Faecal proteinases of the fungus-growing ant, Atta texana: Their fungal origin and ecological significance

The fluid contained within the mycelium of the fungus cultured by the attine ant, Atta texana, contains three proteolytic enzymes. One enzyme is a DFP-sensitive alkaline proteinase; the other two are metal-chelator-sensitive neutral proteinases. These three enzymes are indentical by all criteria examined to the three proteinases previously isolated from the faecal fluid of A. texana. It is concluded that the faecal enzymes of the fungus-growing ants are derived from the mycelial fluid upon which they feed. The basis for the symbiosis between the attine ants and the fungi which they cultivate in their nests is reinterpreted in the context of this finding.     

Dynamics of predominant microbiota in the human gastrointestinal tract and change in luminal enzymes and immunoglobulin profile during high-altitude adaptation

High-altitude (HA) visitors like pilgrims, trackers, scientists and military personnel face a group of nonspecific gastrointestinal (GI) symptoms during acclimatization to hypobaric hypoxia. In order to investigate the alteration of indigenous gastrointestinal microbiota in the development of such GI symptoms, an experiment was conducted for the enumeration of dominant cultivable faecal microbiota of 15 soldiers at base level (Delhi) and during their 15-day acclimatization at 3,505 m HA (Leh). At HA, faecal microbiota analysis revealed that total aerobes decreased significantly with increase of total and facultative anaerobes. The strict anaerobes like Bifidobacterium sp., Bacteroidetes sp. and Lactobacillus sp. exhibited positive growth direction index (GDI) like other predominant obligate anaerobes Clostridium perfringens and Peptostreptococcus sp. Different enzymes like amylase, proteinase and polyphenol hydrolase produced by different bacterial populations showed positive GDI, whereas phosphatase producers exhibited negative GDI. The levels of microbe-originated enzymes like amylase, proteinase, alkaline phosphatase and β-glucuronidase were also elevated during HA acclimatization. In addition, in vitro gas production ability was enhanced with increase of faecal immunoglobulins IgA and IgG. We demonstrated that hypoxic environment at HA had the potential to alter the gut microbial composition and its activities that may cause GI dysfunctions.     

Digestive Secretion of Dionaea muscipula (Venus's Flytrap)

The digestive fluid of Dionaea muscipula has been studied with respect to its protein content as a function of time after entrapment of protein material and some enzymes of the secretion. Maximum secretion of enzyme occurs within the first 3 days of the digestive cycle and protein reaches its maximum at 4 days. Phosphatase, proteinase, nuclease and amylase have been observed in the secretion. The enzymes have acid pH optima and the proteinase has a molecular weight of about 40,000.     

Alpha glucosidases in white blood cells, with reference to the detection of acid alpha 1-4 glucosidase deficiency.

The iron-containing violet acid phosphatases from beef spleen and pig allantoic fluid have been purified to homogeneity. Molecular weight determinations by zonal gel filtration, SDS-gel electrophoresis, and ultracentrifugation support values close to 40,000 for both enzymes, necessitating reappraisal of literature values. Similarly, the equivalent weight for iron is close to 20,000 for both enzymes, indicating the presence of two iron atoms per molecule of enzyme. The enzymes also have very similar ultraviolet and visible spectra, with λ_max values close to 550 nm, and ?₅₅₀ values(in terms of iron) of 2.04 × 10³ and 2.00 × 10³ for the beef spleen and pig allantoic fluid enzymes respectively.     

A titration approach to identify the capacity for starch digestion in milk-fed calves

