Hugo Aréchiga

Morphological and biochemical characterization of Procambarus clarki blood cells

Morphological and cytochemical analysis of Procambarus clarki hemocytes demonstrated three cell types: hyaline, semigranular, and granular. Hyaline cells showed a higher nuclear/cytoplasmic ratio with few small electron-dense granules in the cytoplasm. Semigranular cells presented numerous round or oval eosinophilic granules (0.40-0.78 micron). Granular cell contained large eosinophilic granules (1.79-3.05 microns). Ultrastructurally, all cells showed microtubules near the borders, a poorly developed Golgi complex, and secretory-type electron-dense particles. No mitotic figures were seen. Cell monolayers showed three morphologically distinct cell types (composed of flattened and well-spread cells) depending on the presence and size of granules (hyaline, semigranular, and granular). No sex-related differences could be documented in cell features or proportions. Cytochemical studies showed that the three cell types were positive for acid phosphatase. Granular and semigranular cells were also positive for nonspecific esterase. Phenoloxidase activity was localized only in granular and semigranular hemocytes, and peroxidase activity was observed only in the granular hemocytes. These results may suggest that the semigranular and granular hemocytes participate in the prophenoloxidase system and also in phagocytic or cytotoxic function.

Prophenoloxidase system activation in the crayfish Procambarus clarki

The prophenoloxidase system (proPO) was studied in primary cultures of hemocytes of the crayfish Procambarus clarki. Both zymosan and lipopolysaccharide (LPS) separately induced rapid degranulation and lysis of semigranular hemocytes, with concurrent release of proPO. ProPO could be demonstrated in the hemocyte lysate supernatant (HLS) obtained by a freeze/thaw method, and was specifically activated by LPS and zymosan. Phenoloxidase activity was blocked by serine protease inhibitors, such as soybean trypsin inhibitor (STI), leupeptin, and phenylmethyl-sulphonylfluoride (PMSF), and substantially increased by cysteine protease inhibitors (N-methylmaleimide, N-ethylmaleimide, and iodoacetamide). This enhancement was observed only when the proPO system was activated. Incubation without activators or preincubation with STI prevented the induced enhancement. Electrophoretic analyses of HLS treated with zymosan or LPS showed that three bands at 41, 39, and 37 kDa were specifically modified when the system was activated. These results suggest that a serine protease is involved in the activation of the proPO system in P. clarki, and a mechanism susceptible to cysteine protease inhibitors could be related to its regulation.

Circadian clock function in isolated eyestalk tissue of crayfish

Electrical mass response of crayfish photoreceptors (electroretinogram) was recorded continuously for up to seven days in isolated preparations that consisted of the retina and lamina ganglionaris. Electroretinogram amplitude varied in a circadian manner with a nocturnal acrophase and a period of 22–23h in preparations kept in darkness. Acclimatization of animals to reversed light/dark cycles resulted in a phase reversal of the rhythm in vitro. The per (period) gene of Drosophila has been implicated in the genesis of rhythms in insects and in vertebrates. Immunocytochemical staining with an antibody against the PER gene product revealed immunoreactivity in the retinal photoreceptors, as well as in cell bodies in the lamina ganglionaris. Labelled axons run distally towards the photoreceptors and proximally to other areas of the lamina.

Ionic dependence of screening pigment migrations in crayfish retinal photoreceptors

SummaryThe ionic dependence of the screening pigment migrations in crayfish (Procambarus bouvieri) retinula cells was studied by incubation of isolated eyes in solutions of different compositions. The pigment aggregation that normally occurs in the dark was inhibited by high potassium in a concentration dependent manner. Inhibition of aggregation was observed also with high external sodium or when sodium was replaced by lithium. Pigment aggregation is favored in the presence of high calcium, cobalt ions or in sucrose solution. The pigment dispersion induced by light is partly inhibited by lithium and in sucrose solution. Neither movement was affected by high magnesium, EDTA or EGTA. Agents expected to increase the intracellular activity of sodium or calcium, as ouabain, caffeine, ruthenium red and the ionophore A23187, all inhibited pigment aggregation. These observations assert the view that the crustacean retinula cells are independent pigmentary effectors, and agree with the notion of an engagement between membrane events and pigment position. It is suggested that sodium influx induced by light results in calcium release from internal stores, which in turn triggers dispersion of the pigment granules within the retinula cells.

Transformations in the cytoplasmic structure of crayfish retinula cells during light- and dark-adaptation

SummaryExisting descriptions of morphological correlates of light- and dark-adaptation in crayfish photoreceptors are primarily concerned with changes of the rhabdom or components derived from it, and little is known about mechanisms involved in screening pigment migration and other transformations that take place in the cytoplasm of the retinula cells. In the present study, electron microscopy was used to examine the effects of light and darkness on the cytoplasmic structure of the retinula cells.In the light-adapted state a multitude of pigment granules and many small vacuoles are dispersed throughout the cells (Figs. 1 a and 2a). On dark-adaptation the granules are withdrawn towards the axon and many of the small vacuoles in the cell body are replaced by large cisternae (Figs. 1b and 2b). The cisternae attain maximal development in the distal cytoplasm, close to the nucleus, and they do not show a preferential association with the rhabdom. Vacuoles of intermediate size and some pigment granules are seen in partly-adapted retinula cells (Fig. 3). A decrease in the density of the ground substance is observed in the cell body of dark-adapted specimens. At least part of the ground substance is constituted by a fuzzy filamentous material that in thin-sections appears surrounding and often interconnecting the pigment granules and other structures (Fig. 4). In thick-sections observed with stereo high-voltage electron microscopy, this material is revealed as a threedimensional fibrous matrix in which the pigment particles are suspended (Fig. 5). This matrix appears more compact where the granules aggregate, suggesting that it moves with the pigment.The transformation of small vacuoles into large cisternae, interpreted as equivalent to the growth of a subrhabdomal palisade in other compound eyes, is proposed to represent a structural adjustment related to the withdrawal of a cytoskeletal complex of pigment granules and their associated matrix during dark-adaptation (Fig. 6).

Neurosecretory Role of Crustacean Eyestalk in the Control of Neuronal Activity

From early histological work with methylene blue staining, the existence of neurosecretory cells was postulated in different regions of the eyestalk. Histochemical work rendered similar results (see Gabe, 1966). The most conspicuous system is that composed by the sinus gland, a neurohemal organ located in the distal part of the eyestalk, between the medulla externa and the medulla interna in many species (Fig. 1A). Its basic structure, as seen in Fig. 1B, is that of a bunch of neurosecretory endings, which are the dilated terminals of axons coming from other regions of the eyestalk, to end in apposition to a blood sinus. From morphological and physiological work, the notion was evolved of the sinus gland as the common end of secretory neurons all over the eyestalk and even of incoming fibers from other central ganglia. However, more recently, from experiments with cobalt backfills, a more restricted origin has been advocated, limiting the source of neurosecretory fibers to the sinus gland, to a group of 100–150 cell bodies clustered in the medulla terminalis and known since long ago as the X organ, or Hanstrom’s organ (Andrew et al., 1978; Jaros, 1978). Only a small number of cells outside this cluster were backfilled from the sinus gland.

Synaptic Regulation of Neurosecretory Cell Activity in the Crayfish Eyestalk

The neurosecretory cells in the crayfish X organ receive an excitatory synaptic input mediated by GABA. GABA can be released from the medulla terminalis in a Ca++-dependent manner. Topical application of GABA and baclofen mimick the synaptically evoked excitation. This is blocked by Picrotoxin. Light induces GABA release from the eyestalk. GABA-like immunoreactive neurones are present in the medulla terminalis