Differentiation of important and closely related cereal plant species (Poaceae) in food by hybridization to an oligonucleotide array

We report the development of an oligonucleotide microarray for the simultaneous detection of six important cereal food plant species from the Poaceae based on the chloroplast trnL intron sequence. We used universal primers to amplify the trnL intron from wheat, rye, barley, oat, rice, and maize, followed by a cyclic labeling of oligonucleotides probes and subsequent hybridization to an oligonucleotide microarray. In single taxon analyses, positive signals were produced with a high signal-to-noise ratio. The assay also enabled the analysis of mixed samples. The results obtained for real food samples were in agreement with the ingredient labels, but positive results for grains not declared on the ingredients list were observed in three out of 10 samples, which indicates that the final products and/or the declared ingredients were probably botanically impure or contaminated. The combination of the sensitivity of a universal polymerase chain reaction with the specificity of the labeling reaction allows this protocol to be applied in routine analyses of food samples, as demonstrated by successful analysis of processed composite food products.     

Seed oil and fatty acid content in okra (Abelmoschus esculentus) and related species

Approximately 1100 genebank accessions of okra (Abelmoschus esculentus) and 540 additional accessions that included six of its related species-A. caillei, A. crinitis, A. esculentus, A. ficulneus, A. manihot, A. moschatus and A. tuberculatus-were evaluated for seed oil content using time domain NMR (TD-NMR). Oil content in seed of A. caillei, A. esculentus, A. ficulneus, A. manihot, A. moschatus and A. tuberculatus was in the ranges 2.51-13.61%, 12.36-21.56%, 6.62-16.7%, 16.1-22.0%, 10.3-19.8% and 10.8-23.2%, respectively. Accession PI639680 (A. tuberculatus) had the highest seed oil content (～23%). Accessions of A. esculentus with high seed oil content included PI nos. PI274350 (21.5%), PI538082 (20.9%) and PI538097 (20.9%). Values for the three accessions of A. manihot with the highest seed oil content were PI nos. PI639673 (20.4%), PI639674 (20.9%) and PI639675 (21.9%), all representing var. tetraphyllus. Average percent seed oil in materials of A. esculentus from Turkey and Sudan (17.35% and 17.36%, respectively) exceeded the averages of materials from other locations. Ninety-eight accessions (total of six species) were also examined for fatty acid composition. Values of linoleic acid ranged from 23.6-50.65% in A. esculentus. However, mean linoleic acid concentrations were highest in A. tuberculatus and A. ficulneus. Concentrations of palmitic acid were significantly higher in A. esculentus (range of 10.3-36.35%) when compared to that of other species, and reached a maximum in PI489800 Concentrations of palmitic acid were also high in A. caillei (mean = ～30%). Levels of oleic acid were highest in A. manihot, A. manihot var. tetraphyllus and A. moschatus.     

N-acyl-homoserine lactones from Enterobacter sakazakii (Cronobacter spp.) and their degradation by Bacillus cereus enzymes

A chemical study of acyl-homoserine lactones (acyl-HSLs) produced by Enterobacter sakazakii resulted in the identification of three molecules: (S)-N-heptanoyl-HSL, (S)-N-dodecanoyl-HSL and (S)-N-tetradecanoyl-HSL. Mixed cultures of E. sakazakii and Bacillus cereus depleted E. sakazakii acyl-HSLs, suggesting acyl-HSL degradation by B. cereus hydrolases (hydrolysis of the lactone or amide moiety). The expression of B. cereus acyl-HSL lactonase and acyl-homoserine acylase was confirmed by monitoring the biotransformation of (S)-N-dodecanoyl-HSL into (S)-N-dodecanoyl-homoserine, dodecanoic acid and homoserine in the presence of B. cereus whole cells, using electrospray-mass spectrometry (ESI-MS).     

Local population dynamics of adult butterflies and the conservation status of two closely related species

Intensive mark-recapture studies have become standard in attempts to elucidate the conservation status of butterfly populations. In order to test the usefulness of this approach, such mark-recapture studies were conducted with two Euphydryas species: E. editha, a highly endangered resident of grasslands on serpentine soils, and E. chalcedona, a ubiquitous resident of widespread coastal chaparral. Detailed statistical analysis of the resulting data revealed few differences between the two species in most adult population parameters that have been measured in previous studies, despite the two species' disparate conservation status. This suggests that standard mark-recapture statistics alone are insufficient to reveal adult population features that distinguish species susceptible to extinction from those that are comparatively more secure. New approaches both to mark-recapture analysis and the study of endangered species in general are needed to generate more useful information concerning the conservation status of invertebrate populations.     