Calf milk replacers (MR) commonly contain 40% to 50% lactose. For economic reasons, starch is of interest as a lactose replacer. Compared with lactose, starch digestion is generally low in calves. It is, however, unknown which enzyme limits the rate of starch digestion. The objectives were to determine which enzyme limits starch digestion and to assess the maximum capacity for starch digestion in milk-fed calves. A within-animal titration study was performed, where lactose was exchanged stepwise for one of four starch products (SP). The four corn-based SP differed in size and branching, therefore requiring different ratios of starch-degrading enzymes for their complete hydrolysis to glucose: gelatinised starch (α-amylase and (iso)maltase); maltodextrin ((iso)maltase and α-amylase); maltodextrin with α-1,6-branching (isomaltase, maltase and α-amylase) and maltose (maltase). When exceeding the animal’s capacity to enzymatically hydrolyse starch, fermentation occurs, leading to a reduced faecal dry matter (DM) content and pH. Forty calves (13 weeks of age) were assigned to either a lactose control diet or one of four titration strategies (n=8 per treatment), each testing the stepwise exchange of lactose for one SP. Dietary inclusion of each SP was increased weekly by 3% at the expense of lactose and faecal samples were collected from the rectum weekly to determine DM content and pH. The increase in SP inclusion was stopped when faecal DM content dropped below 10.6% (i.e. 75% of the average initial faecal DM content) for 3 consecutive weeks. For control calves, faecal DM content and pH did not change over time. For 87% of the SP-fed calves, faecal DM and pH decreased already at low inclusion levels, and linear regression provided a better fit of the data (faecal DM content or pH v. time) than non-linear regression. For all SP treatments, faecal DM content and pH decreased in time (P<0.001) and slopes for faecal DM content and pH in time differed from CON; P<0.001 for all SP), but did not differ between SP treatments. Faecal DM content of SP-fed calves decreased by 0.57% and faecal pH by 0.32 per week. In conclusion, faecal DM content and pH sensitively respond to incremental inclusion of SP in calf MR, independently of SP characteristics. All SP require maltase to achieve complete hydrolysis to glucose. We therefore suggest that maltase activity limits starch digestion and that fermentation may contribute substantially to total tract starch disappearance in milk-fed calves.     

Determinants of Antimicrobial Resistance in Escherichia coli Strains Isolated from Faeces and Urine of Women with Recurrent Urinary Tract Infections

For women with recurrent urinary tract infections (rUTI), the contribution of antibiotic use versus patient-related factors in determining the presence of antimicrobial resistance in faecal and urinary Escherichia coli, obtained from the same patient population, has not been assessed yet. Within the context of the ‘Non-antibiotic prophylaxis for recurrent urinary tract infections’ (NAPRUTI) study, the present study assessed determinants of antimicrobial resistance in E. coli isolated from urinary and faecal samples of women with rUTIs collected at baseline. Potential determinants of resistance were retrieved from self-administered questionnaires. From 434 asymptomatic women, 433 urinary and 424 faecal samples were obtained. E. coli was isolated from 146 (34%) urinary samples and from 336 (79%) faecal samples, and subsequently tested for antimicrobial susceptibility. Multivariable analysis showed trimethoprim/sulfamethoxazole (SXT) use three months prior to inclusion to be associated with urine E. coli resistance to amoxicillin (OR 3.6, 95% confidence interval: 1.3–9.9), amoxicillin-clavulanic acid (OR 4.4, 1.5–13.3), trimethoprim (OR 3.9, 1.4–10.5) and SXT (OR 3.2, 1.2–8.5), and with faecal E. coli resistance to trimethoprim (OR 2.0, 1.0–3.7). The number of UTIs in the preceding year was correlated with urine E. coli resistance to amoxicillin-clavulanic acid (OR 1.11, 1.01–1.22), trimethoprim (OR 1.13, 1.03–1.23) and SXT (OR 1.10, 1.01–1.19). Age was predictive for faecal E. coli resistance to amoxicillin (OR 1.02, 1.00–1.03), norfloxacin and ciprofloxacin (both OR 1.03, 1.01–1.06). In conclusion, in women with rUTI different determinants were found for urinary and faecal E. coli resistance. Previous antibiotic use and UTI history were associated with urine E. coli resistance and age was a predictor of faecal E. coli resistance. These associations could best be explained by cumulative antibiotic use.     

