Nutritional value of the cryptophyte Rhodomonas lens for Artemia sp.

Juvenile or adult Artemia sp. are often used as live prey for the rearing of early life stages of some crustacean, fish and cephalopod species. The improvements of both Artemia growth and its biochemical composition are key issues for the suitable use of Artemia biomass in these rearing processes. In this study we evaluated the growth and survival rates of Artemia fed with the cryptophyte Rhodomonas lens in comparison with different microalgal species commonly used in aquaculture: the prasinophyte Tetraselmis suecica, the prymnesiophyte Isochrysis galbana Parke, and the eustigmatophyte Nannochloropsis gaditana. Microalgae were cultured semi-continuously in nutrient saturated conditions and with a daily renewal rate of 30% of the volume of cultures, to obtain biomass of controlled and optimized composition. Considerable differences in Artemia growth were observed, as well as in the survival rate. At day 8 of rearing, Artemia fed R. lens had the highest length (4.9 ±0.6 mm, P < 0.001), followed by individuals fed T. suecica (4.2 ± 0.7 mm), I. galbana (3.6 ± 0.7 mm) and finally those fed N. gaditana (1.5 ± 0.2 mm). The survival rate of Artemia fed N. gaditana (18 ± 3%) was much lower (P < 0.001) than values found for the remaining groups (69 to 88%). The growth rate of Artemia obtained with R. lens was in general much higher than with other microalgal diets previously reported in the literature. The higher protein content of R. lens could explain the higher growth obtained with this species, but differences of Artemia growth with the different diets could not be explained solely on the basis of the gross composition of microalgae. Factors such as cell size and digestibility all seem to contribute to the results observed. Another trial was carried out to investigate differences in Artemia growth and on its biochemical composition when fed the best two diets: R. lens or T. suecica. The fatty acid (FA) and total amino acid (AA) composition of both microalgal species and the composition of Artemia were assessed as well. As found in the first experiment individuals fed R. lens (group ARHO) grew faster than those fed T. suecica (group ATET), attaining 3.6 ± 0.3 mm and 3.2 ± 0.4 mm (P < 0.001), respectively, after 5 days of rearing. The much higher AA content obtained in R. lens may be on the basis of the higher growth obtained with this species. Protein and carbohydrate levels in Artemia juveniles were very similar in both groups (64-68% of dry weight, and 8-10%, respectively). Lipid was slightly lower in ARHO (12%) than in ATET (15%, P < 0.01). Regarding the FA composition, juveniles from group ARHO contained higher levels of eicosapentaenoic acid (EPA, 6.2%) than juveniles from ATET (4.1%, P < 0.01), whereas docosahexaenoic acid (DHA) was only found in juveniles from ARHO (1.1%). Taking into account that the daily productivity of R. lens culture was higher than, or at least equal, the remaining microalgal species this cryptophyte is confirmed as an excellent diet to optimize the growth of Artemia, as well as to improve its biochemical composition.     

Use of selected bacteria and yeast to protect gnotobiotic Artemia against different pathogens

To evaluate the potential probiotic effect of two bacterial strains towards Artemia cultured in different gnotobiotic conditions, challenge tests were performed with a virulent Vibrio campbellii or with an opportunistic Vibrio proteolyticus strain. For that purpose, three feed sources (different isogenic Saccharomyces cerevisiae mutant strains) were chosen, yielding distinct Artemia culture performances. Both bacterial strains, selected from previous well-performing Artemia cultures, were able to protect against the opportunistic V. proteolyticus, while, generally, these bacteria could not protect Artemia against V. campbellii. The quality of the feed provided (in the form of the isogenic mnn9 yeast mutant) to Artemia had a stronger influence on nauplii protection against the opportunistic and the virulent Vibrio than the addition of beneficial bacteria. This feed has a higher nutritional value for Artemia, but contains also more cell wall bound β-glucans and chitin. Data suggest that the change in the cell wall composition, rather than the overall better nutritional value, of the mnn9 strain is responsible for the protection against both Vibrios.     

Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a protozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decomposing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseni antibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2 kDa. Immunofluorescent assay indicated that the rabbit anti-P. olseni IgG was specific to all life stages, including the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where P. olseni-associated clam mortality has recurred for the past decade. Purified cells were positively stained with the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time the presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1-2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes.     

Laboratory evaluation of the chemosterilant lufenuron against the fruit flies Ceratitis capitata, Bactrocera dorsalis, B. cucurbitae, and B. latifrons

Four species of tephritid fruit flies, Ceratitis capitata, Bactrocera dorsalis, B. cucurbitae, and B. latifrons were evaluated for toxic, developmental, and physiological responses to the chemosterilant lufenuron. No significant mortality of laboratory strains of the first three species was observed after their exposure up to 50 μg/mL of lufenuron in agar adult diet, whereas B. latifrons adults fed with 50 μg/mL of lufenuron in the diet caused significant mortality compared to the control. Fertility of C. capitata adults fed on 50 μg/mL lufenuron-fortified diet between 7 and 12 days of age was approximately 46% of the no lufenuron control. Fertility of B. dorsalis and B. latifrons adults fed on 50 μg/mL lufenuron-incorporated diet was about 45% and 62% of the control, respectively. Lufenuron did not significantly affect fertility of B. cucurbitae adults. Lufenuron did not affect fecundity of C. capitata and B. dorsalis. Fecundity of B. cucurbitae and B. latifrons was not evaluated due to difficulty to count the eggs laid deep in the agar diet. Larvae fed on a liquid larval diet with ≤ 0.1 μg/mL of lufenuron were also evaluated. Pupal recovery, adult emergence, adult fliers, mating, egg hatch, and egg production of C. capitata were significantly decreased, while for B. dorsalis, pupal recovery, larval duration and adult emergence were affected. No effect of lufenuron on B. cucurbitae larvae was observed. B. latifrons was not performed because shortage of eggs at the time of this research. Lufenuron is a potential agent for management and control of C. capitata and B. dorsalis.     

Babesia bigemina: In vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells

This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.     

Phenoloxidase and trypsin in germ-free larvae of Artemia fed with cooked unicellular diets: Examining the alimentary and protective effects of putative beneficial bacterium, yeast and microalgae against vibriosis

Three putative beneficial unicellular organisms, the marine bacterium Roseobacter sp, the yeast Saccharomyces cerevisiae mnn9 strain (SC-mnn9) and the microalga Tetraselmis suecica were cooked and offered separately as diets to developing germ-free (GF) Artemia larvae, in order to analyze their alimentary and protective effects. GF Artemia larvae were able to grow with cooked Tetraselmis and SC-mnn9 but failed with cooked Roseobacter. In spite of its high dietary quality, Tetraselmis failed to provide protection against Vibrio proteolyticus infection, while Roseobacter failed as food as well to provide protection. Cooked SC-mnn9 appeared to possess both values, dietary for growth and protective against Vibrio infection. GF Artemia larvae were apparently rapid adapted to dietary swaps; from yeast to algal and from algal to yeast. While the diets swap from algal or yeast, to bacterial diet appeared to be detrimental. Phenoloxidase-L (PO-L) and trypsin-L were used as biochemical indicators of defense and digestive functions, respectively. Developmental trypsin-L patterns were similar when fed on yeast and microalgae diets, suggesting a good digestive adaptation to plant or fungal substrates at early stages. On the contrary, diets swap or Vibrio infection affected PO-L and trypsin-L suggesting a sort of ‘alteration’ of digestive and defense functions.     

Chemically induced predator avoidance behaviour in the burrowing bivalve Macoma balthica

The responses of the burrowing bivalves Macoma balthica and Cerastoderma edule to chemical cues emitted by feeding shore crabs Carcinus maenas were investigated. M. balthica held in the laboratory and exposed to chemical signals in effluent water discharging from tanks containing C. maenas fed 20 M. balthica day^− 1 reacted by increasing their burial depths from approximately 30 mm to depths of > 60 mm, over a period of several days. When the signal was removed the bivalves gradually returned to their original depth over 5 days. C. edule similarly exposed to effluent from crabs feeding on conspecifics showed no response. In an attempt to identify the signal inducing this burrowing response, M. balthica were exposed to a variety of chemical signals. Crabs fed M. balthica elicited the strongest response, followed by crabs fed C. edule. There were also small responses to effluent from crabs fed on fish, crabs previously fed on M. balthica and to crab faeces, but no responses to starved crabs, crushed M. balthica, or controls. We conclude that increased burrowing depth of M. balthica is induced by some as yet unidentified chemical cue produced by feeding crabs and is strongest when the crabs were fed on M. balthica. Unexpectedly, neither the presence of crabs themselves, nor of damaged conspecifics, was effective in eliciting a burrowing response. The mortality rates of M. balthica and C. edule selected by crabs when burrowed at normal depths and after exposure to effluent from feeding crabs were different. Crabs selected 1.5 times more C. edule than M. balthica when both species were burrowed at their normal depths, but 15 times more after the tanks had been exposed to effluent from feeding crabs for 5 days. The burrowing response of M. balthica thus appears to reduce mortality significantly by displacing predation pressure on to the more accessible C. edule.     

Evaluation of different yeast cell wall mutants and microalgae strains as feed for gnotobiotically grown brine shrimp Artemia franciscana

The nutritional value of isogenic yeast strains and two microalgal species for gnotobiotically grown Artemia was examined. Yeast cell wall mutants were always better feed for Artemia than their respective wild type. Yeast cells harbouring null mutants for enzymes involved early in the biochemical pathway for cell wall mannoproteins synthesis performed best as feed for Artemia. Yeast cells defective in chitin or β-glucan production were scored in second order. The mnn6 isogenic yeast mutant, harbouring a null mutation for mannoprotein phosphorylation, performed poorly as feed for Artemia, although with good growth. These results suggest that any mutation affecting the yeast cell wall scaffolding by reducing the amount of covalent links between the major components of yeast cell wall, namely mannoproteins, β-glucans and chitin, is sufficient to improve the digestibility for Artemia. The results with microalgae indicated that within one species, strains can have different nutritional value under gnotobiotic conditions. The growth phase was another parameter influencing feed quality, although here it was not possible to reveal the exact cause. It is anticipated that the standard Artemia gnotobiotic growth test is an excellent tool to study the mode of action of bacteria, with a probiotic as well as with a pathogenic character.     

Australian dingoes are definitive hosts of Neospora caninum

To provide objective data on the potential role of dingoes (Canis lupus dingo) in the life cycle of Neospora caninum in Australia, the production of N. caninum oocysts by experimentally infected canids was investigated. Three dingo pups raised in captivity and three domestic dogs were fed tissue from calves infected with an Australian isolate of N. caninum, Nc-Nowra. Oocysts of N. caninum, confirmed by species-specific PCR, were shed in low numbers by one dingo pup at 12-14 days p.i. The remaining animals did not shed oocysts. Furthermore, the blood from two out of three dingoes tested positive for DNA of N. caninum using PCR tests at 14 and 28 days p.i. Oocyst shedding from the intestinal tract of a dingo demonstrates that dingoes are definitive hosts of N. caninum and horizontal transmission of N. caninum from dingoes to farm animals and wildlife may occur in Australia.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

Hematodinium sp. infection of red Paralithodes camtschaticus and blue Paralithodes platypus king crabs from the Sea of Okhotsk, Russia

