Purification of vitellin from grass shrimp Palaemonetes pugio, generation of monoclonal antibodies, and validation for the detection of lipovitellin in Crustacea

Much effort has been put into developing vitellogenin antibodies against a wide variety of aquatic vertebrate species to study potential estrogen or anti-estrogen endocrine disrupters. Little work has been done on endocrine disruption in aquatic invertebrates. Although some antibodies have been produced against blue crab and penaeid shrimp lipovitellin, they have only poor cross-reactivity with the important estuarine grass shrimp, Palaemonetes pugio. Vitellin was purified from eggs, monoclonal antibodies were produced using standard techniques, and hybridoma supernatants were screened by ELISA. Western blots were done using extracts from male and female grass shrimp to verify specificity of the monoclonal antibodies. Two low molecular mass bands in the range of 68-85 kD and two high molecular mass bands in the range of 190-221 kD were found. In addition to grass shrimp, several other crustacean species were screened and cross-reactivity found, including blue crab (Callinectes sapidus), mud crab (Rhithropanopeus harrisii), red swamp crayfish (Procambarus clarkii ) and Daphnia magna. To further investigate the use of the antibody, we performed a chronic 6-week pyrene exposure study. We found that vitellin was upregulated in females after 6 weeks and that this may be a protective measure against lipophilic xenobiotics.     

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.     

Prevalence,geographic distribution,and fitness effects of Microphallus turgidus (trematoda: Microphallidae) in grass shrimp (Palaemonetes spp.) from coastal Georgia

Grass shrimp, Palaemonetes pugio Holthuis and P. vilgaris (Say), were collected at 11 localities along the coast of Georgia and surveyed for digenetic trematode metacercariae. The effect of trematode infection on grass shrimp fitness was also examined. Microphallus turgidas (Leigh) was the only trematode observed. The prevalence of metacercarial cysts of this parasite in P. pugio (75%) was higher than in P. vulgaris (24%), as were the mean intensity, abundance, and population density (no. cysts/cm host body length) of the parasite. Infected shrimp were found at every collection locality and parasite prevalence and density were greatest in P. pugio from higher salinity localities (> or = 20 parts per thousand [ppt]). There was no relation between host body size and parasite density in P. vulgaris, and parasite density increased with host body size in P. pugio, suggesting that the parasite does not affect host survival. There was no relation between parasite density and shrimp egg mass, but nonovigerous female P. pugio were more heavily infected than ovigerous ones. In addition, 1 metacercaria of M. turgidus in each of 2 specimens of P. vulgaris was parasitized by the haplosporidian Urosporidium crescens De Turk. This represents a new shrimp host record for this hyperparasite.     

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.     

Serological estimation of prey-protein gut-residence time and quantification of meal size for grass shrimp consuming meiofaunal copepods

A series of experiments using serological reagents was conducted to examine predation, ingestion and digestion in a model predator-prey system. The harpacticoid copepod Amphiascoides atopus, obtained from mass culture, was used as prey and the grass shrimp, Palaemonetes pugio, as predator. Bulk-gut passage time in P. pugio was measured by visualization of latex beads and ranged from 0.5 to 4 h in starved and continuously-fed grass shrimp. A polyclonal antibody was prepared from crude extracts of A. atopus; cross reactions with P. pugio and three other crustaceans were either negligible or not detected using slide agar-gel-double-immunodiffusion (AGID) and Western blot preparations. The presence of A. atopus antigens was detected with great sensitivity (e.g., seven copepods, 35 μg dry weight, gave positive results) in grass shrimp gut contents even when proteins of other crustacean prey were present. Prey-proteins could be detected for as long as 4 h with AGID and 8 h with Western blot techniques. Individual grass shrimp that were fed A. atopus and consumed from 0 to 98 copepods h(-1) were subjected to Western-blot preparation with chemiluminescence detection and densitometric evaluation. There was a significant curvilinear relationship between protein content and the number of copepod prey ingested. Results suggest that serological techniques can be modified to estimate the mass or abundance of standard-sized prey ingested by field-collected predators.     