Potential Wildlife Sentinels for Monitoring the Endemic Spread of Human Buruli Ulcer in South-East Australia

The last 20 years has seen a significant series of outbreaks of Buruli/Bairnsdale Ulcer (BU), caused by Mycobacterium ulcerans, in temperate south-eastern Australia (state of Victoria). Here, the prevailing view of M. ulcerans as an aquatic pathogen has been questioned by recent research identifying native wildlife as potential terrestrial reservoirs of infection; specifically, tree-dwelling common ringtail and brushtail possums. In that previous work, sampling of environmental possum faeces detected a high prevalence of M. ulcerans DNA in established endemic areas for human BU on the Bellarine Peninsula, compared with non-endemic areas. Here, we report research from an emergent BU focus recently identified on the Mornington Peninsula, confirming associations between human BU and the presence of the aetiological agent in possum faeces, detected by real-time PCR targeting M. ulcerans IS2404, IS2606 and KR. Mycobacterium ulcerans DNA was detected in 20/216 (9.3%) ground collected ringtail possum faecal samples and 4/6 (66.6%) brushtail possum faecal samples. The distribution of the PCR positive possum faecal samples and human BU cases was highly focal: there was a significant non-random cluster of 16 M. ulcerans positive possum faecal sample points detected by spatial scan statistics (P<0.0001) within a circle of radius 0.42 km, within which were located the addresses of 6/12 human cases reported from the area to date; moreover, the highest sample PCR signal strength (equivalent to ≥10⁶ organisms per gram of faeces) was found in a sample point located within this cluster radius. Corresponding faecal samples collected from closely adjacent BU-free areas were predominantly negative. Possums may be useful sentinels to predict endemic spread of human BU in Victoria, for public health planning. Further research is needed to establish whether spatial associations represent evidence of direct or indirect transmission between possums and humans, and the mechanism by which this may occur.     

Investigating zoonotic infection barriers to ape Plasmodium parasites using faecal DNA analysis

African apes are endemically infected with numerous Plasmodium spp. including close relatives of human Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Although these ape parasites are not believed to pose a zoonotic threat, their ability to colonise humans has not been fully explored. In particular, it remains unknown whether ape parasites are able to initiate exo-erythrocytic replication in human hepatocytes following the bite of an infective mosquito. Since animal studies have shown that liver stage infection can result in the excretion of parasite nucleic acids into the bile, we screened faecal samples from 504 rural Cameroonians for Plasmodium DNA. Using pan-Laverania as well as P. malariae- and P. vivax-specific primer sets, we amplified human P. falciparum (n?=?14), P. malariae (n?=?1), and P. ovale wallikeri (n?=?1) mitochondrial sequences from faecal DNA of 15 individuals. However, despite using an intensified PCR screening approach we failed to detect ape Laverania, ape P. vivax or ape P. malariae parasites in these same subjects. One faecal sample from a hunter-gatherer contained a sequence closely related to the porcupine parasite Plasmodium atheruri. Since this same faecal sample also contained porcupine mitochondrial DNA, but a matching blood sample was Plasmodium-negative, it is likely that this hunter-gatherer consumed Plasmodium-infected bushmeat. Faecal Plasmodium detection was not secondary to intestinal bleeding and/or infection with gastrointestinal parasites, but indicative of blood parasitaemia. Quantitative PCR identified 26-fold more parasite DNA in the blood of faecal Plasmodium-positive than faecal Plasmodium-negative individuals (P?=?0.01). However, among blood-positive individuals only 10% - 20% had detectable Plasmodium sequences in their stool. Thus, faecal screening of rural Cameroonians failed to uncover abortive ape Plasmodium infections, but detected infection with human parasites, albeit with reduced sensitivity compared with blood analysis.     

Faecal microbiome sequences in relation to the egg-laying performance of hens using amplicon-based metagenomic association analysis