Estimating the proportions of closely related species: Performance of the two-phase ratio estimator

In many ecological field contexts, accurately classifying closely related species based on phenotypic characteristics may be difficult. In such cases, definitive classification of species may require expensive genetic analysis of tissues or extensive quantitative measurements. If inexpensive phenotype-based species classifications are highly correlated with expensive but definitive classifications, however, then estimating the proportion of a target species in a mixed species complex using a two-phase ratio estimator may prove cost-efficient. In two-phase ratio estimation, a first-phase sample is randomly selected, and phenotype is used to classify all individuals in the sample. A smaller, second-phase sample is then randomly selected from the first-phase sample, and a definitive method is used to classify the individuals in this subsample. Net relative efficiency (i.e., cost-effectiveness) of the optimally allocated two-phase ratio estimator of a proportion depends on the relative costs of classification at the first and second phases, on the phenotypic classification accuracy for the target species (sensitivity) and nontarget species (specificity), and on the magnitude of the target species proportion. Results are presented that allow assessment of the circumstances in which this two-phase estimation approach can be recommended over an equal-cost single-phase approach based only on expensive but definitive classification. An illustrative application of these methods is provided using an example of two closely related, sympatric, anadromous salmonid species: steelhead (Oncorhynchus mykiss) and coastal cutthroat trout (O. clarki clarki).     

The RAPD evidence for the phylogenetic relationship of the closely related species of cultivated apple

The phylogenetic relationship of cultivated apple and its closely related species is still not clear in the taxonomy of genus Malus. To try to find new evidence for the origin and evolution of the cultivated apple, random amplified polymorphic DNA (RAPD) markers of 14 taxa of Malus, among which a reference species (M. toringoides) and six presumably ancestral species of cultivated apple in the genus were investigated. The RAPD data obtained were used to construct both unrooted and rooted trees using TREECON software package. The result showed in our rooted tree that M. sieversii from the Xinjiang Autonomous Region of China is the species which is most closely related to the cultivated apple, M. domesticacv. `Golden Delicious'. The phylogenetic relationships among the species studied are discussed.     

Bacterial diversity in the rhizosphere of two phylogenetically closely related plant species across environmental gradients

Purpose

Rhizosphere soil bacterial communities are crucial to plant growth, health, and stress resistance. In order to detect how bacterial communities associated with the rhizosphere of phylogenetically related plant species vary in terms of composition, function, and diversity, we investigated the rhizosphere bacterial community structure of two perennial shrub species, Caragana jubata and Caragana roborovskyi, under natural field conditions in northwest China and analyzed the influence of soil properties and environmental factors.

Materials and methods

Eighteen root samples, eight for C. jubata, and ten for C. roborovskyi, along with any adherent soil particles, were collected from multiple sites in northwest China. The rhizosphere soil was washed from the roots, and bacterial communities were analyzed using Illumina MiSeq sequencing of 16S rRNA gene amplicons. Then, α-diversity and β-diversity were calculated using QIIME.

Results and discussion

Across species, Proteobacteria (29 %), Actinobacteria (15 %), Chloroflexi (10 %), Acidobacteria (10 %), Bacteroidetes (8 %), Firmicutes (8 %), Planctomycetes (7 %), Gemmatimonadetes (4 %), and Verrucomicrobia (3 %) were the most abundant phyla in the rhizosphere of C. jubata and C. roborovskyi. However, principal co-ordinates analysis indicated strong interspecific patterns of bacterial rhizosphere communities. Further, the richness of Proteobacteria, Acidobacteria, Bacteroidetes, Verrucomicrobia, Firmicutes, and Nitrospirae was significantly higher in the rhizosphere of C. jubata compared with C. roborovskyi, while the opposite was found for Actinobacteria and Cyanobacteria. However, the Shannon index showed no significant difference in α-diversity between C. jubata and C. roborovskyi. Distance-based redundancy analysis indicated that soil properties and environmental factors exerted strong influences on the structure of the rhizosphere bacterial community and explained 47 and 46 % of community variances between samples, respectively.

