Toxicity and effects of mosquito larvicides methoprene and surface film (Agnique® MMF) on the development and fecundity of the tadpole shrimp Triops newberryi (Packard) (Notostraca: Triopsidae)

We investigated the interactions of tadpole shrimp, a mosquito biological control agent, with the juvenile hormone analog methoprene and a monomolecular surface film. In laboratory assays, the tadpole shrimp (TPS) Triops newberryi (Packard) was able to tolerate high concentrations of methoprene without negative impacts on its growth, longevity, and fecundity when exposed to 1 to 10 mg/liter, or 90–900 fold, of the IE₉₀ levels against a laboratory colony of Culex quinquefasciatus Say. The same held true in field trials when the habitats were treated with Altosid® Liquid Larvicide (Altosid® LL, 5% methoprene) at 0.3–1.2 liters/ha. or 1–4 fold of the label rates for mosquito control. However, some significant impacts on the TPS occurred when they were exposed to Agnique® Monomolecular Film (Agnique® MMF) at the label rates for mosquito control ranging from 1.89–9.45 liters/ha. under laboratory and field conditions. To avoid the negative impact of Agnique MMF on tadpole shrimp, it appears that 1.89 liters/ha. would be the maximum rate when Agnique MMF is used to control mosquitoes in the habitats where the TPS is employed as a biological control agent, or prevailing in the aquatic habitats with potential for suppressing mosquito larval populations.     

Susceptibility of the brine shrimp Artemia and its pathogen Vibrio parahaemolyticus to chlorine dioxide in contaminated sea-water

Adults and nauplii of the brine shrimp, Artemia , together with Vibrio parahaemolyticus , were placed in sewage-contaminated sea-water which had been treated with chlorine dioxide (Hallox E-100^TM) to test its potential as a disinfectant for salt water aquaculture. The nauplii were very susceptible to low concentrations of chlorine dioxide (47 μg/1 Cl^-), but the adults were slightly more resistant. Sterile sea-water treated with lower concentrations of chlorine dioxide (less than 47 μg/1 Cl^-) had no effect on the shrimp, but inhibited the growth of V. parahaemolyticus. In sewage-contaminated sea-water, chlorine dioxide levels of 285–2850 μg/1, necessary for the inactivation of V. parahaemolyticus and any native bacteria, destroyed the Artemia culture. Hallox E-100^TH persisted in sea-water for 18 h, but later decayed. We conclude that: (i) Artemia nauplii are a sensitive and convenient test-organism to determine low concentrations of chlorine dioxide in sea-water; (ii) chlorine dioxide is efficient for controlling V. parahaemolyticus in sea-water; and (iii) chlorine dioxide should be further evaluated as a potential disinfectant for aquaculture, but, for higher organisms than Artemia.     

Biomass production and nutritional value of Artemia sp. (Anostraca: Artemiidae) in Campeche, México

Biomass of the crustacean Artemia sp. has multiple uses. The biochemical composition and biomass production of Artemia grown from cysts produced by a native population from Real de Salinas were evaluated under laboratory conditions. Nauplii (instar I) were stocked at density of 10 nauplii/ml in 1.5 l tanks, fed with rice bran from day 2 to day 6, and with the microalgae Tetraselmis suecica from day 7 to day 15. At the end of the trial (day 15) the average length was 5.34 mm, biomass production was 15.72 g/l (wet weight), and survival was 79%. The proximal analysis and biochemical composition of Artemia biomass indicated that its nutrient percentages are closely similar to Artemia from other regions, making this species a suitable food for cultured fish and crustacean.     

Function and cellular localization of farnesoic acid O-methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis.

The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum. FAMeT was localized in the neurosecretory cells of the X-organ-sinus gland complex of the eyestalk. As shown by RT-PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeT activity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.     

Susceptibility of the brine shrimp Artemia and its pathogen Vibrio parahaemolyticus to chlorine dioxide in contaminated sea-water.

