Ultrastructure of the eyes of the grass shrimp,Palaemonetes pugio

Summary The cone cells and corneagenous cells possess extensive networks of smooth tubular endoplasmic reticulum that may be involved in optical reflectance and light-adaptational responses, respectively. The extracellular basal lamina of the basement membrane is confluent with glial cell capillary walls and may prove to be a viaduct for the transmission of hemolymph-borne substances to the retina or of retinal degradation products to the hemolymph. In addition to dense pigment granules, the distal pigment cells are shown for the first time to contain migratory reflecting platelets that are usually polymorphic in light-adapted eyes but are rectangular in dark-adapted eyes. In the latter these plates become aligned against the crystalline cones and presumably contribute to the reflection superposition optics of the grass shrimp. Dark-adapted retinular cells possess well-developed perirhabdomal cisternae, oblong or ovoid mitochondria, generally vesicular rough endoplasmic reticulum, and occasional, spherical, calcium-like intrarhabdomal inclusions. Light-adapted retinular cells possess poorly developed perirhabdomal cisternae, lamelliform rough endoplasmic reticulum, and condensed mitochondria frequently associated with lipid droplets and pigment granules. The cytoplasmic boundaries of the reflecting pigment cells expand into the extracellular spaces between individual ommatidial retinular cells during dark adaptation and recede to the interommatidial extracellular spaces during light adaptation. Cytoplasmic microfilament bundles found only at the bases of partially light-adapted rhabdomeric microvilli may be involved in microvillar shortening.     

Antioxidants and total oxyradical scavenging capacity during grass shrimp, Palaemonetes pugio, embryogenesis

During embryogenesis in grass shrimp the capacity to scavenge oxyradicals increased as measured by the Total Oxyradical Scavenging Capacity (TOSC) assay. The increase in TOSC during embryogenesis was associated with increasing concentrations of a number of antioxidants, including coenzyme Q (ubiquinone), alpha-tocopherol and reduced glutathione. Glutathione concentrations ranged from 0.004 to 0.005 nmol/embryo in early embryo stages and reached concentrations between 0.16 to 0.23 nmol/embryo in late embryo stages. Ascorbate remained essentially constant (0.16-0.20 nmol/embryo) throughout embryogenesis and may provide the preponderance of TOSC during early embryo development. Carotenoids were associated with yolk lipovitellin and these antioxidants decreased as yolk was absorbed during embryogenesis. Astaxanthin and beta-carotene were identified in embryos with astaxanthin always the principal carotenoid. In early embryo stages there are maternally derived antioxidants but as embryogenesis proceeds there is an assembly of a complex antioxidant system by newly formed cells and tissues.     

Effects of cyclic hypoxia on gene expression and reproduction in a grass shrimp, Palaemonetes pugio

Cyclic changes in dissolved oxygen occur naturally in shallow estuarine systems, yet little is known about the adaptations and responses of estuarine organisms to cyclic hypoxia. Here we examine the responses of Palaemonetes pugio, a species of grass shrimp, to cyclic hypoxia (1.5-8 mg/l dissolved oxygen; 4.20-22.42 kPa) at both the molecular and organismal levels. We measured alterations in gene expression in hepatopancreas tissue of female grass shrimp using custom cDNA macroarrays. After short-term (3-d) exposure to cyclic hypoxia, mitochondrial manganese superoxide dismutase (MnSOD) was upregulated and 70-kd heat shock proteins (HSP70) were downregulated. After 7-d exposure, nuclear genes encoding mitochondrial proteins (ribosomal protein S2, ATP synthase, very-long-chain specific acyl-CoA dehydrogenase [VLCAD]) were downregulated, whereas mitochondrial phosphoenol pyruvate carboxykinase (PEP Cbk) was upregulated. After 14 d, vitellogenin and apolipoprotein A1 were upregulated. Taken together, these changes suggest a shift in metabolism toward gluconeogenesis and lipid export. Long-term (77-d) exposure to hypoxia showed that profiles of gene expression returned to pre-exposure levels. These molecular responses differ markedly from those induced by chronic hypoxia. At the organismal level, cyclic hypoxia reduces the number of broods and eggs a female can produce. Demographic analysis showed a lower estimated rate of population growth in grass shrimp exposed to both continuous and short-term cyclic hypoxia, suggesting population-level impacts on grass shrimp.     