Changes in caridac output and hemolymph flow during hypoxic exposure in the gravid grass shrimp, <Emphasis Type="Italic">Palaemonetes pugio</Emphasis>

The cardiovascular response of decapod crustaceans to hypoxic exposure is well documented; however, information is limited concerning the influence of reproductive state on cardiovascular demands during hypoxic exposure. Given the additional metabolic demand of reproduction, we investigated the cardiovascular adjustments employed by gravid grass shrimp Palaemonetes pugio to maintain oxygen delivery during hypoxic stress. Cardiac output values were elevated in gravid compared to nongravid grass shrimp. Gravid grass shrimp were exposed to hypoxia and the stroke volume, heart rate, cardiac output and hemolymph flow were determined using video-microscopy and dimensional analysis. Oxygen consumption rates were determined using respirometry. There where no changes in the cardiac output values of gravid females until reaching 6.8 kPa O₂, with a significant redistribution of hemolymph flow at 13.7 kPa O₂. Flow was significantly decreased to the anterior lateral arteries that supply the ovaries and hepatopancreas, the anterior aorta and the posterior aorta. The redistribution of hemolymph flow away from these vessels results in an enhanced hemolymph flow to the sternal artery that supplies the ventral segmental system, the gills, the buccal apparatus and the ventral nerve cord. The data suggest that during hypoxic stress, gravid females place a priority on survival.     

Studies of the Phoront of Hyalophysa chattoni (Ciliophora,Apostomatida) Encysted on Grass Shrimp1

ABSTRACT. The apostomatous ciliate Hyalophysa chattoni, an ectosymbiont of the grass shrimp Palaemonetes pugio, encysts and dedifferentiates within 48 h from the migratory tomite to a phoretic stage devoid of complex ciliary fields. The presettlement crawling and pivoting of the tomite may play a role in its initial attachment to the shrimp. Metamorphosis of exuviotrophic apostomes has been previously observed to take place immediately prior to host ecdysis. The study has found that Hyalophysa's metamorphosis to the feeding stage on grass shrimp is initiated by a cue from the premolt host and begins during earlier stages of the molt cycle (D₀ and D₁). Due to the long premolt stage of the host's diecdysic molt cycle, metamorphosis is initiated well before ecdysis (over six days). Hyalophysa was able to encyst and metamorphose within 41/4 h when exposed to shrimp in a late premolt stage, indicating that the control of apostome metamorphosis is solely host-dependent.     

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.     

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.     

Life cycle of Calyptospora funduli (Apicomplexa: Calyptosporidae)

The taxonomic status of the extraintestinal piscine coccidium Calyptospora funduli is based in part on its requirement of an intermediate host (the daggerblade grass shrimp Palaemonetes pugio). In the present study, grass shrimp fed livers of Gulf killifish (Fundulus grandis) infected with sporulated oocysts of C. funduli exhibited numerous sporozoites suspended in the intestinal contents when fresh squash preparations were examined by light microscopy. Using this method, sporozoites were not seen in intestinal epithelial cells of the grass shrimp or in any other cell types. Ultrastructural examination, however, revealed sporozoites in the cytoplasm of the gut basal cells. Cross-sections of 1-13 sporozoites were seen within a single cell, and those sporozoites each appeared to be situated in individual membrane-bound vesicles, rather than in a single parasitophorous vacuole. These ultrastructural observations indicate that in the grass shrimp intermediate host, sporozoites that develop into an infective stage probably undergo that development in gut mucosal basal cells. Prior studies revealed that these sporozoites modified their structure over 4-5 days and that before that time, they were not infective to the fish host. Following ingestion of an infected shrimp by a killifish, the infective sporozoites apparently reach the liver of their killifish definitive hosts through the bloodstream. Sporozoites were seen in blood smears from the longnose killifish, Fundulus similis, 4 hr after fish were fed experimentally infected grass shrimp. Additionally, coccidian trophozoites and early meronts were seen in hepatocytes from several longnose killifish at 48, 72, and 96 hr postinfection. This study, in conjunction with previous findings, clearly confirms that a true intermediate host is required in the life cycle of C. funduli, that a developmental period of about 5 days in grass shrimp is necessary for sporozoites to become infective to killifishes, and that sporozoites do occur intracellularly in gut basal cells of the grass shrimp.     

