Carbon dioxide combining properties of the blood of the shore crab Carcinus maenas (L): carbon dioxide solubility coefficient and carbonic acid dissociation constants.

The carbon dioxide solubility coefficient, alphaCO2, and the apparent carbonic acid dissociation constants, K'1 and K'2 were estimated in the serum of the crab Carcinus maenas at various temperatures and ionic strengths. At 15 degrees C, the indirectly determined alphaCO2 value is 0-0499 m-mole l-1 torr-1 for crabs living in normal sea water (salinity ca. 35 percent). It is apparently independent of the serum protein concentration and of the stage of the moulting cycle. For crabs living in undiluted sea water, the mean pK'1 value, determined either gasometrically or titrimetrically, is 6-027 at 15 degrees C. At the same temperature, pK'2=9-29. These values approximate to those of sea water at 35 percent salinity. pK'1 drops as temperature rises; the measured deltapK'1/deltat is -0-0053 pH unit degrees C-1 between 10 and 30 degrees C. PK'1 rises as the ionic strength is lowered. Alignment nomograms have been constructed for the determination of alphaCO2, pK'1 and pK'2 values in relation to various conditions of temperature and salinity.     

Stability of human enteroviruses in estuarine and marine waters.

Studies of the effects of temperature and salinity on the survival of three enteric viruses (poliomyelitis type 1, echovirus-6, and coxsackievirus B-5) under controlled laboratory conditions and in situ indicate that temperature rather than salinity is the critical factor affecting their stability, in that the higher the temperature the more rapid was the loss of viral infectivity. In the laboratory studies, all three viruses were quite stable at 4 degrees C, with infectious virus still detectable after 46 weeks of incubation. In situ studies on virus survival in free-flowing estuarine or marine waters showed that, although the viruses were more labile in natural waters than in the laboratory studies, they persisted for several months, in some cases during the winter months. At all temperatures and salinities, coxsackievirus B-5 was the most stable, echovirus-6 was intermediate, and poliovirus 1 was the least stable of the viruses tested.     

Stability of human enteroviruses in estuarine and marine waters.

Studies of the effects of temperature and salinity on the survival of three enteric viruses (poliomyelitis type 1, echovirus-6, and coxsackievirus B-5) under controlled laboratory conditions and in situ indicate that temperature rather than salinity is the critical factor affecting their stability, in that the higher the temperature the more rapid was the loss of viral infectivity. In the laboratory studies, all three viruses were quite stable at 4 degrees C, with infectious virus still detectable after 46 weeks of incubation. In situ studies on virus survival in free-flowing estuarine or marine waters showed that, although the viruses were more labile in natural waters than in the laboratory studies, they persisted for several months, in some cases during the winter months. At all temperatures and salinities, coxsackievirus B-5 was the most stable, echovirus-6 was intermediate, and poliovirus 1 was the least stable of the viruses tested.     

Long-term survival of hepatitis A virus and poliovirus type 1 in mineral water.

The survival in mineral water of hepatitis A virus (HAV) and poliovirus type 1 was compared, under controlled experimental conditions, at 4 degrees C and room temperature. Viral infectivity titers were determined by cell culture titration, while HAV antigenicity was monitored by radioimmunoassay-endpoint titration. Both viruses persisted longest at 4 degrees C. At this temperature, after 1 year of exposure, the inactivation of either HAV or poliovirus type 1 was not important. At room temperature, poliovirus type 1 was not detected after 300 days, whereas HAV was still infectious. For both temperatures, the computed regression coefficients of best-fit lines for inactivation rates for the two viruses were significantly different. The survival of HAV was also studied at 4 degrees C and room temperature in mineral water with 5- and 50-micrograms/ml protein concentrations (i.e., purity of the virus suspension) for 120 days. As shown by a comparison of the regression coefficients for the inactivation rates, the stability of HAV in mineral water depends on protein concentration and temperature. Radioimmunoassay-endpoint titration results showed inactivation patterns similar to those of cell culture titration, with the most significant reduction in HAV antigenicity at room temperature. At the two temperatures, the infectivity of HAV declined at a faster rate than the antigenicity.     