A disease caused by a parasitic dinoflagellate of the genus Hematodinium was identified in red, Paralithodes camtschaticus, and blue, Paralithodes platypus, king crabs from the north-east region of the Sea of Okhotsk, Russia, during annual stock surveys. No carapace color change was observed even in heavily infected crabs, but diseased crabs possessed creamy-yellow hemolymph, which was visible through the arthrodial membranes of the abdomen and appendages. Several stages of the parasite’s life history, including trophonts, plasmodia, sporonts and macrodinospores, were observed in tissues of infected king crabs. Numerous parasite cells were observed in the lumina of the myocardium, the gills, the connective tissue of antennal glands and the sinuses of nerve ganglia, eyestalks and gastrointestinal tract of king crabs with gross signs of infection. Based on sequencing of the 18S rDNA, it appears that the Hematodinium sp. found in red and blue king crabs is identical or closely related to Hematodinium sp. isolated from crabs of the genera Chionoecetes and Lithodes. Observed prevalences were 0.33% in sublegal male red king crabs, 0.18% in female red king crabs, 0.34% in sublegal male blue king crabs and 0.31% in female blue king crabs.     

Partial cloning and production of polyclonal antiserum against recombinant capsid protein of Hepatopancreatic Parvovirus (HPV) and its application for diagnostics in penaeid shrimp

Hepatopancreatic Parvovirus (HPV) causes infection in the early stages of shrimp leading to retarded growth, ultimaltely resulting in monetary loss to the shrimp farmers. To over come this situation screening of post-larvae (PL) by immunology-based diagnostics is required. Hence, the specific gene of capsid protein for HPV was cloned into pRSET B expression vector and rHCP overexpressed with 6-histidine tagged fusion protein in Escherichia coli BL21(DE3). Immunology-based methods like Western blot, dot blot and ELISA techniques were employed to detect HPV in infected samples using the antiserum raised in rabbits against recombinant HCP of HPV. The dot blot assay using anti-rHCP was found to be capable of detecting HPV in HPV infected post-larvae as early as at 24 h post infection. The antiserum could detect the HPV in the infected samples at 1 ng of total protein. HPV infection estimated by ELISA using anti-HCP and pure r-HCP as a standard was found to increase gradually during the course of infection from 24 h post infection. The sensitivity of antibody-based diagnostics employed in the present study was compared with that of PCR diagnostic method to screen the post-larvae for the detection of HPV.     

Artemia as a possible vector for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus transmission (XSV) to Macrobrachium rosenbergii post-larvae

Five developmental stages of Artemia were exposed to Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) by immersion and oral routes in order to investigate the possibility of Artemia acting as a reservoir or carrier of these viruses. The second objective was to determine if virus-exposed Artemia were capable of transmitting the disease to post-larvae (PL) of M. rosenbergii. There was no significant difference in percent mortality between Artemia control groups and groups challenged with these viruses. On the other hand, all the developmental stages of Artemia were positive for both viruses by nested RT-PCR, regardless of the challenge route. In horizontal transmission experiments, 100% mortality was observed in M. rosenbergii PL fed with Artemia nauplii exposed to MrNV and XSV by either challenge route. However, no mortality was observed in PL fed with virus-free Artemia. RT-PCR analysis of the M. rosenbergii PL confirmed the presence of MrNV and XSV in the challenge group and absence in the control group.     

The resistance to ammonia stress of Penaeus monodon Fabricius juvenile fed diets supplemented with astaxanthin

This study was aimed at determining if the increase of body astaxanthin content through dietary supplementation in tiger prawn Penaeus monodon juvenile could enhance its antioxidant defense capability and resistance to ammonia stress. Haemolymph total antioxidant status (TAS) and superoxide dismutase (SOD) were chosen as parameters of shrimp antioxidant capacity. Resistance to chemical stress was evaluated by shrimp survival rate, and haemolymph aspartate aminotransferase (AST) and alanine aminotransferase (ALT). P. monodon 5-day postlarvae were fed diets supplemented with 0 and 71.5 mg kg⁻¹ astaxanthin for 8 weeks. Shrimps were then subjected to 72-h exposure of ammonia at 0.02, 0.2, 2 and 20 mg l⁻¹. The survival rates of the astaxanthin-fed (AX) shrimp were higher than those of the control shrimp under all levels of ammonia except 20 mg l⁻¹, showing that the shrimp's resistance to ammonia stress had been improved by dietary astaxanthin. AX shrimp had higher TAS than control shrimp at ammonia levels higher than 0.02 mg l⁻¹ and lower SOD at all ammonia levels suggested that antioxidation capability had been greatly enhanced. AST in AX shrimp was lower than that in control shrimp under all levels of ammonia stress. ALT in AX shrimp was either lower than or equal to that in control shrimp under various levels of ammonia. Both AST and ALT reflected that shrimp hepatopancreatic function had been improved by dietary astaxanthin. Astaxanthin can become essential for P. monodon when the animal is under ammonia stress.     