Ecdysteroid responses of estuarine crustaceans exposed through complete larval development to juvenile hormone agonist insecticides

Fenoxycarb and pyriproxyfen are insecticides that gain theirtoxicity by specifically acting as insect juvenile hormone agonists(JHA), and so are endocrine disruptors by design and effectivelyprevent larvae from maturing into adults. Efforts to assessthe environmental effects of JHAs on nontarget populations ofinvertebrates have resulted in the utilization of several establishedestuarine crustacean models. This work was conducted to testthe hypothesis that the mortality, inhibition of developmentand decreased fecundity reported previously in these animalsfrom JHA exposure coincides with abnormal circulating titersof ecdysteroids. Gravid female grass shrimp (Palaemonetes pugio)and mud crabs (Rhithropanopeus harrisii), species with differentdevelopmental plasticity and JHA tolerances, were collectedand held at wet lab conditions (20 ppt salinity, 25°C) untillarval release. Larvae were collected <12 hr after hatchand exposed to JHAs during a static renewal test through endof development with seawater or nominal concentrations of JHApreviously shown to induce significant developmental delaysand/or decreased body weights. Larvae were subsampled (10 larvae/sample,n = 2 to 8) at each developmental stage, lyophilized, and ecdysteroidsextracted by homogenization in 80% methanol and elution fromC18 Sep-Pak cartridges with 25%, 60% and 100% methanol to capturethe polar, free, and apolar conjugates, respectively, and thenquantified by ELISA. As was expected significant differencesin successful completion of development (larval survival), developmentalduration, and growth (dry weight) were observed. These physiologicalperturbations were linked with significantly altered ecdysteroidtiters, supporting a newly emerging theory that juvenoids possiblyact as anti-ecdysteroids through a novel molecular mechanisminvolving inhibition of ecdysteroid signaling.     

Ecdysteroids in nemerteans: Presence and physiological role

Ecdysteroids were detected in the phylum Nemertea and their physiological role was studied. Radioimmunoassay (RIA) measurements showed ecdysteroid concentrations ranging from 1–47 pg/mg wet weight in several nemertean species from the orders Palaeonemertea, Heteronemertea, and Hoplonemertea. High-performance liquid chromatographic (HPLC) analysis of Paranemertes peregrina displayed peaks of RIA activity with retention times similar to those of authentic ecdysone and 20-hydroxyecdysone standards. Fluctuating ecdysteroid titers were observed in the various life stages of Carcinonemertes errans with the highest concentrations (47 pg/mg wet weight) found in gravid females. RIA of HPLC fractions of Carcinonemertes errans eggs indicated the presence of ecdysteroids (105 pg/mg wet weight). Alterations in the growth of juvenile, male, or female C. errans were not observed when the worms were exposed to 10^–7. 10^–6, or 10^–5 M 20-hydroxyecdysone. However, the eggs of C. errans appeared to be stimulated by 20-hydroxyecdysone. Shorter hatching times were observed in the egg strings exposed to hormone (10^–7 to 10^–5 M) compared to sea water and cholesterol (10^–11 and 10^–9 M) controls. Possible physiological roles and the evolutionary significance of ecdysteroids in nemerteans are discussed.     

