Fungal diversity in larval diets of Meliponainterrupta: Impacts on queen development andsurvival

BackgroundTo analyze the effects of different cultivation patterns on the structure and diversity of the microbial community in the rhizosphere soil of Trichosanthes kirilowii (T. kirilowii) arms to establish reasonable and effective strategies to mitigate the continuous cropping barriers and promote the high-quality cultivation of T. kirilowii.MethodsThree distinct cultivation patterns were investigated: open-field cultivation (TM1), film-mulched cultivation (TM2), and soybean intercropping cultivation (TM3). High-throughput sequencing and bioinformatic analyses were employed to evaluate the rhizosphere microbiome, and redundancy analysis was utilized to investigate the relationship between the microbial communities and soil nutrient indicators.ResultsTM2 and TM3 increased soil bacterial community diversity, reduced fungal community diversity, elevated the relative abundance of beneficial bacterial genera, and reduced the abundance of detrimental fungal genera in the rhizosphere soil. The relative abundance of Pseudarthrobacter, unclassified_Steroidobacteraceae, and Nocardioides in TM2 and TM3 was markedly higher than in TM1. Conversely, the relative abundance of Fusarium, Rhizoctonia, Ceratobasidium, and Plectosphaerella in TM2 and TM3 was significantly reduced compared to TM1. The contents of available potassium (AK), total nitrogen (TN), total phosphorus (TP), and pH in the rhizosphere soil of TM2 and TM3 were significantly higher than those in TM1. The distribution of soil bacterial genera was significantly influenced by the contents of TN and AK, while the distribution of soil fungal genera was significantly or extremely significantly impacted by the contents of TP, total potassium (TK), soil organic matter (SOM), and pH. The content of AK was extremely significantly positively correlated with the relative abundance of Nocardioides, whereas the content of TK showed an extremely negative correlation with the relative abundance of Ceratobasidium. Similarly, pH demonstrated an extremely negative correlation with the relative abundance of Rhizoctonia and Ceratobasidium.ConclusionsFilm-mulched cultivation and soybean intercropping cultivation altered the soil nutrients, as well as the structure and diversity of soil microbial communities. Thus, in agricultural production, film-mulched cultivation and soybean intercropping cultivation can serve to regulate soil nutrients and microbial communities, thereby mitigating the barriers of continuous cropping of T. kirilowii.

Fungal diversity in the soil may be limited under natural conditions by inappropriate environmental factors such as: nutrient resources, biotic and abiotic factors, tillage system and microbial interactions that prevent the occurrence or survival of the species in the environment. The aim of this paper was to determine fungal genetic diversity and community level physiological profiling of microbial communities in the soil under long-term maize monoculture. The experimental scheme involved four cultivation techniques: direct sowing (DS), reduced tillage (RT), full tillage (FT), and crop rotation (CR). Soil samples were taken in two stages: before sowing of maize (DSBS-direct sowing, RTBS-reduced tillage, FTBS-full tillage, CRBS-crop rotation) and the flowering stage of maize growth (DSF-direct sowing, RTF-reduced tillage, FTF-full tillage, CRF-crop rotation). The following plants were used in the crop rotation: spring barley, winter wheat and maize. The study included fungal genetic diversity assessment by ITS-1 next generation sequencing (NGS) analyses as well as the characterization of the catabolic potential of microbial communities (Biolog EcoPlates) in the soil under long-term monoculture of maize using different cultivation techniques. The results obtained from the ITS-1 NGS technique enabled to classify and correlate the fungi species or genus to the soil metabolome. The research methods used in this paper have contributed to a better understanding of genetic diversity and composition of the population of fungi in the soil under the influence of the changes that have occurred in the soil under long-term maize cultivation. In all cultivation techniques, the season had a great influence on the fungal genetic structure in the soil. Significant differences were found on the family level (P = 0.032, F = 3.895), genus level (P = 0.026, F = 3.313) and on the species level (P = 0.033, F = 2.718). This study has shown that: (1) fungal diversity was changed under the influence different cultivation techniques; (2) techniques of maize cultivation and season were an important factors that can influence the biochemical activity of soil. Maize cultivated in direct sowing did not cause negative changes in the fungal structure, even making it more stable during seasonal changes; (3) full tillage and crop rotation may change fungal community and soil function.