Conclusions

Our results showed strong interspecific clustering of the bacterial rhizosphere communities of C. roborovskyi and C. jubata. Altitude explained most of the variation in the composition of bacterial rhizosphere communities of C. roborovskyi and C. jubata, followed by soil pH, water content, organic matter content, total nitrogen content, and mean annual rainfall.

     

Characterization of cider apples on the basis of their fatty acid profiles

In the current study, the fatty acids composition of 30 monovarietal apple juices from six cider apple varieties belonging to two categories was analyzed. The different apple juices were obtained from three consecutive harvests (1997, 1998, and 1999). The fatty acids concentration in apple juice together with chemometric techniques such as principal components analysis (PCA), soft independent modeling of class analogy (SIMCA), and linear discriminant analysis (LDA), allowed us to differentiate apple juices on the basis of the sweet or sharp category to which the cider apple variety belongs. Fatty acids such as the unsaturated oleic and linoleic acids, and saturated caprylic, capric, stearic, and palmitic acids were related to the sweet cider apple category, while pentadecanoic acid is related to the sharp class.     

Use of fatty acid profiles to identify food-borne bacterial pathogens and aerobic endospore-forming bacilli

Capillary gas chromatography (GC) with flame ionization detection was used to determine the cellular fatty acid profiles of various food-borne microbial pathogens and to compare the fatty acid profiles of spores and vegetative cells of the same endospore-forming bacilli. Fifteen bacteria, representing eight genera (Staphylococcus, Listeria, Bacillus, Yersinia, Salmonella, Shigella, Escherichia, and Vibrio) and 11 species were used to compare the extracted fatty acid methyl esters (FAMEs). Endospore-forming bacilli were processed to obtain pure spores and whole cell FAMEs for GC analysis. A data set for each bacterial agent was prepared using fatty acid profiles from five replicates prepared on different days. The results showed that these fatty acid intensity profiles were unique for each of the 11 species and that they could be used as a fingerprint for the organisms. The cellular fatty acid profiles for Bacillus anthracis and Bacillus cereus show that there are two branched chain fatty acids, iso 17:1 omega10c and 17:1 anteiso, which are unique in these species. Iso 17:1 omega10c is present in B. cereus vegetative cells and spores but is not observed in B. anthracis. The 17:1 anteiso fatty acid is present in B. anthracis cells but not in B. cereus cells. Fatty acids 16:0 2OH and 17:0 iso 3OH are present in B. anthracis and B. cereus spores but not in the vegetative cells. In summary, analysis of FAMEs from bacteria and spores can provide a sensitive procedure for the identification of food-borne pathogens.     

Effect of added caffeic acid and tyrosol on the fatty acid and volatile profiles of camellia oil following heating

Camellia oil is widely used in some parts of the world partly because of its high oxidative stability. The effect of heating a refined camellia oil for 1 h at 120 degrees C or 2 h at 170 degrees C with exogenous antioxidant, namely, caffeic acid and tyrosol, was studied. Parameters used to assess the effect of heating were peroxide and K values, volatile formation, and fatty acid profile. Of these, volatile formation was the most sensitive index of change as seen in the number of volatiles and the total area count of volatiles in gas chromatograms. Hexanal was generally the dominant volatile in treated and untreated samples with a concentration of 2.13 and 5.34 mg kg(-1) in untreated oils heated at 120 and 170 degrees C, respectively. The hexanal content was significantly reduced in heated oils to which tyrosol and/or caffeic acid had been added. Using volatile formation as an index of oxidation, tyrosol was the more effective antioxidant of these compounds. This is contradictory to generally accepted antioxidant structure-activity relationships. Changes in fatty acid profiles after heating for up to 24 h at 180 degrees C were not significant.     

