Intracoelomic Injection Research Articles

Dietary Beta‐Glucans and Nucleotides Enhance Resistance of Red‐Tail Black Shark (Epalzeorhynchos bicolor, fam. Cyprinidae) to Streptococcus iniae Infection

Abstract Two experiments were conducted to examine the effect of commercially available beta‐glucan (Macrogard) and nucleotide (Aquagen) on the resistance to Streptococcus iniae infections in vaccinated or nonvaccinated juvenile red‐tail black sharks (Epalzeorhynchos bicolor, RTB) (1.4 ± 0.4 g weight total (WT), 5.6 ± 0.5 cm total length (TL)). The immunostimulants were added to a control diet formulated without any yeast source following the recommended doses of 1 g/kg feed for the beta‐glucan and 2 g/kg feed for the nucleotide. Beginning 4 d after introduction into tanks, fish were fed the experimental diets for 24 d, at 3% body weight per day, divided into two feedings. At the end of this period, fish were challenged by an intracoelomic injection of S. iniae. In the first experiment, both vaccinated and nonvaccinated fish were fed one of the two immunostimulants. In the second experiment, only vaccinated fish were fed the immunostimulants. Fish were vaccinated a week after being introduced into the system and challenged by intracoelomic injection with 1.5 × 105 S. iniae colony‐forming units /fish after three additional weeks; mortality was recorded for 2 wk after the bacterial challenge. In the first experiment, the mortality of both vaccinated and nonvaccinated fish fed beta‐glucan (23 ± 7% and 82 ± 1%, respectively) or nucleotide (28 ± 6% and 86 ± 5%, respectively) was significantly lower than the mortality of the control groups (35 ± 4% and 93 ± 5%, respectively), but there was no significant difference in mortality between fish fed beta‐glucan or nucleotide. In the second experiment, the mortality of vaccinated fish fed beta‐glucan (25 ± 7%) or nucleotide (43 ± 9%) was significantly lower than that of vaccinated fish fed the control diet (69 ± 7%). In both experiments, there was no significant difference in growth rate among fish fed immunostimulants or the control diet. The results of this investigation demonstrated the efficacy of beta‐glucans and nucleotides in increasing resistance to S. iniae in RTB sharks.

Cross-immunity and antibody responses to different immobilisation serotypes of Ichthyophthirius multifiliis

Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.

Nuclear abnormalities in erythrocytes of parrots (Aratinga canicularis) related to genotoxic damage

Nuclear abnormalities in erythrocytes, as micronuclei and nuclear buds (BE), are considered potential biomarkers of genotoxic exposure. We described previously the frequency of spontaneous micronucleated erythrocytes (MNE) in the species Aratinga canicularis. Here, we have used this species to evaluate the induction of MNE and BE by mitomycin-C. Animals were given a single intracoelomic injection of 0, 2, 3 or 4 mg/kg mitomycin-C on two consecutive days. A drop of blood was obtained after 0, 24, 48 and 72 h, and stained smears were used to count micronucleated polychromatic erythrocytes (MNPCE) and polychromatic erythrocytes with buds (BPCE)/1000 polychromatic erythrocytes. The number of MNE and BE in 10 000 total erythrocytes was also counted. MNPCE and BPCE frequencies were elevated at 24, 48, and 72 h after the administration of the lower dose (P<0.03). At a 3 mg/kg dose, the frequency of MNPCE increased at 48 and 72 h (P<0.04) whereas the number of BPCE increased, but not significantly. Administration of 4 mg/kg mitomycin-C increased the number of MNE observed at 72 h (P<0.03), the number of MNPCE at 48 h (P<0.01) and 72 h (P<0.006), the BE frequency at 72 h (P<0.05), and the frequency of BPCE at 48 and 72 h (P<0.001). While mitomycin-C appears to produce a parallel increase in MNPCE and BPCE frequencies, the MNE seemed to be a more sensitive indicator of genotoxicity than the BE. This suggests that evaluating BE and MNE in routine haematological analysis should be considered to evaluate environmental genotoxic exposure.

