Integrated treatment of shrimp effluent by sedimentation, oyster filtration and macroalgal absorption: a laboratory scale study

Effluent water from shrimp ponds typically contains elevated concentrations of dissolved nutrients and suspended particulates compared to influent water. Attempts to improve effluent water quality using filter feeding bivalves and macroalgae to reduce nutrients have previously been hampered by the high concentration of clay particles typically found in untreated pond effluent. These particles inhibit feeding in bivalves and reduce photosynthesis in macroalgae by increasing effluent turbidity. In a small-scale laboratory study, the effectiveness of a three-stage effluent treatment system was investigated. In the first stage, reduction in particle concentration occurred through natural sedimentation. In the second stage, filtration by the Sydney rock oyster, Saccostrea commercialis (Iredale and Roughley), further reduced the concentration of suspended particulates, including inorganic particles, phytoplankton, bacteria, and their associated nutrients. In the final stage, the macroalga, Gracilaria edulis (Gmelin) Silva, absorbed dissolved nutrients. Pond effluent was collected from a commercial shrimp farm, taken to an indoor culture facility and was left to settle for 24 h. Subsamples of water were then transferred into laboratory tanks stocked with oysters and maintained for 24 h, and then transferred to tanks containing macroalgae for another 24 h. Total suspended solid (TSS), chlorophyll a, total nitrogen (N), total phosphorus (P), NH4+, NO3-, and PO43-, and bacterial numbers were compared before and after each treatment at: 0 h (initial); 24 h (after sedimentation); 48 h (after oyster filtration); 72 h (after macroalgal absorption). The combined effect of the sequential treatments resulted in significant reductions in the concentrations of all parameters measured. High rates of nutrient regeneration were observed in the control tanks, which did not contain oysters or macroalgae. Conversely, significant reductions in nutrients and suspended particulates after sedimentation and biological treatment were observed. Overall, improvements in water quality (final percentage of the initial concentration) were as follows: TSS (12%); total N (28%); total P (14%); NH4+ (76%); NO3- (30%); PO43-(35%); bacteria (30%); and chlorophyll a (0.7%). Despite the probability of considerable differences in sedimentation, filtration and nutrient uptake rates when scaled to farm size, these results demonstrate that integrated treatment has the potential to significantly improve water quality of shrimp farm effluent. (C) 2001 Elsevier Science B.V. All rights reserved.

ENCORE: The effect of nutrient enrichment on coral reefs. Synthesis of results and conclusions