Growth of Nyctiphanes (Euphausiacea) on different diets

To determine the effects of diet on the growth rate of Nyctiphanes australis (Euphausiacea), metanauplii were reared to mature adults in the laboratory. Sibships (siblings from the same mother) were raised on different food items collected from the field and cultured in the laboratory. A sibship was divided at the calyptopis stage and 50% of the siblings were fed one of three experimental diets (Thalassiosira, Heterocapsa, or Phaeocystis); the balance of the siblings were fed a control diet of Tetraselmis chuii and Artemia larvae.The growth rate of siblings was not altered by the different diets. Siblings developed asynchronously, however, from egg to adult.Some animals were always at a more advanced developmental phase and by day 100, up to 25% larger than their siblings (p<0.001). A possible implication of this result is that the larval growth of N. australis is strongly influenced by genotype.     

Culex pipiens affected by joint infection of a mosquito iridescent virus and Strelkovimermis spiculatus

Dual infections with a mosquito iridescent virus (MIV) and the mermithid nematode, Strelkovimermis spiculatus were recorded in natural Culex pipiens populations around La Plata city, Argentina. S. spiculatus was detected in 82% of samples that were positive for MIV infection. Dissected larvae of Cx. pipiens with patent MIV infection presented 42% infection with S. spiculatus. Larvae of Cx. pipiens exposed to MIV and S. spiculatus under laboratory conditions produced a high joint infection rate (82.5%) while no infection was recorded on larvae exposed to virus suspension only. Field and laboratory results suggest a strong association between S. spiculatus and MIV in natural populations of Cx. pipiens, in which S. spiculatus could be a mode of entry for the virus into the mosquito hemocele.     

Molecular and biological characterization of molt-inhibiting hormone of Penaeus monodon

The action of molt-inhibiting hormone (MIH) on the inhibition of ecdysone release from the Y-organ of decapod crustacean keeps the animal in the intermolt stage that dominates its molting cycle. MIH is thus one of the major keys in mediating growth and reproduction. This study has isolated cDNA encoding two types of MIH, Pem-MIH1 and Pem-MIH2, from the black tiger shrimp, Penaeus monodon on the basis of sequence homology to MIH from two other shrimp species. The full-length cDNA of Pem-MIH1 was characterized. Pem-MIH1 cDNA harbored 318 bp open reading frame that coded for a translated product containing 28 amino acids of the signal peptide and a putative mature Pem-MIH of 77 amino acids. The recombinant Pem-MIH1 was expressed in Pichia pastoris as a secreted protein. After purification by gel filtration, the purified Pem-MIH1 exhibited the ability to extend molting duration of P. monodon from 11.8 days to 16.3 days suggesting that Pem-MIH1 be responsible for molt-inhibiting function in the shrimp. The attempt to clone Pem-MIH1 and Pem-MIH2 genes was achieved by direct PCR amplification and PCR-based genome walking strategy, respectively. The structure of both Pem-MIH genes, containing three exons interrupted by two introns, was similar to each other and also to that of MIH genes of other crustaceans reported so far. Expression study of Pem-MIH1 and Pem-MIH2 in various tissues of P. monodon revealed the difference in expression patterns. Pem-MIH1 expressed in both the eyestalk and the thoracic ganglia whilst Pem-MIH2 expression was limited to the eyestalk. The expression of MIH in non-eyestalk tissue may suggest additional role of this hormone.     

New evidence for the involvement of Paracartia grani (Copepoda,Calanoida) in the life cycle of Marteilia refringens (Paramyxea)

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.