Catabolic response and phospholipid fatty acid profiles as microbial tools to assess soil functioning

Microbial properties may help to provide an integrated view of changes in soil functioning associated with soil management or soil status. The fatty acid profiles of membrane phospholipids (PLFA) can give the composition of ecophysiological groups of soil microbial communities, while catabolic response profiles (CRP) estimate the heterotrophic functional diversity in soils, both relevant to the understanding of the role of micro‐organisms in the functioning of the soil. The objectives of this study were (i) to evaluate the CRP and PLFA as microbial tools to characterize changes in soil functioning and (ii) clarify the relation among these microbial measurements, with other physical, chemical and biochemical soil properties. We compare the same soil subjected to different managements and degrees of erosion. An undisturbed soil (UN), an old pasture soil (OP) and soils under continuous cultivation (NT) with four different depth of A horizon: 25 cm (NT 25), 23 cm (NT 23), 19 cm (NT 19) and 14 cm (NT 14) were tested. Substrate‐induced respiration of most substrates diminished when cropping pressure increased (UN > OP > NT), and soil catabolic evenness, as a diversity index, decreased by increasing production pressure and soil erosion. The correlation found among most of the measured physical, chemical and biochemical soil properties with the catabolic evenness showed the potential of this measurement to provide an integrated view of soil functioning. The PLFA analysis showed that the composition of microbial community denoting a partial recovery after 10 yr under grazed grassland. The stress indicators showed that farming practices increased microbial stress with the highest values found in the most eroded soils.     

Crop rotation and seasonal effects on fatty acid profiles of neutral and phospholipids extracted from no‐till agricultural soils

Analysis of phospholipids (PLFA) and neutral lipids fatty acids (NLFA) was used to characterize no‐till productive agricultural soils associated with different crop rotation levels, replicated across a 400 km transect in the Argentinean pampas, during two sampling seasons, summer and winter. High rotation (HR) management consisted in maize–wheat–soybean intense rotation including cover crops. Low rotation (LR) management trend to soybean monocultures. Soils from nearby natural environments (NEs) were used as references. Fatty acids concentration in soils (nmol/g) decreased c.a. 50% from summer to winter differentially according to soil treatment being the smallest decrease in HR management 35%. Both PLFA and NLFA profiles showed strong potential to discriminate between different land uses. In winter samples, some rare or unknown fatty acids were relevant for the discrimination of agricultural practices while NLFA 20:0 appears to be a good marker of HR soils despite season or location. The PLFA‐based taxonomic biomarkers for total bacteria, Gram‐negative bacteria and arbuscular mycorrhiza showed a significant trend NE>HR>LR in the winter sampling. HR management was also characterized by high levels of NLFA in winter samples as if high crop rotation improves lipids reserves in soil during winter more than in monocropping soil management. In conclusion, PLFA and particularly NLFA profiles appear to provide useful and complementary information to obtain a footprint of different soil use and managements, improving soil biochemistry characterization tools.     

Correlation studies on oil content and fatty acid profile of some Cruciferous species

Mean individual fatty acid concentration among differentspecies of the family Cruciferae are not similar because of the presence ofdifferent kind of significant relationship among the individual fatty acids.Brassica napus L., Sinapis alba L.,Raphanus caudatus L.f. Raphanus sativusL. and B. nigra (L.) Koch arecomparatively low erucic acid (<40%) containing species. Crambeabyssinica Hochst. ex. R. E. Fries collections gave as high as 60%erucic acid. Low erucic acid concentration was found associated with thepresence of high level of palmitic and oleic acid concentrations. Erucic acidconcentrations were found varied within the range of 42 to 57% among thecollections of Brassica rapa L. and B.carinata A. Braun. All the collections ofB. nigra (L.) Koch were found to havehigher proportion of linoleic acid than the corresponding oleic acidconcentration. Linolenic acid concentrations of B.nigra (L.) Koch was also higher in comparison to otherspecies of the Cruciferae. Raphanus collections were foundto contain higher levels of total saturated fatty acids as well as eicosenoicacid. Oil content of the total collections under present study did not show anysignificant relationship either with linolenic acid or with erucic acidconcentration. But significant positive relationship was observed between totaloil and erucic acid level within the collections of B.juncea Czern. & Coss. Similarly significant negativecorrelation was observed between total oil and linolenic acid content inBrassica rapa L. ssp. trilocularis (Roxb.) Hanelt, yellow sarson.     

