Donald L. Lovett

Ontogenetic Change in Digestive Enzyme Activity of Larval and Postlarval White Shrimp Penaeus setiferus (Crustacea, Decapoda, Penaeidae)

Whole specimens of developmental stages of Penaeus setiferus (Linnaeus, 1767) were homogenized and assayed for activities of digestive enzymes. In all developmental stages, activities were present for trypsin, carboxypeptidase A and B, amylase, and non-specific esterase; none for pepsin or lipase were detected. Activities assayed with substrates for chymotrypsin and aminopeptidase are not apparently due to the presence of these enzymes in the gut. Peak activities for all enzymes occurred during late zoeal or early mysis larval stages; low activities occurred at metamorphosis. During postlarval development, amylase activity increased steadily (by a tenfold increase over five weeks), whereas most other enzyme activities were relatively constant until the fifth week of postlarval development. Although it alters enzyme activity, diet does not appear to be the primary effector of ontogenetic change in digestive enzyme activity. Instead, ontogenetic change in digestive enzyme activity may reflect either a developmentally cued change in enzyme synthesis, or a secondary effect of change in the function and relative size of the midgut during its differentiation.

Application of regression techniques to studies of relative growth in crustaceans

ABSTRACT Studies of relative growth in crustaceans are often based, in whole or in part, upon the practice of log transformation of all linear morphometric data prior to the calculation of a least squares estimate linear regression for a broad size class of specimens. This and other practices may result in misinterpretation of the regression analysis. We propose that analysis of untransformed data with a Model II regression technique such as the reduced major axis is appropriate for description of relative growth in crustacean linear morphometric features, and that data for broad size-class samples should be examined as iteratively divided subsets, rather than assuming a priori that the entire sample is adequately represented by a single continuous function. A statistical test for evaluation of regression functions on the basis of randomness of residuals is provided.

Ontogeny of gut morphology in the white shrimp Penaeus setiferus (Decapoda, Penaeidae)

Ontogeny of the gut in Penaeus setiferus was investigated by reconstruction of serial sections examined by light microscopy. Development of the gut into the adult form is protracted over several weeks beyond metamorphosis in steps that may be directly related to the unique postlarval life history of Penaeus. The gastric mill is lacking in larval stages of P. setiferus. In protozoeal stages Z1‐Z3, the pyloric ampullae are blind sacs that do not communicate with the midgut. The gland filter first appears in mysis stage M2. The gastric mill in early postlarval (PL) stages consists of poorly chitinized lobes with flexible setae. By PL21 the ossicles of the gastric mill are rigid and setae are replaced by spine‐like denticles, but even by PL35 the gastric mill is neither as massive nor heavily chitinized as in adults. During the mysis stages and early PL stages, the hepatopancreas communicates freely with both the foregut and the midgut trunk. By PL35 the hepatopancreatic ducts are essentially isolated from the remainder of the midgut by foregut ossicles.

Hemolymph levels of methyl farnesoate increase in response to osmotic stress in the green crab, Carcinus maenas

The salinity of estuarine environments can vary widely, exposing resident organisms to considerable osmotic stress. The green crab Carcinus maenas is well known for its ability to osmoregulate in response to such stress. Therefore, we tested the relationship between osmoregulation and hemolymph levels of methyl farnesoate (MF), a compound previously shown to rise in response to various types of environmental stresses. When crabs were transferred from 100% seawater to dilute (hypo-osmotic) seawater, hemolymph osmolality dropped rapidly, reaching an acclimation level 48 h after transfer. Hemolymph levels of MF also rose in these animals after a delay of 6 h, and reached a maximum level at 48 h. MF levels remained elevated as long as the crabs were maintained in dilute seawater, and quickly returned to basal levels when the animals were returned to full strength seawater. In most (but not all) animals, MF levels were elevated when hemolymph osmolality fell below the isosmotic point (approx. 800 mOsm/kg). These data suggest that MF may have a role in osmoregulation by this species. In addition, the elevation of MF by hypo-osmotic seawater suggests an experimental strategy for manipulating MF levels in crustaceans.

ONTOGENY OF KINEMATICS IN THE GUT OF THE WHITE SHRIMP PENAEUS SETIFERUS (DECAPODA: PENAEIDAE)