Absence of neurotoxic effects in leopard sharks, Triakis semifasciata, following domoic acid exposure

Domoic acid (DA), a potent neurotoxin produced by select species of algae and diatoms, kills neurons bearing kainic acid-type glutamate receptors. Studies have shown that DA bioaccumulates in invertebrates and fish that consume the diatoms. In every vertebrate species tested or observed in the wild, dietary or systemic DA causes neuronal damage or clinical signs of neurotoxicity. Sharks, like marine birds and mammals, are exposed to DA through their diet; however, no research has demonstrated the effect of DA on shark behavior or physiology. In this study, juvenile leopard sharks ( Triakis semifasciata) were given DA by intracoelomic injection at doses of 0, 1, 3, 9, and 27 mg/kg and observed for 7 days. The sharks failed to demonstrate behavioral or histological changes in response to the toxin. We identified putative brain glutamate receptors by probing western blots with an antibody specific for kainic acid-type glutamate receptors and demonstrated receptor localization in the cerebellum with immunohistochemistry. Blood levels of DA in three sharks dosed at 9 mg/kg fell rapidly within 1.5 h of injection. We show that leopard sharks possess the molecular target for DA but are resistant to doses of DA known to be toxic to other vertebrates.

Characterization of Streptococcus iniae isolated from ornamental cyprinid fishes and development of challenge models

Streptococcus iniae was isolated from red-tail black shark(RTB shark) Epalzeorhynchos bicolor, and rainbow shark, E. erythrurus (fam: Cyprinidae), obtained from a local freshwater ornamental fish farm in Hillsborough county (FL). Darkening of the skin and lethargy were the first signs observed in infected sharks. Hemorrhages were observed on the ventral side of the body, on the head, and at the base of the pelvic and pectoral fins. Exophthalmia was observed in a low percentage of fish (∼ 10%). Moribund fish demonstrated the characteristic spinning, swimming pattern observed during streptococcal infections. Histological analysis revealed leukocyte infiltration in the intestinal area, spleen, posterior kidney, and brain. Necrosis and tissue degeneration were observed in the same organs in addition to degeneration of the renal tubules. Gram positive cocci were isolated from the cultures of brain and kidney tissues. Bacterial identification was obtained with the BIOLOG MicroLog3 version 4.00 System (Biolog, Inc., Hayward, California) and with standard microbiological tests. S. iniae-free RTB and rainbow sharks were used for developing a challenge model with the isolated bacteria strain. The experiments were conducted in two separate recirculating systems each with 25 tanks, 38 L volume per tank. In all experiments, the water in the system was maintained at 25 ± 1 °C. For each treatment, there were 3 replicate tanks, each stocked with 25 fish. Tanks were randomly assigned to treatment or control groups. Sharks were challenged by intracoelomic injection of S. iniae, and mortality was recorded for 14 days. Moribund fish showed classical signs of streptococcosis and S. iniae was isolated from moribund and dead fish. Differences in susceptibility to S. iniae were observed between the two species. An injection of 1.5 × 10 4 colony forming units (CFUs)/fish caused mortality close to 70% in RTB shark, and a higher mortality rate in rainbow shark. Mortalities ceased in 11–12 days after bacterial challenge in RTB shark, and in 3–4 days in rainbow shark. In this report, the pathology of S. iniae infections in two ornamental freshwater sharks and the development of a challenge model are described.

