelagic habitats.

First detection of neuropeptide Y (NPY)-like immunoreactivity in the lateral line: presence and distribution in the neuromasts of the Antarctic notothenioid fish Trematomus bernacchii.

The mechanosensory lateral line (LL) is involved in many fish and amphibian behaviors, however little is known about the molecules involved in the signal transmission. Neuropeptide Y (NPY) has a number of functions in vertebrate physiology and also plays important roles in different sensory systems. The Antarctic nototheniods are a monophyletic radiation of fishes that have evolved under the extreme environmental conditions of low light and cold, where non-visual sensory structures, such as LL, are of importance. In this study we describe the presence of NPY-like immunoreactivity (IR) in LL of the Antarctic nototheniod fish, Trematomus bernacchii Boulenger. Differences in size and cellular composition between the two neuromasts were in compliance with previous descriptions of these sensory organs. Despite structural and functional differences between canal and superficial neuromasts, the distribution of NPY-like IR was similar within both the receptors classes. In particular, NPY IR was observed in all three cell types which constitute these sensory organs, allowing us to hypothesize the involvement of this molecule in the processing of the sensory information.

GnRH immunodetection in the brain of the holocephalan fish Chimaera monstrosa L.: correlation to oocyte maturation.

Chimera monstrosa (rabbit fish) like other holocephalans is a rare, delicate deep sea fish. Owing to the difficulty of sampling individuals in good shape, there is a paucity of information available on the morphology and physiology of this species especially concerning reproduction. In holocephalans, a hypothalamus-pituitary-gonadal axis has been postulated and a GnRH molecule identical to cGnRH II has been identified. The aim of the present study was to correlate the presence of steroidogenic enzymes in the ovarian follicles with the presence of GnRH in the hypothalamus. Estrogens, the steroids that trigger the accumulation of yolk (vitellogenesis) in the oocytes are synthesized by the somatic cells of the follicle in the vitellogenic stages via a cascade of steroid dehydrogenases involving 3 beta-hydroxysteroid-dehydrogenase (3 beta-HSD; in the inner thecal layer) and aromatase cytochrome (P450; granulosa layer). Our results showed that 3 beta-HSD is present concomitant with the presence of cGnRH II in the preoptic area and in the ventral hypothalamus. Another form of immunoreactive GnRH, mGnRH is also present in the brain of C. monstrosa. It is localized in the ventral telencephalon and in the midbrain caudal diencephalon (boundary between ventral thalamus and tegmentum of the mesencephalon). This form of GnRH is probably correlated with sexual behaviour.