In vitro fertilization of the rock shrimp, Rhynchocinetes typus (Decapoda, Caridea): a review

This review compiles all the research done on gametes and fertilization in the rock shrimp, R. typus, and describes the sequence of events from the first gamete interaction to zygote formation and the first cleavage of the embryo, with light, fluorescence confocal and electron microscopes. Early studies showed that sperm from the vas deferens have a tack-shape with a "needle-like process" or rigid spike (RS) that extends from a semi-spherical body that contains the arms with chromatin and spines. Upon contact with seawater and by action of Na +, the arms and spines extend, producing an inverted umbrella form of the spermatozoa. The first sperm-oocyte interaction occurs between protein receptors type lectins of the sperm RS and oocyte chorion sperm ligands. These ligands contain residues of a-Glu, Man (a 1-3) Man, a and p-GlcNAc and a-GalNA terminal residues. It was found that a-Man and GlcNAc residues are the ligands that are directly related to the adhesion process and further penetration of sperm. After this first interaction, the RS enters the oocyte envelope by the action of a trypsin-like enzyme, rhynchocinecine, present in the acicular process. Later, arms and spines penetrate the oocyte cytoplasm, where the chromatin of the arms begin to migrate to the central area of the sperm, condensing in a cup-shaped structure near the connecting piece, which forms the male pronucleus.

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.

Relationship between DAPI-fluorescence fading and nuclear DNA content: An alternative method to DNA quantification?

In observations by confocal or conventional fluorescence microscopy, important factors should be considered in order to obtain accurate images. One of them, such as the fluorescence bleaching from highest intensity to lowest signal of fluorescence is a common problem with several DNA fluorochromes and especially for DAPI stain. The fluorescence of DAPI fades rapidly when it is exposed to UV light, under optimal conditions of observation. Although the fading process can be retarded using a mounting medium with antifading reagents, the photochemical process underlying the fluorescence decay has not yet been fully explained. In addition, no relationship between fluorescence fading and nuclear DNA content has been tested. In order to test this relationship, we measured by means of image analysis the DAPI-fluorescence intensity in several cellular types (spermatozoa, erythrocytes and haemocytes) during their fluorescence bleaching. An algorithm specifically built in MATLAB software was used for this approach. The correlation coefficient between nuclear DNA content and DAPI-fluorescence fading was found equal to 99 percent. This study demonstrates the feasibility to measure nuclear DNA content by fluorescence fading quantification, as an alternative method concurrently with image analysis procedures.