A quantitative real-time RT-PCR assay to measure TGF-β mRNA and its correlation with hematologic, plasma chemistry and organo-somatic indices responses in triamcinolone-treated Atlantic menhaden, Brevoortia tyrannus

A quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay was developed to measure transforming growth factor-beta (TGF-beta) in Atlantic menhaden (Brevoortia tyrannus), an estuarine-dependent species plagued by ulcerative skin lesions in the estuaries along the eastern United States. Atlantic menhaden were acclimated in a closed system for two weeks prior to initiation of the study. The synthetic glucocorticoid, triamcinolone acetonide (10mg/kg body weight) was administered by intracoelomic injection and its effect on the splenic mononuclear cell TGF-beta mRNA transcription, liver-somatic index, spleno-somatic index, hematology, and plasma chemistry were compared to untreated fish at 48 and 96h post-treatment. Triamcinolone-treated Atlantic menhaden showed suppression of TGF-beta mRNA production, neutrophilia, monocytosis, lymphopenia, and an increase in blood glucose concentrations. The health indices used in this study may help us interpret some of the changes observed during the development of ulcerative skin lesions in wild-caught menhaden.

Cytochemical and functional characterization of blood and inflammatory cells from the lizard Ameiva ameiva

The fine structure and differential cell count of blood and coelomic exudate leukocytes were studied with the aim to identify granulocytes from Ameiva ameiva, a lizard distributed in the tropical regions of the Americas. Blood leukocytes were separated with a Percoll cushion and coelomic exudate cells were obtained 24 h after intracoelomic thioglycollate injection. In the blood, erythrocytes, monocytes, thrombocytes, lymphocytes, plasma cells and four types of granulocytes were identified based on their morphology and cytochemistry. Types I and III granulocytes had round intracytoplasmic granules with the same basic morphology; however, type III granulocyte had a bilobued nucleus and higher amounts of heterochromatin suggesting an advance stage of maturation. Type II granulocytes had fusiformic granules and more mitochondria. Type IV granulocytes were classified as the basophil mammalian counterpart based on their morphology and relative number. Macrophages and granulocytes type III were found in the normal coelomic cavity. However, after the thioglycollate injection the number of type III granulocyte increased. Granulocytes found in the coelomic cavity were related to type III blood granulocyte based on the morphology and cytochemical localization of alkaline phosphatase and basic proteins in their intracytoplasmic granules. Differential blood leukocyte counts showed a predominance of type III granulocyte followed by lymphocyte, type I granulocyte, type II granulocyte, monocyte and type IV granulocyte. Taken together, these results indicate that types I and III granulocytes correspond to the mammalian neutrophils/heterophils and type II to the eosinophil granulocytes.

Virulence and Molecular Typing of Vibrio harveyi Strains Isolated from Cultured Dentex, Gilthead Sea Bream and European Sea Bass

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.

Tissue distribution and neurotoxic effects of domoic acid in a prominent vector species, the northern anchovy Engraulis mordax

The planktivorous northern anchovy is a prominent vector of the phycotoxin domoic acid (DA) to organisms at higher trophic levels, including fish-eating seabirds and mammals. Although there are abundant data reporting DA-induced excitotoxic symptoms in higher vertebrates, to date there has been no reported evidence of neurotoxic effects in lower vertebrate vectors such as fish. To explain this apparent lack of toxicity, it has been suggested that DA may not reach the brain in anchovies and/or that fish are not as sensitive neurologically to DA. In the present study, intracoelomic (IC) injection of DA, at doses ranging from 1 to 14 μg DA g−1 total fish weight, resulted in severe neurotoxic symptoms such as spinning, disorientation, inability to school, and mortality, indicating that anchovies are neurologically susceptible and that DA crosses the blood–brain barrier in fish. An ED50 of 3.2 μg DA g−1 total body weight was determined via IC injection of DA in 83 anchovies. Comparable intraperitoneal injection studies with mice, rats, and monkeys report similar DA-induced neurotoxic symptoms at doses near 3.2 μg DA g−1, suggesting a similar neurologic sensitivity and mechanism of toxicity between anchovies and mammals. DA tissue distribution measurements from freshly collected field-exposed anchovies and orally gavaged anchovies indicate that DA uptake from the gastrointestinal tract does occur. Levels as high as 1,175 μg DA g−1 were measured in anchovy viscera, while muscle and brain tissue DA levels were 3 orders of magnitude lower, indicating low but measurable DA uptake. Further evidence is needed to confirm that uptake is sufficient during field events to induce symptoms in anchovies. Our work provides the first reported evidence of neurotoxic symptoms in fish and suggests that anchovies may be affected by DA during toxic diatom blooms. If sufficient uptake occurs, DA-induced neurotoxic symptoms and mortality may make fish easier prey targets, thereby selecting for the highest toxin levels transferred, as well as providing a possible pathway for the transfer of DA to benthic communities.