Effects of the trematode Microphallus turgidus on locomotion and prey capture in the grass shrimp Palaemonetes pugio

The grass shrimp, Palaemonetes pugio, is common in estuaries and marshes along the east coast of the USA and is frequently infected with metacercariae of the trematode, Microphallus turgidus. To test whether or not M. turgidus has an effect on intermediate host behaviour, the length of time spent swimming and walking over 1 min and 3 min intervals and prey (Artemia) capture rates of uninfected grass shrimp and those infected with 1-10, 11-20 or 21-30 metacercarial cysts were compared. Uninfected shrimps spent significantly more time swimming than infected shrimps during the first minute of observation. There were no differences between the control and infected groups in terms of swimming at 3 min, walking at 1 and 3 min, or in numbers of prey captured. These results indicate that M. turgidus may induce little or no change in grass shrimp locomotion nor in prey capture behaviour. This is in contrast to other parasites that modify intermediate host behaviour to enhance their transmission to definitive hosts. Furthermore, these data support earlier studies indicating that M. turgidus does not affect the growth and survival of P. pugio.     

Salinity tolerance in Hyalophysa chattoni (Ciliophora, Apostomatida), a symbiont of the estuarine grass shrimp Palaemonetes pugio

The apostome ciliate Hyalophysa chattoni, a symbiont of the estuarine grass shrimp Palaemonetes pugio, was tested for its growth and reproductive ability in a wide range of salinities from 0.1 to 55 ppt. Shrimp, with their attached ciliates, were slowly acclimated to different salinities in order to assess protozoan cell size and division. The trophont and tomont stages of the ciliate life cycle were analyzed. In both stages, cell size increased with salinity from 0.1 to 20 ppt. Cell size leveled in the 20-35 ppt range, and decreased at higher salinities. The number of daughter cells produced per tomont cyst correlated with increased cell size, and also correlated with increased salinity. Additionally, increased salinity correlated with an increase in the percentage of cells able to divide and excyst as tomite stages. These results indicate that H. chattoni is able to grow and divide more effectively at salinities closer to seawater than in the estuarine environment from which they were collected. Though able to survive salinities from 0.1 to 55 ppt, the species is better adapted for an existence in the higher salt concentrations.     

Compensatory partitioning of carbon budgets by the grass shrimp (Palaemonetes pugio) and implications for predator prey relationships

Carbon utilization and allocation were examined between two populations of shrimp (Palaemonetes pugio) to determine the possible effects of living in an area of high anthropogenic impact. Carbon assimilation has been studied in P. pugio, but no study has looked at how assimilation might be influenced by contaminants. Anthropogenic effects on carbon assimilation in grass shrimp represent a major unmeasured impact on the carbon budget of multi-cellular organisms in estuaries and near shore environments. The influence of anthropogenic contamination on carbon assimilation has implications for predicting the environmental impact of contaminants, for models of estuarine function, and trophic transfer from the dominant macroscopic detritus processor to species of direct economic importance. Shrimp budgets were compared between two populations, one from a highly polluted marsh creek system in Northern New Jersey, and one from a clean reference site in Southern New Jersey. All components of the carbon budgets were measured directly including carbon allocated to reproduction. Carbon lost to respiration was lower in shrimp from the polluted system allowing them to have increased reproductive output. This is examined in the context of previous studies that show lowered predation by a piscine predator at the polluted site, resulting in a trophic cascade and changes in ecosystem function due to anthropogenic impacts.     

Rosette glands in the gills of the grass shrimp,Palaemonetes pugio. I. Comparative morphology,cyclical activity,and innervation

Two types of exocrine rosette glands (called type A and type B), located in the gill axes of the grass shrimp Palaemonetes pugio, are described. The type A glands are embedded within the longitudinal median septum of the gill axes, whereas the type B glands typically project into the efferent hemolymph channels of the gill axes. Although both glands have certain common characteristics (i.e., a variable number of radially arranged secretory cells, a central intercalary cell, and a canal cell that forms the cuticular ductule leading to the branchial surface), they differ in the following respects. The type B gland is innervated, but the type A gland is not; axonal processes, containing both granular (ca. 900–1300 Å) and agranular (ca. 450–640 Å) vesicles, occur at a juncture between adjacent secretory cells and the central cell of the type B gland. The secretory cells of type A and type B glands differ in their synthetic potential and membrane specializations. These differences are more pronounced in well-developed, mature glands, most frequently encountered in larger (24–28 mm, total length) grass shrimp, than in the underdeveloped, immature glands that are most abundant in smaller (14–18 mm, total length) grass shrimp. Thus, in mature glands, the secretory cells of the type A rosette glands are characterized by extensive RER, abundant Golgi, and numerous secretory granules, whereas the secretory cells of the type B gland are characterized by extensively infolded and interdigitated basal plasmalemmas and by the presence of numerous mitochondria. In general, both types of glands exhibit increased secretory activity soon after ecdysis. The central and canal cells in both glands seem to have a role in the modification of the secreted materials. The possible functions assigned to the type A gland and the type B gland include phenol-oxidase secretion and osmoregulation, respectively.     