Growth of epizootic hemorrhagic disease, akabane, and ephemeral fever viruses in Aedes albopictus cells maintained at various temperatures

The growth curves of one epizootic hemorrhagic disease (EHD) virus serotype (Reoviridae), two Akabane virus strains (Bunyaviridae) and three bovine ephemeral fever (BEF) group viruses (Rhabdoviridae) were determined in Aedes albopictus cells maintained at 15, 20, 28 and 33 degrees C. Ae albopictus cells supported the growth of all the viruses although not necessarily at all temperatures. Because none of the viruses exhibited cytopathic effect in Ae albopictus cells, growth was assayed in baby hamster kidney 21 (BHK21) cells maintained at 37 degrees C. The temperature at which the Ae albopictus cells were maintained had a marked effect on the growth and yield for each virus studied. EHD virus was heat-stable and grew after 4 days at 28 and 33 degrees C, and after 8 days at 20 degrees C. No growth was recorded up to 12 days at 15 degrees C. The two Akabane viruses were heat-sensitive and exhibited different growth patterns. One strain (B8935) showed no growth at 15 degrees C and only minimal growth at 20, 28 and 33 degrees C. The other strain (CSIRO 16) showed growth after 1-2 days at all temperatures with higher titres reached at 15 and 20 degrees C than at 28 and 33 degrees C. The BEF group viruses grew to approximately the same titres at all temperatures. At the higher temperatures (28 and 33 degrees C) most of BEF group viruses had disappeared within 9 days. In contrast at the lower temperatures (15 and 20 degrees C), there was still virus present 18 days after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adaptive significance of hatching rhythms and dispersal patterns of estuarine crab larvae: avoidance of physiological stress by larval export?

Estuarine crabs commonly display two larval dispersal patterns in which larvae are either exported from or retained within estuaries. The semiterrestrial fiddler crab Uca minax (LeConte, 1855) hatches on nocturnal spring high tides in the upper estuary and larvae are rapidly transported downstream. The mud crab Rhithropanopeus harrisii (Gould, 1841) hatches on nocturnal high tides of any amplitude and larvae are retained behaviorally in the upper estuary throughout development. If larvae are exported from the estuary to avoid environmental stress, then exported larvae should be less tolerant of high temperatures and low salinities than retained larvae. Larvae of these two species of estuarine crabs were hatched at 20‰ and 25 °C and subjected to salinities of 0, 5, 10,20, and 30‰, temperatures of 25 and 35 °C, and exposure times of 2, 6, 12, and 48 h. Larvae of both species reared at 30 or 20‰ survived well, while those reared in fresh water all died within 2 h regardless of temperature. Mud crab larvae reared at 5 and 10‰ survived better at the lower temperature (25 °C), higher salinity, and shorter exposure times. There was no significant effect of temperature or salinity on the survival of fiddler crab larvae, although survival decreased with increasing exposure time. Thus, the hypothesis that fiddler crab larvae are exported into stable coastal waters to reduce physiological stress is not supported. However, fiddler crab larvae may have evolved to be very tolerant of extreme temperature and salinity stress because they, unlike mud crabs, often release their larvae into shallow creeks. Most fiddler crab larvae are released on nocturnal spring high tides, which facilitates dispersal from tidal creeks. However, freshwater runoff and heat transferred from the marsh surface to flooding waters may still create stressful conditions for larvae soon after they are released. Larval release on spring high tides may facilitate dispersal from tidal creeks.     