Spatial Control of Ca2+ Signaling by Nicotinic Acid Adenine Dinucleotide Phosphate Diffusion and Gradients

Intracellular Ca(2+) is able to control numerous cellular responses through complex spatiotemporal organization. Ca(2+) waves mediated by inositol trisphosphate or ryanodine receptors propagate by Ca(2+)-induced Ca(2+) release and therefore do not have an absolute requirement for a gradient in either inositol trisphosphate or cyclic ADP-ribose, respectively. In contrast, we report that although Ca(2+) increases induced by nicotinic acid adenine dinucleotide phosphate (NAADP) are amplified by Ca(2+)-induced Ca(2+) release locally, Ca(2+) waves mediated by NAADP have an absolute requirement for an NAADP gradient. If NAADP is increased such that its concentration is spatially uniform in one region of an egg, the Ca(2+) increase occurs simultaneously throughout this area, and only where there is diffusion out of this area to establish an NAADP gradient is there a Ca(2+) wave. A local increase in NAADP results in a Ca(2+) increase that spreads by NAADP diffusion. NAADP diffusion is restricted at low but not high concentrations of NAADP, indicating that NAADP diffusion is strongly influenced by binding to immobile and saturable sites, probably the NAADP receptor itself. Thus, the range of action of NAADP can be tuned by its concentration from that of a local messenger, like Ca(2+), to that of a global messenger, like IP(3) or cyclic ADP-ribose.

Cortical Granule Exocytosis and Fertilization Coat Elevation is Reduced in Aging Sea Urchin Eggs

Abstract Cortical granules are specialized Golgi-derived membrane-bound secretory granules that are located beneath the plasma membrane in unfertilized sea urchin eggs. Upon fertilization cortical granules discharge in a reaction induced by calcium and release their contents between the plasma membrane and a thin vitelline layer that lines the plasma membrane. Microvilli at the plasma membrane elongate incorporting cortical granule membranes during elongation. The vitelline layer elevates and becomes the egg's fertilization coat that hardens and serves as physical block to polyspermy. While we do not understand the precise mechanisms that participate in cortical granule discharge it is believed that actin plays a role in this process. Because actin and calcium metabolism is affected in aging cells we investigated if cortical granule secretion is affected in aging sea urchin eggs. Lytechinus pictus eggs were obtained by intracoelomic injection of 0.5M KCI to release the eggs into sea water at 23°C.

Isolation and characterization of earthworm hemolysins and agglutinins

Hemolytic activity in coelomic fluid of Eisenia fetida (ECF) is due to three proteins H 1, H 2, H 3 with molecular weights of 46, 43 and 40 kD, respectively. These proteins were isolated by preparative PAGE. H 1 and H 2 were shown to be stable in SDS and α-2-ME whereas H 3 splits into two fragments with molecular weights of 18 and 21 kD after SDS treatment. IEF indicates that each protein consists of different isoforms with pIs between 5.1 and 6.2. H 3 was demonstrated to be a bifunctional protein that can lyse and agglutinate erythrocytes. At 56°C hemolytic activity of all three proteins was inactivated, but the agglutination activity of H 3 was stable. Intracoelomic injection of erythrocytes reduced the number of hemolysins from three to two. Monospecific antisera were raised against the isolated hemolysins H 1,2 and 3. The use of these antibodies and of carbohydrates as inhibitors of the biological acivity of the molecules demonstrates the close structural relationship of agglutinins and hemolysins in the CF of E. fetida.