Evidence suggests that there is a rich and diverse microbial community in the semen of mammals, which may be important in reproductive health and fertility. However, the composition of ram seminal microbiota remains under-characterized, with factors shaping it still largely unknown. The objectives of this study were to; 1) characterize the seminal microbiota of mature rams and their ram lambs using 16S rRNA gene sequencing; 2) evaluate whether managing the mature rams on divergent planes of nutrition can influence their seminal microbiota and that of their male offspring; and 3) compare the seminal microbiota between mature rams and ram lamb cohorts to identify age-related microbiota characteristics. For this, mature rams (n = 24) were assigned to one of the 3 nutritional planes: 1) Positive (POS), to gain 12% of initial body weight (BW) (n = 8), 2) maintenance (MAINT), to maintain BW (n = 8), and 3) negative (NEG), to lose 12% BW (n = 8) over an 84-d period. Semen samples were collected from the mature rams (F0) after 28-d, 56-d, and 84-d from the start of the trial. Following the 84-d period, the 24 rams were used to breed 240 mature ewes over 28-d. After lambing, the ram lambs (F1) sired by POS, MAINT, and NEG rams were maintained on the same diet until 11 months of age, at which semen samples were collected. Genomic DNA was extracted from the semen, and the microbiota was analyzed using 16S rRNA gene (V3-V4) sequencing. Overall, there was a relatively diverse and dynamic bacterial microbial community in the ram semen, mainly dominated by Actinobacteriota, Bacillota, Bacteroidota, and Proteobacteria phyla. The predominant genera identified included Fastidiosipila, Corynebacterium, Trueperella, Arthrobacter, Dietzia, and Bifidobacterium. The seminal microbial community structure, composition, and alpha diversity of F0 rams was influenced by diet during the first 28-d, but these diet influences later diminished. The paternal plane of nutrition did not influence the seminal microbiota of offspring ram lambs. The mature rams and ram lambs had distinct seminal microbiota, with young rams showing greater microbial richness and diversity (P < 0.005). Our results suggest that there is a relatively diverse and dynamic microbial community present in the semen of both mature rams and ram lambs, and that this microbiota is transiently influenced by diet and age. Managing rams on divergent planes of nutrition may not affect their offspring's seminal microbiota.

Meliponiculture is a traditional activity in northern and northeastern Brazil, where it represents a complementary source of income (Ueira-Vieira et al. 2013). Indigenous Brazilian people have domesticated some species of stingless bees, such as the Melipona genus, due to their heartiness and honey production. Rational rearing for commercialization of the Melipona scutellaris bee is common in northeastern Brazil. Their hive structure is different from honeybee mainly for lower number of workers than Apis mellifera . The honey and pollen are stored in a pot. The larval food is provisioned by workers, and after the queen lays eggs, the cell is totally closed by the worker. Therefore, there is no contact between the workers and larvae like in the honeybee. The caste determination mechanism in the Melipona genus is based on the interaction between environmental and genetic components, which is different from other bees (Kerr and Nielsen 1966). Recently, our research group has received a large amount of information from beekeepers about a decrease in the stingless bee population as well as an increase in the number of dead stingless bees. Therefore, we performed reverse transcription polymerase chain reaction (RT-PCR) screening of seven viruses in M. scutellaris sampled from two meliponaries in Brazil. Workers of M. scutellaris were collected from the meliponary at the Federal University of Uberlandia, Minas Gerais State (S 180 55′/W 450 17′) (n=10 hives) and a meliponary at Igarassu City, Pernambuco State (07° 50′ 03′′ S 34° 54′ 23′′W) (n=5 hives). Only forager bees were used for molecular analysis, and they were collected after arriving back to the hive. The total RNAwas extracted from five stingless bees per hive using the TRIzol reagent (Invitrogen), according to manufacturer’s recommendations. The total RNA was treated with DNAse enzyme (Promega) and used for reverse transcription using M-MLV reverse transcriptase (USB), according to the manufacturer’s recommendations. The rp49 gene was used to test the quality of complementary DNA (cDNA). Seven RNA viruses were analyzed using specific primers synthesized for each type of virus (see supplementary material, Table S1). DNA amplification of the different viruses was performed using Taq polymerase Platinum (Invitrogen) according to the manufacturer’s instructions. For in silico analysis, the CAP3 sequence assembly software was used to form a contig among sequencing repetitions (three replicate sequences for each amplicon). Phylogenetic analysis was performed using the Maximum Parsimony Method Software Mega 6 with the statistical analysis based on 1000 bootstrap replications. After observing stingless bee death in our meliponary (Minas Gerais State in Brazil), we analyzed whether viruses were present inM. scutellaris stingless bees. Among the seven viruses screened by RT-PCR, positive results were only obtained for the acute bee paralysis virus (ABPV), and surprisingly, the infection was present in all 10 of the hives tested (Figure 1). The hives were observed weekly and the population was Electronic supplementary material The online version of this article (doi:10.1007/s13592-015-0353-2) contains supplementary material, which is available to authorized users.