不同肥力水平和利用历史的红壤磷脂脂肪酸图谱

Analysis of phospholipid fatty acids(PLFAs) was used to estimate the microbial community structures of eight Chinese red soils with different fertility levels and land use histories.The total amounts of PLFAs in the soils were significantly correltaed with soil organic carbon, total nitrogen,microbial biomass C and basal respiration,indicating that total PLFA was closely related to fertility and sustainbility in these highly weathered soils.Soils of the eroded wastelan were rich in Gram-positive species .When the eroded soils were planted with citrus trees,the soil microbial population had changed little in 4 years but took up to 8-12 yearss before it reached a significantly different population,Multivariate analysis of PLFAs demonstrated that land use history and plant cover type had a significant impact on microbial community structure.Howver,the difference of soil microbial community structure in the paddy field compared to other land uses was not larger than expected in this experiment.     

Comparison of fatty acid and polar lipid contents of tubers from two potato species, Solanum tuberosum and Solanum phureja

Total and individual fatty acid contents were determined in raw tubers of four genotypes from each of the two species Solanum phureja and S. tuberosum. The four S. phureja genotypes contained statistically significantly higher concentrations of total fatty acids, with their average value being 37% greater than that for the mean of the four S. tuberosum cultivars. In both species, a total of 17 fatty acids were detected in quantifiable amounts, and in all genotypes the predominant fatty acid was linoleic followed by alpha-linolenic and palmitic acids. Unusually, 15-methyl hexadecanoate was present as a minor acid in both species. Although a number of statistically significant differences in the fatty acid percentage compositions were found between and within the two species, these were generally small. Averaged over all species and genotypes, tuber storage resulted in an initial small but statistically significant increase in total fatty acid content, but prolonged storage resulted in a fall to the initial values detected close to harvest. The same trend was evident for S. phureja alone (for mean values of all genotypes), but for S. tuberosum the total fatty acid content remained constant over the whole storage period. For both species, the contents (both as absolute levels and as percent compositions) of linoleic acid decreased and alpha-linolenic acid increased in tubers over the whole storage period, and possible mechanisms are discussed. Also, the absolute levels of these two acids were greater in S. phureja than in S. tuberosum, and this is discussed in relation to the development of flavor-related compounds during cooking. The polar lipids of one representative of S. tuberosum and of S. phureja were qualitatively similar. There were only minor differences in the polar lipid percentage compositions and in the corresponding fatty acid compositions of the individual polar lipids between the two species, although the absolute levels of the total, and of some individual, polar lipids were higher in S. phureja.     

Changes in fatty acid composition with age and environment in different types of peat profiles in Japan

Fatty acid composition of the crude lipid fraction of peat was investigated using several typical peat profiles in Japan. Fatty acid composition varied with the peat layers accumulated in a peat profile since 32,000 years BP. Deposition of long-range transported volcanic ash tephras also affected the composition remarkably due to the acceleration of decomposition. Fatty acid composition differed among high moor, transitional moor, and low moor peat profiles sampled in several locations in Hokkaido, Japan. The difference in the plants involved in the formation of peat was considered to be a very important factor determining the fatty acid composition. For example, arachidic acid was considered to be a good indicator for the presence of reed, while the percentage of stearic acid tended to be high in the high moor peat profiles dominated by sphagnum. As the fatty acids with a longer chain length were more stable than those with a shorter chain length, the percentage of longer fatty acids (C24 and C26) tended to increase and that of shorter fatty acids (CI4 and CI6) to decrease in the lower peat profiles. The drying of peatland also exerted a similar effect on the fatty acid composition.     

Assessment of oil content and fatty acid composition variability in two economically important Hibiscus species

The Hibiscus genus encompasses more than 300 species, but kenaf (Hibiscus cannabinus L.) and roselle (Hibiscus sabdariffa L.) are the two most economically important species within the genus. Seeds from these two Hibiscus species contain a relatively high amount of oil with two unusual fatty acids: dihydrosterculic and vernolic acids. The fatty acid composition in the oil can directly affect oil quality and its utilization. However, the variability in oil content and fatty acid composition for these two species is unclear. For these two species, 329 available accessions were acquired from the USDA germplasm collection. Their oil content and fatty acid composition were determined by nuclear magnetic resonance (NMR) and gas chromatography (GC), respectively. Using NMR and GC analyses, we found that Hibiscus seeds on average contained 18% oil and seed oil was composed of six major fatty acids (each >1%) and seven minor fatty acids (each <1%). Hibiscus cannabinus seeds contained significantly higher amounts of oil (18.14%), palmitic (20.75%), oleic (28.91%), vernolic acids (VA, 4.16%), and significantly lower amounts of stearic (3.96%), linoleic (39.49%), and dihydrosterculic acids (DHSA, 1.08%) than H. sabdariffa seeds (17.35%, 18.52%, 25.16%, 3.52%, 4.31%, 44.72%, and 1.57%, respectively). For edible oils, a higher oleic/linoleic (O/L) ratio and lower level of DHSA are preferred, and for industrial oils a high level of VA is preferred. Our results indicate that seeds from H. cannabinus may be of higher quality than H. sabdariffa seeds for these reasons. Significant variability in oil content and major fatty acids was also detected within both species. The variability in oil content and fatty acid composition revealed from this study will be useful for exploring seed utilization and developing new cultivars in these Hibiscus species.     