Enema infusion technique inappropriate for evaluating viral competence of ticks.

To determine whether anal infusion of virus simulates the natural route of infection rather than intracoelomic injection, we compared the course of Venezuelan equine encephalitis (VEE) virus infection in Amblyomma cajennense (F.) ticks that had been exposed to virus by enema infusion with that in ticks fed on a viremic host or exposed by intracoelomic inoculation. Although virus was detected in virtually all ticks 14 d after exposure, orally exposed ticks contained significantly less virus (10(1.9) plaque-forming units [PFU] per tick) than did ticks infected by enema (10(4.1) PFU per tick) or intracoelomically (10(4.2) PFU per tick). At 42 d after virus exposure, only 1% of 512 orally exposed ticks contained virus, but most enema (77%, n = 43) or intracoelomically (79%, n = 29) exposed ticks were infected. Replication of VEE virus in A. cajennense ticks exposed to virus by enema infusion, therefore, appeared more similar to that of ticks inoculated intracoelomically than to those exposed orally. Thus, because enema infusion may bypass potential midgut infection and escape barriers, this procedure may not be appropriate for determining vector competence in ixodid ticks.

Nereis virens (Annelida: Polychaeta) is not an adequate sentinel species to assess the genotoxic risk (comet assay) of PAH exposure to the environment.

Polychaetes, because of their bioturbation capacity, play an important role in the distribution of anthropogenic contaminants (including polycyclic aromatic hydrocarbons [PAHs]) throughout the sediments. In this work the use of Nereis virens (Annelida: Polychaeta) as a bioindicator to assess the genotoxic risk of PAH exposure for the environment was evaluated. For this purpose the alkaline single cell gel electrophoresis [comet] assay was applied on the coelomocytes of in vivo exposed Nereis virens. Benzo[a]pyrene (B[a]P) was chosen because it is classified by the IARC (International Agency for Research on Cancer) as "probably carcinogenic to humans" and because its mechanisms of action are well-known. Nereis virens was exposed to B[a]P in concentrations of 0.3, 0.6, 10, 20, 35 and 45 mg/ml by an intracoelomic injection of B[a]P (20 microliters) dissolved in dimethyl sulphoxide (DMSO). A solvent control with DMSO, a positive control with ethyl methane sulphonate (EMS) (12.1 mg/ml) and a negative control were included in each experiment. For each treatment four animals were analysed. After 1 hr treatment coelomocytes were harvested by puncturing the coelomic cavity with a sharpened Pasteur pipette, mixed with 0.5% low melting point agarose and sandwiched between two other gel layers. Ethidium bromide stained nuclei were analysed for tail length and tail moment. 12.1 mg/ml EMS, pure DMSO (98.9%) and B[a]P in all tested concentrations induced a statistically significant increase of DNA single strand breaks in the comet assay. The effect of B[a]P, however, was only at the highest concentration (45 mg/ml) significantly stronger than the effect of DMSO alone. Although a relatively large heterogeneity in the results could be observed, these experiments clearly showed that Nereis virens is not suited as a sentinel species for the assessment of the genotoxic risk of PAH exposure because this species seems to be very resistant to benzo[a]pyrene.