The influence of insect juvenile hormone agonists on metamorphosis and reproduction in estuarine crustaceans

Comparative developmental and reproductive studies were performedon several species of estuarine crustaceans in response to threejuvenile hormone agonists (pyriproxyfen, methoprene and fenoxycarb).Larval development of the grass shrimp, Palaemonetes pugio,was greater than two orders of magnitude more sensitive to disruptionby methoprene and fenoxycarb than was embryonic development.Developing larvae of the mud crab, Rhithropanopeus harrisii,exhibited reduced metamorphic success at lower concentrationsof methoprene and pyriproxyfen than grass shrimp larvae. Theseresponses suggest that the more rigidly controlled metamorphicprocess in crabs is more sensitive to compounds acting as endocrinedisruptors than is the more flexible metamorphic pattern inshrimp. The final crab larval stage, the megalopa, was moresensitive to methoprene and fenoxycarb exposure than earlierzoeal stages. Mud crab larvae exposed to fenoxycarb had reducedbiomass and lipid content, particularly triglycerides and sterols.Concentrations of fenoxycarb which reduced the reproductivecapacity in single life-cycle exposures of the estuarine mysid,Americamysis bahia, were similar to those concentrations whichinhibited metamorphosis in grass shrimp. Juvenile mysids releasedby exposed adults and reared through maturation without furtherexposure produced fewer young and had altered sex ratios (lowerpercentages of males) at lower parental-exposure concentrationsthan directly affected parental reproduction. These transgenerationalresponses may well be a product of irreversible effects duringdevelopmental exposures which become apparent following maturationand initiation of reproduction. These findings support usinga functional approach as an appropriate screening procedureto evaluate potential environmental endocrine-disrupting chemicalsin aquatic environments.     

True Intermediate Hosts for Eimeria funduli (Apicomplexa) from Estuarine Fishes1

Grass shrimp (Palaemonetes pugio) fed liver containing sporulated oocysts of Eimeria funduli permitted development of sporozoites that became infective to a variety of killifishes. The shrimp's gastric mill mechanically ruptured the oocysts. Sporozoites then excysted through an opening in the sporocyst, and by 12 and 13 h postinfection (p.i.) numerous empty sporocysts and free sporozoites occurred extracellularly in the intestine of the grass shrimp. Even at 5, 7, 8, 11, 46, 79, and 83 days p.i., and presumably for many months, numerous sporozoites still occurred free in the alimentary tract or between intestinal cells. The coccidium did not infect killifish at either 2 or 4 days p.i., but did at 5 days; after release from the sporocyst, it became more elongate with a distinct nucleus and two relatively large refractile bodies. Infections of E. funduli resulted in about one half of the fish that were fed either entire hepatopancreas or tips of hepatopancreas from experimentally infected shrimp. Feeding either the entire alimentary tract proximal to the first abdominal segment or any portion of that section from experimentally infected shrimp produced infections in nearly all tested fish. Feeding portions of the cephalothorax without any attached hepatopancreas or alimentary tract failed to produce an infection. Feeding killifish with wild grass shrimp from an enzootic area produced infections in only a fourth of the fish sample; however, feeding experimentally infected wild, laboratory-reared, and juvenile grass shrimp produced infections in nearly all fish. Palaemonid shrimps other than P. pugio also can serve as intermediate hosts for E. funduli, and these shrimps include Palaemonetes vulgaris, P. paludosus, P. kadiakensis, and Macrobrachium ohione. In contrast, a penaeid shrimp, mysidacean, amphipod, and crab fed liver with sporulated oocysts did not produce infections when fed to killifish.     