Motility of the gut in developmental stages of Penaeus setiferus (Linnaeus) was monitored in vivo with video microscopy. Oral drinking, anal drinking, and propagation of contractile waves in the midgut trunk (MGT) were observed in larval and postlarval stages. A gastric mill and functional gland filter were absent in larvae. In early postlarval stages, the gastric mill did not function in trituration. The gland filter appeared to function in emulsification rather than in exclusion of large-sized particles from the hepatopancreas. Chyme from within the peritrophic membrane, as far posteriad as abdominal segment 2, was moved at times anteriad into the hepatopancreas. Both antiperistaltic and peristaltic waves mixed and transported chyme within the midgut. Substantial ontogenetic change in function of the foregut and midgut occurred (1) at metamorphosis, (2) at onset of motility in the median tooth, and (3) with differentiation of hepatopancreatic tubules. We propose that antiperistalsis along the MGT, anal drinking, oral drinking, and contractions of the foregut maintain positive hydraulic pressure within the midgut. This pressure expands regions of the midgut following contractions of intrinsic muscles. Hydraulic pressure, particularly that propagated by antiperistalsis of the MGT, is responsible for expansion of contracted hepatopancreatic tubules. We further propose that the peritrophic membrane separates stored chyme and rejected fecal matter from water taken up by anal drinking and pumped anteriad by antiperistaltic waves. In P. setiferus hydraulic pressure in the extraperitrophic space of the midgut lumen, together with pressure exerted by constriction of the MGT in abdominal segments 3 and 4, compresses the peritrophic membrane around its contents. This compression expels fluids anteriad and compacts feces before the fecal strand reaches the hindgut.

Expression profiles of Na+,K+-ATPase during acute and chronic hypo-osmotic stress in the blue crab Callinectes sapidus.

During acclimation to dilute seawater, the specific activity of Na+,K+-ATPase increases substantially in the posterior gills of the blue crab Callinectes sapidus. To determine whether this increase occurs through regulation of pre-existing enzyme or synthesis of new enzyme, mRNA and protein levels were measured over short (< 24 h) and long (18 days) time courses. Na+,K+-ATPase expression, both mRNA and protein, did not change during the initial 24-h exposure to dilute seawater (10 ppt salinity). Thus, osmoregulation in C. sapidus during acute exposure to low salinity likely involves either modulation of existing enzyme or mechanisms other than an increase in the amount of Na+,K+-ATPase enzyme. However, crabs exposed to dilute seawater over 18 days showed a 300% increase in Na+,K+-ATPase specific activity as well as a 200% increase in Na+,K+-ATPase protein levels. Thus, it appears that the increase in Na+,K+-ATPase activity during chronic exposure results from the synthesis of new enzyme. The relative amounts of mRNA for the α-subunit increased substantially (by 150%) during the acclimation process, but once the crabs had fully acclimated to low salinity, the mRNA levels had decreased and were not different from levels in crabs fully acclimated to high salinity. Thus, there is transient induction of the Na+,K+-ATPase mRNA levels during acclimation to dilute seawater.

Evaluation of the rotifer Brachionus plicatilis as a substitute for Artemia in feeding larvae of Macrobrachium rosenbergii

Abstract Addition of the rotifer Brachionus plicatilis Muller to an Artemia -based diet yielded no significant increase in larval survival or rate of larval development in Macrobrachium rosenbergii (de Man). B. plicatilis apparently contributed little to the nutrition of M. rosenbergii larvae and provided no benefit to their culture. Larvae fed on a diet restricted to Brachionus had significantly lower survival and rate of development than did larvae fed on a diet that included Artemia . Larvae fed only rotifers did not have significantly different survival from that of starved larvae. Development of larvae fed only rotifers and reared in artificial sea water was arrested at a mean stage of 2.33; in natural sea water development was arrested at a mean stage of 2.98.

Salinity-sensitive alkaline phosphatase activity in gills of the blue crab, Callinectes sapidus Rathbun

Abstract A levamisole-sensitive (Ki = 0.72 mM) alkaline phosphatase (pH optimum 9.1) and a levamisole-insensitive alkaline phosphatase (pH optimum 7.1) are present in gills of the blue crab Callinectes sapidus. Both enzymes are distinct from ouabain-sensitive ATPase. Specific activity for either phosphatase is greatest in the acinar tissue, which lines the branchial vessels. Histochemical localization of the enzymes confirmed this distribution. Activity of levamisole-sensitive alkaline phosphatase is affected by acclimation salinity. Vmax of the levamisole-sensitive alkaline phosphatase is greater in high-salinity crabs than in low-salinity crabs; apparent Km is not significantly different. The levamisole-sensitive alkaline phosphatase associated with the acinar tissue lining the branchial vessels may modulate the osmoregulatory response in blue crabs.

Changes in polyamine levels in response to acclimation salinity in gills of the blue crab Callinectes sapidus rathbun

Abstract The concentration of putrescine in gills 3, 4, 6 and 7 was approximately two-fold higher in gills acclimated to 35 ppt salinity than in those acclimated to 10 ppt salinity for 28 days ( c . 90 nmol/g wet weight vs 40 nmol/g wet weight, respectively). Spermine concentration was significantly higher in gills 6 and 7 than in gills 3 and 4 at 10 ppt salinity, and also was significantly higher than in gills 6 and 7 of crabs acclimated to 35 ppt salinity. Putrescine concentrations varied inversely and spermine concentrations varied directly with specific activity of Na + , K + -ATPase in all gills. On the basis of these data and other recent reports, we suggest that polyamines are involved in the regulation of osmotic and ionic homeostasis by interacting directly with the Na + , K+-ATPase enzyme.

D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus.

Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis.