Intracellular pH and the metabolic status of dormant and developing Artemia embryos

Encysted embryos of the brine shrimp Artemia salina have long been known to enter profound dormancy under anaerobic conditions. Utilizing in vivo³¹P nuclear magnetic resonance, we show that the reversible transitions between anaerobic dormancy and aerobic development are accompanied by large (>1 unit) intracellular pH changes, the more acid pH being associated with the dormant state. Furthermore, dormant cyst intracellular pH is independent of that of the buffer, suggesting the potential for pH-mediated regulation of enzyme activities during dormancy. An example concerning cyst nucleotide metabolism is discussed.     

Mixture of parthenogenetic and zygogenetic brine shrimp Artemia (Branchiopoda: Anostraca) in commercial cyst lots from Great Salt Lake, UT, USA

The brine shrimp Artemia is one of the most studied animals in the world. A large part of the knowledge of this crustacean is based on cysts harvested from two main sources; the Great Salt Lake, UT (GSL), and the San Francisco Bay salterns, CA (SFB), USA. Artemia populations from these habitats are recognized to belong to a single zygogenetic species, Artemia franciscana Kellogg, 1906. However, the GSL Artemia has been in doubt for more than a century about the existence of parthenogenetic reproduction. By using morphological, reproductive, and molecular analyses, we report that commercial GSL cyst lots contained two different brine shrimp species; a parthenogenetic (60%) and a zygogenetic (A. franciscana) (40%). From this finding, at least three hypotheses can be drawn. The parthenogenetic Artemia is native of GSL, or it was introduced to GSL, or foreign parthenogenetic cysts were mixed with A. franciscana cysts and canned for commercial distribution. Researchers using brine shrimp cysts from GSL should therefore pay careful attention to the correct identity of the species under study. The potential of an easy and unnoticed introduction of parthenogenetic Artemia into America is discussed.     

Administration of Probiotics Improves the Brine Shrimp Production and Prevents Detrimental Effects of Pathogenic <Emphasis Type="Italic">Vibrio</Emphasis> Species

In this study, we evaluated a consortium of probiotic bacteria as an environmentally-friendly strategy for controlling pathogenic Vibrio species during the brine shrimp incubation period. Probiotic strains were initially selected on basis of (i) their ability to colonize the cyst surfaces, (ii) their absence of cross-inhibitory effects, and (iii) no detrimental effect on cyst hatching. The cysts and nauplius surfaces were immediately colonized after the application of selected probiotic strains, without detrimental effects on survival. Ten probiotic strains were mixed at similar proportions (probiotic consortium) and evaluated at different concentrations into brine shrimp cultures during incubation and early stages of development. Subsequently, these cultures were challenged with Vibrio parahaemolyticus and Vibrio harveyi. The probiotic consortium was effective to reduce the abundance of pathogenic Vibrio species and to prevent the mortality during Vibrio challenges; however, its effect was concentration-dependent and was successful at a starting concentration of 1.8?×?10⁶ CFU/ml. Our results suggest that this probiotic consortium offers an alternative to antimicrobial agents routinely used to reduce the incidence and prevalence of pathogenic Vibrio species in brine shrimp production.     

Histochemical and ultrastructural studies on the metacercarial cyst of Echinostoma revolutum (Trematoda).

Histochemical and ultrastructural studies were made on the metacercarial cyst of Echinostoma revolutum obtained from the kidney of experimentally infected Physa and Lymnaea snails. Ultrastructural studies revealed three cyst walls, an outer, middle and inner. The outer wall was more electron-dense than the middle, and contained coarser granules than those found in the middle layer. The inner wall was lamellated and contained membranous whorls. Collagenous fibers presumably of host origin surrounded the outer cyst wall. The outer and middle cyst walls stained identically with all histochemical procedures used. These walls contained acid mucopolysaccharides and glycoprotein, whereas the inner cyst wall contained glycoprotein. All cyst walls stained positively with a variety of protein stains.     

A smaller particle size improved the oral bioavailability of monkey head mushroom, Hericium erinaceum, powder resulting in enhancement of the immune response and disease resistance of white shrimp, Litopenaeus vannamei

The effects of different particle sizes (100–150, 74–100, and <74 μm) of powder of the dried and ground stipe from the monkey head mushroom, Hericium erinaceum, on the immune response and disease resistance of white shrimp, Litopenaeus vannamei, against the pathogen, Vibrio alginolyticus, were examined. Mushroom powder with a particle size of <74 μm had a significantly higher effect on the disease resistance of shrimp compared to particle sizes of >74 μm. Mortality of shrimp after being injected with V. alginolyticus was particle size-dependent, increasing from 66.7% ± 3.3%–93.3% ± 3.3% with diets containing stipe particle sizes of <74 and 100–150 μm, respectively. The mortality of shrimp fed the diet containing <74-μm stipe powder for 28 days was significant lower than that of shrimp fed with the control diet and the diet containing 74–100-μm stipe powder after being challenged by V. alginolyticus. The optimal concentration of the <74-μm mushroom powder for enhancing the immune response and disease resistance of shrimp was 0.2 μg (g shrimp)^?1 day^?1. No significant change in the total hemocyte count, differential hemocyte count, glutathione reductase, or phagocytic activity was found in shrimp fed the control diet and mushroom powder-containing diet at a level of up to 0.2 μg (g shrimp)^?1 day^?1. Shrimp fed 0.2 μg (g shrimp)^?1 day^?1 of a mushroom-containing diet had a significantly higher disease resistance to V. alginolyticus via an increase in phenoloxidase activity, respiratory bursts, superoxide dismutase activity, and glutathione peroxidase activity. Therefore, a diet containing the stipe powder of monkey head mushroom with a particle size <74 μm at a level of 0.2 μg (g shrimp)^?1 day^?1 was found to enhance the immunity and disease resistance of shrimp.     

Encapsulation of the metacercariae of two species of trematodes in the skin and musculature of the stone cockscomb <Emphasis Type="Italic">Alectrias alectrolophus</Emphasis> (Pallas, 1814)

The structure of the coating surrounding the metacercaria of Cryptocotyle lingua in the skin and the metacercaria of Liliatrema skrjabini in the muscles of the stone cockscomb was studied. Metacercariae of these trematodes were surrounded by a cyst adjacent to the helminth and by a capsule. In both species, the cyst consisted of electron- dense homogeneous material, which was probably synthesized by the parasite. The capsules were formed from host tissues; in C. lingua, they mostly consisted of ordered layers of collagen fibers and in L. skrjabini they were formed from fibroblasts and, presumably, phagocytes. The differences in capsule structure of the studied metacercariae may be related to the physiological features of surrounding tissues or to the taxonomic attribution of the trematodes.     

The stable isotope of nitrogen in an experimental culture of Ulva spp. and its assimilation in the nutrition of white shrimp Litopenaeus vannamei, Baja California Sur, Mexico

Stable nitrogen isotope ratios have been used to study the incorporation of nitrogen into the food webs of marine systems. Some species of algae can be cocultured with shrimp, resulting in a sustainable alternative to reduce or eliminate the use of commercial food. One option is the development of Ulva spp. in open-air ponds under a rigorous control of water quality. Recently, the coculture of Ulva spp. and juvenile shrimp (in aquaria and open-air ponds) has shown, under stereomicroscope observation, that the crustaceans were feeding on the Ulva spp. The consumption of commercial food and Ulva spp. by juvenile shrimp has been evaluated to establish the uptake of nitrogen into tissues of this crustacean. The muscle tissue of juvenile shrimp initially assimilated nitrogen from commercial feed and later the cocultured shrimp assimilated nitrogen from the Ulva spp., which demonstrated the potential application of live and fresh diets and the optimization of their use in diets containing very low levels or no commercial food.     

Clip domain serine protease and its homolog respond to Vibrio challenge in Chinese white shrimp,Fenneropenaeus chinensis

Clip domain serine proteases and their homologs are involved in invertebrate innate immunity, including hemolymph coagulation, antimicrobial peptide synthesis, cell adhesion, and melanization. Recognition of pathogens by pattern recognition receptors can trigger activation of a serine protease cascade. We report here the cDNA cloning of a serine protease (FcSP) and a serine protease homolog (FcSPH) from Chinese white shrimp, Fenneropenaeus chinensis. Both FcSP and FcSPH possess a clip domain at the N-terminal and an SP or SP-like domain at the C-terminal. In contrast to FcSP, FcSPH lacks a catalytic residue and is catalytically inactive. Tissue distribution and time course qRT-PCR analysis indicates that FcSP and FcSPH can respond to Vibrio anguillarum challenge in hemocytes, hepatopancreas and intestine. In situ hybridization analysis shows that FcSP is distributed in hemocytes and gills, and originated mainly from the hemocytes. FcSPH protein is expressed in gills and stomach of non-challenged shrimp. Its expression in gill mainly originates from the hemocytes in it. Two immunoreactive bands of FcSP can be detected in gills and stomach of non-challenged shrimp. FcSP protein is partially cleaved in non-challenged shrimp, while FcSPH protein is unprocessed in unchallenged shrimp and is partially cleaved after V. anguillarum challenge. Our results suggest that this Clip domain serine protease and its homolog may be involved in the serine protease cascade and play an important role in innate immunity of the shrimp.     

Possible toxic effects of the marine blue-green alga, Spirulina subsalsa, on the blue shrimp, Penaeus stylirostris.

Blooms of a marine species of blue-green algae identified as Spirulina subsalsa (Cyanophyta, Oscillatoriacae) were found to be related to a particular disease syndrome in raceway-reared blue shrimp, Penaeus stylirostris. The disease was characterized by necrosis of the lining epithelium of the midgut, dorsal cecum, and hindgut gland, and a consequent hemocytic enteritis. Bacterial infections due predominately to Vibrio alginolyticus were common in affected shrimp and presumed to be a secondary condition resulting from necrosis of the gut epithelium. These bacterial infections were expressed as local abscesses near or on the gut or as fulminating septicemias.     

Microhabitat distribution and behaviour of Branchiobdellidan Holtodrilus truncatus found on the freshwater shrimp Neocaridina spp. from the Sugo River, Japan

A study was performed on the microhabitat distribution and some aspects of behaviour of the ectosymbiotic branchiobdellidan Holtodrilus truncatus (Annelida, Clitellata) found on the freshwater shrimp that inhabit the Sugo River, Hyogo Prefecture, western Japan. Observations on shrimp that were collected from the Sugo River (2003 to 2011) confirmed that the host shrimp is Neocaridina spp. (Atyidae). The attachment location on the host shrimp was predominately between the 1^st pleopod and the 5th pereopod (55.3%). The reproductive method of H. truncatus is hemaphroditism. The cocoon was found only inside the carapace of the host shrimp. The cocoon was transparent and contained a maximum of 14 juvenile worms (developing embryos). When hatching approached, H. truncatus’s worms became elongated and slender, and only one worm hatched out at a time. When Holtodrilus truncatus was removed from its host and was maintained in river water without any food, it survived for a maximum of 46 days. In a host exchange experiment, where we provided several other freshwater shrimp species, Palaemonidae fed on H. truncatus. Moreover, Palaemon paucidens and Macrobrachium nipponense from Lake Biwa also preyed upon H. truncatus. The possible symbiotic relationship between H. truncatus and Neocaridina spp. (family Atyidae) is further discussed.     

Observations on the pathogenesis of the imperfect fungus,Fusarium solani,in the California brown shrimp,Penaeus californiensis

The pathogenesis of Fusarium solani infections was studied using a highly susceptible species, Penaeus californiensis. In two experiments, F. solani infections were established in artificially wounded and infected juvenile and adults of P. californiensis, while similar control groups were wounded but not artificially infected. In the first experiment, the progress of F. solani infections in 15-g cultured juveniles of P. californiensis was followed by gross inspection and by light and electron microscopical study of the wound area. Developing F. solani infections were produced with a success rate of 100% within 14 days postinfection, and deaths due to disease of greater than 50% of the artificially infected group had occurred by 24 days postinfection. Infections were histologically characterized by (1) expansion of lesions into the tissue surrounding the point of entry, (2) destruction of invaded tissues by host granuloma formation and possibly by fungal enzymes and toxins, and (3) hemocyte encapsulation of hyphae with eventual melanization of the encapsulating hemocytes. In the second study, wild adult P. californiensis were artificially infected with F. solani, and changes in several of their hemolymph constituents [alkaline phosphatase, serum glutamic oxaloacetic transaminase (SGOT), glucose, total protein, hemocyte count, and hematopoietic tissue mitotic index] were determined as the disease developed. Significant differences occurred in these hemolymph parameters of shrimp with advanced F. solani infections compared to those of uninfected, unwounded control shrimp or those with early F. solani infections. Hemolymph from severely infected shrimp was hypoproteinemic, contained lower numbers of circulating hemocytes, and frequently failed to coagulate.     

