Beneficial Effect of Verminephrobacter Nephridial Symbionts on the Fitness of the Earthworm Aporrectodea tuberculata

Almost all lumbricid earthworms (Oligochaeta: Lumbricidae) harbor species-specific Verminephrobacter (Betaproteobacteria) symbionts in their nephridia (excretory organs). The function of the symbiosis, and whether the symbionts have a beneficial effect on their earthworm host, is unknown; however, the symbionts have been hypothesized to enhance nitrogen retention in earthworms. The effect of Verminephrobacter on the life history traits of the earthworm Aporrectodea tuberculata (Eisen) was investigated by comparing the growth, development, and fecundity of worms with and without symbionts given high (cow dung)- and low (straw)-nutrient diets. There were no differences in worm growth or the number of cocoons produced by symbiotic and aposymbiotic worms. Worms with Verminephrobacter symbionts reached sexual maturity earlier and had higher cocoon hatching success than worms cured of their symbionts when grown on the low-nutrient diet. Thus, Verminephrobacter nephridial symbionts do have a beneficial effect on their earthworm host. Cocoons with and without symbionts did not significantly differ in total organic carbon, total nitrogen, or total hydrolyzable amino acid content, which strongly questions the hypothesized role of the symbionts in nitrogen recycling for the host.Symbiosis has long been recognized as a source of evolutionary innovation (24), and the acquisition of symbionts can enable animal hosts to exploit previously inaccessible niches (3). The phylum Annelida is no exception to this; chemosynthetic symbionts in marine annelids (e.g., the giant tubeworm Riftia sp. and other gutless marine oligochaetes [9]) gain energy from the oxidation of reduced sulfur compounds and fix CO₂ and supply their animal host with fixed carbon. A more obscure partnership is known from the bone-eating annelid Osedax sp., where endosymbionts help degrade the bones of whale carcasses, the only known habitat of the worms (33). The medicinal leech Hirudo sp., like other blood-feeding animals (3, 7), has symbionts that are thought to produce essential vitamins missing from a blood meal (13). In addition, leeches have a number of symbionts of unknown function in their nephridia (excretory organs) (18). Earthworms (Oligochaeta: Lumbricidae) have also long been known to harbor symbiotic bacteria in their nephridia (19, 36). The function of this symbiosis, however, is still not known, but the stability of the symbiosis over evolutionary time (23) suggests that the symbionts benefit the host.The earthworm symbionts reside in the nephridia and have therefore been proposed to be involved in internal recycling of nitrogen in the host (29). The earthworm nephridia play an important role in both nitrogenous waste excretion and osmoregulation (20). The nephridia are found in pairs in each segment of the worm and consist of a coiled tube leading from the coelom to the exterior (Fig. ​(Fig.1).1). The tube forms three loops, and the symbiotic bacteria are situated in the ampulla in the second loop, where they form a dense population lining the lumen wall (19, 36).Open in a separate windowFIG. 1.The nephridia are found as paired organs in each segment of the worm. The nephridostome (the inlet to the nephridia) protrudes into the previous segment. The nephridial tube forms three loops and finally empties out through the body wall via the nephridopore. The symbionts (black) reside in the ampulla in the second loop. (Modified from reference 23 with permission.)The symbionts form the monophyletic genus Verminephrobacter (Betaproteobacteria) (30, 36); they are species specific and present in almost all lumbricid earthworms (23). The Verminephrobacter symbionts are transmitted vertically via the cocoon, where they are deposited along with eggs and sperm (5). During embryogenesis, the symbionts migrate into the developing nephridia, and after the worm hatches, the symbionts can no longer infect (5, 6). By taking advantage of the vertical transmission mode, it has been possible to establish symbiont-free earthworm cultures in the laboratory through controlled antibiotic treatment of newly deposited cocoons (5; this study). Separation of the symbiotic partners allows studies of the effect of the symbionts on their earthworm hosts.Pandazis (29) hypothesized that the symbionts enhance earthworm nitrogen retention by excreting proteolytic enzymes that will degrade peptides and proteins lost in urine; this would allow the earthworm to reabsorb the resulting amino acids. As a consequence, earthworms cured of their symbionts should have a lower fitness level than control worms when grown under nitrogen-limiting conditions. To test this hypothesis, growth, development, fecundity, and cocoon hatching success were compared for symbiotic and aposymbiotic earthworms of the species Aporrectodea tuberculata (Eisen) under high and low nutrient availability conditions.     