Understanding the extent of heterogeneity in soil microbial community structure and function at different scales within vegetation communities is critical to designing appropriate sampling protocols. Environmental factors (e.g. disturbance) make sampling in the riparian zone particularly challenging as vegetation communities are highly heterogeneous. To assess whether heterogeneity in soil and vegetation factors is reflected in microbial communities, a study was conducted in a riparian area in southern Australia. Nine quadrats were established encompassing different environmental conditions. Within quadrats physical, chemical and biological soil properties were analysed at two depths (top-soil = 0–10 cm and sub-soil = 20–30 cm), and floristic composition of ground cover, sub-canopy and canopy vegetation assessed. Soil biological analyses included microbial community composition (genetic analysis using ITS and 16S regions), and function (microbial metabolic activity using EcoPlates). Variation in soil microbial communities (fungi, bacteria, archaea) was related to differences in vegetation factors, particularly sub-canopy, and to a lesser extent, soil chemical properties. Relationships between variation in microbial communities and vegetation composition were stronger in top-soil than sub-soil. These observations were consistent for fungal communities excluding the phylum Glomeromycota, where the relationship was stronger with ground cover and only for top-soil. Variation in soil microbial community function was not related to variation in microbial community composition, soil physicochemical properties or vegetation factors. Our findings suggest there is little variation in the composition of soil microbial communities within areas with similar vegetation, and a small sampling effort would be needed to adequately describe the characteristics of such soil communities.

Stingless bees are a diverse group with a relevant role in pollinating native species. Its diet is rich in carbohydrates and proteins, by collecting pollen and nectar supplies the development of its offspring. Fermentation of these products is associated with microorganisms in the colony. However, the composition of microorganisms that comprise this microbiome and its fundamental role in colony development is still unclear. To characterize the colonizing microorganisms of larval food in the brood cells of stingless bees Frieseomelitta varia, Melipona quadrifasciata, Melipona scutellaris, and Tetragonisca angustula, we have utilized molecular and culture-based techniques. Bacteria of the phyla Firmicutes, Proteobacteria, Actinobacteria, and fungi of the phyla Ascomycota, Basidiomycota, Mucoromycota, and Mortierellomycota were found. Diversity analysis showed that F. varia had a greater diversity of bacteria in its microbiota, and T. angustula had a greater diversity of fungi. The isolation technique allowed the identification of 189 bacteria and 75 fungi. In summary, this research showed bacteria and fungi associated with the species F. varia, M. quadrifasciata, M. scutellaris, and T. angustula, which may play an essential role in the survival of these organisms. Besides that, a biobank with bacteria and fungus isolates from LF of Brazilian stingless bees was created, which can be used for different studies and the prospection of biotechnology compounds.