Prevalence of the bopyrid isopod Probopyrus pandalicola in the grass shrimp, Palaemonetes pugio, in four tidal creeks on the South Carolina-Georgia coast

The grass shrimp, Palaemonetes pugio, is an important food source for many invertebrate and fish species, including several of commercial importance. The bopyrid Probopyrus pandalicola prevents reproduction in P. pugio by sexual sterilization. The purpose of our research was to determine bopyrid prevalence in grass shrimp over the course of a year. Shrimp were collected from 2 estuarine systems in South Carolina and 2 estuarine systems in Georgia and examined for parasite presence, sex, and gravidity. Site-specific monthly prevalence ranged from 0 to 6.3%. Country Club Creek had the maximum mean +/- SE prevalence of 3.1 +/- 0.3%, and Harbour Town had the minimum of 1.3 +/- 0.3%. Maximum prevalence was concurrent with peak gravidity for Moon River; thus, at this site the negative effect of this parasite on reproductive output may be greater. Reduced egg production may affect grass shrimp abundance and ultimately the recruitment success of its predators.     

Effects of Microphallus turgidus (Trematoda: Microphallidae) on the predation, behavior, and swimming stamina of the grass shrimp Palaemonetes pugio

The effect of the trematode Microphallus turgidus on its second intermediate host, the grass shrimp, Palaemonetes pugio, was tested. To do so, we measured the susceptibility of infected and uninfected shrimp to predation by the mummichog, Fundulus heteroclitus. Shrimp behavior was compared in the presence and absence of a fish predator, and the swimming stamina and backthrust escape responses of infected and uninfected shrimp were measured. Infected shrimp were more likely to be eaten by a predator than uninfected shrimp, had lower swimming stamina, and spent more time swimming and less time motionless in the presence of a predator. There was no difference between backthrust distances traveled in response to a stimulus by either infected or uninfected shrimp. Thus, M. turgidus may increase the predation of P. pugio in the wild, possibly by affecting the swimming stamina and predator avoidance responses of the shrimp.     

Some factors affecting size distribution and density of grass shrimp (Palaemonetes pugio) populations in two New Jersey estuaries

Mature Palaemonetes pugio, from a polluted estuary, Piles Creek (PC) are larger than those in a more pristine estuary in Tuckerton (TK). Possible causes for the differences in size-structure could be differences in environmental factors at the two creeks, differential competition at the two sites, inherent factors causing a greater growth rate in the PC population, differences in reproductive timing, or differential predation at the two sites. Lab microcosm studies were used to examine the possibility of inherent population differences or environmental conditions (including differences in salinity) causing faster growth in PC. There was no evidence that PC shrimp grew faster than TK shrimp, nor that PC conditions fostered greater growth compared to TK. Salinity adjustments in PC and TK tanks also had no effect on growth. Therefore, it appears that genetic and environmental factors did not play a significant role in greater shrimp growth in PC. In the field, data on relative abundances of Fundulus heteroclitus and P. pugio, and size-frequency distributions of P. pugio were collected from the two estuaries. Gravid females and recruits of young shrimp physically appeared at approximately the same time within both systems, eliminating earlier reproduction as a cause of the size discrepancy. There were three times as many shrimp in PC than in TK (eliminating the possibility of increased population density and competition at TK as a cause) and three times as many F. heteroclitus present in TK than in PC. Since it has already been established that the Fundulus at PC are smaller than at TK and that they are poor predators, differences in Fundulus predation would appear to be an important factor in determining the number and size-frequency of the grass shrimp.     

A new species of Pleistophora (Microsporida: Pleistophoridae) parasitic in the shrimp Palaemonetes pugio

Pleistophora sp. Sprague, 1970, in the muscle of Palaemonetes pugio was studied. Formalin fixed spores were ellipsoidal, 2.5–3.3 × 1.4–2.0 μm, and with large anterior and posterior clear areas representing the polaroplast and posterior vacuole. This species, after comparison with four other species of Pleistophora in decapod crustacea, was found to be new. It is named Pleistophora lintoni n. sp.     