Adults and nauplii of the brine shrimp, Artemia, together with Vibrio parahaemolyticus, were placed in sewage-contaminated sea-water which had been treated with chlorine dioxide (Hallox E-100TM) to test its potential as a disinfectant for salt water aquaculture. The nauplii were very susceptible to low concentrations of chlorine dioxide (47 micrograms/l Cl-), but the adults were slightly more resistant. Sterile sea-water treated with lower concentrations of chlorine dioxide (less than 47 micrograms/l Cl-) had no effect on the shrimp, but inhibited the growth of V. parahaemolyticus. In sewage-contaminated sea-water, chlorine dioxide levels of 285-2850 micrograms/l, necessary for the inactivation of V. parahaemolyticus and any native bacteria, destroyed the Artemia culture. Hallox E-100TM persisted in sea-water for 18 h, but later decayed. We conclude that: (i) Artemia nauplii are a sensitive and convenient test-organism to determine low concentrations of chlorine dioxide in sea-water; (ii) chlorine dioxide is efficient for controlling V. parahaemolyticus in sea-water; and (iii) chlorine dioxide should be further evaluated as a potential disinfectant for aquaculture, but, for higher organisms than Artemia.     

Solvent extracts of the red seaweed Gracilaria fisheri prevent Vibrio harveyi infections in the black tiger shrimp Penaeus monodon

Vibriosis is a common bacterial disease that can cause high mortality and morbidity in farmed shrimp. Since compounds from seaweed have been reported to have anti-bacterial and immunostimulant activity, this study was conducted to determine whether solvent extracts from the red seaweed Gracilaria fisheri might be a possible alternative for prevention and treatment of shrimp vibriosis caused by Vibrio harveyi. Seaweed extracts prepared using ethanol, methanol, chloroform and hexane were evaluated for anti-V. harveyi activity by the disc-diffusion method. The ethanol, methanol and chloroform extracts showed activity against a virulent strain of V. harveyi with potency (minimal inhibitory concentrations in the range of 90-190 μg ml(-1)) equivalent to the antibiotic norfloxacin. The ethanol extract was not toxic to the brine shrimp Artemia salina when it was fed to them for enrichment prior to their use, in turn, as feed for postlarvae of Penaeus monodon. Postlarvae fed with these enriched Artemia gave significantly lower mortality than control postlarvae after challenge with V. harveyi. In addition, P. monodon juveniles injected with the ethanol extract showed a significant increase in the total number of haemocytes and an increased proportion of semi-granulocytes and granulocytes when compared to control shrimp. The activities of phenoloxidase and superoxide dismutase were also increased, with an accompanying increase in superoxide anion production. When these juvenile shrimp were challenged with V. harveyi, mortality was markedly reduced compared to that of control shrimp. The results indicated that ethanol extracts of G. fisheri had immunostimulant and antimicrobial activity that could protect P. monodon against V. harveyi.     

The shrimp FAMeT cDNA is encoded for a putative enzyme involved in the methylfarnesoate (MF) biosynthetic pathway and is temporally expressed in the eyestalk of different sexes

Methylfarnesoate (MF), an analogue of the insect juvenile hormone III, has been implicated to play a vital role in the regulation of the growth and reproductive development in crustaceans. Farnesoic acid O-methyltransferase (FAMeT) is the key enzyme involved in catalyzing the final step in the MF biosynthetic pathway. In this study, we report the cloning and characterization of the cDNA encoding the putative FAMeT of the shrimp Metapenaeus ensis. FAMeT comprises 280 amino acid residues with a predicted molecular weight of 32kDa. The predicted putative FAMeT protein reveals a high degree of structural conservation of FAMeT with the lobsters. It shares 79 and 70% sequence identities with the putative FAMeTs of Homarus americanus and Panulirus interruptus, respectively. As revealed by the Southern blot analysis and genomic PCR, only one gene exists in the shrimp genome and the gene is uninterrupted in the coding region. The shrimp FAMeT mRNA is widely distributed in many tissues with the highest expression level observed in the central nervous system. A constant level of FAMeT expression is recorded in the ventral nerve cord of the juveniles and the mature females during the reproductive cycle. Unlike the ventral nerve cord, the eyestalk of the juvenile male, but not the female, expresses FAMeT. Further study shows that the eyestalk of the mature female expresses FAMeT during all stages of ovarian maturation. We speculate that FAMeT may be important for the regulation of eyestalk neuropeptides. This is the first extensive study on the molecular characterization, structural analysis and expression of the crustacean FAMeT.     