Exploring the composition and structure of the faecal microbial community improves the understanding of the role of the gut microbiota in the gastrointestinal function and the egg-laying performance of hens. Therefore, detection of hen–microbial interactions can explore a new breeding marker for the selection of egg production due to the important role of the gut microbiome in the host’s metabolism and health. Recently, the gut microbiota has been recognised as a regulator of host performance, which has led to investigations of the productive effects of changes in the faecal microbiome in various animals. In the present study, a metagenomics analysis was applied to characterise the composition and structural diversity of faecal microbial communities under two selections of egg-laying performance, high (H, n = 30) and low (L, n = 30), using 16S rRNA-based metagenomic association analysis. The most abundant bacterial compositions were estimated based on the operational classification units among samples and between the groups from metagenomic data sets. The results indicated that Firmicutes phylum has higher significant (P < 0.01) in the H group than in the L group. In addition, higher relative abundance phyla of Bacteroides and Fusobacteria were estimated in the H group than the L group, contrasting the phyla of Actinobacteria, Cyanobacteria and Proteobacteria were more relative abundance in the L group. The families (Lactobacillus, Bifidobacterium, Acinetobacter, Flavobacteriaceae, Lachnoclostridum and Rhodococcus) were more abundant in the H group based on the comparison between the H and L groups. Meanwhile, three types of phyla (Proteobacteria, Actinobacteria and Cyanobacteria) and six families (Acinetobacter, Avibacterium, Clostridium, Corynebacterium, Helicobacter and Peptoclostridium) were more abundant in the L group (P < 0.01). Overall, the selection of genotypes has enriched a relationship between the gut microbiota and the egg-laying performance. These findings suggest that the faecal microbiomes of chickens with high egg-laying performance have more diverse activities than those of chickens with low egg-laying performance, which may be related to the metabolism and health of the host and egg production variation.     

Short-Chain Fructo-Oligosaccharides Modulate Intestinal Microbiota and Metabolic Parameters of Humanized Gnotobiotic Diet Induced Obesity Mice

Prebiotic fibres like short-chain fructo-oligosaccharides (scFOS) are known to selectively modulate the composition of the intestinal microbiota and especially to stimulate Bifidobacteria. In parallel, the involvement of intestinal microbiota in host metabolic regulation has been recently highlighted. The objective of the study was to evaluate the effect of scFOS on the composition of the faecal microbiota and on metabolic parameters in an animal model of diet-induced obesity harbouring a human-type microbiota. Forty eight axenic C57BL/6J mice were inoculated with a sample of faecal human microbiota and randomly assigned to one of 3 diets for 7 weeks: a control diet, a high fat diet (HF, 60% of energy derived from fat)) or an isocaloric HF diet containing 10% of scFOS (HF-scFOS). Mice fed with the two HF gained at least 21% more weight than mice from the control group. Addition of scFOS partially abolished the deposition of fat mass but significantly increased the weight of the caecum. The analysis of the taxonomic composition of the faecal microbiota by FISH technique revealed that the addition of scFOS induced a significant increase of faecal Bifidobacteria and the Clostridium coccoides group whereas it decreased the Clostridium leptum group. In addition to modifying the composition of the faecal microbiota, scFOS most prominently affected the faecal metabolome (e.g. bile acids derivatives, hydroxyl monoenoic fatty acids) as well as urine, plasma hydrophilic and plasma lipid metabolomes. The increase in C. coccoides and the decrease in C. leptum, were highly correlated to these metabolic changes, including insulinaemia, as well as to the weight of the caecum (empty and full) but not the increase in Bifidobacteria. In conclusion scFOS induce profound metabolic changes by modulating the composition and the activity of the intestinal microbiota, that may partly explain their effect on the reduction of insulinaemia.     

Cold-Adapted Digestive Aspartic Protease of the Clawed Lobsters Homarus americanus and Homarus gammarus: Biochemical Characterization

Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K _M value at 4°C, 10°C, and 25°C, and had 20-fold higher k _cat /K _M values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature.     

Evaluation of titanium dioxide and chromic oxide as digestibility markers in ponies fed alfalfa hay in relation to marker dosing frequency