Nereis virens (Annelida: Polychaeta) is not an adequate sentinel species to assess the genotoxic risk (comet assay) of PAH exposure to the environment

Polychaetes, because of their bioturbation capacity, play an important role in the distribution of anthropogenic contaminants (including polycyclic aromatic hydrocarbons [PAHs]) throughout the sediments. In this work the use of Nereis virens (Annelida: Polychaeta) as a bioindicator to assess the genotoxic risk of PAH exposure for the environment was evaluated. For this purpose the alkaline single cell gel electrophoresis [comet] assay was applied on the coelomocytes of in vivo exposed Nereis virens. Benzo[a]pyrene (B[a]P) was chosen because it is classified by the IARC (International Agency for Research on Cancer) as "probably carcinogenic to humans" and because its mechanisms of action are well-known. Nereis virens was exposed to B[a]P in concentrations of 0.3, 0.6, 10, 20, 35 and 45 mg/ml by an intracoelomic injection of B[a]P (20 microliters) dissolved in dimethyl sulphoxide (DMSO). A solvent control with DMSO, a positive control with ethyl methane sulphonate (EMS) (12.1 mg/ml) and a negative control were included in each experiment. For each treatment four animals were analysed. After 1 hr treatment coelomocytes were harvested by puncturing the coelomic cavity with a sharpened Pasteur pipette, mixed with 0.5% low melting point agarose and sandwiched between two other gel layers. Ethidium bromide stained nuclei were analysed for tail length and tail moment. 12.1 mg/ml EMS, pure DMSO (98.9%) and B[a]P in all tested concentrations induced a statistically significant increase of DNA single strand breaks in the comet assay. The effect of B[a]P, however, was only at the highest concentration (45 mg/ml) significantly stronger than the effect of DMSO alone. Although a relatively large heterogeneity in the results could be observed, these experiments clearly showed that Nereis virens is not suited as a sentinel species for the assessment of the genotoxic risk of PAH exposure because this species seems to be very resistant to benzo[a]pyrene.

Haemocytes in Cerastoderma edule (Mollusca, Bivalvia): distinct cell types engage in different responses to sheep erythrocytes

Three types of circulating haemocytes were identified in the haemolymph of the cockle Cerastoderma edule with light microscopy (LM), and electron microscopy (EM) and enzyme cytochemistry. Type I cells are large spreading haemocytes, with a ruffled surface, eccentric nucleus and distinct cytoplasmic organelles. These cells are esterase- and acid phosphatase- positive. Type II cells are spherical, with a smooth surface and central nucleus surrounded by a cytoplasm devoid of organelles. They are negative for esterase and acid phosphatase reactions. Type III cells, which have not been described before in molluscs, have a round shape, a small eccentric nucleus and a large vacuole filled with granular material (TEM) occupying the majority of the cytoplasm. These cells are peroxidase positive and morphologically resemble the serous cells of bivalves or the signet ring cells of urochordates. The cellular response of Cerastoderma edule to the intracoelomic injection of sheep red blood cells (SRBC) was studied. The cell types involved in E-rosette formation and phagocytosis were documented by LM, TEM and SEM. In light microscopy and ultrastructural analysis the E-rosette forming cell was identified as Type II cells. The Type I cells phagocytosed SRBC, autologous cells and remnants thereof. Red blood cells with disrupted membranes and different electron densities were observed not only inside but also in close vicinity of phagocytic cells, which may suggest that the cells produce a cytolytic factor. The function of Type III cells remains obscure.

D-mannitol, a specific hydroxyl free radical scavenger, reduces the developmental toxicity of hydroxyurea in rabbits.