Survival and development of horseshoe crab (Limulus polyphemus) embryos and larvae in hypersaline conditions

The horseshoe crab Limulus polyphemus spawns in the mid- to upper intertidal zone where females deposit eggs in nests below the sediment surface. Although adult crabs generally inhabit subtidal regions of estuaries with salinities from 5 to 34 ppt, developing embryos and larvae within nests are often exposed to more extreme conditions of salinity and temperature during summer spawning periods. To test whether these conditions have a negative impact on early development and survival, we determined development time, survival, and molt cycle duration for L. polyphemus embryos and larvae raised at 20 combinations of salinity (range: 30-60 ppt) and temperature (range: 25-40 degrees C). Additionally, the effect of hyperosmotic and hypoosmotic shock on the osmolarity of the perivitelline fluid of embryos was determined at salinities between 5 and 90 ppt. The embryos completed their development and molted at salinities below 60 ppt, yet failed to develop at temperatures of 35 degrees C or higher. Larval survival was high at salinities of 10-70 ppt but declined significantly at more extreme salinities (i.e., 5, 80, and 90 ppt). Perivitelline fluid remained nearly isoosmotic over the range of salinities tested. Results indicate that temperature and salinity influence the rate of crab development, but only the extremes of these conditions have an effect on survival.     

Behavioral thermoregulation in Hemigrapsus nudus,the amphibious purple shore crab

The thermoregulatory behavior of Hemigrapsus nudus, the amphibious purple shore crab, was examined in both aquatic and aerial environments. Crabs warmed and cooled more rapidly in water than in air. Acclimation in water of 16 degrees C (summer temperatures) raised the critical thermal maximum temperature (CTMax); acclimation in water of 10 degrees C (winter temperatures) lowered the critical thermal minimum temperature (CTMin). The changes occurred in both water and air. However, these survival regimes did not reflect the thermal preferences of the animals. In water, the thermal preference of crabs acclimated to 16 degrees C was 14.6 degrees C, and they avoided water warmer than 25.5 degrees C. These values were significantly lower than those of the crabs acclimated to 10 degrees C; these animals demonstrated temperature preferences for water that was 17 degrees C, and they avoided water that was warmer than 26.9 degrees C. This temperature preference was also exhibited in air, where 10 degrees C acclimated crabs exited from under rocks at a temperature that was 3.2 degrees C higher than that at which the 16 degrees C acclimated animals responded. This behavioral pattern was possibly due to a decreased thermal tolerance of 16 degrees C acclimated crabs, related with the molting process. H. nudus was better able to survive prolonged exposure to cold temperatures than to warm temperatures, and there was a trend towards lower exit temperatures with the lower acclimation (10 degrees C) temperature. Using a complex series of behaviors, the crabs were able to precisely control body temperature independent of the medium, by shuttling between air and water. The time spent in either air or water was influenced more strongly by the temperature than by the medium. In the field, this species may experience ranges in temperatures of up to 20 degrees C; however, it is able to utilize thermal microhabitats underneath rocks to maintain its body temperature within fairly narrow limits.     

Preliminary Observations on the Uptake of Poliovirus by West Coast Shore Crabs

West Coast shore crabs (Pachygrapsus sp. and Hemigrapsus sp.), when in seawater contaminated with poliovirus or allowed to feed on virus-contaminated mussels (Mytilus californianus), were found to accumulate high titers of virus.     

Inactivation of polioviruses and coxsackieviruses in surface water.

Inactivation rates of polioviruses 1 and 3 and coxsackieviruses A-13 and B-1 were determined in situ in the Rio Grande in southern New Mexico, using membrane dialysis chambers. Inactivation of the viruses was exponential, and the rates of inactivation were apparently affected principally by the water temperature. Stability of the viruses in river water differed, with poliovirus 1 and coxsackie B-1 being most stable. Typically 1-log reductions of infectivity at water temperatures ranging between 23 and 27 degrees C required 25 h for poliovirus 1, 19 h for poliovirus 3, and 7 h for coxsackie virus A-13. At water temperatures of 4 to 8 degrees C, the log reduction times for poliovirus 1 and coxsackievirus B-1 were 46 and 58 h, respectively. Results obtained with labeled poliovirus 1 and coxsackievirus B-1 and with infectious ribonucleic acid indicate that inactivation was due to damage to viral ribonucleic acid. Virus-inactivation rates were also affected by heat sterilization of river water, indicating the presence of a heat-labile or volatile inactivating factor. The inactivating factor in Rio Grande water was apparently present at a constant concentration over a 1-year period.     