Differentiation of several geographical origins in single-strength Valencia orange juices using quantitative comparison of carotenoid profiles.

Pure Valencia orange (Citrus sinensis) juices (64 samples) from Spain, Israel, Belize, Cuba, and Florida, harvested during two seasons (1996-1997 and 1997-1998), were analyzed for their carotenoid profiles. The detection of saponified carotenoid pigments has been achieved and quantitated using a photodiode array detection monitored at 350, 430, and 486 nm. Carotenoid pigments commonly found in the orange variety Valencia have been separated on a polymeric C-30 column using a ternary gradient as eluent. Pure Valencia juices from oranges grown in Mediterranean regions (Israel and Spain) have a high carotenoid content, expressed in beta-carotene (5-18 and 14-35 mg L(-)(1), respectively), compared to those grown in tropical and subtropical regions (Cuba, Belize, and Florida) (4-10, 2-8, and 5-10 mg L(-)(1), respectively). Quantitative results allowed the differentiation of Valencia variety geographical origins, in particular, the Mediterranean area from tropical and subtropical areas, using multidimensional analyses of carotenoid contents.     

Differentiation of species from the Penicillium roqueforti group by volatile metabolite profiling

Species from the Penicillium roqueforti group were differentiated by volatile metabolite profiling primarily of sesquiterpenes. A total of 24 isolates from species P. roqueforti, Penicillium carneum, and the recently described species Penicillium paneum were inoculated on yeast extract sucrose agar. Volatile metabolites were collected by diffusive sampling onto tubes containing Tenax TA, overnight between the fifth and sixth days of incubation. Volatiles were thermally desorbed and analyzed by gas chromatography coupled to mass spectrometry. The sesquiterpene area of the chromatogram was investigated, and potential sesquiterpenes were tabulated by comparison of their Kovats retention index and mass spectrum. In general, P. carneum isolates produced the lowest number of sesquiterpenes, all of which were unique for P. carneum within the P. roqueforti group. P. roqueforti and P. paneum produced a larger variety of volatile metabolites, some of which they have in common and some of which are unique for the two species. (+)-Aristolochene was found in samples from P. paneum and P. roqueforti. Other Penicillium species in which (+)-aristolochene was also detected were P. commune, P. glandicola, and P. solitum.     

Changes in the fatty acid profiles through the digestive tract of the earthworm Lumbricus terrestris L.

The gut of many soil arthropods contains a complex and mutualistic microbial community that usually assists the host with digestion. The same is probably true for earthworms, but the nature and function of the microbiota inhabiting their gut are virtually unknown. In this paper, we studied the microbial community in the gut content of the earthworm Lumbricus terrestris L. and in the bulk soil by assessing their fatty acid (FA) profiles. Our results indicated that the total FA concentration in the earthworm gut was about two orders of magnitude greater than in bulk soil, with higher concentration of bacteria (up to 500-fold), fungal and metazoan-derived FAs. Several FAs appearing in the gut were not present in bulk soil. PCA analysis revealed that the microbial community in the gut was different from that in the bulk soil, and that significant changes occurred between midgut, hindgut and proctodeum. Cluster analysis of bacterial and fungal-derived FA profiles grouped the bulk soil samples apart from the gut samples, where the hindgut profiles were more closely related to those from the proctodeum than those from the midgut. We showed important changes in the FA concentration and composition occurring at very small spatial scales inside the gut of the earthworm L. terrestris. These results have implications for understanding earthworm digestion, and they suggest that the microbial community in the earthworm gut is not a casual combination of microorganisms already present in the soil. Further study is needed to determine how these gut microbial communities are involved in earthworm digestion processes.