An enzootic nuclear polyhedrosis virus of pink shrimp: Ultrastructure,prevalence, and enhancement

A nuclear polyhedrosis virus exists in pink shrimp, Penaeus duorarum, from waters of the northern Gulf of Mexico. This virus is rod-shaped, 269 nm long, and possesses an outer envelope surrounding its nucleocapsid. The nucleocapsid is 50 nm in diameter. The virus occurs in nuclei of host hepatopancreatic and midgut cells, and is both free in the nucleus and occluded within pyramidal-shaped polyhedral inclusion bodies (PIB's). Histochemically and ultrastructurally, the shrimp PIB's appear to be ribonucleoprotein and in fine structure bear close resemblance to polyhedral inclusion bodies of Baculovirus species from insects. However, the lattice line-to-line spacing is greater than that usually reported for insect PIB's. Crowding and chemical stress of shrimp in aquaria may enhance and increase the virus infection and prevalence. In limited experiments, shrimp fed heavily infected hepatopancreatic tissues had much higher mortality than controls fed only fish. The virus appears to be enzootic in pink shrimp in nature. Cytopathological changes in infected cells of shrimp appear similar to those in insects infected with certain species of Baculovirus. The name Baculovirus penaei n.sp. is proposed for the shrimp virus.     

Intra-specific variability in the thirteen known populations of the fairy shrimp Chirocephalus ruffoi (Crustacea: Anostraca): resting egg morphometrics and mitochondrial DNA reveal decoupled patterns of deep divergence

Chirocephalus ruffoi is a fairy shrimp endemic to the Italian peninsula, where it is known only from thirteen high mountain locations. Twelve of these are in the Northern Apennines while the thirteenth is about 700 km away in the Calabrian Apennines (Southern Italy). We quantified degree of genetic divergence within the species by sequencing a fragment of the mitochondrial DNA encoding for Cytochrome Oxidase I. We then combined genetic data with the re-analysis of two different datasets on the morphometrics of the resting eggs (cysts) produced by the species as an adaptation to survive seasonal droughts. Genetic data revealed profound divergence; we identified four clusters of haplotypes within the species phylogeography, three in the Northern Apennines and one in the Calabrian Apennines with most of the genetic variation (≈70%) being apportioned among haplogroups. We found high variability in cyst morphometrics, especially in cyst size and height of the spines ornamenting the surface. Genetic and morphometric data are decoupled suggesting that cyst morphology is either under selection or a plastic trait. We, therefore, caution against using cyst morphology for taxonomic purposes in anostracans.     

Evidence of a store of informational RNA in encysted embryos of Artemia salina

RNA prepared from dormant cysts and developmental stages of the brine shrimp Artemia salina stimulated the incorporation of ¹⁴C-leucine into polypeptide by a cell-free Escherichia coli system. Preparations from cysts were about as active as those from hatching embryos or nauplii. When analysed by density gradient centrifugation the activity of cyst RNA showed a heterodisperse distribution, not quantitatively related to the absorbance profile. These results and evidence from similar experiments with crude ribosome preparations indicated that the contribution of 18S and 28S ribosomal RNA to the template-like activity was fairly limited. The experiments suggest that RNA with latent messenger activity is present in Artemia cysts during the resting stage.     

