Identification and functional analysis of the doublesex gene in the redclaw crayfish, Cherax quadricarinatus.

DM-domain (Zn-finger motif domain) genes play an important role in the sex determination and differentiation among animal kingdom. In the present study, the gene of Doublesex (Cqdsx) was identified and characterized for the first time in the redclaw crayfish, Cherax quadricarinatus. The full-length cDNA was 1271 bp, comprising a 155 bp 5'-untranslated region (5'-UTR), an 885 bp predicted open reading frame (ORF) encoding 294 amino acid polypeptides, and a 231 bp 3'-UTR. The deduced amino acid sequence of Cqdsx was predicted to contain a highly conserved DM domain and shared nearly 50% identity to DM-peptides from other species. The results of quantitative Real-time PCR in various tissues revealed that Cqdsx was strongly expressed in gonads, while was almost undetectable in gill, heart, hepatopancreas, muscle and intestine. Comparing expression level in different embryonic stages found that Cqdsx was gradually increased with the development of the embryos. In situ hybridization to gonad sections showed that intensive hybridization signals were mainly observed in oocytes and ovarian lamellae and weak signals were detected in spermatocyte. Additionally, Cqdsx gene exhibited higher transcript levels in the early stage of ovarian development. Furthermore, RNAi-targeting Cqdsx silencing induced a decrease of Cq-IAG trascripts, which regulate the male sexual differentiation in crustaceans. Taken together, these findings strongly suggest an essential role for Cqdsx in the female ovarian development/differentiation of the redclaw crayfish.

Transcriptomic characterisation of neuropeptides and their putative cognate G protein-coupled receptors during late embryo and stage-1 juvenile development of the Aotearoa-New Zealand crayfish, Paranephrops zealandicus.

We de novo assembled a transcriptome for early life-stages of the Aotearoa-New Zealand crayfish, Paranephrops zealandicus, establishing the first genetic resource for this under-developed aquaculture species and for the Paranephrops genus. Mining of this transcriptome for neuropeptides and their putative cognate G protein-coupled receptors (GPCRs) yielded a comprehensive catalogue of neuropeptides, but few putative neuropeptide GPCRs. Of the neuropeptides commonly identified from decapod transcriptomes, only crustacean female sex hormone and insulin-like peptide were absent from our trinity de novo transcriptome assembly, and also RNA-sequence reads. We identified 63 putative neuropeptide precursors from 43 families, predicted to yield 122 active peptides. Transcripts encoding 26 putative neuropeptide GPCRs were identified but were often incomplete. Putative GPCRs for 15 of the neuropeptides identified here were absent from our transcriptome and RNAseq reads. These data highlight the diverse neuropeptide systems already present at the early development life stages sampled here for P. zealandicus.

Effects of metazachlor and its major metabolite metazachlor OA on early life stages of marbled crayfish.

The effects of the herbicide metazachlor and its major metabolite metazachlor OA at two concentrations, including environmentally relevant concentrations of metazachlor (0.0115 µmol/l and 0.0790 µmol/l) and metazachlor OA (0.0117 µmol/l and 0.0805 µmol/l), respectively, were evaluated on early ontogeny, growth, behaviour, oxidative stress, antioxidant enzyme levels, histology, and mortality of marbled crayfish Procambarus virginalis. Both tested concentrations of metazachlor and metazachlor OA were associated with significantly lower growth and delayed ontogenetic development compared to controls. Exposure of metazachlor at 0.0115 µmol/l and metazachlor OA at 0.0117 µmol/l and 0.0805 µmol/l resulted in significantly lower activity of total superoxide dismutase (SOD), catalase (CAT), glutathione s-transferase (GST), glutathione reductase (GR), and reduced glutathione (GSH) compared with control and resulted in gill anomalies ranging from wall thinning to focal disintegration of branchial structure. Metazachlor at the environmentally relevant concentration of 0.0790 µmol/l was associated with significant alterations of crayfish distance moved and walking speed. The potential risk associated with metazachlor use in agriculture related to effects on non-target aquatic organisms.