In equines, Cr₂O₃ is widely accepted as an indigestible marker, but there are health concerns regarding the carcinogenic properties of Cr₂O₃. Recently, TiO₂ has been suggested to be an alternative digestibility marker in equines. However, a comparison between Cr₂O₃ and TiO₂ has not been made in equines. Six Welsh pony geldings (initial BW: 254±3 kg; 7 years of age) fed chopped alfalfa hay were used to evaluate the use of TiO₂ (Ti) and Cr₂O₃ (Cr) as markers for calculating apparent digestibility and to investigate the effect of frequency of marker administration on the measurement of digestibility values. Diets contained 4.65 kg dry matter (DM) chopped alfalfa hay supplemented with minerals, vitamins, TiO₂ (3.3 g Ti/day) and Cr₂O₃ (3.2 g Cr/day). Ponies were dosed with either 3.3 g Ti and 3.2 g Cr once daily (DF1) or with 1.65 g Ti and 1.60 g Cr twice daily (DF2). After adaptation to the diets and procedures for 14 days, voluntary voided faeces were collected quantitatively over 7 days and analysed for moisture, ash, Ti and Cr. Apparent total tract DM digestibility (DMD) and organic matter digestibility (OMD) were calculated using the total faecal collection (TFC) and marker method (Ti and Cr). The overall mean cumulative faecal recovery of Cr and Ti (as % of intake) were 102.0% and 96.6%, respectively. Mean daily faecal recoveries of Cr as well as of Ti were not different (P=0.323; P=0.808, respectively) between treatments. Overall daily faecal recovery of Cr differed (P=0.019) from 100% when the marker was dosed once daily, whereas overall daily faecal recovery was similar to 100% for both administration frequencies when Ti was used as a marker. For both markers, the coefficient of variation of the mean faecal marker recovery between horses was lower when the markers were administrated twice per day. Across treatments, cumulative DMD and OMD estimated with Ti were similar (P=0.345; P=0.418, respectively) compared with those values determined by TFC method. When Cr was used, the calculated cumulative DMD tended (P=0.097) to be greater compared with those estimated with TFC, and cumulative OMD values were overestimated (P=0.013). Orally supplemented Ti recovery in the faeces of ponies fed chopped alfalfa hay with Ti administered once or twice daily was close to 100%, making it the preferred marker for digestibility trials in equines.     

An alternative hypothesis for the primary function of a proposed mate assessment behaviour in red-backed salamanders

Using a laboratory experiment, we tested the hypothesis that faecal inspection and squashing behaviours by female red-backed salamanders, Plethodon cinereus, function in a context other than mate assessment, in this case foraging. Because only female P. cinereus squash faecal pellets, other studies have hypothesized that the faecal squashing is a mate assessment behaviour. During the courtship season, we exposed gravid female P. cinereus maintained on a ‘high-quality’ or ‘low-quality’ diet, to the faecal pellets of male and female conspecifics fed ‘high-quality’ diets. Female diet had no effect on activity level, and, overall, P. cinereus showed greater interest in male-marked substrates than female-marked substrates. However, female diet had a large effect on faecal inspection and squashing behaviours, and females showed similar inspection and squashing rates towards both male and female faecal pellets. These results suggest that faecal squashing does not function primarily in mate assessment, but rather as a means for female P. cinereus to gain information about prey availability. We propose that male P. cinereus have evolved to exploit female faecal inspection and squashing behaviours (sensory exploitation) to attract foraging females into areas to increase male encounter rates with females.     

Comparison of the bacterial community structure within the equine hindgut and faeces using Automated Ribosomal Intergenic Spacer Analysis (ARISA)

The horse’s hindgut bacterial ecosystem has often been studied using faecal samples. However few studies compared both bacterial ecosystems and the validity of using faecal samples may be questionable. Hence, the present study aimed to compare the structure of the equine bacterial community in the hindgut (caecum, right ventral colon) and faeces using a fingerprint technique known as Automated Ribosomal Intergenic Spacer Analysis (ARISA). Two DNA extraction methods were also assessed. Intestinal contents and faeces were sampled 3 h after the morning meal on four adult fistulated horses fed meadow hay and pelleted concentrate. Irrespective of the intestinal segment, Principal Component Analysis of ARISA profiles showed a strong individual effect (P<0.0001). However, across the study, faecal bacterial community structure significantly (P<0.001) differed from those of the caecum and colon, while there was no difference between the two hindgut communities. The use of a QIAamp® DNA Stool Mini kit increased the quality of DNA extracted irrespective of sample type. The differences observed between faecal and hindgut bacterial communities challenge the use of faeces as a representative for hindgut activity. Further investigations are necessary to compare bacterial activity between the hindgut and faeces in order to understand the validity of using faecal samples.     