亚洲玉米螟卵黄原蛋白基因的克隆、表达谱及对UV-A胁迫的响应

【目的】本研究克隆亚洲玉米螟Ostrinia furnacalis卵黄原蛋白(vitellogenin, Vg)基因,分析其表达模式,旨在探究UV-A胁迫对亚洲玉米螟Vg基因表达及生殖力的影响。【方法】采用RT-PCR和RACE技术克隆亚洲玉米螟Vg基因的全长,并用生物信息学方法分析其特征;利用RT-qPCR检测亚洲玉米螟不同发育阶段(卵、1-5龄幼虫、蛹和成虫)、雌成虫不同组织(头、足、表皮、卵巢、中肠和脂肪体)中和雌成虫在UV-A胁迫不同时间(0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5和4 h)下该基因的相对表达量。测定UV-A照射不同时长(0, 1, 2, 3和4 h/d)后亚洲玉米螟成虫的生殖及F_1代发育情况。【结果】克隆获得亚洲玉米螟Vg基因的序列,命名为OfVg(GenBank登录号:MK782978)。该基因全长5 760 bp,开放阅读框(ORF)长5 331 bp,编码1 776个氨基酸,蛋白相对分子量为202.10 kD,等电点为9.06。OfVg包含Vg-N, DUF 1943D和VWD 3个功能结构域,无跨膜区结构。系统发育分析表明,OfVg与鳞翅目其他昆虫的Vg的亲缘关系最近。发育表达谱表明,OfVg基因在雌成虫中表达量显著高于其他龄期的表达量,且在雌虫羽化24 h时OfVg表达量最高;组织表达谱表明,OfVg基因在雌成虫脂肪体中特异表达。UV-A照射能诱导雌成虫OfVg的表达,随着照射时间的延长,其表达量先下降再快速上升,在照射3.5 h时其表达量最高,然后骤降;UV-A处理1, 2和3 h/d雌成虫的产卵量显著增加,且F_1代幼虫发育历期显著延长。【结论】OfVg基因在亚洲玉米螟不同发育阶段、雌成虫不同组织中和UV-A照射不同时间的雌成虫中差异表达。本研究为深入研究UV-A胁迫对亚洲玉米螟发育和繁殖的影响奠定了基础。     

Effects of a methanolic extract of the plant Haplophyllum tuberculatum and of teflubenzuron on female reproduction in the migratory locust, Locusta migratoria (Orthoptera: Oedipodinae)

The effects of a methanolic extract of the plant Haplophyllum tuberculatum (ME-Ht) and of teflubenzuron (TFB) were compared on several reproductive variables and ecdysteroid titers in the females of Locusta migratoria. The test products were administered orally to newly emerged females at doses of 1500 (ME-Ht) and 10μg/female (TFB). The methanolic extract and TFB had comparable effects on several of the variables examined. Both significantly delayed the first oviposition and reduced fecundity and fertility. ME-Ht and TFB also displayed similar effects on ovarian growth, vitellogenesis and ecdysteroid titers. Both treatments induced a drop in hemolymph protein levels as well as a reduction in vitellogenin uptake by oocytes. This delay in oogenesis was accompanied by a resorption of terminal oocytes. However, whereas TFB completely blocked egg hatch, ME-Ht only had a modest inhibitory effect on this variable. Hemolymph and ovarian ecdysteroid titers, as measured by radioimmunoassay, were similar and low in both control and treated females, except for a peak observed only in control females at the end of vitellogenesis. We discuss the functional significance of the observed effects in the context of the putative modes of action of the methanolic plant extract and TFB.     

Hemolymph ecdysteroids do not affect vitellogenesis in the lubber grasshopper

The role of hemolymph ecdysteroids in the reproduction of non-dipteran insects is unclear. We examine the role(s) of hemolymph ecdysteroids during egg production in the lubber grasshopper, Romalea microptera. In all individuals, hemolymph ecdysteroids rose to a sharp peak with similar maxima and then fell to undetectable levels. The time from the adult molt to the maximum ecdysteroid titer (E(max) titer) varied in response to food availability, whereas the time from E(max) titer to oviposition was unrelated to food availability. Because both the timing of egg production and the timing of E(max) responded similarly to environmental changes, ecdysteroids may be involved in egg production. We hypothesized that this role is the stimulation of vitellogenesis. Ovariectomized females had vitellogenin but no ecdysteroids, so ecdysteroids are not necessary for vitellogenin production. In addition, treatment of females with ecdysteroids altered neither Vg titers nor ovarian growth. Ovarian ecdysteriods increased at the same age in development as hemolymph ecdysteroids. In contrast to hemolymph ecdysteroids, ovarian ecdysteroids persisted until oviposition. Despite this, [(3)H]ecdysone injected into the hemolymph was detected later only at very low levels in the ovary, suggesting that hemolymph ecdysteroids are not sequestered by the ovary. In summary, our studies indicate that hemolymph ecdysteroids in adult females of the lubber grasshopper are associated with the timing of egg production, but they neither regulate vitellogenesis nor act as a source of ecdysteroids for the ovary.