Immunolocalization of Neurotransmitters and Neuromodulators in the Developing Crayfish Brain.

In the field of neurosciences, the crayfish nervous system is an important model for understanding how arthropods process sensory stimuli and generate specific behaviors. Furthermore, crayfish embryos have been important study objects for well over 200 years. Immunohistochemistry against neurotransmitters, neuromodulators, and neurohormones is widely used to analyze the ontogeny of neurons in the emerging brain of several crustacean species and to date represents one of the most powerful approaches to analyze aspects of brain development in this group of organisms. In recent years, the analysis of brain development in crustaceans has gained new momentum by the establishment of the Marmorkrebs Procambarus virginalis (Marbled Crayfish), a parthenogenetic crayfish, as new model system. The embryonic development of marbled crayfish is well characterized and these animals can be easily cultivated in the lab. This chapter describes protocols for immunolocalization of neuroactive substances in the developing crayfish brain.

Effect of prometryne on early life stages of marbled crayfish (Procambarus fallax f. virginalis).

OBJECTIVES: The aim of this study is to assess the toxicity of prometryne in early life stages of marbled crayfish (Procambarus fallax f. virginalis) on the basis of mortality, early ontogeny, growth rate, and histopathology during and at the end of the test. DESIGN: The early life stages of marbled crayfish were exposed to prometryne at four concentrations, 0.51, (reported concentration in Czech rivers), 144, 1440, and 4320 µg x l(-1) for 53 days and compared to crayfish in a non-treated control group. RESU LTS: Prometryne in concentration 144, 1444 and 4320 µg x l(-1) caused decrease of weight and specific growth rates of crayfish. Crayfish exposed the highest concentration 4320 µg x l(-1) showed delay in ontogeny development. All crayfish groups exposed to prometryne showed histopathological changes in gill. On the basis of histopathological changes the values of LOEC = 0.51 µg x l(-1) and NOEC = for 0.10 µg x l(-1) of prometryne for marbled crayfish juveniles was estimated. CONCLUSIONS: Chronic exposure of prometryne on early life stages of crayfish has affected their mortality, growth rate, and histology. Some of the changes were observed only at higher exposures (144, 1444 and 4320 µg x l(-1)), but histopathological changes in gills were observed also in crayfish exposed to the real environmental concentration in Czech rivers (i.e. 0.51 µg x l(-1)), which is about 9 times lower than maximal concentration (4.40 µg x l(-1)) reported in surface waters of Greece. Concentrations of prometryne in World rivers have been reported to generally vary in the range of 0.1-4.40 µg x l(-1).

A developmental study of serotonin-immunoreactive neurons in the embryonic brain of the marbled crayfish and the migratory locust: evidence for a homologous protocerebral group of neurons.

It is well established that the brains of adult malacostracan crustaceans and winged insects display distinct homologies down to the level of single neuropils such as the central complex and the optic neuropils. We wanted to know if developing insect and crustacean brains also share similarities and therefore have explored how neurotransmitter systems arise during arthropod embryogenesis. Previously, Sintoni et al. (2007) had already reported a homology of an individually identified cluster of neurons in the embryonic crayfish and insect brain, the secondary head spot cells that express the Engrailed protein. In the present study, we have documented the ontogeny of the serotonergic system in embryonic brains of the Marbled Crayfish in comparison to Migratory Locust embryos using immunohistochemical methods combined with confocal laser-scan microscopy. In both species, we found a cluster of early emerging serotonin-immunoreactive neurons in the protocerebrum with neurites that cross to the contralateral brain hemisphere in a characteristic commissure suggesting a homology of this cell cluster. Our study is a first step towards a phylogenetic analysis of neurotransmitter system development and shows that, as for the ventral nerve cord, traits related to neurogenesis in the brain can provide valuable hints for resolving the much debated question of arthropod phylogeny.

Neurogenesis in the central olfactory pathway of adult decapod crustaceans: development of the neurogenic niche in the brains of procambarid crayfish.