Faecal pellets as burrow markers: intra- and interspecific odour recognition by western plethodontid salamanders

Chemical communication is widespread among plethodontid salamanders (Caudata: Plethodontidae), but little information exists on the role of odours in interspecific competition in general and among species in western North America in particular. The present study examined the potential of faecal odours for intra- and interspecific communication among sympatric plethodontids in two-choice tests in the laboratory. In experiment 1, Plethodon vehiculum and Aneides ferrus from Vancouver Island, B.C., Canada, were presented with two burrows marked with (1) a faecal pellet of male P. vehiculum versus a control clay pellet, and (2) a faecal pellet of male A. ferreus versus a control pellet. In experiment 2, responses of P. vehiculum, P. dunni and P. vandykei from Washington, U.S.A., were tested towards faecal pellets of conspecific males and females, and towards pellets of congeneric males. Both P. vehiculum and P. dunni distinguished odours of conspecific males and females, based on their behavioural responses towards faecal and control pellets. Only P. dunni, however, avoided burrows marked with faeces of conspecific individuals. In contrast, neither A. ferreus nor P. vandykei appeared to distinguish faecal odours of conspecific salamanders from control pellets. In tests with odours of heterospecific individuals, P. dunni and P. vehiculum distinguished odours of each other, and P. vandykei distinguished odours of P. vehiculum. The data from these two experiments suggest that P. dunni use faecal pellets as territorial markers both in intraspecific communication and in interspecific encounters with P. vehiculum.     

Abundance and germination capability of resting cysts of Alexandrium spp. (Dinophyceae) from faecal pellets of macrobenthic organisms

The Alexandrium spp. resting cysts were found abundantly in faecal pellets collected from the bottom sediments at two stations in Hiroshima Bay. It is considered that these faecal pellets were excreted by the macrobenthos, such as polychaeta and mollusca, based on their size and morphology. Polychaeta was the most dominant macrobenthos, and mollusca was the second most dominant group in Hiroshima Bay. The resting cysts of Alexandrium spp. in the bottom sediments at the two stations were counted in both the faecal pellets of macrobenthos and in the surrounding sediment. As a result, the number of cysts in the faecal pellets accounted for 28.9-35.2% of total cysts. In addition, cysts isolated from faecal pellets had almost the same germination ability as those in the sediment. Thus, Alexandrium cysts are tolerant to the predation and digestive processes of macrobenthic organisms.     

Molecular identification of Eimeria species in Spanish bats

This is the first study reporting the detection and molecular characterization of Eimeria in bats in Spain, specifically in 12 of 32 chiropteran species described in the Iberian Peninsula. A total of 76 faecal samples were collected from different bat roosting sites across Spanish territory. The DNA was extracted from the samples and sequenced by targeting the Eimeria SSU-rRNA gene. Two Eimeria species were detected in 29 of the 76 faecal samples (38%), and the bat-specific Eimeria rioarribaensis and rodent-specific Eimeria jerfinica were detected in 4 (5%) and 25 (33%) of the samples, respectively. This is the first report of E. rioarribaensis in the bats Rhinolophus euryale, Myotis myotis and Nyctalus lasiopterus, extending the host and geographical ranges for this bat coccidian parasite. The identification of the rodent-specific parasite species E. jerfinica in bats indicates the occurrence of this species in Spain, although its presence has not previously been reported in wild rodents in this country. Considering that most of the Eimeria spp. reported in bats were described only on the basis of morphometric data, molecular studies are required to determined which Eimeria species occur in bats, to complete the identification of these species and to clarify the phylogeny.     

Bacteriolytic enzymes from the blood plasma of the sheep

Bacteriolytic factors from the blood plasma of healthy sheep have been studied. Three enzymes not described earlier in the literature have been identified and characterized. Two enzymes exhibit activity toward Escherichia coli and have molecular weights of 15 ± 2 kDa. The third enzyme that exhibits activity toward E. coli and Micrococcus luteus has a molecular weight of 34 ± 4 kDa. The kinetic parameters of bacterial lysis for all enzymes have been determined; in particular, optimal pH values for each of the substrates used have been found. For the identification of the enzymes, trypsinolysis and a mass-spectroscopic study of fragments have been carried out. The results were compared with the data on sheep proteins available in the Swiss-Prot, NCBI, and MSDB databases.     