Soil microorganisms are greatly affected by their microenvironment. To reveal the influence of different land use patterns on the composition and diversity of soil bacterial and fungal communities, this study analyzed microbial (bacteria and fungi) community composition and diversity under different land use patterns (vegetable land, wasteland, woodland, cultivated land) based on 16S rRNA, 18S rRNA, and high-throughput sequencing method in the Taojia River Basin. Spearman analysis and redundancy analysis (RDA) were used to explore the correlation between soil physicochemical properties and soil fungal and bacterial community composition, and a partial least squares path model (PLS-PM) was constructed to express the causal relationship between soil physicochemical properties and soil bacterial and fungal community diversity. The results showed that the soil bacterial species richness was highest in vegetable land and the lowest in the wasteland. Proteobacteria is the dominant phylum (20.69%–32.70%), and Actinobacteria is the dominant class (7.99%–16.95%). The species richness of fungi in woodland was the highest, while was the lowest in cultivated land. The dominant phylum of fungi in vegetable land, woodland, and cultivated land is Mucoromycota, 29.39%, 41.36%, and 22.67%, respectively. Ascomycota (42.16%) is the dominant phylum in wasteland. Sordariomyetes of Ascomycota is the dominant class in wasteland and cultivated land. Mortierellomycetes and Glomeromycetes of Mucoromycota are the dominant class in vegetable land and woodland. The results of the Spearman analysis revealed that the dominant groups in the bacterial and fungal communities had significant correlations with soil pH, clay, and sand (p &lt; 0.01). The RDA results showed that soil clay, pH, and moisture were the key environmental factors affecting the diversity of soil microbial communities. Fungal diversity is more affected by different land use patterns than bacteria. These results provided a theoretical basis for the changes in soil microbial community composition and diversity in river basins.

Laboratory microcosm experiments were conducted to evaluate effects of bacteria isolated from senescent white oak leaves on the growth and survivorship of larval Aedes albopictus (Skuse). Larvae hatched from surface-sterilized eggs were reared in microcosms containing individual bacterial isolates, combined isolates (Porphyrobacter sp., Enterobacter asburiae, Acidiphilium rubrum, Pseudomonas syringae, and Azorhizobium caulinodans), a positive control containing a microbial community from an infusion of white oak leaves, and a negative control consisting of sterile culture media. Experiments were conducted for 21 d after which microcosms were deconstructed, larval survivorship was calculated, and bacteria contained in pupae, and adults that developed were quantified to determine rates of transstadial transmission. Positive control microcosms containing diverse microbial communities had an average (±SE) pupation rate of 89.3 (±5.8)% and average larval survivorship of 96.0 (± 2.3)%. Pupation in microcosms with bacterial isolates only occurred twice among all experimental replications; average larval survivorship ranged from 19 to 56%, depending on treatment. Larval growth was not found to be dependent on bacterial isolate density or isolate species, and larval survivorship was dependent on bacterial isolate density, not on isolate species. Potential mechanisms for failed development of larvae in microcosms with bacterial isolates are discussed. Bacterial isolates alone did not support larval development. High larval survivorship in positive control microcosms suggests that a diverse microbial community is required to complete larval development. Additional studies are needed to evaluate larval growth and survivorship on nonbacterial microbes, such as fungi and protozoa.

Queen production in stingless bees with fusion of neighboring brood cells occurs by the perforation of the adjacent brood cell or construction of an auxiliary one. This study describes the auxiliary brood cell building behavior in queenless colonies of Plebeia lucii. Queenright and queenless (orphan) colonies were monitored, and auxiliary cell construction was video-recorded in orphan colonies. Brood cells with auxiliary cells added were analyzed with X-rays to identify the amount of food and the larval behavior into the brood cells. Plebeia lucii had specific behavioral sequence in auxiliary cell building. The addition of auxiliary cells is the main strategy to produce queens in P. lucii, mainly for the production of emergency queens in orphan colonies because queen absence triggered a high production of auxiliary cells. X-ray analyses showed that auxiliary cell addition occurred when the food in the larval brood cells had been completely eaten and showed changes in dorsoventral position of the larvae. Larvae of males did not perforate auxiliary cells, indicating that sex-related factors affect this behavior. The wax handling by workers and the fused thin and concave-shaped wall between the auxiliary and larval brood cells seems to facilitate wall perforation by the larvae.