BACKGROUND: In the decapod crustacean brain, neurogenesis persists throughout the animal's life. After embryogenesis, the central olfactory pathway integrates newborn olfactory local and projection interneurons that replace old neurons or expand the existing population. In crayfish, these neurons are the descendants of precursor cells residing in a neurogenic niche. In this paper, the development of the niche was documented by monitoring proliferating cells with S-phase-specific markers combined with immunohistochemical, dye-injection and pulse-chase experiments. RESULTS: Between the end of embryogenesis and throughout the first post-embryonic stage (POI), a defined transverse band of mitotically active cells (which we will term 'the deutocerebral proliferative system' (DPS) appears. Just prior to hatching and in parallel with the formation of the DPS, the anlagen of the niche appears, closely associated with the vasculature. When the hatchling molts to the second post-embryonic stage (POII), the DPS differentiates into the lateral (LPZ) and medial (MPZ) proliferative zones. The LPZ and MPZ are characterized by a high number of mitotically active cells from the beginning of post-embryonic life; in contrast, the developing niche contains only very few dividing cells, a characteristic that persists in the adult organism. CONCLUSIONS: Our data suggest that the LPZ and MPZ are largely responsible for the production of new neurons in the early post-embryonic stages, and that the neurogenic niche in the beginning plays a subordinate role. However, as the neuroblasts in the proliferation zones disappear during early post-embryonic life, the neuronal precursors in the niche gradually become the dominant and only mechanism for the generation of new neurons in the adult brain.

[Dynamics of glucocorticoids in the ontogenesis of freshwater crawfish Astacus leptodactylus Esch].

Dynamics of changes in glucocorticoid (hydrocortisone and corticosterone) levels was studied in the ontogenesis of freshwater crawfish Astacus Leptodactylus Esch. It was shown that steroid concentrations increase during the embryogenesis period. Decrease in the glucocorticoid levels during postembryonic development is most probably related to the stabilization of young fish growth and the attainment of hormone levels typical for adult individuals. The correlation ofglucocorticoid levels with physiological-biochemical changes at different stages of ontogenesis indicates that these steroids play an essential role in the regulation of freshwater crawfish vital functions.

Muscle development in the marbled crayfish--insights from an emerging model organism (Crustacea, Malacostraca, Decapoda).

The development of the crustacean muscular system is still poorly understood. We present a structural analysis of muscle development in an emerging model organism, the marbled crayfish--a representative of the Cambaridae. The development and differentiation of muscle tissue and its relation to the mesoderm-forming cells are described using fluorescent and non-fluorescent imaging tools. We combined immunohistochemical staining for early isoforms of myosin heavy chain with phallotoxin staining of F-actin, which distinguishes early and more differentiated myocytes. We were thus able to identify single muscle precursor cells that serve as starting points for developing muscular units. Our investigations show a significant developmental advance in head appendage muscles and in the posterior end of the longitudinal trunk muscle strands compared to other forming muscle tissues. These findings are considered evolutionary relics of larval developmental features. Furthermore, we document the development of the muscular heart tissue from myogenic precursors and the formation and differentiation of visceral musculature.

Expression of immune-related genes in one phase of embryonic development of freshwater crayfish, Pacifastacus leniusculus.

Crayfish do not have larval stage as other crustacean such as penaeid shrimp they spawn their eggs until hatching and what hatches out from the eggs are miniature crayfish known as juveniles. In order to address the question whether immune genes are initially expressed during the embryo development in the egg stage, the expression of some immune-related genes: prophenoloxidase (proPO), peroxinectin, hemocyanin, anti-lipopolysaccharide factor (ALF), plcrustin, astakine-1, 2 and transglutaminase (TGase) were determined in the middle phase of crayfish embryo development. Furthermore, immune challenge was used to determine the immune response of eggs by immersing them in a solution of the highly pathogenic bacterium Aeromonas hydrophila. Semi-quantitative RT-PCR analysis showed that all tested genes are present except proPO in this phase of crayfish embryo development and none of the genes tested changed their expression following immersion in A. hydrophila. The proPO transcript has been reported from hemocytes in crustaceans and it plays crucial roles in crustacean immune response. This may indicate that the development of immune-competent hemocytes in this stage of crayfish embryo is not completed and the egg shell as such plays an important role as a shield in protecting the embryo from bacteria and maybe also other pathogens.