Fate of resting cysts of Alexandrium spp. ingested by Perinereis nuntia (Polychaeta) and Theola fragilis (Mollusca)

The ingestion of resting cysts of Alexandrium spp. by Perinereis nuntia (Polychaeta) and Theola fragilis (Mollusca) was experimentally examined in the laboratory. P. nuntia and T. fragilis were cultured in bottom sediment containing a high density of Alexandrium cysts under dark conditions. Moreover, to evaluate the degree and consequence of being ingested, the density of cysts in the control sediment (no macrobenthic organisms) and the germination capability of the cysts in the faecal pellets of the two species of macrobenthos were examined.Cysts in the culture sediment were found to be ingested by both P. nuntia and T. fragilis. No difference in the density of cysts between the sediments cultured with and without P. nuntia was observed. However, the density of cysts in the sediments with T. fragilis decreased by 24% compared to the density in the control sediment. It is possible that most of the cysts ingested were digested by T. fragilis. The rate of Alexandrium cyst digestion by this species is estimated 594 cysts/individual/day. It is estimated that 91% of the cysts ingested by T. fragilis were partially or totally digested and only 9% were excreted in a viable state during the experiment. Thus, T. fragilis has a stronger affect on the abundance of Alexandrium cysts compared with P. nuntia.No significant difference was observed between the germination success of the cysts from faecal pellets of P. nuntia and T. fragilis compared to the cysts in the control sediment. If, however, the necessary light for the cysts to germinate is cut off by being enclosed within the faecal pellet, the germination rate of cysts from the faecal pellets may be suppressed.     

Recent advances in studies of the mechanisms of microbial degradation of lignins

Major advances in our understanding of the biochemical and enzymological mechanisms of lignin biodegradation have been made in the past three years. Research has principally involved two ligninolytic microorganisms, the white rot fungus Phanerochaete chrysosporium and the actinomycete Streptomyces viridosporus. Research has been centred on attempts to identify the microbial catalysts that mediate lignin decay in these two microbes. Emphasis has been on studies concerned with isolating specific lignin catabolic enzymes and/or reduced forms of oxygen involved in attacking the lignin polymer. The possibility that lignin degradation might be non-enzymatic and mediated by extracellular reduced oxygen species such as hydrogen peroxide (H₂O₂), superoxide (O₂∪c-|_.), hydroxyl radical (·OH) or singlet oxygen (¹O₂) has been investigated with both microorganisms. Using methods which have not always been unequivocal, the question of involvement of reduced oxygen species in lignin degradation by P. chrysosporium has been examined exhaustively. Evidence for the involvement of H₂O₂ is conclusive. However, there is little evidence to support the involvement of other extracellular reduced oxygen species, including ·OH, directly in the process of lignin degradation. Scavenger studies have been inconclusive because of questions of their specificity. If activated oxygen species are involved, the activated oxygen is probably held within the active site of an enzyme molecule. With S. viridosporus, scavenger studies also strongly indicate that extracellular reduced oxygen species are not involved in lignin degradation since scavengers generally do not significantly affect the ligninolytic system. The involvement of specific enzymes in lignin degradation by both P. chrysosporium and S. viridosporus has now been confirmed. With P. chrysosporium, ligninolytic enzymes recently discovered include extracellular non-specific peroxidases and oxygenases. They show numerous activities including dehydrogenative, peroxidatic, oxygenative and C_α?C_β cleavages of lignin side chains. At least one P. chrysosporium enzyme, a unique H₂O₂-requiring oxygenase, has been purified to homogeneity. Evidence has been presented to show that S. viridosporus also produces a ligninolytic enzyme complex involved in demethylation of lignin's aromatic rings and in the oxidation of lignin side chains and cleavage of β-tether linkages within the polymer. The combined activites of these enzymes generate water-soluble polymeric modified lignin fragments, which are then slowly degraded further by S. viridosporus. The β-ether cleaving enzyme complex is probably membrane associated, but it is not extracellular. These first isolations of ligninolytic enzymes have changed the course of basic research on lignin biodegradation. New research priorities are already emerging and include enzyme purifications, kinetic studies, enzyme reaction mechanism studies and screenings for more enzymes. In addition, genetic studies are being carried out with both P. chrysosporium and S. viridosporus. Genetic manipulations include not only classical mutagenesis techniques, but also recombinant DNA techniques such as protoplast fusion. This latter technique has already been used to generate overproducers of the ligninolytic enzyme complex in S. viridosporus and it has been successfully used to recombine mutant strains of P. chrysosporium.