Coral reef degradation resulting from nutrient enrichment of coastal waters is of increasing global concern. Although effects of nutrients on coral reef organisms have been demonstrated in the laboratory, there is little direct evidence of nutrient effects on coral reef biota in situ. The ENCORE experiment investigated responses of coral reef organisms and processes to controlled additions of dissolved inorganic nitrogen (N) and/or phosphorus (P) on an offshore reef(One Tree Island) at the southern end of the Great Barrier Reef, Australia. A multi-disciplinary team assessed a variety of factors focusing on nutrient dynamics and biotic responses. A controlled and replicated experiment was conducted over two years using twelve small patch reefs ponded at low tide by a coral rim. Treatments included three control reefs (no nutrient addition) and three + N reefs (NH4Cl added), three + P reefs (KH2PO4 added), and three + N + P reefs. Nutrients were added as pulses at each low tide (ca twice per day) by remotely operated units. There were two phases of nutrient additions. During the initial, low-loading phase of the experiment nutrient pulses (mean dose = 11.5 muM NH4+; 2.3 muM PO4-3) rapidly declined, reaching near-background levels (mean = 0.9 muM NH4+; 0.5 muM PO4-3) within 2-3 h. A variety of biotic processes, assessed over a year during this initial nutrient loading phase, were not significantly affected, with the exception of coral reproduction, which was affected in all nutrient treatments. In Acropora longicyathus and A. aspera, fewer successfully developed embryos were formed, and in A. longicyathus fertilization rates and lipid levels decreased. In the second, high-loading, phase of ENCORE an increased nutrient dosage (mean dose = 36.2 muM NH4+; 5.1 muM PO4-3 declining to means of 11.3 muM NH4+ and 2.4 muM PO4-3 at the end of low tide) was used for a further year, and a variety of significant biotic responses occurred. Encrusting algae incorporated virtually none of the added nutrients. Organisms containing endosymbiotic zooxanthellae (corals and giant clams) assimilated dissolved nutrients rapidly and were responsive to added nutrients. Coral mortality, not detected during the initial low-loading phase, became evident with increased nutrient dosage, particularly in Pocillopora damicornis. Nitrogen additions stunted coral growth, and phosphorus additions had a variable effect. Coral calcification rate and linear extension increased in the presence of added phosphorus but skeletal density was reduced, making corals more susceptible to breakage. Settlement of all coral larvae was reduced in nitrogen treatments, yet settlement of larvae from brooded species was enhanced in phosphorus treatments. Recruitment of stomatopods, benthic crustaceans living in coral rubble, was reduced in nitrogen and nitrogen plus phosphorus treatments. Grazing rates and reproductive effort of various fish species were not affected by the nutrient treatments. Microbial nitrogen transformations in sediments,were responsive to nutrient loading with nitrogen fixation significantly increased in phosphorus treatments and denitrification increased in all treatments to which nitrogen had been added. Rates of bioerosion and grazing showed no significant effects of added nutrients, ENCORE has shown that reef organisms and processes investigated ill situ were impacted by elevated nutrients. Impacts mere dependent on dose level, whether nitrogen and/or phosphorus mere elevated and were often species-specific. The impacts were generally sub-lethal and subtle and the treated reefs at the end of the experiment mere visually similar to control reefs. Rapid nutrient uptake indicates that nutrient concentrations alone are not adequate to assess nutrient condition of reefs. Sensitive and quantifiable biological indicators need to be developed for coral reef ecosystems. The potential bioindicators identified in ENCORE should be tested in future research on coral reef/nutrient interactions. Synergistic and cumulative effects of elevated nutrients and other environmental parameters, comparative studies of intact vs. disturbed reefs, offshore vs, inshore reefs, or the ability of a nutrient-stressed reef to respond to natural disturbances require elucidation. An expanded understanding of coral reef responses to anthropogenic impacts is necessary, particularly regarding the subtle, sub-lethal effects detected in the ENCORE studies. (C) 2001 Published by Elsevier Science Ltd.

Experimental infection of Australian brushtail possums, Trichosurus vulpecula (Phalangeridae : Marsupialia), with Ross River and Barmah Forest viruses by use of a natural mosquito vector system

Brushtail possums, Trichosurus vulpecula Kerr, were experimentally infected with Ross River (RR) or Barmah Forest (BF) virus by Aedes vigilax (Skuse) mosquitoes. Eight of 10 animals exposed to RR virus developed neutralizing antibody, and 3 possums developed high viremia for < 48 hr after infection, sufficient to infect recipient mosquitoes. Two of 10 animals exposed to BF virus developed neutralizing antibody. Both infected possums maintained detectable neutralizing antibody to BF for at least 45 days after infection (log neutralization index > 2.0 at 45 days). Eight possums did not develop neutralizing antibody to BF despite exposure to infected mosquitoes. These results suggest that T. vulpecula may potentially act as a reservoir species for RR in urban areas. However, T. vulpecula infected with BF do not develop viremia sufficient to infect mosquitoes and are unlikely to be important hosts for BF.

Resolution of natural groups using iterative assignment tests: an example from two species of Australian native rats (Rattus)

Sympatric individuals of Rattus fuscipes and Rattus leucopus, two Australian native rats from the tropical wet forests of north Queensland, are difficult to distinguish morphologically and are often confused in the field. When we started a study on fine-scale movements of these species, using microsatellite markers, we found that the species as identified in the field did not form coherent genetic groups. In this study, we examined the potential of an iterative process of genetic assignment to separate specimens from distinct (e.g. species, populations) natural groups. Five loci with extensive overlap in allele distributions between species were used for the iterative process. Samples were randomly distributed into two starting groups of equal size and then subjected to the test. At each iteration, misassigned samples switched groups, and the output groups from a given round of assignment formed the input groups for the next round. All samples were assigned correctly on the 10th iteration, in which two genetic groups were clearly separated. Mitochondrial DNA sequences were obtained from samples from each genetic group identified by assignment, together with those of museum voucher specimens, to assess which species corresponded to which genetic group. The iterative procedure was also used to resolve groups within species, adequately separating the genetically identified R. leucopus from our two sampling sites. These results show that the iterative assignment process can correctly differentiate samples into their appropriate natural groups when diagnostic genetic markers are not available, which allowed us to resolve accurately the two R. leucopus and R. fuscipes species. Our approach provides an analytical tool that may be applicable to a broad variety of situations where genetic groups need to be resolved.