Engrailed-like immunoreactivity in the embryonic ventral nerve cord of the Marbled Crayfish (Marmorkrebs).

The homeobox transcription factor Engrailed is involved in controlling segmentation during arthropod germ band formation but also in establishing individual neuronal identities during later embryogenesis. In Crustacea, most studies analysing the expression of Engrailed so far have focussed on its function as segment polarity gene. In continuation to these previous studies, we analysed the neuronal expression of the Engrailed protein by immunohistochemistry in the embryonic nerve cord of a parthenogenetic crustacean, the Marbled Crayfish (Marmorkrebs). We paid particular attention to the individual identification of Engrailed expressing putative neuroblasts in the crayfish embryos. Engrailed positive cells in the neuroectoderm were counted, measured and mapped from 38 to 65% of embryonic development. That way, several Engrailed positive putative neuroblasts and putative neurons were identified. Our findings are compared with earlier studies on Engrailed expression during germ band formation in Crustacea. Recent data on neurogenesis in an amphipod crustacean have provided compelling evidence for the homology of several identified neuroblasts between this amphipod and insects. The present report may serve as a basis to explore the question if during crustacean neurogenesis additional communalities with insects exist.

Embryonic development of the histaminergic system in the ventral nerve cord of the Marbled Crayfish (Marmorkrebs).

The embryonic development of neurotransmitter systems in crustaceans so far is poorly understood. Therefore, in the current study we monitored the ontogeny of histamine-immunoreactive neurons in the ventral nerve cord of the Marbled Crayfish, an emerging crustacean model system for developmental studies. The first histaminergic neurons arise around 60% of embryonic development, well after the primordial axonal scaffold of the ventral nerve cord has been established. This suggests that histaminergic neurons do not serve as pioneer neurons but that their axons follow well established axonal tracts. The developmental sequence of the different types of histaminergic neurons is charted in this study. The analysis of the histaminergic structures is also extended into adult specimens, showing a persistence of embryonic histaminergic neurons into adulthood. Our data are compared to the pattern of histaminergic neurons in other crustaceans and discussed with regard to our knowledge on other aspects of neurogenesis in Crustacea. Furthermore, the possible role of histaminergic neurons as characters in evolutionary considerations is evaluated.

Daily and circadian expression of cryptochrome during the ontogeny of crayfish.

Cryptochromes (CRY) are proteins with a dual role in the circadian function of different animals, participating in phototransduction and light signaling to the clock and as a transcriptional repressor that provides negative feedback in the clock feedback loop. Here we characterize functional expression of CRY as a marker of the functionality of the circadian pacemaker of crayfish (Procambarus clarkii) throughout post-embryonic development. Using different experimental light protocols and by means of immunofluorescence and biochemical methods, we report that, as in the adult, in young crayfish from the first embryonic stage CRY is present in cells adjacent to the eyestalk hemiellipsoidal body and the anterior margin of the brain protocerebrum. In the brain, CRY cycles after 72 h darkness, entraining to LD cycles. Meanwhile, as in the adult eye, in juveniles CRY is driven by light, showing an arrhythmic pattern in DD and cycling under LD. These results, as well as the completely different period length found in the brain circadian oscillations of 2nd post-embryonic stage and juvenile animals, suggest important changes in the properties of the crayfish pacemaker through the development. Therefore these data support a previous idea about the functionality of the circadian system from hatching.

The engrailed-expressing secondary head spots in the embryonic crayfish brain: examples for a group of homologous neurons in Crustacea and Hexapoda?