Andosols comprise one of the most important soil groups for agricultural activities in Japan because they cover about 46.5% of arable upland fields. In this soil group, available phosphorus (P) is accumulated by application of excessive fertilizer, but little is known about the influence of increasing P availability on microbial community diversity at large scales. We collected soil samples from 9 agro-geographical sites with Andosol soils across an available P gradient (2048.1-59.1 mg P2O5·kg(-1)) to examine the influence of P availability on the fungal community diversity. We used polymerase chain reaction - denaturing gradient gel electrophoresis to analyze the fungal communities based on 18S rRNA genes. Statistical analyses revealed a high negative correlation between available P and fungal diversity (H'). Fungal diversity across all sites exhibited a significant hump-shaped relationship with available P (R(2) = 0.38, P < 0.001). In addition, the composition of the fungal community was strongly correlated with the available P gradient. The ribotype F6, which was positively correlated with available P, was closely related to Mortierella. The results show that both the diversity and the composition of the fungal community were influenced by available P concentrations in Andosols, at a large scale. This represents an important step toward understanding the processes responsible for the maintenance of fungal diversity in Andosolic soils.

Variations in microbial community and di-(2-ethylhexyl) phthalate (DEHP) dissipation in different rhizospheric compartments between low- and high-DEHP accumulating cultivars of rice (Oryza sativa L.)

Relatively little is known about the microbial communities adapted to soil environments contaminated with aged complex hydrocarbon mixtures, especially in the subsurface soil layers. In this work we studied the microbial communities in two different soil profiles down to the depth of 7 m which originated from a 30-year-old site contaminated with petroleum hydrocarbons (PHCs) and from a clean site next to the contaminated site. The concentration of oxygen in the contaminated soil profile was strongly reduced in soil layers below 1 m depth but not in the clean soil profile. Total microbial biomass and community composition was analyzed by phospholipid fatty acid (PLFA) measurements. The diversity of fungi and actinobacteria was investigated more in detail by construction of rDNA-based clone libraries. The results revealed that there was a significant and diverse microbial community in subsoils at depth below 2 m, also in conditions where oxygen was limiting. The diversity of actinobacteria was different in the two soil profiles; the contaminated soil profile was dominated by Mycobacterium -related sequences whereas sequences from the clean soil samples were related to other, generally uncultured organisms, some of which may represent two new subclasses of actinobacteria. One dominating fungal sequence which matched with the ascomycotes Acremonium sp. and Paecilomyces sp. was identified both in clean and in contaminated soil profiles. Thus, although the relative amounts of fungi and actinobacteria in these microbial communities were highest in the upper soil layers, many representatives from these groups were found in hydrocarbon contaminated subsoils even under oxygen limited conditions.

In human societies, not everyone is born equal. The phrase `born with a silver spoon in the mouth' refers to the fact that, at birth, some people find themselves in positions of privilege and wealth whereas others do not. Similar inequalities exist within animal societies and, in the case of some insects such as ants and bees, are taken to great extremes. Ants and bees have evolved eusociality – several generations live together in colonies, only one or a few individuals (queens) have offspring, and non-reproductive colony members, the workers, care for these offspring. Becoming a queen is not something that workers can aspire to. Although some sneaky workers do try to have offspring themselves, individuals born as workers cannot become queens. Nutrition is an important determinant of social class in many eusocial insect species. In some species, larvae destined to become queens are fed a particular substance (royal jelly in honey bees), whereas in other species queen larvae get more food than worker larvae. This latter system is found in the stingless bee Schwarziana quadripunctata. Now, an international team of researchers from Belgium, Britain and Brazil led by Tom Wenseleers has discovered that some stingless bees manage to beat the system and change their fate.The team investigated previous reports that dwarf queens occur in stingless bee colonies in addition to the normal, large queens. Wenseleers and his co-workers weighed the dwarf queens and showed that they are indistinguishable in weight from regular worker bees. In addition, dwarf queen larvae are raised in cells that are identical to the cells in which worker larvae are raised,which are smaller than the large specialized cells in which the queen larvae develop. Dwarf queens aren't a rare occurrence; when the team assessed the social class of 11 574 individual stingless bees from 19 colonies, they found six times more dwarf queens than normal queens. The team used a series of morphological measurements to identify adult dwarf queens and distinguish them from normal queens. They showed that the dwarf queens headed 12 out of a further 54 colonies, suggesting that the dwarf queens are able to reproduce. Although this may seem like a large proportion of colonies, it actually suggests that dwarf queens aren't as successful as normal queens – 86%of queens reared were dwarf queens, but they only headed 22% of the colonies that the team studied. Wenseleers and his co-workers suggest that this discrepancy may be due to workers actively discriminating against the dwarf queens.Many questions remain about this intriguing system in which individuals seem able to choose whether to become queens. In particular, it would be interesting to know exactly which mechanisms determine the fate of stingless bee larvae. What role does nutrition play in determining their fate? If nutrition determines whether an individual becomes a queen or a worker, then some larvae destined to become workers are apparently overcoming nutritional limits and becoming queens despite their limited food supply. What physiological mechanisms control nutritional responses in this system? Recent advances in Drosophila have given us some places to start looking for answers, such as insulin receptor pathways. Perhaps these or other, as yet unknown, pathways play a role in the production of dwarf queens in stingless bee colonies. Whatever the case, this system has the potential to keep ecologists and physiologists busy for many years to come.

