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.

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.

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.

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.