Assessment of fluorescent-labeled bacteria for evaluation of in vivo uptake of bacteria (Vibrio spp.) by crustacean larvae

Available methods to study crustacean digestive tract colonization by bacteria are laborious, time-consuming, and do not permit in vivo assays and observation. This paper reports on a rapid and consistent technique to apply a fluorescent label to bacteria, which can then be presented to filter-feeding crustacea such as Artemia and penaeid larvae for later in situ bacterial distribution observation. Three luminescent Vibrio spp. were stained and observed inside Artemia nauplii, shrimp zoea and mysis stages, Vibrio harveyi type strain ATCC 14126, M(1) (pathogenic) and Ea (non-pathogenic). Factors such as dye (DTAF) concentration, exposure time/temperature and sonication time were evaluated. Viability of the dye and stained bacteria were tested at 4, -20 and -70 degrees C storage temperatures for up to 81 days. Results show that 4 and -20 degrees C storage temperatures are not recommended. At -70 degrees C, both bacteria and dye are optimally preserved. Monodispersed fluorescent-labeled bacterial cells can be observed inside the digestive tract of crustacean larvae at a density of inoculation as high as 5.2 x 10(6) CFU ml(-1). After 2 to 4 h, some leaching occurs, increasing difficulty in observation, although after 24 h, it is still possible to observe monodispersed FLB inside the digestive tract of crustacean larvae. Autofluorescence may complicate observation when filter-feeding crustacean larvae are co-fed with microalgae.     

The effects of surimi and pelleted diets on the laboratory survival, growth, and feeding rate of the cuttlefish Sepia officinalis L.

The effects of feeding a prepared surimi diet (fish-based) and a prepared pelleted diet (shrimpbased) on the survival, growth and feeding rate of the cuttlefish Sepia officinalis L. were evaluated during a 45-day experiment. One hundred and twenty juveniles of laboratory cultured cuttlefish (74.5 ± 12.36 g) were divided into three treatments and were fed thawed shrimp (control), pellets or surimi. Survival rates on these diets were 95.0%, 67.5% and 22.5%, respectively. Preliminary data indicated that the low survival of cuttlefish fed surimi may have been caused by low levels of copper in their blood (131 vs 244 μg/ml) since copper is required for their respiratory blood pigment, hemocyanin. Instantaneous growth rates were 2.71 % body weight BW/day for cuttlefish fed raw shrimp, 0.33% BW/day for cuttlefish fed pellets, and 0.54% BW/day for cuttlefish fed surimi. The feeding rate of cuttlefish fed shrimp was high (6–8% BW/day). The feeding rate on pellets increased with time (from < 1 to 3% BW/day) but never reached the level for raw shrimp. The feeding rate on surimi increased to equal the rate for raw shrimp during days 1–30 (8 to 9% BW/day) and thereafter decreased (<4% BW/day). In conclusion, there was a major distinction between the palatability of a prepared diet and the ability of that diet to support growth. Surimi was highly palatable but resulted in poor survival, suggesting low nutritional quality. In contrast, pellets were less palatable but produced maintenance growth. Development of practical surimi diets will require supplementation of the surimi with soluble micro- and macronutrients.     

Accumulation, distribution and toxicology of dietary nickel in lake whitefish (Coregonus clupeaformis) and lake trout (Salvelinus namaycush)

An 18-day experiment was conducted to investigate the uptake and sublethal toxicity of dietary Ni in adult lake whitefish (LWF, Coregonus clupeaformis) and lake trout (LT, Salvelinus namaycush) fed diets containing 0, 1000 and 10000 microg Ni/g, prepared with and without brine shrimp. The results of this experiment were used to design an experiment of longer duration in which one of the fish species was selected and exposed to lower dietary Ni doses. In the present study feed refusal was observed in LT and LWF fed 10000 microg Ni/g, after three and 4-5 feedings, respectively. LT fed Ni-contaminated diets exhibited different patterns of Ni accumulation than LWF. Increased Ni concentrations in all LWF tissues, except the intestine, were associated with increased doses of Ni. Copper and Zn concentrations in kidney and liver of LWF were altered. Metallothionein concentrations in kidneys of LT fed 1000 microg Ni/g and 10000 microg Ni/g and LWF fed 10000 microg Ni/g and in livers of LWF fed 10000 microg Ni/g (diet without shrimp only) increased significantly. Increased lipid peroxide production in the plasma of LT fed 10000 microg Ni/g was observed. Blood glucose and electrolytes were affected by Ni exposure. Histopathological alterations were observed in kidneys of LWF fed low and high dose diets, livers of whitefish fed high dose diets, and intestines of LWF fed high dose diets and LT fed low and high dose diets. LT fed high dose diets exhibited significant decreases in weight.     

Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults

Letrozole is a synthetic aromatase inhibitor and interferes in the committed step in the synthesis of endogenous estrogens from androgens. To evaluate potential effects on the early life stages of Japanese medaka, larvae and fertilized eggs were exposed to letrozole for 96 h and 14 days, respectively. No larvae died and no adverse effects were found on embryonic development at concentrations up to 3125 microg/L. Reproductive effects were assessed by exposing adults to 1, 5, 25, 125 and 625 microg/L letrozole for 21 days. A dose-dependent decrease in fecundity (>25 microg/L) and fertility (>5 microg/L) accompanied by histological changes suggested the inhibition of oocyte growth and possibly maturation. At 625 microg/L, the fish ceased spawning during the last week of exposure. Letrozole (>5 microg/L) reduced plasma vitellogenin levels in females in a dose-dependent manner. Transgenerational effects were evaluated by removing freshly-laid F1 eggs from letrozole-contaminated water and raising them to 15 days post-hatching in control water. Hatchability and time to hatching were detrimentally affected (>5 microg/L), but no morphological deformities were observed. Furthermore, a dose-dependent increase in the proportion of genotypic F1 males was found (>5 microg/L).     

Ascorbate concentration in fish ontogeny

The ontogenetic trend of ascorbate has been quantified in three freshwater fishes: roach ( Rutilus rutilus ), whitefish ( Coregonus lavaretus ) and Arctic charr ( Salvelinus alpinus ). Total ascorbate (reduced and oxidized) declined from 150 to μg g⁻¹ as newly hatched larvae grew to become several-months-old juveniles. Declining total ascorbate with increasing size of metamorphosing fish could not be reversed by feeding on brine shrimp, Artemia salina nauplii, zooplanktonic food containing > 74μg g⁻¹ total ascorbate. The proportion of reduced ascorbate in total ascorbate increases with fish size/age. The physiological mechanism of the changes in transferable ascorbate forms remains unknown, but high dehydroascorbate concentrations suggest high vulnerability of larval fish to oxidation stress. This is the first report on quantity of vitamin C retained in actively-feeding larval and juvenile fish. The efficiency of ascorbate transfer from zooplankters to larval fish amounted to 5–20%. The ecological significance of larval fish feeding on various zooplankters and/or phytoplankton may reflect a trend toward maximum transfer of this vitamin in freshwater food webs.     

Possible role of non-classical chromatophorotropins on the regulation of the crustacean erythrophore.

Two neuropeptides, the pigment dispersing hormone (PDH) and the pigment concentrating hormone (PCH), are well known to respectively promote centrifugal and centripetal granule translocation in the freshwater shrimp Macrobrachium potiuna erythrophores. Herein, we demonstrate for the first time the effects of crustacean non-classical chromatophorotropins on the pigment migration in M. potiuna erythrophores. Although proctolin, 20-hydroxyecdisone (20HE), and melatonin were ineffective, the crustacean cardioactive peptide (CCAP) was a full agonist, inducing pigment dispersion in a dose-dependent manner with EC(50) of 9.5. 10(-7) M. In addition, concentrations of CCAP lower than the minimal effective dose (10(-8) and 10(-7) M) decreased the PCH-induced aggregation, shifting rightward the dose-response curve (DRC) to PCH 2.2- and 29-fold, respectively. Surprisingly, melatonin (10(-7) and 10(-6) M) also shifted to the right 8.7- and 46.5-fold, respectively, the DRC to PCH. In conclusion, our data demonstrate that besides PCH and PDH, CCAP and melatonin also regulate the pigment migration within the crustacean erythrophore. J. Exp. Zool. 284:711-716, 1999.Copyright 1999 Wiley-Liss, Inc.     

Hyperglycemic and osmotic effects of dopamine and recombinant hormone CHH-B1 in the Pacific white shrimp Litopenaeus vannamei

The effects of dopamine (DA) on crustacean hyperglycemic hormone (CHH) release and osmoregulation were investigated in the white shrimp Litopenaeus vannamei. Application of 2 μg of the recombinant CHH-B1_His hormone or 2?×?10^?6 mol L^?1 of DA to intact shrimp caused an increase in the hemolymph glucose levels 1 h post-injection, suggesting that DA might stimulate hyperglycemia through CHH release from the sinus glands. This assumption was supported by similar experiments using bilaterally eyestalk-ablated shrimp. Additionally, rCHH-B1_His restored the osmoregulatory capacity (OC) of shrimp under hyperosmotic conditions to basal values 2 h post-injection, whereas DA led to an OC decrease in shrimp at all sampling times. These neuroendocrine factors may be involved in the control of metabolism and osmoregulation in L. vannamei and could be important for its adaptation to different environmental conditions.     