Dendritic cells rapidly undergo apoptosis in vitro following culture with activated CD4(+) V alpha 24 natural killer T cells expressing CD40L

Human V alpha 24 natural killer T (V alpha 24NKT) cells are activated by -glycosylceramide-pulsed dendritic cells (DCs) in a CDld-dependent and T-cell receptor-mediated manner. There are two major subpopulations of V alpha 24NKT cells, CD4(-) CD8(-) V alpha 24NKT and CD4(+) V alpha 24NKT cells. We have recently shown that activated CD4(-) CD8 V alpha 24NKT cells have cytotoxic activity against DCs, but knowledge of the molecules responsible for cytotoxicity of V alpha 24NKT cells is currently limited. We aimed to investigate whether CD4(+) V alpha 24NKT cells also have cytotoxic activity against DCs and to determine the mechanisms underlying any observed cytotoxic activity. We demonstrated that activated CD4(+) V alpha 24NKT cells [CD40 ligand (CD40L) -positive] have cytotoxic activity against DCs (strongly CD40-positive), but not against monocytes (weakly CD40-positive) or phytohaemagglutinin blast T cells (CD40-negative), and that apoptosis of DCs significantly contributes to the observed cytotoxicity. The apoptosis of DCs following culture with activated CD4(+) V alpha 24NKT cells, but not with resting CD4(+) V alpha 24NKT cells (CD40L-negative), was partially inhibited by anti-CD40L mAb, Direct ligation of CD40 on the DCs by the anti-CD40 antibody also induced apoptosis of DCs. Our results suggest that CD40-CD40L interaction plays an important role in the induction of apoptosis of DCs following culture with activated CD4+ Va24NKT cells. The apoptosis of DCs from normal donors. triggered by the CD40-CD40L interaction, may contribute to the homeostatic regulation of the normal human immune system, preventing the interminable activation of activated CD4(+) V alpha 24NKT cells by virtue of apoptosis of DCs.

The natural history of Hendra and Nipah viruses

Pteropid bats (flying foxes), species of which are the probable natural host of both Hendra and Nipah viruses, occur in overlapping populations from India to Australia. Ecological changes associated with land use and with animal husbandry practices appear most likely to be associated with the emergence of these two agents. (C) 2001 Editions scientifiques et medicales Elsevier SAS.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV Proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles. At times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Interaction between natural and sexual selection during the evolution of mate recognition

The interaction between natural and sexual selection is central to many theories of how mate choice and reproductive isolation evolve, but their joint effect on the evolution of mate recognition has not, to my knowledge, been investigated in an evolutionary experiment. Natural and sexual selection were manipulated in interspecific hybrid populations of Drosophila to determine their effects on the evolution of a mate recognition system comprised of cuticular hydrocarbons (CHCs). The effect of natural selection in isolation indicated that CHCs were costly for males and females to produce. The effect of sexual selection in isolation indicated that females preferred males with a particular CHC composition. However, the interaction between natural and sexual selection had a greater effect on the evolution of the mate recognition system than either process in isolation. When natural and sexual selection were permitted to operate in combination, male CHCs became exaggerated to a greater extent than in the presence of sexual selection alone, and female CHCs evolved against the direction of natural selection. This experiment demonstrated that the interaction between natural and sexual selection is critical in determining the direction and magnitude of the evolutionary response of the mate recognition system.