Inactivation of polioviruses and coxsackieviruses in surface water.

Inactivation rates of polioviruses 1 and 3 and coxsackieviruses A-13 and B-1 were determined in situ in the Rio Grande in southern New Mexico, using membrane dialysis chambers. Inactivation of the viruses was exponential, and the rates of inactivation were apparently affected principally by the water temperature. Stability of the viruses in river water differed, with poliovirus 1 and coxsackie B-1 being most stable. Typically 1-log reductions of infectivity at water temperatures ranging between 23 and 27 degrees C required 25 h for poliovirus 1, 19 h for poliovirus 3, and 7 h for coxsackie virus A-13. At water temperatures of 4 to 8 degrees C, the log reduction times for poliovirus 1 and coxsackievirus B-1 were 46 and 58 h, respectively. Results obtained with labeled poliovirus 1 and coxsackievirus B-1 and with infectious ribonucleic acid indicate that inactivation was due to damage to viral ribonucleic acid. Virus-inactivation rates were also affected by heat sterilization of river water, indicating the presence of a heat-labile or volatile inactivating factor. The inactivating factor in Rio Grande water was apparently present at a constant concentration over a 1-year period.     

Uptake of Bacteriophage and Their Subsequent Survival in Edible West Coast Crabs After Processing

Edible West Coast crabs (Cancer magister and Cancer antennarius), when in seawater contaminated with coliphage T4, were found to accumulate high titers of this virus. To study the extent of the hazard presented by crabs contaminated with virus, samples of edible crabs were contaminated with coliphage T4 and then processed by boiling. Results indicated that virus in crabs withstood this method of processing. The survival rate varied from 2.5 to 20%, depending upon the processing time. Heat penetration studies showed that, although internal temperature in the crabs was sufficient to inactivate virus, the processing times normally used to cook crabs were not. These results suggest that processed crabs could serve as vectors for the dissemination of virus diseases if the crabs are harvested from a polluted area.     

Stability of feline caliciviruses in marine water maintained at different temperatures

Since human caliciviruses are responsible for viral gastroenteritis transmitted by contaminated foods and the viruses barely propagate in cell culture, feline caliciviruses were employed as a model for the measurement of their stability in marine water. Survival of four strains of feline calicivirus in marine water was measured when the seed viruses were diluted 1/10 with marine water and maintained at 4 degrees C, 10 degrees C, and 20 degrees C respectively. Among the virus strains studied, a considerable amount of infective viruses remained at 10 degrees C or lower temperature conditions even for a period of 30 days.     

Enterovirus inactivation in soil.

The inactivation of radioactively labeled poliovirus type 1 and coxsackievirus B 1 in soils saturated with surface water, groundwater, and septic tank liquor was directly proportional to temperature. Virus persistence was also related to soil type and the liquid amendment in which viruses were suspended. At 37 degrees C, no infectivity was recovered from saturated soil after 12 days; at 4 degrees C, viruses persisted for at least 180 days. No infectivity was recovered from dried soil regardless of temperature, soil type, or liquid amendment. Additional experiments showed that evaporation of soil water was largely responsible for the decreased recovery of infectivity from drying soil. Increased rates of virus inactivation at low soil moisture levels were also demonstrated.     

Enterovirus inactivation in soil.