Hexapoda have been traditionally seen as the closest relatives of the Myriapoda (Tracheata hypothesis) but molecular studies have challenged this hypothesis and rather have suggested a close relationship of hexapods and crustaceans (Tetraconata hypothesis). In this new debate, data on the structure and development of the arthropod nervous system contribute important new data ("neurophylogeny"). Neurophylogenetic studies have already provided several examples for individually identifiably neurons in the ventral nerve cord that are homologous between insects and crustaceans. In the present report, we have analysed the emergence of Engrailed-expressing cells in the embryonic brain of a parthenogenetic crayfish, the marbled crayfish (Marmorkrebs), and have compared our findings to the pattern previously reported from insects. Our data suggest that a group of six Engrailed-expressing neurons in the optic anlagen, the so-called secondary head spot cells can be homologised between crayfish and the grasshopper. In the grasshopper, these cells are supposed to be involved in establishing the primary axon scaffold of the brain. Our data provide the first example for a cluster of brain neurons that can be homologised between insects and crustaceans and show that even at the level of certain cell groups, brain structures are evolutionary conserved in these two groups.

Desarrollo embrionario del camarón de río Cryphiops caementarius (Decapoda: Palaemonidae) en condiciones de laboratorio / Embryonic development of the freshwater shrimp Cryphiops caementarius (Decapoda: Palaemonidae) under laboratory conditions

We describe seven stages for the embryonic development of Cryphiops caementarius (Molina, 1782) using both light and scanning electron microscopy. Thirty ovigerous females were captured in Limarí River, Chile, and maintained separately in three 35 liter tanks with temperatures of 15, 22 and 28 ºC. At the same time, some embryos were separated from the females and placed in a 100 ml beaker for in vitro culture at 15 ºC. Duration of development at 15, 22, and 28 ºC was of 36-39, 30-32 and 25-28 days, respectively, with no differences observed between in vitro and in vivo cultures. Seven development stages were observed: (I) homogeneously distributed vitellum; the embryo cleavage leads to the morulae stage; (II) first embryonic stage; antennule, antenna and ocular globe are present; (III) primordium lengthens, and the outlines of the pereiopods can be observed; ocular pigmentation appears as a curved line; (IV) ocular pigmentation in hemispherical form; the antennae develop, and the first red pigmentation appears near the eye; heartbeat is visible; (V) the pereiopods touch the base of the antennae; the ocular globe is more evident, in a oval form; (VI) eyes are faceted, and both antenna and third abdominal segment with chromatophores; ocular pigmentation as spherical form; (VII) ocular globe with spherical eye pigmentation and facets on the surface; star-shaped chromatophores on the appendages. Egg volume increased from 0.082 mm3 at the beginning of the development to 0.125 mm3 close to hatching, representing a rise of 65.6 %. The diameter of the embryo and eye pigmentation may be used as growth parameters.

Se describen siete estados del desarrollo embrionario de Cryphiops caementarius Molina, 1782), tanto a microscopía de luz, como electrónica de barrido, y se establece el tiempo de desarrollo en embriones cultivados a tres temperaturas. Además se realizó un cultivo in vitro a 15 ºC en vasos precipitados de 100 ml. Hembras ovígeras capturadas en el río Limarí, Chile se ubicaron en estanques de 35 litros a temperaturas de 15, 22 y 28ºC. La duración del desarrollo a 15, 22 y 28 ºC fue de 36-39, 30-32 y 25-28 días, respectivamente. El cultivo in vitro a 15 ºC no presentó diferencias con los cultivos in vivo. Se establecieron siete estados de desarrollo. (I) vitelo distribuido homogéneamente, durante este estado comienza dividirse llegando a estado de mórula; (II) aparece el primordio embrionario con los esbozos de la anténula, antena y mandíbula; (III) se observan los esbozos de los pereiópodos y abdomen; pigmentaciónocular en forma de línea curva; (IV) pigmentación ocular en forma de una semiesfera, aparece pigmentación roja en las antenas, cerca del ojo; se observa latido cardíaco; (V) los pereiópodos se extienden hasta la base de las antenas; la pigmentación ocular es oscura y con forma ovalada; (VI) pigmentación ocular esférica y con algunas facetas en la superficie; cromatóforos en la antena y tercer segmento abdominal; (VII) la pigmentación ocular tiene forma esférica y aparecen cromatóforosestrellados en los pereiópodos. El embrión aumenta su volumen en 65.6 %, desde 0.082 mm3 en el estado I hasta 0.125 mm3 en del estado VII. El diámetro del embrión y la pigmentación del ojo pueden ser utilizados como parámetros de crecimiento en esta especie.