Diving behaviour of two Australian bimodally respiring turtles, Rheodytes leukops and Emydura macquarii, in a natural setting

Time-depth recorders were used to investigate the diving performance and behaviour of two bimodally respiring turtle species, Rheodytes leukops and Emydura niacquarii, known to have a high and low reliance on aquatic respiration, respectively. Significant differences in diving performance between R. leukops and E. macquarii were observed in the number of dives/day (39.3 +/- 5.38 vs 112.2 +/- 11.73 dives/day; mean +/- SE), mean dive length (33.1 +/- 7.33 min vs 9.6 +/- 2.26 min) and maximum dive length (623 +/- 104.74 min vs 67.1 +/- 8.14 min), respectively. Differences in diving performance between R. leukops and E macquarii are attributed to the species' reliance (or lack thereof) upon aquatic respiration. Rheodytes leukops displayed a weak bimodal pattern of increased surfacing frequency in the early morning (05:00-07:00) and late afternoon (14:00-18:00), while E. macquarii displayed a strong bimodal pattern of elevated surfacing frequency over similar time periods. Daily patterns of increased surfacing frequency for both species failed to correlate with fluctuating aquatic Po-2 levels or water temperature, and may instead be explained by the heightened activity levels of both species during twilight.

N-15 natural abundance of fossil peat reflects the influence of animal-derived nitrogen on vegetation

delta(15)N signatures of fossil peat were used to interpret past ecosystem processes on tectonically active subantarctic Macquarie Island. By comparing past vegetation reconstructed from the fossil record with present-day vegetation analogues, our evidence strongly suggests that changes in the delta(15)N signatures of fossil peat at this location reflect mainly past changes in the proportion of plant nitrogen derived from animal sources. Associated with uplift above sea level over the past 8,500 years, fossil records in two peat deposits on the island chronicle a change from coastal vegetation with fur and elephant seal disturbance to the existing inland herbfield. Coupled with this change are synchronous changes in the delta(15)N signatures of peat layers. At two sites N-15-enriched peat delta(15)N signatures of up to +17parts per thousand were associated with a high abundance of pollen of the nitrophile Callitriche antarctica (Callitrichaceae). At one site fossil seal hair was also associated with enriched peat delta(15)N. Less N-15 enriched delta(15)N signatures (e.g. -1.9parts per thousand to +3.9parts per thousand) were measured in peat layers which lacked animal associated C. antarctica and Acaena spp. Interpretation of a third peat profile indicates continual occupation of a ridge site by burrowing petrels for most of the Holocene. We suggest that N-15 signatures of fossil peat remained relatively stable with time once deposited, providing a significant new tool for interpreting the palaeoecology.

N-15 natural abundance studies in Australian commercial sugarcane

The measurement of natural N-15 abundance is a well-established technique for the identification and quantification of biological N-2 fixation in plants. Associative N-2 fixing bacteria have been isolated from sugarcane and reported to contribute potentially significant amounts of N to plant growth and development. It has not been established whether Australian commercial sugarcane receives significant input from biological N-2 fixation, even though high populations of N-2 fixing bacteria have been isolated from Australian commercial sugarcane fields and plants. In this study, delta(15)N measurements were used as a primary measure to identify whether Australian commercial sugarcane was obtaining significant inputs of N via biological N-2 fixation. Quantification of N input, via biological N-2 fixation, was not possible since suitable non-N-2 fixing reference plants were not present in commercial cane fields. The survey of Australian commercially grown sugarcane crops showed the majority had positive leaf delta(15)N values (73% >3.00parts per thousand, 63% of which were

Geographic variation of natural products of tropical nudibranch Asteronotus cespitosus