Although the taxonomical/phylogenetic diversity of microbial communities in biological heap leaching systems has been investigated, the diversity of functional genes was still unclear, and, especially, the differentiation and the relationships of diversity and functions of microbial communities in leaching heap (LH) and leaching solution (LS) were also still unclear. In our study, a functional gene array (GeoChip 5.0) was employed to investigate the functional gene diversity, and 16S rRNA gene sequencing was used to explore the taxonomical/phylogenetic diversity of microbial communities in LH and LS subsystems of Dexing copper mine (Jiangxi, China). Detrended correspondence analysis (DCA) showed that both functional gene structure and taxonomical/phylogenetic structure of microbial communities were significantly different between LH and LS. Signal intensities of genes, including genes for sulfur oxidation (e.g., soxB), metal homeostasis (e.g., arsm), carbon fixation (e.g., rubisco), polyphosphate degradation (e.g., ppk), and organic remediation (e.g., hydrocarbons) were significantly higher in LH, while signal intensities of genes for carbon degradation (e.g., amyA), polyphosphate synthesis (e.g., ppx), and sulfur reduction (e.g., dsrA) were significantly higher in LS. Further inspection revealed that microbial communities in LS and LH were dominated by Acidithiobacillus and Leptospirillum. However, rare species were relatively higher abundant in LH. Additionally, diversity index of functional genes was significantly different in LS (9.915±0.074) and LH (9.781±0.165), and the taxonomical/phylogenetic diversity index was also significantly different in LH (4.398±0.508) and LS (3.014±0.707). Functional tests, including sulfur-oxidizing ability, iron-oxidizing ability, and pyrite bioleaching ability, showed that all abilities of microbial communities were significantly stronger in LH than those in LS. Further studies found that most key genes (e.g., soxC and dsrA), rather than functional gene diversity index, were significantly correlated with abilities of microbial communities by linear regression analysis and Pearson correlation tests. In addition, the abilities were significantly correlated with taxonomical/phylogenetic diversity index and some rare species (e.g., Ferrithrix).

As a polymicrobial disease, sour rot decreases grape berry yield and wine quality. The diversity of microbial communities in sour rot-affected grapes depends on the cultivation site, but the microbes responsible for this disease in eastern coastal China, has not been reported. To identify the microbes that cause sour grape rot in this important grape-producing region, the diversity and abundance of bacteria and fungi were assessed by metagenomic analysis and cultivation-dependent techniques. A total of 15 bacteria and 10 fungi were isolated from sour rot-affected grapes. High-throughput sequencing of PCR-amplicons generated from diseased grapes revealed 1343 OTUs of bacteria and 1038 OTUs of fungi. Proteobacteria and Firmicutes were dominant phyla among the 19 bacterial phyla identified. Ascomycota was the dominant fungal phylum and the fungi Issatchenkia terricola, Colletotrichum viniferum, Hanseniaspora vineae, Saprochaete gigas, and Candida diversa represented the vast majority ofmicrobial species associated with sour rot-affected grapes. An in vitro spoilage assay confirmed that four of the isolated bacteria strains (two Cronobacter species, Serratia marcescens and Lysinibacillus fusiformis) and five of the isolated fungi strains (three Aspergillus species, Alternaria tenuissima, and Fusarium proliferatum) spoiled grapes. These microorganisms, which appear responsible for spoiling grapes in eastern China, appear closely related to microbes that cause this plant disease around the world.