The inactivation of radioactively labeled poliovirus type 1 and coxsackievirus B 1 in soils saturated with surface water, groundwater, and septic tank liquor was directly proportional to temperature. Virus persistence was also related to soil type and the liquid amendment in which viruses were suspended. At 37 degrees C, no infectivity was recovered from saturated soil after 12 days; at 4 degrees C, viruses persisted for at least 180 days. No infectivity was recovered from dried soil regardless of temperature, soil type, or liquid amendment. Additional experiments showed that evaporation of soil water was largely responsible for the decreased recovery of infectivity from drying soil. Increased rates of virus inactivation at low soil moisture levels were also demonstrated.     

Survival of enteric viruses on environmental fomites.

The survival of human enteric viruses on several porous (paper and cotton cloth) and nonporous (aluminum, china, glazed tile, latex, and polystyrene) environmental surfaces has been evaluated. Viruses persisted for extended periods on several types of materials commonly found in institutions and domestic environments. The stability of the viruses was generally influenced by environmental factors such as relative humidity (RH), temperature, and the type of surface contaminated. Overall, hepatitis A virus (HAV) and human rotavirus (HRV) were more resistant to inactivation than enteric adenovirus (ADV) and poliovirus (PV). The resistance to the desiccation step appears to be of major significance in determining the survival of a virus dried on fomites. ADV and PV showed a pronounced decrease in titer at this stage, whereas HAV and HRV displayed little decay at the desiccation step. HAV and HRV persistence was not affected by the presence of fecal material. On nonporous surfaces, PV and ADV persisted better in the presence of feces. However, on porous fomites the presence of fecal material had a negative influence on the survival of PV and ADV. Except for HRV, greater virus survival was observed at 4 degrees than at 20 degrees C. PV and HAV survival was enhanced at high RH; the survival of the latter was enhanced at least for nonporous materials. When dried on porous materials, HRV also exhibited greater persistence at high RH. The survival of ADV was not affected by RH. The validity of using bacteriophages of Bacteroides fragilis as indicators of human viruses dried on fomites was evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of gamma irradiation on human cortical bone transplants contaminated with enveloped and non-enveloped viruses.

In the production of bone grafts intended for transplantation, basic safety measures to avoid the transmission of pathogens are selection and serological screening of donors for markers of virus infections. As an additional safety tool we investigated the effect of gamma irradiation on the sterility of human bone diaphysis transplants and evaluated its impact on the virus safety of transplants. Model viruses were included in the study to determine the dose necessary to achieve a reduction factor for the infectivity titres of at least 4 log(10) at a temperature of -30+/-5 degrees C. The following viruses were used: human immunodeficiency virus type 2 (HIV-2), hepatitis A virus (HAV), and poliovirus (PV-1), and the following model viruses: pseudorabies virus (PRV) as a model for human herpesviruses, bovine viral diarrhoea virus (BVDV) for HCV, and bovine parvovirus (BPV) for parvovirus B19. A first approach was to determine the D(10) values (kGy) for the different viruses (virus inactivation kinetics: BPV 7.3; PV-1 7.1; HIV-2 7.1; HAV 5.3; PRV 5.3; BVDV <3.0 kGy). Based on these results, inactivation of these viruses was studied in experimentally contaminated human bone transplants (femoral diaphyses). For BPV, the most resistant one of the viruses studied, a dose of approximately 34 kGy was necessary to achieve a reduction of infectivity titres of 4 log(10). We therefore recommend a dose of 34 kGy for the sterilisation of frozen bone transplants.     

The Effect of Environmental Factors on Development of the Land-Grab, Cardisoma guanhumi Latreille