Extracts of the dorid nudibranch Asteronotus cespitosus from two geographically separate regions of Australia and from the Philippines were compared using thin-layer, high-performance liquid and gas chromatography and H-1 NMR analysis. Halogenated metabolites were detected in all mollusk specimens. The major component detected in digestive tissue of specimens from the Great Barrier Reef in northeastern Australia was 4,6-dibromo2-(2',4'-dibromophenoxy)phenol (1), with minor amounts of 3,5-dibromo-2(3',5'-dibromo-20-methoxyphenoxy)phenol (2). In a specimen collected from northwestern Australia, only 3,5-dibromo-2-(3',5'-dibromo-2'-methoxyphenoxy)phenol was found. The specimen from the Philippines contained 2,3,4,5-tetrabromo-6-(2'-bromophenoxy) phenol (3) together with a novel chlorinated pyrrolidone (4). In addition, the sesquiterpenes dehydroherbadysidolide (5) and spirodysin (6) were detected in the digestive organs and mantle tissue of the nudibranchs from the Great Barrier Reef and from the Philippines, whereas these chemicals were not found in the specimen from northwestern Australia. All of the chemicals (1-3,5, and 6) have previously been isolated from the sponge Dysidea herbacea, as have chlorinated metabolites related to 4. This is the first time the characteristic halogenated metabolites that typify Dysidea herbacea have been reported from a carnivorous mollusk, which implies a dietary origin as opposed to de novo synthesis.

Cloud-point extraction and preconcentration of cyanobacterial toxins (microcystins) from natural waters using a cationic surfactant

A new cloud-point extraction and preconcentration method, using a cationic, surfactant, Aliquat-336 (tricaprylyl-methy;ammonium chloride), his-been developed for the determination of cyanobacterial toxins, microcystins, in natural waters. Sodium sulfate was used to induce phase separation at 25 degreesC. The phase behavior of Aliquat-336 with respect to concentration of Na2SO4 was studied. The cloud-point system revealed a very high phase volume ratio compared to other established systems of nonionic, anionic, and cationic surfactants: At pH 6-7, it showed an outstanding selectivity in ahalyte extraction for anionic species. Only MC-LR and MC-YR, which are known to be predominantly anionic, were extracted (with averaged recoveries of 113.9 +/- 9% and 87.1 +/- 7%, respectively). MC-RR, which is likely to be amphoteric at the above pH range, was. not cle tectable in.the extract. Coupled to HPLC/UV separation and detection, the cloud-point extraction method (with 2.5 mM Aliquat-336 and 75 mM Na2SO4 at 25 degreesC) offered detection limits of 150 +/- 7 and 470 +/- 72 pg/mL for MC-LR and MC-YR, respectively, in 25 mL of deionized water. Repeatability of the method was 7.6% for MC-LR and 7.3% for MC-YR: The cloud-point extraction process can be. completed within 10-15 min with no cleanup steps required. Applicability of the new method to the determination of microcystins in real samples was demonstrated using natural surface waters, collected from a local river and a local duck pond spiked with realistic. concentrations of microcystins. Effects of salinity and organic matter (TOC) content in the water sample on the extraction efficiency were also studied.

Sponge halogenated natural products found at parts-per-million levels in marine mammals

Several unknown, abundant brominated compounds (BCs) were recently detected in the blubber of dolphins and other marine mammals from Queensland (northeast Australia). The BC were interpreted as potential natural products due to the lack of anthropogenic sources for these compounds. This study investigated whether some of the BCs accumulated by diverse marine mammal species are identical with natural BCs previously isolated from sponges (Dysidea sp.) living in the same habitat. Isolates from sponges and mollusks (Asteronotus cespitosus) were compared with the signals detected in the mammals' tissue. Mass spectra and gas chromatography retention times on four different capillary columns of the isolates from sponges and mammals were identical in all respects. This proves that the chemical name of the compound previously labeled BC-2 is 4,6-dibromo-2-(2'-dibromo)phenoxyanisole and that the chemical name of BC-11 is 3,5-dibromo-2-(3',5'-dibromo-2'-methoxy)phenoxyanisole. Using a quantitative reference solution of BC-2, we established that the concentrations of the brominated metabolies found in the marine mammals are frequently >1 mg/kg. The highest concentration (3.8 mg/kg), found in a sample of pygmy sperm whale (Kogia breviceps), indicates that BC-2 is a bioaccumulative, natural organohalogen compound. This is supported by the concentrations of the BCs in our samples being equal to the highest concentrations of anthropogenic BCs in any environmental sample. The quantitative determination of BC-2 in blubber of marine mammals from Africa and the Antarctic suggests that BC-2 is wide-spread. These results are direct proof that marine biota can produce persistent organic chemicals that accumulate to substantial concentrations in higher trophic organisms.