ArHsp22, a developmentally regulated small heat shock protein produced in diapause-destined Artemia embryos, is stress inducible in adults

Diapause embryos of the crustacean Artemia franciscana exhibit extreme stress tolerance, a property thought to involve molecular chaperones known as small heat shock proteins. To further explore this idea, the structure, function and synthesis of ArHsp22, an Artemia small heat shock protein, were characterized. ArHsp22 contains amino-terminal WXDPF motifs, an alpha-crystallin domain with a highly conserved arginine, and a carboxy-terminal I/VXI/V motif, all typical of small heat shock proteins. ArHsp22 formed large oligomers and exhibited molecular chaperone activity in vitro, protecting citrate synthase and insulin from denaturation. Quantitative PCR and immunoprobing of western blots revealed that ArHsp22 synthesis is restricted to diapause-destined Artemia embryos and that the protein is degraded during post-diapause development. ArHsp22 was observed in cyst nuclei, a location shared by p26 but not ArHsp21, which are two other diapause-specific Artemia small heat shock proteins. ArHsp22 production was enhanced by thermal stress, but only in adults, thus representing the first crustacean small heat shock protein whose synthesis is known to be both developmentally regulated and stress inducible. The results demonstrate that expression of the gene for ArHsp22 is modulated by multiple cues that vary with life history stage. Such findings are of importance in understanding diapause maintenance in Artemia embryos and the survival of adult animals experiencing environmental insult.     

Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection

Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp (Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1h after the rLV-CHH injection and then returned to normal after 3h. In addition to the effect of rLV-CHH administration (25 μg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBS-injected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection.     

Regulation of the Crustacean Mandibular Organ

The crustacean mandibular organ (MO) produces methyl farnesoate(MF), a juvenile hormone-related compound thought to have rolesin crustacean reproduction and development. Therefore, the controlof MF production by the MO has been of considerable interest.Current evidence indicates that the MO is negatively regulatedby peptides present in the eyestalk (MO inhibiting factor, MO-IH).Several eyestalk neuropeptides have been identified that inhibitMF synthesis by MO incubated in vitro. The amino acid sequencesof these MO-IH peptides are similar to peptides in the crustaceanhyperglycemic hormone (CHH) family of neuropeptides. In addition,there appears to be a compound in the eyestalk that lowers hemolymphlevels of MF in vivo but does not directly affect the MO invitro. The inhibition of MF synthesis by eyestalk peptides involvesthe inhibition of farnesoic acid O-methyl transferase, the lastenzyme in the MF biosynthetic pathway. The activity of thisenzyme is affected by cyclic nucleotides, suggesting that thesecompounds may be involved in the signal transduction pathwaymediating the effects of MO-IH.     

Priming the prophenoloxidase system of Artemia franciscana by heat shock proteins protects against Vibrio campbellii challenge

Like other invertebrates, the brine shrimp Artemia franciscana relies solely on innate immunity, which by definition lacks adaptive characteristics, to combat against invading pathogens. One of the innate mechanisms is melanisation of bacteria mediated by the activation of the prophenoloxidase (proPO) system. The 70 kDa heat shock proteins (Hsp70) derived from either prokaryote (Escherichia coli) or eukaryote (Artemia), well conserved and immune-dominant molecules, protect Artemia against Vibrio campbellii. However, the molecular mechanisms by which these proteins protect Artemia against Vibrio campbellii infection are unknown. Here we demonstrated that feeding gnotobiotically grown Artemia with either Artemia Hsp70 or the E. coli Hsp70 equivalent DnaK, each overproduced in E. coli, followed by V. campbellii challenge enhanced the proPO system, at both mRNA and protein activity levels. Additionally, the Artemia fed with these proteins survived well in a Vibrio challenge assay. These results indicated that Hsp70s derived from either prokaryotic or eukaryotic sources generate protective immunity in the crustacean Artemia against V. campbellii infection by priming the proPO system. This is apparently the first in vivo report on priming activity of Hsp70 in an invertebrate.