Hydroxyurea (HU) is a potent mammalian teratogen. Within 2-4 hours after maternal injection, HU causes 1) a rapid episode of embryonic cell death and 2) profound inhibition of embryonic DNA synthesis. A variety of antioxidants delays the onset of embryonic cell death and reduces the incidence of birth defects. Antioxidants do not block the inhibition of DNA synthesis, indicating that early embryonic cell death is not caused by inhibited DNA synthesis. We have suggested that some HU molecules may react within the embryo to produce H2O2 and subsequent free radicals, including the very reactive hydroxyl free radical. The free radicals could cause the early cell death; antioxidants are believed to terminate the aberrant free radical reactions resulting in lessened developmental toxicity. To investigate whether hydroxyl free radicals cause the early episode of cell death, pregnant New Zealand white rabbits were injected subcutaneously on gestational day 12 with a teratogenic dose of HU (650 mg/kg) in the presence or absence of 550 mg/kg of D-mannitol (Man), a specific scavenger of hydroxyl free radicals. Osmotic control rabbits received HU plus 550 mg/kg of xylose (Xyl, a nonactive aldose). At term, the teratologic effects of HU were ameliorated by Man as evidenced by decreased incidences of the expected limb malformations. Xyl exerted no demonstrable effect on HU teratogenesis. Histological examination of limb buds at 3-8 hours after maternal injection, showed that Man delayed the onset of HU-induced cell death by as much as 4 hours. Xyl had no effect. That Man acts within the embryo was shown by performing intracoelomic injections on alternate implantation sites with Man, Xyl, or saline followed by subcutaneous injection of the pregnant doe with HU. Embryos were harvested 3-8 hours later. Limb buds from saline- and Xyl-injected embryos exhibited the typical pattern of widespread HU-induced cell death at 3-4 hours, whereas Man-injected embryos did not exhibit cell death until 5-8 hours. These results are consistent with those reported for antioxidant-mediated amelioration of HU-induced developmental toxicity and with the hypothesis that hydroxyl free radicals are the proximate reactive species in HU-induced early embryonic cell death.

Clearance and breakdown of pathogenic bacteria injected into the body cavity of the earthworm Eisenia fetida andrei

1. Early mechanisms of defense initiated by an intracoelomic injection of bacteria were investigated in earthworms. 2. Bacteria rapidly lost their capacity to proliferate and were segregated in multicellular aggregates. 3. A system of two lectins of M r 40,000 and 45,000, synthesized by chloragocytes, seemed to be a chief mediator of antibacterial defense. 4. Certain bacteria escaped this defense by lodging in tissues which surround the coelom, especially in the longitudinal muscles of the body wall.

A simple and efficient procedure for the oral inoculation of Trichoplusia ni larvae with polyhedrin-negative recombinant baculovirus

Current procedures for inoculating lepidopteran larvae with polyhedrin-negative recombinant baculovirus, i.e. intracoelomic injection or coinfection with wild type virus, are laborious and can compromise final yields of recombinant protein. Herein is described a simple and efficient method for oral inoculation. Up to 100% infection was obtained when individual early fifth instar Trichoplusia ni larvae were fed a small piece of a formaldehyde-free insect diet to which 4.2 × 10 5 PFU of a polyhedrin-negative recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) containing the gene for β-galactosidase was applied. Infected larvae were identified by assaying hemolymph for β-galactosidase activity. The maximum levels of β-galactosidase detected in these hemolymph samples were identical to those obtained for larvae infected by intracoelomic injection. The dose of polyhedrin-negative recombinant virus recommended for intracoelomic injection of T. ni was efficacious for the oral route of inoculation.

Absence of fever in planarian: Turbellaria: Phogocata gracilis

1. 1. Groups of 5 to 10 planarians (Turbellaria: Phogocata gracilis) were placed in a water gradient of temperature extending from 0 to 40°C over 0.8 m. In control sessions, the planarians avoided water at less than 9°C and more than 23°C. 2. 2. In the 4 hr following intracoelomic injection of E. coli endotoxin (2.5, 5 and 10 ng/mg), human interleukin-2 (0.5 and 2 mU/mg), interferon a (0.02,0.2, and 1 U/mg), and vehicle the thermopreferendum was not different from control. 3. 3. The pyrogenic drugs were toxic at higher concentrations, therefore active. 4. 4. The diffusion of injected substances to the whole body was verified with a dye. 5. 5. These negative results are not in favor of fever in Platyhelminthes. The absence of fever in both Platyhelminthes and Molluscs, while Arthropods and Annelids can have fever, might be explained by a common phylogenetic origin of the former.