Development and Validation of a Continuous In Vitro System Reproducing Some Biotic and Abiotic Factors of the Veal Calf Intestine

Following the January 2006 European ban of antibiotics used as growth promoters in the veal calf industry, new feed additives are needed in order to maintain animal health and growth performance. As an alternative to in vivo experiments in the testing of such additives, an in vitro system modeling the intestinal ecosystem of the veal calf was developed. Stabilization of the main cultured microbial groups and their metabolic activity were tracked in an in vitro continuous fermentor operated under anaerobiosis, at pH 6.5, and at a temperature of 38.5°C and supplied with one of three different nutritive media (M1, M2, or M3). These media mainly differed in their concentrations of simple and complex carbohydrates and in their lipid sources. In vitro microbial levels and fermentative metabolite concentrations were compared to in vivo data, and the biochemical composition of the nutritive media was compared to that of the veal calf intestinal content. All three nutritive media were able to stabilize anaerobic and facultative anaerobic microflora, lactate-utilizing bacteria, bifidobacteria, lactobacilli, enterococci, and Bacteroides fragilis group bacteria at levels close to in vivo values. The microbiota was metabolically active, with high concentrations of lactate, ammonia, and short-chain fatty acids found in the fermentative medium. Comparison with in vivo data indicated that M3 outperformed M1 and M2 in simulating the conditions encountered in the veal calf intestine. This in vitro system would be useful in the prescreening of new feed additives by studying their effect on the intestinal microbiota levels and fermentative metabolite production.European regulations introduced in January 2006 banned the use of antibiotics as growth promoters (AGP) at subtherapeutic levels in animal feed (regulation EC 1831/2003), particularly for veal calves. AGP generated significantly enhanced growth performance via complex processes. The mechanism of growth promotion is still speculative, but many studies suggest the involvement of the intestinal microbiota (7, 9). First of all, AGP did not promote the growth of germfree animals (6). Moreover, they strongly inhibited the bacterial catabolism of urea and amino acids and the fermentation of carbohydrates both in vitro and in vivo (10, 28, 35). AGP treatment thus provided the animal with higher nutrient availability and led to a decrease in the toxic metabolites produced by bacteria, like ammonia or amines, limiting the energy needed by the animal to detoxify the organism. Some authors also argue that another beneficial effect of AGP results from improved control of intestinal pathologies, such as necrotic enteritis in poultry (12). The January 2006 ban is thus expected to have an impact on veal calf health by leading to more frequent digestive disorders, as previously observed in pigs and poultry in the Nordic countries (36), where AGP have been totally prohibited since the 1990s. Even though no scientific study has yet been done on calves, there have already been reports of higher death rates on experimental commercial farms subsequent to the withdrawal of AGP. The main digestive diseases leading to veal calf deaths are enteritis and enterotoxemia, which are mainly triggered by pathogenic strains of Escherichia coli and Clostridium perfringens (22, 30).Veal calf producers are looking for new feed additives to allay the consequences of the AGP ban. Alternative approaches include the use of prebiotics, probiotics, or plant extracts. Several studies have reported both consistent improvements in weight gain and feed conversion and a reduction of the incidence of diarrhea with the addition of such additives to the veal calf diet (1, 11, 14). One of the hypotheses used to explain these beneficial effects involves the modulation of the intestinal microbiota. In particular, oligosaccharides containing mannose or fructose are known to selectively increase the growth of beneficial intestinal bacteria, including lactobacilli and bifidobacteria (21). Timmerman et al. (33) showed that a calf-specific probiotic containing six Lactobacillus species reduced the fecal counts of E. coli. Green tea extracts also improved the intestinal microbial balance by maintaining high fecal levels of Bifidobacterium and Lactobacillus spp. and decreasing those of C. perfringens (16).As indicated above, it is important to assess the action of newly developed feed additives on the veal calf intestinal microbiota. High interindividual variability makes it difficult and expensive to carry out in vivo studies. Alternatively, experiments can be conducted via in vitro systems modeling the intestinal environment of the animals, provided the model has been checked as pertinent. This approach should allow an economical and ethical way to prescreen feed additives by studying their effects on the intestinal microbiota cultured in the in vitro system and its metabolic activity. With this objective in mind, a necessary requirement is knowledge of the veal calf intestinal ecosystem. Thus, the bacterial and biochemical composition of the jejunoileal chyme of calves was previously characterized (13).The aims of the present study were (i) to set up an in vitro system where the main cultured microbial groups identified in the veal calf intestinal chyme are reproducibly stabilized and metabolically active and (ii) to validate our model by comparing the in vitro and in vivo levels of selected biotic and abiotic variables.     

Diversity and aboveground biomass in three tropical forest types in the Dja Biosphere Reserve,Cameroon

We present tree community diversity, species composition, basal area and aboveground biomass of three forest types in the Dja Biosphere Reserve, in South‐East Cameroon, part of the contiguous tropical forest of the Congo Basin. A total of fourteen, 1 ha, plots were established in heterogeneous terra firme forests (TFF), Gilbertiodendron dewevrei forests (GDF) and periodically flooded forests (PFF). A total of 281 tree species with diameter ≥10 cm were recorded. The Shannon diversity index was significantly higher in TFF (5.7 ± 0.28) and PFF (5.6 ± 0.23) than in GDF (2.29 ± 0.48) (ANOVA, F_2,11 = 139.75, P < 0.001). While tree density did not differ between forest types (F_2,11 = 3.50, P = 0.06), basal area differed significantly (F_2,11 = 7.38, P = 0.009), as did aboveground biomass (F_2,11 = 17.95, P < 0.001). Mean AGB values were respectively, 596.1 ± 62.24, 401.67 ± 58.06 and 383.14 ± 61.91 Mg ha^?1 in GDF, TFF and PFF. Variation in the abundance of trees with large diameter was the main reason for these differences. Few dominant species made the greatest contribution to the AGB. G. dewevrei, accounted for 83% of AGB in GDF, Penthaclethra macrophylla for 9.9% in TFF and Uapaca heudolotii for 10.6% in PFF. The importance of preserving G. dewevrei forest in the context of ‘Reducing Emissions from Deforestation and forest Degradation’ (REDD) policies is discussed.     

Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates

Fungi belonging to the Fusarium solani Species Complex (FSSC) are well known plant pathogens. In addition to being the causative agent of some superficial infections, FSSC has recently emerged as a group of common opportunistic moulds, mainly in patients with haematological malignancies. Molecular typing methods are essential in order to better understand the epidemiology of such opportunistic agents with the final goal of preventing contamination. A three-locus typing scheme has thus been developed for FSSC; based on polymorphisms in the domains of the ITS, EF-1 alpha, and RPB2 genes. This method is now considered to be a useful reference for phylogenetic and taxonomic studies. In other significant clinical fungi (e.g., Candida sp., Cryptococcus neoformans, and Aspergillus fumigatus), genes coding for metabolic enzymes have been widely used and proven to be very informative for diagnosis and epidemiology. The contribution of these genes has never been evaluated for Fusarium sp. and more specifically for F. solani Species Complex.Here, we have evaluated the contribution of 25 genes for diagnosis and epidemiological purposes. We then report a new five-locus MLST scheme useful for diagnosis and typing of clinical FSSC isolates. The method has been validated on 51 epidemiologically unrelated strains of FSSC and presents a high power of discrimination calculated at 0.991.     

Phylogenetic affiliation of the desert truffles <Emphasis Type="Italic">Picoa juniperi</Emphasis> and <Emphasis Type="Italic">Picoa lefebvrei</Emphasis>

The molecular phylogeny and comparative morphological studies reported here provide evidence for the recognition of the genus Picoa, an hypogeous desert truffle, in the family Pyronemataceae (Ascomycota, Pezizales). Picoa juniperi and Picoa lefebvrei were reassigned to the genus Picoa based on large subunit (LSU) sequence (28S) rDNA and internal transcribed spacer (ITS) rDNA (including the partial 18S, ITS1, ITS2, 5.8S gene, and partial 28S of the nuclear rDNA) data. Morphological studies of spores, asci, perida, and gleba revealed high similarities between P. lefebvrei and P. juniperi, thereby confirming the membership of both species in the genus Picoa. These two species were primarily distinguishable based on ascospore ornamentation.     

Altered SK3/KCa2.3-mediated migration in adenomatous polyposis coli (Apc) mutated mouse colon epithelial cells

Lost of adenomatous polyposis coli gene (Apc) disturbs the migration of intestinal epithelial cells but the mechanisms have not been fully characterized. Since we have demonstrated that SK3/KCa2.3 channel promotes cancer cell migration, we hypothesized that Apc mutation may affect SK3/KCa2.3 channel-mediated colon epithelial cell motility. We report evidence that SK3/KCa2.3 channel promotes colon epithelial cells motility. Following Apc mutation SK3/KCa2.3 expression is largely reduced leading to a suppression of the SK3/KCa2.3 channel mediated-cell migration. Our findings reveal a previously unknown function of the SK3/KCa2.3 channel in epithelial colonic cells, and suggest that Apc is a powerful regulator SK3/KCa2.3 channel.     

Characterization of oil and starch accumulation in tubers of Cyperus esculentus var. sativus (Cyperaceae): A novel model system to study oil reserves in nonseed tissues

? Premise of the study: Storage oil (triacylglycerol) accumulates in tissues such as the embryo and endosperm of seeds and the fruit mesocarp, but seldom in underground organs. As a rare exception, cultivated variants of yellow nutsedge (Cyperus esculentus) contain high amounts of both oil and starch in the mature tubers. ? Methods: Biochemical analyses and light and electron microscopy were used to study the accumulation patterns of storage nutrients in developing nutsedge tubers. ? Key results: During the initial phase of tuber development, the conducting rhizome tissue is transformed into a storage compartment, then massive storage reserves accumulate in the tuber. At the beginning of tuber development, a large sugar load coincided with the onset of starch accumulation. Oil accumulation started later, concomitant with a substantial drop in the sugar content. Initially, oil accumulated at a lower rate compared to starch, but the rate later increased; after 6 wk, oil made up 24% of tuber dry mass, while starch made up 32%. Protein concentration changed only a small amount throughout this development. Oil and starch accumulated in the same cells throughout the tubers in a sequential fashion during tuber development. ? Conclusions: The developmental pattern in the build up of storage nutrients in the tubers highlights nutsedge as a novel model plant, having potential to significantly widen our understanding on how synthesis of storage reserves, and in particular oils, is regulated and directed in nonseed tissues such as tubers and roots.