Cardiac development in crayfish: ontogeny of cardiac physiology and aerobic metabolism in the red swamp crayfish Procambarus clarkii.

The cardiovascular system performs key physiological functions even as it develops and grows. The ontogeny of cardiac physiology was studied throughout embryonic and larval development in the red swamp crayfish Procambarus clarkii using videomicroscopic dimensional analysis. The heart begins to contract by day 13 of development (at 25 degrees C, 20 kPa O2). Prior to eclosion, heart rate (fH) decreases significantly. Previous data suggests that the decrease in cardiac parameters prior to hatching may be due to an oxygen limitation of the embryo. Throughout development, metabolizing mass and embryonic oxygen consumption primarily increased while egg surface area remains constant. The limited area for gas exchange of the egg membrane, in combination with the increasing oxygen demand of the embryo could result in an inadequate diffusive supply of oxygen to developing tissues. To determine if the decrease in cardiac function was the result of an internal hypoxia experienced during late embryonic development, early and late stage embryos were exposed to hyperoxic water (PO2 = 40 kPa O2). The fH in late stage embryos increased significantly over control values when exposed to hyperoxic water suggesting that the suppression in cardiac function observed in late stage embryos is likely due to a limited oxygen supply.

Ontogeny of the Marmorkrebs (marbled crayfish): a parthenogenetic crayfish with unknown origin and phylogenetic position.

Development, growth, and egg production of the Marmorkrebs (marbled crayfish), a crayfish with parthenogenetic reproduction, uncertain geographic origin, and taxonomic position, was studied under laboratory conditions. Length and weight increments strongly depended on temperature being highest at 30 degrees C, and lowest at 15 degrees C. At 25 degrees C, cephalothorax length and weight increased by 17.5 mm and 1700 mg, respectively, in the course of 150 d, whereas at 15 degrees C these parameters increased by only 7 mm and 100 mg during the same period of time. Photoperiod slightly affected growth at 25 degrees C. During growth experiments, mortality was lower at 20 degrees C compared to higher (25 degrees , 30 degrees C) or lower temperatures (15 degrees C), and lower under short-day than under long-day conditions. Females matured early (at an age of 141-255 d, a cephalothorax length of 14-21.5 mm, and a weight of 0.63-2 g) compared to other crayfish species. Reproductive females with a cephalothorax length of between 25-35 mm produced large clutches (up to 416 eggs) and brooding periods varied between 22 and 42 d. In order to establish a staging scheme for Marmorkrebs embryos, embryos were photographed, externally visible ontogenetic events charted, and dissected embryos stained with a nuclear dye. These experiments indicate that their development is virtually identical to that of other crayfish. In conclusion, these results and others show that the Marmorkrebs may be taken as a representative valid model organism for future developmental studies on Crustacea.

Immunolocalization of Na+,K+-ATPase in the branchial cavity during the early development of the crayfish Astacus leptodactylus (Crustacea, Decapoda).

The ontogeny of osmoregulation was examined in the branchial cavity of embryonic and early post-embryonic stages of the crayfish Astacus leptodactylus maintained in freshwater, at the sub-cellular level through the detection of the sodium-potassium adenosine triphosphatase (Na(+),K(+)-ATPase). The embryonic rate of development was calculated according to the eye index (EI) which was 430-450 microm at hatching. The distribution of the enzyme was identified by immunofluorescence microscopy using a monoclonal antibody IgGalpha5 raised against the avian alpha-subunit of the Na(+),K(+)-ATPase. Immunoreactivity staining, indicating the presence of Na(+), K(+)-ATPase appeared in the gills of late embryos (EI>/=400 microm), i.e. a few days before hatching time, and steadily increased throughout the late embryonic and early post-embryonic development. The appearance of the enzyme correlates with the ability to osmoregulate which also occurs late in the embryonic development at EI 410-420 microm and with tissue differentiation within the gill filaments. These observations indicate that the physiological shift from osmoconforming embryos to hyper-regulating late embryos and post-hatching stages in freshwater must originate partly from the differentiation in the gill epithelia of ionocytes which are the site of ion pumping, as suggested by the location of Na(+),K(+)-ATPase. Only the gills were immunostained and a lack of specific staining was noted in the lamina and the branchiostegites. Therefore, osmoregulation through Na(+)active uptake is likely achieved in embryos at the gill level; all the newly formed gills in embryos function in ion regulation; other parts of the branchial chamber such as the branchiostegites and lamina do not appear to be involved in osmoregulation.

Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): anatomical and cell differentiation.

The ontogeny of the antennal glands was studied during the embryonic and post-embryonic development of Astacus leptodactylus. The future glands arising from undifferentiated columnar cells were detectable at the metanauplius stage EI 150 microm (EI: eye index; approximately 440 microm at hatching). The tubule and labyrinth differentiated in embryos at EI 190 microm, and the bladder and coelomosac at EI 250 microm. At EI 350 microm, the tubule lengthened and divided into proximal and distal sub-regions. In later stages, the gland retained the same morpho-anatomy but the differentiation and size of each part increased. The cells of the coelomosac displayed the cytological features of podocytes in late embryonic development at EI 440 microm. Only small apical microvilli and a few mitochondria were observable in the labyrinth cells at EI 250 microm; by EI 440 microm, these cells presented well-shaped apical microvilli, formed bodies, basal infoldings and mitochondria. In the cells of the tubules and bladder, mitochondria and basal infoldings occurred at EI 440 microm and EI 250 microm, respectively. The differentiation of the tubules and bladder cells suggested that they were involved in active transport at EI 440 microm. Following hatching, the differentiation of the cells and the size of the glands increased. The ontogeny of the antennal glands thus starts in early embryos, the specific cellular functional features being differentiated in the various parts of the glands by EI 440 microm. The antennal glands are probably functional just before hatching, i.e., before the juveniles are confronted with the low osmolality of freshwater.

Brood care in freshwater crayfish and relationship with the offspring's sensory deficiencies.

Prolonged brood care is one of the evolutionary clues for the successful colonization of freshwater habitats by freshwater crayfish (Astacida). By means of macrophotography, light microscopy, and scanning electron microscopy we investigated all phases of brood care in freshwater crayfish, with particular emphasis on the morphological structures involved. We selected the recently discovered parthenogenetic marbled crayfish (species identity not yet known) as a model organism due to its fast reproduction and high resistance to handling stress. In order to examine if there is a causal relationship between brood care and the developmental status of the offspring's sensory apparatus, we additionally investigated major sense organs of juvenile Stages 1-5 in comparison with those of the adults. Brood care in the marbled crayfish is characterized by initial and final "active" phases dominated by specific maternal or juvenile behavior and a medial "passive" phase based more on the action of temporarily developed structures rather than on behavior. The most remarkable feature of this period, which includes permanent carrying of the eggs and the first two juvenile stages under the mother's abdomen, is safeguarding of hatching by a telson thread that keeps the helpless newborn hatchlings linked to the egg cases on the maternal pleopods and thus prevents them from being lost. Further important transient structures are the recurved hooks on the first pereiopods of Stage 1 and 2 juveniles that are used to firmly attach these nonfeeding stages to the mother's abdomen. In hatchlings all sense organs necessary for an independent life, such as eyes, olfactory aesthetascs, gustatory fringed setae, hydrodynamic receptor hairs, and statocysts are not developed or are underdeveloped, making brood care indispensable. Most of these sense organs appear in Stage 2 juveniles, but only from Stage 3, the first freelancing and feeding stage, are all sense organs well developed and operating, thus reducing brood care in this final period to temporary provisioning of shelter. Brooding of the eggs and postembryonic brood care are to some extent also found in other freshwater Decapoda like freshwater crabs and aeglid anomurans, but safeguarding of hatching is confined to the Astacida only. This sophisticated mode of passive brood care is unique in the animal kingdom and is apparently related to the sensory deficiencies of the first juvenile stage.