Ecology of the brine shrimp Artemia in the Yucatan, Mexico, Salterns

One year of field data on the Yucatan, Mexico, North Coast hypersalinepools and salterns revealed extreme alkalinity, salinity andtemperature conditions, hypoxia and in some instances, totaldesiccation. The four locations were similar in water temperature,nitrites, and phosphates, but differed in salinity, oxygen content,water depth, nitrates, ammonia, silicates, carbonates, sulfates,and pH. The dominant multicellular organism in these hypersalineecosystems is Artemia, the brine shrimp. Artemia abundance andpopulation dynamics were significantly correlated with specificenvironmental conditions, most often water temperature, salinity,and oxygen concentration. The different schedules of Artemiaand cyst production at each location suggest habitat partitioningamong the brine shrimp populations across the North Coast ofthe Yucatan Peninsula. Our results provide an ecological basisfor rational management of these endangered hypersaline ecosystems.     

Enhancement of immunity and disease resistance in the white shrimp,Litopenaeus vannamei,by the probiotic,Bacillus subtilis E20

Effects of Bacillus subtilis E20 isolated from fermented soybean on immune parameters and the disease resistance of the white shrimp (Litopenaeus vannamei) after 98 days of B. subtilis E20 feeding were evaluated in this study. Shrimp fed B. subtilis E20-containing diets at concentrations of 10⁶ (E206), 10⁷ (E207), and 10⁸ (E208) cfu kg^?1, respectively, had significantly increased survival rates of 13.3%, 16.7%, and 20%, compared to the control (fed no probiotic) after being challenged with Vibrio alginolyticus. There were no significant differences in the total hemocyte count, respiratory burst, or superoxide dismutase glutathione peroxidase among all treatments. Shrimp fed a higher concentration of the probiotic (E208) exhibited significant increases in phenoloxidase activity, phagocytic activity, and clearance efficiency compared to control shrimp. In addition, B. subtilis E20 showed a weaker inhibitory effect against the growth of Aeromona hydrophila with around a 0.3-cm inhibitory zone, but showed no inhibitory effects against other selected pathogens, such as white shrimp pathogens: V. alginolyticus and Vibrio vulnificus. These results suggest that the increased resistance of shrimp after B. subtilis E20 consumption occurs through immune modifications, such as increases in phenoloxidase activity, phagocytic activity, and clearance efficiency against V. alginolyticus.     

Influence of peat substrate on the distribution and behaviour patterns of sand shrimp, Crangon septemspinosa, under experimental conditions

Peat harvesting is a lucrative industry in New Brunswick, Canada, and is for the most part located along the coast. Current methods of mining peatlands are such that high levels of peat fibers are transported by runoff into fresh and marine waters. To understand better the influence of peat substrate on aquatic organisms living in peat‐impacted habitats, the sand shrimp (Crangon septemspinosa) was used as a bioindicator. A series of laboratory experiments was conducted in which shrimp were provided with a choice between sand and peat. Long‐term studies on starved shrimp confirmed that whereas foraging activities took place on both substrates, shrimp display an overall preference for sand (73% of all shrimp observed were on sand, n=60). When food was deposited on each of the substrates, shrimp generally opted to feed on sand (75% of all shrimp observed were on sand, n=60). After all food on sand was consumed, shrimp tended to move to peat. This was followed by a short period of foraging on both sand and peat with an eventual return to sand in most instances. The presence of food on both substrates did not influence their preference for sand. However, shrimp will move onto a peat substrate to forage if food is present only there. This observation suggests that, although there is a marked preference for sand, peat is not completely repellent to them. Finally, histological investigations of the shrimp digestive system revealed that peat was ingested with food particles and processed in the gastric mill. There was, however, no discernible discrimination between the two substances during ingestion. Finally, starved shrimp on peat substrate did not ingest peat fibers, suggesting that peat is not perceived as an alternative food source.     

A new fungus disease of the white shrimp Penaeus setiferus

A primary mycosis of larvae of the white shrimp, Penaeus setiferus, is described. The disease first became apparent in larvae in the second protozoeal stage and disappeared as the shrimp reached the first mysis stage. Affected shrimp became immobilized by near complete tissue destruction and replacement by the expanding mycelium. The fungus was found to be Lagenidium sp. and was infective to larval brown shrimp, Penaeus aztecus.