Behavioral chemoattractants for the shrimp, Palaemonetes pugio: identification of active components in food extracts and evidence of synergistic mixture interactions

We are studying chemoattractants for the shrimp, Palaemonetespugio, to identify the active components of stimulatory foodextracts, and to determine whether the mixtures of substancesoccurring in extracts express themselves in an additive or aninteractive manner. The results of quantitative analyses ofthe specific amino acids, quaternary ammonium compounds, organicacids, nucleotides and related substances in extracts of fourorganisms (crab, shrimp, oyster, mullet) were used to formulateartificial mixtures based on the composition of each organism.Quantitative behavioral bioassays with P. pugio showed thatthe artificial mixtures based on the composition of the craband shrimp extracts were virtually as effective as the respectivenatural extracts, whereas artificial mixtures based on the compositionof the oyster and mullet extracts were far less effective. Sincethe same types of substances were included in all of the mixtures,it is apparent that the substances serving as chemoattractantsin different extracts are not constant but can vary dependingupon the source of the extract. To examine for interactionsamong the components of the four artificial mixtures, responsesto the mixtures and to their individual components were analyzedusing the additive methods of stimulus summation and responsesummation. Synergistic interactions were evident since eachmixture was markedly more effective than predicted on the basisof these additive models. The degree of synergism varied withthe composition of the mixture.     

Development of monoclonal antibodies for detection of necrotizing hepatopancreatitis in penaeid shrimp

Monoclonal antibodies (MAbs) were produced against necrotizing hepatopancreatitis bacteria (NHP-B) of penaeid shrimp. The MAbs tested in dot-immunoblot (D-IB) assays were capable of detecting the NHP-B in hepatopancreas samples collected from moribund juvenile Litopenaeus vannamei during an experimentally induced NHP-B infection. The MAbs were also screened by immunohistochemistry (IHC) using case submissions that were determined to be infected not only by histology, but also polymerase chain reaction (PCR) and in situ hybridization (ISH) assays using specific digoxigenin (DIG)-labeled probes on histological sections prepared from naturally infected shrimp. Two of the MAbs were chosen for development of detection methods for NHP. The MAbs were tested using IHC methods on Davidson's alcohol-formalin-acetic acid (AFA) fixed tissue sections and identified NHP-B infected cells and tissues in a pattern similar to that seen with DIG-labeled NHP-specific gene probes. None of the MAbs reacted with tissue from specific pathogen-free (SPF) shrimp or with shrimp tissues infected with a rickettsia-like bacteria, Vibrio sp., Campylobacter sp., and Spiroplasma sp. The MAbs were found to be negative against these other organisms, demonstrating that they are species specific and useful for rapid diagnostic detection of NHP-B.     

Ultrastructure of an exocrine dermal gland in the gills of the grass shrimp,Palaemonetes pugio: Occurrence of transitory ciliary axonemes associated with the sloughing and reformation of the ductule

Exocrine dermal glands, comparable to the class 3 glandular units of insects, are found in the gills of the grass shrimp, Palaemonetes pugio. The dermal glands are composed of three cells: secretory cell, hillock cell and canal cell. Originating as a complex invagination of the apical cytoplasm of the granular secretory cell, a duct ascends through the hillock and canal cells to the cuticular surface. The duct is divisible into four regions: the secretory apparatus in the granular secretory cell, the locular complex, the hillock region within the hillock cell and the canal within the canal cell. A tubular ductule is contained within the latter two regions. As the ductule ascends to the cuticular surface, its constitution gradually changes from one of a fibrous material to one which possesses layers of epicuticle. During the proecdysial period, the ductule is extruded into the ecdysial space and this is followed by the secretion of a new ductule. Temporary ciliary structures, located near the secretory apparatus of the secretory cell, are associated with the extrusion and reformation of the ductule. Characterized only by a basal body and rootlets throughout most of the intermolt cycle, the ciliary organelles give rise to temporary axonemic processes which ascend through the ductule toward the ecdysial space at the onset of proecdysis. Subsequently, the old ductule is sloughed off and a new ductule is reformed around the ciliary axonemes. Following this reformation, the ciliary axonemes degenerate. The function of cytoplasmic processes, derived from the apical cytoplasm of the secretory cell, is also discussed.     

Purification and characterization of vitellin from the estuarine mysid Neomysis integer (Crustacea; Mysidacea)

Invertebrates account for roughly 95% of all animals, yet surprisingly, little effort has been invested to understand their value in signaling potential environmental endocrine disruption. There has been, however, much recent attention on vitellogenin induction in egg-laying invertebrates and vertebrates as indicators of exposure to estrogenic xenobiotics. Mysid shrimp (Crustacea: Mysidacea) have been put forward by several researchers and regulatory bodies (e.g., US-EPA) as suitable test organisms for the evaluation of environmental endocrine disruption. In view of developing sensitive assays to study endocrine disruption in the estuarine mysid Neomysis integer, we isolated and characterized vitellin, the major yolk protein in eggs. Vitellin was purified using gel filtration and characterized by electrophoresis using different staining procedures. Specific (as shown by Western blotting) polyclonal antibodies were produced in rabbit against the purified vitellin of N. integer. These antisera will be used to develop immunoassays to study vitellogenesis in mysids and to detect potential stimulatory or inhibitory effects of endocrine disruptors on the production of vitellin.