Larvae of the land-crab, Cardisoma guanhumi, Latreille. weremaintained in 24 different combinations of salinity and temperaturefrom the time of hatching. Survival to the first crab occurredin salinities of 15–45 p.p.t., 25° and 30°C. Durationof the five zoeal and one megalops stages was similar in salinitiesof 20–40 p.p.t., but at 15 and 45 p.p.t. a greater periodof time was required for total development. Mortality of allthe larvae at 20°C suggests that temperature plays a moreimportant role in survival and distribution of the larvae ofC. guanhumi than salinity. Increments of size in crabs during the first seven post-larvalmolts were similar in salinities of 5–35 p.p.t., 25°C,but in fresh water increase in size at the time of molting wasreduced. Although there was no apparent relationship betweenfrequency of molting and salinities of 5–35 p.p.t., theduration of intermolt was reduced in crabs maintained in freshwater, and survival was also lower. From the present study there is no indication that the morphologicaland physiological processes that are associated with adaptationof the adult crab to the terrestrial environment are initiatedduring larval development. Although the adult crabs have successfullypenetrated the terrestrial environment, the pelagic larvae arestill subject to the numerous ecological variables of the estuarineand marine environments.     

In situ monitoring of heart rates in shore crabs Carcinus maenas in two tidal estuaries: effects of physico-chemical parameters on tidal and diel rhythms

Heart rates were monitored in situ in the shore crab, Carcinus maenas, in relation to variations in depth, salinity, oxygen tension, temperature, light intensity and pH. Experiments were performed in the Looe Estuary, Cornwall, England and in Batson Creek in the Salcombe-Kingsbridge Estuary, Devon, England. Experiments in the Looe Estuary were conducted in the vicinity of a storm water storage discharge whereas the experiments in Batson Creek were performed on a clean site. Tidal rhythms in heart rates were commonly detected but diel rhythms in heart rate were also observed frequently. Both types of rhythm were more evident in animals from Batson Creek than from Looe. In Batson Creek, 12 out of 15 crabs expressed tidal rhythms in heart rate, whereas 6 out of 15 crabs expressed diel rhythms. In the two studies in the Looe Estuary, 6 out of 15 crabs and 3 out of 15 crabs expressed tidal and diel rhythm in heart rate, respectively. At both experimental sites, heart rates were positively correlated with increasing changes in depth and salinity, whereas heart rates were negatively correlated with light intensity. In addition, heart rates appeared to be positively correlated with increasing oxygen tension in the experiments performed in the Looe Estuary. The study suggests that depth and oxygen availability are more important to in situ heart rates in shore crabs within tidal estuaries than are salinity, light intensity and pH. Also, sewage discharge appears to cause an acute increase in heart rate, which may affect expression of biological rhythms in shore crabs.     

Effects of eyestalk ablation on carbonic anhydrase activity in the euryhaline blue crab Callinectes sapidus: neuroendocrine control of enzyme expression

Carbonic anhydrase (CA) activity in the gills of the euryhaline blue crab, Callinectes sapidus, was measured in response to acute low-salinity transfer and treatment with eyestalk ablation (ESA) in an attempt to elucidate potential regulatory mechanisms of salinity-mediated CA induction. ESA alone resulted in an approximate doubling of CA activity in the posterior, ion-transporting gills of crabs acclimated to 35 ppt. Transfer of intact crabs to 28 ppt, a salinity at which the blue crab is still an osmotic and ionic conformer, had no effect on CA activity, but treatment with ESA prior to transfer resulted in a 5-fold increase. Hemolymph osmolality was unaffected by ESA. There was a 7-fold induction of CA activity in posterior gills of intact crabs transferred from 35 to 15 ppt, and this was potentiated by about 100% by ESA. Hemolymph osmolality was slightly elevated in the ESA-treated crabs. CA activity in anterior gills did not increase in response to any treatment. Hemolymph concentrations of methyl farnesoate (MF) were measured for all experimental animals. MF concentrations were undetectable in all intact crabs, regardless of salinity. Treatment with ESA resulted in elevated levels of hemolymph MF, but these levels were still relatively low and unrelated to salinity. These results suggest that CA induction is under the control of a regulatory substance located in the eyestalk. This substance appears to be a CA repressor, keeping CA expression at low levels in the gills of crabs acclimated to high salinity. Exposure to low salinity, or treatment with ESA, removes the effects of this putative repressor and allows CA induction to occur.